Molecular Interactions Between Childhood Acute Lymphoblastic Leukaemia Cells And The Microenvironment

Ana Markovic

A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy

Children’s Institute Australia for Medical Research

School of Women’s and Children’s Health THE UNIVERSITY OF NEW SOUTH WALES 2009

THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: MARKOVIC First name: ANA Other name/s: Abbreviation for degree as given in the University calendar: PhD School: Women’s & Children’s Health Faculty: MEDICINE Title: Molecular Interactions Between Childhood Acute Lymphoblastic Leukaemia Cells And The Bone Marrow Microenvironment.

Abstract 350 words maximum Acute lymphoblastic leukaemia (ALL) is the most common cause of death from disease in children. Whilst cure rates over the last 30 years have drastically improved, children that do go on and relapse have a very poor prognosis. Additionally, the ones that do survive can have significant long term side effects from existing treatments. Understanding the molecular mechanisms of the relationship between leukaemia and its microenvironment is essential for the identification of novel targets for treatment and/or the manipulation of existing treatments.

Our previous work has established a panel of childhood ALL xenografts from patient in NOD/SCID mice. Several samples secrete vascular endothelial growth factor (VEGF), an integral component of neovascularisation and normal , and express FMS-like tyrosine kinase-3 (FLT-3), a tyrosine kinase, which plays an essential role in regulating normal haematopoiesis. This thesis builds on previous work by examining the relationship between VEGF and FLT-3, two widely, yet independently studied molecules in leukaemia, with the aberrant expression of either having adverse outcomes for patients.

The results show that the high expression and activation of FLT-3, significantly increases secretion of VEGF. To assess whether VEGF secretion is triggered by FLT-3 signalling, we measured VEGF in the absence and presence of an inhibitor (SU11657), humanised anti-FLT-3 blocking antibodies as well as decreasing the receptors with siRNA. All of these manipulations were able to decrease the secretion of VEGF in leukaemia cells. To further investigate this relationship, we examined the phosphorylation status of FLT-3 and the downstream signalling pathway. Our results indicate that FLT-3 signalling may be an important factor in the induction of VEGF secretion in a sub-type of leukaemia cells, and in turn, VEGF secretion can be attenuated by an FLT-3 specific inhibitor. Two separate microarray studies were also used to assess simultaneous expressions between the leukaemia and bone marrow microenvironment, and to examine the effects of FLT-3 ligand on ALL xenograft cells. The results of the microarray studies confirm the previously observed results regarding the manipulation of the microenvironment by the leukaemic cells.

Inhibition of the FLT-3/VEGF pathway may disrupt paracrine signalling between leukaemia cells and the bone marrow microenvironment, and future studies into how this disruption may influence leukaemia cell responses to conventional are warranted.

Declaration relating to disposition of project thesis/dissertation

I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only).

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Declaration

ORIGINALITY STATEMENT

‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’

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Declaration iii



Acknowledgements

This thesis would not be possible without the support, belief and guidance of many people. I thank my supervisor A/Prof. Richard Lock for the opportunity he provided to undertake this work, along with Dr. Karen Mackenzie for her support. I have learned a great deal during my time at the Children's Cancer Institute Australia.

I would also like to acknowledge the Berry family together with the Australian Academy of Science and the Foundation of the National Institutes of Health for establishing the fellowship which enabled me to experience working at the NIH. This includes, in particular, Dr. Javed Khan and his lab for accepting me for the short time I was there and teaching me about microarrays.

I thank the staff at the faculty of Science at the University of NSW, for giving me the opportunity to experience the joy of teaching. Also, I want to thank the friends that have stayed by me in this madness, otherwise known as a PhD; especially Dr. Marina Pajic, Dr. Scott Melville and Laura Veas.

Thank you to Dr. Andrew Kinsela, who has learned more about leukaemia, more than he ever thought he would or even wanted to know. Thank you, I am very grateful for all your love and support. Looks like I too have made it to the end.

A thank you also needs to go to my grandparents. I am just sorry my grandfather is not here to see the completion of this thesis. Finally my mum and dad and my brother Ivan – thank you, I love you.

Acknowledgements v



Conferences, Publications and Awards

Conferences

Markovic A, MacKenzie KL, Lock RB. (2007) Induction of vascular endothelial growth factor secretion in acute cells via the FLT-3 signaling pathway, AACR- NIH-EORTC Conference “Molecular Targets and Cancer Therapeutics: Discovery, Biology, and Clinical Applications, San Francisco, USA.

Markovic A, MacKenzie KL, Lock RB. (2007) Induction of VEGF Secretion in Acute Leukaemia Cells Through the FLT-3 Signalling Pathway, 19th Lorne Cancer Conference, Lorne, Victoria, Australia.

Markovic A, MacKenzie KL, Lock RB. (2006) Novel Function of FLT-3: Induction of the Vascular Endothelial Growth Factor in Acute Leukaemia, Australian Society for Medical Research annual NSW Meeting, Sydney, Australia.

Markovic A. (2005) Experience at the NIH as the Inaugural Recipient of the Adam J. Berry Memorial Fellowship, Annual Meeting of the Australian Academy of Science, Canberra, Australia.

Conferences, Publications and Awards vii Publications

Markovic A, MacKenzie KL, Lock RB. FLT-3: a new focus in the understanding of acute leukemia. Int J Biochem Cell Biol. 37(6):1168-72.

Markovic A, MacKenzie KL, Lock RB. Induction of VEGF in leukaemia cells (manuscript in preparation)

Awards

2007 - AACR Eli-Lilly Travel Award, for the conference AACR-NIH-EORTC Conference “Molecular Targets and Cancer Therapeutics: Discovery, Biology, and Clinical Applications, San Francisco, USA.

2005 - Australian Academy of Science & the Foundation for the National Institutes of Health USA - Inaugural Recipient of the Adam J. Berry Memorial Fellowship

Conferences, Publications and Awards viii 

Abstract

Acute lymphoblastic leukaemia (ALL) is the most common cause from death of disease in children. Whilst cure rates over the last 30 years have drastically improved, the children that do go on and relapse have a very poor prognosis. Additionally, the ones that do survive can have significant long term side effects from existing treatments. Understanding the molecular mechanisms of the relationship between leukaemia and its microenvironment is essential for the identification of novel targets for treatment and/or the manipulation of existing treatments.

The role that vascular endothelial growth factor (VEGF), an integral component of both neovascularisation and normal haematopoiesis, plays in the progression and invasiveness of solid tumours is well established. However, its function in haematological malignancies has been a more recent and thus less considered observation. leukaemia cells secrete VEGF, which may act in a paracrine manner with the bone marrow microenvironment to promote the survival and proliferation of leukaemia cells. In addition to VEGF being produced by leukaemias, it also increases vascularity in the bone marrow and lymph nodes of patients.

Our previous work has established a panel of 10 childhood acute lymphoblastic leukaemia xenografts from patient biopsies in NOD/SCID mice. Several of these secrete VEGF, and express the FMS-like tyrosine kinase-3 (FLT-3). FLT-3, a receptor tyrosine kinase (RTK), and its ligand, play an essential role in regulating normal haematopoiesis. This thesis builds on the previous work by examining the relationship between VEGF and FLT-3, two widely, yet independently studied molecules in leukaemia, with the aberrant expression of either having adverse outcomes for patients.

Abstract ix The results show that the high expression and activation of FLT-3, significantly increases the secretion VEGF. To assess whether VEGF secretion is triggered by FLT-3 signalling, we measured VEGF in the absence and presence of a class III receptor tyrosine kinase (RTK) inhibitor (SU11657), humanised anti-FLT-3 blocking antibodies as well as decreasing the receptors with siRNA. All of these manipulations were able to decrease the secretion of VEGF in leukaemia cells. To further investigate this relationship, we examined the phosphorylation status of FLT-3 and the downstream signalling pathway. Our results indicate that FLT-3 signalling may be an important factor in the induction of VEGF secretion in a sub-type of leukaemia cells and in turn, VEGF secretion can be attenuated by an FLT-3 specific inhibitor. Two separate microarray studies were also used to assess simultaneous gene expressions between the leukaemia and bone marrow microenvironment, and to examine the effects of FL on ALL xenograft cells. The results of the microarray studies confirm the previously observed results regarding the manipulation of the microenvironment by the leukaemic cells.

Inhibition of the FLT-3/VEGF pathway may disrupt paracrine signalling between leukaemia cells and the bone marrow microenvironment, and future studies into how this disruption may influence leukaemia cell responses to conventional chemotherapy are warranted.

Abstract x 

Table of Contents

DECLARATION ...... III ACKNOWLEDGEMENTS ...... V CONFERENCES, PUBLICATIONS AND AWARDS ...... VII ABSTRACT ...... IX LIST OF TABLES ...... XVI LIST OF FIGURES ...... XIX ABBREVIATIONS ...... XXIII CHAPTER ONE: INTRODUCTION ...... 1 1.1 PREFACE ...... 1 1.2 LEUKAEMIA ...... 4 1.2.1 Introduction to Leukaemia: The Disease (ALL & AML) and Prognoses ...... 4 1.2.2 Chromosomal Translocations ...... 13 1.2.3 Mixed Lineage Leukaemia (MLL) ...... 15 1.3 BONE MARROW MICROENVIRONMENT ...... 18 1.3.1 Normal Haematopoiesis ...... 18 1.3.2 Function of the BM with regard to Leukaemia ...... 20 1.4 LEUKAEMIA AND ANGIOGENESIS ...... 22 1.4.1 Angiogenesis ...... 22 1.4.2 Leukaemia and Angiogenesis ...... 24 1.5 HYPOXIA AND THE HYPOXIA INDUCIBLE FACTOR ...... 25 1.5.1 Hypoxia Inducible Factors ...... 26 1.6 VASCULAR ENDOTHELIAL GROWTH FACTOR ...... 30 1.6.1 VEGF-A ...... 31 1.6.2 VEGF Receptors ...... 36 1.6.3 Role of VEGF in Leukaemia ...... 41 1.7 FMS-LIKE TYROSINE-3 ...... 44 1.7.1 Receptor Tyrosine Kinases ...... 44 1.7.2 FLT-3 ...... 45 1.7.3 Mutations in FLT-3 and Expression in Leukaemia ...... 48

Table of Contents xi 1.7.4 Signalling Cascade of FLT-3 ...... 49 1.8 SUMMARY ...... 51

CHAPTER TWO: MATERIALS AND METHODS ...... 53 2.1 TISSUE CULTURE ...... 53 2.1.1 Reagents ...... 53 2.1.2 In Vitro Cell Culture ...... 54 2.2 ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) ...... 54 2.2.1 Reagents ...... 54 2.2.2 ELISA ...... 55 2.3 FLOW CYTOMETRY ...... 56 2.3.1 Reagents ...... 56 2.3.2 Cell viability ...... 56 2.3.3 Receptor levels ...... 56 2.4 3-(4,5-DIMETHYLTHIAZOL-2-YL)-2,5-DIPHENYL TETRAZOLIUM BROMIDE (MTT) CYTOTOXICITY ASSAY ...... 57 2.4.1 Reagents ...... 57 2.4.2 MTT Assay ...... 57 2.5 RNA ISOLATION ...... 58 2.5.1 Reagents ...... 58 2.5.2 RNA Isolation: With Trizol® ...... 58 2.5.3 Modified RNA Isolation: With Trizol® and the RNeasy Kit ...... 59 2.5.4 Formaldehyde Agarose Gel Electrophoresis for Determining RNA Integrity ...... 59 2.6 COMPLEMENTARY DNA SYNTHESIS AND REAL-TIME REVERSE TRANSCRIPTASE -POLYMERASE CHAIN REACTION (RT-PCR)...... 61 2.6.1 Reagents ...... 61 2.6.2 cDNA synthesis ...... 62 2.6.3 Real-Time RT-PCR ...... 63 2.7 SEMI-QUANTITATIVE RT-PCR OF VEGF ISOTYPES ...... 64 2.7.1 Reagents ...... 64 2.7.2 RT-PCR ...... 65 2.7.3 Agarose Gel Elecrophoresis ...... 65 2.8 VEGF MRNA STABILITY ...... 66 2.8.1 Reagents ...... 66 2.8.2 Actinomycin D Experiment ...... 66 2.9 GENOMIC DNA PCR OF THE FLT-3 RECEPTOR ...... 67 2.9.1 Reagents ...... 67 2.9.2 Isolation of genomic DNA (gDNA) ...... 68 2.9.3 PCR of the FLT-3 Receptor ...... 68 2.9.4 Enzyme Digestion ...... 69 2.10 IMMUNOPRECIPITATION AND IMMUNOBLOT ANALYSIS ...... 70

Table of Contents xii 2.10.1 Reagents ...... 70 2.10.2 Isolation and Quantification ...... 71 2.10.3 Immunoprecipitation of the FLT-3 Receptor ...... 72 2.10.4 Electrophoresis and Protein Transfer onto PVDF ...... 73 2.10.5 Western Immunoblotting ...... 73 2.11 SIRNA TRANSFECTION INTO ALL XENOGRAFT CELLS AND LEUKAEMIA CELL LINES ...... 74 2.11.1 Reagents ...... 74 2.11.2 Nucleofection ...... 74 2.12 MICROARRAY ...... 75 2.12.1 Reagents ...... 75 2.12.2 Slide Preparation ...... 76 2.12.3 Total RNA Extraction ...... 77 2.12.4 RNA Amplification ...... 78 2.12.5 Reverse Transcription Reaction of the RNA ...... 81 2.12.6 RNA Labelling ...... 82 2.12.7 Cy Dye coupling ...... 83 2.12.8 Probe Purification ...... 84 2.12.9 Prehybridisation ...... 84 2.12.10 Hybridisation ...... 85 2.12.11 Slide Washing ...... 85 2.12.12 Normalising and Filtering of the Slides ...... 86 2.13 CHROMATIN IMMUNOPRECIPITATION (CHIP) ...... 86 2.13.1 Reagents ...... 86 2.13.2 HIF ChIP ...... 87 2.13.3 PCR amplification of the VEGF promoter ...... 89 2.14 STATISTICAL ANALYSIS ...... 89

CHAPTER THREE: VEGF SECRETION BY LEUKAEMIA CELLS INDUCED BY FLT-3 ACTIVATION ...... 91 3.1 INTRODUCTION ...... 91 3.1.1 ALL Xenograft Mouse Model ...... 92 3.2 RESULTS ...... 95 3.2.1 Expression of FLT-3 on Leukaemia Cells ...... 95 3.2.2 Secretion of VEGF by Leukaemia Cells ...... 98 3.2.3 Inhibition of VEGF Secretion with a FLT-3 Small Molecule Inhibitor ...... 103 3.2.4 VEGF mRNA ...... 109 3.2.5 Inhibition of VEGF in ALL Xenograft cells with anti-FLT-3 Antibodies ...... 113 3.2.6 siRNA Knockdown of FLT-3 and effects on VEGF ...... 115 3.3 DISCUSSION ...... 116

CHAPTER FOUR: ACTIVATION OF THE FLT-3 SIGNALLING PATHWAY AND ITS INDUCTION OF VEGF ...... 125

Table of Contents xiii 4.1 INTRODUCTION ...... 125 4.2 RESULTS ...... 128 4.2.1 Mutation and Phosphorylation Status of The FLT-3 Receptor ...... 128 4.2.2 FLT-3 Signalling Pathway and the Effects of SU11657 ...... 131 4.2.3 Effects of RTK Inhibitors on the FLT-3 - VEGF Relationship ...... 135 4.2.4 Effects of Signalling Pathway Inhibitors on the FLT-3 - VEGF Relationship ...... 141 4.2.5 Effects of Anti-FLT-3 Antibodies ...... 146 4.3 DISCUSSION ...... 147

CHAPTER FIVE: GENE DETERMINATION OF SELECT ALL XENOGRAFTS USING MICROARRAY ...... 153 5.1 INTRODUCTION ...... 153 5.2 RESULTS ...... 157 5.2.1 Effects of BM Stromal Cells on in ALL Xenograft cells ...... 157 5.2.2 Effects of FL on Gene Expression in ALL Xenograft cells ...... 170 5.2.3 Gene confirmation analyses ...... 183 5.3 DISCUSSION ...... 187 5.3.1 Gene Expression Profiles of Microarray Studies ...... 187 5.3.2 Specific confirmations ...... 190

CHAPTER SIX: GENERAL DISCUSSION AND FUTURE RESEARCH DIRECTIONS ...... 197 REFERENCES ...... 203 APPENDICES ...... 259 APPENDIX A ...... 259 A.1 EFFECTS OF BM STROMAL CELLS ON ALL XENOGRAFT CELLS ...... 259 A.1.1 T-Test ...... 259 APPENDIX B ...... 269 B.1 TOP 67 GENES BY T-TEST ...... 269 APPENDIX C ...... 275 C.1 GENESPRING ...... 275 C.1.1 GeneSpring filtering by fold change ...... 275 APPENDIX D ...... 291 D.1 SUMMARY OF GENES OBTAINED FROM ALL THREE METHODOLOGIES ...... 291 APPENDIX E ...... 295 E.1 EFFECTS OF FL ON GENE EXPRESSION IN ALL XENOGRAFT CELLS...... 295 E.1.1 Xenograft: ALL-3 ...... 295 E.1.2 Xenograft: ALL-17 ...... 297 E.1.3 Xenograft: ALL-19 ...... 298 APPENDIX F ...... 299 F.1 GENES TWO-FOLD DIFFERENTIALLY REGULATED BY FL IN ALL-3 XENOGRAFT CELLS ...... 299 APPENDIX G ...... 335 G.1 GENES TWO-FOLD DIFFERENTIALLY REGULATED BY FL IN ALL-17 XENOGRAFT CELLS ..... 335

Table of Contents xiv APPENDIX H ...... 361 H.1 GENES TWO-FOLD DIFFERENTIALLY REGULATED BY FL IN ALL-19 XENOGRAFT CELLS..... 361 APPENDIX I ...... 379 I.1 HIF CHIP ...... 379

Table of Contents xv 

List of Tables

Table 1.1. Classification of childhood acute leukaemias...... 8 Table 1.2. Genetic abnormalities found in ALL and their proportions in children and adults...... 9 Table 1.3. Side effects of drugs used to treat leukaemia...... 12 Table 1.4. Subtypes of childhood leukaemia and their associated chromosomal changes...... 14 Table 1.5. Some of the current strategies manipulating VEGF and its receptors in the treatment of different ...... 40 Table 1.6. Some of the compounds in use against FLT-3...... 48 Table 2.1. 10X MOPS Buffer...... 60 Table 2.2. 1X MOPS Running Buffer...... 60 Table 2.3. 5X RNA Loading Buffer...... 61 Table 2.4. EF1 primer and probe sequence...... 62 Table 2.5. List of gene expression assays...... 62 Table 2.6. cDNA RT master mix reagents...... 63 Table 2.7. Real-time master mix...... 63 Table 2.8. Real-time cycle times...... 64 Table 2.9. VEGF primer sequences...... 64 Table 2.10. PCR master mix for VEGF isotypes...... 65 Table 2.11. PCR running conditions for both VEGF and EF1...... 65 Table 2.12. PCR master mix for Actinomycin D experiments...... 67 Table 2.13. Primer sequences...... 67 Table 2.14. PCR master mix FLT-3...... 69 Table 2.15. PCR conditions for FLT-3 and GAPDH...... 69 Table 2.16. EcoRV digestion of FLT-3...... 70 Table 2.17. Table of all immunoblotting antibodies and their conditions...... 71 Table 2.18. Cell lysis buffer for protein extraction...... 72 Table 2.19. PBS without magnesium or calcium for use in microarray experiments...... 76 Table 2.20. Slide cleaning solution...... 76 Table 2.21. Slide coating solution...... 77 Table 2.22. Blocking solution for 60 slides...... 77 Table 2.23. RNA Amplification 1st strand synthesis enzyme reaction...... 79

Abbreviations xvi Table 2.24. RNA Amplification 2nd strand synthesis reaction...... 79 Table 2.25. Reaction mixture for in vitro transcription reaction...... 81 Table 2.26. RNA and primer hybridisation for RT reaction...... 81 Table 2.27. Reverse transcription reaction for cDNA probes...... 82

Table 2.28. 1 M KPO4 buffer ...... 82 Table 2.29. Wash buffer...... 83 Table 2.30. Elution buffer...... 83 Table 2.31. Carbonate buffer...... 83 Table 2.32. Prehybridisation solution...... 84 Table 2.33. 1X Hybridisation solution...... 85 Table 2.34. Probe solution...... 85 Table 2.35. Slide washing procedure ...... 85 Table 2.36. Primer sequences for the VEGF promoter...... 87 Table 2.37. ChIP washing procedure ...... 88 Table 2.38. PCR master mix for the VEGF HRE...... 89 Table 2.39. PCR running conditions for VEGF HRE...... 89 Table 3.1. Characteristics of the ALL xenograft panel, including patient clinical data...... 94 Table 3.2. Leukaemia cell lines and their characteristics...... 95 Table 3.3. Expression of FLT-3 and VEGF secretion on Day 3 by ALL xenograft cells...... 102 Table 4.1. List of inhibitors and their specificity...... 127 Table 5.1. Summary of the 20 discriminating genes as identified by the t test...... 161 Table 5.2. The 14 most significant genes as scored by SAM...... 166 Table 5.3. Data generated by the GeneSpring individual approach analysis...... 168 Table 5.4. List of the 18 discriminating genes as identified by the individual approach analysis...... 169 Table A.1. Descriptions of functions for the 20 discriminating genes as identified by a t test...... 259 Table B.1. Descriptions of functions for the 67 discriminating genes as identified by a t test...... 270 Table C.1. Sample and gene clustering following GeneSpring average approach analysis...... 277 Table C.2. Fold change cut off, genes and dendrograms for the individual approach GeneSpring analysis...... 280 Table C.3. The top 28 discriminating genes as identified by GeneSpring analysis...... 281 Table C.4. Descriptions of functions for the 28 discriminating genes as identified by GeneSpring analysis...... 283 Table D.1. Gene lists by t-test...... 291 Table D.2. Gene lists by SAM...... 292 Table D.3. Gene lists by GeneSpring...... 293 Table E.1. Top genes by slope in ALL-3 xenograft cells...... 295 Table E.2. Top genes by slope in ALL-17 xenograft cells...... 297 Table E.3. Top genes by slope in ALL-17 xenograft cells...... 298 Table F.1. Genes changed by greater than 2-fold in ALL-3 xenograft cells after 2 hrs...... 299 Table F.2. Genes changed by greater than 2-fold in ALL-3 xenograft cells after 6 hrs...... 307

Abbreviations xvii Table F.3. Genes changed by greater than 2-fold in ALL-3 xenograft cells after 24 hrs...... 321 Table G.1. Genes changed by greater than 2-fold in ALL-17 xenograft cells after 2 hrs...... 335 Table G.2. Genes changed by greater than 2-fold in ALL-17 xenograft cells after 6 hrs...... 343 Table G.3. Genes changed by greater than 2-fold in ALL-17 xenograft cells after 24 hrs...... 352 Table H.1. Genes changed by greater than 2-fold in ALL-19 xenograft cells after 2 hrs...... 361 Table H.2. Genes changed by greater than 2-fold in ALL-19 xenograft cells after 6 hrs...... 366 Table H.3. Genes changed by greater than 2-fold in ALL-19 xenograft cells after 24 hrs...... 372

Abbreviations xviii 

List of Figures

Figure 1.1. Leukaemic transformation on a simplified haematopoiesis tree...... 5 Figure 1.2. Schematic diagram of normal haematopoiesis...... 19 Figure 1.3. Regulation of HIF-1...... 28 Figure 1.4. VEGF growth factors and their receptors...... 31 Figure 1.5. Schematic representation of seven different VEGF isotypes...... 32 Figure 1.6. Schematic representation of the possible paracrine and autocrine loops between leukaemia and the BM microenvironment...... 41 Figure 1.7. Structure of FLT-3...... 47 Figure 1.8. The FLT-3 signalling cascade...... 50 Figure 3.1. Diagrammatic representation of the ALL continuous xenograft model...... 93 Figure 3.2. Flow cytometry histograms of FLT 3 expression levels...... 98 Figure 3.3. VEGF secretion by ALL-3 cultured on MS5 cells...... 99 Figure 3.4. VEGF secretion by ALL xenograft cells...... 100 (A) FL increased VEGF secretion ( ) compared with no FL addition ( ) in ALL-3 and P-14 xenograft cells, *P = 0.0002 a ...... 101 Figure 3.5. Secretion of VEGF by ALL-3 xenograft cells after 72 hrs of culture...... 101 ALL-3 cells were cultured without or with an MS5 stromal support layer, the CM was collected and VEGF was measured by ELISA. The amount of VEGF secretion was normalised to the amount of live cells at the time of harvest, as measured by flow cytometry, described in Section 2.3.2. FL increased the VEGF secretion ( ) compared without FL addition ( ). This secretion increased 10-fold with culturing on a MS5 stromal support layer. FL increases this further, by 2.5-fold without MS5s and 4.5-fold in cells grown with MS5s (P < 0.01 for both). Results are the mean ± SE of at 17 separate experiments...... 101 Figure 3.6. VEGF secretion by leukaemia cell lines after 3 days of culture...... 103 Figure 3.7 A & B. Cell survival in increasing concentrations of SU11657 measured by MTT assay. .... 104 Figure 3.8. Effects of SU11657 on VEGF secretion by ALL-3 xenograft cells...... 105 Figure 3.9. Effects of SU11657 on VEGF secretion by different xenograft cells...... 107 Figure 3.10. Effects of SU11657 on VEGF secretion by different leukaemia cell lines...... 108 Figure 3.11. Isotypes of VEGF mRNA expressed in ALL-3 xenograft and MV4;11 cells...... 109 Figure 3.12. VEGF mRNA in ALL-3 measured over time...... 110

Abbreviations xix Figure 3.13 A & B. Effects of SU11657 on VEGF mRNA expression...... 111 Figure 3.14 A & B. Stability of VEGF mRNA...... 112 Figure 3.15. Effects of FLT-3 blocking antibodies on VEGF secretion by ALL-3 xenograft cells...... 113 Figure 3.16. Effects of FLT-3 blocking antibodies on VEGF secretion by ALL-3 xenograft cells cultured on MS5 stromal cells...... 114 Figure 3.17. Western blots showing FLT-3 knockdown by siRNA...... 115 Figure 3.18 A & B. VEGF secretion after siRNA knockdown of FLT-3...... 116 Figure 3.19. The structures of SU11657 and SU11248...... 121 Figure 4.1. Analysis of the FLT-3 gene for known mutations...... 129 Figure 4.2. Activation of FLT-3 with its ligand and the effects of SU11657...... 131 Figure 4.3. Effects of SU11657 on the FLT-3 pathway in ex vivo cultured ALL xenograft cells...... 132 Figure 4.4. Effects of SU11657 on the FLT-3 signalling pathway in the leukaemia cell lines...... 133 Figure 4.5. Western blot analyses of STAT5 activation...... 134 Figure 4.6. Immunoprecipitation of STAT5 from ALL-2 and -3 xenograft cells...... 135 Figure 4.7. Effect of different inhibitors on VEGF secretion in the MV4;11 cell line...... 136 Figure 4.8 A & B. Effects of RTK inhibitors on FLT-3 phosphorylation in the MV4;11 cell line...... 137 Figure 4.9. Effects of a range of inhibitors on the FLT 3 signalling pathway in MV4;11 cells...... 138 Figure 4.10. Effects of different inhibitors on VEGF secretion by ALL-3 xenograft cells...... 139 Figure 4.11 A & B. Effects of RTK inhibitors on the activation of FLT-3 in ALL-3 xenograft cells...... 140 Figure 4.12. Effects of RTK inhibitors on AKT and ERK1/2 in ALL-3 xenograft cells...... 141 Figure 4.13. Effects of different pathway inhibitors on VEGF secretion in the MV4;11 cell line...... 142 Figure 4.14. Effects of different pathway inhibitors on FLT-3 signalling in MV4;11 cells...... 143 Figure 4.15. Effects of different pathway inhibitors on VEGF secretion by ALL-3 xenograft cells...... 144 Figure 4.16. Effects of different inhibitors on AKT and ERK1/2 in ALL-3 xenograft cells...... 145 Figure 4.17. Correlation between the decrease in VEGF versus the decrease in ERK1/2 phosphorylation...... 145 Figure 4.18. Effects of blocking antibodies on FLT-3 phosphorylation in ALL-3 xenograft cells...... 146 Figure 4.19. Effects of blocking antibodies on the FLT-3 signalling pathway...... 147 Figure 5.1. Two possible pooling strategies for microarray samples...... 155 Figure 5.2. An unsupervised centred cluster...... 158 Figure 5.3. Top 20 discriminating genes as identified by the t test...... 160 Figure 5.4. Pearson centred cluster of the 20 discriminating genes as identified by the t test...... 162 Figure 5.5 A, B & C. The top 20 differentially expressed genes categorised into ontology groups...... 164 Figure 5.6. Pearson centred cluster of the top 1547 genes according to t test...... 165 Figure 5.12. Global gene expression heat map and hierarchical clustering...... 171 Figure 5.13 A, B & C. groups of the differentially expressed genes in ALL-3 ...... 173 Figure 5.14 A, B & C. Gene ontology groups of the differentially expressed genes in ALL-17 ...... 174 Figure 5.15 A, B & C. Gene ontology groups of the differentially expressed genes in ALL-19 ...... 175 Figure 5.16. Venn diagrams of common genes across time points in ALL-3...... 176 Figure 5.17. Venn diagrams of common genes across time points in ALL-17...... 177

Abbreviations xx Figure 5.18. Venn diagrams of common genes across time points in ALL-19...... 178 Figure 5.19. Venn diagrams of common genes across all xenografts at the 2 hr time point...... 179 Figure 5.20. Venn diagrams of common genes across all xenografts at the 6 hr time point...... 180 Figure 5.21. Venn diagrams of common genes across all xenografts at the 24 hr time point...... 181 Figure 5.22 A, B, C & D. Expression profiles of ALL-17 and -19 of the differentially expressed genes identified in ALL-3...... 182 Figure 5.24. Time course for real-time RT-PCR of HIF1 expression for six ALL xenograft cells...... 183 Figure 5.25. Time course for real-time RT-PCR of TERF2 expression for six ALL xenograft cells ...... 184 Figure 5.26. Time course for real-time RT-PCR of P4HA1 expression for six ALL xenograft cells ...... 185 Figure 5.27. Time course for real-time RT-PCR of EGR1 expression for six ALL xenograft cells ...... 186 Figure 5.28. Time course for real-time RT-PCR of EGR1 expression examining the effects of FL ...... 186 Figure 5.29. Western blot of EGR1 expression in ALL xenograft cells...... 187 Figure B.1. Gene set of 67 genes considered to be discriminating according to t test...... 272 Figure B.2 A, B & C. The top 70 differentially expressed genes clustered into ontology groups by function, process and component...... 273 Figure C.1. Schematic diagram of the two different approaches used by GeneSpring to analyse gene expression data...... 276 Figure C.2 A & B. GeneSpring average approach genes at 15-fold cut off...... 279 Figure C.3. Pearson centred cluster of 28 genes found to be discriminating by GeneSpring...... 282 Figure I.1 ChIP assay of the HIF-1 binding capacity to the VEGF promoter...... 379

Abbreviations xxi



Abbreviations   Ab antibody FA Formaldehyde agarose aFGF acidic fibroblast growth factor FAB French-American-British ALL acute lymphoblastic leukaemia FCS foetal calf serum AML acute myeloid leukaemia FDR false discovery rate APC allophycocyanin FGF fibroblast growth factor ATP adenosine triphosphate FGFR FGF receptor  FL FLT-3 ligand FLT-3 BCA bicinchoninic acid FMS-like tyrosine kinase-3 Bcl B-cell  B-CLL B-cell chronic lymphocytic leukaemia G-CSF CSF BD Becton Dickinson gDNA genomic DNA bFGF basic fibroblast growth factor GM-CSF granulocyte-macrophage CSF BM bone marrow GTP guanosine triphosphate BMEC BM endothelial cells  bp HCL hydrochloric acid BSA Bovine Serum Albumin HRE hypoxia response elements  HRP Strepavidin-Horseradish Peroxidase cDNA complementary DNA hrs hours ChIP Chromatin Immunoprecipitation  CM conditioned media IC50 half maximal inhibitory concentration CML chronic myelocytic leukaemia IFN interferon CNS central nervous system Ig Immunoglobulin CSF colony-stimulating factor IGF insulin-like growth factor Ct threshold cycle IL interleukin  IMDM Iscove’s Modified Dubbecco’s Medium DMSO dimethylsulfoxide ITD internal tandem duplication DNA Deoxynucleic acid ITS Insulin, Transferrin and Selenium dNTPs deoxynucleoside triphosphates IFN interferon DTT dithiothreitol Ig immunoglobulin   ECM extracellular matrix JM juxtamembrane Ethylenediaminetetraacetic Acid EDTA  Tetrasodium Salt KDR kinase insert domain receptor EF elongation factor KI kinase insert EFS event free survival KDRi KDR inhibitors EGF epidermal growth factor KL KIT ligand EGR early growth response ELISA Enzyme Linked Immunosorbent Assay   ERK extracellular signal-regulated kinase LPS lipopolysaccharide ESTs expressed sequence tags

Abbreviations xxiii  MAPK mitogen-activated protein kinase M-CSF macrophage CSF MEKi MEK inhibitor MEM- minimum essential medium alpha MgCl2 magnesium chloride mixed lineage leukaemia or myeloid MLL lymphoid leukaemia MMLV Moloney murine leukaemia virus MMP matrix metalloproteinase [N-morpholino] propanesulfonic MOPS acid 3-(4,5-Dimethylthiazol-2-yl)-2,5- MTT diphenyltetrazolium bromide mRNA messenger Ribonucleic acid 

Na(VO2)3 sodium orthovanadate

NaCl2 sodium chloride NaF sodium fluoride National Center for Biotechnology NCBI Information non-obese diabetic/severe combined NOD/SCID immunodeficient NP-40 nonidet P  procollagen-proline, 2-oxoglutarate P4HA1 4-dioxygenase (proline 4- hydroxylase), alpha polypeptide I PBS phosphate buffered saline PCR polymerase chain reaction PDGF derived growth factor PDGFR PDGF receptor PE phycoerythrin pg picograms PKC protein kinase-C PLG Phase Lock Gel PlGF placenta growth factor penicillin G sodium; streptomycin PSG sulfate, L-glutamine PVDF polyvinylidene difluoride  QBSF-60 Quality Biologicals Serum Free-60  RFI relative fluorescence intensity rhVEGF recombinant human VEGF Rn run rpm revolutions per minute Roswell Park Memorial Institute- RPMI-1640 1640 RT room temperature RT-PCR reverse transcriptase - PCR RTK receptor tyrosine kinase

Abbreviations xxiv  SAM significance analysis of microarray SCF stem-cell factor SDF stromal cell-derived factor SDS sodium dodecyl sulfate SF steel factor SMP skim milk powder SS DNA Salmon Sperm DNA signal transducer and activator of STAT5 transcription 5  TBS Tris Buffered Saline TBST TBS Tween-20 TERF telomeric repeat binding factor TGF transforming growth factors tissue inhibitor of matrix TIMP metalloproteinase TKD tyrosine kinase domain TMB 3,3’,5,5’-Tetramethylbenzidine TNF tumour necrosis factor Tris Trizma base UTR untranslated region  VEGF vascular endothelial growth factor VEGFR VEGF receptors VHL von Hippel-Lindau  w/v weight/volume WBC white  xg times gravity

Abbreviations xxv

 

CHAPTER ONE: Introduction

1.1 Preface

Cancer is the most common cause of death in children second only to injuries. Acute lymphoblastic leukaemia (ALL) is the most common childhood malignancy; and it is also the most common cause of death from disease in children (Pui et al. 1998b). Whilst current treatments have substantially improved the survival of children diagnosed with this disease, for those who relapse the likelihood for survival is poor. Indeed, the majority of patients who suffer an early bone marrow relapse eventually succumb to the disease, usually due to the leukaemia being drug resistant.

In normal haematopoiesis there is a dynamic relationship between haematopoietic cells and the bone marrow (BM) microenvironment (Campana et al. 1996). Since leukaemia cells originate from their normal counterparts and also reside in the BM microenvironment, the same is true for haematopoietic malignancies (Bradstock et al. 1996). Therefore, it logically follows that stromal cells will influence the proliferation and apoptosis of leukaemia cells through the production and secretion of a variety of cytokines and growth factors (Itoh et al. 1989; Jazwiec et al. 1998), which can enhance the survival and proliferation of leukaemia cells thereby affecting apoptosis (Konopleva et al. 2002). This interaction can either be provided by direct contact between the leukaemias and the stroma, or alternatively by the secretion of growth factors into the microenvironment. Leukaemia cells have also been shown to produce a variety of different cytokines and growth factors that influence their surroundings. For example, several studies have shown an increased vascularity in the bone marrow of patients

Chapter One: Introduction 1 suffering ALL, acute myeloid leukaemia (AML), chronic myelocytic leukaemia (CML), and in the lymph nodes of patients with B-cell non- and B-cell chronic lymphocytic leukaemia (B-CLL).

Understanding the molecular mechanisms of the relationship between leukaemia and its microenvironment is essential for the identification of targets for treatment and/or the manipulation of existing treatments. Ultimately, this research should provide the basis for a better understanding of the biology of leukaemia cell proliferation and survival, which in turn may lead to the development of targeted, more effective and less toxic therapies. One aspect of this study, which examines the interaction of leukaemia with the BM microenvironment has crossed into the field of angiogenesis. The traditional focus of angiogenesis research has been its role in solid tumours. However, over the last 10 years there has been a great deal of research examining the role that angiogenesis may play in the progression of haematological malignancies, and as such, this has been a concomitantly increasing area for drug development. Small molecule inhibitors are being developed in an attempt to disrupt the ‘cross-talk’ between the leukaemia cells and the microenvironment in which they reside and proliferate. These small molecule inhibitors specifically target malignant cells through their aberrant expression of growth factor receptors. As such, these targeted inhibitors have a two fold purpose: one is to inhibit the specific receptors on the leukaemia surface, modulating their function and allowing other drugs to be more effective at killing the cells, and the other is to be cytotoxic themselves towards leukaemia cells. Of interest in this thesis are two such targets, the angiogenic growth factor, vascular endothelial growth factor (VEGF) and the receptor tyrosine kinase, FMS-like tyrosine kinase-3 (FLT-3). They have both, yet separately been associated with leukaemia.

Chapter One: Introduction 2 The primary focus of this thesis is to enhance our understanding of the relationship between childhood ALL cells and the BM microenvironment. This was examined on three different levels generating the following broad aims of my thesis;

(1.) To examine the relationship between FLT-3 signalling and VEGF secretion; deciphering whether VEGF is a target of the FLT-3 signalling pathway in ALL cells, and if so, through which intermediary pathway this signal is conferred.

(2.) To identify the effects of FLT-3 activation on gene expression in ALL cells. The understanding of its effects and potential identification of new target genes will give a better understanding of, not just the FLT-3 pathway in general, but also its specific role in leukaemia proliferation and survival.

(3.) To understand the effects of BM stormal cells on the gene expression profile of ALL cells. The determination of the effects of the BM on a diverse panel of ALL cell types should assist with identifying genes that are important in supporting leukaemic growth. A key aspect of this aim will be to examine specific effects that ALL cells exert on their environment. This will have an additional benefit of potentially identifying novel targets for future manipulation in the treatment of this disease.

Chapter One: Introduction 3 1.2 Leukaemia

1.2.1 Introduction to Leukaemia: The Disease (ALL & AML) and Prognoses Leukaemia arises from the disregulated clonal expansion of immature and non- functional lymphoid or myeloid progenitor cells that are blocked at a particular stage of differentiation (Pui 1995). The type of leukaemia is dependent upon the point at which the transformation during haematopoiesis occurs. As such these cells share features with normal lymphoid or myeloid progenitors. Using the differences in surface antigens and cytoplasmic components, the distinction is generally based upon ALL cells more closely resembling the lymphoid precursors (Pui et al. 1993; Pui 1995), and AML cells more closely resembling the myeloid lineage (Lowenberg et al. 1999). Figure 1.1 is a simplified diagrammatic view of haematopoiesis, with the arrows highlighting the points of transformation in the generation of ALL and AML.

There are of course exceptions, for example, some infant acute leukaemias exhibit both myeloid and lymphoid characteristics (Russell 1997). Another important exception is Philadelphia positive ALL. The Philadelphia is formed by a translocation that joins part of the BCR gene on chromosome 22 with the c-ABL proto-oncogene on chromosome 9. Multiple lineages can be involved, indicating that the cell of origin is a common multipotent myeloid-lymphoid stem cell (Secker-Walker et al. 1993; Russell 1997).

Chapter One: Introduction 4 AML AML / Secondary AML Granulocyte

Erythrocyte

Ph + ALL Megakaryocyte

c-ALL Ph + ALL

B- T-ALL

T-lymphocyte

Pluripotent Lineage committed Mature cells stem cells progenitors

Figure 1.1. Leukaemic transformation on a simplified haematopoiesis tree. A pictorial representation of the cells at which leukaemiac transformations are thought to occur. Modified from Russel (1997).

The uncontrolled growth and accumulation of leukaemic blasts in the patient’s bone marrow leads to a failure of normal haematopoiesis resulting in a deficiency of erythrocytes (anaemia), (thrombocytopaenia), and leukocytes (especially neutrophils i.e., neutropenia) within the blood (Redaelli et al. 2005). If left untreated, the leukaemic blasts infiltrate other organs, such as the liver, , kidneys, lymph nodes and testes. In rare cases, usually in infant leukaemias, there is also infiltration of the central nervous system (CNS) at the time of diagnosis (Pui et al. 1998c). Despite low occurrences, all patients receive intrathecal treatment in current treatment protocols, as the incidence of CNS infiltration increases at relapse (Pui et al. 1998c). The disease manifests itself with the patients presenting with: fever, fatigue, pallor and weakness as a result of anaemia; bleeding nose and gums, and bruising from thrombocytopenia; in the respiratory and urinary tracts from leukopenia, and pain, as well as abdominal swelling due to hepatic and splenic enlargement. The disease progresses in a relatively rapid fashion and if left untreated, ultimately leads to death (Pui et al. 1998a; Uckun et al. 1998b).

Chapter One: Introduction 5 Leukaemia is classified based on morphologic and immunologic characteristics, and over the last 20 years, the study of the biological and genetic characteristics of childhood leukaemias has led to an increased understanding of the disease (Yeoh et al. 2002). These advances have led not only to the revision and to a point uniformity in the classification of leukaemias (Smith et al. 1996), but also to the development of new approaches to treatments and clinical management of the disease (Pui et al. 1998a). Certain genetic alterations are associated with particular types of leukaemia. Therefore identifying the specific cytogenetic lesions can determine the pathogenesis and prognosis of the disease (Look 1997). For example, ALL with either BCR-ABL or MLL- AF4 fusion genes have a high risk of treatment failure and are recommended for haematopoietic stem cell transplantation in first remission (Gishizky et al. 1992). The TEL-AML1 fusion gene on the other hand, is associated with low risk of treatment failure and extended survival (Shurtleff et al. 1995; McLean et al. 1996).

AML cells can be readily identified in most instances by the presence of Auer rods, myeloperoxidase, or monocyte-associated esterases. Leukaemic lymphoblasts however, lack specific morphologic or cytochemical features, meaning the diagnosis of ALL depends on immunophenotyping. Even though white blood counts can show anaemia thrombocytopenia and granulacytopenia, ALL diagnosis is only confirmed by bone marrow , and immunophenotyping.

Immunophenotype analysis initially subdivides ALL into two classes: B- and T-cell lineage. The B-cell lineage is further differentiated into B-precursor, common ALL (c-ALL) and the more mature B-ALL, and also transitional pre-B. Most of B-cell leukaemias, arising particularly in children, are thought to occur in committed, early B-lymphoid progenitor cells (Pui 1995). These cells are found in low numbers in the bone marrow. B-precursor ALL is the more common form of ALL in children, comprising about 80 - 85% of all cases. Generally, children diagnosed with B-precursor ALL have a relatively good prognosis. T-lineage ALL can be subclassified depending on the the expression of T-cell recptor or stage of thymocyte differentiation (Campana et al. 1991), but with limited clinical value (Pui et al. 1990a). T-cell ALL occurs in the cortical thymocytes (Russell 1997) and T-lineage ALL only accounts for 10 - 15% of childhood ALL, although it historically has a far less favourable prognosis and is more frequent in older (1 10 years), male ALL patients (Pui et al. 2001). In more recent years

Chapter One: Introduction 6 though, with the introduction of risk-adapted combination chemotherapy protocols, the overall survival rates for patients with T-ALL has increased and now approaches that for B-lineage ALL (Goldberg et al. 2003).

Whilst ALL is differentiated into only two primary classes, AML is a more of heterogenous group of malignant haematologic disorders. It is divided into eight sub- groups designated M0 to M7 with differences principally based on the morphological and cytochemical criteria of the French-American-British (FAB) classification (Bennett et al. 1976). The classification and frequencies of childhood leukaemias (ALL and AML) are summarised in Table 1.1.

AML is an aggressive disorder characterised by an uncontrolled growth and accumulation of immature and abnormal haematopoietic cells in the bone marrow, with egression into the peripheral blood (Lowenberg et al. 1999; Estey et al. 2006). AML does not have as favourable survival statistics when compared to ALL (Arceci 2002; Loeb et al. 2002). If left untreated, the mean survival of an adult with AML is only 3 - 4 months. In adults, the high proliferative rate of the blasts and increased vascularisation found in the BMs of patients are thought to be responsible for the rapid development of this disease (Lee et al. 2001). In the case of children, angiogenesis is likely to be as important, although results from initial studies of the pro-angiogenic VEGF are conflicting (De Bont et al. 2002; Jeha et al. 2002). Whilst there have been advances in the diagnosis and treatment of AML, most patients will eventually succumb to the disease. In adults the 5-year survival rate has not changed since the 1970s and remains between 15 - 30% globally (Lowenberg et al. 1999). Conversely, the long-term survival rate of children with AML is reaching 65%, which is a substantial increase over the past decades (Kaspers et al. 2007). However, the intensive chemotherapy used to achieve this also results in a high degree of treatment-related deaths and significant late-onset adverse effects.

Chapter One: Introduction 7 Table 1.1. Classification of childhood acute leukaemias. (Pui 1995) Type of leukaemia Frequency (%)

ALL

Immunophenotype

Early pre–B cell 57

pre–B cell 25

Transitional pre–B cell 1

B cell 2

T cell 15

Ploidy

Hypodiploid 7

Diploid 8

Pseudodiploid 42

Hyperdiploid 47 to 50 15

Hyperdiploid 50 chromosomes 27

Triploid or tetraploid 1

AML

Morphology

M0 — minimal myeloid differentiation 1

M1 — poorly differentiated 13

M2 — myeloblastic with differentiation 28

M3 — promyelocytic 6

M4 — myeloblastic and monoblastic 19

M5 — monoblastic 21

M6 — erythroleukemic 1

M7 — megakaryoblastic 10

Whilst the disease phenotypes of ALL may show certain similarities, there are stark differences in the biological characteristics, especially regarding the response to treatment between adults and children. The cure rate for childhood ALL is reaching 80%, in comparison to adults with ALL, where the cure rate is approximately 30 - 40% (Cortes et al. 1995). This difference may be partially attributed to a higher rate of

Chapter One: Introduction 8 genetic abnormalities associated with poor treatment outcome in the leukaemic lymphoblasts of adults (Table 1.2) (Copelan et al. 1995; Pui 1995; Chessells et al. 1998), or to the difference in drug sensitivity of leukaemic blasts between children and adults ( et al. 1991; Kaspers et al. 1997). The latter point has been examined in several studies using in vitro drug sensitivity assays, where it was demonstrated that adult ALL cells were more resistant to a range of drugs (prednisolone, dexamethasone, cytarabine, daunorubicin and methotrexate), than those of children (Maung et al. 1995; Styczynski et al. 2000; Styczynski et al. 2002).

Table 1.2. Genetic abnormalities found in ALL and their proportions in children and adults. Abnormality Childhood ALL Adult ALL

Random 28% 41%

ETV6–CBFA2D t(12;21) 22% 2%

E2A–PBX1 t(1;19) 5% 3%

MYC t(8;14), t(2;8), t(8;22) 2% 4%

TCR 14q11 4% 6%

MLL rearrangements t(4;11), t(11;19), t(1;11) 6% 7%

BCR–ABL t(9;22) 4% 25%

Hypodiploidy (<45 chromosomes) 1% 4%

Hyperdiploidy (>50 chromosomes) 25% 6%

TCR! 7q35 3% 2%

The incidence of all acute leukaemias is about 4 in every 100 000 people world-wide (Jaffe et al. 2001) and there is a marked difference in the spectrum of haematological malignancies between children and adults. For example AML is more common in adults, with 60% of cases occurring in patients over 60, whereas 80% of ALL arises in children under the age 15 years (Jaffe et al. 2001; Foa et al. 2002; Godwin et al. 2003). ALL is the most common malignancy in children, accounting for 25% of all and 75% of all leukaemias (Li et al. 2008). Before the 1960s, ALL was almost uniformly fatal. However, since the 1970s, the cure rate for childhood ALL has been increasing dramatically with the event-free survival currently reaching over 80% (Gaynon et al. 1997; Gaynon et al. 1998; Pui et al. 1998a; Gaynon 2000; Pui et al. 2001; Pui et al. 2006). Some of the reasons for the increase in cure rates include; the

Chapter One: Introduction 9 identification and development of a range of more effective drugs and drug combinations in treating the disease, the application and intensification of treatments (i.e. CNS treatment), and the development of patient risk-stratification with therapy given accordingly (Smith et al. 1996).

While treatment protocols will inevitably vary depending on institution and country, the conventional ALL therapy generally consists of induction, consolidation, maintenance and CNS preventative therapies (Pui et al. 1998b). The purpose of induction therapy is to shift the patients into a state of remission and to restore normal haematopoiesis. It consists of a combination of drugs: a (dexamethasone or prednisone), vincristine and L-asparaginase, and may also include an and methotrexate. Consolidation, or intensification therapy, is given to patients who have successfully achieved remission, and who have resumed normal haematopoiesis. Its purpose is to make sure all the remaining leukaemic blasts are eliminated. Once again a combination of drugs is used in consolidation therapy, generally including: high-dose methotrexate with or without 6-mercaptopurine; high-dose L-asparaginase given for an extended period; an epipodophyllotoxin plus cytarabine; vincristine, dexamethasone, asparaginase, , and thioguanine with or without cyclophosphamide. A further treatment stage is maintenance therapy, consisting of weekly methotrexate and daily 6-mercaptopurine treatments. The treatment also includes regular pulses of vincristine and prednisolone. This continuing therapy has been shown to be beneficial but only in ALL (Burnett et al. 1997; Pui et al. 1998b). While this therapy has been shown to be effective in the majority of childhood leukaemia, there are still children, young adults (16 - 21 years), and adults that are considered at high risk and fail therapy. Therefore the intensification of this treatment may not be the answer and alternative methods of treatment need to be explored. This is likely to be achieved with increased understanding of the molecular mechanisms leading to the proliferation of leukaemic cells, and the mechanisms of drug resistance (Pui et al. 2006).

The identification of uniform prognostic factors has become an important part in the treatment and research of the disease. Initially, patient age and their (WBC) count at diagnosis have been used to define prognostic groups. These are easy to determine, and show prognostic significance for B-precursor ALL (Smith et al. 1996). The blast karyotype has also been added as a prognostic factor, and several studies have

Chapter One: Introduction 10 also added the initial response to treatment as an additional prognostic factor (Riehm et al. 1987; Gaynon et al. 1990; Reiter et al. 1994; Gajjar et al. 1995; Schrappe et al. 1996; Steinherz et al. 1996; Gaynon et al. 1997). Although childhood ALL can be classified into several groups based on the number of chromosomes (ploidy) present in cells, only two seem to have any clinical significance. One is hyperdiploidy (50 chromosomes per cell), and it is associated with a better prognosis than is indicated by a decreased WBC count and an age range between 1 and 10 years (Pui et al. 1990c; Trueworthy et al. 1992). The other is hypodiploidy (<45 chromosomes), and it is associated with an exceptionally poor prognosis (Pui et al. 1990b).

Unfortunately, the children that do go on to relapse have a very poor prognosis usually due to the development of resistance to the drug treatment. Risk of treatment failure is divided into three groups; standard/low, intermediate or high. The criteria are based on age, WBC count, initial response to treatment, immunophenotype, genotype, and in cases of B-cell precursor disease the presence or absence of extramedullary disease (Pui et al. 1998b). Patients who are at higher risk of relapse are given a more intensive treatment regime than patients classified as standard risk (Pui et al. 1998a). For those children who do fall into this category (i.e. ALL with Philadelphia-positive chromosome, infants, children with hypodiploidy (<44 chromosomes), or children who have relapsed or have not been able to reach initial complete remission), the intensification of current therapies may not necessarily improve the cure rates (Winick et al. 2004).

Owing to the severity of current treatments, even those children who are cured often experience both short and long term side-effects. In the short term, patients can suffer from: nausea, fatigue, increased risk of , diarrhoea and hair loss (Redaelli et al. 2005). The long term effects of treatment can range from heart and problems, osteoporosis, learning difficulties, growth retardation from irradiation, infertility, and increased risk of developing secondary cancers. A detailed list of the side-effects of the various treatments is shown in Table 1.3. As the frequency of children that are cured and reach adulthood increases, there is the possibility that other chronic health conditions have yet to be identified and/or linked to paediatric cancer treatments (Oeffinger et al. 2006). This is partially because there have only been a few studies looking at the long term health effects of leukaemia treatment on children (Armstrong et

Chapter One: Introduction 11 al. 2007). Such adverse effects have been previously justified by a balance between immediate survival and long-term health.

Table 1.3. Side effects of drugs used to treat leukaemia. Adapted from Redaelli (2004). Type of Side Effects treatment

Hair loss, post-treatment somnolence, seizures, neurological/endocrine Irradiation dysfunction, hair loss, osteoporosis, brain tumours

Hyperglycaemia, hypertension, change in mood, acne, weight gain, Glucocorticoid hepatomegaly avascular necrosis of bone, osteoporosis Nausea/vomiting, liver dysfunction, myelosuppression, sun sensitivity, Antimetabolite leukoencephalopathy, osteoporosis, fever, rashes, mucositis, hepatodysfunction, conjunctivitis, decreased fertility Peripheral neuropathy, constipation, seizures, hair loss, chemical Alkaloid cellulites Nausea/vomiting, hair loss, mucositis, myelosuppression, chemical Anthracycline cellulites, cardiomyopathy from high cumulative doses (especially in women) Enzyme Nausea/vomiting, allergic reactions, hyperglycemia, pancreatitis, (L-Asparaginase) hepatodysfunction, thrombosis, encephalopathy

Nausea/vomiting, mucositis, myelosuppression, sun sensitivity, Purine Analogue hepatodysfunction, osteoporosis

Nausea/vomiting, hair loss, mucositis, myelosuppression, allergic Podophyllotoxin reactions, AML Nausea/vomiting, haemorrhagic cystitis, myelosuppression, increased Alkylating agent antidiuretic hormone secretion, hair loss, bladder cancer, acute myelogenous leukaemia, decreased fertility

While the molecular analysis of genetic abnormalities has advanced the understanding of the pathogenesis, prognosis and treatment of ALL (Pui et al. 2004b), there is clearly more work remaining to understand the molecular basis of the disease. This knowledge will allow for the development of more targeted treatments with better clinical outcomes and less adverse side-effects. Identifying new subtypes of ALL, novel tumour targets and genes that may be relevant and important for the development and proliferation of leukaemia can further improve current treatments which although effective, are associated with high toxicity and morbidity (Pui et al. 1995; Greaves 1997; Pui et al. 1998a; Armstrong et al. 2002; Maia et al. 2005). Drugs that can more accurately modify or block pathways that have been transformed or enhanced and have led to the rapid

Chapter One: Introduction 12 growth and drug resistance of leukaemic cells is one method by which such outcomes may be achieved (Druker et al. 2001a). One example of such a target is BCR/ABL and the specific potent inhibitor STI571 which was designed to selectively target the tyrosine kinase activity of BCR/ABL. BCR/ABL is a fusion protein caused by the . It is the genetic alteration that causes CML, and is also found in some ALL patients (20% of adults and 5% of children). The breakpoints in ALL differ from that in CML, with the protein formed in ALL being smaller (185 kDa compared to 210 kDa in CML), but both still have aberrant tyrosine kinase activity. STI571 has both very good tolerance by patients and high activity against the leukaemia cells which contain the BCR/ABL fusion, as the cells are dependent on BCR/ABL for their survival.

Gene expression profiles of childhood ALL can be potentially used as a tool for the risk stratification at diagnosis (Cario et al. 2005), and also for defining the biological features of the disease and for the distinguishing between different subclasses of the disease (De Pitta et al. 2005). The leukaemic cell genetic profile can also help in the assessment of a patient’s risk of failing therapy within already identified subgroups (Yeoh et al. 2002). For example, it is important to identify patients with B-cell ALL who have the t(8;14) translocation as they have exceptionally poor prognosis with standard therapy. However they can be cured if they get intensified short-term therapy for six months which includes high-dose cyclophosphamide, cytarabine, and methotrexate, as well as repeated intrathecal chemotherapy to prevent central nervous system leukaemia (Reiter et al. 1992). Another example is the the group of patients diagnosed with pre-B-cell ALL. Within which it is important to distinguish between patients with t(1;19) translocations and patients without. Those with the translocation require intensified chemotherapy, while the patients without the translocation can be treated successfully with the regimens for lower-risk ALL (Crist et al. 1990; Raimondi et al. 1990). The t(1;19) translocation can also be found in early pre-B-cell ALL, but in those cases no E2APBX1 chimeric protein is formed and so it is not associated with a poor prognosis (Pui et al. 1994b).

1.2.2 Chromosomal Translocations Translocations have been shown to be one of the three main types of cytogenic changes present in tumours (alongside deletions and inversions). Advances in molecular biology Chapter One: Introduction 13 have enabled the identification and cloning of a number of chromosomal translocations which are important in the diagnosis and prognosis of the disease, as well as in the identification of the specific molecular lesions involved. Ultimately, this has further enhanced our understanding of the molecular biology of cancer. Chromosomal translocations are common in leukaemias, being identified in 80 - 90% of acute childhood leukaemia cases (Uckun et al. 1998a; Hrusak et al. 2002; Pui et al. 2003; Pui et al. 2004a). Different chromosomal abnormalities can be associated with particular subtypes of the disease [e.g. Philadelphia chromosome is associated with CML; and t(4;11) is associated with infant leukaemias (Chen et al. 1993; Biondi et al. 2000)]. These genetic changes in haematopoietic cells can contribute to leukaemic transformation, as many of the genes affected by these translocations have been shown to code for transcription factors which regulate genes involved in haematopoietic growth and differentiation (Sawyers 1997). Chromosomal aberrations can also create gene fusions which result in chimeric . For example, the fusion of c-ABL with the BCR gene gives rise to the BCR/ABL fusion protein which exhibits increased tyrosine kinase activity (Bartram et al. 1983; De Klein et al. 1986), and is associated with high-risk B-lineage ALL.

Table 1.4. Subtypes of childhood leukaemia and their associated chromosomal changes. Adapted from Greaves et al. (2003). Chromosomal Subtype Cell type Molecular lesion Frequency abnormality B-cell MLL-AF4, -ENL & ~85% (of infant ALL) ALL 11q23 progenitor other fusions ~5% (of total ALL) B-cell Increased gene Hyperdiploidy ~35% precursor dosage B-cell t(12;21)(p13;q22) TEL-AML1 fusion ~20% precursor B-cell t(1;19)(q23;p13) E2A-PBX1 fusion ~5% precursor B-cell t(9;22)(q34;q11) BCR-ABL fusion ~5% precursor T-cell 1q deletion or SIL-SCL fusion ~25% precursor t(1;14)(p32;q11) MLL-AF6, -AF9, - AML 11q23 ~50% (infant AML) AF10 or others

t(8;21)(q22;q22) AML1-ETO fusion ~15% (total AML)

Chapter One: Introduction 14 A more recent discovery has been the identification of another chromosomal aberration which has been associated with the particular leukaemia subtype MLL (mixed lineage leukaemia or myeloid lymphoid leukaemia). The connected gene (MLL) is located at a chromosomal breakpoint, and was identified and cloned in 1991 from the translocations occurring on the chromosome band 11q23 (Ziemin-van der Poel et al. 1991). This translocation has been of interest due to its recurring association with leukaemia, occurring in about 5% of AML patients, and up to 10% of patients with ALL (Kaneko et al. 1986). Of particular relevance to this project, however, is the high frequency observed in infant leukaemia (up to 80%), for both ALL and AML (Heerema et al. 1994; Rowley 1998). Table 1.4 lists the frequency of chromosomal changes associated with the different subtypes of leukaemia.

1.2.3 Mixed Lineage Leukaemia (MLL) The MLL gene, also known as ALL-1 or HRX, was identified and cloned from translocations on chromosome 11 at band q23 (Ziemin-van der Poel et al. 1991). MLL encodes a 430 kDa protein, which is proteolytically processed into 2 fragments of 300 and 180 kDa that heterodimerise with each other (Nakamura et al. 2002; Yokoyama et al. 2002; Hsieh et al. 2003a; Hsieh et al. 2003b). The MLL protein is a homeotic regulator that shares homology with Drosophila trithorax (trx). Its normal function is thought to be as a transcription regulator, with evidence suggesting a role in HOX gene expression during embryogenesis. It is thought that MLL is required for proper segment identity in the axioskeletal system (Yu et al. 1995; Milne et al. 2005).

Deletions of MLL in mice are embryonic lethal, while heterozygous mice (MLL+/-) display aberrations in segment identity, as well as having small birth weights, retarded growth, along with anaemia and decreased platelets (Yu et al. 1995). Hox genes are expressed in specific haematopoietic lineages, and their disruption has been shown to inhibit differentiation. Since MLL is required for the maintenance of Hox gene expression (Yu et al. 1998), one of the consequences of MLL disruption could lead to the disruption of haematopoiesis, and leukaemia (Hess et al. 1997).

MLL rearrangements and amplification are found in ALL, AML and MDS and are associated with abnormal Hox gene regulation (Yu et al. 1995). Unlike other gene translocations, which have a distinct fusion partner, MLL is considered much more Chapter One: Introduction 15 promiscuous as over 60 gene- and region-fusion partners have been identified (Meyer et al. 2006). AML patients with MLL translocations (3 - 10%) have an intermediate prognosis, while B-lineage ALL patients with MLL translocations (8 - 10%) have a poor prognosis. In T-lineage ALL, MLL translocations occur very rarely and are associated with a very good prognosis (Rubnitz et al. 1999).

Armstrong et al. (2002) demonstrated that ALL with MLL gene rearrangements (MLL) had a unique gene profile that is significantly different from both ALL and AML. As part of this study, the authors were able to correctly distinguish MLL from both ALL and AML in 95% of their samples - purely by their gene expression differences. This prompted a call to classify MLL as a separate disease. Gene expression analysis has identified a high level of expression of both FLT-3 and certain Hox genes, as well as the silencing of the tumour suppressor FHIT, in MLL (Armstrong et al. 2002; Stam et al. 2005; Stam et al. 2006). Of particular interest to this project is the association of high expression of FLT-3, the gene most consistently overexpressed in MLL, compared to other types of leukaemia.

As mentioned previously, another point of interest regarding 11q23 translocations and the MLL gene, is their high frequency in two distinct types of acute leukaemias. About 80% of infant (< 1 year) acute leukaemias (Kaneko et al. 1988; Chen et al. 1993; Heerema et al. 1994; Janssen et al. 1994; Nagayama et al. 2006) and 25% of chemotherapy-induced leukaemias express this translocation. Chemotherapy-induced leukaemia is usually associated with patients who were treated with topoisomerase II inhibitors. Although it is suggested in some studies that exposure during pregnancy to dietary bioflavonoids and natural topoisomerase II inhibitors may contribute to the MLL translocations, the sample sizes were too small to make any definitive conclusions (Wiemels et al. 1999; Strick et al. 2000).

The most common MLL translocations occurring in infant ALL are: t(4;11)(p22;q23) (~60%), t(11;19)(q23;p13.3) (~20%) and t(9;11)(p22;q23) (~5%) (Meyer et al. 2006). As outlined above, MLL is biologically and genetically distinct (Armstrong et al. 2002) and thus requires supplementary molecular understanding to improve diagnosis along with its treatment. Leukaemia cells from infants with 11q23 have been shown to contain distinct biological properties to those from other children with ALL as: a) they grew

Chapter One: Introduction 16 better on a stomal layer in vitro (Kumagai et al. 1996); b) when injected in to SCID mice the cells had a high rate of recovery (Uckun et al. 1995); and, c) after serum deprivation in vitro they were more resistant to cell death (Kersey et al. 1998). This is all consistent with infant leukaemias having lower cure rates. The ability to engraft in immunodeficient mice can also be a strong and independent predictor of relapse. Their ability to grow in the bone marrow is also expected to influence the level of tissue infiltration. The ability of these cells to long periods of growth factor deprivation, would allow their survival longer outside the BM microenvironment in the extramedullary sites (Kersey et al. 1998).

Infants diagnosed with ALL who have the disrupted MLL gene, respond poorly to conventional ALL-therapy (>50% failure rate) and have a very poor prognosis, with their 5-year event free survival (EFS) being only ~35% (Pui et al. 1994a; Pui et al. 1995; Greaves 1996), compared to infants with wild-type MLL who have a far better outcome (up to 80%) (Heerema et al. 1999; Nagayama et al. 2006). While t(4;11) translocations primarily occur in infants, they have also been found in older children, with their incidence in childhood ALL cases up to the age of 15 years and excluding infancy being about 2% (Mann et al. 2007). It is currently thought that ALL patients with t(4;11) who are older than 1 year, have a better disease outcome (Biondi et al. 2000; Pui et al. 2002; Pui et al. 2003; Mann et al. 2007). This is a substantial change compared to as recently as a decade ago, when it was thought that the prognosis was the same as for infants (Behm et al. 1996).

Of all the MLL translocations, the t(4;11)(q21;q23) chromosomal translocation where the MLL gene is fused to AF4 (resulting in the MLL-AF4 fusion), appears to be the most common (Gu et al. 1992; McCabe et al. 1992; Domer et al. 1993). The AF4 gene encodes a serine/proline-rich protein which contains a nuclear localisation sequence and a guanosine triphosphate (GTP) binding domain. AF4 localises to the nucleus (Li et al. 1998) and may be involved in the control of gene transcription. AF4-deficient mice exhibit imperfect T-cell development and modest alterations in B-cell development with only the lymphoid compartment being affected (Isnard et al. 2000). The MLL-AF4 fusion is mainly associated with high-risk ALL. Infants comprise roughly 50% of ALL with this fusion and they have been shown to have a particularly poor prognosis (Heerema et al. 1985; Pui et al. 1991; Hilden et al. 1995; Behm et al. 1996; Reaman et

Chapter One: Introduction 17 al. 1999; Pui et al. 2002). However, infants with an 11q23 translocation, but a different fusion partner other than AF4, have a better prognosis to some extent (Heerema et al. 1994). The MLL-AF4 fusion may be important in the maintenance of the leukaemia, as shown by a study conducted by Thomas and colleagues (2005). Cells with downregulated MLL-AF4 had reduced proliferation and clonogenicity but showed an increase in apoptosis, and their ability to engraft into SCID mice was hindered (Thomas et al. 2005).

1.3 Bone Marrow Microenvironment

1.3.1 Normal Haematopoiesis The communication between the BM and haematopoietic cells has been shown to play an essential role in both normal haematopoiesis and haematologic malignancies, such as leukaemias (Yamazaki et al. 1989; Jiang et al. 1998; Punzel et al. 2003). This system is maintained by a constant production of maturing cells which are released from the bone marrow to the peripheral blood and secondary organs.

The BM microenvironment is a three-dimensional structure consisting of a heterogeneous population of cells in tight association with the extracellular matrix (laminin, fibronectin and collagen). The cell types include: haematopoietic stem cells, progenitor and precursor cells, endothelial cells, fibroblasts, chondroclasts, osteoblasts and osteoclasts, natural killer cells, , , dendritic cells and erythrocytes (Weiss et al. 1984). These cells are a source of a variety of growth factors that regulate the proliferation and differentiation of the progenitor cells (Weiss et al. 1984). Cytokines are either provided by direct contact with the cells or by secretion into the microenvironment (Roberts et al. 1988; Jiang et al. 1998). Some of the cytokines involved are granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), Interleukin (IL)-3, IL-4, IL-6, IL-8, KIT ligand (KL) and FLT-3 ligand (FL) (Pelletier et al. 2000). A schematic diagram of haematopoiesis is shown in Figure 1.2.

The extracellular matrix proteins are strong adhesive components for cells in the BM, but they can also be utilised by tumour cells (Gluck et al. 1989; Edelmann et al. 2008).

Chapter One: Introduction 18 In particular, the BM stroma provides both mechanical and metabolic support: it sustains self-renewal of haematopoietic stem cells, and also regulates haematopoiesis and the proliferation and migration of blood cells by providing distinct microenvironments or niches for the development of different blood cell types (Metcalf et al. 1971; Weiss 1976; Trentin 1978; Lichtman 1981; Lambertsen et al. 1983; Weiss et al. 1984).

Self renewing NEGATIVE POSITIVE TFG! KL TNF IL-1,-3,-6,-11 IFN Pluripotent TPO Haematopoietic Mast cell G-CSF thymus Stem cell KL Myeloid Lymphoid Thymocyte IL-3, -4 Progenitor Progenitor CFU-GEMM FL KL IL-7 GM-CSF Myelomonocytic IL-3,-9 Progenitor IL-11 CFU-GM Erythroid KL pre-B cell Progenitor FL IL-3,-6 IL-1,-2,-4 BFU-E KL G-CSF IL-3,-6,-11 GM-CSF IL-1, -6 GM-CSF TPO IL-2,-4,-6,-9,-12 T-cell IL-13,-14,-18 NK cell IL-3 KL GM-CSF IL-3 GM-CSF IL-3, B-cell GM-CSF EPO IL-3,-5 IL-9,-12, -13 KL M-CSF IL-15, -16, -18 GM-CSF IL-1,-3,-5 TPO

CFU-E GM-CSF M-CSF TNF- Megakaryocyte IL-4

Monocyte G-CSF Dendritic cell EPO KL IL-3

Normoblast Neutrophil IL-5 Basophil Macrophage Platelets Eosinophil IL-8 G-CSF GM-CSF Erythrocytes SDF-1 IL-1,-2,-3,-4,-6 IL-7,15,-16,-17 IL-5,-16 GM-CSF M-CSF IL-10 (-ve)

Figure 1.2. Schematic diagram of normal haematopoiesis. Haematopoietic cell differentiation and cytokines, which are for the main part provided by the BM microenvironment, are depicted. The diagram is from KL MacKenzie and MAS Moore, pers. comm. BFU-E - Burst forming unit erythroid; CFU-E - CFU-erythroid; CFU-GEMM - CFU- granulocyte erythrocyte monocyte macrophage; CFU-GM – CFU-granulocyte macrophage; CFU-MEG - Megakaryocytic CFU; EPO – erythropoietin; TPO - Thrombopoietin.

Chapter One: Introduction 19 In engraftment models of both normal and malignant cells, the cells are injected into mice from where they migrate to the bone marrow to engraft. The cells subsequently proliferate and egress into the circulation, resulting in the repopulation of normal haematopoietic cells or the spread of leukaemia - depending on the initial cell type. Homing and engraftment of normal stem and progenitor cells is tightly regulated by the chemokine stromal cell-derived factor-1 (SDF-1), which is expressed in the BM and also by its receptor which is present on the surface of progenitor cells (Peled et al. 1999; Kollet et al. 2001).

1.3.2 Function of the BM with regard to Leukaemia The BM microenvironment, in a similar manner to the functioning of haematopoietic cells, is a critical determinant in the success of tumour cells. There exists a dynamic interaction between leukaemia cells and stromal cells in the BM, which in many ways mimics that of normal haematopoietic cells. One example of this is the survival and growth factors provided by the stromal support for leukaemia cells.

A tumour microenvironment plays a critical role in determining the fate of tumour cells. Leukaemia cells, like their normal counterparts reside in the BM. Therefore just like the relationship between haematopoietic cells and the BM microenvironment, the relationship between leukaemia cells and the BM is crucially important. There is a dynamic interaction between leukaemia cells and stromal cells in the BM, which in many ways mimics that of normal haematopoietic cells. Normal CD34+ cells and haematopoietic precursors are able to secrete a variety of angiogenic and haematopoietic growth factors (such as VEGF), hepatocyte growth factor, fibroblast growth factor (FGF)-2, KL, FL, thrombopoietin, IL-16, insulin-like growth factor (IGF)-1, transforming growth factor-beta (TGF-!)-1 and -2, Regulated upon Activation Normal T-cell Expressed and Secreted, Macrophage inflammatory protein-1 and !, IL- 8, and platelet factor-4, angiopoietin-1 and-2), which enable cross-talk between themselves and the BM microenvironment. This suggests that these cells can regulate both haematopoiesis and angiogenesis (Majka et al. 2001; Pomyje et al. 2003). Leukaemia cells proliferate in close contact with the extracellular matrix and the stromal cells. The stromal cells provide survival and growth factors for the leukaemia cells, and in turn, the cells have an effect on the bone marrow microenvironment themselves. As the leukaemia progresses the environment of the BM changes becoming more hypoxic Chapter One: Introduction 20 and acidic, this hampers normal haematopoiesis and promotes leukaemia growth, as shown in rat studies (Mortensen et al. 1998). Acute leukaemia cells secrete angiogenic factors such as VEGF (Zhang et al. 2005), and this leads to neovascularisation which has been observed in ALL (Aguayo et al. 2000). In another study it was shown that BM plasma taken from leukaemia patients induced the BM endothelium to form capillary- like structures in vitro, in a similar manner to a cocktail of pro-angiogenic cytokines of FGF, VEGF, epidermal growth factor (EGF) and IGF-1. This was compared to control media which had no effect on capillary formation (Veiga et al. 2006). This finding indicates that ALL cells can actively influence BM cells to enhance proliferation and migration.

Apart from their effects on angiogenesis, proangiogenic factors also have a direct effect on the proliferation of leukaemic blasts. Depending on the growth factors expressed and their combinations, the resultant effects can be divergent. However, in most cases a growth enhancement is observed (Bruserud et al. 2003). Different cell types in the BM stroma can produce growth factors such as GM-CSF, G-CSF, IL-6, IL-8, and SCF (Taichman et al. 1996), that can be provided to the tumour cells by both physical contact and the secretion of soluble factors. These factors have been shown to induce cell proliferation and even drug resistance (Nefedova et al. 2003).

With leukaemia cells originating from haematopoietic cells, and also residing in the BM microenvironment, it is expected that they interact with the BM stromal cells in a similar manner to their normal counterparts (Rafii et al. 1995; Bradstock et al. 1996). This interaction between the two cell types can influence their proliferation and the survival of leukaemia cells. Leukaemia cells secrete angiogenic cytokines such as FGF and VEGF (Perez-Atayde et al. 1997), which promote endothelial cell growth. In turn the BM stroma secrete cytokines (e.g IL-7) which have been shown to promote survival of leukaemia cells (Veiga et al. 2006). The specific niche-environment that is provided by the BM stromal cell in vivo can, to some extent, be replicated in vitro with long term BM cultures that allow for the growth and analysis of both haematopoietic and leukaemic cells in vitro (Wolf 1979; Weiss et al. 1984; Johnson et al. 1986; Roberts et al. 1987; Itoh et al. 1989; Manabe et al. 1992; Suzuki et al. 1992; Kim et al. 1998; Winter et al. 2000; Winter et al. 2002; Nefedova et al. 2003).

Chapter One: Introduction 21 Physical interaction between B-cells and stromal cells within the BM microenvironment is critical to the survival of normal and malignant B-cells. Precursor B-ALL cells isolated from patients undergo apoptosis if cultured in media alone. However, whilst in the presence of BM stromal cells, apoptosis does not occur due to the expression of anti- apoptotic genes (such as B-cell lymphoma (Bcl)-2 family members) (Veiga et al. 2006). Veiga and colleagues (2006) showed that by co-culturing ALL cells with human BM endothelial cells, the expression of both Bcl-2 and Bcl-xL increased, compared to culturing the leukaemia cells without BM support. It has also been shown that the survival of ALL cells in culture is prolonged if cultured on BM stromal cells, through the interaction of the !1 integrins (Manabe et al. 1992; Manabe et al. 1994; Campana et al. 1996). Additionally, stromal cells can support the growth and proliferation of B-ALL and AML cells and regulate B-ALL cell survival during chemotherapy (Damiano et al. 1999; Hazlehurst et al. 2000; Mudry et al. 2000; De La Fuente et al. 2002; Astier et al. 2003; Wang et al. 2004). Similar effects have also been reported in studies of T-ALL (Winter et al. 2002). Studies with AML and CML have described similar results of stromal and leukaemia cell interaction, whereby the adhesion of leukaemic cells through their integrins gives cells protection from chemotherapy and apoptosis (Lundell et al. 1996; Damiano et al. 2001). Culturing AML cells on a stromal support has been found to increase the expression of anti-apoptotic molecules (such as Bcl-2 and Bcl-xL) as well as decreasing their sensitivity to the chemotherapeutic drug cytarabine (Lagneaux et al. 1998; Dankbar et al. 2000; Konopleva et al. 2002).

1.4 Leukaemia and Angiogenesis

1.4.1 Angiogenesis Angiogenesis is a physiological process in which new capillaries are formed from existing blood vessels. This process is required for the formation of a vascular supply to both normal and neoplastic tissue. It involves the stimulation of endothelial cells to degrade their basement membrane and invade the surrounding stroma, eventually resulting in the formation of functioning capillaries. Many growth factors, and also naturally occurring inhibitors are involved in angiogenesis (Folkman 1995a; Carmeliet 2003). Some of these growth factors are: VEGF, acidic and basic FGF (aFGF, bFGF), platelet derived growth factor (PDGF), EGF, G-CSF, GM-CSF, IGF-1, IL-1, IL-2, IL-

Chapter One: Introduction 22 6, IL-8, angiogenin, angiopoietin-1, transforming growth factors (TGF)- and -!), and tumour necrosis factor (TNF)-. Some known inhibitors are: endostatin fragment of XVIII collagen, thrombospondin, fibronectin, prolactin, angiostatin plasminogen fragments, peptides of type 1 collagen, tissue inhibitor of matrixmetalloproteinase (TIMP), IL-12 and -, !- and "-interferon (IFN) (Ribatti et al. 2003). From the range of growth factors listed, VEGF is a particularly strong inducer of angiogenesis and will be discussed in more detail in Section 1.6.

Beside the role it plays in normal vascularisation (embryogenesis, placenta development and ovulation), angiogenesis also plays a role in solid tumour growth and metastasis (Folkman 1971; Folkman 1995a; Ellis et al. 1996). Normal vessels are composed of: a monolayer of endothelial cells that are in close contact with each other, the vascular basement membrane, and surrounding pericytes. During physiological development, the vessels mature quickly becoming stable. This process is tightly controlled by a balance between pro- and anti-angiogenic factors (Carmeliet 2003). Unlike that observed in normal vessels, the balance between pro- and anti-angiogenic factors is less tightly regulated in tumour growth, leading to tumour vessels being highly disorganised, dilated, having an uneven diameter, excessive branching and shunts, along with numerous openings and widened interendothelial junctions, and endothelial cells that are of abnormal shape (Dvorak et al. 1999; Hashizume et al. 2000). All these features make the vessels ‘leaky’, enhancing capillarity (Carmeliet et al. 2000).

As mentioned previously, for most tumours to continue to grow it is essential that they are able to recruit new blood vessels. This process is activated through the actions of VEGF, which results in a reorganisation of existing vasculature into de novo microvessels (Folkman et al. 1987). Such microvessels can freely infiltrate newly growing tumours and thereby supply it with nutrients and oxygen. However, only a small proportion of these vessels actually become functional and stable. They are typically characterised by haemorrhaging, poorly formed endothelial tubes, absence of pericytes and endothelial cell permeability (Dvorak et al. 1999). Further discussion of VEGF is contained in Section 1.6.

A number of cytokines can induce the expression of VEGF and bFGF by vascular smooth muscle cells and as such, indirectly stimulate endothelial cell proliferation

Chapter One: Introduction 23 (Brogi et al. 1994). Hypoxia is another cause that has been found to increase and/or induce VEGF expression, a point which is discussed further in Section 1.6.

1.4.2 Leukaemia and Angiogenesis Over the past decade there has been growing evidence of the interaction between the tumour and stromal cells in haematologic malignancies (Hideshima et al. 2002). Under normal conditions the BM microenvironment forms a network of support for normal haematopoietic cells (Section 1.3). Several studies have observed a higher microvessel density in the bone marrow of children with ALL and patients with AML, compared to normal controls (Perez-Atayde et al. 1997; Aguayo et al. 2000; Hussong et al. 2000; Lee et al. 2001; Pule et al. 2002). This data implies that as leukaemia cells proliferate they produce angiogenic growth factors, such as VEGF, which in turn influence the BM microenvironment. As the disease progresses and the cells proliferate there is an increase in the number of vessels, supporting the ideas that angiogenesis may be important in the progression of leukaemia.

The microvasculature forms an important component of the stroma. It provides an oxygen and nutrient supply and it is also a means for waste disposal. The BM endothelial cells (BMEC), which form part of the vasculature are involved in autocrine and paracrine interactions with the tumour cells through the secretion of a range of molecules including; VEGF, bFGF, matrix metalloproteinase (MMP)-2, MMP-9 and monocyte chemoattractant-1. These growth factors can not only affect the endothelial cells that secrete them in an autocrine manner, but they also may affect the tumour cells which reside in the BM, in a paracrine manner. The tumour cells can secrete their own growth factors which result in the secretion of more cytokines (stem-cell factor (SCF), FL, GM-CSF, IL-6 and IL-7) by the BMECs, which will again stimulate the tumour cells (Yamaguchi et al. 1996; Fiedler et al. 1997; Bellamy et al. 1999; Dankbar et al. 2000; Ribatti et al. 2001). Furthermore, activators and inhibitors of angiogenesis operate only at a local level, meaning the levels of these mediators may not always be detectable in the serum and/or plasma.

Angiogenesis and increased microvascular density in the BM is not confined to ALL (or AML); it has also been being observed in multiple myeloma (Vacca et al. 1999a), non-hodgkins lymphoma (Ribatti et al. 1996) (Pruneri et al. Chapter One: Introduction 24 1999; Korkolopoulou et al. 2001), chronic lymphocytic (Molica et al. 2002) and chronic myeloproliferative leukaemias (Lundberg et al. 2000; Mangi et al. 2000; Moehler et al. 2003; De Raeve et al. 2004). In AML patients BMs, there have been several reports of an increased number of vessels compared to normal BM (Hussong et al. 2000). Similarly, BMs from patients with AML were evaluated for the amount of vessels (per millimetre) at the time of diagnosis and it was found to be increased compared to normal controls. These patients’ BMs were assessed after induction therapy, and there was a significant decrease in the number of vessels in the BM of patients without residual blasts compared to patients with residual blasts. Vascularity returned to normal levels in patients who achieved complete remission (Padro et al. 2000; De Bont et al. 2001).

Importantly angiogenesis seems to be vital to the initial development of AML (Aguayo et al. 2000). AML tumours have been shown to secrete quantities of pro-angiogenic VEGF (Glenjen et al. 2005), and intracellular VEGF has been shown to be an independent indicator of disease outcome (Aguayo et al. 1999). Development of the clinical disease is associated with leukaemic blasts infiltrating throughout the BM. This infiltration requires the interaction of between the blast and non-leukaemic cells and is characterised by local angiogenesis and increased bone marrow vessel density (Hussong et al. 2000; Padro et al. 2000).

1.5 Hypoxia and the Hypoxia Inducible Factor

Hypoxia is important in the regulation of blood vessel formation and structure. It is also a vital component of many pathological processes and is a major pathophysiological condition which regulates angiogenesis. Increased angiogenesis, as a result of hypoxia, is an adaptive response with the purpose of increasing the delivery of oxygen and nutrients to tissues. In mammalian cells the response to hypoxia is regulated by the hypoxia-inducible factor (HIF)-1. It was first identified in studies investigating the hypoxia-inducible expression of the growth factor erythropoietin (Semenza et al. 1992). Semenza and colleagues (1992) found that under hypoxic conditions, the activation of the erythropoietin gene was dependent on the binding of a nuclear factor to the promoter region of the gene. This nuclear factor was found to be HIF.

Chapter One: Introduction 25

One of the main stimuli of angiogenesis in tumours is hypoxia (Carmeliet et al. 2000). Once a tumour reaches 2 mm3, the transfer of oxygen and nutrients via diffusion alone is insufficient for its continued growth. The partial pressure of oxygen (pO2) is lower in tumour tissue than in normal tissue, passively inducing hypoxia. In addition to HIF being strongly activated in the tumour microenvironment by hypoxia (Iyer et al. 1998a), the hypoxic effects are further amplified by HIF induction through growth factors and mutations in oncogene and tumour suppressors (Semenza 2000a). Interestingly, it has been suggested that the HIF pathway may also activate genes which inhibit cell growth and have pro-apoptotic effects during hypoxic conditions (Maxwell et al. 1999). Hypoxia can also overcome the lack of exogenous addition of growth factors needed for endothelial cell growth and sprouting in culture (Calvani et al. 2006).

HIF is involved in the induction and transcription of a variety of genes through its binding to other hypoxia response elements (HRE) in the promoter region of genes. Examples of cell responses include; release of angiogenic growth factors and vasomotor regulators, and molecules involved in energy metabolism, changes in pH, neurotransmitter release, matrix remodelling, as well as changes to iron transport, and regulation of apoptosis and cell proliferation (Liu et al. 1995; Carmeliet et al. 1998; Dang et al. 1999; Calvani et al. 2006; Lum et al. 2007). Other responses to hypoxia include cells switching to anaerobic metabolism and the induction of angiogenesis and erythropoiesis (Semenza et al. 1992; Ivanov et al. 1998; Wykoff et al. 2000). It should be noted that HIF-1 can play a role in stress responses beyond that of hypoxia, as it has been shown that HIF-1 can be activated by insulin and IGFs under normoxic conditions (Zelzer et al. 1998; Feldser et al. 1999; Fukuda et al. 2002; Stiehl et al. 2002; Bilton et al. 2003).

1.5.1 Hypoxia Inducible Factors The hypoxia-inducible factor is a key transcriptional regulator of angiogenic growth factors occurring through an oxygen sensing process (Maxwell et al. 1997; Semenza 1999; Semenza 2000b; Harris 2002; Semenza 2003). Collectively HIFs are a group of heteromeric transcription factors consisting of an oxygen regulated -subunit and a constitutively expressed !-subunit - both of which have basic helix-loop-helix domains which bind to DNA (Wang et al. 1995; Ema et al. 1997; Iyer et al. 1998b). It is Chapter One: Introduction 26 primarily through the -subunit in which HIFs are regulated (Pugh et al. 1997). The !- subunit is constitutively expressed and localised to the nucleus. HIFs can also associate with other transcription factors not necessarily involved with hypoxia (Semenza 2003).

In vertebrates three different -subunits have been identified (HIF-1, 2 and 3). Even though they are all thought to be regulated by oxygen in a similar manner, their tissue distribution, and in turn target genes, may differ. For example, unlike HIF-1 which is ubiquitously expressed, HIF-2’s expression is more restricted, being limited to certain cell types (endothelial cells, epithelial cells, fibroblasts and neurons)(Tian et al. 1997). In mammalian cells the hypoxic response is principally mediated through the HIF-1 pathway (Semenza 1998). Cell specificity has been used to describe the function of HIF-1 and HIF-2. Even though HIF-2 was initially identified in endothelial cells and thought to be specific to endothelial cells (Wiesener et al. 1998). Calvani et al. (2006) have found that it is actually HIF-1 that is involved in the hypoxia-dependent survival and sprouting of endothelial cells.

Of relevance to this project, HIF-1 is a major regulator of VEGF gene expression (Wang et al. 1995; Forsythe et al. 1996; Semenza 1999). It not only contributes to hypoxia induced VEGF production, but it may also play a role in oncogene-dependent expression of VEGF (Jiang et al. 1997; Blancher et al. 2001). HIF-2 shares 48% amino acid sequence identity with HIF-1, and it can also dimerise with HIF-1! and bind HREs (Hu et al. 2003).

Chapter One: Introduction 27 Figure 1.3 schematically depicts the hypoxia-induced regulation of angiogenesis. In the presence of oxygen or under normoxic conditions,  subunits are degraded by the ubiquitin-proteasome system. The mechanism involves the hydroxylation of two prolyl residues by iron-dependent oxygenases in the central degradation region of the protein; which is then recognised by the product of the von Hippel-Lindau (VHL) tumour suppressor gene, pVHL. This forms part of an E3 ubiquitin ligase complex and is targeted for degradation by the proteaosome (Epstein et al. 2001; Ivan et al. 2001; Calvani et al. 2006).

Polyubiquitination  Activation of Destruction VHL E3 ligase of HIF complex  VHL

OXYGEN HIF expression  HYPOXIA  Stabilisation    Nuclear entry   p300   ! HRE Complex assembly & DNA binding

Target genes activated

Metabolic Pro- adaptation / apoptotic Enhanced Increased effects? glycolysis oxygen delivery / Angiogenesis

Figure 1.3. Regulation of HIF-1. Modified from Semenza (2003) and Giaccia (2003).

Chapter One: Introduction 28 Under hypoxic conditions there is an accumulation of HIF-1 as well as increased transcriptional activity. This is due to the reduction in proline hydroxylation, nuclear localisation, heterodimerisation, DNA binding and recruitment of the co-activator p300 (Arany et al. 1996). This stabilisation leads to the aforementioned increase in its transcriptional activity. The complex then binds specific HREs in target genes. Levels of HIF-1 are also influenced by genetic alterations to the VHL gene (which are tumour suppressor proteins that increase HIF-1 protein synthesis by a pathway involving PI3K/AKT/mTOR and MAPK), and cytokines produced by both tumour and stromal cells (Blancher et al. 2001; Jiang et al. 2001; Laughner et al. 2001; Kurmasheva et al. 2007).

The HIF system is induced or amplified by oncogenic pathways and suppressor mutations (Jiang et al. 1997; Maxwell et al. 1999; Zundel et al. 2000; Fatyol et al. 2001; Maxwell et al. 2001; Wiesener et al. 2001). These effects can act at several levels; transcription, translation, stabilisation and activation of HIF-1. From the study of solid tumours there is some evidence that drug and radiation resistance can be mediated through HIF-1 and as such, may offer a new therapeutic target (Semenza 2003). Its role in haematologic malignancy is less well known. Also within the BM, normal haematopoiesis occurs under relatively low oxygen (hypoxic) conditions in regions isolated from the blood supply (Caldwell et al. 2001). These regions are crucial for the normal proliferation and differentiation of haematopoietic cells. Experiments with HIF-1/Rag2-null mice showed that HIF-1 has an important role in B-cell development (Kojima et al. 2002), with the loss of HIF-2 leading to pancytopenia. Considering the strong circumstantial evidence outlined above, along with the fact that an increase in HIF-1 protein has been detected in the BM of ALL patients compared with normal BM (Wellmann et al. 2004), one can infer that HIF may also play an, as yet unidentified, role in leukaemia. Interestingly, VEGF is a target gene of HIF-1, and it has been shown to be expressed by leukaemia cells and has also been suggested to be an independent predictor of outcome (as discussed in Section 1.6). Therefore a possible function of HIF may be through the induction of VEGF.

Chapter One: Introduction 29 1.6 Vascular Endothelial Growth Factor

The vascular endothelial growth factor (VEGF) belongs to a family of secreted glycoproteins which are involved in the growth and development of blood and lymphatic vessels. It was originally identified as a secreted polypeptide, having specific and direct effects on endothelial cell proliferation (Ferrara et al. 1989; Leung et al. 1989; Ferrara et al. 1992).

There are five members of the VEGF family encoded by five different genes; VEGF-A (also referred to as just VEGF), VEGF-B, VEGF-C, VEGF-D and placenta growth factor (PlGF). There are three types of VEGF receptors (VEGFR) which belong to the class V receptor tyrosine kinase (RTK) family through which the five VEGF members can act. This is summarised in Figure 1.4. Additionally, VEGFs can interact with two co-receptors called neuropilins. There is another VEGF member, VEGF-E, which is a viral VEGF homologue which binds VEGFR-2 and which is encoded by the parapoxvirus (Lyttle et al. 1994). VEGF-B is closely related to VEGF and binds VEGFR-1, but its physiological role is less clear. VEGF-B knockdown mice show no conspicuous abnormalities, although it is speculated that VEGF-B may have a role in cardiac development (Aase et al. 2001). VEGF-C and -D bind both VEGFR-2 and VEGFR-3 and have been shown to act as lymphangiogenic factors (Enholm et al. 2001; Veikkola et al. 2001) through their binding of VEGFR-3. It is likely that they aid in the spread of tumours via lymphatic vessels. PlGF is induced by hypoxia and is expressed in the placenta throughout pregnancy, and also in the heart, , brain and skeletal muscle (Hauser et al. 1993; Persico et al. 1999). PlGF is a poor inducer of angiogenesis and has only a weak mitogenic effect on endothelial cells, but can act as an angiogenic amplifier by signalling through VEGFR-1 (Park et al. 1994). Tissues of PlGF-knockout mice show inhibited angiogenesis and arteriogenesis (Carmeliet et al. 2001).

Chapter One: Introduction 30 VEGF145 VEGF121 VEGF121 VEGF165 VEGF165 VEGF 145 VEGF-A VEGF189 145 VEGF-B VEGF165 VEGF165 VEGF-A165 VEGF-C PlGF-2 PlGF-1 VEGF-C VEGF-B PlGF VEGF-B VEGF-C PlGF-2 VEGF-D VEGF-D PlGF-2

VEGF Binding Ig like Loop-1 Domain C1r/s Domains Dimerization Domain FV/VIII Domains Ig like Loop-7 MAM Domain

Split Tyrosine Kinase Domain

Heparan- VEGFR-1 VEGFR-2 VEGFR-3 Sulfate Neuropilin-1 Neuropilin-2 (flt-1) (KDR/flk-1) (flt-4) Proteoglycan

AQA Proliferation; Proliferation; VEGFR-2 co- VEGFR-2 VEGFR-3 migration; migration; receptor; release co-receptor; co-receptor survival; survival; of angiogenic regulator of angiogenesis lymph- factors from the blood vessel angiogenesis extracellular development matrix; extracellular storage of VEGFs

Figure 1.4. VEGF growth factors and their receptors. The three VEGF receptors, two neuropilins and heparin-sulfate proteoglycan are shown, along with the VEGF member to which they bind. For VEGF-A the isoforms are also shown. The receptors depict only the main structural features. (Adapted from (Neufeld et al. 1999) and (Ria et al. 2003))

1.6.1 VEGF-A The human VEGF gene is located on chromosome 6 (p12-p21) and consists of eight exons. Multiple isoforms of VEGF exist and are generated by alternate splicing of the pre-VEGF mRNA (Houck et al. 1991; Tischer et al. 1991). The five most common splice variants are; VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206 (Figure 1.5), and they have all been shown to be proangiogenic.

Chapter One: Introduction 31

Exon6 Exon7

Exon1 Exon2 Exon3 Exon4 Exon5 6a 6b 7a 7b Exon8 5’UTR 3’UTR VEGF206

VEGF189

VEGF183

VEGF165

VEGF148

VEGF145

VEGF121

Figure 1.5. Schematic representation of seven different VEGF isotypes. VEGF is made up of 8 exons; the first 4 of which are common to all isotypes. Variations occur with differing combinations of the last four exons. (Adapted from (Keyt et al. 1996; Woolard et al. 2004)).

Native VEGF is a basic, heparin-binding glycoprotein with a disulfide-linked homodimer with subunits of 45 kDa. VEGF121 is a weakly acidic polypeptide that does not bind heparin and is freely diffusible. The mitogenic activity of VEGF is significantly reduced with the loss of the heparin-binding domain (Keyt et al. 1996).

Recombinant VEGF145 induces endothelial cell proliferation and enhances the angiogenic activity of endothelial cells in vivo. It interacts with heparin-like molecules as well as VEGFR-2 and has an affinity similar to VEGF165. The expression of VEGF145 appears to be restricted to reproductive tissue (Cheung et al. 1995; Poltorak et al. 1997;

Krussell et al. 2001). VEGF165 has intermediate properties and is the most commonly expressed isotype. It is secreted, but it can also bind with the extracellular matrix (ECM), heparin, and heparan-sulfate. When it is secreted however, a significant proportion is retained on the ECM. An inhibitory form of VEGF has also been identified, termed VEGF165b (Woolard et al. 2004). VEGF189 and VEGF206 are highly basic and bind heparin with high affinity and are almost completely sequestered within the ECM. Even though they are incorporated in the ECM, they can be released in a diffusible form by several agents (heparin and plasmin), and as such, are bioactive (Park et al. 1993). The differences in VEGF isotypes and their varying affinity for heparin,

Chapter One: Introduction 32 affects their bioavailability. The bioavailability of VEGF is highly regulated, through transcription at the mRNA level (through mRNA turnover), and stability at the protein level (by proteolysis) (Houck et al. 1992).

The VEGF gene can be regulated at both the transcriptional and translational level. VEGF is transcriptionally regulated by hypoxia through HIF-1 binding. During oxygen deprivation or hypoxia, HIF-1/! is the most important driving the production of VEGF mRNA. A HIF-1 binding site, a 5’-CGTG-3’ HRE consensus region was identified in the promoter region of the gene (Forsythe et al. 1996). Enhancer elements are also positioned in the 5’ and 3’ untranslated region (UTR) of the gene. However, HIF-independent VEGF induction has also been reported (Duyndam et al. 2003). Several growth factors and inflammatory cytokines (IGF-I, EGF, TNF, TGF-, TGF-!1, FGF, PDGF, IL-1! and IL-6) have been associated with the induction of VEGF mRNA (Cohen et al. 1996; Ferrara et al. 1997; Neufeld et al. 1999), along with mutations or amplification of the Ras oncogene (Grugel et al. 1995; Okada et al. 1998). Heparanase has been shown to induce VEGF production through p38 and Scr activation (Zetser et al. 2006). Also, VEGF has been shown to be translationally regulated through the 5’-UTR, which effectively stabilises the mRNA. In macrophages, VEGF mRNA is stabilised by lipopolysaccharide (LPS) (Du et al. 2006). Hypoxia was also shown to control VEGF gene expression by increasing both the transcription and the steady state concentration of mRNA (Levy et al. 1996).

The requirement of VEGF in embryonic development has been shown in mouse experiments where the deletion of a single VEGF allele, lead to the abnormal development of blood vessels and embryonic lethality (Carmeliet et al. 1996; Ferrara et al. 1996). VEGFR-1 and -2 knockout mice have shown similar defects compared to VEGF deficient ones, with both receptors being essential for normal vascular development (Fong et al. 1995; Shalaby et al. 1995; Fong et al. 1999a).

The induction of angiogenesis by VEGF occurs by regulating several endothelial cell functions including proliferation, migration and lumen formation (Ferrara et al. 1993). Growth induction of endothelial cells is mediated through the activation of the Raf- MEK-ERK pathway (Xu et al. 2008). VEGF also regulates mitogenesis as well as increases microvascular permeability, vascular tone and the production of vasoactive

Chapter One: Introduction 33 molecules (Senger et al. 1990). However, it is only the mitogen that acts specifically on endothelial cells, and in so doing, alters their gene expression (Nor et al. 1999). Although angiogenesis is a complex process involving numerous different growth factors, the role that VEGF plays in its functioning appears to be paramount as VEGF is involved in both physiological (embryonic and placental development) and pathological (cancer, diabetes and cardiovascular disease) angiogenesis (Bortoloso et al. 2004). Originally identified as an endothelial cell specific mitogen (Leung et al. 1989), VEGF is an important factor in the function, growth and survival of cancer cells (Relf et al. 1997; Graells et al. 2004; Santos et al. 2004; Cong et al. 2005). It is also vital in a variety of non-cancerous cells, assisting their function, migration and growth of neurons, migration of monocytes and the survival of haematopoietic stem cells (Barleon et al. 1996; Hidaka et al. 1999; Zisch et al. 2003; Scavelli et al. 2004).

VEGF has been demonstrated to have an additional role in haematopoiesis, by acting through an internal autocrine loop to regulate haematopoietic cell survival, as observed by Gerber and colleagues (2002). This particular study showed that the conditional knockout of the VEGF gene in BM mononuclear cells reduced the rate of repopulation of haematopoietic cells in mice. It was also observed that VEGF deficient haematopoietic progenitor cells had reduced survival and colony formation in vitro (Gerber et al. 2002). This study has revealed a role for VEGF in the survival of haematopoietic stem cells, as well as for VEGF receptors. This may also give an insight into their role in haematopoietic malignancies which have been shown to express both VEGF and its receptors.

In the context of the BM microenvironment, VEGF has been shown to inhibit dendritic cell maturation (Gabrilovich et al. 1996), increase osteoclastic bone-resorption activity (Nakagawa et al. 2000) and osteoclast chemotaxis (Henriksen et al. 2003), modulate immune responses by enhancing NK cells adhesion to the tumour endothelium (Melder et al. 1996), trigger circulating endothelial cell differentiation, and also recruit monocytes (Barleon et al. 1996) and endothelial cells to the vasculature (Lyden et al. 2001). It has also been shown to not only protect endothelial cells from apoptosis (Gerber et al. 1998; Nor et al. 1999), but a similar effect has also been observed in leukaemia cells (Dias et al. 2002b). Together these studies suggest that the mechanism of cell survival is the same in both endothelial and leukaemia cells. The cell survival

Chapter One: Introduction 34 effect of VEGF is mediated through induction of Bcl-2 (Gerber et al. 1998; Nor et al. 1999; Dias et al. 2002b).

Numerous studies have shown both high levels of expression of VEGF, and high levels of mRNA expression of VEGF receptors in tumours (Aguayo et al. 1999; Bellamy et al. 1999; Kumar et al. 2003; Zhang et al. 2005). Notably this expression has been adjacent to tumour necrotic areas, leading to the model that low oxygen tension or hypoxia associated with tumour necrosis, induced VEGF expression (Shweiki et al. 1992; Maxwell et al. 1997; Giavazzi et al. 2003). This in turn, stimulated the proliferation of vascular endothelial cells ultimately leading to the sprouting of new capillaries. Liu et al. (1995) showed that endothelial cells exposed to hypoxic conditions (0% O2) for 18 hours, had elevated VEGF mRNA expression compared to cells incubated under normoxic (21% O2) conditions. Furthermore, this work led to the identification and characterisation of a hypoxia-responsive enhancer, including a 28-bp element, in the promoter region of the VEGF gene which is responsible for the hypoxia induced increase of VEGF expression (Liu et al. 1995).

The possible role of VEGF and its receptors in haematological malignancies has been further implicated by a study of multiple myeloma patients. This research identified plasma cells in the BM of multiple myeloma patients which expressed VEGF, while the normal myeloid and monocytic cells surrounding the tumour (in the BM) expressed elevated levels of the two high affinity VEGF receptors (VEGFR-1 and -2) (Bellamy et al. 1999). This suggests that by secreting VEGF, the leukaemia cells induce angiogenesis in the BM, as they may be dependent on it. Other studies have shown that the level of angiogenesis in the BM of patients with multiple myeloma correlated with the severity of disease (Vacca et al. 1994; Kumar et al. 2003), implying that the secretion of angiogenic factors, such as VEGF, aside from increasing angiogenesis, also increases the severity-grading of the tumour. Further evidence has been shown by an increase in the number of microvessels observed in the lymph nodes of B-cell non-Hodgkin’s lymphoma patients (Ribatti et al. 1996; Vacca et al. 1999b). Similarly, patients with myeloproliferative disorders have also shown increased levels of VEGF in their serum (Di Raimondo et al. 2001) and BM (Wrobel et al. 2003).

Chapter One: Introduction 35 VEGF, together with its two receptors (VEGFR-1 and -2), are key regulators of not only vascular, but also haematopoietic development (Risau et al. 1995; Carmeliet et al. 1996; Carmeliet et al. 1999) and adult haematopoiesis (Ziegler et al. 1999; Gehling et al. 2000). VEGF promotes the proliferation and survival of endothelial cells, which appears to be mediated through VEGFR-2 (Ferrara 1999a). The inhibition of VEGF and/or its receptors leads to a reduction in the number of haematopoietic progenitors in vivo (Gerber et al. 2002). For tumour angiogenesis to occur, marrow precursors need to be recruited from the BM (Hattori et al. 2001). VEGF has the capacity to do this, with the recruitment of haematopoietic cells demonstrated to be primarily through VEGFR-1, and endothelial progenitors primarily through VEGFR-2. While endothelial cells are targets for VEGF, they can also express VEGF and regulate their growth and permeability in an autocrine manner (Liu et al. 1995).

1.6.2 VEGF Receptors VEGF acts by binding and activating two receptor tyrosine kinases, VEGFR-1 (or FLT-1; which is important for regulating endothelial cell-cell and cell-matrix interactions), and VEGFR-2 (also known as Flk-1 or KDR; which mediates the mitogenic and chemoattractant actions of VEGF). Although VEGF can only bind to VEGFR-1 and -2, it can also interact with neuropilins. Neuropilin-1 is another VEGF receptor which can act as a co-receptor that enhances the function of VEGF through VEGFR-2 (Larrivee et al. 2000; Zachary et al. 2001). VEGFR-2 has been shown to heterodimerise with VEGFR-1 (Kendall et al. 1996). These binding receptors belong to the receptor tyrosine kinase (RTK) class V family. As is common to all class V receptors, they both have an extracellular region consisting of seven immunoglobulin- like domains, a single transmembrane domain and a tyrosine kinase sequence interrupted by a kinase insert domain (Shibuya et al. 1990; Terman et al. 1991). Their expression was originally thought to be restricted to endothelial cells; however, it is now known that they are also present on bone-marrow derived cells, monocytes and haematopoietic precursors ( et al. 1993; Ferrara et al. 1997; Sawano et al. 2001). Although VEGFR-3 (or FLT-4) belongs to the same class of RTKs, it does not bind to VEGF, being only a receptor for VEGF-C and VEGF-D (Pajusola et al. 1992; Finnerty et al. 1993).

Chapter One: Introduction 36 VEGF binding induces receptor dimerisation, triggering kinase activation of the receptor, followed by the release of their associated signal-transducing molecules (Takahashi et al. 2001). VEGF signalling is mediated through the MAP- and PI3-kinase pathways (Meadows et al. 2001). Additionally, receptor activity can be regulated by naturally occurring soluble receptors (Hornig et al. 2000). Binding affinities for VEGF to VEGF receptors were measured using iodinated VEGF (as described in Mori et al. (1991)). The affinity of VEGF for VEGFR-2 (Kd: 75-770 pM) (Terman et al. 1992a; Millauer et al. 1993; Quinn et al. 1993; Waltenberger et al. 1994) is from 10 to 90 times lower than for VEGFR-1 (Kd: 10 pM) (De Vries et al. 1992; Waltenberger et al. 1994).

Disruption of either the VEGFR-1 or -2 genes, results in lethal embryonic vascular and haematopoietic abnormalities. VEGFR-1 (FLT-1) promotes cell migration, but has no effect on proliferation (De Vries et al. 1992). Apart from binding VEGF, it is also a receptor for VEGF-B and PlGF (Park et al. 1994; Olofsson et al. 1998). Knocking out VEGFR-1 in mice has been shown to be embryonic lethal, with the resultant embryos found to have high levels of endothelial cells and hemangioblast formation, as well as disorganisation of the vascular system (Fong et al. 1995; Fong et al. 1999a). VEGFR-1 may in fact regulate haematopoiesis by increasing haematopoietic cell motility and recruitment. The tyrosine kinase domain of VEGFR-1 is required for the VEGF-induced migration of monocytes (Barleon et al. 1996; Hiratsuka et al. 1998). In lung endothelial cells, VEGFR-1 has been shown to induce MMP-9 and thus facilitate lung metastasis (Hiratsuka et al. 2002). Hattori et al. (2002) demonstrated that VEGFR-1 was not only present on bone marrow repopulating cells, but that it was also essential for the reconstitution of bone marrow. In ALL, VEGFR-1 activation results in a distinct localisation of cells in the BM (Fragoso et al. 2006). VEGFR-1-expressing-cells localise in the epiphysis of long bones whereas cells not expressing VEGFR-1, or those whose receptor was blocked, localise in the diaphysis in the BM (Fragoso et al. 2006). This data suggests that VEGFR-1 is important for leukaemia cells to exit the bone marrow and enter into the circulation. It also shows that VEGFR-1 and -2 have different biological effects in leukaemia cells. Therefore, leukaemia cells producing their own VEGF may be driving their own migration into the circulation through an autocrine loop. A splice variant of the VEGFR-1 mRNA forms a soluble receptor, consisting of the extracellular immunoglobulin-like domains and a unique C-terminal extension derived from an intron sequence. It has been suggested that it acts as an inhibitor of

Chapter One: Introduction 37 VEGF activity as it has very high affinity for VEGF (Kendall et al. 1993; Hornig et al. 2000).

VEGFR-2 (KDR or FLK-1), is required for cell differentiation and has a key role in angiogenesis and haematopoiesis (Terman et al. 1992a; Terman et al. 1992b; Millauer et al. 1993; Quinn et al. 1993; Mitchell et al. 1998). It has been shown that Flk-1-null mice fail to develop organised blood vessels and blood islands (Shalaby et al. 1995). VEGF has a high affinity for VEGFR-2, and via this process, VEGF mediates its angiogenic and permeability enhancing effects. VEGFR-2 is mainly expressed on endothelial cells, being essential for endothelial cell development (Shalaby et al. 1995), and is the major mediator of mitogenic, angiogenic and permeability-enhancing effects of VEGF. The central role played by VEGFR-2 in the maintenance of haematopoiesis by the promotion of survival of haematopoietic progenitors, was established in a study by Larravée et al. (2003). The unique signalling effects of VEGFR-2 were studied without the enhancing effects of either neuropilin-1 or VEGFR-1. However, they were able to show that the activation of VEGFR-2 under conditions of cytokine starvation was able to adequately maintain the haematopoietic progenitor population. This increased survival rate was mediated through the PI3K/AKT pathways, although the involvement of ERK1/2/MAPK signalling could not be ruled out.

VEGF is the most widely studied angiogenic growth factor, owing to its multifaceted role in a variety of tumours alongside its functioning in inflammatory disease (rheumatoid arthritis), wound healing and diabetic retinopathy (Aiello et al. 1994; Malecaze et al. 1994). Due to such a prominent role in cancer progression, VEGF and its receptors have been the target of different strategies to block and inhibit their function. A multitude of clinical investigations with the aim of developing treatments for both solid tumours and haematologic malignancies are taking place, some of which are already in clinical trials for solid cancers. The treatments range from both anti-VEGF and anti-VEGF receptor antibodies to small molecule inhibitors. Table 1.5 summarises some of the current strategies, with a focus on the study of haematologic malignancies. While small molecule inhibitors can be very potent they can also have some off target effects. Inhibitors against VEGFR-1 can also inhibit other VEGFRs and other members of the Class V and III RTKs family. Monoclonal antibodies on the other hand have a high specificity toward their targets and numerous antibodies have been

Chapter One: Introduction 38 developed against both VEGFR-1 and -2. Several agents have progressed to clinical trials (SU11248 and ZD6474 phase II, PTK-787 phase III, and bevacizumab (avastin) which has been approved by the Food and Drug Administration in the USA for therapy).

Chapter One: Introduction 39 Table 1.5. Some of the current strategies manipulating VEGF and its receptors in the treatment of different cancers. Treatment Type Target Reference (Sweeney et al. 2001; Bevacizumab Kabbinavar et al. antibody VEGF (Avastin) 2003; Zondor et al. 2004; Marshall 2005) Cyclo-VEGI peptide VEGF (Zilberberg et al. 2003) (CBO-P11) VEGF-Trap R1R2 (Huang et al. 2003; Fusion protein VEGF, PlGF, VEGF-B (AVE-0005) Saishin et al. 2003) (Jayson et al. 2002; HuMV833 antibody VEGF , VEGF 121 165 Jayson et al. 2005) VEGF , VEGF , (Brekken et al. 2000; 2C3 antibody 121 165 VEGFR-2 Zhang et al. 2002) AEE788 VEGFRs, EGFR, HER-2 (Park et al. 2005) (Weng et al. 2001; Angiozyme ribozyme VEGFR-1 Weng et al. 2005) 4- ZD4190 VEGFR-1, VEGFR-2 (Wedge et al. 2000) anilinoquinazoline VEGFR-1, VEGFR-2, (Gingrich et al. 2003; CEP-7055 pyrrolocarbazole VEGFR-3 Ruggeri et al. 2003) VEGFR-1, VEGFR-2, (Matsunaga et al. KRN633 quinazoline VEGFR-3 2006) AAL993 VEGFR-1, VEGFR-2, N-benzylaniline (Lee et al. 2000) (ZK260253) VEGFR-3 indole-ether VEGFR-1, VEGFR-2, AZD2171 (Miller et al. 2006) quinazoline VEGFR-3, PDGFR! VEGFR-1, VEGFR-2, (Liu et al. 2005; Rugo AG-013736 indazole VEGFR-3, PDGFR!, KIT et al. 2005) PTK787/ZK222584 VEGFR-1, VEGFR-2, aminophthalazines (Bold et al. 2000) (Vatalanib) VEGFR-3,PDGFR, KIT SU5416 VEGFR-1, VEGFR-2, indolinone (Kuenen et al. 2003) (Semaxanib) VEGFR-3, PDGFR! GW786034 indazolylpyrimidine VEGFR-2 (Dredge 2004) (Neovastat) IMC-1C11 chimeric antibody VEGFR-2 (Zhu et al. 1998)

IMC-2C6 antibody VEGFR-2 (Zhu et al. 1999)

IMC-1121 antibody VEGFR-2 (Zhu et al. 2003)

CP-547,632 isothiazole VEGFR-2, FGFR, EGFR (Beebe et al. 2003) (Petti et al. 2005; OSI-930 thiophene VEGFR-2, KIT, PDGFR! Garton et al. 2006) VEGFR-2, PDGF, SU6668 indolinone (Kuenen et al. 2005) bFGFR heteroaromatic- ZD6474 VEGFR-2, VEGFR-3, substituted (Ciardiello et al. 2003) (Vandetanib) EGFR/FGFR-1 anilinoquinazoline Bay 43-9006 VEGFR-2, VEGFR-3, tosylate salt (Yano et al. 2000) (Sorafenib) PDGFR, KIT, FLT-3

Chapter One: Introduction 40 1.6.3 Role of VEGF in Leukaemia The role of VEGF in supporting the growth of solid tumours has been much more vigorously studied when compared to investigations into liquid tumours, such as leukaemias. However, there are a growing number of reports which imply that patients with haematological malignancies show an increase in BM angiogenesis (Aguayo et al. 2000; Molica et al. 2002; Kumar et al. 2004). Not only are haematological malignancies associated with an increased number of vessels in the BM, but the vessels also appear to have larger diameters. It has also been put forward that such increases in the proliferating BM endothelial mass releases growth factors, which further support leukaemic growth in a paracrine manner (Fiedler et al. 1997). Expression of the two VEGF receptors has been reported in both patient samples and leukaemia cell lines (Bellamy et al. 2001; Fragoso et al. 2007), suggesting that VEGF and VEGF receptors can act in both an autocrine and a paracrine manner to promote leukaemia cell growth (Figure 1.6).

Autocrine Loop VEGF

c-Kit+ FLT-3+ VEGFR+

Paracrine Loop

SCF Leukaemia cells VEGF FLT-3L

VEGFR+

Bone marrow stroma Figure 1.6. Schematic representation of the possible paracrine and autocrine loops between leukaemia and the BM microenvironment. An example of the possible VEGF autocrine loop in leukaemia cells and the paracrine loop between leukaemia cells and the BM.

Chapter One: Introduction 41 The release of angiogenic factors by leukaemia cells (such as VEGF), infers a poor patient outcome and a rapid progression of the disease (Aguayo et al. 1999). Dias et al. (2000) showed that leukaemia cells not only produce VEGF, but also functional VEGFRs, thereby forming an autocrine loop of growth induction. This process can potentially result in an autocrine loop that can both support leukaemia survival and migration in vivo. After examining the effects of both autocrine and paracrine signalling pathways on leukaemic growth in vivo, using neutralising antibodies against human and mouse VEGFR-2, Dias and colleagues (2001) were able to show that inhibiting only one receptor slowed the growth of the HL60 cell line in NOD/SCID mice. However, only when both pathways were blocked, did they achieve long term remission. It has been demonstrated that the inhibition of VEGF in leukaemia cells can lead to a decrease in cell proliferation and a decrease in angiogenesis (He et al. 2003). This was shown by He and colleagues (2003) when they downregulated VEGF mRNA in a leukaemia cell line. Supernatants from the transfected cells had a decreased induction of migration on proliferation on endothelial cells and the cells showed slower growth in vitro and in vivo in nude mice (He et al. 2003).

In another example, Glenjen (2005) found that the cross-talk between AML blasts and local non-leukaemic cells in the BM resulted in an increase in VEGF levels. Co- culturing AML cells with either fibroblasts or osteoblastic sarcoma cells also resulted in an increase in VEGF production, an enhancement that did not require cell-cell contact. In multiple myeloma, VEGF is secreted by both the malignant cells as well as the BM stromal cells (Dankbar et al. 2000; Gupta et al. 2001). Also, the secreted VEGF has been shown to trigger proliferation and migration of multiple myeloma cells through an autocrine loop, mediated through the MEK-ERK pathway and protein kinase-C (PKC) cascade (Podar et al. 2001). Multiple myeloma patients who exhibited elevated VEGF levels have been shown to have increased angiogenesis in the BM and a higher plasma cell proliferation rate (Vacca et al. 1999a; Rajkumar et al. 2000).

AML patient samples have been shown to express VEGF mRNA, as well as secret VEGF (Fiedler et al. 1997; Aguayo et al. 1999). In a study by Aguayo and colleagues (1999), cellular VEGF levels in AML samples were higher when compared to normal BM samples, showing that in this study, cellular levels of VEGF are an independent prognostic marker of the disease. It is similarly thought that this secretion has both

Chapter One: Introduction 42 autocrine and paracrine effects (Fiedler et al. 1997; Hussong et al. 2000). As these cells express VEGFR-1 and -2, there is the possibility of autocrine effects. Conversely, the BM microenvironment contains endothelial cells expressing VEGF receptors (Larrivee et al. 2003; Narendran et al. 2003; Larrivee et al. 2005). Therefore, a paracrine loop may not only contribute to the growth stimulation of AML blasts in the bone marrow, but it may also give a growth advantage to circulating AML cells and facilitate their expansion into the peripheral blood. The circulation of AML cells may result in a growth stimulation from extramedullary sites, which leaves open the possibility that paracrine growth stimulation may not be restricted to the BM (Fiedler et al. 1997). Bone marrow samples from AML patients, compared to normal controls, show an increase in the expression of both VEGF and VEGFR-2 (Padro et al. 2002). This increase in VEGFR-2, and not of VEGFR-1, was shown to be related to the degree of angiogenesis in the BM. Padro and colleagues (2002) compared the expression of VEGF, VEGFR-1 and -2 in the BM of AML by looking at microvessel densities. At the time of diagnosis, expression of both VEGF and VEGFR-2 correlated with higher microvessel density in the BM, but not so with VEGFR-1. This was followed up with those patients that reached complete remission, and BM biopsies which subsequently demonstrated significantly lower VEGFR-2 levels.

Downregulating VEGF, together with BCR/ABL using antisense oligonucleotides in CML cells has been shown to have an additive tumour suppressing effect (Cong et al. 2005). Cong and colleagues (2005) showed that transfected cells were more sensitive to apoptotic stimuli, and that the tumours formed in nude mice by the transfected cells were significantly smaller further suggesting that VEGF promotes leukaemic survival. High VEGF levels correlate with shorter survival in CML (Verstovsek et al. 2002) and adverse prognosis in Non-Hodgkin’s Lymphoma (Salven et al. 2000). In CLL, the leukaemia cells express both VEGF and its receptors, with high intracellular VEGF levels correlating with poor prognosis (Dias et al. 2002a). The autocrine pathway formed between VEGF and its receptors in CLL is important for the pathogenesis of the disease as it aids in the motility of the cells through the endothelium (Till et al. 2005). Autocrine VEGF also has antiapoptotic effects on CLL cells (Farahani et al. 2005).

VEGF in the serum of paediatric AML patients was shown to be an independent prognostic factor of event-free survival. Patients classified in the low-risk group, exhibit

Chapter One: Introduction 43 significantly lower VEGF serum levels (De Bont et al. 2002). A similar result was found for children with ALL by the Children’s Oncology Group study CCG-1962, where it was shown that standard-risk patients with high or increasing VEGF serum levels during induction therapy, correlated with relapse and poor survival (Avramis et al. 2006). ALL patient samples also express VEGF, and this expression is elevated in recurrent ALL cases, when compared to the diagnostic samples. As such, VEGF can also be used as a prognostic indicator in childhood ALL (Koomagi et al. 2001). Whilst cellular VEGF levels are lower in paediatric cases compared to adult AML cases, there is no difference in VEGFR-2 expression (Jeha et al. 2002).

As a result of autocrine VEGF stimulation, the VEGFR-2 receptor is expressed both on the cell surface as well as internally. Santos et al. (2004) showed that the majority of the receptor is actually nuclear and constitutively active. VEGF neutralising antibodies were able to inhibit VEGFR-2 accumulation to a greater extent than specific VEGFR-2 inhibitors, suggesting the VEGF/VEGFR-2 autocrine loop is external and requires the VEGF to be secreted in contrast to normal haematopoietic cells (Gerber et al. 2002). When anti-VEGF antibody and anti-VEGFR-2 antibodies were used against localised AML, they were able to reduce tumour size in mice (Reichert et al. 2005). Small molecule inhibitors have also been used to target VEGF receptors on AML cells in an attempt to disrupt the possible paracrine loop formed between the BM and AML cells (Smolich et al. 2001).

1.7 FMS-Like Tyrosine-3

1.7.1 Receptor Tyrosine Kinases Receptor tyrosine kinases (RTKs) are a large family of transmembrane proteins which are important in the proliferation, differentiation and survival of many cell types including haematopoietic cells (Rosnet et al. 1993a). The proteins consist of a ligand binding extracellular domain and a catalytic intracellular kinase domain. Their tyrosine kinase activity in normal cells is tightly regulated by the binding of their ligand, which is usually a growth factor. RTKs are often deregulated in malignancy by; over- expression of the receptor and/or its ligand (e.g. FL, VEGF, PDGF), a mutation in the kinase rendering it constitutively active (e.g. FLT-3, KIT, PDGFR).

Chapter One: Introduction 44 Of relevance to this project are the class III and V RTKs. They share common structural features, being composed of an extracellular region made up of several immunoglobulin-like sub-domains (class III have five, class V have seven), a juxtamembrane (JM) domain, and two intracellular tyrosine kinase domains divided by a kinase insert. Class III consists of FMS-like tyrosine kinase-3 (FLT-3), KIT, FMS and the platelet derived growth factor (PDGF) receptors, while class V is composed of the VEGF receptors (discussed previously). FMS, KIT and FLT-3 share sequence and structural similarities (Agnes et al. 1994) and the same is true for their ligands, CSF, SCF and FL, with SCF and FL sharing 4 conserved cystine residues. FMS is a receptor for either the colony-stimulating factor-1 (CSF1) or macrophage CSF (M-CSF) and it is involved in the proliferation and differentiation of macrophage precursors, as well as mononuclear phagocytes and osteoclasts. KIT is a receptor for stem cell factor (SCF) or steel factor (SF). The SCF–KIT axis plays an important role in the development of many cell types, including primordial germ cells, melanoblasts and haematopoietic cells. They also play a role in the survival, proliferation and differentiation of multipotential progenitors, lymph haematopoietic progenitors, erythrocytes, mast cells and in early B and T lymphopoiesis. FLT-3 is expressed on haematopoietic progenitors and lymphoid precursors, and thus is an essential component in haematopoiesis.

1.7.2 FLT-3 The crucial role that FLT-3 performs in haematopoiesis has been demonstrated by disruption studies. More specifically, disruption of the murine FLT-3 gene, or its ligand (FL), results in a deficiency in the development of hematopoietic progenitors (Mackarehtschian et al. 1995; McKenna et al. 2000). FLT-3 knockout mice survive, are generally healthy and have a normal number of peripheral blood cells. They do however have a reduced number of very primitive B lymphoid progenitors, and in transplantation experiments, the progenitor cells from FLT-3 deficient mice result in an impaired developmental capacity. This is because the cells are unable to repopulate, with the greatest impact being seen in the lymphoid lineage (Mackarehtschian et al. 1995). As with FLT-3 knockout mice, FL knockout animals have a healthy appearance and a defect in early B-cell development. Nevertheless, one difference is reduced cellularity in the peripheral blood, spleen and BM (McKenna et al. 2000).

Chapter One: Introduction 45 The human FLT-3 gene is located on chromosome 13q12 and it is composed of 24 exons. The gene encodes for a membrane-bound N-glycosylated protein of 993 amino acids with a molecular weight of 158-160 kDa, and a non-glycosylated protein of 130-143 kDa, that is not associated with the plasma membrane (Rosnet et al. 1993b). In common with other members of the class-III RTK family, FLT-3 is composed of an extracellular region containing five immunoglobulin-like sub-domains, a transmembrane domain, a juxtamembrane (JM) domain, and two intracellular tyrosine kinase domains divided by a kinase insert (as shown in Figure 1.7). It has been suggested that both the juxtamembrane region and the activation loop of the tyrosine kinase domain are important in mediating an autoinhibitory function of the receptor (Yokota et al. 1997; Brown et al. 2004).

FLT-3 is expressed by primitive CD34+ haematopoietic cells, dendritic cells, B- progenitors and natural killer cells, as well as on neural tissues, the gonads and the placenta (Rosnet et al. 1993b). In contrast, FL is expressed in a wide variety of tissues, suggesting that FLT-3 is the limiting factor in determining specificity (Stirewalt et al. 2003).

Chapter One: Introduction 46

Figure 1.7. Structure of FLT-3. FLT-3 is composed of an extracellular region containing five immunoglobulin-like sub-domains, a transmembrane domain, a juxtamembrane (JM) domain, and two intracellular tyrosine kinase domains divided by a kinase insert. Both the JM region and the activation loop of the tyrosine kinase domain appear to be important in mediating an autoinhibitory function of the receptor (Markovic et al. 2005).

Aside from its role in regulating the expansion of normal haematopoietic progenitors, FLT-3 is also highly expressed in several haematological malignancies, including AML (over 90% of cases), B-acute ALL (up to 100% of cases) and T-ALL (27-87% of cases) (Birg et al. 1992; Brasel et al. 1995; Meierhoff et al. 1995; Carow et al. 1996; Drexler 1996). Mutations of the receptor, resulting in its constitutive activation, have also been observed in these malignancies, the majority of which occur in AML cases (up to 45%), making it the most commonly mutated gene in AML (Meshinchi et al. 2001; Stirewalt et al. 2003). A number of inhibitors have been developed against FLT-3 which act against both the wild-type and mutant receptors (Table 1.6).

Chapter One: Introduction 47 Table 1.6. Some of the compounds in use against FLT-3. Refer to (Markovic et al. 2005)

Compound Name Clinical Derivation Reference

SU11248 indolinone (O'Farrell et al. 2003b; Fiedler et al. 2005)

CT53518 quinoxaline (Kelly et al. 2002c) (Miknyoczki et al. 1999; Levis et al. 2002; CEP-701 indolocarbazole Smith et al. 2004) AG1295 quinoxaline (Levis et al. 2001)

PKC412 indolocarbazole (Kocic et al. 2001; Armstrong et al. 2003) tricyclic benzofurano- GTP-14564 (Murata et al. 2003) indazolo

1.7.3 Mutations in FLT-3 and Expression in Leukaemia Two distinct types of mutations have been found in patients with AML, the most common being an internal tandem duplication (ITD) in the JM, which is encoded by exons 14 and 15 (Nakao et al. 1996). Even though these insertions vary in length, they always maintain a head-to-tail orientation and preserve the reading frame. ITDs cause ligand-independent dimerisation and thereby give rise to constitutive receptor activation, enabling cytokine-independent proliferation of haematopoietic cells. It has been suggested that it is a conformational change in the JM domain of the FLT-3 receptor, rather than a specific duplication, that leads to dimerisation and its activation (Meshinchi et al. 2001). ITDs have been reported to be associated with both leukaemocytosis (Kiyoi et al. 1997) and leukaemic transformations (Horiike et al. 1997). FLT-3/ITDs occur in 30-40% of AMLs, 5-10% of MDS cases, and in a small number of T-ALL cases (Xu et al. 1999; Rombouts et al. 2000; Stirewalt et al. 2003; Jonsson et al. 2004). Unfortunately, the presence of an ITD confers a poor prognosis in paediatric AML cases, and is an independent indicator of poor clinical outcome (Yokota et al. 1997; Xu et al. 1999; Meshinchi et al. 2001; Minami et al. 2003).

The second most common FLT-3 mutation in AML is a missense point mutation in the activation loop within exon 20. Almost all mutations involve an aspartate to tyrosine substitution at codon 835, but changes to histidine or glutamic acid have also been observed, as have other mutations. These occur either as deletions (Asp835 - Ser838) or insertions (Asp835, Leu836, Glu837) (Shih et al. 2004; Takahashi et al. 2004). Such

Chapter One: Introduction 48 mutations cause a conformational change, which may then disrupt the autoinhibitory function, effectively rendering the receptor constitutively active. These have been found to occur in 5-10% of AML cases, 2-5% of MDS cases, and in 1-3% of ALL patients. These mutations also show ligand-independent activation of the receptor, which increases the intrinsic tyrosine-kinase activity. However, it still remains unclear whether dimerisation of the receptors is required or if this intrinsic activity has a different signalling cascade to the wild-type receptor, as is the case with ITDs (Stirewalt et al. 2003). The TKD mutations have not been found to have a significant influence on clinical outcome, which may be due to the low frequency of this mutation and the relatively small number of patients evaluated (Mead et al. 2007).

Mutations in FLT-3, both ITDs and point mutations at Asp835, have rarely been observed in patients with ALL. Recently however, several studies have observed high levels of FLT-3 expression, as well as mutations in the receptor, in infant ALL with MLL translocations (Xu et al. 1999; Armstrong et al. 2004; Taketani et al. 2004). ALL/MLL show markers of both lymphoid and myeloid lineage and also have consistently very high expression of FLT-3. This over-expression of the receptor has led to further investigations into its status, with the discovery that up to 18% of samples examined had an activation mutation of FLT-3 (Taketani et al. 2003; Armstrong et al. 2004). These were found to be point mutation in the activation loop of FLT-3.

1.7.4 Signalling Cascade of FLT-3 The binding of FLT-3 with FL causes the receptor to dimerise, leading to tyrosine kinase activation and receptor autophosphorylation, thus initiating the phosphorylation of downstream proteins (Lyman 1995). The binding affinity of FL to its receptor has been estimated to be 200 to 500 pM (Turner et al. 1996). The process of activation, internalisation and the subsequent degradation of FLT-3 occurs in a similar fashion to other members of the class III RTK family such as KIT. After ligand binding and dimerisation, the receptors are internalised and degraded (Stirewalt 2004). The wild type FLT-3 transduces its signalling cascade through the phosphorylation of the receptor and activation of intracellular kinases. Wild-type FLT-3 transduces its signaling cascade principally via the PI3K and Ras pathways, leading to activation of

Chapter One: Introduction 49 AKT and ERK1/2 (Yokota et al. 1997). FLT-3 appears not to bind directly to the p85 subunit of PI3K, but forms a complex with other proteins such as SHC, SHP, SHIP, GRB2 and GAB2, which then acts on the RAS and PI3K pathways. The pro-survival function of FLT-3 may be mediated by phosphorylation of the pro-apoptotic Bad protein, by induction of the anti-apoptotic Bcl-2, or by preventing induction of the pro- apoptotic Bax (Meshinchi et al. 2001; Karlsson et al. 2003; Minami et al. 2003).

FL

RAS PI3K GRB2 GRB2 SHP PDK1 p p SHIP p p RAF1 GAB1/2 SHC AKT

MEK1/2 BAD BCL2 FOXO3 STAT5 ERK1/2 BAX BIM

Survival & proliferation

Figure 1.8. The FLT-3 signalling cascade. (Markovic et al. 2005)

The signalling mechanisms of both wt and mutant, FLT-3/ITD have been shown to possess similar and unique pathways (Minami et al. 2003; Quentmeier et al. 2003). FLT-3/ITD initiates tyrosine phosphorylation and activates Ras/MAPK, AKT and signal transducer and activator of transcription (STAT) 5 (Stirewalt et al. 2003). Several groups have demonstrated that STAT5 is phosphorylated by the constitutively activated

Chapter One: Introduction 50 receptor, but only weakly by the wt receptor after signalling with FL (Spiekermann et al. 2003). Murata et al. (2003) proposed that STAT5 plays a more critical role in FLT-3/ITD signalling compared to the wt receptor. The signalling through STAT5 may also prevent the upregulation of apoptotic genes, such as Bax (Kohl et al. 2007). The mutant FLT-3/ITD receptor has also been shown to inhibit the function of the Forkhead transcription factor FoxO3, thereby inhibiting induction of the pro-apoptotic Bim protein. Therefore their phosphorylation by FLT-3/ITD inhibits their function and promotes their translocation to the nucleus. By this process FLT-3/ITD can suppress apoptosis and advance cell survival and proliferation (Jonsson et al. 2004; Scheijen et al. 2004). Pathways considered to be preferentially activated by FLT-3/ITD are shown in blue in Figure 1.8.

1.8 Summary

In summary, whilst the two facets of leukaemia development (VEGF and FLT-3) have been widely studied independently, the interactions between the two have yet to be examined in ALL. The evidence from the literature would suggest that both play a role in the progression of the disease. A link between the two components is compelling because both have already been targets of study and drug development, therefore the identification of a link may give new insights into the mechanisms of these already known targets.

The subsequent chapters address the relationship between VEGF and FLT-3 in ALL cells, followed by the analysis of the FLT-3 signalling cascade. Finally, microarray studies of ALL xenograft cells is used to attempt to; 1) identify the effects of FLT-3 activation on gene expression in ALL cells other than its effect on VEGF, and 2) the effects of BM cells on the gene expression profile of ALL cells.

Chapter One: Introduction 51

 

CHAPTER TWO: Materials and Methods

2.1 Tissue Culture

2.1.1 Reagents Quality Biologicals Serum Free-60 (QBSF-60) was purchased from Quality Biologicals, (Rockville, USA). The amount of FLT-3 ligand (FL) used in all experiments was 20 ng/ml, and was kindly provided by Amgen Inc., (Thousand Oaks, USA). Roswell Park Memorial Institute-1640 (RPMI-1640) medium, Iscove’s Modified Dubbecco’s Medium (IMDM), Minimum essential medium alpha (MEM-), Dulbecco’s phosphate buffered saline (PBS), antibiotic-antimycotic solution (100 U/mL penicillin G sodium; 100 )g/mL streptomycin sulfate), L-glutamine (2 mM) (PSG), trypsin solution (0.25% weight/volume (w/v) trypsin in Hank’s solution), foetal calf serum (FCS), and Insulin, Transferrin and Selenium Supplement (100 X) liquid solution (ITS) were all purchased from Invitrogen Life Technologies (Gaithersburg, USA). Tissue culture flasks were obtained from Corning (Lowell, USA) and all tissue culture plates (6-, 12-, 24- and 96-well) were from Greiner Bio One (Frickenhausen, Germany). Cells were visualised using an inverted microscope from Olympus Optical Company (Tokyo, Japan). SU11657 was a gift from Pfeizer (La Jolla, USA). SU11657 powder was dissolved in dimethylsulfoxide (DMSO) (Sigma, St Louis, USA). The FLT-3 blocking antibodies (EB10 and D43) were kindly provided by ImClone Systems Incorporated (New York, USA).

Chapter Two: Materials and Methods 53 2.1.2 In Vitro Cell Culture Childhood ALL xenografts were derived from patient biopsies as described previously by Lock et al (2002). For all experiments performed in these studies, xenograft cells were retrieved from cryostorage. Briefly, cells were thawed rapidly and resuspended in RPMI-1640 medium containing 10% FCS and PSG following centrifugation (500 xg for 5 min). They were then washed in RPMI-1640, with cell viability estimated by trypan blue exclusion. Cells were re-centrifuged, resuspended in QBSF-60 medium supplemented with PSG, and used for all subsequent experiments.

All human leukaemia cell lines and ALL xenograft cells were maintained at 37°C with

5% CO2 in humidified incubators. The MV4;11 cell line was purchased from the American Tissue Culture Collection (Manassas, USA) and maintained in IMDM supplemented with 20% FCS, PSG and 1X ITS solution. The RS4;11 cell line was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures), GmbH, (Braunschweig, Germany) and was maintained in MEM- with 10% FCS and PSG. All other leukaemia cells lines (REH, NB4, NALM6 and HL60) were maintained in RPMI-1640 medium with 10% FCS and PSG. This general method of cell culture was undertaken for all the experiments outlined below. The murine stromal cell line MS5 was maintained in MEM- with 10% FCS and PSG.

For experiments in which ALL xenograft cells were co-cultured with stromal cells, MS5 cells were seeded into 96 well, U-bottomed plates and grown until confluent. Wells were washed twice with PBS, with ALL xenograft cells (2x106 cells/ml) subsequently added and cultured for the required time.

2.2 Enzyme Linked Immunosorbent Assay (ELISA)

2.2.1 Reagents High binding flat bottom 96-well plates were purchased from Greiner Bio One. Acetate plate seals, Sucrose and Tween®-20, were supplied by MP Biomedicals (Seven Hills, Australia). Bovine Serum Albumin (BSA) fraction V; 3,3’,5,5’-Tetramethylbenzidine (TMB) tablets and the phosphate citrate buffer with sodium perborate capsules were Chapter Two: Materials and Methods 54 from Sigma. While the anti-human polyclonal VEGF antibody, anti-human biotinylated VEGF antibody, recombinant human VEGF (rhVEGF) and the Strepavidin-Horseradish Peroxidase (HRP) were all purchased from R&D Systems (Minneapolis, USA), Sulfuric

Acid (H2SO4) was from Ajax Finechems (Taren Point, Australia).

2.2.2 ELISA ALL xenograft cells and the MV4;11 cell line were cultured in several different treatment conditions for 72 hours (hrs) in 96-well U-bottomed plates. The conditioned media (CM) was collected and stored at -80°C until it was assayed. The secretion of VEGF by the cells was assayed by ELISA, using commercially available reagents. High binding 96-well plates were coated with polyclonal anti-human VEGF antibody (100 )l at 1 )g/ml) in sterile PBS, sealed and incubated overnight at room temperature (RT), then were washed 3X with 300 )l wash buffer (PBS with 0.1% Tween®). They were then blocked for 1 hr with 300 )l PBS containing 5% sucrose and 5% BSA (w/v) and the washing step was repeated. After washing, rhVEGF was serially diluted to form a standard curve for the determination of VEGF concentrations in the CM. This was added to the wells, along with the CM previously collected. The plates were sealed and incubated at 37°C for 1.5 hrs. Plates were again washed and the biotinylated anti-VEGF antibody added (100 )l/well). The biotinylated anti-VEGF antibody was diluted in 1% BSA in PBS; the plates were again incubated for 1.5 hrs at 37°C . After the incubation and another round of washing, avidin-HRP (in 1% BSA/PBS) was added for 30 min and the colour developed with TMB in Phosphate Citrate buffer using Perborate.

The reaction was stopped with 2 M H2SO4, and absorbance read at 450 nm, using 655 nm as a reference.

Cells were also harvested at the same time as the media was collected and viable cells counted in each well, in order to determine the amount of VEGF secreted per viable cells. Cell viability was measured using flow cytometry (Section 2.3.2). VEGF was expressed as picograms (pg) VEGF per 1 million viable cells.

Chapter Two: Materials and Methods 55 2.3 Flow Cytometry

2.3.1 Reagents Normal mouse serum was purchased from the Veterinary Services Division at the Institute of Medical and Veterinary Science (Adelaide, Australia). Standard, L10, Nominal 10 μm, Latex Particle (flow cytometry counting beads) were purchased from Beckman Coulter (Fullerton, USA). All antibodies, unless otherwise stated, were purchased from Becton Dickinson (BD), (Franklin Lakes, USA). All samples were analysed on the BD FACSCalibur™ system.

2.3.2 Cell viability After removal of CM from wells, the cells were harvested and stained with antihuman CD45 antibody conjugated with allophycocyanin (APC). Cells were then analysed using flow cytometry, and only CD45 positive cells were gated. Viable cells were assessed by flow cytometry according to the following:

Number of viable & CD45+ cells Number of cells / well = × Number of beads added Number of beads

2.3.3 Receptor levels Cells were incubated with antihuman FLT-3 antibody conjugated to phycoerythrin (PE) or Immunoglobulin (Ig) G isotype control. The levels of FLT-3 expression were compared to the isotype control on overlaying histograms, and relative fluorescent intensity (RFI) was determined as the geometric mean between FLT-3 and the geometric mean of the isotype control.

Chapter Two: Materials and Methods 56 2.4 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

(MTT) Cytotoxicity Assay

2.4.1 Reagents MTT was from Sigma, sodium dodecyl sulfate (SDS) from ICN Biomedicals (Aurora, USA) and hydrochloric acid (HCL) from Ajax Finechem. The solubilisation solution was made from 10% SDS in 0.01 M HCl (SDS/HCl). MTT solution was prepared from powder to 5 mg/ml in sterile PBS.

2.4.2 MTT Assay The MTT assay was used to test the in vitro cytotoxicity of the small molecule inhibitor SU11657 on ALL xenograft cells and cell lines. In this particular assay, MTT is reduced to a formazan product. The crystals are solubilised with SDS/HCl and the absorbance measured.

ALL xenograft cells or cell lines were suspended in their appropriate medium at optimal cell densities and 100 μl of the cell suspension was added to each well in a sterile

U-bottomed 96-well plate and equilibrated overnight in a 37°C, 5% CO2 incubator. Increasing concentrations of SU11657 (1 nM to 1 μM) were then added in triplicate. Additional wells were set up containing medium only, for blanks and cells with solvent (DMSO) only for cell viability controls. After 48 hrs, 12 μl of MTT labelling reagent (5 mg/ml) (Sigma) was added to all wells and plates, which were then incubated for an additional 6 hrs (37°C, 5% CO2). Solubilisation solution was added at 100 )l per well and the plates were placed in a 37°C, 5% CO2 incubator overnight for the formazan crystals to dissolve. The optical density of each well was measured at 560 nm using a spectrophotometric plate reader (GE Healthcare, Buckinghamshire, UK). Cell survival at each concentration was assessed by mitochondrial metabolic activity relative to controls. Data was expressed as percentage cell viability and calculated using the following formula:

Average absorbance of treated wells % cell viability = × 100 Average absorbance of control wells

Chapter Two: Materials and Methods 57 Data was compared by the best fit to the sigmoidal dose response curve with variable slope (GraphPad Software, Version 4, San Diego, USA) and the IC50 value determined as the concentration that inhibited cell growth by 50%. Sublethal concentrations (less then the IC50) of SU11657 were used for all subsequent experiments.

2.5 RNA Isolation

2.5.1 Reagents Trizol® reagent, RNase/DNase free water and the RNA ladder were all purchased from Invitrogen, RNeasy RNA isolation kit was from QIAGEN (Valencia, USA), DNA Grade Absolute Ethanol, Chloroform, Isopropanol, Sodium Acetate, Ethylenediaminetetraacetic Acid Tetrasodium Salt (EDTA), [N-morpholino] propanesulfonic acid (MOPS) was purchased from Sigma. Formaldehyde from Ajax Chemicals (Sydney, Australia), DNA Grade Agarose from Progen Industries (Darra, Australia), Ethidium Bromide, Formamide, Bromophenol blue were from Merck (Kilsyth, Australia), and the glycerol from ICN Biochemicals.

2.5.2 RNA Isolation: With Trizol® After treatment, total cellular RNA was isolated from cell suspensions. Cell suspensions were collected, washed in ice-cold PBS, and pelleted (500 xg; 5 min; 4°C). Cell pellets were resuspended in 1 ml Trizol® reagent. Conversely, adherent cells were washed in ice-cold PBS after which 1 ml Trizol® reagent was added. Cells were then scraped with a cell scraper and collected into a 1.5 ml centrifuge tube. The remainder of the procedure was undertaken in accordance with the manufacturer’s instructions.

Samples in Trizol® reagent were vortexed for 1 min. Lysate was incubated at room temperature for 5 minutes. Chloroform was added (200 )l) and the samples were shaken vigorously for 15 sec followed by a further 5 min incubation at room temperature. Samples were then centrifuged (12000 xg for 15 min at 4°C,) and the upper aqueous layer collected. RNA in the aqueous layer was precipitated using an equal volume of isopropanol. After isopropanol addition samples were mixed by vortexing and incubated for 1 hour at -20°C. Samples were then centrifuged (12000 xg for 15 min at 4°C) and

Chapter Two: Materials and Methods 58 RNA pellets washed twice with 75% ethanol by vortexing. RNA pellets were collected by centrifugation (7500 xg for 5 min at 4°C), after which they were air-dried and dissolved in RNase/DNase free water.

The concentration of RNA was determined using spectrophotometric absorbance, measured at 260 nm, with one A260 OD unit taken as the equivalent to 40 )g/ml of

RNA. A260/A280 ratios 1 2.0 indicated that samples were considered free of contaminating protein.

2.5.3 Modified RNA Isolation: With Trizol® and the RNeasy Kit RNA isolation procedure was followed as described above until the precipitation of RNA. Instead, the aqueous layer was collected and the RNA was precipitated using an equal volume of 70% ethanol. Ethanol was added drop by drop while vortexing the sample slowly. Samples were then loaded onto a QIAGEN RNeasy spin column following the manufacturer’s instructions.

Briefly, the RNeasy columns were centrifuged for 5 min at RT at 3000 xg. The flowthrough was discarded and 700 )l of buffer RW1 added. RNeasy columns were centrifuged for 5 min at RT at 3000 xg. The flowthrough and collection tube were discarded. The spin column was placed into a new collection tube and 500 )l of buffer RPE added and centrifuged for 5 min at RT at 3000 xg. This step was repeated and the flowthrough and collection tube discarded. The spin column was placed in a 1.5 ml centrifuge tube and 20 )l H2O was added to elute the RNA. The tubes were incubated for 1 min at RT after which they were centrifuged at 3000 xg for 5 min. RNA concentration was quantified by NanoDrop (Wilmingtion, USA).

2.5.4 Formaldehyde Agarose Gel Electrophoresis for Determining RNA Integrity Formaldehyde agarose (FA) gel electrophoresis was used to determine RNA integrity because of its ability to maintain RNA in its denatured condition. RNA retains much of its secondary structure during electrophoresis unless it is first denatured, and formaldehyde is added to the agarose gel for this reason. A 1.2% FA gel was prepared

Chapter Two: Materials and Methods 59 as per the following. To agarose powder (0.55 g), 5 ml of 10X MOPS buffer and 45 ml RNase/DNase-free water were added.

Table 2.1. 10X MOPS Buffer. Reagent Amount Final Concentration

MOPS, pH 7 41.8 g 200 mM

Sodium Acetate (1 M) 20 ml 50 mM

EDTA (0.5 M), pH 8 20 ml 10 mM

H2O To 1 L

The agarose powder was melted by heating in a microwave oven and subsequently cooled ‘to touch’ prior to adding 900 )l of 37% formaldehyde and 0.5 )l of ethidium bromide (10 mg/ml) into the gel solution. Agarose was then poured into a casting tray and allowed to solidify with a well-forming comb set in place. The FA gel was equilibrated in 1X MOPS running buffer (Table 2.2) prior to the addition of samples, for at least 30 minutes by running at 30 V.

Table 2.2. 1X MOPS Running Buffer. Reagent Amount

10X MOPS buffer 100 ml

37% formaldehyde 20 ml

RNase/DNase free water 880 ml

Total RNA (10 )l) was mixed with 5X loading buffer (2.5 )l). This mixture was incubated at 65°C for 5 min to denature the RNA and then placed on ice. This mixture was then loaded onto the equilibrated FA gel. The gel was run using 1X MOPS running buffer at 75 V for 2 hrs. RNA was visualized and imaged on the Bio-Rad Versa Doc apparatus.

Chapter Two: Materials and Methods 60 Table 2.3. 5X RNA Loading Buffer. Reagent Amount

saturated aqueous bromophenol blue solution 16 )l

500 mM EDTA pH 8.0 80 )l

37% formaldehyde 720 )l

100% glycerol 2 ml

formamide 3.084 ml

10X MOPS buffer 4 ml

RNase/DNase free water To 10 ml

2.6 Complementary DNA synthesis and Real-Time Reverse

Transcriptase -Polymerase Chain reaction (RT-PCR)

2.6.1 Reagents Moloney murine leukaemia virus (MMLV) reverse transcriptase, 5X first strand buffer

(50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2), 0.1 M dithiothreitol (DTT) and RNase/DNase free water were from Invitrogen Life Technologies; RNasin ribonuclease inhibitor from Promega (Madison, USA); deoxynucleoside triphosphates (dNTPs) and random hexamer primers from GE Healthcare (Buckinghamshire, UK), EF1alpha primers were purchased from both Sigma-Genosys and Invitrogen. The following were all ordered from Applied Biosystems (Foster City, USA): Elongation factor (EF)1 probe, gene expression assays for all genes of interest (VEGF, HIF1, telomeric repeat binding factor (TERF)-2, early growth response (EGR)-1, procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha polypeptide I (P4HA1), 2X Taqman mastermix, as well as real-time PCR plates, optical caps and plate seals. The reactions were performed on either the ABI Prism 7000 or ABI Prism 7500 (Applied Biosystems).

Chapter Two: Materials and Methods 61 Table 2.4. EF1 primer and probe sequence. Primer Sequence

Forward CTG AAC CAT CCA GGC CAA AT

Reverse GCA GTG TGG CAA TCC AAT

Probe 5’VIC - AGC GCC GGC TAT GCC CCTG - TAMRA 3’

Table 2.5. List of gene expression assays. Gene Assay code

VEGF Hs00173626_m1

HIF1 Hs00153153_m1

EGR1 Hs00152928_m1

P4HA1 Hs00168575_m1

TERF2 Hs00194619_m1

2.6.2 cDNA synthesis cDNA was prepared based on a previously published protocol (Noonan et al. 1990), from RNA isolated and quantitated as described above (section 2.5). For a single aliquot of cDNA, 2 )g of total cellular RNA was added to a thin walled PCR tube with 1 )l

(0.1 )g) random primers and made up to 5.3 )l with RNase/DNase free H2O. The reaction was heated to 65°C for 10 min., and then cooled on ice for 2 min. To each aliquot 4.7 )l RT mix (Table 2.6 below) was added and incubated for 60 min at 37°C. To confirm that all contaminating DNA had been removed, each RNA sample was subjected to a mock reverse transcription by omitting the reverse transcriptase from the reaction mix. The reaction mix was incubated for 60 min at 37°C. After the incubation the reaction was diluted 1:5 with RNase/DNase free water and stored at -20°C.

Chapter Two: Materials and Methods 62 Table 2.6. cDNA RT master mix reagents. Reagent Volume

RT + RT -

5X first strand buffer 2 μl 2 μl

0.1M DTT 0.33 μl 0.33 μl

RNasin (40U/μl) 0.33 μl 0.33 μl

2.5mM dNTPs 1 μl 1 μl

200U MMLV 1 μl 0 μl

RNase/DNase free H2O 0 μl 1 μl

Total 4.7 μl 4.7 μl

2.6.3 Real-Time RT-PCR For every single reaction 1 )l of the diluted cDNA was added in triplicate to 96-well PCR plated. To that 19 )l of the reaction master mix (Table 2.7) was added. Each reaction was run in triplicate.

Table 2.7. Real-time master mix. Reagent Volume

2X Taqman master mix 12.5 )l

Gene of interest probe & primer mix 1 )l

EF1 forward primer (10 )M) 0.6 )l

EF1 reverse primer (10 )M) 1 )l

EF1 probe (10 )M) 0.4 )l

RNase/DNase free water to 20 )l

The plate was sealed with a clear optical plate seal. Plates were vortexed to mix the DNA and mastermix and centrifuged at 500 xg for 5 min. Plates were subsequently run on the ABI prism real-time PCR machine (7000 or 7500) using a standard protocol. The running conditions are shown in Table 2.8.

Chapter Two: Materials and Methods 63 Table 2.8. Real-time cycle times. Cycle Temperature Time Repeat

1 50°C 10 min 1 times

2 95°C 2 min 1 times

95°C 15 sec 3 40 times 60°C 1 min

4 72°C 10 min 1 times

An amplification threshold value or threshold cycle (Ct), defined as the PCR cycle number at which the run (Rn) crosses a fluorescence signal threshold, was calculated for each reaction. Each reaction had probes of both the genes of interest as well as the EF1 housekeeping gene. Therefore the Ct value of the gene of interest was normalised to the EF1 control, giving the Ct for each reaction. Relative change of each samples mRNA due to treatment were determined by Ct analysis that involved taking the Ct value at the treatment point and normalising it to the Ct of the untreated control.

2.7 Semi-Quantitative RT-PCR of VEGF Isotypes

2.7.1 Reagents AmpliTaq® Gold DNA polymerase, AmpliTaq® Gold 10X PCR buffer and 10 mM magnesium chloride (MgCl2) solution were all purchased from Applied Biosystems, dNTPs were from GE Healthcare. The primer sequences for EF1 are shown in Table 2.4 above, and primer sequences for VEGF are shown in the Table 2.9 below. Primers were purchased from SigmaGenosys. DNA Grade Agarose from Progen Industries, Ethidium Bromide, Bromophenol blue were from Merck, 10X TAE buffer was from Invitrogen.

Table 2.9. VEGF primer sequences. Primer Sequence

Forward CTG AAC CAT CCA GGC CAA AT

Reverse GCA GTG TGG CAA TCC AAT

Chapter Two: Materials and Methods 64 2.7.2 RT-PCR RNA was isolated and reverse transcribed as previously described, Section 2.6.2. For the PCR reaction 1 )l of the cDNA was used and mixed with 24 )l of the appropriate RT mix (given in Tables 2.10 and 2.11).

Table 2.10. PCR master mix for VEGF isotypes. Reagent Volume

10X Buffer 2.5 )l

10 mM MgCl2 1 )l

Taq GOLD Polymerase 1 )l

dNTPs 4 )l

VEGF / EF1 forward primer (10 )M) 2 / 0.6 )l

VEGF / EF1 reverse primer (10 )M) 2 / 1 )l

RNase/DNase free water to 25 )l

All the reagents were mixed in a thin-walled PCR tubes, they were mixed by vortexing and quickly spun down. The PCR was run in a standard PCR machine (Bio Rad, Hercules, USA) under the conditions in Table 2.11 below.

Table 2.11. PCR running conditions for both VEGF and EF1. Cycle Temperature Time Repeat

1 95°C 5 min 1 times

94°C 20 sec

2 60°C 1 min times

72°C 1 min

3 72°C 10 min 1 times

2.7.3 Agarose Gel Elecrophoresis PCR products were separated by electrophoresis on 1% agarose gels. Agarose gels comprising of agarose (1 g) dissolved in 1X TAE buffer, by boiling in the microwave, then the solution was allowed to cool to prior to the addition of ethidium bromide

Chapter Two: Materials and Methods 65 (1 mg/ml, 10 )l). The agarose was poured into a casting tray and allowed to solidify with well-forming comb set in place. The gel was then submerged in 1X TAE buffer and aliquots of the PCR which were previously combined with 10X gel loading buffer (0.25% bromophenol blue; 25% sucrose) loaded into the wells and electrophoresed at 80 V for approximately 45 min or until the dye front travelled half way down the gel. Under ultraviolet transillumination (302 nm) the DNA bands were visualized and imaged on the Bio-Rad VersaDoc apparatus.

2.8 VEGF mRNA Stability

2.8.1 Reagents MMLV reverse transcriptase, 5X first strand buffer (50 mM Tris-HCl pH 8.3, 75 mM

KCl, 3 mM MgCl2), 0.1 M DTT AND RNase/DNase free water were from Life Technologies (Grand Island, USA); RNasin ribonuclease inhibitor from Promega (Madison, USA); dNTPs and random hexamers primers from GE Healthcare. The following were all ordered from Applied Biosystems: gene expression assays for VEGF mRNA and 18S ribosomal RNA, 2X Taqman mastermix, as well as real-time PCR plates, optical caps and plate seals. The reactions were performed on either the ABI Prism 7000 Sequence Detection System from Applied Biosystems. Actinomycin D was purchased from Sigma.

2.8.2 Actinomycin D Experiment Cells were cultured in 6-well plates under standard culture conditions, after 24 hrs cells were treated with 15 μg/ml actinomycin D. The treated cells were harvested after 1 and 2 hrs of actinomycin D addition. RNA was isolated from each flask (see 2.3.3) and cDNA generated from 2 μg starting RNA as described in section 2.4.2, the reagents and their quantities used in the PCR are given in Table 2.12. Quantitative PCR reactions were performed using the ABI Prism 7000 Sequence Detection System. VEGF mRNA was compared to the 18S RNA, under similar conditions as previously. VEGF mRNA was quantified against the 18S ribosomal RNA and the levels were expressed as the fold changed compared to the untreated controls.

Chapter Two: Materials and Methods 66 Table 2.12. PCR master mix for Actinomycin D experiments. Reagent Volume

2X Taqman master mix 12.5 )l

VEGF mix 1 )l

18S mix 1 )l

RNase/DNase free water to 20 )l

2.9 Genomic DNA PCR of the FLT-3 Receptor

2.9.1 Reagents Blood DNA isolation kit and the QIAquick gel extraction kit for purification of PCR products from agarose gels were purchased from QIAGEN (Valencia, USA), RNase/DNase Free water, AmpliTaq® Gold DNA polymerase, AmpliTaq® Gold 10X

PCR buffer, 10 mM MgCl2 solution were all from Applied Biosystems. Primers for the FLT-3 gene and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Sigma-Genosys. The sequences are given in Table 2.13. DNA grade agarose Progen Industries; 50X TAE Buffer from Invitrogen Life Technologies, Ethidium Bromide, EcoRV enzyme, enzyme Multi-CoreTM buffer and BSA were all purchased from Promega (Madison, USA).

Table 2.13. Primer sequences. Primer Sequence

FLT-3 Exon 14 Forward GCAATTTAGGTATGAAAGCCAGC

FLT-3 Exon 14 Reverse CTTTCAGCATTTTGACGGCAACC

FLT-3 Exon 20 Forward TCACCGGTACCTCCTACTG

FLT-3 Exon 20 Reverse AAATGCACCACAGTGAGT

GAPDH Forward CCCAACTTTCCCGCCTCTC

GAPDH Reverse CAGCCGCCTGGTTCAACTG

Chapter Two: Materials and Methods 67 2.9.2 Isolation of genomic DNA (gDNA) Total cellular DNA was isolated from 5 million ALL xenograft cells or cell lines. The blood DNA isolation kit from QIAGEN was used and manufacturer’s instructions were followed. Briefly, 20 )l of proteinase K, 200 )l of the cell suspension and 200 )l of buffer AL were added to a 1.5 ml centrifuge tube and pulse-vortexed for 15 sec. Samples were incubated for 10 min at 56°C. After the incubation 200 )l ethanol (96 - 100%) was added and pulse-vortexes for 15 sec.

The whole mixture was applied to a spin mini column and centrifuged at 6000 xg for 1 min. The collection tube with the filtrate was discarded and the spin column was then placed into a new collection tube. 500 )l buffer AW1 was added and centrifuged at 6000 xg for 1 min. The collection tube with the filtrate was discarded and the spin column was then placed into a new collection tube. Next, 500 )l buffer AW21 was added and centrifuged at full speed (20000 xg) for 3 min. The collection tube with the filtrate was discarded; the spin column was then placed into a new collection tube and centrifuged at full speed for another 1 min. the spin column was placed into a clean 1.5 ml centrifuge tube, 200 )l of buffer AE added. The columns were then incubated for 5 min at RT and then centrifuged at 6000 xg for 1 min.

The concentration of DNA was determined using spectrophotometric absorbance measured at 260 nm, with 1 A260 OD unit taken as the equivalent of 50 )g/ml RNA.

A260/ A280 ratios 1 1.8 indicated that samples were free of contaminating protein.

2.9.3 PCR of the FLT-3 Receptor For the PCR of the FLT-3 gene 100 ng of gDNA was used. The gDNA was mixed with PCR master mix (Table 2.14) and made up to 25 )l with RNase/DNase free water. GAPDH gene was used as a control the concentrations for this reaction are also given in Table 2.14.

Chapter Two: Materials and Methods 68 Table 2.14. PCR master mix FLT-3. Reagent Volume

10X Buffer 2.5 )l

10 mM MgCl2 (1.5 nM) 1.56 )l

Taq GOLD Polymerase 0.5 )l

dNTPs 4 )l

FLT-3 / GAPDH forward primer (10 )M) 1 / 1.25 )l

FLT-3 / GAPDH reverse primer (10 )M) 1 /1.25 )l

RNase/DNase free water to 25 )l

The PCR was performed under conditions outlined in Table 2.15 in a standard PCR machine from BioRad. The PCR products then mixed with 6X gel loading dye and electrophoresed on an agarose gel, as described in section 2.7.3.

Table 2.15. PCR conditions for FLT-3 and GAPDH. Cycle Temperature Time Repeat

1 95°C 5 min 1 times

94°C 45 sec

2 56°C 45 sec 35 times

72°C 1 min

3 72°C 7 min 1 times

2.9.4 Enzyme Digestion Part of the PCR reaction from above was mixed in thin walled PCR tubes, with 10X reaction buffer D and BSA and were mixed by pipetting, then the EcoRV enzyme was added (Table 2.16) and incubated at 37°C overnight. After the digestion 10 )l was mixed with 2X DNA loading buffer and run on an 1% agarose gel and the bands visualised on the VersaDoc, as described in section 2.7.3. The PCR for exon 14 (TKD) amplifies a 263-bp product, the wild-type product subsequently can be digested with the enzyme EcoRV producing two bands. The PCR for exon 20 (JM) amplifies a 324-bp sized product.

Chapter Two: Materials and Methods 69 Table 2.16. EcoRV digestion of FLT-3. Reagent Volume

PCR product 2 )l

EcoRV enzyme (1 U) 0.5 )l

10X reaction buffer D 2 )l

Acetylated BSA (10 )g/ml) 0.2 )l

H2O to 20 )l

2.10 Immunoprecipitation and Immunoblot Analysis

2.10.1 Reagents NuPAGE® 4-12% Bis-Tris gradient polyacrylamide gels, NuPAGE® 20X MES SDS running buffer, NuPAGE® transfer buffer, antioxidant, reducing agent, SeeBlue II protein markers were all from Invitrogen, as well as the gel running tanks and transfer apparatus; methanol was from Univar (Ajax, Seven Hills, Australia), Ponceau S sodium salt, protease inhibitor cocktail, Tris.HCl, ethylenediaminetetraacetic acid tetrasodium salt (EDTA), sodium chloride (NaCl), sodium fluoride (NaF), sodium orthovanadate

(Na(VO2)3), BSA fraction V were all from Sigma; MgCl2 and Trizma base (Tris) were from Astral Bioscientific (Gymea, Australia), Tween®-20; and SDS was from ICN Biomedicals (Aurora, USA); Nonidet P (NP)-40 from Fluka (Buchs, Switzerland); glycerol from Ajax Chemicals); from Dutch Jug (Melbourne, Australia) branded skim milk powder (SMP) was used for blocking; polyvinylidene difluoride (PVDF) membrane, Immobilon-P was supplied by Millipore (Bedford, USA), bicinchoninic acid (BCA) protein assay kit, SuperSignal® Westdura chemiluminescence substrate and Western Blot Stripping Buffer were obtained from (Rockford, USA); 3 mm Chromatography blotting paper from Whatman (Maidstone, UK); and protein A Sepharose beads from GE Healthcare .

SU11657, SU5416 and SU6668 were provided by Pfizer, other signalling inhibitors were commercially available; the KDR inhibitors (KDRi), U0126, PD98059 and the MEK inhibitor (MEKi) were purchased from Merck (Whitehouse Station, USA), LY294002 and Wortmannin were purchased from Sigma, FLT-3 blocking antibodies (EB10 and D43) and the IgG control were a gift from ImClone. Chapter Two: Materials and Methods 70 Primary antibodies were obtained from various vendors, the antibodies and their specifications are given in Table 2.17:

Table 2.17. Table of all immunoblotting antibodies and their conditions. Antibody Species Concentration Buffer Condition Company Phospho- Mouse 1 )g/ml 5% BSA O/N; RT Upstate Tyrosine FLT-3 Rabbit 1:500 1% SMP 2hrs; RT Santa Cruz

Phospho-STAT5 Rabbit 1:1000 5% BSA O/N; RT Cell Signaling

Phospho-AKT Rabbit 1:1000 5% BSA O/N; RT Cell Signaling

Phospho-ERK1/2 Rabbit 1:1000 5% BSA O/N; RT Cell Signaling

STAT5 Rabbit 1:1000 5% BSA O/N; RT Cell Signaling

AKT Rabbit 1:1000 5% BSA O/N; RT Cell Signaling

ERK1/2 Rabbit 1:1000 5% BSA O/N; RT Cell Signaling

EGR1 Rabbit 1:1000 1% SMP 2 hrs; RT Santa Cruz

Actin Rabbit 1:2000 1% SMP 2 hrs; RT Sigma

Anti-rabbit Goat 1:2000 As 1°Ab 2 hrs; RT Pierce

Anti-mouse Goat 1:2000 As 1°Ab 2 hrs; RT Pierce O/N - overnight; Ab - Antibody; Upstate - Millipore, Billerica, USA; Santa Cruz Biotechnology, Inc. Santa Cruz, USA; Cell Signalling, Danvers, USA; Pierce, Rockford, USA.

Blots were stripped with a commercial stripping buffer from Pierce. The signals were quantified by phosphoimaging using a VersaDoc 5000 Imaging System (BioRad) and data were analysed using Quantity One software also from BioRad.

2.10.2 Protein Isolation and Quantification ALL xenograft cells and cell lines were treated for 2 hrs with different inhibitors at standard culturing conditions. ALL xenograft cells after the initial 2 hr incubation were treated for an additional 15 min with FL (20 ng/ml). Total cellular protein preparations were obtained from both ALL xenograft cells and cultured cell lines. Cell suspensions were collected in 15 ml falcon tubes and centrifuged at 500 xg for 5 min at 4°C. They were subsequently washed with 15 ml of ice-cold PBS and re-centrifuged. PBS was aspirated and cell pellets were snap frozen in liquid nitrogen At the time of lysis, pellets

Chapter Two: Materials and Methods 71 were thawed on ice and lysed in lysis buffer (Table 2.18) supplemented with a protease inhibitor cocktail.

For every 10 million cells, 100 )l lysis buffer was added and incubated for 30 min on ice. The samples were votexed for 30 sec every 10 min during the incubation, following which the lysates were centrifuged (10000 xg; 10 min, 4°C). The supernatant was then transferred to a new microcentrifuge tube and stored at -80°C for further use. The protein concentration of the supernatant was determined using the BCA protein assay kit. A standard curve was established from dilutions of a BSA stock solution (1.5 mg/ml) provided with the kit, and the BSA amounts ranging from 0-1200 )g. Absorbances were determined with a microplate reader (GE Healthcare) from which the yield in )g for protein corresponded to a particular absorbance value. This reading was converted to a concentration value ()g/ml) taking into account the dilution factor of the protein sample.

Table 2.18. Cell lysis buffer for protein extraction. Reagent Concentration

Tris.HCl 200 mM

NaCl 150 mM

EDTA 10 mM

NaF 100 mM

Na(VO2)3) 1 mM

NP-40 1%

2.10.3 Immunoprecipitation of the FLT-3 Receptor Either 500 )g of ALL xenograft cells or 1 mg of the MV4;11 cell line protein lysate were immunoprecipitated with FLT-3 (1 )g) antibody overnight at 4°C, then captured using protein A Sepharose beads for 2 hrs at 4°C. The beads with the captured antibody and receptor were washed 3X in lysis buffer. After that the bead slurry was suspended with 2X gel loading buffer containing reducing agent, and heated to 70°C for 10 min. Samples were loaded onto precast 4-12% BisTris polyacrylamide gels, see section 2.10.4.

Chapter Two: Materials and Methods 72 2.10.4 Electrophoresis and Protein Transfer onto PVDF Prior to electrophoresis, total protein preparations (ranging from 20-100 )g) were mixed with of 4X sample buffer and NuPAGE® sample reducing agent, and reducing reagent and heated to 70°C for 10 min. Lysates or immunoprecipitates were loaded onto precast 4-12% BisTris polyacrylamide gels and separated in 1X NuPAGE® MES SDS running buffer. Gels were run at 160-200 V for approximately 1 hour. When the dye front had run of the gel, they were stopped and electrotransferred to a PVDF membrane.

The separated proteins were then transferred using the NuPAGE® transfer module containing 1X NuPAGE® transfer buffer. The gel was sandwiched between 2 blotting pads and 2 sheets of Whatman blotting paper which were pre-soaked in 1X transfer buffer. The gel was also positioned on the anode side of the methanol activated PVDF membrane. Proteins were transferred for 2 hrs at 30 V.

2.10.5 Western Immunoblotting Prior to probing, the PVDF membranes were blocked for 1 hr at RT in Tris Buffered Saline (TBS; 20mM Tris, pH 8; 150mM NaCl) with 0.05% Tween-20 (TBST), containing either 5% (w/v) SMP or BSA. Membranes were then washed 3X for 10 min in TBST prior to incubating in primary antibody.

Membranes were probed with antibodies for phospho-Tyrosine, phospho-STAT5, phospho-AKT or phospho-ERK1/2 antibodies (Table 2.17). Secondary antibodies used were horseradish peroxidase conjugates of either anti-mouse or anti-rabbit IgG (Pierce, Rockford, IL). The membranes were washed as described above then incubated with anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated IgG secondary antibody for 2 hrs at room temperature. The membrane was then washed again, followed by a 5 min incubation in SuperSignal®ULTRA Substrate Working Solution (1 part ULTRA Luminol/Enhancer solution, 1 part ULTRA Stable Peroxide solution). Light emission was captured on X-ray film and multiple exposure times were used to ensure the linear range of the film was not exceeded. Protein bands were then also quantified by densitometric analysis using Quantity One software version 4.4.1 (Bio Rad, Richmond, USA).

Chapter Two: Materials and Methods 73 To confirm equal loadings membranes were subsequently stripped with a commercial stripping buffer (Pierce) whilst rocking at 37oC for 30 minutes, after which they were blocked and re-probed with FLT-3, total-STAT5, total-AKT or total-ERK1/2 antibody (Table 2.17). They were developed as described above. Differences between samples or groups of samples in terms of their densitometric values were determined using Student’s t-tests (GraphPad Prism 4, La Jolla, USA).

2.11 siRNA Transfection into ALL Xenograft Cells and Leukaemia

Cell Lines

2.11.1 Reagents Amaxa transfection solutions were purchased from Amaxa Biosystems (Cologne, Germany). For the MV4;11 cells the Cell Line Nucleofector® Kit L was used and for the ALL xenograft cells the Human B Cell Nucleofector® Kit was used. Scrambled and FLT-3 siRNA were purchased from Dharmacon (Chicago, USA). Media and other tissue culture reagents were as stated in section 2.1.

2.11.2 Nucleofection Manufacturer’s instructions were followed. For ALL xenograft cells 2.5 million cells per cuvette were used. Xenograft cells were thawed on the day of nucleofection. For the MV4;11 leukaemia cell line 2 million cells per cuvette were used. The cells were passaged the day before nucleofection. Before the tranfection the Nucleofector Solutions were warmed to RT.

Cells were pelleted by centrifugation (90 xg for 10 min at RT) at the required cell number. The supernatant was completely discarded so that no residual medium covered the cell pellet. Cells were then resuspended in 100 )l RT Cell Line Nucleofector solution. The cell suspension was mixed with 1.5 )g appropriate siRNA. The whole sample was then transferred to an amaxa certified cuvette and the cap was closed. Each cuvette was placed into the cuvette holder of the Amaxa Nucleofector, one at a time, and the appropriate Nucleofector program selected (Q-001 for MV4;11 cells and U-15

Chapter Two: Materials and Methods 74 for ALL xenograft cells). After the program had finished (30 – 60 sec) cells were removed from the cuvettes immediately to avoid damage by adding 500 )l pre-warmed medium and transferred with a plastic pipette into culture vessels containing warm medium, and taken to a humidified incubator (37°C and 5% CO2). Cells were harvested 24, 48, and 72 hrs after nucleofection.

Whole cell lysates were analysed by western blot to assess the amount of FLT-3 knockdown. The cell lysis and western blot procedure was described in section 2.10.

2.12 Microarray

2.12.1 Reagents The reagents used in the microarray experiments were as follows: Gold Seal slides from Becton Dickinson, Poly-L-lysine solution (0.1% w/v), 1-methyl-2-pyrrolidinone, succinic anhydride, ammonium acetate (7.5 M), aa-dUTP (100 mM), EDTA and NaOH, Sodium acetate were all from Sigma-Aldrich; Trizol® was from Invitrogen as well as, Superscript choice system, Superscript II RT, E. coli DNA Polymerase I, E. coli DNA Ligase, E. coli RNase H, T4 DNA Polymerase, 5X second strand buffer, dNTP Set (10 mM) and random hexamers. Phase Lock Gel was purchased from Eppendorf (Westbury, USA) and Microcon® YM-30 was from Millipore (Bedford, USA). QIAGEN RNeasy® RNA isolation kit and the PCR purification kit were purchased from

QIAGEN. The T7-(dT)24 Primer (HPLC purified (DNA-5’-GGC-CAG-TGA-ATT- GTA-ATA-CGA-CTC-ACT-ATA-GGG-AGG-CGG-(dT)24-3’) was from GENSET Corp (San Diego, USA), all water used was Molecular Biology Grade and RNase/DNase free. The RNasin was from Promega. From Ambion (Austin, USA) were RnaseZap®, Phenol/Chloroform/isoamyl alcohol, linear acrylamide and the T7 Megascript Kit. The Cy dyes were purchased from Amersham Pharmacia (Piscataway, USA). Ethanol was 200 proof Ethyl Alcohol from USP, (Cockeysville, USA). It is important that this ethanol was used as 200 proof ethanol from other manufacturers may contain fluorescent contaminants. Borate buffer was made by adjusting the pH of boric acid (ACS grade) with sodium hydroxide (ACS grade), from Sigma. PBS was without magnesium or calcium (Table 2.19).

Chapter Two: Materials and Methods 75 Table 2.19. PBS without magnesium or calcium for use in microarray experiments. Reagent Amount

NaCl 8 g

KCl 0.2 g

Na2HPO4 (anhydrous) 1.44 g

KH2PO4 (anhydrous) 0.24 g

H2O Bring to 1 L

2.12.2 Slide Preparation

2.12.2.1 Slide Coating Gold Seal slides were inspected to make sure they were clean and free from scratches, dirt or any other imperfections. Then they were racked into metal racks. The racks were submerged into the cleaning solution (Table 2.20) in glass tanks and shaken for 2 hrs.

Table 2.20. Slide cleaning solution. NaOH was dissolved in water, then ethanol was added. The solution was stirred until clear. Reagent Volume

NaOH pellets 200 g

Water 800 ml

95% Ethanol 1200 ml

The slides were rinsed in distilled (DI) water for 2 – 5 min, 5 times. Slides were transferred to 25 slide racks. They were submerged into coating solution (Table 2.21) and shaken for 1 hr. they were rinsed in deionised water for 1 min, and centrifuged at 700 rpm in a swinging holder to remove the water.

Slides were transferred to a clean slide box and stored at RT in a dry place, and allowed to age for 2 weeks. Then they were coated for a second time as described above. Slides were then ready for printing. The cDNA was printed onto slides using a BioRobotics MicroGrid II spotter (Harvard Bioscience, Holliston, USA)

Chapter Two: Materials and Methods 76 Table 2.21. Slide coating solution. NaOH was dissolved in water, then the ethanol was added. The solution was stirred until clear. Reagent Volume

Poly-L-lysine solution 140 ml

Tissue Culture PBS (formula below) 140 ml

H2O 1120 ml

2.12.2.2 Slide Blocking After DMSO printing, microarray slides were UV cross-linked in Strategene Stratalinker (Agilent Technologies, Santa Clara, USA) with a dose of 450 milli Joules. Then the slides racked into metal racks and put into glass containers.

After the blocking solution (Table 2.22) was made it was immediately poured into the slide container, and shaken for 30 min. Slides were removed from the passivation reaction and immediately dunked into water. After 2 min of shaking in water, they were transferred into 95% ethanol. After 2 min shaking in ethanol, the racks with the slides were centrifuge at 700 rpm for 5 min. The slides were allowed to dry overnight in a dust-free cabinet, and then racked back into slides box. The slides were then ready for hybridisation.

Table 2.22. Blocking solution for 60 slides. Reagent Amount

Succinic Anhydride 8.6 g

1-methyl-2-pyrrolidinone 464 ml

1 M NaBorate (pH 8.0) 36 ml

Total volume 500 ml

2.12.3 Total RNA Extraction There were two aims for the microarray experiments; (1) to identify differentially expressed genes in ALL xenograft cells when they are cultured in the absence or presence of the stromal cell line MS5; and (2) to identify FLT-3 induced genes in ALL cells. Chapter Two: Materials and Methods 77 Therefore, for aim (1) the stromal cells were grown to confluence, the media was washed off with sterile PBS and ALL xenograft cells, suspended in QBSF-60 with FL were seeded on them. The MS5 cells which did not have ALL xenograft cells cultured on them had their media also changed to QBSF-60 + FL, so that the media was the same in all the flasks. Cells were cultured for 24 hrs after which they were harvested as described in section 2.5.2. As the RNA extracted from MS5 + ALL xenograft cells had both mouse and human RNA, the cells which have been grown separately were combined before the RNA extraction procedure so that there was the same ratio of human to mouse RNA in both samples, to compensate for the species contamination.

For aim (2), ALL xenografts cell were also seeded on a confluent layer of MS5 cells. Cells were cultured in QBSF-60 media, with and without FL (20 ng/ml). This time point was considered to be ‘time 0’. The cells were subsequently harvested at 1, 3, 6, 12 and 24 hrs. Again the cells were harvested in Trizol® reagent as described in section 2.5.2.

The RNA extraction procedure was carried out as in section 2.5.3.. After extraction and quantification of the RNA, all samples were assessed for the RNA integrity on the 2100 Bioanalyzer Lab-On-A-Chip system for RNA, from Agilent Technologies (Santa Clara, USA).

2.12.4 RNA Amplification

2.12.4.1 Primer Hybridisation and First Strand Synthesis RNA (5 )g) was concentrated to a final volume of 8 )l, with a Microcon-30. To the

RNA, 1 )l of the T7-(dT)24 primer (0.5 μg/μl) was added, the RNA and primer mix was incubated at 70°C, after 5 min, it was quickly spun down and put on ice. 11 )l of the reaction mix (Table 2.23) was added, mixed well and incubated for 1 hr at 42°C. After the incubation the reaction tube was centrifuged and put on ice.

Chapter Two: Materials and Methods 78 Table 2.23. RNA Amplification 1st strand synthesis enzyme reaction. Reagent Volume

5X First strand cDNA buffer 4 )l

0.1M DTT 2 )l

10mM dNTP mix 2 )l

RNasin 1 )l

Superscript II 2 )l

Total reaction volume 20 )l

2.12.4.2 Second Strand Synthesis The samples were ready for the second round synthesis. To the samples, 130 )l of second strand synthesis reaction (Table 2.24) was added; the tubes and solutions were mixed by gentle tapping and briefly centrifuged. The reaction was incubated for 2 hrs at 16°C.

Table 2.24. RNA Amplification 2nd strand synthesis reaction. Reagent Volume

DEPC treated H20 91 )l Second strand buffer 30 )l

10 mM dNTP mix 3 )l

DNA Polymerase I (10 U/)l) 4 )l

DNA Ligase (10 U/)l) 1 )l

RNase H (2 U/)l) 1 )l

Total reaction volume 150 )l

After the incubation 2 )l of T4 DNA Polymerase (10 U) was added and the reaction incubated for another 5 min at 16°C. The reaction was stopped with the addition of first 10 )l of 0.5 M EDTA, and then 10 )l of 1 M NaOH and incubated for 10 min at 65°C, then neutralised with 25 μl Tris-HCl (pH 7.5).

Chapter Two: Materials and Methods 79 2.12.4.3 Clean Up of Double Stranded cDNA Phase Lock Gel (PLG) was pelleted in a microcentrifuge at maximum speed for 30 sec. To the final DNA synthesis preparation (198 μl), equal volume (198 μl) of (25:24:1) Phenol:chloroform:isoamyl alcohol (saturated with 10 mM Tris-HCl (pH 8.0)/1 mM EDTA) was added (final volume 396 μl) and vortexed. The whole mixture was transferred to the PLG tube, and centrifuged at maximum speed for 2 min. The aqueous supernatant was transferred to a new 1.5 ml centrifuge tube and 1 )l linear acrylamide. Half a volume of 7.5 M Ammonium Acetate (100 μl) and 2.5 volumes (including the added Ammonium Acetate, 750 μl) of ice cold 95% ethanol was added to the samples and vortexed. Tubes were centrifuged at maximum speed in a microcentrifuge at 4°C for 20 min. Supernatants were removed, the pellets washed with 0.5 ml of cold 75% ethanol. The tubes were centrifuged again at maximum speed for 5 min at RT and the ethanol removed carefully. The wash was repeated with cold 75% ethanol wash once again. The pellets were air dried (~ 15 min), and pellets resuspend in 13 μl of Nuclease- free water. 8 μl of sample was taken for in vitro transcription, and the rest (5 μl) of sample stored at -80°C for future use.

2.12.4.4 In vitro transcription Ambion T7 Megascript kit was used and manufacturer’s instructions were followed for a total 20 μl reaction volume. The in vitro reaction was set up at RT so that the spermidine in the buffer did not precipitation. The reaction was set up as per Table 2.25, and incubated at 37°C for 15 hrs. After the 15 hr incubation 80 μl of Nuclease free water was added to bring the total volume up to 100 μl. RNA was purified using the QIAGEN RNeasy kit. The protocol for the RNA clean-up was followed.

Chapter Two: Materials and Methods 80 Table 2.25. Reaction mixture for in vitro transcription reaction. Reagent Volume

Double stranded DNA template 8 )l

10X Reaction Buffer 2 )l

ATP solution (75 mM T7) 2 )l

CTP solution (75 mM T7) 2 )l

GTP solution (75 mM T7) 2 )l

UTP solution (75 mM T7) 2 )l

Enzyme Mix 2 )l

Total 20 )l

Briefly, after the reaction was adjusted to 100 μl, 350 μl of RTL buffer was added and mixed well. Then 250 μl 100% ethanol was added and mixed by pipeting. The sample was transferred to the RNeasy Mini spin column placed in a 2 ml collection tube, then centrifuged for 15 sec at 8000 xg and the flowthrough discarded. 500 )l of RPE buffer was added to the spin column, again the tubes were centrifuged for 15 sec at 8000 xg and the flowthrough discarded. Another 500 )l of RPE buffer was added to the spin column, tubes were centrifuged for 15 sec at 8000 xg and the flowthrough discarded. The RNeasy Mini spin column was placed into a new 1.5 ml centrifuge tube and the RNA was eluted twice with 30 )l of Nuclease free water. The RNA was quantified using the NanoDrop.

2.12.5 Reverse Transcription Reaction of the RNA After RNeasy purification 2 )g RNA was used for labelling. Amplified RNA was mixed with random hexamers (3 )g/ml, 2 )l), as in Table 2.26 the reaction was incubated at 70°C for 5 min then cooled to 42°C.

Table 2.26. RNA and primer hybridisation for RT reaction. Reagent Volume

RNA 22.2 )l

Random Hexmer 3 )g/)l 2 )l

Total 24.2 )l

Chapter Two: Materials and Methods 81 Next, 15.8 )l of the RT reaction (Table 2.27) was added, and incubated at 42°C for 30 minutes. One μl of SSII RT enzyme was added and the reaction incubated at 42°C for an additional 30 min and cooled to RT.

Table 2.27. Reverse transcription reaction for cDNA probes. Reagent Volume

RNase Inhibitor RNasin 1 )l

5X First Strand Buffer 8 )l

0.1 M DTT 4 )l

50X aa-dNTP mix 0.8 )l

SSII Reverse Transcriptase 2 )l

Volume per reaction 15.8 )l

2.12.6 RNA Labelling To each labelling reaction, 5 )l of 0.5 M EDTA (pH 8.0) was added, mixed well and 10 )l of 1 M NaOH added. The reactions were incubated at 65°C for 20 min then cooled to RT. To neutralise the NaOH, 10 )l of 1 M HCl was added. Probes were then purified with the QIAGEN PCR spin column following manufacturer’s instructions. With the following exceptions; the provided wash and elution buffer were not used, they were freshly made to avoid Tris, as it interferes with the labelling reaction. The 1 M KPO4 in the phosphate wash buffer was made from two potassium phosphate solutions solution:

1 M KH2PO4 and 1 M K2HPO4 (Table 2.28).

Table 2.28. 1 M KPO4 buffer Reagent Volume

1M K2HPO4 9.5 ml

1 M KH2PO4 0.5 ml

pH should be 8.5 – 8.7

Briefly the purification procedure, 350 μl PB buffer was added to the RT reaction and transferred to a QIAquick column. Tubes were centrifuged at maximum speed in a table top microfuge for 1 min and flowthrough discarded. Then 750 )l phosphate wash buffer Chapter Two: Materials and Methods 82 was added to the spin column (Table 2.29). Tubes were centrifuged at maximum for 1 min and flowthrough discarded. The phosphate buffer wash was repeated. The collection tubes were emptied and the spin columns recentrifuged at maximum speed for 1 min. The spin columns were then transferred to a new 1.5 ml centrifuge tube and the cDNA eluted with 30 )l elution buffer (Table 2.30). Tubes were incubated for 1 min at RT before they were centrifuged at maximum speed for 1 min. This step was repeated. The eluted samples were dried by speedvac (~ 40 min).

Table 2.29. Wash buffer. The solutions were mixed in the order written in the table. This solution was slightly cloudy. Reagent Volume

H2O 15.25 ml

95% Ethanol 84.25 ml

1 M KPO4 pH 8.5 0.5 ml

Total 100 ml

Table 2.30. Elution buffer. Reagent Volume

1 M KPO4 pH 8.5 0.4 ml

H2O 99.6 ml

Total 100 ml

2.12.7 Cy Dye coupling

The cDNA was resuspended in 4.5 μl 0.1 M Carbonate buffer (Table 2.31). Then, 4.5 μl NHS-Cy dye was added (resuspended in 73 )l of DMSO) and the reaction was incubated in the dark for 1 hr at RT.

Table 2.31. Carbonate buffer. Reagent Amount

Na2CO3 0.27 g

H2O 20 ml

Adjust pH to 9.0 with 6 N HCl

Total with H2O 25 ml

Chapter Two: Materials and Methods 83 2.12.8 Probe Purification

After the coupling reaction 35 μl of 100 mM sodium acetate (pH 5.2), was added to the reaction. The probes were purified using the QIAquick PCR purification Kit. 250 )l PB buffer was added and the QIAquick PCR purification protocol followed as described in section 2.12.6. The provided wash buffer was used. The samples were eluted twice with

30 )l H2O. Two )l of the sample was taken for dye incorporation analysis. The Probes of Cy3 and Cy5 were mixed and dried with the speedvac (~40 minutes).

For the dye incorporation analysis the 2 )l of sample was diluted 1:12 in 50 )l of water and absorbance measured at 260 nm, and also at 550 nm for Cy3 and 650 nm for Cy5. The optimal amount for hybridisation is 150 pmole per slide.

The nucleotide to dye ratio should be <50, and was calculated the following way:

OD260 For Cy3 = 17.1 x OD550

OD260 For Cy5 = 28.5 x OD650

2.12.9 Prehybridisation Slides to be hybridised were put into a staining dish filled with prehybridisation buffer (Table 2.32), and incubated at 42°C for 45 min. Slides were then washed in a Wheaton slide rack with DIH2O at RT with agitation for 2 min. Slides were then dipped into RT isopropanol and dried by centrifugation at 700 rpm for 5 min. Slides were inspected to make sure there were no protein marks from the lysine coating visible and then the water and isopropanol washes were repeated. Slides were used within 1 hr following prehybridisation

Table 2.32. Prehybridisation solution. Reagent

5X SSC

0.1% SDS

1% BSA Chapter Two: Materials and Methods 84 2.12.10 Hybridisation The probes were redissolved in 27 )l hybridization solution (Table 2.33) and combined with COT1-DNA, Yeast tRNA and Poly(A)-DNA (Table 2.34) to block any nonspecific hybridization. This solution was incubated at 95°C for 2 min to denature the probes.

Table 2.33. 1X Hybridisation solution. Reagent Volume

H2O 240 )l 20X SSC 250 )l

SDS 10 )l

formamide 500 )l

Total 1 ml

Table 2.34. Probe solution. Reagent Volume

Cy5/Cy3 labelled probes 27 )l

Poly dA (8mg/ml) 1.3 )l

Yeast tRNA (4mg/ml) 1.3 )l

CoT-1 DNA (10mg/ml, concentrated) 1.3 )l

Total 30.9 )l

Next, 14 )l of 5X SSC was added into each modified hybridisation chamber to prevent drying. Probes were applied to the cover slips that were big enough for the printed area. Then the prehybridised microarray slide was put on top of the cover slip, invert, and hybridised in a humidified chamber at 42°C for 16-24 hours.

2.12.11 Slide Washing After hybridisation the slides were removed from the humidified chambers, put into metal racks and washed 4 times. The washing procedure is described in Table 2.35.

Table 2.35. Slide washing procedure Chapter Two: Materials and Methods 85 Wash Temperature Time Solution

1 RT until cover slip falls off 0.2% SDS + 1X SSC

2 42°C 4 min 0.2% SDS + 1X SSC

3 RT 4 min 0.2% SDS + 0.1X SSC

4 RT 4 min 0.06X SSC

Following the last wash, slides were centrifuged immediately (700 rpm for 5 min at RT). Slides were then ready to be read. Images were acquired by an Agilent DNA microarray scanner (Palo Alto, CA). They were analysed using the Microarray Suite program coded in IPLab (Scanalytics, Fairfax,VA).

2.12.12 Normalising and Filtering of the Slides The expression ratios between test sample RNA and reference RNA on each microarray were normalised using a pinbased normalization method modified from Chen et al. (Chen et al. 1997). The quality of the individual cDNA spots was calculated at the oncogenemics laboratory at the NIH, according to Chen (Chen et al. 2002).

The filtered genes were required to have a red intensity greater than 20 across all experiments. The relative red intensity for each gene was defined as the mean intensity of that spot divided by the mean intensity of filtered genes. The natural logarithm (ln) of relative red intensity was used as a measure of the expression levels.

2.13 Chromatin Immunoprecipitation (ChIP)

2.13.1 Reagents The protease inhibitors mix and glycine were purchased from Sigma. The 37% formaldehyde was from Ajax Finechem, the ChIP kit was from Upstate (Millipore, Befored, USA) the HIF1- antibody was purchased from Abcam (Cambridge, USA) and the PCR purification Kit was from QIAGEN. Primers used for the VEGF HRE were purchased from Invitrogen, they are shown in Table 2.36.

Chapter Two: Materials and Methods 86 Table 2.36. Primer sequences for the VEGF promoter. Primer Sequence

VEGF HRE Forward CAGGTCAGAAACCAGCCAG

VEGF HRE Reverse CGTGTGATTCAAACCTACC

2.13.2 HIF ChIP For the ChIP experiments 10 million xenograft cells per ChIP were used. Cells were cultured as described previously. After 24 hrs in culture protein and DNA was crosslinked by adding 37% formaldehyde directly to the culture medium at a final concentration of 1% and incubating for 10 min at 37°C (135 )l of 37% formaldehyde per 5 ml of media). After which 1 ml of 1.25 M glycine was added for another 5 min and incubated at RT. Cells were then harvested and washed twice with ice cold PBS (containing protease inhibitors). Cells were pelleted by centrifugation at 500 xg for 4 min at 4°C.

The Upstate ChIP kit was used for the immunoprecipitation and manufacturer’s procedures were followed. When SDS lysis buffer was warmed to RT to dissolve precipitated SDS a protease inhibitor cocktail was added. Cells were resuspended in 300 )l SDS lysis buffer and incubated on ice for 10 min. The lysate was sonicated to shear the DNA to lengths between 200 and 1000 bp. Samples were kept on ice at all times. Xenograft cells were sonicated 6X at 15 sec pulses at output 3 - 4, with 1 min between rounds to avoid sample overheating.

Sonicated samples were centrifuged for 10 min at 13000 rpm at 12°C (to avoid SDS precipitating) and 200 )l of the supernatant transferred to a new 15 ml tube and the pellet was discarded. The sonicated supernatant was diluted 10-fold in ChIP dilution buffer containing protease inhibitors, also 50 )l of the diluted sample was kept for evaluation as the input sequence.

For non-specific background to be reduced, the supernatant was precleared with 60 )l of Salmon Sperm DNA (SS DNA)/Protein A agarose 50% slurry for 30 min at 4°C on a rotating platform. The samples were centrifuged, to pellet the agarose protein A beads,

Chapter Two: Materials and Methods 87 at 2000 xg for 3 min and the supernatant fraction transferred into a new tube. The immunoprecipitating antibody (HIF-1) was then added to the supernatant and incubated overnight at 4°C with rotation.

The next day 60 )l of SS DNA/Protein A slurry was added and incubated for 1 hr at 4°C with rotation. The agarose was then pelleted by centrifugation at 2000 xg for 3 min 4°C. The supernatant was removed and the protein A agarose washed 5X for 5 min on a rotating shaker with 1 ml each of the following buffers in the order listed below in Table 2.37. The tubes were centrifuged at 2000 xg for 3 min and the supernatant discarded.

Table 2.37. ChIP washing procedure Wash Volume Time Solution

1 1 ml 5 min Low salt immune complex wash buffer

2 1 ml 5 min High salt immune complex wash buffer

3 1 ml 5 min LiCl immune complex wash buffer

4 1 ml 5 min 1X TE

5 1 ml 5 min 1X TE

The DNA was eluted from the agarose protein A beads with 250 )l of elution buffer

(1% SDS, 0.1 M NaHCO3). After the buffer was added the samples were briefly vortexed to mix and incubated at RT for 15 min with rotation. The tubes were then centrifuged for 3 min at 2000 xg at RT and the supernatant transferred to a new tube. The elution step was repeated and a total volume of eluate of 500 )l obtained. 20 )l of 5 M NaCl was added to the eluates and incubated at 65°C for 4 hrs to reverse the histone-DNA crosslinks. The input material was included in this step. The DNA was then purified with the QIAGEN QIAquick PCR purification kit and manufacturer’s instructions were followed, as described in section 2.12.6. All the provided wash buffers were used. The DNA was eluted in 28 )l of H2O.

Chapter Two: Materials and Methods 88 2.13.3 PCR amplification of the VEGF promoter The PCR procedure was performed as described previously. For the PCR amplification of the VEGF promoter 5 )l of the eluate were used in the PCR. The rest of the reagents for PCR and concentrations used are in Table 2.38 and the conditions in Table 2.39. The PCR products were combined with 10X gel loading buffer and loaded on to a 1% agarose as described in section 2.7.3.

Table 2.38. PCR master mix for the VEGF HRE. Reagent Volume

ChIP 5 )l

10X Buffer 2.5 )l

10 mM MgCl2 1 )l

Taq GOLD Polymerase 0.5 )l

dNTPs 4 )l

VEGF forward primer (10 )M) 1 )l

VEGF reverse primer (10 )M) 1 )l

RNase/DNase free water to 25 )l

Table 2.39. PCR running conditions for VEGF HRE. Cycle Temperature Time Repeat

1 95°C 4 min 1 times

95°C 45 sec

2 56°C 45 sec 25 times

72°C 1 min

3 72°C 7 min 1 times

2.14 Statistical Analysis

To assess the statistical significance of the differences observed between the levels of gene and protein expression in various groups of treatments, the Mann-Whitney-U test was used. The statistical software GraphPad Prism (GraphPad Software, version 4, CA, USA) was used for these calculations. All experiments were performed at least three times.

Chapter Two: Materials and Methods 89

Chapter Two: Materials and Methods 90  

CHAPTER THREE: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation

3.1 Introduction

As identified in Chapter 1, VEGF is an integral component of both neovascularisation and normal haematopoiesis. VEGF has been shown to induce blood vessel formation in solid tumours (lung, breast, prostate, etc.) (Dvorak et al. 1991; Relf et al. 1997; Bachelder et al. 2002), and may contribute to metastasis by enhancing the migration, permeability, and mitogenic activity of endothelial cells. However, its pathological role in a range of haematological malignancies (Santos et al. 2004; Farahani et al. 2005), is a more recent observation. Avramis and colleagues (2006) demonstrated this in a recent study, which reported that high VEGF serum levels in ALL patients correlated with a worse prognosis. Therefore, understanding the mechanism of VEGF secretion is an important initial step in deciphering the underlying biology of the disease, which will allow for progression towards novel and improved treatments.

FLT-3 and its ligand FL play an essential role in regulating normal haematopoiesis (Mackarehtschian et al. 1995). FLT-3 is frequently expressed and often mutated in AML (Gilliland et al. 2002), which is associated with a poor prognosis (Rombouts et al. 2000). It is also expressed in a large proportion of ALL patients; in B-ALL, up to 100% and in T-ALL, between 27 - 87% of cases (Birg et al. 1992; Carow et al. 1996; Drexler 1996). Mutations in ALL however, are not very common; occurring in less than 5% of cases (Yamamoto et al. 2001).

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 91 The aim of this study was to investigate the relationship between FLT-3 and VEGF in ALL cells. In experiments conducted in our laboratory, FL is routinely added to the in vitro culture conditions of our ALL xenograft cells. Interestingly, this addition of FL increased the secretion of VEGF by the cells (as detailed further in the following results section). Therefore, it is postulated that FLT-3 signalling can induce the secretion of VEGF by leukaemia cells. In this study I investigated the mechanism by which FL induced VEGF secretion in ALL xenografts, and further characterised the FLT-3 signalling pathway in ALL xenograft cells. Examination into the exact mechanism of FLT-3 induction of VEGF secretion is necessary for a better understanding of the importance of FLT-3 expression on leukaemia cells. This could potentially lead to the development of novel FLT-3/VEGF targets, through the use of small molecule inhibitors (Sohal et al. 2003) and/or blocking antibodies, to combat the disease.

3.1.1 ALL Xenograft Mouse Model Our laboratory has previously established mouse models of childhood ALL from patient biopsies (Lock et al. 2002), which accurately reflect the clinical disease of the patients from whom the xenografts were derived. The patient characteristics are listed in Table 3.1. The xenograft panel assessed in this chapter also includes an infant leukaemia sample with an MLL translocation, designated P-14 (Henderson et al. 2008). The P-14 sample was provided by the Telethon Institute (Perth, Australia) and while not a part of the standard ALL xenograft panel from our laboratory, was nonetheless engrafted into NOD/SCID mice. The P-14 xenograft cells were obtained from a MLL patient with a non-classical MLL gene rearrangement. This continuous xenograft model is summarised in Figure 3.1. A brief description of the overall procedure is provided in the Figure 3.1 caption. Lock et al. (2002) should be consulted for further details.

Importantly, previous research has also shown that these xenograft cells retain the cytological and immunophenotypic characteristics of the original patient samples, as well as the blast morphology and genotype profile through serial passages in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice (Borgmann et al. 2000; Lock et al. 2002). The response to chemotherapeutic drugs of these xenografts, correlates with the patient clinical outcome from which the xenografts were derived (Liem et al. 2004). It has also been identified that the ALL cells infiltrate the same

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 92 organs in NOD/SCID mice as they do in children. Therefore, the conditions by which the xenografts are established in the mice, i.e. the organ infiltration profile, reflect the clinical condition of the patient (Lock et al. 2002).

In addition, the in vitro sensitivity of the ALL xenograft cells to dexamethasone can be correlated with both the in vivo responses and patient clinical outcomes (Liem et al. 2004). Whiteford and colleagues (2007) demonstrated through gene expression analyses, that tumour xenografts were more closely related (i.e. in their mRNA profile) to primary tumour cells than cell lines, further emphasising their biological relevance in the study of childhood ALL.

Apart from this system being a model of ALL that is useful for the testing of new agents, it is also the only method by which these cells can be propagated for in vitro experiments. For example, if 5 million cells are injected into the mice, an average of 700 million can be isolated from the spleen after engraftment, providing an abundant and renewable source of cells for biological experiments.

7-week old 250 cGy NOD/SCID

2.5 -10 x 106 mononuclear cells

Purified mononuclear cells

Cryostorage Used for additional in vivo and in vitro experiments

Figure 3.1. Diagrammatic representation of the ALL continuous xenograft model. Leukaemia cells isolated from patient biopsies are injected into irradiated NOD/SCID mice. Human cell engraftment is monitored by tail vein bleed. The blood is stained for human and mouse CD45+. When the human CD45+ cells reach >75% in the peripheral blood, the mice are sacrificed, with and BMs being collected, and xenograft cells isolated by ficoll gradient centrifugation. Purified cells are stored in liquid nitrogen for future in vitro and in vivo use.

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 93 Unlike ALL cell lines, ALL xenograft cells proliferate only to a minor extent in culture (Liem et al. 2004). Whilst the same is true of our xenografts, experiments previously conducted in our lab (unpublished data) have shown that when cultured in vitro the ALL xenograft cells did not require serum in the media. Indeed, the opposite was true, with the xenograft cells surviving better without the presence of FCS. Interestingly, it was shown that the addition of the haematologic growth factor FL, to the QBSF-60 media, increased the secretion of the growth factor VEGF by these cells.

Table 3.1. Characteristics of the ALL xenograft panel, including patient clinical data. Survival Disease Age at Length of Current After Xenograft Subtype status at Sex Diagnosis CR1 Clinical Relapse biopsy (months) (months) Status (months) ALL-2 c-ALL Relapse F 65 30 46 D.O.D.

ALL-3 Pre-B Diagnosis F 154 38 No relapse Alive

ALL-4 Ph+ c-ALL Diagnosis M 105 10 1 D.O.D.

ALL-7 Biphen. Diagnosis M 88 7 6 D.O.D.

ALL-8 T-ALL Relapse M 152 17 1 D.O.D.

ALL-10 c-ALL Diagnosis M 48 No relapse Alive

ALL-11 c-ALL Diagnosis F 37 No relapse Alive

ALL-16 T-ALL Diagnosis F 122 No relapse Alive

ALL-17 c-ALL Diagnosis F 107 25 No relapse Alive

ALL-18 c-ALL Relapse F 30 43 No relapse Alive

ALL-19 c-ALL Relapse M 194 4 11 D.O.D.

P-14 MLL Diagnosis F 2 14 126 Alive D.O.D. = Dead of Disease; c-ALL = common (CD10+) ALL; Ph+ = Philadelphia chromosome positive; Biphen = Biphenotypic ALL

The cell line MV4;11, a myelomonocytic leukaemia, was also utilised as a model system as part of these experiments. It was chosen because it secretes high levels of VEGF, as well as having a mutated and constitutively active FLT-3 receptor (Quentmeier et al. 2003). This makes it of specific interest for this study, to examine the FLT-3/VEGF relationship. Several other leukaemia cell lines were investigated to ascertain whether they also expressed FLT-3 and secreted VEGF. The cell lines and their characteristics are summarised in Table 3.2.

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 94

Table 3.2. Leukaemia cell lines and their characteristics. Cell line Type of Leukaemia Patient Age (months) MV4;11 biphenotypic B myelomonocytic 10

REH B cell precursor 15

NB4 acute promyelocytic 23

RS4;11 B cell precursor 32

NALM6 B cell precursor 19

HL60 acute myeloid 35

3.2 Results

3.2.1 Expression of FLT-3 on Leukaemia Cells FLT-3 expression levels, represented as the relative fluorescence intensity (RFI) (which is the ratio of the fluorescence geometric mean of FLT-3 antibody to the matched IgG control), on ALL xenograft cells were measured by flow cytometry. The FLT-3 expression histograms for each of the xenografts are shown in Figure 3.2 and Table 3.3. ALL xenograft cells showed varying levels of FLT-3 cell surface expression. Xenografts ALL-2, -3 and -17 and P-14 showed relatively high cell surface expression of the FLT-3 receptor (RFI values: 5.2, 6.8, 4.7 and 7.9, respectively). ALL-3 xenograft cells also had the highest levels of FLT-3 expression (RFI = 6.8) from our ALL xenograft panel. Conversely, FLT-3 expression in ALL-4 and -7 cells was relatively low (RFI 2.8 and 2.6, respectively). Expression levels of FLT-3 on the MV4;11 cell line was also investigated. The levels of FLT-3 are relatively low compared to some of the xenograft cells (e.g. ALL-2 and ALL-3). However, it has a mutated receptor with an ITD, resulting in the constitutive activation of the receptor.

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 95

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 96

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 97

Figure 3.2. Flow cytometry histograms of FLT 3 expression levels. Expression levels of FLT 3 on the surface of ALL xenograft cells and the MV4;11 cell line was examined by flowcytometry. ALL xenograft cells have variable levels of FLT 3 expression. MV4;11 cells have a relatively low expression of FLT 3 on their surface. The pink line depicts FLT 3 and the blue line depicts the IgG control.

3.2.2 Secretion of VEGF by Leukaemia Cells As identified in Chapter 1, many studies have shown that leukaemia cells produce and secrete VEGF e.g. De Bont et al. (2002). Leukaemia cell lines however, can become genetically unstable over time. One example of this has been shown in the expression of the tumour suppressor gene , where mutations were detected in approximately 60% of T-ALL cell lines, whilst gene mutations in patients are relatively uncommon (Jonveaux et al. 1991). Indeed, our ALL xenograft cells have been shown to be genetically closer to the primary disease from which they are derived, compared to leukaemia cell lines (Whiteford et al. 2007). Therefore, we analysed VEGF secretion into the conditioned media (CM) by ALL xenograft cells. The secretion of VEGF by ALL xenograft cells was measured by ELISA as described in Section 2.3. The expression of bFGF, another potent angiogenic growth factor, was also examined. As bFGF has been shown to be expressed by leukaemia cells (Aguayo et al. 2000), and in some leukaemias (e.g CML), be associated with clinical stage (Gabrilove 2001). However no expression was detected using an ELISA, the focus was remained solely on the expression of VEGF, owing to its large expression by some ALL xenograft cells.

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 98 BM stromal cells have been shown to enhance the in vitro survival of normal and malignant haematopoietic cells (Wang et al. 2004). ALL xenograft cells are routinely cultured on a layer of the murine stromal cell line MS5, as they have been shown to survive for longer periods (Manabe et al. 1994). MS5 cells were seeded in U-bottomed 96-well plates and cultured to confluence before ALL xenograft cells were added. Cells were cultured with and without the addition of the cytokine FL (20 ng/ml). After 72 hrs of culturing ALL-3 xenograft cells with FL there was a clear and significant increase in VEGF in the CM, compared to VEGF secreted by the cells cultured without FL, (from 1220 + 206 pg/ml to 4458 + 680 pg/ml, P = 0.0002) (Figure 3.3). No consistent or significant differences in cell viability were detected with the addition of FL (data not shown). Even when cultured on an MS5 stromal support, the ALL xenograft cells progressively died over time. As such, in all subsequent experiments, cells were harvested and CM collected after 72 hrs of culturing as this time point provided the optimal balance between having readily detectable levels of VEGF in the CM by ELISA and the viability of the xenografts.

ALL-3 + MS5 120000 FL- 95000 FL+ 70000 cells) 6 45000 20000 15000

10000 * 5000 VEGF per(pg/ml 10 VEGF 0 Day 1 Day 2 Day 3 Day 4 Day 7

Figure 3.3. VEGF secretion by ALL-3 cultured on MS5 cells. VEGF secretion was measured over time, ALL-3 xenografts were cultured on a layer of MS5 stromal cells, with and without FL (20 ng/ml). VEGF levels in the CM were assessed by ELISA. VEGF secretion was normalised to the number of live cells at the time of harvest, as measured by flow cytometry, described in Section 2.3.2. Therefore, the amount of VEGF given is given as pg/106 live cells. Results are the mean ± SE of ten separate experiments.

VEGF in the CM was detectable in 4 other ALL xenograft cells (ALL-2, ALL-4, ALL-7 and ALL-17), as well as the MLL xenograft P-14 (Figure 3.4A). Interestingly, the

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 99 addition of FL to P-14 xenograft cells significantly increased VEGF secretion (944.9 + 187.0 pg/ml to 2164.1 + 473.0 pg/ml, P = 0.008), in a similar manner to that observed with the ALL-3 xenograft cells. While the addition of FL also produced an increase in VEGF secretion in some of the other xenograft cells, it proved not to be significant at P = 0.05 (Figure 3.4A). In ALL-2 cells, VEGF increased from 1060 + 311 pg/ml to 1456 + 556 pg/ml (P = 0.6), in ALL-4 from 213 + 78 pg/ml to 242 + 41 pg/ml (P = 1), ALL-7 from 3776 + 713 pg/ml to 4584 + 797 pg/ml (P = 0.4) and in ALL-17 from 1052 + 315 pg/ml to 1334 + 273 pg/ml (P = 0.49), (Table 3.3). The linear regression between FLT-3 receptor expression and VEGF secretion induced by FL has a correlation coefficient of R2=0.35, which significantly deviated from zero. The linear regression between FLT-3 receptor expression and VEGF secretion without the addition of FL has a correlation coefficient of R2=0.2, which did not show significant deviation from zero (Figure 3.4B).

A 5000 * FL- FL+ 4000 cells) 6 3000 *

2000

1000 VEGF (pg/mlVEGF per 10 0 ALL-2 ALL-3 ALL-4 ALL-7 ALL-17 P-14 Xenograft Cells

B 5000 FL+ FL- 4000 R2 = 0.35 3000 P = 0.04

2000 R2 = 0.2 VEGF Secretion 1000 P = 0.16 0 1 2 3 4 5 6 7 8 FLT-3

Figure 3.4. VEGF secretion by ALL xenograft cells. Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 100 (A) FL increased VEGF secretion ( ) compared with no FL addition ( ) in ALL-3 and P-14 xenograft cells, *P = 0.0002 and 0.04, respectively. *P < 0.05 versus control. Results are the mean ± SE of at least three separate experiments. (B) Linear regression analysis between VEGF secretion by all ALL xenografts ± FL and their FLT-3 expression.

It is interesting to note that by culturing ALL-3 xenograft cells without a MS5 stromal layer, the cells secreted detectable levels of VEGF into the conditioned media. Observation after 72 hrs of culturing, in the presence of the cytokine FL (20 ng/ml), showed levels of VEGF secreted by ALL-3 to have increased approximately 4.5-fold, from 337 + 221 pg/ml to 1036 + 416 pg/ml (P = 0.0086), in a manner similar to that observed when these cells where cultured on MS5 cells. The secretion of VEGF by ALL-3 xenograft cells in the presence of both MS5 cells and FL was more than additive, compared to both of these conditions alone (Figure 3.5). There were no detectable levels of VEGF in the other xenografts tested when cultured without MS5 stromal cells (ALL-2, -4, -7, -8, -10, -11, -16, -17, -18 and -19, as well as P-14) (Table 3.3).

* 5000 FL- FL+ 4000 cells) 6

3000

2000 *

1000 VEGF (pg/ml per 10 (pg/ml VEGF 0 No MS5 MS5 ALL-3

Figure 3.5. Secretion of VEGF by ALL-3 xenograft cells after 72 hrs of culture. ALL-3 cells were cultured without or with an MS5 stromal support layer, the CM was collected and VEGF was measured by ELISA. The amount of VEGF secretion was normalised to the amount of live cells at the time of harvest, as measured by flow cytometry, described in Section 2.3.2. FL increased the VEGF secretion ( ) compared without FL addition ( ). This secretion increased 10-fold with culturing on a MS5 stromal support layer. FL increases this further, by 2.5-fold without MS5s and 4.5-fold in cells grown with MS5s (P < 0.01 for both). Results are the mean ± SE of at 17 separate experiments.

A summary of the data generated from the ELISA showing the secretion of VEGF by ALL xenograft cells is shown in Table 3.3. It should be noted, that although levels of VEGF below 10 pg/ml were delineated, the detection limit of the ELISA was 10 pg/ml

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 101 VEGF, and thus any secretion below this level was considered as zero. It should be noted that the heterogeneity of the xenograft panel is reflected in the varying levels of both FLT-3 expression profile as well as the levels of VEGF secreted by the cells.

Table 3.3. Expression of FLT-3 and VEGF secretion on Day 3 by ALL xenograft cells. RFI VEGF Secretion Xenograft of Without MS5s With MS5s FLT-3 FL- FL+ FL- FL+ ALL-2 5.2 <10 <10 1060 + 311 1456 + 556

ALL-3 6.8 227 + 182 1036 + 348 1220 + 206 4458 + 680 *

ALL-4 2.8 <10 <10 213 + 78 242 + 141

ALL-7 2.6 <10 <10 3776 + 713 4584 + 797

ALL-8 1.7 <10 <10 <10 <10

ALL-10 1.9 <10 <10 <10 <10

ALL-11 1.7 <10 <10 <10 <10

ALL-16 1.2 <10 <10 <10 <10

ALL-17 4.7 <10 <10 1052 + 315 1334 + 273

ALL-18 1.9 <10 <10 <10 <10

ALL-19 2.5 <10 <10 <10 <10

P-14 7.9 <10 <10 878.1 + 187.3 1979.4 + 479.4 * *Significant increase with the addition of FL; VEGF is expressed as pg/106 viable cells; RFI = relative fluorescent intensity

Several leukaemia cell lines were tested for VEGF secretion and FLT-3 expression, to be used as a cell model to further characterise the relationship between FLT-3 and VEGF in leukaemia cells. Details of the cell lines used, including their FLT-3 expression patterns, are displayed in Figure 3.6.

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 102

20000 RFI=2.24 RFI=2.24

cells) 15000 6

10000 .77 RFI=7.12 0

9

5000 RFI=1 RFI=7.4 VEGF per(pg/ml 10 VEGF 0

1 4 1 6 0 ;1 ;1 NB REH S4 ALM HL6 MV4 R N Leukaemia Cell Lines

Figure 3.6. VEGF secretion by leukaemia cell lines after 3 days of culture. The levels of FLT-3 expression are also shown on the graph. The NALM6 and HL60 cell line do not express FLT-3. RFI - relative fluorescent intensity. VEGF levels were assessed by ELISA, and normalised to the amount of live cells. The VEGF secretion results are expressed as the mean ± SE of at least three separate experiments.

3.2.3 Inhibition of VEGF Secretion with a FLT-3 Small Molecule Inhibitor The relationship between VEGF and FLT-3 was explored further by using the small molecule RTK inhibitor, SU11657, to assess whether VEGF secretion is triggered by the activation of FLT-3 signalling. SU11657 acts by attaching to the ATP binding site of FLT-3, effectively inhibiting phosphorylation of the receptor. In the first instance, the sensitivity of both ALL xenograft cells and leukaemia cell lines to SU11657 was measured by MTT (Figure 3.7A & B). Sub-lethal concentrations (100 nM and 1 )M) of the inhibitor were chosen based on the toxicity on the ALL xenograft cells. Such concentrations were used as the experimental intent was to inhibit FLT-3 phosphorylation and examine the down stream effect, rather than using cytotoxic SU11657 concentrations and causing cell death. As shown in Figure 3.7A, ALL-3 is the most sensitive of the xenograft cells tested, with an IC50 of 1.38 )M. Examination of the cell lines showed that MV4;11 was the most sensitive, with an IC50 of 25.4 nM (Figure 3.7B). The inhibitor concentrations of 100 nM and 1 )M, were kept constant for all experiments in order to allow comparative effects to be assessed. It is for this reason

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 103 that future references to VEGF expression are as ‘amount per live cells’, due to the differing sensitivities between the ALL xenograft cells and the cell lines.

A Xenografts

125 ALL-3 ALL-2 100 ALL-4 ALL-7 75 ALL-17 ALL-19 50 % of Control of %

25

0 -10 -9 -8 -7 -6 -5 -4 -3 Concentration of SU11657 (M)

B Cell Lines

150 NALM-6 REH 125 NB4 MV4;11 100 HL60

75

% of of Control % 50

25

0 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 Concentration of SU11657 (M)

Figure 3.7 A & B. Cell survival in increasing concentrations of SU11657 measured by MTT assay. ALL Xenograft cells (A) and Leukaemia cell lines (B) were analysed for their sensitivity to SU11657. The results are expressed as the mean ± SE of at three separate experiments.

ALL-3 xenograft cells were treated with 100 nM of the RTK inhibitor SU11657 and the effects on VEGF secretion were examined (Figure 3.8). When this experiment was conducted without a MS5 stromal layer support, SU11657 had no effect on basal VEGF

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 104 secretion; a decrease in VEGF was observed in the cells cultured with FL and SU11657, but it proved not to be significant (P = 0.5, by the Mann-Whitney unpaired t-test).

ALL-3 2000 FL- FL+

cells) 1500 6

1000

500 VEGF (pg/ml per 10 per (pg/ml VEGF 0 0 nM 100 nM SU11657

Figure 3.8. Effects of SU11657 on VEGF secretion by ALL-3 xenograft cells. Secretion of VEGF was measured in leukaemia xenografts after treatment with SU11657. ALL-3 showed a reduction in VEGF secretion after treatment when cultured without MS5 stromal cells. VEGF was assessed by ELISA, and normalised to the amount of live cells at the time of harvest. Results are the mean ± SE of 16 separate experiments.

The same experiment was then conducted with a MS5 stromal cell support. Exposure of ALL-3 cells to 100 nM or 1 )M of SU11657 significantly reduced FL-induced VEGF secretion by 2- and 4-fold respectively (Figure 3.9A). In ALL-2, SU11657 significantly reduced VEGF secretion in cells cultured both with and without FL (Figure 3.9B). P-14 xenograft cells showed a similar SU11657 response compared to ALL-3, significantly reducing its FL-induced VEGF secretion at 1 )M (Figure 3.9C). No statistically significant decreases in VEGF secretion by ALL-4, -7 and -17 were detected after exposure to SU11657, both with and without FL (Figures 3.9D, E & F).

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 105 A ALL-3 6000 FL- 5000 FL+ cells) 6 4000

3000 * P = 0.037

2000 * P = 0.012 1000 VEGF (pg/ml per 10 VEGF 0 0 nM 100 nM 1 μM n = 18 SU11657

ALL-2 B 2000 FL- FL+

cells) 1500 6 * P = 0.0062

1000 P = 0.003

* P = 0.045 500 VEGF (pg/ml per 10 per (pg/ml VEGF 0 0 nM 100 nM 1 μM n = 8 SU11657

C P-14

2500 FL- FL+ 2000 cells) 6

1500

1000 * P = 0.027

500 VEGF (pg/ml per 10 per (pg/ml VEGF 0 0 nM 100 nM 1 μM n = 9 SU11657

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 106 D ALL-4 400 FL- FL+

cells) 300 6

200

100 VEGF (pg/ml per (pg/ml 10 VEGF 0 0 nM 100 nM 1 μM n = 7 SU11657

E ALL-7 6000 FL- FL+ 5000 cells) 6 4000

3000

2000

1000 VEGF (pg/ml per 10 per (pg/ml VEGF 0 0 nM 100 nM 1 μM n = 3 SU11657

ALL-17 F 2000 FL- FL+

cells) 1500 6

1000

500 VEGF (pg/ml perVEGF 10 0 0 nM 100 nM 1 μM n = 4 SU11657

Figure 3.9. Effects of SU11657 on VEGF secretion by different xenograft cells. Cells were treated with the RTK inhibitor SU11657; concentrations are shown on the graph. VEGF in the CM was measured by ELISA after 72 hrs, as previously described. ALL-3 (A) showed a significant reduction in VEGF secretion after treatment, as did ALL-2 (B) and P-14 (C) xenograft cells. The effects of SU11657 on ALL-4 (D), -7 (E) and -17 (F) - the other xenografts which secreted detectable VEGF, did not have significant reduction, but there was an observable trend in VEGF reduction in ALL-17. Results are the mean ± SE of at least three separate experiments.

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 107 Several cell lines (MV4;11, NALM6, NB4, HL60 and RS4;11) were used to further characterise the possible importance of FLT-3 signalling and its induction of VEGF secretion. The addition of 100 nM and 1 )M SU11657, to the MV4;11 cells, significantly reduced VEGF secretion from 15007 + 4285 pg/ml to 730 + 351 pg/ml (P < 0.001) and 597 + 299 pg/ml (P < 0.001) respectively (Figure 3.10). Cell death occurred in a percentage of the samples analysed as a result of the addition of SU11657, despite being used at overall sub-lethal concentrations. This would obviously lead to a reduction in the levels of VEGF secretion. However, when expressed as a percentage of live cells, Figure 3.10 shows a decrease in VEGF secretion in excess of that which could be explained by cell death. The cell line RS4;11 is known to have wild-type FLT-3, and secreted detectable amounts of VEGF. However, the presence of FL had no observable effect on VEGF secretion, nor did it decrease with the addition of SU11657 (Figure 3.10). Whilst the NALM6 cell line does not express FLT-3, VEGF protein was measured in the conditioned media. This secretion however, was not abrogated by the addition of SU11657. Similarly, SU11657 was not able to reduce VEGF secretion in NB4 or HL60 cell lines (Figure 3.10).

20000 No SU11657 18000 100 nM SU11657 1 μM SU11657 16000 14000 cells)

6 12000 10000 8000 6000 VEGF(pg/10 4000 * 2000 * 0

1 ;1 B4 4;11 LM6 N V S4 A HL60 M R N Cell lines

Figure 3.10. Effects of SU11657 on VEGF secretion by different leukaemia cell lines. MV4;11 cells have a constitutively active receptor and we have shown that they secrete VEGF. VEGF secretion was reduced with SU11657 (100 nM & 1 μM, *p < 0.01). SU11657 had no effect on VEGF secretion in RS4;11, NB4, HL60 or NALM6 cells. Results are the mean ± SE of at least three separate experiments.

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 108 3.2.4 VEGF mRNA The ELISA used to detect VEGF secretion in the media identified two different VEGF splice variants of VEGF; VEGF121 and VEGF165. While the probe and primers used to analyse VEGF mRNA span the boundary of exon 1 and 2, the first 4 exons are present in all 7 splice variants of the mRNA (refer to Figure 1.5), and therefore cannot be distinguished from one another. Primers were designed to identify the specific variants that were expressed in our ALL xenografts and the MV4;11 cell line. As can be seen in

Figure 3.11 only 2 bands were observed, the sizes of which correspond to the VEGF121 and VEGF165 isotypes; which match the splice variants that the ELISA is able to detect.

ALL-3 ALL-3 MV4;11

165 VEGF 121

 EF1

Figure 3.11. Isotypes of VEGF mRNA expressed in ALL-3 xenograft and MV4;11 cells. A standard, non-quantitative PCR was performed to asses the isotypes of VEGF expressed in the two cell types. EF1 was used as a control for the PCR.

VEGF mRNA levels in ALL-3 xenograft cells were measured by real time RT-PCR, and shown over time in Figure 3.12. In ALL-3 xenograft cells, FL increased VEGF protein secretion, as demonstrated in Section 3.2.2, but VEGF mRNA also increased after 24 hrs in culture with FL. The culturing of ALL-3 on MS5 stromal cells without FL also increased VEGF mRNA, but to a lesser extent (2-fold). The effects of both MS5 and FL culturing appeared to be additive (Figure 3.12).

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 109 ALL-3+FL 16 ALL-3+MS5 ALL-3+MS5+FL 14 12 10 8 6 4

fold change to untreated to change fold 2 0 1 hr 2 hr 6 hr 24 hr 48 hr 72 hr Hours in culture

Figure 3.12. VEGF mRNA in ALL-3 measured over time. As observed at the protein level, FL also induced VEGF mRNA production. There was a steady increase in VEGF mRNA with FL over time. MS5s also increase VEGF mRNA compared to untreated cells, however, the combination of FL and MS5s has the biggest effect on mRNA production, being slightly more than additive. Results are the mean ± SE of at three separate experiments.

The effects of both FL and SU11657 (100 nM) on VEGF mRNA, at the 24 hr time point, were also analysed. In a similar manner to that observed with the secreted protein, FL significantly increased VEGF mRNA expression (P = 0.0038) after 24 hrs in ALL-3 xenograft cells (Figure 3.13A). SU11657 by itself had no effect on VEGF mRNA expression. It did however, attenuate the effect of FL. Treating ALL-3 xenograft cells with both FL (20 ng/ml) and SU11657 (100 nM), resulted in no change from basal VEGF mRNA expression. mRNA expression with FL was significantly higher than the expression in cells treated with both FL and SU11657 (P = 0.017) (Figure 3.13A).

MV4;11 cells were treated with varying concentrations of SU11657 over a 72 hr period. With the addition of SU11657 there was a decrease of VEGF mRNA (Figure 3.13B). After 24 hrs of SU11657 treatment there was a 28, 31 and 48% decrease with 10, 100 nM and 1 )M, respectively. The reduction in VEGF mRNA was enhanced after 48 hrs, but stayed at the same level at 72 hrs. The greatest decrease was observed at 48 hrs with 100 nM, when the levels were reduced by 66% compared to the untreated sample. Reductions of 38 and 62% were also evident at 10 nM and 1 )M after 48 hrs, and 39 and 61% after 72 hrs, respectively.

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 110

A * 3.5

3.0

2.5

2.0

1.5

1.0

fold change to untreated fold 0.5

0.0 ALL-3 FL SU FL + SU 100nM SU11657

B MV4;11 24 hrs 1.25 48 hrs 72 hrs 1.00

0.75

0.50

0.25 fold change to untreated change fold

0.00 0 nM 10 nM 100 nM 1 μM SU11657

Figure 3.13 A & B. Effects of SU11657 on VEGF mRNA expression. In ALL-3 xenograft cells, 100 nM SU11657 decreased FL-induced VEGF mRNA back to basal levels (A). MV4;11 cells were incubated with different concentrations of SU11657. The SU11657 was shown to be able to decrease VEGF mRNA compared to the untreated cells, by over 60% with 100 nM and 1 )M SU11657 after both 48 and 72 hrs of treatment (B). Results are the mean ± SE of at four separate experiments.

VEGF expression is controlled at many levels. Similarly VEGF mRNA can be stabilised through different treatments. In macrophages, it has been shown that VEGF mRNA has a longer half-life after treatment with LPS (Du et al. 2006). This can be achieved through a consensus region in the 5’-UTR. In ALL-3, VEGF mRNA levels increased over time with treatments. As the increase was only seen after 24 hrs (Figure 3.12), it was important to investigate whether mRNA stabilisation had a role to

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 111 play in the increase of VEGF mRNA. ALL-3 xenograft cells and the MV4;11 cell line were treated as described in Figure 3.14, and after 24 hrs in culture, actinomycin D (15 μg/ml) was added, with the cells being harvested after 1 and 2 hrs. The time was chosen based on the half-life of VEGF mRNA (Du et al. 2006). In the ALL-3 xenograft cells which were treated with FL, the VEGF mRNA degraded significantly slower than in the cells which were cultured without FL (P = 0.028), after one hour of ActinomycinD treatment (Figure 3.14A). The MV4;11 cells were only treated with SU11657, no FL was added. In the treated sample, the VEGF mRNA degraded at a slightly faster rate, although it was not significantly different from the untreated cells (Figure 3.14B).

A 1.4 ALL-3 ALL-3+FL 1.2 * P = 0.028 1.0

0.8

0.6

0.4 fold change to 0 hrs to change fold 0.2

0.0 0 hr 1 hr 2 hr hours with ActinomycinD

B 1.0 No treatment 100 nM SU11657 0.8

0.6

0.4 fold to change 0 hrs fold

0.2 0 hr 1 hr 2 hr hours with ActinomycinD

Figure 3.14 A & B. Stability of VEGF mRNA. Cells were treated for 24 hrs as indicated after which 15 )g/ml Actinomycin D was added. Cells were harvested after 1 and 2 hrs and the mRNA was extracted. VEGF mRNA was quantified against the 18S ribosomal RNA and the levels were expressed as the fold changed compared to the untreated controls. Results are the mean ± SE of at four separate experiments.

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 112

3.2.5 Inhibition of VEGF in ALL Xenograft cells with anti-FLT-3 Antibodies The FLT-3 blocking antibodies ImClone EB10 and D43 were used to further explore the relationship between FLT-3 signalling and VEGF secretion. Above it is shown that FLT-3 is activated by its ligand in ALL-3 xenograft cells, and as such these cells were used to examine the downstream effect of FLT-3 signalling and VEGF secretion. As MV4;11 cells have a constitutively active receptor and do not require FL binding for activation, they were not included in this set of experiments.

The two ImClone FLT-3 antibodies had similar effects on VEGF secretion, with the effects of D43 being slightly more inhibitory. The addition of EB10 decreased FL- induced VEGF secretion in ALL-3 cells by 50% and 33% at 10 and 50 μg/ml, respectively. The D43 antibody, at the same concentrations, reduced FL-induced VEGF secretion by 56 and 43% respectively. None of these reduction proved significant at P = 0.05, comparing to the non-treated and the Ab control. The effect on basal VEGF secretion was negligible (Figure 3.15).

5500 FL- 5000 FL+ 4500 4000 3500 cells) 6 3000 2500 2000

VEGF(pg/10 1500 1000 500 0 l l l -3 ml ml L 657 /m / / 1 g g g/m g/m g AL μ μ μ μ μ 0 0 SU1 1 50 1 50 0 b 10 43 A B B1 D D43 50 l E E ro 100 nM ont C

Figure 3.15. Effects of FLT-3 blocking antibodies on VEGF secretion by ALL-3 xenograft cells. Results are the mean ± SE of at three separate experiments.

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 113 The addition of EB10 and D43 to ALL-3 xenograft cells cultured on MS5 stromal cells, and their effects were also investigated. The EB10 antibody at 10 )g/ml decreased VEGF secretion by 23%, and an increase in concentration to 50 )g/ml, did not have any additional effect (Figure 3.16). The addition of both antibodies to the cells, without FL, slightly increased VEGF secretion. This result is not entirely unexpected as these antibodies have been shown to, in certain cases, induce the phosphorylation of FLT-3 through their binding (Piloto et al. 2005). With 10 )g/ml of D43, a reduction in VEGF secretion by 36% was observed, compared to the FL-induced secretion by ALL-3 xenograft cells. However, this reduced the secretion to the same level as the cells which were treated with D43-with no FL added. Therefore, D43 reduced the secretion back to the basal levels of ALL-3. At a higher concentration (50 )g/ml) a significant reduction (52%, P = 0.028) occurred, when compared to the FL-induced VEGF secretion. Humanised non-binding antibodies were used for controls in all experiments. When comparing the secretion of VEGF to just the antibody control there was a 61% reduction (P = 0.032).

14000 P = 0.032 FL- 12000 FL+

10000

cells) P = 0.028 6 8000

6000 * 4000 VEGF(pg/10

2000

0 3 7 l - ml m 65 /ml / / LL 1 g/ml g/ml g g g/ml g A μ μ μ μ μ μ U1 0 0 S 1 5 3 3 50 b M 4 A n D4 D l EB10 10 EB10 50 o 00 tr 1 n Control AbCo 10

Figure 3.16. Effects of FLT-3 blocking antibodies on VEGF secretion by ALL-3 xenograft cells cultured on MS5 stromal cells. Results are the mean ± SE of at seven separate experiments.

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 114 3.2.6 siRNA Knockdown of FLT-3 and effects on VEGF Previously, the manipulation of FLT-3 activation was achieved using a small molecule inhibitor (SU11657) to inhibit FLT-3 pharmacologically, after which it was blocked by specific antibodies (EB10 & D43). Further to this, a FLT-3 specific siRNA was utilised along with transfected cells (using the Amaxa nucleofector), to knock down the receptor in ALL-3 xenograft cells and the MV4;11 cell line. Representative blots of the FLT-3 knockdown are shown in Figure 3.17. A stronger knockdown in ALL-3 xenograft cells (~45% knockdown of FLT-3) was observed, when compared with the MV4;11 cell line (~40% knockdown of FLT-3).

ALL-3 MV4;11

Scrambled siRNA siRNAFLT-3 Scrambled siRNA siRNAFLT-3

FLT-3

Actin

Figure 3.17. Western blots showing FLT-3 knockdown by siRNA. After 72 hrs of culture, whole cells lysates were examined for FLT-3 expression. Actin was used as a loading control (representative blot).

VEGF secretion by both cell types was also measured after transfection. ALL-3 xenograft cells were seeded into wells which had a layer of MS5 stromal cells in them, FL was also added to the culture conditions. The CM was collected at 24, 48 and 72 hrs. The cells transfected with the FLT-3 siRNA, exhibited consistently lower levels of VEGF secretion over the time course tested. After 72 hrs a 30% decrease in VEGF secretion was detected in ALL-3 xenograft cells (Figure 3.18A) and 27% in MV4;11 cells (Figure 3.18B).

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 115 A Scrambled siRNA 3000 FLT3 siRNA cells) 6 2000

1000 VEGF per(pg/ml 10 VEGF 0 24 hrs 48 hrs 72 hrs Hours in culture after transfection

B Scrambled siRNA 5000 FLT-3 siRNA

4000 cells) 6 3000

2000

1000 VEGF (pgper 10

0 24 hrs 48 hrs 72 hrs Hours in culture after transfection

Figure 3.18 A & B. VEGF secretion after siRNA knockdown of FLT-3. Conditioned media was also collected and VEGF was measured by ELISA as previously described. ALL-3 xenograft cells are shown in (A), and the ELISA for MV4;11 cells is shown in panel (B). Results are expressed as a range of two separate experiments.

3.3 Discussion

VEGF has been detected in the serum of leukaemia patients and is frequently associated with poor outcome for those suffering from ALL (Aguayo et al. 1999; Koomagi et al. 2001; Avramis et al. 2006), as well as other haematological malignancies (Salven et al. 2000). It has been shown that an increased level of VEGF in the serum of patients may not necessarily be cancer derived, and that other cells, such as platelets and

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 116 megakaryocytes may be a potential source (Verheul et al. 1997; Banks et al. 1998). However, unequivocal evidence also demonstrates that leukaemia cells do produce and secrete VEGF (Bellamy et al. 1999; Koomagi et al. 2001; Jeha et al. 2002; Wellmann et al. 2004). The results from this chapter confirm such an assertion, with several of our panel of childhood ALL xenograft cells secreting VEGF.

The secretion by leukaemia cells of endogenous VEGF, a potent angiogenic growth factor, infers an explicit alteration of the BM microenvironment by these cells. This progression has been widely demonstrated in the invasion and metastasis of solid tumours (see review paper Folkman (1995a) and references contained within). Although capillary formation by endothelial cells in the BM has been reported in leukaemia (Aguayo et al. 2000), it has not been as widely established compared to its solid tumour counterparts. Whilst other studies have demonstrated the induction of VEGF by various growth factors, e.g. IGF-I (Miele et al. 2000; Fukuda et al. 2002); GM-CSF and IL-5 (Horiuchi et al. 1997), the results from this chapter show the production and secretion of VEGF with the exogenous addition of the cytokine FL. Aside from the novel finding of FL-induction of VEGF within childhood ALL, the haematopoietic growth factor FL is also secreted by BM stromal cells (Lisovsky et al. 1996), suggesting a paracrine interaction between the microenvironment and these cells.

While, these findings were limited to one ALL xenograft and the infant MLL tested, it exemplifies the heterogeneity of the disease. The ALL biopsies used to establish this panel were obtained at either the diagnosis or relapse from patients who encompassed a broad range of disease subtypes and treatment outcomes (Lock et al. 2002). Interestingly, these two xenografts which secreted the largest levels of VEGF also had the highest expression of FLT-3 on their surface (ALL-3 & P-14). Notably, these two xenografts have abnormalities at 11q23: whilst the xenograft ALL-3 does not have a 4;11 translocation at 11q23, it does have an abnormality in the region, in the form of a truncation at 11q23 (R. Lock, unpublished observations); moreover, the P-14 xenograft sample used for this study is an infant leukaemia, containing a non-classical, more complex translocation at 11q23. It is still classified however as MLL (Henderson et al. 2008) This suggests that the high levels of FLT-3 are related to the chromosomal abnormality, as has previously shown by microarray studies (Armstrong et al. 2002; Tsutsumi et al. 2003).

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 117

Due to the association of FLT-3 expression with poor patient progression, there has been a proliferation of drug development targeting this receptor, e.g. Tse et al. (2001), Kelly et al. (2002c), Levis et al. (2002), Weisberg et al. (2002), Murata et al. (2003). One of these small molecule inhibitors, the Pfizer compound SU11657, which was designed with the intent to inhibit FLT-3, was used in this study. The relationship between FLT-3 and VEGF secretion was further examined using concentrations below the IC50 of SU11657. As it was shown previously that SU11657 inhibited FLT-3 phosphorylation, the resulting effect on VEGF secretion provides further information on this association. Statistically significant reductions occurred in the ALL-2 and ALL-3 xenograft cells (at 100 nM), and in P-14 samples at higher SU11657 concentrations (1 μM). Notably, the addition of FL induced significant increases in VEGF secretion in both ALL-3 and P-14 xenograft cells. The inhibition of the receptor with SU11657, as expected, reversed the effects of FL-induced VEGF secretion in ALL-3 and P-14 xenograft cells. These results would appear to confirm the above findings that the activation of FLT-3, by its ligand, induced the secretion of VEGF in these ALL xenograft cells.

In ALL-2, the presence or absence of FL in the conditioned media resulted in disparate reductions in the VEGF expression upon introduction of the inhibitor. Such a finding leaves open the possibility that FLT-3 signalling may still play a role in VEGF secretion in ALL-2 xenograft cells. Since there was no observed increase in VEGF secretion upon the addition of FL with regards to ALL-4, -7 and -17 xenograft cells, it was expected that there would be no corresponding change with the addition of SU11657, which proved to be the case. It is entirely plausible that the secretion of VEGF in these xenografts could be through a different mechanism, compared to ALL-3. Examples in the literature of VEGF induction have been numerous, ranging from the induction by VEGF receptors (Broggini et al. 2003), to HIF-1 (Jensen et al. 2006; Shinojima et al. 2007), as well as by FGF4 as shown by Deroanne and colleagues (1997).

In order to confirm the observations of our xenograft model, a range of human cell lines were utilised which similarly had variable amounts of both VEGF secretion and FLT-3. Cells lines survive and proliferate in culture and are, under certain circumstances, easier to manipulate. Utilising a combination of both xenograft cells and cell lines may give a

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 118 better overall understanding of the biological mechanisms that occur in primary tumours in vivo. In the case of the cell lines, the focus was on the MV4;11 cell line, which similarly has an abnormality at 11q23 (t4;11). The addition of SU11657 (1100 nM) also decreased VEGF secretion in MV4;11 cells, once again suggesting the importance of FLT-3 in inducing VEGF secretion in this cell line.

Several splice variants of the VEGF gene can be expressed by human cells. Two isoforms were expressed by the leukaemia cells tested, as was shown by RT-PCR and detected in the ELISA. They were the two secreted in their soluble forms VEGF121 and

VEGF165. A study of normal peripheral blood mononuclear cells show the expression of

VEGF121, VEGF165 mRNA, similarly as was shown by our leukaemia cells, but they also showed the expression of VEGF189 (Iijima et al. 1993).

The expression of VEGF has been reported to be regulated through both transcriptional and post-transcriptional mechanisms (Ferrara 1999b). As was observed with VEGF protein, which was increased with FL and decreased with SU11657, VEGF mRNA was altered in a similar fashion in our model systems. VEGF mRNA has a short half-life and can be increased by hypoxia, a process which has also been demonstrated in activated macrophages (Shih et al. 1999; Du et al. 2006). This stabilising of VEGF mRNA is mediated by mRNA binding proteins which act on the cis elements in the 3’-UTR (Xu et al. 1997; Claffey et al. 1998; Levy et al. 1998).

The stability of the mRNA was also affected with the treatments, shown with the addition of the transcription inhibitor actinomycin D. FL increased the half-life of VEGF mRNA in ALL-3 xenograft cells. This result shows that mRNA stabilisation may be one mechanism leading to increased VEGF production in ALL xenograft cells. The opposite effect was observed upon the addition of SU11657 to MV4;11 cells. With the addition of actinomycin D, there was a decrease in VEGF mRNA, suggesting that the mRNA is shorter lived in SU11657 treated cells. Since actinomycin D reduced VEGF mRNA, it is likely that SU11657 blocked the FLT-3 signalling, which was acting to enhance the half-life of VEGF mRNA.

SU11248 is an SU11657 analogue, and a next generation inhibitor which has progressed to clinical trials (Fiedler et al. 2005; Norden-Zfoni et al. 2007). It was designed to have

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 119 the same specificity as the inhibitor used in this study as can be seen by their structural similarities (Figure 3.19). One previous study has shown a decrease in VEGF secretion with this different FLT-3 inhibitor (SU11248) in AML cells and in the MV4;11 cell line (O'Farrell et al. 2003a). While we also showed a reduction in VEGF secretion in the MV4;11 cell line, our study mainly focused on childhood ALL samples where we showed an increase of VEGF secretion with the addition of FL, and a subsequent decrease with the addition of an inhibitor (SU11657).

The study also looked at the effects of SU11248 in vivo. The MV4;11 cell line was injected subcutaneously into athymic mice and tumour volume was monitored. The second employed method was a BM model in which cells were injected into the tail vein of NOD/SCID mice, then after 90 days blood and bone marrows were harvested, and the amount of human cells was assessed. In both models, SU11248 produced observable effects, with a dramatic regression in the tumours in two different mouse models. In one model tumours were injected subcutaneously with the tumour volume measured, while in the other the leukaemia was injected intravenously by which it engrafted in the BM. The in vivo single agent efficacy of SU11248 has been tested in our xenograft model and showed effects on the progression of ALL-2 into NOD/SCID mice (Maris et al. 2008). In this study, ALL-2 was shown to have an objective response, effectively delaying the progression of the leukaemia in the mice (Maris et al. 2008). The in vivo effect of SU11248 on ALL-2 may be related to the effects observed in vitro with SU11657 and the almost complete reduction in VEGF secretion by these cells.

The development of blocking antibodies against different receptors, associated with expression on cancers, has surged over the last few years. Some examples of targets are: the IGF-I receptor, VEGF and human epidermal growth factor receptor 2 (Burger et al. 2007; Haluska et al. 2007; Bender et al. 2008; Lacy et al. 2008; Wilson et al. 2008). One of the many targets of ImClone blocking antibodies has been FLT-3, and two of these were tested on our ALL-3 xenograft cells (EB10 and D43). These FLT-3 blocking antibodies have been shown to significantly decrease the engraftment of AML into NOD/SCID mice, as well as to prolong the survival of mice engrafted with ALL cells lines and primary cells (Piloto et al. 2005; Piloto et al. 2006). These antibodies were specifically designed for the inhibition of FLT-3, as when they bind to the FLT-3 receptor; they also block the binding of its ligand and hence negate its activation. The

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 120 overall benefit of this is that they give a more specific effect than the use of small molecule inhibitors, which may have some unwanted off-target effects. The above results demonstrate that the addition of FLT-3 blocking antibodies to in vitro ALL-3 xenograft cultures reduced the FL-induced VEGF secretion. This would indicate that the activation of FLT-3 by its ligand and the subsequent signal transduction induced the secretion of VEGF in these xenograft cells.

SU11657 SU11248

Hydrogen Carbon Fluorine Nitrogen Oxygen

Figure 3.19. The structures of SU11657 and SU11248.

FLT-3 was inhibited with SU11657 and blocked with FLT-3 specific antibodies, both of which resulted in the decrease in VEGF. To further exemplify the contribution of FLT-3 to VEGF, the receptor was down-regulated with siRNA. While the down-regulation was by no means dramatic, there was a sufficient decrease of VEGF secretion shown with a similar decrease of the receptor.

BM stromal cells have been shown to enhance the in vitro survival of normal and malignant haematopoietic cells (Manabe et al. 1992; Konopleva et al. 2002). These

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 121 effects can be mediated through both direct contact (Manabe et al. 1994) or through the secretion of growth factors into the surrounding environment (Jarvis et al. 1997; Nefedova et al. 2003). In the course of our experiments, ALL xenograft cells were routinely cultured on the murine stromal cell line MS5. This enhanced the secretion of VEGF, by not only ALL-3 xenograft cells, but also by five other different xenografts, which showed VEGF secretion only when cultured on such a stromal support. As MS5 cells do not secrete FL (Keller et al. 2002), the observed effects are obviously due to other cytokines or growth factors secreted by MS5 cells, as well as the direct cell to cell contact. The bone marrow stroma is known to support the proliferation and differentiation of normal cells, and in turn malignant cells residing and proliferating in the BM (Rafii et al. 1995; Rafii et al. 1997). These cells have also been isolated and cultured to be used as supportive cells for the in vitro growth of different tumours, especially those of haematopoietic origin (Johnson et al. 1986; Suzuki et al. 1992). They provide a physical support to which the cells can adhere to and even burrow underneath. These supporting cells express adhesion molecules making the attachment process possible (Jarvis et al. 1997).

The stromal and leukaemia cells form an interactive relationship, by way of a paracrine loop, enabling the tumour cells to receive their required factors necessary for growth (Jiang et al. 1998; Glenjen et al. 2005; Veiga et al. 2006). By examining the survival and proliferation of leukaemia cells when co-cultured with stromal cells, Jiang and colleagues (1998) showed that the stromal cells induced the production of cytokines (IL 1a and IL 11) by direct contact with the leukaemia cells. The expression of these cytokines receptors was increased upon their attachment to the stromal layer. This cross-talk between stromal cells and leukaemia cells has been similarly observed in a study by Glenjen and colleagues (2005), who showed and increase in VEGF production by AML when co-cultured with stromal cells, similar to the observations in our study. In in vitro cultures leukaemia cells actively move towards and interact with BM endothelium in matrigel (Veiga et al. 2006). Considering that ALL xenograft cells have an enhanced survival rate when cultured on MS5 stromal cells compared to growing them on just tissue culture plastic, and that these xenografts exhibit increased VEGF secretion, the results would imply that there is a cross-talk between our xenograft panel and the stromal MS5 cells on which they are cultured. The effects of the BM stroma on

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 122 ALL cells will be explored further in Chapter 5. One of the aspects of this cross-talk is the relationship between FLT-3 and VEGF in ALL cells.

To summarise, the relationship between the induction of VEGF by FLT-3 was able to be disrupted in three distinct ways. Firstly, by pharmacologically inhibiting the receptor with the RTK inhibitor, SU11657; secondly, by blocking it with FLT-3 blocking antibodies; and finally, by decreasing the receptor with specific siRNA. VEGF was previously shown to be a downstream target of Src, and this signalling was able to be inhibited with a tyrosine kinase inhibitor (Mukhopadhyay et al. 1995), and this signalling cascade has been shown to be mediated through the MAPK pathway (Avruch et al. 1994; Daum et al. 1994). The relationship between FLT-3 and VEGF in ALL cells and the effects of the FLT-3 signalling pathway are further explored in the following chapters of this thesis.

These particular results show that in a specific subset of childhood ALL xenograft cells, VEGF message levels and protein secretion can be induced with the addition of FL. What is clear from this research is the heterogeneity of the disease, with the different cell types harbouring differing molecular lesions or abnormalities. As such, different drugs (which have different targets for these specific abnormalities), will more than likely improve patient outcomes. Therefore, there is merit in a more specific, patient- dependent approach in order treat the disease, and the results from this study (regarding the ALL-3 subtype) could potentially add to this specificity of treatment.

Chapter Three: VEGF Secretion by Leukaemia Cells Induced by FLT-3 Activation 123

 

CHAPTER FOUR: Activation of the FLT-3 Signalling Pathway and its Induction of VEGF

4.1 Introduction

In the previous chapter, it was shown that the ALL xenograft cells expressed varying levels of FLT-3. It has previously been well established that the receptor tyrosine kinase, FLT-3, is expressed in early haematopoietic progenitors and plays an important role in haematopoiesis (Mackarehtschian et al. 1995). It is also expressed in several types of acute leukaemias, including ALL. Whilst rarely expressed in adult ALL (Nakao et al. 1996), it has been shown to be highly associated with specific subtypes of paediatric ALL, specifically, both ALL with MLL rearrangements (Armstrong et al. 2003) and also in hyperdiploid ALL (50 or more chromosomes) (Yeoh et al. 2002). Much of the previous research on FLT-3 in acute leukaemias has focussed on the role of activating mutations, and particularly, their association with poor prognosis (e.g. Meshinchi et al. (2006)).

It was therefore necessary to follow up the earlier findings (in Chapter 3) in order to establish whether the FLT-3 expressed in our xenograft panel is wild-type or mutant and thus its pattern of activation. It was also deemed necessary to examine the downstream pathway(s) compared to the published scientific literature. It was also established that our ALL xenograft panel secreted VEGF when cultured in vitro, and this secretion was significantly manipulated by the additions of either FL or FLT-3 inhibitors. As discussed previously, VEGF, like FLT-3, has been shown to be expressed by leukaemia cells and is associated with poor patient prognosis. The novel interrelationship between

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 125 FLT-3 and VEGF, identified in Chapter 3, was followed up by examining the effects on VEGF secretion by further manipulation of FLT-3 activation and its signalling intermediaries. Understanding the relationship between these two factors in leukaemia may identify alternative targets for high risk patients with high FLT-3 expression.

Following FLT-3 activation, it has been previously shown that there is successive downstream phosphorylation of the PI3K and MAPK pathways as shown in leukaemia cell lines (Dosil et al. 1993; Zhang et al. 1999a; Zhang et al. 1999b; Zhang et al. 2000a; Srinivasa et al. 2002). Much of this chapter focuses on the AKT and ERK1/2 pathways, as they have previously been shown to be responsible for FLT-3 signal transduction (Zhang et al. 1999a; Zhang et al. 1999b; Zhang et al. 2000a). Several studies have also shown that mutant FLT-3 receptor (or more specifically FLT-3 with ITDs) also signals through the STAT5 pathway (Hayakawa et al. 2000; Mizuki et al. 2000; Choudhary et al. 2007). Therefore, it was necessary to initially establish whether the ALL xenografts expressed either wild type or mutated FLT-3. The xenograft panel was tested for the two most common mutations in leukaemia, an in-frame ITD in the juxtamembrane domain and a point mutation at Asp835 (refer to Section 1.6.3). The phosphorylation status of FLT-3 was then analysed, followed by the activation of signalling intermediates, AKT and ERK1/2. In an attempt to elucidate the FLT-3 signalling pathway with respect to the induction of VEGF secretion in leukaemia, several RTK and signalling pathway inhibitors were utilised. The effects of the inhibitors on both the secretion of VEGF by leukaemia cells, activation of the FLT-3 receptor, and activation of its downstream effectors, AKT and ERK1/2, were all assessed. The different inhibitors that were used are shown in Table 4.1.

All compounds with the prefix SU were kindly provided by Pfizer (SU11657, SU5416 and SU6668). SU5416 is a small, membrane permeable, indolinone compound, which inhibits the phosphorylation of tyrosine residues on the VEGFR-2 and KIT receptors (Fong et al. 1999b). It has been tested in clinical trials for leukaemia and metastatic colorectal cancer (Saltz et al. 2007). Like SU5416, SU6668 is a small molecule kinase inhibitor, with a broader inhibition profile compared to SU5416, covering the inhibition of VEGFR-2, as well as PDGFR and FGFR (Laird et al. 2000). Both of the above mentioned compounds have also been shown to inhibit the activity of the KIT receptor in AML cell lines (Smolich et al. 2001). As these compounds act in a similar way to

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 126 SU11657, by targeting the ATP binding site of different RTKs, they were used as a comparison to SU11657 and its activity against FLT-3 in leukaemia cells. A commercially available KDR inhibitor, referred to as KDRi, with similar structure to SU5416 and SU6668 was also tested. The signalling pathway inhibitors used were against the MEK/ERK1/2 (PD98059, U0126 and a MEK inhibitor, referred to as MEKi) and PI3K (LY294002 and Wortmannin) pathways. All of the pathway inhibitors were commercially available when the research was undertaken.

Table 4.1. List of inhibitors and their specificity. Inhibitor Specificity Reference Company

SU11657 FLT-3 (Sohal et al. 2003) Pfizer

SU5416 VEGFR-2, KIT (Fong et al. 1999b) Pfizer

SU6668 VEGFR-2, PDGFR, KIT (Laird et al. 2000) Pfizer

KDRi VEGFR-2 (Sun et al. 2000) Merck

U0126 MEK/ERK1/2 (Favata et al. 1998) Merck

PD98059 MEK/ERK1/2 (Dudley et al. 1995) Merck

MEKi MEK/ERK1/2 (Wityak et al. 2004) Merck

LY294002 PI3K (Vlahos et al. 1994) Sigma

Wortmannin PI3K (Powis et al. 1994) Sigma

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 127 4.2 Results

4.2.1 Mutation and Phosphorylation Status of The FLT-3 Receptor

Mutations As indicated above, the two most common mutations in FLT-3 are ITDs occurring in the juxtamembrane domain, and single point mutations in the second tyrosine kinase domain (TKD) of the receptor at position Asp835 (Figure 4.1 part I). These regions can be identified by PCR of genomic DNA. A primer pair specific for exon 14 and 15, the region where ITDs occur, amplifies a 324-base pair (bp) fragment in wild-type FLT-3 (Figure 4.1(II)A). ITDs are in frame duplications and can be easily identified on an agarose gel by a shift in the band size depending on the number of duplications. The MV4;11 cell line has a known ITD, with a 30 bp repeat (Quentmeier et al. 2003), and consequently a small shift can be observed on the PCR gel, indicating the presence of a duplication in the gene. The PCR products for the ALL xenograft cells (ALL-4, ALL-7, ALL-17, ALL-2 & ALL-3) did not appear to have a change in the band size compared to the control cell line RS4;11, which has known wild-type FLT-3. This indicates that the xenograft cells do not contain ITD mutations.

The D835 and I836 codons contain the nucleotide sequence GATATC which form the EcoRV restriction site. To detect if there was a FLT-3 mutation at D835, the region was amplified by PCR, producing a 263-bp product (Figure 4.1(II)B), which was subsequently digested with EcoRV. Wild-type FLT-3 was digested to produce 2 bands (Figure 4.1(II)C). A mutation at codon 835 causes a loss of the EcoRV site, characterised by the presence of an uncut band. No mutations were observed at D835 in any of the xenografts tested compared to the two cell line controls. GAPDH was used as a PCR control (Figure 4.1(II)D).

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 128

I NH2

ECD

TMD JMD Exon14/15 ITD TKD1

KI R D I M CGAGATATCATG wild-type TKD2 D835 EcoRV

CGATATATCATG mutant R Y I M COOH

II

ALL-4 ALL-7 ALL-17 ALL-2 ALL-3 MV4;11 RS4;11 Exon14/15 

Exon20 

Exon20 

GAPDH 

Figure 4.1. Analysis of the FLT-3 gene for known mutations. Part (I), a schematic diagram showing the structure of FLT-3, and regions of know mutations. Part (II), the PCR products of exons 14/15 and 20. (A) Exon 14/15, the JM region of the gene. The band in the MV4;11 lane shows a small shift in size, consistent with presence of an ITD. (B) and (C) PCR products of exon 20, around area around codon D835. (B) shows the PCR of exon 20, with (C) showing the same band after digestion with EcoRV. (D) GAPDH was used as the control gene for the gDNA PCR. ECD - extracellular domain; TMD - transmembrane domain; JMD - juxtamembrane domain; TKD - tyrosine kinase domain; KI - kinase insert.

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 129 Phosphorylation In order to begin to elucidate the pathway between FLT-3 activation and VEGF secretion in our model system, the phosphorylation status of FLT-3 and SU11657- mediated effects thereupon were examined. Ex vivo cultures of ALL xenograft cells were incubated for 2 hrs in QBSF-60 media in the presence or absence of 100 nM SU11657, following which FL (20 ng/ml) was added for 15 min to activate the receptor. FL was not added to MV4;11 cells in this study, due to its constitutively active receptor.

Figure 4.2 shows representative blots of FLT-3 phosphorylation and the effects of SU11657 on cells from the 6 different xenografts which secreted VEGF, as well as the MV4;11 cell line. Across all the xenografts, ALL-2, -3 and P-14 showed basal phosphorylation levels of FLT-3. The addition of FL (20 ng/ml) increased the phosphorylation of FLT-3 receptor in all xenograft cells, as shown by the more pronounced second band in each of the top panels in Figure 4.2. Interestingly, the xenografts with the highest levels of the receptor (ALL-2, -3 and P-14) also showed the most pronounced phosphorylation, as identified previously in Chapter 3. Although the FL-induced activation was much less pronounced in ALL-4, -7 and -17, they also showed a considerably lower level of total receptor, potentially accounting for this observed difference. The small molecule inhibitor SU11567 (at 100 nM), reduced the FL-induced phosphorylation of FLT-3 in all ALL xenograft cells. In ALL-3, -4, -7, -17 and P-14, a reduction to pre-FL-induced activation was observed. ALL-2, although displaying a 57% (av.) reduction in phosphorylation upon the addition of the inhibitor, it did not reach basal levels. In the case of the MV4;11 cells, the phosphorylation of the FLT-3 receptor, was also inhibited with the addition of SU11657 (also at 100 nM) to undetectable levels (Figure 4.2).

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 130

ALL-2 ALL-3 ALL-4

20 ng/ml FL - + + - + + - + + 100 nM SU11657 -- + --+ -- + Phos-FLT-3

Total-FLT-3

ALL-7 ALL-17 P-14 20 ng/ml FL - + + - + + - + + 100 nM SU11657 -- + --+ -- +

Phos-FLT-3

Total-FLT-3

MV4;11 100 nM SU11657 - +

Phos-FLT-3

Total-FLT-3

Figure 4.2. Activation of FLT-3 with its ligand and the effects of SU11657. FLT-3 was immunoprecipitated from whole cell lysates of ALL xenograft cells and the MV4;11 cell line. Blots were probed for phospho-tyrosine (top panels) and then for total FLT-3 (bottom panels). The blots are a representative of at least three biological replicates.

4.2.2 FLT-3 Signalling Pathway and the Effects of SU11657 To further explore the relationship between VEGF secretion and FLT-3 activation in leukaemia cells I examined FL-induced activation of the known FLT-3 downstream targets (AKT, ERK1/2 and STAT5), and the effects of SU11657 on their activation. AKT was examined to explore the role of the PI3K pathway. As shown in Figure 4.3, although AKT appeared to be initially activated, there were no observable changes in its phosphorylation; as a consequence of exposure to the addition of either FL or SU11657.

All ALL xenografts, except ALL-3, showed some basal ERK1/2 activation (Figure 4.3). In contrast to AKT, the FL-induced phosphorylation of ERK1/2 was shown to vary substantially between xenografts. The greatest stimulation occurred with ALL-3 and

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 131 P-14, with smaller increases observed in ALL-2, -7 and -17. ALL-4 showed no change to its basal levels. Again, the addition of 100 nM SU11657 produced varying changes to ERK1/2 phosphorylation, with reductions not readily apparent in ALL-4, -7 and -17. Conversely, in ALL-2, -3 and P-14, ERK1/2 phosphorylation was reduced to basal levels or below with the addition of SU11657. Separation into two distinct categories, based on their response to SU11657, was coincident with their reduction in VEGF levels upon the addition of SU11657 (as shown in Section 3.2.3, Figure 3.9).

ALL-2 ALL-3 P-14

20 ng/ml FL - + + - + + - + +

100 nM SU11657 -- + --+ --+ Phos-AKT

Total-AKT

Phos-ERK1/2 Total-ERK1/2

ALL-4 ALL-7 ALL-17

20 ng/ml FL - + + - + + - + +

100 nM SU11657 -- + --+ -- + Phos-AKT

Total-AKT

Phos-ERK1/2

Total-ERK1/2

Figure 4.3. Effects of SU11657 on the FLT-3 pathway in ex vivo cultured ALL xenograft cells. Whole cell lysates were initially blotted for phospho-AKT and -EKR1/2, after which the blots were stripped and total-AKT and -ERK1/2 examined. The blots are a representative of at least three biological replicates.

In regards to the leukaemia cell lines (MV4;11, HL60 and NB4), as was the case with the ALL xenograft cells, there were observable levels of basal AKT phosphorylation, and once again, the addition of SU11657 (100 nM) produced no observable reductions in this activation (Figure 4.4). Within the MAPK pathway, ERK1/2 was endogenously activated in the MV4;11 cell line, in a manner corresponding to its FLT-3 receptor

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 132 (shown above in Figure 4.2). The addition of SU11657 resulted in a decrease in ERK1/2 phosphorylation in only MV4;11 cells. This observation for MV4;11 is in line with the results for both the ERK1/2 suppression in ALL-2, -3 and P-14 (above), as well as in regards to their decreases in VEGF secretion (shown in Section 3.2.3, Figure 3.10), confirming their similarity in FLT-3 pathway activation.

In order to specifically examine the impact of the RTK inhibitor on the FLT-3 signalling pathway, SU11657 was assessed on two other cell lines HL60 and NB4. Whilst HL60 expresses FLT-3 and NB4 does not, they both secrete VEGF. Unlike that shown in the MV4;11 cell line however, that secretion was not able to be modulated with SU11657. Treatment with SU11657 (also at 100 nM) had no discernible effects on the phosphorylation of AKT or ERK1/2 in the two other leukaemia cell lines tested, both of which exhibit exogenous activation of their associated intermediaries (Figure 4.4).

MV4;11 HL60 NB4 100 nM SU11657 - + - + - + Phos-AKT Total-AKT

Phos-ERK1/2

Total-ERK1/2

Figure 4.4. Effects of SU11657 on the FLT-3 signalling pathway in the leukaemia cell lines. Refer to Figure 4.3, for methodological details. The blots are a representative of at least three biological replicates.

The activation of STAT5 was also examined, as FLT-3 with ITD mutations has also been reported to signal through this pathway (Tse et al. 2000; Zhang et al. 2000b). No changes in STAT5 activation were detected in all ALL xenograft cells tested upon the addition of FL (Figure 4.5). As the xenografts have been previously shown to lack ITDs, this finding, along with the corresponding non-effect of SU11657, was expected. The MV4;11 cell line was used as the positive control in this experiment because of its known ITD, and Figure 4.5 shows the expected STAT5 phosphorylation, and subsequent reduction upon the addition of SU11657 to below detectable levels.

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 133

ALL-2 ALL-3 P-14

FL - + + - + + - + + SU11657 - - + - - + - - + Phos-STAT5

Total-STAT5

ALL-4 ALL-7 ALL-17 FL - + + - + + - + + SU11657 - - + - - + - - + Phos-STAT5 Total-STAT5

MV4;11

M SU11657 ) 0 nM 100 nM 1

Phos-STAT5

Total-STAT5

Figure 4.5. Western blot analyses of STAT5 activation. Cell lysates from ALL xenograft cells and the MV4;11 cell line were analysed to asses the activation status of STAT5 in response to the presence / absence of FL (20 ng/ml) and SU11657 (100 nM). The blots are a representative of at least three biological replicates.

Given that the levels of STAT5 in ALL-3 xenograft cells were extremely low, the protein was immunoprecipitated with an antibody against total STAT5 and immunoblotted for phospho-tyrosine to asses its phosphorylation status. As shown in Figure 4.6, there was no change in the phosphorylation of STAT5 in either ALL-2 or ALL-3 xenograft cells with the addition of FL (20 ng/ml) or SU11657 (100 nM). It should be noted that these two xenograft cells were of primary interest as they showed high levels of VEGF secretion, which was attenuated with SU11657, along with the finding that they also expressed high levels of FLT-3 on their surface.

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 134

ALL-2 ALL-3

20 ng/ml FL - + + - + +

100 nM SU11657 - - + - - + Phos-STAT5

Total-STAT5

Figure 4.6. Immunoprecipitation of STAT5 from ALL-2 and -3 xenograft cells. FLT-3 was immunoprecipitated from whole cell lysates and blotted with anti-phospho-tyrosine Ab, as was performed with the FLT-3. Blots were stripped and probed for total-STAT5. The blots are a representative of at two biological replicates.

4.2.3 Effects of RTK Inhibitors on the FLT-3 - VEGF Relationship To further elucidate the FLT-3 signalling pathway with respect to VEGF secretion in leukaemia, alternative RTK inhibitors were utilised (refer back to Table 4.1). Their effects were tested on the secretion of VEGF, as well as on the activation of FLT-3, STAT5, AKT and ERK1/2. These experiments were performed with the MV4;11 cell line and then confirmed in ALL-3 xenograft cells.

Effects in the MV4;11 cell line Across all the inhibitors tested, the effects of SU11657 were distinctly the most potent on VEGF secretion (Figure 4.7). Even at 10 nM there was a 43% (P = 0.038) decrease in VEGF secretion. At higher concentrations of SU11657 (100 nM & 1 )M), VEGF secretion was decreased by 63% (P = 0.0008) and 84% (P < 0.0001), respectively. At 10 nM, the other inhibitors tested did not show any decreases in VEGF secretion by the MV4;11 cell line. A 27% decrease in VEGF secretion occurred at 100 nM SU5416, with a further decrease to 50% at 1 )M. The other RTK inhibitors only showed observable effects at 1 )M. Looking at the overall results, SU11657 showed the strongest inhibition of VEGF secretion followed by SU5416 (inhibitor of VEGFR-2 and KIT), compared with SU6668 (inhibitor of VEGFR-2, PDGFR and KIT) (7%) and KDRi (inhibitor of VEGFR-2) (28%), which only caused a marginal reduction.

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 135 9000 SU11657 SU5416 8000 SU6668 KDRi 7000

6000

5000

4000

3000 VEGF(pg/10^6 cells) 2000

1000

0 0 nM 10 nM 100 nM 1 μM Concentration

Figure 4.7. Effect of different inhibitors on VEGF secretion in the MV4;11 cell line. VEGF levels in the CM were assessed by ELISA. The amount of VEGF secretion was normalised to the amount of live cells at the time of harvest, as measured by flow cytometry. Therefore, the amount of VEGF given is given as pg/106 live cells. The VEGF secretion results are expressed as the mean ± SE of at least three separate experiments.

The effects of these inhibitors were subsequently tested on FLT-3 and its downstream mediators. The decrease in VEGF secretion that was observed in Figure 4.7 corresponded with a comparable decrease in FLT-3 receptor phosphorylation by these compounds (Figure 4.8A). There proved to be a significant correlation between the decrease in VEGF secretion by the inhibitors (100 nM and 1 )M) and the decrease in the tyrosine phosphorylation of the FLT-3 receptor, R2 = 0.77 and P = 0.02 (Figure 4.8B).

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 136

A

No Inhibitors Inhibitors No SU11657 SU5416 SU6668 KDRi

Phospho-FLT-3

Total-FLT-3

B 125

100

75

50

25

0

% decrease in FLT-3 phosphorylation FLT-3 in decrease % 0 25 50 75 100 % decrease in VEGF

Figure 4.8 A & B. Effects of RTK inhibitors on FLT-3 phosphorylation in the MV4;11 cell line. (A) FLT-3 was immunoprecipitated and the tyrosine phosphorylation examined. The blots were subsequently stripped and blotted for total FLT-3. Concentrations of the inhibitors were all 1 )M. (B) The decrease in VEGF was calculated compared to basal VEGF secretion by MV4;11 cells, as was the decrease in FLT-3 phosphorylation. The effects of the inhibitors at two concentrations (100 nM and 1 )M) were used in the correlation analysis. The Spearmann correlation was used for the statistical analysis, and results are described in text. The blots are a representative of at least three biological replicates.

The effects of the RTK inhibitors on the phosphorylation of the downstream signalling mediators are shown in Figure 4.9, all of which were compared to those seen by SU11657. The phosphorylation of STAT5 varied in response to the different inhibitors, with the greatest inhibition occurring with SU11657, followed by SU5416 (at 1 )M). As was shown previously, the endogenous activation of AKT was not attenuated by SU11657, nor any of the other RTKs examined. In terms of the MAPK pathway, ERK1/2 phosphorylation was decreased by SU11657 (at > 100 nM) and SU5416 (at 1 )M) by 68 and 22% respectively. SU11657 was the only inhibitor to markedly decrease the phosphorylation of ERK1/2, with minor decreases observed with the addition of 1 )M SU5416 and SU6668 (22 and 15% respectively).

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 137

MV4;11 only SU11657100 nM μM 1 SU11657 SU5416100 nM μM 1 SU5416 SU6668100 nM μM 1 SU6668 KDRi100 nM 1 μM KDRi

Phospho-STAT5

Total-STAT5

Phospho-AKT Total-AKT

Phospho-ERK1/2

Total-ERK1/2

Figure 4.9. Effects of a range of inhibitors on the FLT 3 signalling pathway in MV4;11 cells. Whole cell lysates were blotted for anti-phospho-STAT5, AKT and ERK1/2. They were then stripped and blotted for total STAT5, AKT and ERK1/2. The panel shows the effects of RTK inhibitors (SU11657, SU5416, SU6668 and KDRi) compared to untreated cells. The blots are a representative of at least three biological replicates.

Effects in ALL-3 xenograft cells The ALL-3 xenograft cells were chosen as the ALL experimental model, to elucidate the FLT-3 signalling pathway involved in VEGF secretion. These ALL xenograft cells proved the obvious choice because of their VEGF secretion upon the addition of FLT-3. As an initial step, the effects on VEGF secretion of the various inhibitors were compared to those observed with the addition of SU11657 (Figure 4.10). At equivalent concentrations (1 )M), SU11657 had the highest potency (80%) in the reduction of VEGF secretion. In ALL-3 xenograft cells, a limited reduction (35 - 40%) in VEGF secretion was observed with other RTK inhibitors (SU5416, SU6668 and KDRi) (Figure 4.10). The only statistically significant reduction in VEGF secretion occurred with SU11657 at both 100 nM (which showed a 59% reduction in VEGF) and 1 )M (80%) (P = 0.032 and P = 0.015, respectively). The remainder of the inhibitors had P-values of > 0.14.

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 138 7000 FL- FL+ 6000

5000 cells) 6 4000

3000

2000 VEGF(pg/10

1000

0 L-3 57 57 16 68 Ri AL 116 116 U54 U66 KD SU SU μM S μM S μM nM μM 1 1 1 100 1

Figure 4.10. Effects of different inhibitors on VEGF secretion by ALL-3 xenograft cells. VEGF levels in the CM were assessed by ELISA. The amount of VEGF secretion was normalised to the amount of live cells at the time of harvest, as measured by flow cytometry. Therefore, the amount of VEGF given is given as pg/106 live cells. The VEGF secretion results are expressed as the mean ± SE of at least three separate experiments.

The effects of the RTK inhibitors were next analysed on the activation of FLT-3 in ALL-3 xenograft cells. As observed with VEGF secretion, the addition of SU11657 (at both 100 nM, P = 0.032 and 1 )M, P = 0.015), significantly decreased the FL-induced phosphorylation of FLT-3 (Figure 4.11A). The other RTKs; SU5416, SU6668 and KDRi, which had previously shown minor decreases to VEGF secretion (Figure 4.10), induced concomitant changes in FLT-3 phosphorylation (Figure 4.11A). As was the case in the MV4;11 cell line, the decrease in VEGF secretion by different RTK inhibitors significantly correlated with their effects on FLT-3 activation (P = 0.017, Figure 4.11B).

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 139

A

ALL-3 only 20 ng/ml FL 100SU11657 nM 1 μM SU11657 1 μM SU5416 1 μM SU6668 1 μM KDRi

Phospho-FLT-3

Total-FLT-3

B

100 2 R = 0.94 P = 0.017

50

0

-50

% decrease in FLT-3 phosphorylation FLT-3 decrease in % 0 10 20 30 40 50 60 70 80 90 % decrease in VEGF

Figure 4.11 A & B. Effects of RTK inhibitors on the activation of FLT-3 in ALL-3 xenograft cells. (A) FLT-3 was immunoprecipitated and the tyrosine phosphorylation examined. The blots were subsequently stripped and blotted for total FLT-3. (B) The decrease in VEGF was calculated compared to the FL-induced VEGF secretion by the ALL-3 xenograft cells, as was the decrease in FLT 3 phosphorylation. The effects of the inhibitors were used in the correlation analysis. Effects of SU11657 were assessed at two concentrations (100 nM and 1 )M). The Spearmann correlation was used for the statistical analysis, and results are described in text. The blots are a representative of at least three biological replicates.

Following on, the effects of these same inhibitors were also examined on the downstream signalling intermediaries of FLT-3. As was observed with the MV4;11 cell line, there were no detectable changes in the activation status of AKT under any of the conditions tested (Figure 4.12). This was also the case for STAT5 (data not shown). Conversely, the decreased levels of VEGF secretion in the presence of SU11657 would appear to be associated with a corresponding reduction in ERK1/2 phosphorylation, as demonstrated in Figure 4.12.

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 140

ALL-3 only only ALL-3 FL ng/ml 20 100SU11657 nM 1 μM SU11657 1 μM SU5416 1 μM SU6668 1 μM KDRi

Phospho-AKT

Total-AKT

Phospho-ERK1/2

Total-ERK1/2

Figure 4.12. Effects of RTK inhibitors on AKT and ERK1/2 in ALL-3 xenograft cells. The experiment was preformed as described in Figure 4.9. The blots are a representative of at least three biological replicates.

4.2.4 Effects of Signalling Pathway Inhibitors on the FLT-3 - VEGF Relationship Extending the work from the last section; signalling pathway inhibitors were also used to assess their effects on VEGF secretion and the FLT-3 pathway intermediaries, to further probe the relationship. The signalling pathway inhibitors are also described in Table 4.1.

Effects in the MV4;11 cell line In comparison to SU11657, only minor changes to VEGF secretion were observed with the addition of the inhibitors to MV4;11 cells. Initially at 100 nM, VEGF secretion appeared to increase marginally from basal levels. At 1 )M, U0126 (inhibitor of MEK and ERK1/2) decreased VEGF secretion by 26%, LY294002 (inhibitor of PI3K) by 20%, and the remaining two inhibitors (PD98059 and Wortmannin, inhibitors of MEK and ERK1/2 and PI3K, respectively) demonstrated no suppression within the MV4;11 cell line.

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 141 7000 SU11657 U0126 6000 PD98059 LY294002 Wortmannin 5000

4000

3000

VEGF(pg/10^6 cells) 2000

1000

0 0 nM 100 nM 1 μM Concentration

Figure 4.13. Effects of different pathway inhibitors on VEGF secretion in the MV4;11 cell line. VEGF levels in the CM were assessed by ELISA. The amount of VEGF secretion was normalised to the amount of live cells at the time of harvest, as measured by flow cytometry. Therefore, the amount of VEGF given is given as pg/106 live cells. The VEGF secretion results are expressed as the mean ± SE of at least three separate experiments.

The effects of signalling pathway inhibitors on the activation of the downstream signalling mediators (STAT5, AKT and ERK1/2) were examined by western blot (Figure 4.14). No changes to the phosphorylation status of either STAT5 or AKT were observed with all of the pathway inhibitors. However, it was shown that U0126 caused a decrease (45%) in ERK1/2 phosphorylation. This however, did not translate to a dramatic decrease in VEGF secretion (Figure 4.13). The effects observed with SU11657 were the most potent compared to other inhibitors assessed and all the parameters tested, on the MV4;11 cell line.

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 142

11 ;

MV4 PD98054 U0126 LY294002 Wortmannin SU11657

Phospho-STAT5

Total-STAT5

Phospho-AKT

Total-AKT

Phospho-ERK1/2

Total-ERK1/2

Figure 4.14. Effects of different pathway inhibitors on FLT-3 signalling in MV4;11 cells. Whole cell lysates were blotted for anti-phospho-STAT5, -AKT and -ERK1/2. Then they were stripped and blotted for total STAT5, AKT and ERK1/2. The panel shows the effects of signalling pathway inhibitors (PD98054, U0126, LY294002 and Wortmannin) at 1 )M, compared to untreated and SU11657 treated MV4;11 cells. The blots are a representative of at least three biological replicates.

Effects in ALL-3 xenograft cells The effects of the signalling pathway inhibitors also showed minimal reduction to the secretion of VEGF when compared to those observed with the addition of SU11657 (Figure 4.15). Indeed, the only statistically significant reduction in VEGF secretion occurred with SU11657 at both 100 nM (which showed a 59% reduction in VEGF) and 1 )M (80%) (P = 0.032 and P = 0.015, respectively). Although PD98059, U0126 and MEKi reduced VEGF levels by 12%, 25% and 42%, respectively, at equivalent concentrations to SU11657 (1 )M), the decreases proved not to be significant (at P = 0.05).

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 143 9000 FL- 8000 FL+ 7000 6000 cells) 6 5000 4000 3000 VEGF(pg/10 2000 1000 0 3 7 7 9 6 i 2 n LL- 65 65 05 12 EK 00 nni A U11 U11 D98 U0 μM M 294 ma S S P μM 1 LY ort nM μM μM 1 μM μM W 100 1 1 1 1

Figure 4.15. Effects of different pathway inhibitors on VEGF secretion by ALL-3 xenograft cells. VEGF levels in the CM were assessed by ELISA. The amount of VEGF secretion was normalised to the amount of live cells at the time of harvest, as measured by flow cytometry. Therefore, the amount of VEGF given is given as pg/106 live cells. The VEGF secretion results are expressed as the mean ± SE of at least three separate experiments.

The above mentioned inhibitors were also engaged to clarify the downstream signalling of FLT-3. The resulting effects on downstream signalling are shown in Figure 4.16. As per the previous results, no changes in AKT phosphorylation were observed, regardless of the treatment. This was also the case for STAT5 (data not shown). ERK1/2 phosphorylation was decreased with the addition of U0126 (45%) and to an even greater extent with the MEK inhibitor (61%), both at 1 )M. The decrease in VEGF secretion from the combined observations of the signalling inhibitors significantly correlated with the decrease in FL-induced ERK1/2 phosphorylation (P < 0.0001, Figure 4.17).

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 144

ALL-3 only FL ng/ml 20 1 μM SU11657 1 μM PD98054 1 μM U0126 1 μM MEKi μM LY294009 1 1 μM Wortmannin

Phospho-AKT

Total-AKT

Phospho-ERK1/2

Total-ERK1/2 Figure 4.16. Effects of different inhibitors on AKT and ERK1/2 in ALL-3 xenograft cells. Whole cell lysates were blotted for anti-phospho-AKT and -ERK1/2. They were then stripped and blotted for total AKT and ERK1/2.

2 125 R = 0.89 P = 0.0002

100

75

50

25

0 0 10 20 30 40 50 60 70 80 90 % decrease in ERK1/2 phosphorylation ERK1/2 in decrease % % decrease in VEGF

Figure 4.17. Correlation between the decrease in VEGF versus the decrease in ERK1/2 phosphorylation. Correlation between the decrease in VEGF secretion by ALL-3 xenograft cells versus the decrease in ERK1/2 phosphorylation by the same inhibitors. The decrease in VEGF was calculated compared to the FL-induced VEGF secretion by the ALL-3 xenograft cells, as was the decrease in FLT-3 phosphorylation. The Spearmann correlation was used for the statistical analysis, and results are described in text. The blots are a representative of at least three biological replicates.

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 145 4.2.5 Effects of Anti-FLT-3 Antibodies FLT-3 specific blocking antibodies (EB10 and D43) were utilised to block FL-induced phosphorylation of the receptor. In Chapter 3, it was shown that VEGF secretion can be induced by the addition of FL in ALL-3 xenograft cells. As such, these cells were used to examine the effect of these antibodies on FLT-3 phosphorylation, along with their impacts on downstream targets of FLT-3 signalling. The MV4;11 cell line, with its constitutively active receptor, could not be included in this set of experiments.

The inhibitory effects of both EB10 and D43 blocking antibodies were compared to those observed by SU11657, with representative blots shown in Figure 4.18. Both antibodies showed a substantial inhibition of FL-induced phosphorylation of FLT-3 in ALL-3 xenografts cells. EB10 inhibited FLT-3 activation by 45% at 10 )g/ml, and this increased to 68% with the addition of 50 )g/ml of the antibody. The second blocking antibody assessed (D43) resulted in the inhibition of FLT-3 activation by 53% with 10 )g/ml and 55% with 50 )g/ml.

ALL-3 only only ALL-3 FL ng/ml 20 SU11657 nM 100 10 μg/ml EB10 50 μg/ml EB10 10 μg/ml D43 50 μg/ml D43 10 μg/ml Control 50 μg/ml Control

Phospho-FLT-3

Total-FLT-3

Figure 4.18. Effects of blocking antibodies on FLT-3 phosphorylation in ALL-3 xenograft cells. In order to examine the effects of both blocking antibodies, as with the inhibitor experiments, ALL xenograft cells were incubated for 2 hrs with the appropriate antibody, and subsequently 20 ng/ml FL was added for an additional 15 min. Cells were then harvested and lysed and FLT-3 was immunoprecipitated and assessed based on tyrosine phosphorylation. The blots are a representative of at least three biological replicates.

Whole cell lysates were also analysed for STAT5, AKT and ERK1/2 phosphorylation, and the representative blots of the effects of the blocking antibodies (10 and 50 μg/ml) on ALL-3 xenograft cells is shown in Figure 4.19. As with previous results, no detectable changes were observed in either AKT or STAT5 upon the addition of the antibodies. With respect to ERK1/2 phosphorylation, the D43 antibody had a marginally Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 146 stronger effect than EB10 at both 10 and 50 )g/ml, with an 80 and 88% reduction in FL-induced phosphorylation, respectively. This compared to EB10, which displayed a minor decrease of 71 and 83% at the two concentrations. This level of reduction is equivalent to that seen by the action of SU11657 at 100 nM (84%). This finding is consistent with their induced effects observed on VEGF secretion (Section 3.2.5), as well as that on FLT-3 phosphorylation. Control antibodies showed no effect on the activation of ERK1/2 by FL (Figure 4.19).

ALL-3 only only ALL-3 20 ng/ml FL 100SU11657 nM μg/ml 10 EB10 μg/ml 50 EB10 μg/ml 10 D43 μg/ml 50 D43 μg/ml 10 Control μg/ml 50 Control Phospho-STAT5

Total-STAT5

Phospho-AKT

Total-AKT

Phospho-ERK1/2

Total-ERK1/2

Figure 4.19. Effects of blocking antibodies on the FLT-3 signalling pathway. The western immunoblot was performed as previously described. The blots are a representative of at least two biological replicates.

4.3 Discussion

It was demonstrated in Chapter 3 that the addition of FL to ALL xenograft cells with high FLT-3 expression can increase the secretion of VEGF by these cells and that the subsequent addition of a FLT-3 inhibitor attenuated this secretion. In order to examine this relationship in more detail, it was determined that ALL-2 and ALL-3 xenograft cells (along with the other VEGF secreting ALL cells) do not harbour the two most common FLT-3 mutations (ITDs and point mutation at Asp835) usually found in acute

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 147 leukaemias. The results would indicate that the ALL xenograft cells tested express a wild-type receptor which is activated by the addition of its ligand, FL. Such an observation was not unexpected, as mutations in childhood ALL cells are not as common as in either infant ALL (Armstrong et al. 2002), or AML cells for that matter (Yokota et al. 1997). These findings indicate that our ALL xenograft cells are similar to primary paediatric ALL cells in this regard.

Owing to their induction of VEGF secretion following FLT-3 activation, ALL-3 xenograft cells were used as a model to further elucidate this relationship in childhood leukaemia. It should be remembered that ALL-3 was not isolated from an infant patient. However, it does contain a truncation at 11q23 at the MLL gene and has high levels of FLT-3 expression, which are typical features of infant ALL (Armstrong et al. 2002).

The expression of FLT-3 in leukaemia and the activation of its signalling pathway have been shown to be important in the progression of leukaemia (Mizuki et al. 2000; Tse et al. 2000; Kelly et al. 2002a; Kelly et al. 2002b; Chung et al. 2005). Although, FLT-3 is expressed in leukaemias of both lymphoid and myeloid lineage (Birg et al. 1992), the research into FLT-3 has previously been undertaken for its association with AML with a clear focus on its mutated receptor. The primary reason for this interest is that the expression of FLT-3 with mutations in AML is associated with a poor prognosis for both adult (Rombouts et al. 2000) and paediatric patients (Meshinchi et al. 2001). The presence of FLT-3 mutations in ALL and its prognostic significance, like in AML, is still under investigation. Regardless of this prospect as a prognostic indicator, many compounds which target FLT-3 activation have been synthesised (Levis et al. 2001; Fiedler et al. 2005; De Angelo et al. 2006), in the hope that they may have similar therapeutic potential to the inhibitors of BCR/ABL in CML (Druker et al. 2001a; Druker et al. 2001b; Savage et al. 2002). One such inhibitor is SU11657 which has been used to aid in the analysis of the FLT-3 pathway in our model system.

Several RTK inhibitors were utilised to elucidate the FLT-3 signalling pathway and its induction of VEGF. Their effects were compared to SU11657, which was designed to target FLT-3, and showed strong inhibitory effects against VEGF secretion. A study by Cain et al. (2004) showed a number of potential ‘off-target’ effects resulting from RTK inhibition. SU11657 was able to inhibit the kinase activity of the fusion between TEL

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 148 and the PDGF!R in 32D transfected cells (Cain et al. 2004). The alternative RTK inhibitors were not as effective as SU11657 in reducing VEGF secretion, and the minor decreases that were observed could be accounted for by the concurrent decrease in FLT-3 phosphorylation itself. It follows that the activation of FLT-3, rather than VEGFRs, contributes to the secretion of VEGF in ALL cells, since VEGFR inhibitors did not exert significant inhibitory effects. The deactivation of ERK1/2 by U0126 and a MEK inhibitor showed a corresponding decrease in VEGF secretion. This would indicate that the FLT-3 signal indeed proceeds through the MAPK pathway.

Wild-type FLT-3 is known to transduce its signalling cascade principally via ERK1/2 (Yokota et al. 1997), as well as through the PI3K pathway, leading to activation of AKT (protein kinase B). However, there have been reports of AKT being activated only by FLT-3 with ITDs in AML cells (Brandts et al. 2005). The results show that after activation of the FLT-3 receptor with its ligand, the MAPK signalling pathway was stimulated when compared to the other possibility (AKT). This indirectly demonstrates that the pathway by which FLT-3 induces VEGF secretion is more likely to occur through ERK1/2 phosphorylation rather than AKT, owing to the observed concomitant decrease in ERK1/2 deactivation with SU11657. Such an observation would indicate that AKT signalling is not primarily involved in FLT-3 induction of VEGF, and is confirmed with the lack of alteration detected in either ALL xenograft cells or the MV4;11 cell line. Mutant FLT-3 with ITDs, akin to the one present in the MV4;11 cell line, initiate the activation of Ras/MAPK and AKT, in a similar manner to the wild-type receptor (Stirewalt et al. 2003). However, it has been shown that STAT5 plays a role in FLT-3-ITD signalling (Mizuki et al. 2000; Murata et al. 2003). MV4;11 cells did appear to have phosphorylated STAT5, with the subsequent addition of 100 nM SU11657, reducing levels to below detection. In contrast, the ALL xenograft cells did not appear to have any detectable levels of STAT5 phosphorylation. This observation is consistent with wild-type receptor signalling, with George et al. (2004) demonstrating that STAT5 is activated by FLT-3 with an ITD and not by wild-type FLT-3 and its ligand. Thus, when considered in combination with our previous results, it would appear that STAT5 does not play a direct role in the signalling leading to VEGF secretion. Despite these findings, it may still play a role in the signalling of FLT-3 with ITDs as has already been reported (Tse et al. 2000).

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 149 To further study the link between FLT-3 signalling in the secretion of VEGF by ALL cells, humanised blocking antibodies were used to specifically inhibit activation of the receptor. This approach was taken as Piloto et al. (2005; 2006) demonstrated that FLT-3 antibodies were able to decrease the phosphorylation of FLT-3 and its downstream effectors in the mouse cell line Ba/F, transfected with human FLT-3, and primary AML samples with wild-type FLT-3. The methodological direction for this thesis is vindicated with our observation in ALL-3 xenograft cells having a wild-type receptor. Piloto and colleagues (2005) also revealed that the antibodies were not as effective at inhibiting the phosphorylation of FLT-3 with an ITD. The use of antibodies for targeting receptor activation is a growing area in cancer research, as their activity is more specific compared to chemical compounds. An additional advantage of using blocking antibodies is that the prolonged use of alternative chemical inhibitors (such as SU11657) can select for resistant cells. One such example is the observation in CML patients, who are beginning to show resistance to the BCR/ABL inhibitor STI-571 (Gorre et al. 2001). These observations have also been reported by Piloto and colleagues (2007), within the in vitro treatment of AML cells using FLT-3 inhibitors. Cells were able to overcome the inhibitory effect of RTK inhibitors by activating parallel signalling pathways (Piloto et al. 2007).

Another inherent benefit of the use of antibodies is that they have the potential to recruit the immune system to kill leukaemia cells by antibody-dependent cell-mediated cytotoxicity in vivo (Piloto et al. 2005). As the research in this thesis focuses on the in vitro effects of the antibodies, a possible future direction stemming from this work may be the use of these antibodies in NOD/SCID mice engrafted with ALL cells expressing high levels of FLT-3. Nonetheless, our results show, although not to the same degree as that observed with SU11657, that the ImClone FLT-3 antibodies reduce FL-induced FLT-3 and ERK1/2 activation, and subsequently VEGF secretion.

The decrease in VEGF secretion by MV4;11 cells has been previously demonstrated with SU11248 (O'Farrell et al. 2003a), an analogous compound to SU11657, as discussed in Chapter 3. Our results regarding the decrease in VEGF in MV4;11 cells are consistent with this particular study. However, our study additionally demonstrates that SU11657 also decreases FLT-3 phosphorylation, as well as its downstream targets ERK1/2 and STAT5 in MV4;11 cells. Moreover, our results were also undertaken

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 150 within a different leukaemia subset (ALL). This research clearly demonstrates that VEGF secretion can be decreased by manipulating the FLT-3 receptor; by pharmacological deactivation (via SU11657), or alternatively through the use of FLT-3 blocking antibodies (EB10 and D43). This signal also appears to involve the MAPK pathway through its activation of ERK1/2 and not via the PI3K / AKT pathway. This contrasts with the findings of Zhang et al. (2000a) who showed the dominance of the PI3K pathway in FLT-3 signalling. In addition to the work of O’Farrell et al. (2003a), my research also shows that VEGF secretion can be enhanced in ALL cells with the exogenous addition of FL.

Considering the clinical relevance of both of these molecules (FLT-3 and VEGF) in leukaemia and their association with adverse patient outcomes, this novel finding may have potential clinical benefits for the inclusion of FLT-3 inhibitors in the treatment against ALL. It also gives an insight into the downstream target of this receptor in leukaemia cells and novel relationship between two highly targeted and relevant molecules. Furthermore, there may be substantial benefit in combining this molecular target with current therapies, which would potentially reduce the toxicity associated with existing treatment regimes, whilst not impinging on their effectiveness in providing a cure.

Single therapy treatment directed against FLT-3 may not necessarily be effective against all subtypes of ALL. It was shown that SU11248 produced an ‘objective response’ in one of the eight xenografts tested (Maris et al. 2008). It should be recognised that the panel of ALL xenografts used by Maris and colleagues (2008), was the same as that used in this thesis, and as such, expressed varying levels of FLT3. Such a finding would suggest that these types of inhibitors are probably the most effective against leukaemias with high expression of FLT-3. In the case of ALL, it would be limited to the two subsets with such a characteristic: those with MLL rearrangements and those with high hyperdiploidy (> 50 chromosomes). As such, the results presented here add support to suggestions that FLT-3 inhibitors should be incorporated into conventional therapy for specific ALL subtypes (Brown et al. 2005).

Chapter Four: Activation of the FLT-3 Signalling Pathway & Induction of VEGF 151

 

CHAPTER FIVE: Gene Determination of Select ALL Xenografts Using Microarray

5.1 Introduction

Microarray is a powerful tool in generating vast amounts of data, as it allows for the simultaneous measurement of expression of many genes. In doing so, it allows for the development of a comprehensive overview of the interplay between several processes whilst still maintaining a reference to a specific molecule or pathway (Staunton et al. 2001; Gunther et al. 2003). This technology has been an important advance in the field of molecular biology, and has in a way, revolutionised the study of gene expression by allowing for the analysis of genome-wide gene expression from a single sample.

The advance in microarray technology has led to its application across a diverse range of study areas. One of the primary ways in which this technology has been used in recent years has been to differentiate the gene expression within a disease. An example of this in paediatric acute leukaemia was the identification of a unique leukaemia subgroup using microarray, designated MLL, by Armstrong et al. (Rosenwald et al. 2002), owing to its distinct gene expression profile from both ALL and AML. It has been also used to test drug efficacy (Van't Veer et al. 2002; Van De Vijver et al. 2002), as well as to predict the outcome of patients in a variety of diseases, including; B-cell lymphoma (Yeoh et al. 2002), breast cancer (Wei et al. 2004), paediatric ALL (Passioura et al. 2005), and neuroblastoma (Tusher et al. 2001; Pan 2002; Troyanskaya et al. 2002).

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 153 Perhaps of more relevance to this thesis, has been the use of microarray techniques for in vitro comparisons of gene expression profiles in cells due to biological manipulation or environmental changes. This holistic approach has the potential to elucidate unidentified relationships between genes and pathways, which would not be possible in a ‘one-gene, one-expression’ approach. An example of this is the induced expression of an oncogene (N-ras), which was shown to activate a tumour suppression pathway in AML cells. In this case, microarray was used to identify several genes which were involved in this growth suppression, leading to the determination of a novel pathway, independent to that previously described (Al-Shahrour et al. 2004; Beissbarth et al. 2006; Chute et al. 2006).

Despite its many benefits, microarray is not without its limitations. One of the most commonly cited problems with this technique is the analysis of the vast amounts of data generated by this experimental method. Indeed, this is of utmost importance, as a single microarray slide can have over twenty thousand spots per slide which represent several thousands of genes. This has meant that a significant amount of work has, and is continuously being devoted to the computational filtering and statistical analysis of such data. The development of microarray analysis has resulted in a burgeoning disciplinary cross-over. Numerous techniques have been developed to assist in the identification of differentially expressed genes due to specifically designed conditions [e.g. (Waring et al. 2001; Kendziorski et al. 2003; Peng et al. 2003; Kendziorski et al. 2005)]. Several methods were assessed for their potential use in analysing the microarray data, including using; a t-test, the significance analysis of microarray (SAM) analysis, as well as the program GeneSpring.

The gene ontologies of the microarray samples were also looked at as part of this chapter. Gene ontology classifications provide a controlled vocabulary which categorises and describes the gene attributes in an organism, and can be divided into three distinct areas of cell biology; biological process, molecular function and cellular component. This allows for an exploration of common functional attributes of selected samples, and as such, ontologies have been frequently used as an efficient method of garnering functional profiling information on such co-expressed genes (Tusher et al. 2001).

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 154 All of the microarray experiments were performed at the Oncogenomics section of the Pediatric Oncology Branch at the National Cancer Institute at the National Institutes of Heath in Bethesda, Maryland, USA. Owing to time restrictions, only a limited number of samples could be analysed, which in itself, is a common problem in the field of microarray experimentation. This restriction also occurs because of the high cost of analysis (and time) involved, along with the comparatively large volume of sample required for the microarray procedure. A common way of tackling these issues (as undertaken in this study) is to pool samples, which effectively, reduces the volume of microarrays required. There are two possibilities with regards to the pooling of samples; the pooling of biological replicates, or the pooling of distinct biological samples, as depicted in Figure 5.1.

Type A Type B

ALL-2 ALL-2 ALL-2 ALL-2 ALL-2 ALL-2

ALL-3 ALL-3 ALL-3 ALL-3 ALL-3 ALL-3 ALL-7 ALL-7 ALL-7 ALL-7 ALL-7 ALL-7

ALL-17 ALL-17 ALL-17 ALL-17 ALL-17 ALL-17

ALL-19 ALL-19 ALL-19 ALL-19 ALL-19 ALL-19

Figure 5.1. Two possible pooling strategies for microarray samples.

In both methods described above, the biological samples (Type A) were pooled rather than different leukaemia samples (Type B). Even though this resulted in the use of a greater number of chips, the primary motivation was that it enabled the subset-specific examination of gene profiles. There is a certain amount of division regarding the size of sub-pools as well as the effects of pooling versus complete sample analysis. Indeed, some previous research discourages pooling owing to the decrease in variance, which primarily affects low-expressing genes. However, the use of pooling is widely used and has been proven to be an established technique for the analysis of biological samples (Jiang et al. 1998; Khatri et al. 2005; Veiga et al. 2006).

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 155 As part of this study, two separate microarray studies were performed. The first method was a hypothesis-generating approach to define the genes which were involved in the interaction between leukaemia cells and BM stroma cells. As the leukaemia cells were human and the stromal cells were derived from mice, this experiment proved an ideal way to look at genes which were specific to leukaemia (on human arrays), whilst also allowed the examination of genes which were differentially expressed in the stromal cells due to leukaemia (on mouse arrays). The arrays are species specific, therefore there should be limited cross-reactivity. Five different leukaemias xenograft cells were used (ALL-2, -3, -7, -17 and -19) each having a distinct patient profile (refer back to Table 3.1). Despite the tested group being composed of different subtypes, with the leukaemias having different characteristics (e.g. aggressiveness, chemosensitivity and phenotype), they all represent ALL, a heterogeneous disease. This enabled the examination of genes common to ALL, rather than subtypes of the disease.

The second method was a time-course experiment analysing the patterns in gene expression due to the effects of FL on three different xenografts displaying varying expressions of FLT-3. The previous two chapters have demonstrated that the addition of FL induces the secretion of VEGF. However, the experiments in this chapter expand on the previous research by looking at the global gene expression due to the effects of FL on leukaemia cells. Therefore; ALL-3 was chosen because of its high levels of FLT-3, ALL-17 represents a xenograft with intermediate expression, and ALL-19 has relatively low expression compared with the overall panel (refer back to Table 3.1). By looking at differences in the gene expression under varying conditions (presence/absence of FL), the time-series experiments performed in this chapter reflect cyclic biological processes.

There is no consensus in the literature about the way to analyse microarray time course data. Initially, the results were examined as distinct time points for each xenograft, with an arbitrary cut-off set at 2-fold expression change in order to compare FL-treated versus -non treated cells. This enabled a comparison of the overall gene expression between the different xenografts. Additionally, to take full advantage of the sequential information generated in time course experiments, rather than looking at the genes at individual time points, the data can be analysed in a different way, namely, ‘slope’ and ‘signed area’. The ‘slope’ is simply taken by selecting for an overall increase or decrease in expression over time and using the least squares regression analysis to

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 156 determine its value. This method is useful for finding genes with a consistent increase or decrease over time. For a ‘signed area’, the area under the time course curve is calculated, counting positive area above the line and negative below the line. The signed area is useful for finding genes that rise and then level off or return to their baseline expression levels.

5.2 Results

5.2.1 Effects of BM Stromal Cells on Gene Expression in ALL Xenograft cells The human array was represented on two microarray slides, together containing, 42,569 spots which represented 25,933 unique genes (13,606 known genes and 12,327 unknown expressed sequence tags ESTs). Gene expression ratios between the control (a pool of seven different cell line RNAs) and our test samples were normalised using a pin-based method modified from DeRisi (1996). The gene names were mapped to their corresponding plate positions for all 42,569 entries using details from the raw microarray results files. Figure 5.2 is an unsupervised cluster of all 42,569 spots from both human slides. The clustering analysis of the normalised results ascribed no scoring or filtering of the genes, thus giving a global view of the expression profiles of the entire gene set. As can be seen in Figure 5.2 the samples become clustered according to their specific xenograft samples.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 157

Figure 5.2. An unsupervised Pearson centred cluster. All 42,569 images combined from the two slides representing the human gene set. Red indicates high expression, green indicates a low expression, and black, an intermediate expression, when compared to the control mRNA. The dendograms indicate the pairing of genes (and samples in the panel) and the branch length is proportional to the distances between the clusters.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 158 In order to find the genes of significance which discriminate between ALL xenograft cells cultured with and without MS5 stromal cells, three scoring and filtering methods were used: • t test • Significance Analysis of Microarrays (SAM) • Genespring’s filtering by fold changes

5.2.1.1 t test A paired t test (i.e. with and without MS5 cells) was performed using MS-excel to obtain P-values. These were then sorted and the least significant (i.e. P > 0.05) genes were filtered out. Figure 5.2 shows the most significant genes as identified by a t test between ALL xenograft cells cultured both with and without MS5s, compared to a pool of mRNA from different cells lines (routinely used by the Oncogenomics group). The changes in gene expression between the two culturing conditions (± MS5s) were then compared across the ALL xenograft samples. Table 5.1 combines the t test values (derived in Figure 5.2), with their associated fold changes, as a result of culturing treatments, for the 20 genes. The fold change was calculated in two ways in order to identify possible outlying samples. The first method was calculated by first averaging all samples of ALL xenograft cells cultured with MS5 cells and those ALL cells without MS5 cells and then taking the MS5/Non-MS5 ratio. The second method was calculated by taking the ratio of ALL xenograft cells with and without MS5s for each corresponding sample and subsequently averaging them. As can be seen in Table 5.1 there are no major differences between the two calculations. The information about the 20 genes (i.e. summaries and Gene ontology) was obtained from the ‘Gene Database’ at the National Center for Biotechnology Information (NCBI), the full details of which can be found in Appendix A.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 159

                                  !  Figure 5.3. Top 20 discriminating genes as identified by the t test. The first 5 columns show the xenograft cells which have been cultured with MS5 stromal cells. The following 5 columns are xenograft cells cultured without stromal cells.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 160 Table 5.1. Summary of the 20 discriminating genes as identified by the t test. All values are given in terms of up-regulation in MS5 supported samples. Thus values of < 1 indicate a down- regulation in samples with MS5 support. Fold up Gene Symbol Name t test value regulation */^ telomeric repeat binding factor 2 TERF2 0.00026 1.16 / 1.16

hypoxia-inducible factor 1 HIF1A 0.00031 1.23 / 1.23 Procollagen-proline, 2-oxoglutarate 4- dioxygenase (proline 4-hydroxylase),  P4HA1 0.00034 2.41 / 2.43 polypeptide I Nedd4 family interacting protein 2 NDFIP2 0.00037 2.20 / 2.36

SCD6 homolog A (S. cerevisiae) LSM14A 0.00041 0.90 / 0.90 signal peptidase complex subunit 3 homolog (S. SPCS3 0.00046 1.41 / 1.42 cerevisiae) BCL2/adenovirus E1B 19kD-interacting protein 3- BNIP3L 0.00067 1.56 / 1.57 like ClpP caseinolytic protease, ATP-dependent, CLPP 0.00074 0.89 / 0.89 proteolytic subunit (E. coli) homolog ESTs HsKG11C5 0.00076 1.42 / 1.44

ESTs R43250a6 0.00160 0.64 / 0.64

ESTs HsKG77E6 0.00182 1.31 / 1.32 open reading frame 50, also know C7orf50 0.00086 1.62 / 1.66 as hypothetical protein MGC11257 mitogen-activated protein kinase kinase 1 MAP2K1 0.00077 1.43 / 1.46 procollagen-proline, 2-oxoglutarate 4-dioxygenase P4HA2 0.00081 1.99 / 2.14 (proline 4-hydroxylase),  polypeptide II heparan--glucosaminide N-acetyltransferase HGSNAT 0.00123 1.45 / 1.49

spectrin repeat containing, nuclear envelope 2 SYNE2 0.00129 1.46 / 1.46

phosphoglycerate kinase 1 PGK1 0.00129 1.56 / 1.60

fibulin 2 FBLN2 0.00129 1.77 / 1.79

RAB17, member RAS oncogene family RAB17 0.00147 1.77 / 1.85 integrin,  L (antigen CD11A (p180), lymphocyte ITGAL 0.00153 0.59 / 0.61 function-associated antigen 1;  polypeptide) " #$%#&'( )* + & ,' -+.- ## /0#'    1'.-  & $'## $%#&% '( 2+&3  $'## .(  2+&3%&  %00 &'( &3'. &4+.- &3' 5..6  &+7 8 #$%#&'( )* &4+.- &3'  2+&3 5 2+&3%&  &+   '$3 $ '0.(+.-/0#'.(&3'.,' -+.-&3'/7

A Pearson centred cluster analysis was again performed this time on only the discriminating genes listed in Table 5.1. The clustering dendogram shows that all xenograft cells cultured with an MS5 support and those without an MS5 support are grouped together, independently of each other (Figure 5.4). This validates the t test discrimination used, as the genes that were selected by this method clearly proved to

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 161 differentiate between the two groups. While the five ALL xenograft cells may have different characteristics, as was described in Table 3.1, the aim was to identify genes that were common across all ALL xenograft cells.

NDFIP2 MAP2K1 BNIP3L ESTs HsKG11C5 FBLN2 SYNE2 PGK1 P4HA2 HIF1A TERF2 SPCS3 C7orf50 RAB17 P4HA1 HGSNAT ESTs HsKG77E6 LSM14A ITGAL CLPP ESTs R43250a6

Figure 5.4. Pearson centred cluster of the 20 discriminating genes as identified by the t test. Once again, red indicates high expression, green indicates a low expression, and black, an intermediate expression, when compared to the control mRNA. The dendograms indicate the pairing of genes (and samples in panel) and the branch length is proportional to the distances between the clusters.

These 20 genes were then categorised according to their ontology, comprising of; molecular function, biological process and cellular component. The analysed genes were primarily grouped (50%) into the ‘protein binding’ category for molecular function (Figure 5.5A). ‘Metabolism’ was shown to be the major biological process in

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 162 which the genes were grouped (Figure 5.5B), which comprised of primary and cellular processes (66.67% each) and macromolecule processes (58.3%). Differential expression was also observed in regards to the ‘cell communication’ (42% of the genes) category of biological processes in the ALL xenografts cells (Figure 5.5B). This subset was dominated by the up-regulated genes; HIF1, MAP2K1, NDFIP2 and RAB17. The two major ‘cell components’ were the cell part (92.31%) and membrane-bound organelle (61.54%) (Figure 5.5C). It should be noted that the analysis process does not classify the genes into exclusive ontology groupings, as some genes can have several functions or be involved in many biological processes.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 163 A protein binding ion binding nucleotide binding transferase activity hydrolase activity nucleic acid binding oxidoreductase activity vitamin binding

Molecular FunctionMolecular extracellular matrix structural constituent receptor activity RNA polymerase II transcription factor activity

0 10 20 30 40 50 % of Genes in Ontology Group

B cellular metabolic process primary metabolic process macromolecule metabolic process cell communication regulation of biological process localization of cell nitrogen compound metabolic process cellular developmental process death defense response response to external stimulus response to stress anatomical structure development behavior catabolic process cell activation Biological Process Biological cell adhesion cell cycle cell proliferation cellular component organization and biogenesis establishment of localization multicellular organismal development protein localization regulation of biological quality response to biotic stimulus response to chemical stimulus 0 10 20 30 40 50 60 70 % of Genes in Ontology Group

C cell part membrane-bound organelle organelle part non-membrane-bound organelle extracellular region part

Cellular Component receptor complex

0 25 50 75 100 % of Genes in Ontology Group

Figure 5.5 A, B & C. The top 20 differentially expressed genes categorised into ontology groups. This comprises molecular function (A), biological process (B) and cellular component (C). The clustering was performed using the FatiGO software.

The remainder of the significantly expressed genes (based on the t test) consisted of 388 unique entries, of which, 258 were known genes and 130 were either ESTs or hypothetical proteins. At this point the genes still differentiated into their culturing conditions (± MS5s). In order to determine the maximum number of genes which still

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 164 resulted in the independent clustering of the two culturing conditions, a lower significance threshold was applied to let broader number of genes to filter though. This was undertaken by sequentially increasing the overall chip entries until the genes no longer clustered based on their culturing conditions. The point at which this occurred was between 2600 and 2650 chip spots, equating to approximately 1547 unique entries, of which 1023 are known genes. Even though the ALL xenograft cells cultured with MS5s still cluster together at this level of analysis, the ALL cells cultured without MS5s do not; with ALL-7 clustering with the MS5 treated cells (shown in Figure 5.6). This suggests that this list of genes (to 2600), although not significant to P < 0.05, still discriminates between the two culturing conditions.

Figure 5.6. Pearson centred cluster of the top 1547 genes according to t test. The clustering shows a divergence in culture-based clustering. The P-value at this point was 0.157.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 165 5.2.1.2 Significance Analysis of Microarrays The second scoring algorithm used was SAM, a free web-based program from the University of Stanford (http://www-stat.stanford.edu/~tibs/SAM/). SAM operates by examining the statistically significant changes (also using t tests) between gene expression groupings whilst factoring in a false discovery rate (FDR) (Tusher et al. 2001). The FDR is the expected proportion (as a percentage) of false positives among the declared significant results. FDR increases as amount of significant genes are allowed through. A delta value which strikes the best balance between FDR and the number of significant genes was used. This balance was at the FDR value of 66.38% for our data, giving 14 genes which are listed in Table 5.2. It can be observed that the 14 genes are a subset of the 20 genes which were obtained from the previously used t test method (Table 5.1), and as such, the clustering and gene ontologies were not repeated. Also, the top 11 ranked genes from SAM are consistent with that from the t test in terms of rank order; instilling a level of confidence when interpreting the results.

Table 5.2. The 14 most significant genes as scored by SAM. Gene Symbol Score ()

TERF2 6.104

HIF1A 6.001

P4HA1 5.916

NDFIP2 5.873

SPCS3 5.633

BNIP3L 5.346

EST HsKG11C5 5.220

MAP2K1 5.219

P4HA2 5.167

C7orf50 5.147

HGSNAT 4.862

FBLN2 4.828

PGK1 4.824

SYNE2 4.821

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 166 5.2.1.3 GeneSpring GeneSpring takes two approaches to find significant genes by simply looking at fold changes when comparing two samples. The first is the average approach, which takes the average values for each gene in ALL xenografts cells with MS5s versus those without MS5s, and the fold change is calculated from these averages for individual genes. The second approach is the individual approach, in which the fold changes between individual xenografts with MS5s and the corresponding cells without MS5s are calculated, after which an average is generated. Diagrammatic explanations of the two approaches are detailed in Appendix C.

Average Approach Fold change cut offs of 2, 3, 4, 12 and 15 were used in this method. The constructed dendograms (Appendix C) show that the samples have not clustered according their MS5-culture conditions across all fold change cut off values. Although the fold change cut off of 15 produced the most representative clustering result, the gene set was still not considered for further analysis as it did not sufficiently discriminate between the two treatments. Additionally, the gene list that was generated by this approach consisted primarily of unknown or hypothetical proteins and ESTs. All this information, including the fold changes and gene lists are shown in Appendix C.

Individual Approach In contrast to the average approach, a two-step process is undertaken for the calculation of the gene lists using the individual approach. Firstly, the genes were filtered according to a certain fold change cut off for a gene set to be obtained. This was repeated for all the five individual xenografts to yield five gene sets. Then were then cross-checked to identify genes which were common within the five gene sets, where the greatest overlap deemed the most significant. As shown in Table 5.3 analyses 3 and 5 generated the best clustering outcome in terms of treatment. The gene list generated in analysis number 5 was shown to be a subset of that of analysis number 3, and as such, this list of 28 genes was examined further. Of this 28, 18 known genes were identified, and are listed in Table 5.4 (the remainder shown in Appendix C).

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 167 Table 5.3. Data generated by the GeneSpring individual approach analysis. The table shows the set parameters, number of genes identified and clustering of the samples represented as dendograms. Analysis Genes Parameters Dendograms No. Clustered

Fold change cut off: 1.25 1 Gene common in at least 5 121 out of 5 samples

Fold change cut off: 1.50 2 Gene common in at least 4 212 samples

Fold change cut off: 1.50 3 Gene common in at least 5 28 samples

Fold change cut off: 2.00 4 Gene common in at least 4 41 samples

Fold change cut off: 2.00 5 Gene common in at least 5 4 samples

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 168 Table 5.4. List of the 18 discriminating genes as identified by the individual approach analysis. All values are given in terms of up-regulation in MS5 supported samples. Thus, values < 1 represent a down-regulation in MS5 supported samples. Gene Fold up Name Symbol regulation cytokine-like protein C17 CYTL1 0.41

butyrophilin, subfamily 3, member A3 BTN3A3 0.53

histidyl-tRNA synthetase HARS 0.27

aryl-hydrocarbon receptor repressor AHRR 0.41

synaptic vesicle glycoprotein 2C SV2C 0.46 procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4- P4HA1 2.43 hydroxylase), alpha polypeptide I Serum amyloid A1 SAA1 3.5

Plasminogen activator, urokinase receptor PLAUR 2.08

collagen, type VI, alpha 3 COL6A3 2.14

Annexin A1 ANXA1 2.08

Antigen identified by monoclonal antibodies 12E7, F21 and O13 CD99 1.77 Phosphodiesterase 4B, cAMP-specific (dunce, Drosophila - PDE4B 1.86 homolog phosphodiesterase E4) Early growth response 1 EGR1 3.3

Triosephosphate isomerase 1 TPI1 1.93

Guanylate binding protein 2, interferon-inducible GBP2 2.21

Serum amyloid A2 SAA2 3.01

CD1C antigen, c polypeptide CD1C 2.48

Arylsulfatase B ARSB 2.35

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 169 5.2.2 Effects of FL on Gene Expression in ALL Xenograft cells To summarise, FL was added to the standard culturing conditions for ALL-3, -17 and -19, with the cells being harvested after 2, 6 and 24 hrs. The experiment was repeated in triplicate and mRNA of the biological replicates was pooled and then hybridised to cDNA microarray slides.

A heat map was generated (shown in Figure 5.12) to illustrate the global gene expression profile for all three xenografts. The resulting hierarchical clustering analysis (also depicted in Figure 5.12) shows the decreasing commonality between the samples treatment, their time points, and finally the actual xenograft. This consistency is evident in the clustering between the 6 and 24 hr time points for all treatments and xenografts (as compared with the 2 hr samples).

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 170

Figure 5.12. Global gene expression heat map and hierarchical clustering. The figure shows the analysis for all three ALL xenograft cells both with and without FL. Hierarchical clustering significance was determined with a variance of > 0.25. The hierarchical structure of the dendogram did not change if all genes were included, and not just those > 0.25.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 171 5.2.2.1 Static Analysis The first step in the analysis of this data was to identify the genes, at each time point and for each ALL xenograft, which displayed a greater than two-fold change in expression (increase or reduction) due to treatment with FL versus non-treatment. Full lists of all differentially expressed genes at the differing time points are presented in Appendix F. These genes were then grouped based on their gene ontologies. The biological processes, molecular functions and cellular components of the genes differentially expressed in ALL-3, -17 and -19 are shown in Figures 5.13, 5.14 and 5.15, respectively. As inferred in Figure 5.13A, out of the total genes up- (291) and down- (123) regulated at the 2 hr time point, the two most common biological processes in ALL-3 were involved in ‘cellular process’ (201 gene up-, and 87 down-regulated), followed by ‘metabolic process’ (112 gene up-, and 56 down-regulated). Although the total numbers of genes expressed at the 6 and 24 hr time points varied, the percentages of both the up- and downwardly regulated genes remained similar. The molecular function of ALL-3 (Figure 5.13B) shows that the genes expressed are involved in ‘binding’ across all three time points, whilst the cell component (Figure 5.13C) is dominated by ‘cell part’ and ‘organelle’. The pattern of gene ontologies was consistent across the remaining two xenografts (-17 and -19), with their biological processes primarily being ‘cellular’ and ‘metabolic’ processes, their molecular function involved in ‘binding’ and cell component being principally ‘cell part’ (shown in Figures 5.14 and 5.15).

Within the individual xenografts, the only significant differences (P > 0.05) in up- and down regulation occurred in ALL-3. This was observed in the biological processes (Figure 5.13A) with regards to both the ‘immune system process’ and ‘response to stimulus’ (at 6 and 24 hrs), as well as within the cell component (Figure 5.13C) ‘protein complex (at 6 hrs). The analysis was assessed using the fatiGO website, the two lists of genes were compared by the Fisher’s exact test. The analysis was performed using the fatiGO website (www.fatigo.org), with the two lists of genes compared by the Fisher’s exact test.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 172 A – Biological Process 2 hr 6 hr 24 hr

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            : '.'+.;.&#-* %0 C – Cellular Component 2 hr 6 hr 24 hr $'## '.,'#0' '1& $'##%# /& +1 '1& $'##%#  '-+. /'/) .'6'.$#'(#%/'.  -.'##' 0 &'+.$/0#'1 * *.0'                : '.'+.;.&#-* %0 Figure 5.13 A, B & C. Gene ontology groups of the differentially expressed genes in ALL-3. The ontology is divided up into; biological process (A), molecular function (B) and cellular component (C), all across three time points. *marks the groups which are statistically significant. Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 173

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Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 174

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Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 175 The differentially expressed genes for individual xenografts were assessed for commonality across the separate time points. These are represented by the Venn diagrams for ALL-3 (Figure 5.16), ALL-17 (Figure 5.17) and ALL-19 (Figure 5.18), with the shared genes listed alongside.

HLA-DRB4; TNK1; DLX4; GHR; MMP16; RAB35; IL17B; CD1C; XPO5; MAP6D1; ME1MS4A6A; GJA3 TMEM158; KRT23; BCL2A1; MARK2; ATP5C1; SC4MOL; 2 hr 224 RGS1; EGR2; CSF1R; ADAM8; GZMA; DUSP5; CRADD; TRAF1; NKG7; NT5E; KCNMB1; THBD; 6 h CCL2; IL1RL1; PRDX6; UBE2N; 10 54 NOLC1; NEK3; CST7; GPR65; 24 hr 3 SH2D2A; HBEGF; MRPL12; CCNA1; LRRC6; GPRC5C; 10 GZMB; CCNA1; IBSP; LPO; 160 RBM25; EGR1; APOBEC3G; HBB; PHB; 378 AQP4; DOK2; PTGS1; TRAF7; RPL28; COQ5; NT5E; CD163L1; RAB37; TMEM49; TMEM158; KCNJ2; GREM1; PLEKHF1 ALL-3 GOLGA1; ITM2C; ME1; Up-regulated NKX2-8; RAPH1; genes CCDC62; TMCO2; GPR68; EPB41; CDKL1

IKZF1; DBT; PRKD3; NBEA; FLRT3; PCDH17; SHMT2; LASS5; SYTL1; DNMT3A; IGF2; LARP6; KIT; CLPB; TSPAN12; MYL9; KLRC2; SLC24A6; PLA2R1; RASAL2; RAB6A; PPP2R5A; ABLIM1; TMEM37; HLA-C; APPBP2; ASPH; ZNF532; EPHA2; FAM83A; SLC25A12; MYH9; 2 hr 61 ANKRD38; CCK; CHRNA5;; CRABP2 EFS; T5A; TMEM30B; GPR161; LY6K; DAB2; NTN4; TANC2; NOTCH3; 7 53 C4BPB; SLC7A2; DSCR8; 24 hr 2 6 h MSC; ALAS2; PAPPA; WFDC2; SASH1; LOXL1; 18 BCL11B; PLOD2; GAL; 459 MGST1; PPP4R1 329

MS4A5; RNF138; ATP6V1C2; NUMB; IL15; FZD1 FCGR3A; SLC25A16; USH1C; EVI2A; USP53; ARRB2; POMT1; EML1; ALL-3 SIX6; EIF4A2; NTRK2; HBZ; Down-regulated PLRG1; OR2A7; genes

Figure 5.16. Venn diagrams of common genes across time points in ALL-3. Cross-sectional overlap represents the shared genes between the time points, which are listed alongside.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 176

ACE2; CAPN14; USH1C; NUSAP1; CD1C; CLEC14A; AHDC1; ZNF662; HLA-DRB4; ICOSLG; ANKRD39; PPARA; IL23A; LLGL1; NFYB; PGK1; SESN1; UBL7; MAFG; MYO18A; MAP2K7; NKG7; RASSF5; TRIM36; WWC2; ZXDB 2 hr 292

16 10 6 h 24 hr 4

12 201 86 ABCC9; CRABP2; GJB2; IKZF1; Canx; KCNJ8; MAF; MB; Mkrn2; JPH2; Smhd1 ALL-17 NEK3; SIX6; Up-regulated WISP1; ZNF3; genes

DRG1; WFDC2; TITF1; ARRB2; DOC2A; SKIV2L2; ADAMDEC1; VTI1A; PSMD14; ARHGAP5; LOC729725 RAB6A; ATP10A; DSCR8; RAB35; FAIM; CCDC90A; MOCS1; DIP2C; ZEB1; PROKR1 RPS4Y1; IL17B; PARD3; LMO3; NEBL; KIF21A; BACE1; PRLR 2 hr 44

8 21 6 h 24 hr 6 31 262

183 IL15; PLA2G2A; AGR3; CECR8; CYP19A1; DLG2; DNAH7; EDG3; ELOVL4; FMO1; IL1R1; KHDRBS3; LIFR; MAGI3; ALL-17 NES; IBSP; MS4A6A; NCALD; NOX4; EIF4A2; BEST3; Down-regulated PAGE2; PCDHA6; PHCA; RIN3; HEYL; SMYD1 genes RNASE11; RP11-151A6.2; SERTAD4; SLC35F5; SLC36A3; SMC1A; SPON1; SPRR2A; SSBP4; ST6GAL2;

Figure 5.17. Venn diagrams of common genes across time points in ALL-17. Cross-sectional overlap represents the shared genes between the time points, which are listed alongside.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 177 HLA-DRB4; PLRG1; ADAMDEC1; WNT5A; CCDC90A; PPP4R1; MUT; CYR61; ZNF649; SLC22A16; POFUT2; C18orf34; NES; RNF150; LOC388323; PKP4; LOC130576

2 hr 63

4 13 6 h 24 hr 3

14 203 180 CRABP2; EPB41L3; FLRT2; FREM1; GALC; ATRN; GJB2; HSF5; IL31RA;; MYO18A; MAGEA4; MEFV; TMEM186; SPAG7; TTC27; UTS2; ALL-19 ZIC3; Up -regulated genes

ATP1A3; Cdkn2c; Csf1; EVI2A; FLJ10781; GHR; GRIN2A; HBB; Hbs1l; Il4ra; KCNA5; LOC148413; MIA3; NIPBL; PLEKHA5; POMT1; Ptpn22; RAD9A; SAC3D1; Samhd1; SLC25A12; SOX1; WFDC2; ANPEP; TITF1; SMAD6; EIF4A2; 2 hr 129

4 23 6 h 24 hr 2

9 95 97 SEPT4; MYH9; TNIP1; Canx; TPD52L3; FAIM; RBM35B BAZ2A; YTHDC1; ALL-19 COQ5; MAP2K7 Down-regulated genes Figure 5.18. Venn diagrams of common genes across time points in ALL-19. Cross-sectional overlap represents the shared genes between the time points, which are listed alongside.

Returning to one of the original aims of the chapter, it was necessary to identify the genes whose expression is changed, due to treatment with FL in ALL-3, and how these compare to the two other xenografts which have different levels of expression of FLT-3. Also using Venn diagrams, Figures 5.19, 5.20 and 5.21 show genes that are shared between the three ALL xenograft cells across the 2, 6 and 24 hr time points. In contrast

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 178 to the figures shown immediately above, which demonstrated a homology within the individual xenografts, there were limited numbers of shared genes between the xenografts at their corresponding time points. Additionally, very few of these commonly expressed genes were consistently expressed between the 2, 6 and 24 hr time points.

AHDC1; ANKH; CCDC62; CL3L3; CD1C; CD93; CGREF1; CLK1; CLTA; EVI2A; HBB; KHDRBS3; TRPM2; FAT; NKG7; NTRK2; NUMB; ATRN; ASB1; PPP1R9B; RAPH1; RPL28; PDE4DIP; SC4MOL; TMCO2; USH1C CDKL1; FAM44B

ALL-3 259

7 21 ALL-17 3

ALL-19 8 289 65

MARK2; HLA-DRB4; PLA2G2A; PHB; VEPH1 MYO18A; TMC6; NUSAP1; SOS1; MUT; 2 hr UBL7 Up-regulated genes

LOC148413; ANKS1A; SLC25A12; DLX4; ATP1A3; LOXL1; TIA1; PPP4R1; RAB6A; GRIN2A; ALL-3 CRABP2; DSCR8; MET; BACE1; IBSP; 107

9 7 ALL-19 3 ALL-17 12 57 134 TITF1; NIPBL; MCM2; PRSS23; RAD9A; MAP3K4; 2 hr EIF4A2; PROKR1; SAC3D1; PLEKHA5; FAIM; FLRT2; WFDC2 Down-regulated PARD3; PRLR genes Figure 5.19. Venn diagrams of common genes across all xenografts at the 2 hr time point. Cross-sectional overlap represents the shared genes between the xenografts, which are listed alongside.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 179

SKIV2L2; MAFG; HLA-DRB4; NEK3; COQ5; RPL28; MYO18A; MAP2K7; CRABP2; PSG10; USP25; BCC9; C18orf34; PPP2R5A; JPH2 IL17B; CD93; MS4A6A ALL-3

208 11 7 ALL-17 ALL-19 1

9 210 212 ATRN; GJB2; PLEK LTBP1; RWDD2; SESN1; 6 hr SLC35E1; Up-regulated ZFPL1; CHES1; genes

AGR3; ANKRD57; ARID2; WFDC2; AYTL1; BEST3; BMPR1A; ANPEP; BNC2; CAMSAP1L1; CCDC110; SMAD6; CD36; CDC14B; CLDN1; EIF4A2 CNNM2; CNTN3; COBLL1; COL11A1; CREB5; CYP2U1; ; ACH1; DNAH5; DNER; DOC2A; DPPA4; EHBP1; EIF4A2; ELOVL4; ERBB4; FAM46A; FGF12; FILIP1; FLRT3; FMO1; ALL-3 FN1; FNTA; GPR126; GPR177; IL15;KLHL3; LIFR; LIPI; MAGI3; 453 MAL2; MAST4; NBEA; NBPF1; NDFIP2; ODZ3; PKIA; PKP4; 4 75 PRRG1; RAPH1; RNF128; ALL-19 1 ALL-17 ROBO1; RP5-875H10.1; SGCD; SLC36A3; SLC36A3; SLC38A4; SLC41A2; SMYD1; SPIN3; 9 212 ST6GAL2; SVEP1; SYNPO2; 105 TAF7L; TBC1D15; THAP10; TMCO2; TMEM56; TNFRSF11B; TPTE; TSPYL6; ARRB2 TYRP1; WWC2; ZNF300

6 hr Down-regulated SEPT4; MYH9; TNIP1; Canx; TPD52L3; genes BAZ2A; YTHDC1; COQ5; MAP2K7

Figure 5.20. Venn diagrams of common genes across all xenografts at the 6 hr time point. Cross-sectional overlap represents the shared genes between the xenografts, which are listed alongside.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 180

ACE2; COL11A1; GRHL1; HLA- ARID2; BAG2; CCDC101; DRB4; KCNJ8; DLX4; EHF; FAM129A; PGAP1; PROKR1; FLRT2; FREM1; GPR68; SLN; THSD3; HSF5; IBSP; KRT19; TRAPPC6B; MAL2; MEFV; MS4A7; WISP1; ZEB1 PPP4R1; PXDNL; RAB3B; RD3; ROD1; SAMD9L; SNHG6; SNX9; SPINK5L3; ZIC3 ALL-3 369

17 12 3 ALL-17 16 ALL-19 87 SAC3D1; Mkrn2; 165 WWC2

Cbfb; CCR5; CD1C; 24 hr CRABP2; GIMAP6; Up-regulated GJB2; ICOSLG; IL23A; MB; MYO18A; NBL1; genes Prnp; Ptpn9; RASSF5; TRAPPC5; Tsc22d1

BCAM; IDH3B; BEST3; CD24; IGLL1; MYH9; DOM3Z; EVI2A; NEK3; PLRG1; FCGR3A; HINT3; PPP2R5A; SIX6 IL15; NIP30; NUMB; NUSAP1; PRLR; PSMD14; ALL-3 STON2; USH1C

331

8 14 ALL-17 ALL-19 4 23 215 77 AGR3; CYR61; DNAH7; EMX2OS; ETV1; IL17B; ITGB8; KCNH8; KIF21A; LMO3; MOCS1; MS4A6A; SMC1A; RAB6A; NES; NLGN4X; PLA2G2A; ACOX2; EIF4A2 24hr PLSCR2; PUNC; SCN2A; Down-regulated SESN1; TNK1; WFDC1; WFDC2; ZNF229 genes

Figure 5.21. Venn diagrams of common genes across all xenografts at the 24 hr time point. Cross-sectional overlap represents the shared genes between the xenografts, which are listed alongside.

In order to further explore the independence of ALL-3, in contrast to its low FLT-3 expressing counterparts (ALL-17 and -19), the expression profile of ALL-3 alone, was

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 181 specifically examined. As such, the differentially expressed (greater than 2-fold change) genes in ALL-3 were filtered and then their expression in ALL-17 and -19 were analysed. Figure 5.22A to D shows that across the entire time course, regardless of their up- or downward regulation, the differentially expressed genes in ALL-3 remained unchanged in the other two xenograft cells.

A M'.'+.6 =(2. '-%#&'(+.6 B M'.'+.6 =%0 '-%#&'(+.6

86% 72% 90% 86% 91% 80%

6% 15% 5% 9% 4% 4%

8% 14% 6% 4% 5% 16%

2 hr 6 hr 24 hr 2 hr 6 hr 24 hr

C M'.'+.6 =(2. '-%#&'(+.6 DM'.'+.6 >=%0 '-%#&'(+.6

85% 85% 92% 91% 87% 89%

7% 12% 4% 3% 5% 7%

9% 3% 4% 6% 8% 5%

2 hr 6 hr 24 hr 2 hr 6 hr 24 hr

Figure 5.22 A, B, C & D. Expression profiles of ALL-17 and -19 of the differentially expressed genes identified in ALL-3. (A) Of the genes down-regulated in ALL-3; the white cones represent the genes which were unchanged in ALL-17, the red cones are genes up-regulated in ALL-17, and the green cones represent the genes down-regulated in ALL-17. (B) Of the genes that were up-regulated in ALL-3; the white cones represent the genes which were unchanged in ALL-17, the red cones are genes up-regulated in ALL-17, and the green cones represent the genes down-regulated in ALL-17. (C) Represent the genes down-regulated and (D) the genes up-regulated in ALL-3 compared to the genes in ALL-19. As in (A) and (B) the white cones are the genes that remained unchanged, red the genes up-regulated and green the genes down-regulated.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 182 5.2.3 Gene confirmation analyses Certain representative genes that were identified in our previous examinations, were selected to be further examined by real-time RT-PCR. These included; HIF1 and telomeric repeat binding factor 2 (TERF2), which have been shown to be up-regulated in certain cancers, procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase),  polypeptide I (P4HA1), a gene thought to regulate extracellular matrix proteins, and early growth response 1 (ERG1), a transcriptional regulator. HIF1 is known to induce VEGF, and although it only displayed a 1.23 up-regulation with the addition of MS5 culture, it was second highest ranked gene in both the t test and SAM gene list (refer back to Tables 5.1 and 5.2). Figure 5.24 shows the mRNA levels of HIF1 in six different xenograft cells when compared to their non-MS5 treated counterparts. While the levels do fluctuate somewhat over time there are no statistically significant changes in the levels of HIF1 mRNA in any of the xenograft cells examined.

2.5 ALL-2 ALL-3 2.0 ALL-4 ALL-7 ALL-17 1.5 ALL-19

1.0

Fold Change to no MS5 Change Fold 0.5

0.0 3 hr 6 hr 24 hr 48 hr Time in Culture

Figure 5.24. Time course for real-time RT-PCR of HIF1 expression for six ALL xenograft cells. Cells were cultured with and without MS5s and harvested at the indicated time points. EF1 was used as an internal control as described in the methods. Each fold change was calculated based on their non-MS5-treated counterpart. Results are the mean ± SE of at least three separate experiments.

The second gene analysed by real-time RT-PCR was TERF2, which came out as the most significantly expressed gene in both the t test (P = 0.00026), and from SAM. Similarly to HIF1, TERF2 had only a minor increase in mRNA levels due to culturing with an MS5 stromal support (1.6-fold increase). This was reflected in the real-time

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 183 RT-PCR time course across six ALL xenografts (Figure 5.25). Five of the six xenografts tested had an increase in the levels of mRNA, with only the ALL-7 xenograft cells showing a downward trend. While the there were observable changes in TERF2 mRNA, none were shown to be significant.

2.0 ALL-2 ALL-3 ALL-4 ALL-7 1.5 ALL-17 ALL19

1.0

0.5 Fold Change to no MS5

0.0 3 hr 6 hr 24 hr 48 hr 72 hr Time in Culture

Figure 5.25. Time course for real-time RT-PCR of TERF2 expression for six ALL xenograft cells. Refer to Figure 5.24 caption for further details.

As was the case with the previous two genes examined, P4HA1 was identified as statistically significant, in both the t test and SAM (again refer to Tables 5.1 and 5.2). In contrast to HIF1 and TERF2, it had a > 2 fold change in mRNA (2.41) across the xenografts with an MS5 stromal support. P4HA1 was also identified in the GeneSpring analysis using the individual approach (section 5.2.1.3). The results of the real time RT PCR are shown in Figure 5.26. The ALL-2 xenograft cells showed a steady increase at 6 and 24 hrs (5 and 8 fold, respectively) with a comparative decrease back to 5 fold after 48 hrs. ALL-4 xenograft cells increased 2 fold at 6 hrs and displayed a subsequent rise to 12-fold after 24-hrs. While the increase in ALL-3 xenograft cells was not as pronounced as in ALL-2 or ALL-4 xenograft cells, the levels of EGR1 increased to ~4.5 fold at 24 hrs and remained at this level until the final 72 hr time point. There were no significant changes in the ALL-7, ALL-17 and ALL-19 xenograft cells.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 184 14 ALL-2 ALL-3 12 ALL-4 ALL-7 10 ALL-17 ALL-19

8

6

4 Fold Change to no MS5 Change Fold

2

0 3 hr 6 hr 24 hr 48 hr 72 hr Time in Culture

Figure 5.26. Time course for real-time RT-PCR of P4HA1 expression for six ALL xenograft cells. Refer to Figure 5.24 caption for further details.

Whilst EGR1 was shown to be differentially expressed (3.3-fold) in ALL xenograft cells when cultured on MS5 cells by GeneSpring, it was not shown to be significant by either the t test or from the SAM. However, it was chosen for a detailed confirmation analysis owing to its up-regulation in ALL-3 (with FL) at 2 and 6 hrs (2.9- and 4.9-fold increases, respectively), whilst being absent in the ALL-17 and ALL-19 xenograft cells. As per the previous analyses, the real-time RT-PCR is shown in Figure 5.27. The results show variable expression depending on the xenograft analysed. Confirming the array data, ALL-3 xenograft cells showed a > 2-fold increased levels of EGR1 mRNA, which was subsequently reduced to only a 2 fold increase at 48 hrs. This pattern was also observed in ALL-2 and ALL-4 xenograft cells. Xenografts cells ALL-7 and -17 do not show a greater then 2 fold increase in EGR1 mRNA levels. Whilst levels of EGR1 mRNA for ALL-19 show substantial fluctuations across the experimental time course, any interpretation on this particular xenograft will most likely be erroneous owing to the large variation within the replicates.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 185 EGR1- MS5

ALL-2 MS5 9 ALL-3 MS5 8 ALL-4 MS5 7 ALL-7 MS5 6 ALL-17 MS5 ALL-19 MS5 5

4

3

2

1

Fold Change to cell cultured with MS5 culturedcell with to Change Fold 0 3 hr 6 hr 24 hr 48hr 72 hr Time in Culture

Figure 5.27. Time course for real-time RT-PCR of EGR1 expression for six ALL xenograft cells. Refer to Figure 5.24 caption for further details.

Additionally, the MS5 stromal support was also removed for some samples in Figure 5.28, to allow for the examination of the effects of FL alone. Figure 5.28 shows a time dependent increase in EGR1 mRNA in ALL-2 (5-fold) and ALL-3 (8-fold) upon the addition of FL by 24 hrs of culture. This increase is independent of the culturing of xenografts on an MS5 support.

EGR1- FL

12

10 ALL-2 ALL-3 ALL-4 8 ALL-2 MS5 ALL-3 MS5 6 ALL-4 MS5

4 FoldChange FL

2

0 1 hr 3 hr 6 hr 24 hr Time in Culture

Figure 5.28. Time course for real-time RT-PCR of EGR1 expression examining the effects of FL. The open symbols represent the ALL xenograft cells which were treated with FL, with the mRNA levels at each time point compared to the non-FL-treated counterparts. The closed symbols represent ALL xenograft cells cultured on MS5 cells treated with FL. Again these were then compared to their non-FL-treated counterparts (also on MS5s) to give a fold change. Results are the mean ± SE of at least three separate experiments. Owing to their small values, the error bars, cannot be distinguished for ALL-4.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 186 Protein levels for EGR1 were analysed for ALL-2, ALL-3 and ALL-4 by western blotting (Figure 5.29). The results show that the EGR1 protein increases with FL in both ALL-2 and ALL-3 xenograft cells. These two xenografts also display an observable enhancement of expression when cultured with an MS5 support alone. However, the maximum increase was observed with both the presence of MS5s and FL. As no changes in EGR1 mRNA were observed in ALL-4 xenograft cells with the addition of FL, protein levels were only analysed ± MS5s.

ALL-2 ALL-3 ALL-4

Jurkat Jurkat + PMA ALL-2 FL MS5 MS5 + FL ALL-3 FL MS5 MS5 + FL ALL-4 MS5 + FL

EGR-1

Actin

Figure 5.29. Western blot of EGR1 expression in ALL xenograft cells. Xenograft cells were cultured for 2 hrs in the specified conditions and the protein extracted. The positive control was the Jurkat cell line treated for 2 hrs with PMA. Actin was used as the loading control. This is a representative blot of two biological replicates. FL – at 20 ng/ml; MS5 – with MS5 co-culture; FL + MS5 – with MS5 co-culture and FL.

5.3 Discussion

5.3.1 Gene Expression Profiles of Microarray Studies The validity, reproducibility and thus the interpretation of microarray data has resulted in numerous methodologies being applied to analyse the information generated. Indeed, an inspection of the scientific literature shows that no standard technique has been universally adopted for the examination of microarray data, with relevant statistical interpretations constantly evolving (Sasaki et al. 1995). The results from this study show a distinct overlap of genes between the conventional t-test and the SAM ranking- method, which incorporates the additional FDR factor. This commonality gives a certain confidence in interpreting the filtered global gene expression profile.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 187 The gene ontology profiles show that the molecular function of the up-regulated genes in all ALL xenograft cells is primarily ‘binding’ (50%). The next level of ontology structure reveals that the largest sub-category is cation binding (28%). This were composed of the up-regulated genes; P4HA1, P4HA2, and FBLN2. As our microarray experiment deals with cell-cell interactions, genes relating to extracellular matrix proteins or other cell proteins involved in cell binding or adhesion, were not unexpected. There is some evidence suggesting a ‘cross-talk’ between leukaemia cells and the cells within the BM microenvironment (Manabe et al. 1992; Konopleva et al. 2002; Nefedova et al. 2003). One major benefit of our experimental model is the combination of two species (human and mouse) enabling the examination of their expression profiles separately. As such, we can solely look at the genes which are differentially regulated on our human-specific arrays due to the stroma.

P4HA1 and -2 are two components of prolyl 4-hydroxylase, a key enzyme essential for the three-dimensional folding of newly synthesised pro-collagen chains, an extracellular matrix (ECM) protein (Stucki et al. 2001; Veiga et al. 2006). FBLN2 is also an ECM protein which has been shown to interact with other ECM proteins such as collagen (Gluck et al. 1989; Mudry et al. 2000). Supporting evidence in the scientific literature advocates an interaction between the BM support and leukaemia cells (Aguayo et al. 2000; Padro et al. 2000; Kini et al. 2001). There have also been suggestions that leukaemia cells can create favourable conditions of their surroundings which promote their adhesion and enhance their proliferation (Konopleva et al. 2002; Wang et al. 2004; Veiga et al. 2006; Edelmann et al. 2008).

The observation of up-regulated metabolism-associated genes dominating the biological process ontology of ALL xenograft cells is somewhat intuitive, with it well established that stromal cells influence the survival and proliferation of leukaemia cells (Manabe et al. 1994; Lagneaux et al. 1998; Winter et al. 2000). Therefore, it logically follows that these leukaemia cells may display an increase in their metabolic processes compared to those cells cultured without a stromal support and which have not been exposed to the same advantageous conditions.

As mentioned previously, the results arising from the GeneSpring analysis need to be treated with a certain level of scepticism. However, it is still worth noting that the

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 188 majority of genes which were up-regulated (and identifiable) show commonality regarding their interactions with the extracellular environment. Despite being different to those genes identified by the t-test and SAM, they still agree with our general hypothesis involving leukaemic interactions with the BM microenvironment. This has been demonstrated in the scientific literature in two ways. Firstly, patients with leukaemia display an increase in vasculature in the BM when compared to normal controls (Shah et al. 2001; Glenjen et al. 2005), which suggests that the leukaemia cells secrete factors which shape their environment to enhance their survival and proliferation (Smith et al. 2005). Secondly, in-vitro studies have shown that not only does the BM increase the survival of leukaemia cells, but the cells which attach to the stomal support also influence their morphology. This can occur both through an attachment process (Lamande et al. 2006), and by culturing BM supernatant on stromal cells (Uhlar et al. 1999; Urieli-Shoval et al. 2000).

The promotion of leukaemia cell survival can be inferred by the up-regulation observation of PDE4B (1.9-fold; GeneSpring). One report suggests that elevated levels of PDE4B may make the diffuse large B-cell lymphocyte cells resistant to apoptosis, resulting in the favourable progression of this disease (Preciado-Patt et al. 1996; Hershkoviz et al. 1997; Ancsin et al. 1999a; Ancsin et al. 1999b).

In terms of cell adhesion and migration, the GeneSpring results demonstrated an up- regulation several genes. Firstly, COL6A3 (> 2.0 fold up-regulation), which is a subunit of the ECM protein collagen VI, which is thought to provide a structural support for migrating cells allowing for the anchorage to the ECM, due to both its cell adhesive properties and its ability to interact with other ECM proteins (Mitchell et al. 1993; Migita et al. 1998). Another up-regulated gene identified by GeneSpring analysis was SAA1 (3-fold), which belongs to a family of proteins often occurring at elevated levels in the blood in response to various conditions, including inflammation and neoplasia (May et al. 1998; May et al. 2000; Chapman et al. 2001). As the major functions of SAA1 relate to the adhesion to ECM components (Lou et al. 2007) and the induction of MMPs (Airoldi et al. 2007), it could be argued that its expression in our ALL xenograft cells allows their attachment to the stromal support, and potentially the modification of their microenvironment. Two other genes fitting into the profile of cell adhesion and migration are; PLAUR which is a receptor shown to interact with, and regulate, cell

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 189 adhesion molecules, such as integrins (Quackenbush 2003); and CD99, which has been shown to be involved in cell transmigration (Al-Shahrour et al. 2004), in a similar fashion to CD31, a major mediator of monocyte and neutrophil transmigration.

In the second microarray study, we aimed to assess the effects of FL on three of our xenografts. As was demonstrated by the heat map of the global gene expression (and associated hierarchical clustering), the greatest difference was between individual xenografts, with ALL-3 clustering separately to ALL-17 and -19. Although expected, this confirmed the previous findings regarding the differences in FLT-3 expression and VEGF secretion. The next clustering level is time-based, although the association between the 6 and 24 hr time periods is most likely due to a culturing artefact. The effects of culture treatment proved the most similar, suggesting that the addition of FL may not have such an important impact on the global gene expression of our ALL xenografts. This is not to say that differences in gene expressions between the samples do not exist, as this finding may not necessarily be replicated in a more specialised array; such as those used by (Smogorzewska et al. 2000; Fairall et al. 2001), who used an angiogenesis-specific array to assess the effects of IL-12 in melanoma cells. The downside of such an approach however, is that they only assess genes that are known to be associated with the specialised pathways, precluding the investigation of novel molecular targets. The homology displayed in the global gene expression heat map was also reflected in the gene ontologies for the individual xenografts across all of the different components.

Despite the similarities in the global gene expression profile, the specific genes that were differentially regulated due to the treatment with FL were markedly dissimilar between ALL-3, -17 and -19. The Venn diagrams show that only small proportions of the genes are common between the xenograft cells, highlighting the impact of receptor activation from FL in ALL-3, as shown in Chapter 4.

5.3.2 Specific genes confirmations As has been demonstrated throughout the entire chapter (along with the additional information contained in the Appendices), the vast amounts of data generated from microarray analyses can be analysed in any number of different ways. Indeed, the list of genes identified as differentially regulated, presents a multitude of potential pathways Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 190 and processes. How best to interpret this information and garner meaningful knowledge from it, is a question that most likely does not have a definitive answer.

As part of these experiments, certain genes were examined in more detail in order to gain some further insight into certain mechanisms or contributing factors involved in ALL behaviour due to external stimuli. The genes were chosen, from a combination of factors, and not based solely on their levels of significance as stated in the results section. The selection process of these further studied genes also involved factoring in a biological context, in what is often termed ‘guilt by association’ (Karlseder et al. 2002). A knowledge of the biological context of genes, although no easy task in itself, can help with the interpretation of the list of genes and is a frequently used technique in such analyses (Klapper et al. 2003). The combined statistical significance, fold change and known biological functions were combined to give a list of four genes, the results of which are now discussed.

TERF2 As identified in the results, the gene TERF2 topped the lists of the most significant genes in both the t test and SAM. This made it a particularly important gene to examine further, especially in light of its biological role in telomere binding. TERF2 is a component of the telomere nucleoprotein complex, playing a key role in the protection of telomeres by directly binding to DNA (Klapper et al. 2003). When TERF2 is over- expressed in human primary cells it can protect critically shortened telomeres and delay senescence (Wang et al. 1995). However, in certain tumours which exhibit shortened telomeres, the up-regulation of telomerase alone may not be sufficient for their continued protection (Zhong et al. 2002). Telomere binding proteins, such as TERF2, may therefore be a critical factor in stabilising telomere length, as suggested by Klapper et al. (Semenza et al. 1992; Liu et al. 1995) with regards to non-Hodgkin lymphoma.

Therefore, we hypothesised that TERF2 may be up-regulated in our xenograft cells in a potential attempt to escape senescence. Although the real-time RT-PCR results showed a certain level of fluctuation in the mRNA expression of TERF2 over time, they proved not to be significant. This finding corresponded to the fold changes observed in the microarray analysis at the 24 hr time point, which had only a 1.16-fold up-regulation. Consequently, the mixed results suggested that no definitive conclusion could be drawn

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 191 on the effect of the stromal support on TERF2 expression in our ALL xenograft cells. This is not to discount its future examination in leukaemia research, as many aspects of its biological functioning; particularly its interaction with the telomere maintenance complex has yet to be fully established.

HIF1 The real-time RT-PCR results reflected the levels of HIF1 mRNA identified by the microarray analysis, as would be expected. Despite a relatively low level of up- regulation (1.23-fold), our previous research of the scientific literature (see Chapter 1) has demonstrated its biological importance in many types of cancer. As such, it was considered that even these small increases in HIF1 mRNA may lead to an enhancement of protein levels. This was pursued by an examination of the protein levels in cell lysates after growing the cells on an MS5 support. However, the HIF1 protein was not detected with the several different antibodies that were tried (data not shown), including those supplied by BD, Novus, Cell Signaling. This finding placed the efficacy of the antibodies into question. In order to address this issue, levels of HIF1 protein were looked at under low-oxygen conditions (1%), as it has been shown that under normoxic (20%) conditions HIF1 is degraded (Gray et al. 2005), as explained in detail in Section 1.5. However, there are studies which have shown the expression of HIF1 protein under normoxic conditions in leukaemia cells (Wellmann et al. 2004). Again, no HIF1 protein was able to be detected either in cytoplasm or in the nucleus (data not shown) even in the positive controls, confirming the doubts over the value in using these antibodies.

A different approach was then taken, which looked at the functional activity of HIF1, as it has been widely reported that it can induce the expression of certain proteins (EPO, VEGF) through its binding to a HRE within their promoter regions (Kukkola et al. 2003). We have already shown that levels of VEGF in our ALL xenografts increase with MS5 culture. Therefore, it could be possible that this up-regulation was HIF1- dependent, therefore we examined HIF1-binding to the VEGF promoter by ChIP analysis as per the method described by Gray et al. (Bruick et al. 2001; Epstein et al. 2001; Ivan et al. 2001). Individual results showed an increased binding level of HIF1 to the VEGF promoter region when cultured on MS5s, compared to those cultured without (refer to Appendix I). The extent of this binding was comparable to those

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 192 (control) cells grown under low-oxygen conditions, suggesting that culturing leukaemia on MS5s can potentially be responsible for the induction of HIF1 binding, which in turn could be a precursor of VEGF secretion by these cells. Unfortunately, this initial experiment was unable to be adequately reproduced, hence its inclusion in the Appendices only. However, the importance of such a potential relationship is worth noting, if only for future research into this area, as the co-expression of HIF1 and VEGF has already been shown in the BM of childhood ALL patients (Holster et al. 2007).

P4HA1 As identified in the first part of the discussion, only P4HA1 was shown to be commonly up-regulated when compared to the most significant genes identified in the t-test, SAM and GeneSpring assessments. P4HA1 has only relatively recently been identified and characterised as a key enzyme involved in the formation of the collagen triple helix (Holster et al. 2007), as distinct from prolyl 4-hydroxylases which are central to the regulation of the HIF transcription factor (Hofbauer et al. 2003; Fahling et al. 2006). Holster et al. (Chen et al. 2006; Fahling et al. 2006; Grimmer et al. 2006) has demonstrated that the knock-down of P4HA1 resulted in embryonic lethal phenotype in mice between E10.5 and E11.5. These mice demonstrated a cumulative developmental delay as well as a rupture of the basement membrane (Hofbauer et al. 2003). Additionally, these basement membranes lacked collagen VI, suggesting that P4HA1 is essential in its formation. P4HA1 also presented a desirable research target owing to its increased synthesis under hypoxic conditions (Baron et al. 2006). Research into P4HA1 has primarily focussed on the modulation of its mRNA expression levels and the resulting downstream impacts of this process, i.e. collagen formation (Thiel et al. 2002). One of the ways in which P4HA1 may be regulated is through hypoxia, as Hofbauer and colleagues (Houston et al. 2001) have shown that there is a time-dependent up- regulation of the P4HA1 subunit under hypoxic conditions. These effects were only seen in cells which expressed HIF1, suggesting that it may be specifically regulated through this protein. The co-expression of both of these genes in our ALL xenografts, although by no means indicates causality, does not preclude their potential interrelatedness with regards to leukaemia / BM interactions.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 193 EGR1 EGR1 is a transcription factor which quickly responds to external stimuli (Chen et al. 2006). Depending on the cellular and environmental context in which it studied, EGR1 has been implicated in growth proliferation, as well as tumour suppression and apoptosis (De Mestre et al. 2005). EGR1 can induce numerous growth factors (EGF, PDGF-A & -B, IGF-II, aFGF and bFGF) and it has been suggested that it may also play a role in angiogenesis (Parish et al. 2001; Vlodavsky et al. 2001).

In relation to the previous results, EGR1 has been shown to have both a strong stimulatory effect on collagen gene expression (Vlodavsky et al. 1987; Folkman et al. 1988), along with being able to regulate heparanase in tumour cells (Aguayo et al. 2000). Heparanase cleaves heparan sulfate, an important component of the ECM and vascular basement membrane. As such, the cleavage of heparan sulfate facilitates the degradation of the ECM and promotes cell invasion, which has been associated with tumour metastasis, angiogenesis and inflammation (De Mestre et al. 2005). It is thought that heparanase releases in angiogenic response through the release of heparan sulfate bound growth factors (VEGF and bFGF) (Pui et al. 2008). Therefore, the identification of EGR1, strengthens the hypothesis that the interaction of leukaemia with its environment induces certain angiogenic signals, which can potentially change the BM microstructure of the patients with leukaemia, in a similar manner to that identified by Aguayo (2006) in acute and chronic leukaemias. It is of particular interest that our xenograft cells have been shown to express heparanase (pers. comm. Dr C. Freeman, Australian National University). Largely based on this finding, a ChIP assay for EGR1 was also attempted, based on the method outlined in de Mestre et al. (2004). Unfortunately the assay did not work (data not shown) and after consultation with the authors of this manuscript, it was not pursued owing to difficulties inherent in the technique (pers. comm. Dr L. Khachigian, UNSW). Nevertheless, increases in EGR1 at both the mRNA and protein level were observed, partially confirming the microarray observations.

It could be put forward that the up-regulation of EGR1 may be a culturing artefact. However, if such an assertion were correct, then we would expect uniform expression of EGR1 across all the xenografts tested regardless of the culturing treatments.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 194 Whilst certain genes, which were identified by their significance, fold-change and biological context, were able to be pursued by alternative analytical methods, the mass of differentially regulated data generated from high-throughput microarray analysis has meant that most of these genes are under-represented in this discussion. This is further confounded by the large numbers of hypothetical or unknown genes, and ESTs. Many of these were highly differentiated, but could not be explored. Even the identifiable genes could not be fully assessed, owing to the lack of available biological primers or antibodies, which was the case with P4HA1.

To summarise, the gene expression profile as identified by microarray, yielded a number of important findings with regards to our ALL xenograft panel. It revealed the differential regulation of genes that are indirectly involved with cell adhesion and tissue remodelling. Such information confirms the observations from the previous chapters, highlighting the molecular interrelatedness between the leukaemia cells and BM microenvironment.

Chapter Five: Gene Determination of Select ALL Xenografts Using Microarray 195

 

CHAPTER SIX: General Discussion and Future Research Directions

Until the advent of chemotherapy in the 1970s, the vast majority of paediatric leukaemias were uniformly fatal. Since this time, childhood ALL has enjoyed vast increases in treatment success, with an 80% remission rate currently witnessed. This improvement has been partly due to the stratification of a patients’ ‘risk of relapse’, as well as addressing their genetic and molecular abnormalities, allowing for an according treatment based on these sub-classifications. Despite this success, there are still a proportion of patients (~20%) who respond poorly to present-day multi-agent treatments. The long term side effects of these treatments are also a concern for those patients surviving the disease.

Simply ‘tweaking’ the current treatments by changing treatment regimes or altering protocols of current agents will not be the panacea for those patients who respond poorly (Rasko et al. 1995; Rosnet et al. 1996). With the current and (inevitable) future advancements in molecular genetics, there will be an increased ability to assess the genetic abnormalities within leukaemia. These include the already identified genetic abnormalities; MLL rearrangements [t(4;11), t(11;19) and t(1;11)], Philadelphia chromosome, and the TEL-AML1 fusion gene t(12;21) to name a few (see Pui and Evans (Suzuki et al. 2007; Sallmyr et al. 2008; Shimada et al. 2008) review article, and references within). The enhanced diagnostic methods identifying new genetic abnormalities enable a better sub-classification of the disease, which opens the door to the development of molecular . This however, brings its own set of problems, which are primarily associated with the costs involved. Although the cost will

Chapter Six: General Discussion and Future Research Directions 197 be high initially, in the long term as suggested by Evans and Relling (1995b), it will prove more effective (including in terms of the cost) by reducing the side effects compared with current treatments and improving patient outcomes.

Solely identifying the molecular lesions, although providing an important first step, should not be the conclusion of this process. Understanding their roles and biological effects on the cell is necessary for a focused molecular-based treatment. This thesis provides an example of one particular pathway which has been shown to respond to targeted treatment. It was demonstrated that a sub-type of ALL cells produce and secrete VEGF, which is inducible through the activation of FLT-3. The inhibition of FLT-3 is a particularly attractive target for a couple of important reasons. Firstly, it is expressed in only a small percentage of cells, mainly those of the haematopoietic system, which would suggest minimal off-target effects (Vacca et al. 1994). This point is of particular relevance for the quality of life for patients who survive the disease. And secondly, FLT-3 has widely been studied as a therapeutic target, principally in the context of its association with poor prognosis and the fact that it is frequently mutated (Ribatti et al. 2004). As such, although the focus has mainly been on AML (and mutations), a large number of receptor-specific inhibitors have been developed, which are available for use in alternative systems (such as ALL).

The demonstration that FLT-3 activation plays a role in the induction of VEGF is a novel and interesting finding. It has been previously established, principally in solid cancers, that VEGF plays a vital role in angiogenesis (see Folkman (Vacca et al. 1999b; Mesa et al. 2000; Kini et al. 2001; Faderl et al. 2005), and references within). Angiogenesis and its role in the growth and survival of haematological malignancies, including leukaemia, has only been demonstrated since the mid-1990s (Avramis et al. 2006). Additionally, the exploration of this link has occurred at a relatively slow pace owing to the somewhat illogical connection in haematological systems between the advancement of angiogenesis and the progression of tumours, when compared to their solid counterparts (Fiedler et al. 2003).

Since its initial identification, there has been a succession of studies demonstrating that the degree of angiogenesis observed in the BM of patients is positively associated with disease progression (Giles et al. 2003a). Additionally, VEGF expression itself has been

Chapter Six: General Discussion and Future Research Directions 198 associated with a poorer outcome for patients (Giles et al. 2003b). Therefore, a better understanding of its induction may present new opportunities for therapeutic treatments which focus on minimising VEGF levels in newly presenting or relapsed ALL patients. Indeed, the current scientific literature shows a clear focus on addressing VEGF suppression in leukaemia [e.g. Fiedler (2006), Giles (Keyhani et al. 2001; Fragoso et al. 2007), Giles (2001), Kang (Dunphy 2006)]. An advantage of this research is the possibility of blocking VEGF through a RTK other then its own VEGFRs, and potentially disrupting the paracrine loop between leukaemia cells and the BM microenvironment (Quackenbush 2003).

It needs to be realised that ALL-3 was the one xenograft showing inducible levels of VEGF. Similarly, ALL-2 and -3 were the only two xenografts which demonstrated that in high FLT-3 expressing leukaemia cells, VEGF secretion can be reduced. The fact that only two ALL xenograft cells exhibited such a relationship should not diminish the resultant findings, as this merely highlights the heterogeneous nature of both our panel, and ALL more generally. Microarray analyses demonstrated a very different gene expression pattern after FLT-3 activation between ALL-3 cells and other xenografts (ALL-17 and -19) with lower receptor levels.

The microarray results of ALL xenograft gene expression had varying degrees of success. Indeed, differing findings between analytical methods, highlight the point made by Quackenbush (Quackenbush 2001), that there is probably no ‘best method’ for assessing the gene expression. Despite the drawbacks associated with the analysis, microarray technology is a powerful tool with many purposes and it cannot be denied that is has aided in the diagnosis of leukaemias and (Maris et al. 2008).

The results from the gene expression profile in the ALL xenografts cultured on BM stromal cells showed a novel, yet not entirely unexpected pattern, with the genes belonging to processes primarily involved in cell-cell interaction (Dosil et al. 1993; Mizuki et al. 2000; Tse et al. 2000; Kelly et al. 2002a; Kelly et al. 2002b). They all warrant further investigation upon the development of antibodies (e.g. P4HA1) and possibly better assays to assess their functional role in leukaemia in response to the environmental conditions (e.g. HIF1).

Chapter Six: General Discussion and Future Research Directions 199 Aside from the points discussed above, the cumulative findings within this thesis also lay an important platform for future studies. Firstly, it is apparent that a larger number of ALL patients or xenograft cells which have high FLT-3 expression need to be tested, in order to assess the applicability or potential role of this treatment approach. Furthermore, ALL cells with abnormalities at 11q23 (not restricted to infant MLL samples) need to be incorporated into the sampling regime, as MLL rearrangements are not exclusive to infants and do occur in childhood ALL (as per ALL-3). Such work would assess the potential role of FLT-3 inhibitors against these biologic subtypes, and help in a better identification of patients who may benefit from treatment with FLT-3 inhibitors, as has also been inferred by Brown et al. (Druker 2002).

The project findings can also be translated to an in vivo experimental setting, to examine the effects on the BM after engraftment with these cells which secrete high levels of VEGF. They would be ideally suited to be subsequently tested with (and without) the addition of FLT-3 inhibitors, such as SU11657 or FLT-3 blocking antibodies. Not only would this enable the examination of the effects of the secreted VEGF on the BM microenvironment, but it would also ascertain whether the induction of VEGF secretion by FLT-3 is crucial for ALL cells survival, proliferation and dissemination.

FLT-3 inhibition is an alternative (and potentially superior) method in which to study the biology of disease, because it is a novel way to inhibit VEGF through a receptor that is not so widely expressed (compared to VEGFRs). However, it is doubtful that FLT-3 inhibitors can be used as a single agent therapy, and will probably be more effective in combination with other drugs. The single agent efficacy of the FLT-3 inhibitor SU11248 has already been tested in vivo in our xenograft model and showed effects on the engraftment of ALL-2 into NOD/SCID mice . With ALL-2 it was shown to have an objective response, effectively delaying the engraftment of the cells in the mice. The in vivo effect of SU11248 on ALL-2 can be related to the effects observed in vitro with SU11657 and the almost complete reduction in VEGF secretion by these cells, which may have been enough to slow the engraftment.

Regardless of whether the results are from the high expression of FLT-3 or the expression of mutant FLT-3, it certainly has been shown to aid leukaemogenesis (Dosil et al. 1993; Mizuki et al. 2000; Tse et al. 2000; Kelly et al. 2002a; Kelly et al. 2002b).

Chapter Six: General Discussion and Future Research Directions 200 It should also be recognised that FLT-3 may not be the single causative factor or the driving force in the progression of the leukaemia, as may be the case with the expression of KIT or BCR/ABL in other malignancies (Druker 2002). Therefore, it should be reiterated that simply inhibiting FLT-3 in ALL may not be the panacean cure for all ALL sub-types. The targeting of FLT-3, which in turn could inhibit VEGF secretion in these cells, may however, slow down its growth. When this is used in combination with other anti-leukaemic drugs, a stronger effect against ALL engraftment may well be observed. This novel finding of a relationship between FLT-3 and VEGF further adds to our growing understanding of the disease as well as presenting alterative research opportunities to combat this disease in the future.

Chapter Six: General Discussion and Future Research Directions 201



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Appendices APPENDIX A

A.1 Effects of BM Stromal Cells on ALL Xenograft cells

A.1.1 T-Test Table A.1. Descriptions of functions for the 20 discriminating genes as identified by a t test. All values are given with terms of up regulation in MS5 supported samples thus for values < 1 there is down regulation in MS5 supported sample. * Calculated by first averaging all MS5 supported and non-MS5 supported samples then taking the ratio ^ Calculated by taking the ratio of each corresponding samples then averaging them Fold up regulation Gene Symbol Name t test value */^ telomeric repeat binding factor 2 TERF2 0.00026 1.16 / 1.16 This gene encodes a telomere specific protein, TERF2, which is a component of the telomere nucleoprotein complex. This protein is present at telomeres in metaphase of the cell cycle, is a Summary second negative regulator of telomere length and plays a key role in the protective activity of telomeres. While having similar telomere binding activity and domain organization, TERF2 differs from TERF1 in that its N terminus is basic rather than acidic. Ontology Function DNA binding protein C-terminus binding single-stranded telomeric DNA binding Process Cell cycle Negative regulation of telomere maintenance Regulation of transcription Telomere maintenance via telomerase Component Chromosome Chromosome, telomeric region Nucleosome Nucleus

Appendices 259 Fold up Gene Symbol Name T test value regulation hypoxia-inducible factor 1 HIF1A 0.00031 1.23 / 1.23 Hypoxia-inducible factor-1 is a transcription factor found in mammalian cells cultured under reduced oxygen tension that plays an essential role in cellular and systemic homeostatic responses to hypoxia. HIF-1 is a heterodimer composed of an  subunit and a ! subunit. The ! subunit has Summary been identified as the aryl hydrocarbon receptor nuclear translocator (ARNT). This gene encodes the ! subunit of HIF-1. Overexpression of a natural antisense transcript (aHIF) of this gene has been shown to be associated with nonpapillary renal carcinomas. Two alternative transcripts encoding different isoforms have been identified. Ontology Function Hsp90 protein binding RNA polymerase II transcription factor activity, enhancer binding Contributes to RNA polymerase II transcription factor activity, enhancer binding histone acetyltransferase binding protein heterodimerisation activity contributes to sequence-specific DNA binding signal transducer activity transcription factor binding Process collagen metabolic process connective tissue replacement during inflammatory response elastin metabolic process epithelial to mesenchymal transition mRNA transcription from RNA polymerase II promoter oxygen homeostasis positive regulation of angiogenesis positive regulation of cell migration positive regulation of chemokine production positive regulation of endothelial cell proliferation positive regulation of erythrocyte differentiation positive regulation of gene-specific transcription positive regulation of glycolysis positive regulation of hormone biosynthetic process positive regulation of nitric-oxide synthase activity positive regulation of transcription positive regulation of transcription transcription from RNA polymerase II promoter positive regulation of VEGFR signalling pathway positive regulation VEGF production regulation of transcription from RNA polymerase II promoter in response to oxidative stress regulation of transcription, DNA-dependent regulation of TGF-!2 production response to hypoxia signal transduction Component Nucleus Transcription factor complex

Appendices 260

Fold up Gene Symbol Name T test value regulation procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline P4HA1 0.00034 2.41 / 2.43 4-hydroxylase), alpha polypeptide I This gene encodes a component of prolyl 4-hydroxylase, a key enzyme in collagen synthesis composed of two identical alpha subunits and two beta subunits. The encoded protein is one of several different types of alpha subunits and provides the major part of the catalytic site of the Summary active enzyme. In collagen and related proteins, prolyl 4-hydroxylase catalyses the formation of 4- hydroxyproline that is essential to the proper three-dimensional folding of newly synthesized procollagen chains. Alternatively spliced transcript variants encoding different isoforms have been described. Ontology Function L-ascorbic acid binding Binding Iron ion binding metal ion binding oxidoreductase activity oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors oxidoreductase activity, acting on single donors with incorporation of molecular oxygen,

incorporation of two atoms of oxygen procollagen-proline 4-dioxygenase activity Process oxidation reduction protein metabolic process Component endoplasmic reticulum endoplasmic reticulum lumen

Fold up Gene Symbol Name t test value regulation Nedd4 family interacting protein 2 NDFIP2 0.00037 2.10 / 2.36 Ontology Function signal transducer activity Process positive regulation of I-kappaB kinase/NF-kappaB cascade Component Golgi apparatus Golgi membrane Endosome Endosome membrane Integral to membrane Membrane

Appendices 261

Fold up Gene Symbol Name T test value regulation SCD6 homolog A (S. cerevisiae) LSM14A 0.00041 0.90 / 0.90 Sm-like proteins were identified in a variety of organisms based on with the Sm protein family (see SNRPD2; 601061). Sm-like proteins contain the Sm sequence motif, which Summary consists of 2 regions separated by a linker of variable length that folds as a loop. The Sm-like proteins are thought to form a stable heteromer present in tri-snRNP particles, which are important for pre-mRNA splicing.

Gene Symbol Name T test value Fold up regulation signal peptidase complex subunit 3 homolog (S. SPCS3 0.00046 1.41 / 1.42 cerevisiae) Ontology Function signal peptidase activity Process signal peptide processing Component endoplasmic reticulum integral to membrane membrane Microsome Signal peptidase complex

Gene Symbol Name T test value Fold up regulation ClpP caseinolytic protease, ATP-dependent, proteolytic CLPP 0.00074 0.89 / 0.89 subunit (E. coli) homolog The protein encoded by this gene belongs to the peptidase family S14 and hydrolyses proteins into Summary small peptides in the presence of ATP and magnesium. The protein is transported into mitochondrial matrix and is associated with the inner mitochondrial membrane. Ontology Function endopeptidase Clp activity peptidase activity Process Proteolysis Component mitochondrion

Appendices 262 Gene Symbol Name T test value Fold up regulation BCL2/adenovirus E1B 19kD-interacting protein 3-like BNIP3L 0.00067 1.56 / 1.57 This gene is a member of the BCL2/adenovirus E1B 19 kd-interacting protein (BNIP) family. It interacts with the E1B 19 kDa protein which is responsible for the protection of virally-induced cell Summary death, as well as E1B 19 kDa-like sequences of BCL2, also an apoptotic protector. The protein encoded by this gene is a functional homolog of BNIP3, a proapoptotic protein. This protein may function simultaneously with BNIP3 and may play a role in tumour suppression. Ontology Function lamin binding protein heterodimerisation activity protein homodimerisation activity Process defence response to virus induction of apoptosis interspecies interaction between organisms negative regulation of apoptosis negative regulation of survival gene product expression positive regulation of apoptosis Component Endoplasmic reticulum Integral to membrane Membrane Mitochondrial envelope Mitochondrion Nuclear envelope Nucleus

Gene Symbol Name T test value Fold up regulation ESTs HsKG11C5 0.00076 1.43 / 1.44 ESTs R43250a6 0.00160 0.64 / 0.64 ESTsHsKG77E6 0.00182 1.31 / 1.32 chromosome 7 open reading frame 50 also know as C7orf50 0.00086 1.62 / 1.66 hypothetical protein MGC11257

Appendices 263 Gene Symbol Name T test value Fold up regulation mitogen-activated protein kinase kinase 1 MAP2K1 0.00077 1.43 / 1.46 The protein encoded by this gene is a member of the dual specificity protein kinase family, which acts as a MAP kinase kinase. MAP kinases, also known as ERKs, act as an integration point for multiple biochemical signals. This protein kinase lies upstream of MAP kinases and stimulates the enzymatic Summary activity of MAP kinases upon wide variety of extra- and intracellular signals. As an essential component of MAP kinase signal transduction pathway, this kinase is involved in many cellular processes such as proliferation, differentiation, transcription regulation and development. Ontology Function ATP binding MAP kinase kinase activity Nucleotide binding Protein binding protein serine/threonine kinase activity protein tyrosine kinase activity transferase activity Process Ras protein signal transduction Cell motility Cell proliferation Chemotaxis Keratinocyte differentiation Mitosis Neuron differentiation protein amino acid phosphorylation response to glucocorticoid stimulus response to oxidative stress Component Golgi apparatus cytosol

Appendices 264 Gene Symbol Name T test value Fold up regulation procollagen-proline, 2-oxoglutarate 4-dioxygenase P4HA2 0.00081 1.99 / 2.14 (proline 4-hydroxylase), alpha polypeptide II This gene encodes a component of prolyl 4-hydroxylase, a key enzyme in collagen synthesis composed of two identical alpha subunits and two beta subunits. The encoded protein is one of several different types of alpha subunits and provides the major part of the catalytic site of the active enzyme. Summary In collagen and related proteins, prolyl 4-hydroxylase catalyses the formation of 4-hydroxyproline that is essential to the proper three-dimensional folding of newly synthesized procollagen chains. Alternatively spliced transcript variants encoding different isoforms have been described. Ontology Function L-ascorbic acid binding electron carrier activity iron ion binding metal ion binding oxidoreductase activity oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, 2-

oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of

two atoms of oxygen procollagen-proline 4-dioxygenase activity protein binding Process oxidation reduction protein metabolic process Component endoplasmic reticulum endoplasmic reticulum lumen

Gene Symbol Name T test value Fold up regulation hypothetical protein FLJ22242 - heparan-alpha- HGSNAT 0.00123 1.45 / 1.49 glucosaminide N-acetyltransferase The protein encoded by this gene belongs to the peptidase family S14 and hydrolyses proteins into Summary small peptides in the presence of ATP and magnesium. The protein is transported into mitochondrial matrix and is associated with the inner mitochondrial membrane. Ontology Function acyltransferase activity heparan-alpha-glucosaminide N-acetyltransferase activity transferase activity Process glycosaminoglycan metabolic process Component Integral membrane Lysosomal membrane membrane

Appendices 265 Gene Symbol Name T test value Fold up regulation synaptic nuclei expressed gene 2 - spectrin repeat SYNE2 0.00129 1.46 / 1.46 containing, nuclear envelope 2 Ontology Function Actin binding Structural molecule activity Component Cytoplasm Cytoskeleton Integral to membrane Membrane Nuclear outer membrane nucleus

Gene Symbol Name T test value Fold up regulation phosphoglycerate kinase 1 PGK1 0.00129 1.56 / 1.60 The protein encoded by this gene is a glycolytic enzyme that catalyses the conversion of 1,3- diphosphoglycerate to 3-phosphoglycerate. The encoded protein may also act as a cofactor for Summary polymerase alpha. A pseudogene of this gene has been found on the X-chromosome and another on . Ontology Function ATP binding Nucleotide binding Phosphoglycerate kinase activity Transferase activity Process Glycolysis phosphorylation Component cytoplasm Integral to membrane Membrane Nuclear outer membrane nucleus

Gene Symbol Name T test value Fold up regulation fibulin 2 FBLN2 0.00129 1.77 / 1.79 This gene encodes an extracellular matrix protein, which belongs to the fibulin family. This protein binds various extracellular ligands and calcium. It may play a role during organ development, in particular, Summary during the differentiation of heart, skeletal and neuronal structures. Alternatively spliced transcript variants encoding different isoforms have been identified. Ontology Function calcium ion binding extracellular matrix structural constituent Component extracellular region proteinaceous extracellular matrix

Appendices 266 Gene Symbol Name T test value Fold up regulation RAB17, member RAS oncogene family RAB17 0.00147 1.77 / 1.85 Ontology Function GTP binding Nucleotide binding Protein binding Process Protein transport small GTPase mediated signal transduction Component intracellular

Gene Symbol Name T test value Fold up regulation integrin, alpha L (antigen CD11A (p180), lymphocyte ITGAL 0.00153 0.59 / 0.61 function-associated antigen 1; alpha polypeptide ITGAL encodes the integrin alpha L chain. Integrins are heterodimeric integral membrane proteins composed of an alpha chain and a beta chain. This I-domain containing alpha integrin combines with the beta 2 chain (ITGB2) to form the integrin lymphocyte function-associated antigen-1 (LFA-1), which Summary is expressed on all leukocytes. LFA-1 plays a central role in leukocyte intercellular adhesion through interactions with its ligands, ICAMs 1-3 (intercellular adhesion molecules 1 through 3), and also functions in lymphocyte co-stimulatory signalling. Two transcript variants encoding different isoforms have been found for this gene. Ontology Function calcium ion binding magnesium ion binding protein binding receptor activity Process Cell adhesion Cell motility Inflammatory response integrin-mediated signalling pathway Signal transduction Component Integral to membrane Integrin complex Membrane Plasma membrane

Appendices 267



APPENDIX B

B.1 Top 67 genes by t-test

In order to determine the maximum number of genes which still resulted in the independent clustering of the two culturing conditions, a lower significance threshold was applied to let broader number of genes to filter though. Of the 67 genes that were let to filter through, 36 of those were known genes, 8 were hypothical proteins and 23 ESTs (Table B.1). When the Pearson centred cluster plot of the discriminating genes was performed, the clustering groups remained as with when only 20 genes were let to filter through, suggesting that this larger gene set is still discriminating between the two groups (Figure B.1).

Appendices 269 Table B.1. Descriptions of functions for the 67 discriminating genes as identified by a t test. All values are given with terms of up regulation in MS5 supported samples thus for values < 1 there is down regulation in MS5 supported sample. Fold up Fold up Gene Symbol Name regulation * regulation ^ ESTs R43335c9 0.051 0.040 KIAA1234 protein AHRR 0.409 0.381 ESTs R43368f4 0.438 0.405 A kinase (PRKA) anchor protein 5 AKAP5 0.529 0.504 EST R43354d10 0.561 0.543 integrin,L (antigen CD11A (p180), lymphocyte function-associated ITGAL 0.609 0.591 antigen 1;  polypeptide ESTs R43250a6 0.641 0.639 ESTs R43304c4 0.645 0.623 myeloid/lymphoid or mixed-lineage leukaemia (trithorax (Drosophila) MLL 0.666 0.666 homolog) protein kinase C binding protein 1 PICK1 0.720 0.720 ESTs R4355a10 0.726 0.720 hypothetical protein FLJ23505 0.764 0.757 ESTs R43135b2 0.792 0.791 ESTs R43144f11 0.804 0.800 NADH dehydrogenase (ubiquinone) 1  subcomplex, 5 (13kD, B13) NDUFA5 0.835 0.835 CREB binding protein (Rubinstein-Taybi syndrome) CREBBP 0.847 0.846 ESTs, Weakly similar to I38022 hypothetical protein 0.886 0.885 ClpP caseinolytic protease, ATP-dependent, proteolytic subunit, E. coli CLPP 0.889 0.888 homolog LSM14A, SCD6 homolog A (S. cerevisiae) LSM14A 0.898 0.897 peroxisomal biogenesis factor 7 PEX7 1.097 1.095 telomeric repeat binding factor 2 TERF2 1.158 1.157 hypothetical protein MGC14353 TXNDC17 1.178 1.177 ubiquitin specific protease 9, Y chromosome (Drosophila fat facets USP9Y 1.188 1.189 related) gem (nuclear organelle) associated protein 4 GEMIN4 1.205 1.198 glypican 4 GPC4 1.211 1.205 ESTs R43141e11 1.211 1.208 p21 (CDKN1A)-activated kinase 3 CDKN1A 1.215 1.208 tachykinin, precursor 1 (substance K, substance P, neurokinin 1, TAC1 1.217 1.218 neurokinin 2, neuromedin L, neuro hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix HIF1A 1.231 1.230 transcription factor) ESTs R4335b11 1.237 1.236 ESTs R4338h10 1.245 1.240 guanine nucleotide binding protein (G protein), alpha transducing GNAT1 1.250 1.236 activity polypeptide 1 ESTs, Weakly similar to ORF2-like protein 1.270 1.262 cornichon homolog 4 CNIH4 1.270 1.270 ESTs R43315c4 1.279 1.266 UFM1-specific peptidase 2 UFSP2 1.306 1.308 kinase insert domain receptor (a type III receptor tyrosine kinase) KDR 1.308 1.278 bobby sox homolog BBX 1.320 1.306 ESTs HsKG77E6 1.323 1.308 EST R43407f7 1.327 1.295 defensin, beta 1 DEFB1 1.389 1.351 CD1B antigen, b polypeptide CD1B 1.406 1.398 signal peptidase complex subunit 3 homolog (S. cerevisiae) SPCS3 1.421 1.414 Homo sapiens cDNA: FLJ23111 fis, clone LNG07835 1.429 1.432 ESTs HsKG11C5 1.442 1.429 multiple PDZ domain protein MPDZ 1.452 1.443 mitogen-activated protein kinase kinase 1 MAP2K1 1.457 1.432

Appendices 270 synaptic nuclei expressed gene 2 SYNE2 1.462 1.459 hypothetical protein FLJ22242 HGSNAT 1.485 1.445 spermatid perinuclear RNA-binding protein STRBP 1.543 1.504 eukaryotic translation initiation factor 5 EIF5 1.565 1.514 BCL2/adenovirus E1B 19kD-interacting protein 3-like BNIP1 1.574 1.560 striatin, calmodulin binding protein 4 STRN4 1.576 1.579 ESTs R43251a4 1.580 1.559 phosphoglycerate kinase 1 PGK1 1.603 1.558 hypothetical protein MGC11257 C7orf50 1.655 1.617 ESTs R43410a5 1.684 1.630 ESTs, Weakly similar to Fuzzy (D.melanogaster) 1.691 1.614 ESTs R4385c9 1.742 1.695 fibulin 2 FBLN2 1.787 1.769 ESTs R43102h11 1.815 1.747 RAB17, member RAS oncogene family RAB17 1.846 1.774 enolase 2, (gamma, neuronal) ENO2 2.012 1.867 procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4- P4HA2 2.141 1.985 hydroxylase), alpha polypeptide II Nedd4 family interacting protein 2 NDFIP2 2.361 2.197 procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4- P4HA1 2.427 2.409 hydroxylase), alpha polypeptide I secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T- SPP1 2.609 2.497 lymphocyte activation 1) " #$%#&'( )* + & ,' -+.- ## /0#'    1'.-  & $'## $%#&% '( 2+&3  $'## .(  2+&3%&  %00 &'( &3'. &4+.- &3' 5.6  &+7 8 #$%#&'( )* &4+.- &3'  2+&3 5 2+&3%&  &+   '$3 $ '0.(+.-/0#'.(&3'.,' -+.-&3'/7

Appendices 271

EST R43368f4 NDUFA5 AKAP5 CLPP MLL EST Weakly similar to I38022 hypothetical protein (H. sapiens) EST R32225c9 Hypothetical protein FLJ23505 - discontinued ITGAL EST R43354d10 LSM14A AHRR R4355a10 ESTs 431441e11 EST R43250a6 CREBBP ESTs R43135b2 PICK1 EST R43304c4 EST R43315c4 HIF1A USP9Y TXNDC17 EST R4338H10 MPDZ EST R4335b11 STRN4 CD1B ESTs 43141e11 GEMIN4 HGSNAT SPCS3 GPC4 FLJ23111 fis, clone LNG07835 CNIH4 P4HA1 TERF2 BBX C7orf50 RAB17 DEFB1 KDR PEX7 NDFIP2 MAP2K1 PGK1 STRBP ELF5 P4HA2 CDKN1A ESTs R43102h11 GNAT1 EST Weakly similar to ORF2-like protein (H. sapiens) EST HsKG11C5 FBLN2 EST Weakly similar to Fuzzy (D. melanogaster) EST R4385c9 UFSP2 SYNE2 TAC1 BNIP1 SPP1 EST R43407f7 ENO2 EST HsKG77E6 EST 43251a4 ESTR43410a5

Figure B.1. Gene set of 67 genes considered to be discriminating according to t test. A Pearson centred cluster of the gene set is shown, which gene names listed. In the diagram, red indicates high expression, green low expression and black, intermediate expression. The dendograms indicate the pairing of genes (and samples in panel) and the branch length is proportional to the distances between the clusters. Appendices 272 Of the 67 discriminating genes 19 of those (28%) were down regulated in ALL xenograft cells when they were cultured with MS5 stromal cells and 48 genes (72%) were upregulated. The genes were then grouped according to their gene ontologies, the graphs are shown in Figure B.3.

A biological adhesion down up biological regulation cellular process developmental process immune system process localization metabolic process multicellular organismal process multi-organism process

process Ontology: biological reproduction response to stimulus

0 20 40 60 80 100 B % of genes in ontology group binding down up catalytic activity enzyme regulator activity signal transducer activity structural molecule activity transcription regulator activity translation regulator activity

Ontology: molecular function 0 20 40 60 80 100 % of genes in ontology group C dow n up cell envelope extracellular matrix extracellular region membrane-enclosed lumen organelle protein complex synapse Ontology: cellular component cellular Ontology: 0 20 40 60 80 100 % of genes in ontology group

Figure B.2 A, B & C. The top 70 differentially expressed genes clustered into ontology groups by function, process and component. The clustering was done using the FatiGO software and it should be noted that some genes are included in more then one ontology group. The down-regulated genes are shown in green and the group of up-regulated genes us in red.

Appendices 273



APPENDIX C

C.1 GeneSpring

C.1.1 GeneSpring filtering by fold change GeneSpring takes two approaches to find significant genes by simply looking at fold changes when comparing two samples. The first is the average approach, which takes the average values for each gene in ALL xenografts cells with MS5s versus those without MS5s, and the fold change is calculated from these averages for individual genes. The second approach is the individual approach, in which the fold changes between individual xenografts with MS5 and the corresponding cells without MS5 are calculated, after which an average is generated (Figure C.1). The genes are then filtered according to a certain fold change cut off for a gene set to be obtained. This was repeated for all the samples to yield five gene sets. Then cross checked to filter genes which were common in n amount of gene sets where the higher the n the more significant the gene can taken to be. In both analyses ALL xenograft cell cultured with MS5 stromal cells were compared to expression of genes in ALL xenograft cells cultured without MS5 cells.

Appendices 275

   

ALL + MS5 Ratio:------ALL - MS5

,' -' '10 '+. ,' -' '10 '+.

ALL-2 ALL-3 ALL-7 ALL-17 ALL-19 ALL-2 ALL-3 ALL-7 ALL-17 ALL-19 + + + + + - - - - - MS5 MS5 MS5 MS5 MS5 MS5 MS5 MS5 MS5 MS5

ALL + MS5 Ratio:------ALL - MS5

,' -'  &+.

   

Figure C.1. Schematic diagram of the two different approaches used by GeneSpring to analyse gene expression data.

C.1.1.1 Average approach Fold change cut offs of 2, 3, 4, 12 and 15 were used. The number of genes and the dendrograms of the samples are shown in Table C.1. From the all the dendrograms shown it can be observed that the samples have not clustered according to with ad without MS5 groups for all the gene sets with different fold change cut off variables.

Appendices 276 Fold change cut off of 15 shows the best clustering result, however is not convincing enough to give confidence in the corresponding gene set.

Table C.1. Sample and gene clustering following GeneSpring average approach analysis. The table shows the cut off, number of gene genes and dendrograms. Analysis Fold change cut off Genes Clustered Dendrograms No.

1 2 984

2 3 605

3 4 497

4 12 85

5 15 39

Nevertheless, Figure C.2A shows the genes and values for the results using fold change cut off of 15, which the corresponding Pearson centred cluster shown in Figure C.2B. From both figures it becomes clear as to why this approach was not as successful as the previous two described. A single extreme value greatly increases or decreases a group’s average thus greatly increasing or decreasing the fold change calculation for that gene. Appendices 277 Most of the genes that filtered though, the spots corresponded to expression sequence tags (ESTs) or hypothetical proteins and not actual know genes.

A

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Appendices 278 B

 =2'4#*+/+# & ? #/+.+.6$3+.A B  ->    -     '             =2'4#+/+# &>3*0&3'&+$#0 &'+.  A    B   D(    $  '    '    >   -   $>     3*0&3'&+$#0 &'+.C0       =2'4#*+/+# &0%&&+,'0 A B   $   D>$   '      -   D    D'    '   >   >$   >>   -    >   D C  D E  D$  

Figure C.2 A & B. GeneSpring average approach genes at 15-fold cut off. Genes and values for fold change cut off 15 (A) and Pearson centred cluster (B) as given by Gene spring.

C.1.1.2 Individual approach The second approach taken by GeneSpring showed more promising results. This approach gave much more promising results, they are all summarised in Table C.2. Analysis number 3 and 5 gave the best result in terms of group clustering. The 4 genes from analysis 5 were shown to be a subset of analysis 3. Thus 28 discriminating genes were obtained from this analysis. They are shown in Table C.3, with the cluster plot shown in Figure C.3. Appendices 279 Table C.2. Fold change cut off, genes and dendrograms for the individual approach GeneSpring analysis. Analysis Parameters Genes Clustered Dendrograms No.

Fold change cut off: 1.25 1 Gene common in at least 5 out of 121 5 samples

Fold change cut off: 1.50 2 Gene common in at least 4 212 samples

Fold change cut off: 1.50 3 Gene common in at least 5 28 samples

Fold change cut off: 2.00 4 Gene common in at least 4 41 samples

Fold change cut off: 2.00 5 Gene common in at least 5 4 samples

Appendices 280 Table C.3. The top 28 discriminating genes as identified by GeneSpring analysis. *Calculated by first averaging all samples of ALL xenograft cells cultured with MS5 cells and ALL without MS5 supported then taking the MS5/Non-MS5 ratio. ^Calculated by taking the ALL with / without MS5 ratio of each corresponding sample and then averaging them. All values are given with terms of up regulation in MS5 supported samples thus for values < 1 there is down regulation in MS5 supported samples. Fold up regulation Name Gene Symbol */^ Homosapiens cDNA FLJ12924 fis, clone NT2RP2004709 R4368f8 0.42 / 0.44 ESTs R43335c9 0.04 / 0.05 ESTs R43323g6 0.13 / 0.39 cytokine-like protein C17 CYTL1 0.42 / 0.41 butyrophilin, subfamily 3, member A3 BTN3A3 0.50 / 0.53 ESTs R43192c6 0.42 / 0.46 histidyl-tRNA synthetase HARS 0.27 / 0.27 aryl-hydrocarbon receptor repressor AHRR 0.38 / 0.41 synaptic vesicle glycoprotein 2C SV2C 0.44 / 0.46 ESTs R43139f11 0.066 / 0.23 ESTs R43236b3 0.22 / 0.37 ESTs R43141d10 0.47 / 0.49 ESTs R43138g8 0.096 / 0.27 ESTs R43148f9 0.47 / 0.44 procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4- P4HA1 2.41 / 2.43 hydroxylase), alpha polypeptide I Serum amyloid A1 SAA1 3.10 / 3 .50 Plasminogen activator, urokinase receptor PLAUR 2.05 / 2.08 collagen, type VI, alpha 3 COL6A3 2.52 / 2.14 Annexin A1 ANXA1 2.13 / 2.08 Antigen identified by monoclonal antibodies 12E7, F21 and O13 CD99 1.80 / 1.77 Phosphodiesterase 4B, cAMP-specific (dunce, Drosophila - homolog PDE4B 1.86 /1.86 phosphodiesterase E4) Early growth response 1 EGR1 3.31 / 3.30 Triosephosphate isomerase 1 TPI1 1.86 / 1.93 Guanylate binding protein 2, interferon-inducible GBP2 2.38 / 2.21 Serum amyloid A2 SAA2 2.99 / 3.01 CD1C antigen, c polypeptide CD1C 2.67 / 2.48 Arylsulfatase B ARSB 2.78 / 2.35

Appendices 281

EST R43141d10 HARS EST R43148f9 EST R43138g8 EST R43236b3 BTN3A3 EST R43192c6 EST R43139f11 EST R43335c9 cDNA FLJ12924 AHRR SV2C EST R43323g6 CYTL1 ANXA1 CD1C P4HA1 TPI1 PLAUR PLAUR EGR1 SAA1 SAA2 ARSB GBP2 COL6A3 CD99 PDE4B

Figure C.3. Pearson centred cluster of 28 genes found to be discriminating by GeneSpring.

Appendices 282 Table C.4. Descriptions of functions for the 28 discriminating genes as identified by GeneSpring analysis. *Calculated by first averaging all samples of ALL xenograft cells cultured with MS5 cells and ALL without MS5 supported then taking the MS5/Non-MS5 ratio. Name Gene Symbol Fold up regulation */^ Cytokine-Like Protein C17 CYTL1 0.416 / 0.414 C17 is a cytokine-like protein specifically expressed in bone marrow and cord blood mononuclear Summary cells that bear the CD34 (MIM 142230) surface marker Ontology Function Receptor Binding Process Signal Transduction Component Extracellular Region Extracellular Space Soluble Fraction

Name Gene Symbol Fold up regulation */^ butyrophilin, subfamily 3, member A3 BTN3A3 0.503 / 0.527 Ontology Component Integral to Membrane Membrane

Name Gene Symbol Fold up regulation */^ histidyl-tRNA synthetase HARS 0.274 / 0.274 Aminoacyl-tRNA synthetases are a class of enzymes that charge tRNAs with their cognate amino acids. The protein encoded by this gene is a cytoplasmic enzyme which belongs to the class II family of aminoacyl-tRNA synthetases. The enzyme is responsible for the synthesis of histidyl- Summary transfer RNA, which is essential for the incorporation of histidine into proteins. The gene is located in a head-to-head orientation with HARSL on chromosome five, where the homologous genes share a bidirectional promoter. The gene product is a frequent target of autoantibodies in the human autoimmune disease polymyositis/dermatomyositis. Ontology Function ATP binding histidine-tRNA ligase activity ligase activity nucleotide binding Process histidyl-tRNA aminoacylation Component cytoplasm

Appendices 283 Name Gene Symbol Fold up regulation */^ aryl-hydrocarbon receptor repressor AHRR 0.381 / 0.409 Dioxin is a teratogen that exerts its effects through the arylhydrocarbon receptor in conjunction with the receptor's binding partner, arylhydrocarbon receptor nuclear translocator. The protein encoded by this gene represses signal transduction by the arylhydrocarbon receptor by competing with the Summary arylhydrocarbon receptor nuclear translocator for binding to the arylhydrocarbon receptor. Expression of the repressor is stimulated by the receptor/translocator heterodimer, thereby regulating receptor function through a negative feedback mechanism. In addition, the encoded protein can bind to nuclear factor kappa-B.

Name Gene Symbol Fold up regulation */^ synaptic vesicle glycoprotein 2C SV2C 0.445 / 0.464 Ontology Function transporter activity Process neurotransmitter transport Component cell junction cytoplasmic vesicle integral to membrane membrane synapse synaptic vesicle

Name Gene Symbol Fold up regulation */^ procollagen-proline, 2-oxoglutarate 4- dioxygenase (proline 4-hydroxylase), P4HA1 2.409 / 2.427 alpha polypeptide I This gene encodes a component of prolyl 4-hydroxylase, a key enzyme in collagen synthesis composed of two identical alpha subunits and two beta subunits. The encoded protein is one of several different types of alpha subunits and provides the major part of the catalytic site of the Summary active enzyme. In collagen and related proteins, prolyl 4-hydroxylase catalyses the formation of 4- hydroxyproline that is essential to the proper three-dimensional folding of newly synthesized procollagen chains. Alternatively spliced transcript variants encoding different isoforms have been described. Ontology Function L-ascorbic acid binding binding iron ion binding metal ion binding oxidoreductase activity oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors oxidoreductase activity, acting on single donors with incorporation of molecular oxygen,

incorporation of two atoms of oxygen procollagen-proline 4-dioxygenase activity Process oxidation reduction protein metabolic process Component endoplasmic reticulum endoplasmic reticulum lumen

Appendices 284 Name Gene Symbol Fold up regulation */^ Serum amyloid A1 SAA1 3.10 / 3.50 Ontology Function G-protein-coupled receptor binding lipid transporter activity Process acute-phase response elevation of cytosolic calcium ion concentration lymphocyte chemotaxis macrophage chemotaxis negative regulation of inflammatory response neutrophil chemotaxis platelet activation positive regulation of cell adhesion positive regulation of interleukin-1 secretion regulation of protein secretion Component Extracellular Region

Name Gene Symbol Fold up regulation */^ Plasminogen activator, urokinase receptor PLAUR 2.05 / 2.08 This gene encodes the receptor for urokinase plasminogen activator and, given its role in localizing and promoting plasmin formation, likely influences many normal and pathological processes related to cell-surface plasminogen activation and localized degradation of the extracellular matrix. It binds both the proprotein and mature forms of urokinase plasminogen activator and permits the activation of the receptor-bound pro-enzyme by plasmin. The protein lacks transmembrane or cytoplasmic Summary domains and may be anchored to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) moiety following cleavage of the nascent polypeptide near its carboxy-terminus. However, a soluble protein is also produced in some cell types. Alternative splicing results in multiple transcript variants encoding different isoforms. The proprotein experiences several post-translational cleavage reactions that have not yet been fully defined. Ontology Function U-plasminogen activator receptor activity enzyme binding Process attachment of GPI anchor to protein blood coagulation cell motility chemotaxis regulation of proteolysis signal transduction Component anchored to membrane cell surface extracellular region extrinsic to membrane integral to membrane plasma membrane

Appendices 285 Name Gene Symbol Fold up regulation */^ collagen, type VI, alpha 3 COL6A3 2.52 / 2.14 This gene encodes the  3 chain, one of the three  chains of type VI collagen, a beaded filament collagen found in most connective tissues. The  3 chain of type VI collagen is much larger than the  1 and 2 chains. This difference in size is largely due to an increase in the number of subdomains, similar to von Willebrand Factor type A domains, found in the amino terminal globular domain of all Summary the alpha chains. These domains have been shown to bind extracellular matrix proteins, an interaction that explains the importance of this collagen in organizing matrix components. Mutations in the type VI collagen genes are associated with Bethlem myopathy. Multiple transcript variants have been identified that encode proteins with N-terminal globular domains of varying sizes. Ontology Function protein binding serine-type endopeptidase inhibitor activity structural molecule activity Process cell adhesion muscle development phosphate transport Component collagen type VI cytoplasm extracellular region proteinaceous extracellular matrix

Name Gene Symbol Fold up regulation */^ CD99 molecule- Antigen identified by monoclonal antibodies 12E7, F21 and CD99 1.80 / 1.77 O13 The protein encoded by this gene is a cell surface glycoprotein involved in leukocyte migration, T- cell adhesion, ganglioside GM1 and transmembrane protein transport, and T-cell death by a caspase-independent pathway. In addition, the encoded protein may have the ability to rearrange Summary the actin cytoskeleton and may also act as an oncosuppressor in osteosarcoma. Cyclophilin A binds to CD99 and may act as a signalling regulator of CD99. This gene is found in the pseudoautosomal region of chromosomes X and Y and escapes X-chromosome inactivation. Two transcript variants encoding different isoforms have been found for this gene. Ontology Function protein binding Process cell adhesion Component cytoplasm Integral to plasma membrane Plasma membrane

Appendices 286 Name Gene Symbol Fold up regulation */^ Annexin A1 ANXA1 2.13 / 2.09 Annexin I belongs to a family of Ca2+-dependent phospholipid binding proteins which have a molecular weight of approximately 35,000 to 40,000 and are preferentially located on the cytosolic face of the plasma membrane. Annexin I protein has an apparent relative molecular mass of 40 Summary kDa, with phospholipase A2 inhibitory activity. Since phospholipase A2 is required for the biosynthesis of the potent mediators of inflammation, prostaglandins and leukotrienes, annexin I may have potential anti-inflammatory activity. Ontology Function calcium ion binding calcium-dependent phospholipid binding phospholipase A2 inhibitor activity phospholipase inhibitor activity protein binding, bridging receptor binding structural molecule activity Process anti-apoptosis arachidonic acid secretion cell cycle cell motility cell surface receptor linked signal transduction inflammatory response keratinocyte differentiation lipid metabolic process peptide cross-linking regulation of cell proliferation Component basolateral plasma membrane cilium cornified envelope cytoplasm membrane nucleus sarcolemma

Appendices 287 Name Gene Symbol Fold up regulation */^ Phosphodiesterase 4B, cAMP-specific (dunce (Drosophila) -homolog PDE4B 1.86 /1.86 phosphodiesterase E4) This gene is a member of the type IV, cyclic AMP (cAMP)-specific, cyclic nucleotide phosphodiesterase (PDE) family. Cyclic nucleotides are important second messengers that regulate and mediate a number of cellular responses to extracellular signals, such as hormones, light, and neurotransmitters. The cyclic nucleotide phosphodiesterases (PDEs) regulate the cellular Summary concentrations of cyclic nucleotides and thereby play a role in signal transduction. This gene encodes a protein that specifically hydrolyses cAMP. Altered activity of this protein has been associated with schizophrenia and bipolar affective disorder. Alternate transcriptional splice variants, encoding different isoforms, have been characterized. Ontology Function 3',5'-cyclic-AMP phosphodiesterase activity 3',5'-cyclic-nucleotide phosphodiesterase activity hydrolase activity Process Signal transduction Component Insoluble fraction Soluble fraction

Name Gene Symbol Fold up regulation */^ Early growth response 1 EGR1 3.31 / 3.30 The protein encoded by this gene belongs to the EGR family of C2H2-type zinc-finger proteins. It is a nuclear protein and functions as a transcriptional regulator. The products of target genes it Summary activates are required for differentiation and mitogenesis. Studies suggest this is a cancer suppressor gene. Ontology Function metal ion binding transcription activator activity transcription factor activity zinc ion binding Process differentiation learning and/or memory negative regulation of transcription from RNA polymerase II promoter positive regulation of transcription positive regulation of transcription from RNA polymerase II promoter regulation of transcription, DNA-dependent Component Intracellular nucleus

Appendices 288 Name Gene Symbol Fold up regulation */^ Triosephosphate isomerase 1 TPI1 1.86 / 1.93 Ontology Function isomerase activity triose-phosphate isomerase activity Process fatty acid biosynthetic process gluconeogenesis glyceraldehyde-3-phosphate metabolic process glycolysis metabolic process pentose-phosphate shunt Component Cytosol

Name Gene Symbol Fold up regulation */^ Guanylate binding protein 2, interferon- GBP2 2.38 / 2.21 inducible Interferons are cytokines that have antiviral effects and inhibit tumour cell proliferation. They induce a large number of genes in their target cells, including those coding for the guanylate-binding Summary proteins (GBPs). GBPs are characterized by their ability to specifically bind guanine nucleotides (GMP, GDP, and GTP). The protein encoded by this gene is a GTPase that converts GTP to GDP and GMP. Ontology Function GTP binding GTPase activity nucleotide binding Process Immune Response Component Plasma membrane

Name Gene Symbol Fold up regulation */^ Serum amyloid A2 SAA2 2.99 / 3.01 Ontology Process Acute-Phase Response Component Extracellular Region

Appendices 289 Name Gene Symbol Fold up regulation */^ CD1C antigen, c polypeptide CD1C 2.67 / 2.48 This gene encodes a member of the CD1 family of transmembrane glycoproteins, which are structurally related to the major histocompatibility complex (MHC) proteins and form heterodimers with beta-2-microglobulin. The CD1 proteins mediate the presentation of primarily lipid and glycolipid antigens of self or microbial origin to T cells. The contains five CD1 family Summary genes organized in a cluster on chromosome 1. The CD1 family members are thought to differ in their cellular localization and specificity for particular lipid ligands. The protein encoded by this gene is broadly distributed throughout the endocytic system via a tyrosine-based motif in the cytoplasmic tail. Alternatively spliced transcript variants of this gene have been observed, but their full-length nature is not known. Ontology Process antigen processing and presentation immune response Component endosome endosome membrane integral to plasma membrane plasma membrane

Name Gene Symbol Fold up regulation */^ Arylsulfatase B ARSB 2.79 / 2.35 Arylsulfatase B encoded by this gene belongs to the sulfatase family. The arylsulfatase B homodimer hydrolyzes sulfate groups of N-Acetyl-D-galactosamine, chondriotin sulfate, and Summary dermatan sulfate. The protein is targeted to the lysozyme. Mucopolysaccharidosis type VI is an autosomal recessive lysosomal storage disorder resulting from a deficiency of arylsulfatase B. Two alternatively spliced transcript variants encoding distinct isoforms have been found for this gene. Ontology Function N-acetylgalactosamine-4-sulfatase activity arylsulfatase activity calcium ion binding catalytic activity hydrolase activity sulfuric ester hydrolase activity Process glycosaminoglycan metabolic process lysosomal transport lysosome organization and biogenesis metabolic process Component Lysosome

Appendices 290  

APPENDIX D

D.1 Summary of Genes Obtained from All Three Methodologies

Table D.1. Gene lists by t-test. The table also indicated with genes appeared in the other data analyses. All values are given with terms of up regulation in MS5 supported samples thus for values < 1 there is down regulation in MS5 supported sample. t test Fold change Range of individual Fold change 1st sample fold changes appearance of the gene in the Gene 1st Calculation other gene sets Calculation * Min Max ^ TERF1 1.157 1.158 1.089 1.221 T-2640, T-70, S HIF1A 1.230 1.231 1.140 1.383 T-2640, T-70, S P4HA1 2.409 2.427 2.003 3.263 T-2640, T-70, S, G-3, G-5 NDFIP2 2.197 2.361 1.295 3.296 T-2640, T-70, S LSM14A 0.897 0.898 0.862 0.964 T-2640, T-70 SPCS3 1.414 1.421 1.196 1.664 T-2640, T-70, S BNIP1 1.560 1.574 1.198 1.918 T-2640, T-70, S CLPP 0.888 0.889 0.810 0.921 T-2640, T-70 ESTs 1.429 1.442 1.219 1.747 T-2640, T-70, S HsKG11C5 MAP2K1 1.432 1.457 1.127 1.648 T-2640, T-70, S P4HA2 1.985 2.141 1.179 3.300 T-2640, T-70, S C7orf50 1.617 1.655 1.235 2.364 T-2640, T-70, S HGSNAT 1.445 1.485 1.161 1.993 T-2640, T-70, S SYNE2 1.459 1.462 1.229 1.609 T-2640, T-70, S PGK1 1.558 1.603 1.187 2.019 T-2640, T-70, S FBLN2 1.769 1.787 1.432 2.125 T-2640, T-70, S RAB17 1.774 1.846 1.182 2.513 T-2640, T-70 ITGAL 0.591 0.609 0.440 0.792 T-2640, T-70 ESTs R43250a6 0.639 0.641 0.531 0.743 T-2640, T-70 ESTs 1.308 1.323 1.112 1.460 T-2640, T-70 HsKG77E6 " #$%#&'()* + &,' -+.-##/0#' 1'.-  &$'##$%#&% '(2+&3 $'##.(2+&3%& %00 &'(&3'.&4+.-&3' 5.6  &+78 #$%#&'()*&4+.-&3'2+&352+&3%&  &+ '$3$ '0.(+.-/0#'.(&3'.,' -+.-&3'/7 6 F&0= -'.' /&3' &'&G6F&0-'.' /&3' &'&G6F&0-'.' /&3' &'&G F  G6F'.'0 +.- .#*+H 7#($%& +.##/0#'G6F'.'0 +.-.#*+H7#($%& +.##/0#'7

Appendices 291 Table D.2. Gene lists by SAM. The table also indicated with genes appeared in the other data analyses. All values are given with terms of up regulation in MS5 supported samples thus for values < 1 there is down regulation in MS5 supported sample. SAM Fold change Range of individual Fold change 1st sample fold changes appearance of the gene in the Gene 1st Calculation other gene sets Calculation * Min Max ^ TERF1 1.157 1.158 1.089 1.221 T-2640, T-70 HIF1A 1.23 1.231 1.140 1.383 T-2640, T-70, T-20 P4HA1 2.409 2.427 2.003 3.263 T-2640, T-70, T-20, G-3, G-5 NDFIP2 2.197 2.361 1.295 3.296 T-2640, T-70, T-20 SPCS3 1.414 1.421 1.196 1.664 T-2640, T-70, T-20 BNIP1 1.56 1.574 1.198 1.918 T-2640, T-70, T-20 ESTs 1.429 1.442 1.219 1.747 T-2640, T-70, T-20 HsKG11C5 MAP2K1 1.432 1.457 1.127 1.648 T-2640, T-70, T-20 P4HA2 1.985 2.141 1.179 3.300 T-2640, T-70, T-20 C7orf50 1.617 1.655 1.235 2.364 T-2640, T-70, T-20 HGSNAT 1.445 1.485 1.161 1.993 T-2640, T-70, T-20 FBLN2 1.769 1.787 1.432 2.125 T-2640, T-70, T-20 PGK1 1.558 1.603 1.187 2.019 T-2640, T-70, T-20 SYNE2 1.459 1.462 1.229 1.609 T-2640, T-70, T-20 " #$%#&'()* + &,' -+.-##/0#' 1'.-  &$'##$%#&% '(2+&3 $'##.(2+&3%& %00 &'(&3'.&4+.-&3' 5.6  &+78 #$%#&'()*&4+.-&3'2+&352+&3%&  &+ '$3$ '0.(+.-/0#'.(&3'.,' -+.-&3'/7 6 F&0= -'.' /&3' &'&G6F&0-'.' /&3' &'&G6F&0-'.' /&3' &'&G F  G6F'.'0 +.- .#*+H 7#($%& +.##/0#'G6F'.'0 +.-.#*+H7#($%& +.##/0#'7

Appendices 292

Table D.3. Gene lists by GeneSpring. The table also indicated with genes appeared in the other data analyses. All values are given with terms of up regulation in MS5 supported samples thus for values < 1 there is down regulation in MS5 supported sample. GENESPRING Fold change Range of individual Fold change 1st sample fold changes appearance of the gene in the Gene 1st Calculation other gene sets Calculation * Min Max ^ R4368f8 0.422 0.44 0.247 0.648 T-2640, G-5 R43335c9 0.04 0.051 0.023 0.115 T-2640, T-70, G-5 R43323g6 0.128 0.39 0.058 0.643 CYTL1 0.416 0.414 0.149 0.624 T-2640 BTN3A3 0.503 0.527 0.346 0.607 T-2640 R43192c6 0.416 0.456 0.237 0.583 T-2640 HARS 0.274 0.274 0.132 0.446 G-5 AHRR 0.381 0.409 0.270 0.550 T-2640, T-70 SV2C 0.444 0.464 0.302 0.621 T-2640 R43139f11 0.066 0.229 0.025 0.652 T-2640 R43236b3 0.224 0.37 0.078 0.583 R43141d10 0.472 0.49 0.410 0.609 T-2640 R43138g8 0.096 0.266 0.031 0.601 R43148f9 0.469 0.441 0.281 0.585 T-2640 P4HA1 2.409 2.427 2.003 3.263 T-2640, T-70, T-20, S, G-5 SAA1 3.101 3.504 1.575 8.357 T-2640 PLAUR 2.05 2.08 1.596 2.587 T-2640 PLAUR 2 2.011 1.521 2.471 T-2640 COL6A3 2.524 2.136 1.620 2.943 ANXA1 2.131 2.085 1.519 3.388 CD99 1.798 1.768 1.554 1.945 T-2640 PDE4B 1.864 1.863 1.680 2.239 T-2640 EGR1 3.305 3.302 2.668 4.388 T-2640, G-5 TPI1 1.855 1.934 1.504 2.599 T-2640 GBP2 2.378 2.208 1.613 3.356 SAA2 2.986 3.013 1.745 5.371 T-2640 CD1C 2.673 2.475 1.517 4.104 T-2640 ARSB 2.784 2.354 1.559 3.571 " #$%#&'()* + &,' -+.-##/0#' 1'.-  &$'##$%#&% '(2+&3 $'##.(2+&3%& %00 &'(&3'.&4+.-&3' 5.6  &+78 #$%#&'()*&4+.-&3'2+&352+&3%&  &+ '$3$ '0.(+.-/0#'.(&3'.,' -+.-&3'/7 6 F&0= -'.' /&3' &'&G6F&0-'.' /&3' &'&G6F&0-'.' /&3' &'&G F  G6F'.'0 +.- .#*+H 7#($%& +.##/0#'G6F'.'0 +.-.#*+H7#($%& +.##/0#'7

Appendices 293

 

APPENDIX E

E.1 Effects of FL on Gene Expression in ALL Xenograft cells

SAM slope method analysis of the gene expression in ALL xenograft cells by FL over time.

E.1.1 Xenograft: ALL-3 Details: FDR = 81% Genes = 1414 total, after removal of ESTs, EST, KIAA contain genes, and blank entries revised total is 1270.

Table E.1. Top genes by slope in ALL-3 xenograft cells. Rank Gene Name Symbol 2H 6H 24H 11 collagen, type XI, alpha 1 COL11A1 1.06 0.36 14.91 16 caveolin 2 CAV2 0.88 0.62 2.10 17 CD19 antigen CD19 1.25 1.13 1.45 20 alkaline phytoceramidase PHCA 1.00 0.54 3.69 calcium channel, voltage-dependent, alpha 1H 24 CACNA1H 0.55 0.56 1.36 subunit 31 platelet-activating factor acetylhydrolase 2 (40kD) PAFAH2 0.88 1.35 2.56 34 neuronal leucine-rich repeat protein-3 1.07 0.62 1.98 35 methylmalonyl Coenzyme A mutase MUT 0.86 0.60 1.66 37 myosin regulatory light chain interacting protein MYLIP 0.97 0.93 1.27 42 Cdc42 effector protein 2 CRLF1 0.47 0.36 1.17 43 cytokine receptor-like factor 1 CDC42EP2 0.64 0.38 2.52 44 transcription factor Dp-1 TFDP1 0.75 0.72 1.33 45 musculin (activated B-cell factor-1) MSC 0.79 0.59 2.72 46 Wolfram syndrome 1 (wolframin) WFS1 0.51 0.36 1.64 49 B9 protein B9D1 0.72 0.65 1.19 adaptor protein containing pH domain, PTB domain 51 APPL1 1.18 0.77 1.92 and motif 53 multiple PDZ domain protein MPDZ 0.59 0.48 1.20 ELAV (embryonic lethal, abnormal vision, Drosophila)- 54 ELAVE4 1.25 0.79 1.69 like 4 (Hu antigen D) transmembrane protein induced by tumour necrosis 55 TMEM120A 0.69 0.62 0.97 factor alpha Appendices 295 62 EphA2 EPHA2 0.46 0.35 1.24 64 WNT1 inducible signaling pathway protein 2 WISP2 0.57 0.66 1.70 71 protein arginine N-methyltransferase 3 1.23 0.97 1.46 VAMP (vesicle-associated membrane protein)- 75 VAPB 0.71 0.60 1.00 associated protein B and C 77 ATG3 autophagy related 3 homolog (S. cerevisiae) ATG3 1.15 0.81 1.70 79 phosphoinositide-3-kinase, class 2, beta polypeptide PIK3C2B 1.15 1.02 1.47 ATPase, H+ transporting, lysosomal (vacuolar proton 80 ATP6V0A1 0.70 0.61 1.35 pump) non-catalytic accessory protein 1A (110/116kD ets variant gene 4 (E1A enhancer-binding protein, 82 ETV4 0.46 0.51 1.42 E1AF) 86 collagen, type IV, alpha 4 COL4A4 0.58 0.58 1.20 89 protease, serine, 16 (thymus) PRSS16 0.53 0.55 0.94 sterile-alpha motif and leucine zipper containing 93 ZAK 1.30 0.34 3.12 kinase AZK 94 CDW52 antigen (CAMPATH-1 antigen) CD52 1.91 1.48 2.64 96 dachshund homolog (Drosophila) DACH1 1.17 0.68 1.88 98 insulin-like growth factor 1 receptor IGF1R 0.58 0.54 1.77 102 GATA-binding protein 6 GATA6 0.79 0.71 1.06 103 insulin-like growth factor 1 receptor IGFR1 0.57 0.55 1.63 104 Mevalonate (diphospho) decarboxylase NVD 0.63 0.42 1.20 105 homeo box C10 HOXC10 1.03 0.73 1.82 111 Synovial sarcoma, X breakpoint 1 SSX1 0.91 0.84 1.88 112 prostaglandin I2 (prostacyclin) synthase PTGIS 1.39 0.81 1.55 116 junctional adhesion molecule 2 JAM2 1.32 0.66 1.84 119 aminoacylase 1 ACY1 0.74 0.44 1.27 MADS box transcription enhancer factor 2, 125 MEF2D 1.22 1.65 5.64 polypeptide D (myocyte enhancer factor 2D) cytochrome b5 outer mitochondrial membrane 126 CYB5B 0.44 0.43 2.28 precursor 128 transglutaminase 4 (prostate) TGM4 1.36 1.02 1.31 129 supervillin SVIL 1.20 0.85 4.74 carboxylesterase 1 (monocyte / macrophage serine 134 CES1 0.60 0.53 1.88 esterase 1) likely ortholog of mouse Hes6 neuronal differentiation 138 TP53 1.45 0.92 1.77 gene tumour necrosis factor receptor superfamily, member 141 TNFRSF11B 1.48 0.49 2.94 11b (osteoprotegerin)

Appendices 296 E.1.2 Xenograft: ALL-17 Xenograft: ALL17 Details: FDR = 75.6% Genes = 1469 total, after removal of ESTs, EST, KIAA contain genes, and blank entries revised total is 631

Table E.2. Top genes by slope in ALL-17 xenograft cells. Rank Gene name Symbol 2H 6H 24H 4 RAB2, member RAS oncogene family-like RAB2A 1.13 1.15 1.41 protein kinase C and casein kinase substrate in 8 PACSIN1 1.21 1.25 1.67 neurons 1 10 enolase 2, (gamma, neuronal) ENO2 1.06 1.09 1.88 LIM domain-containing preferred translocation partner 13 LPP 0.96 0.99 1.31 in lipoma 17 syntaxin binding protein 6 (amisyn) STXBP6 0.99 1.26 13.06 18 phosphoinositide-3-kinase, class 2, beta polypeptide PIK3C2B 1.10 1.00 1.39 Fc fragment of IgG, low affinity IIb, receptor for 19 FCGR2B 1.11 1.31 1.54 (CD32) 21 arrestin, beta 1 ARRB1 1.02 1.07 1.59 24 eukaryotic translation initiation factor 2C, 1 EIF2C1 0.98 0.89 1.55 36 ATPase, Na+/K+ transporting, beta 1 polypeptide ATP1B1 1.24 1.18 1.81 38 calbindin 2, (29kD, calretinin) CALB2 1.06 1.02 1.25 39 CD1A antigen, a polypeptide CD1A 0.92 1.07 1.29 42 myosin regulatory light chain interacting protein MYLIP 0.74 0.92 1.24 lanosterol synthase (2,3-oxidosqualene-lanosterol 50 LSS 1.07 0.90 1.35 cyclase) low density lipoprotein-related protein 1B (deleted in 51 LRP1B 0.93 1.12 1.35 tumours) 56 regulator of G-protein signalling 10 RGS10 1.10 1.26 1.56 60 tensin TNS1 1.02 0.96 1.24 63 pleiotropic regulator 1 (PRL1, Arabidopsis homolog) PLRG1 0.97 0.90 1.27 65 insulin-like 6 INSL6 0.99 1.29 1.80 66 phospholipase C, epsilon 2 PLCL2 0.89 0.91 1.06 69 neuregulin 2 NRG2 0.76 1.09 1.27 70 macrophage receptor with collagenous structure MARCO 0.97 1.00 1.20 72 proprotein convertase subtilisin/kexin type 5 PCSK5 0.91 0.96 1.18 73 fibroblast growth factor 12B FGF12 0.58 0.50 1.60 phosphoinositide-3-kinase, catalytic, delta 74 PIK3CD 0.82 1.04 1.27 polypeptide 75 properdin P factor, complement CFP 1.17 1.02 1.46 proline-serine-threonine phosphatase interacting 79 PSTPIP1 1.01 1.31 1.83 protein 1 solute carrier family 18 (vesicular monoamine), 80 SLC18A2 0.98 0.91 1.24 member 2

Appendices 297 E.1.3 Xenograft: ALL-19

Xenograft: ALL19 Details: FDR = 61% Genes = 1120 total, after removal of ESTs, EST, KIAA contain genes, and blank entries revised total is 480.

Table E.3. Top genes by slope in ALL-17 xenograft cells. Rank Gene name 2H 6H 24H 8 apolipoprotein C-IV APOC4 1.44 1.78 0.33 14 testis specific basic protein BPY2 0.97 1.02 0.47 16 ATPase, H+ transporting, lysosomal V0 subunit a1 ATP6V0A1 1.50 1.61 0.76 20 parathyroid 2 PTH2R 1.12 1.16 0.63 24 small proline-rich protein 3 SPRR3 1.44 1.73 0.52 26 sine oculis (Drosophila) homolog 6 SIX6 1.01 0.94 0.49 29 kaptin (actin-binding protein) KPTN 1.05 0.88 0.58 30 lung type-I cell membrane-associated glycoprotein PDPN 0.95 1.16 0.73 32 semenogelin I SEMG1 1.19 1.15 0.82 Homo sapiens mRNA for ATP-dependent 39 YME1L1 0.79 1.30 0.38 metalloprotease YME1L 45 ribosomal protein S3 RPS3 1.15 1.28 0.87 56 insulin-like growth factor-binding protein 4 IGFBP4 0.63 1.40 0.51 60 NTT5 protein SLC6A16 1.42 1.38 0.84 82 GRB2-associated binding protein 1 GAB1 0.96 1.13 0.90 89 HSPC156 protein STXBP6 2.30 1.28 1.01 90 protein kinase NYD-SP25 TPD52L3 2.28 0.36 0.10 92 f-box and leucine-rich repeat protein 7 FBXL7 1.30 1.43 0.86 93 lymphocyte-activation gene 3 LAG3 1.36 1.31 1.12 96 cell cycle related kinase CCRK 1.31 1.23 0.88 97 neighbour of BRCA1 gene 1 NBR1 1.42 1.38 0.55 99 collagen, type IV, alpha 4 COL4A4 1.73 1.58 0.73 103 complement component 4B C4B 1.05 0.84 0.67 105 neuronal specific transcription factor DAT1 LMO3 1.81 4.47 0.34 solute carrier family 5 (sodium/glucose cotransporter), 114 SLC5A1 1.05 1.17 0.75 member 1 117 cathepsin O CTSO 1.01 0.85 0.81 118 5-hydroxytryptamine (serotonin) receptor 4 HTR4 1.06 1.01 0.71 119 HLA-G histocompatibility antigen, class I, G HLA-G 0.96 0.98 0.93 143 IMAGE:1671163 interferon regulatory factor 4 IRF4 2.06 1.60 0.58 cell adhesion molecule with homology to L1CAM 164 CHL1 2.47 1.11 1.10 (close homologue of L1) chondroitin sulfate proteoglycan 4 (melanoma- 175 CSPG4 3.43 1.09 1.04 associated) 197 nucleolar protein ANKT NUSAP1 2.09 0.91 0.84

Appendices 298

 

APPENDIX F

F.1 Genes Two-Fold Differentially Regulated by FL in ALL-3

Xenograft Cells

Table F.1. Genes changed by greater than 2-fold in ALL-3 xenograft cells after 2 hrs. Gene Name Gene ID 2 hrs WAP four-disulfide core domain 2 WFDC2 0.004 Ubiquitin specific peptidase 24 USP24 0.007 Ubiquitin specific peptidase 53 USP53 0.010 RAB27B, member RAS oncogene family RAB27B 0.019 Cellular retinoic acid binding protein 2 CRABP2 0.021 UDP-N-acetyl-alpha-D-galactosamine:polypeptideN-acetylgalactosaminyltransferase-like 2 GALNTL2 0.023 Yip1 domain family, member 5 YIPF5 0.031 Protein phosphatase 4, regulatory subunit 1 PPP4R1 0.039 SAC3 domain containing 1 SAC3D1 0.066 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1 B4GALT1 0.067 Mitogen-activated protein kinase kinase kinase 4 MAP3K4 0.088 RAB6A, member RAS oncogene family RAB6A 0.090 DNA (cytosine-5-)-methyltransferase 3 alpha DNMT3A 0.094 LAG1 homolog, ceramide synthase 5 (S. cerevisiae) LASS5 0.098 Synaptotagmin-like 1 SYTL1 0.108 Glutamate receptor, ionotropic, N-methyl D-aspartate 2A GRIN2A 0.134 Distal-less homeobox 4 DLX4 0.148 Protein phosphatase 2, regulatory subunit B', alpha isoform PPP2R5A 0.149 IKAROS family zinc finger 1 (Ikaros) IKZF1 0.160 Cellular retinoic acid binding protein 2 CRABP2 0.176 Ankyrin repeat domain 38 ANKRD38 0.188 Interleukin 15 IL15 0.194 Signal transducer and activator of transcription 3 (acute-phase response factor) STAT3 0.204 KRR1, small subunit (SSU) processome component, homolog (yeast) KRR1 0.214 Frizzled homolog 1 (Drosophila) FZD1 0.216 Myeloid/lymphoid or mixed-lineage leukemia 5 (trithorax homolog, Drosophila) MLL5 0.217 Amyloid beta precursor protein (cytoplasmic tail) binding protein 2 APPBP2 0.240 Zinc finger, MYM-type 6 ZMYM6 0.268 Phosphatidylinositol glycan anchor biosynthesis, class X PIGX 0.274 TIA1 cytotoxic granule-associated RNA binding protein TIA1 0.274 Disabled homolog 2, mitogen-responsive phosphoprotein (Drosophila) DAB2 0.278

Appendices 299 Solute carrier family 24 (sodium/potassium/calcium exchanger), member 6 SLC24A6 0.278 Lymphocyte antigen 6 complex, locus K LY6K 0.287 UDP-glucose ceramide glucosyltransferase-like 1 UGCGL1 0.289 Notch homolog 3 (Drosophila) NOTCH3 0.293 Myosin, light chain 9, regulatory MYL9 0.299 Galanin GAL 0.319 La ribonucleoprotein domain family, member 6 LARP6 0.319 Complement component 4 binding protein, beta C4BPB 0.323 ATPase, Na+/K+ transporting, alpha 3 polypeptide ATP1A3 0.324 RAS guanyl releasing protein 3 (calcium and DAG-regulated) RASGRP3 0.325 Pregnancy-associated plasma protein A, pappalysin 1 PAPPA 0.331 Mof4 family associated protein 1 MRFAP1 0.333 Aspartate beta-hydroxylase ASPH 0.334 Dihydrolipoamide branched chain transacylase E2 DBT 0.336 Lysyl oxidase-like 1 LOXL1 0.336 Protocadherin 17 PCDH17 0.339 WD repeat domain 26 WDR26 0.341 Growth arrest-specific 1 GAS1 0.346 Tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2 TANC2 0.348 Ankyrin repeat and sterile alpha motif domain containing 1A ANKS1A 0.349 Phospholipase A2 receptor 1, 180kDa PLA2R1 0.350 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 PLOD2 0.357 Ets variant gene 6 (TEL oncogene) ETV6 0.360 PDZ and LIM domain 5 PDLIM5 0.363 Cholecystokinin CCK 0.365 Embryonal Fyn-associated substrate EFS 0.367 Musculin (activated B-cell factor-1) MSC 0.371 SUB1 homolog (S. cerevisiae) SUB1 0.371 Microsomal glutathione S-transferase 1 MGST1 0.374 Early B-cell factor 3 EBF3 0.376 Lon peptidase 2, peroxisomal LONP2 0.378 Transducin (beta)-like 1X-linked TBL1X 0.384 Leucine rich repeat containing 16 LRRC16 0.385 B-cell CLL/lymphoma 11B (zinc finger protein) BCL11B 0.385 Solute carrier family 44, member 1 SLC44A1 0.386 Asparagine-linked glycosylation 8 homolog (S. cerevisiae, alpha-1,3-glucosyltransferase) ALG8 0.388 critical region gene 8 DSCR8 0.392 Tetraspanin 12 TSPAN12 0.392 Phosphatidylinositol 4-kinase, catalytic, beta polypeptide PIK4CB 0.398 Myosin, heavy chain 9, non-muscle MYH9 0.410 ClpB caseinolytic peptidase B homolog (E. coli) CLPB 0.410 Zinc finger protein 526 ZNF526 0.416 Solute carrier family 25 (mitochondrial carrier, Aralar), member 12 SLC25A12 0.417 Neuronal pentraxin II NPTX2 0.418 Solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 SLC7A2 0.419 Killer cell lectin-like receptor subfamily C, member 2 KLRC2 0.420 Cysteine-rich, angiogenic inducer, 61 CYR61 0.423 Actin binding LIM protein 1 ABLIM1 0.424 Integrin-binding sialoprotein (bone sialoprotein, bone sialoprotein II) IBSP 0.425 Netrin 4 NTN4 0.429 Thrombospondin 3 THBS3 0.431 Insulin-like growth factor 2 (somatomedin A) IGF2 0.432 Mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, isozyme A MGAT4A 0.433 Carcinoembryonic antigen-related cell adhesion molecule 8 CEACAM8 0.435

Appendices 300 Protein tyrosine phosphatase, receptor type, D PTPRD 0.436 Transmembrane protein 30B TMEM30B 0.437 Disabled homolog 2, mitogen-responsive phosphoprotein (Drosophila) DAB2 0.437 Beta-site APP-cleaving enzyme 1 BACE1 0.438 Aminolevulinate, delta-, synthase 2 (sideroblastic/hypochromic ) ALAS2 0.442 G protein-coupled receptor 161 GPR161 0.443 SAM and SH3 domain containing 1 SASH1 0.444 Protein kinase D3 PRKD3 0.444 Vasohibin 1 VASH1 0.447 Cyclin-dependent kinase 5 CDK5 0.447 Mitogen-activated protein kinase 13 MAPK13 0.450 V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog KIT 0.453 Prostaglandin F2 receptor negative regulator PTGFRN 0.453 EPH receptor A2 EPHA2 0.457 Ets variant gene 4 (E1A enhancer binding protein, E1AF) ETV4 0.459 Wingless-type MMTV integration site family, member 5A WNT5A 0.465 Serine hydroxymethyltransferase 2 (mitochondrial) SHMT2 0.465 Met proto-oncogene (hepatocyte growth factor receptor) MET 0.467 Creatine kinase, brain CKB 0.467 RAS protein activator like 2 RASAL2 0.476 Fibronectin 1 FN1 0.478 Fibronectin leucine rich transmembrane protein 3 FLRT3 0.480 Nuclear transcription factor Y, beta NFYB 0.480 Aquaporin 3 (Gill blood group) AQP3 0.485 Neurobeachin NBEA 0.485 Histocompatibility (minor) HA-1 HMHA1 0.486 Major histocompatibility complex, class I, C HLA-C 0.487 Keratin 14 (epidermolysis bullosa simplex, Dowling-Meara, Koebner) KRT14 0.488 Sidekick homolog 1 (chicken) SDK1 0.490 Transmembrane protein 37 TMEM37 0.493 Chloride channel, calcium activated, family member 1 CLCA1 0.494 Growth hormone regulated TBC protein 1 GRTP1 0.495 F-box protein 32 FBXO32 0.496 Cholinergic receptor, nicotinic, alpha 5 CHRNA5 0.498 Zinc finger protein 532 ZNF532 0.500 Heparan sulfate (glucosamine) 3-O-sulfotransferase 3A1 HS3ST3A1 0.500 Gap junction protein, alpha 1, 43kDa GJA1 0.500 Homeobox HB9 HLXB9 2.003 BMX non-receptor tyrosine kinase BMX 2.007 TSC22 domain family, member 1 TSC22D1 2.007 Purinergic receptor P2Y, G-protein coupled, 14 P2RY14 2.009 Calcium binding tyrosine-(Y)-phosphorylation regulated (fibrousheathin 2) CABYR 2.009 Transient receptor potential cation channel, subfamily M, member 2 TRPM2 2.010 Phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, type III PIP5K3 2.012 Phenylalanine hydroxylase PAH 2.013 Prolactin receptor PRLR 2.018 RAP2B, member of RAS oncogene family RAP2B 2.020 Myeloid cell leukemia sequence 1 (BCL2-related) MCL1 2.020 Regulator of G-protein signalling 1 RGS1 2.021 Docking protein 2, 56kDa DOK2 2.022 Exportin 5 XPO5 2.024 CD163 molecule-like 1 CD163L1 2.027 Ankyrin repeat domain 47 ANKRD47 2.038 Glutaminase GLS 2.038

Appendices 301 CD1c molecule CD1C 2.040 G protein-coupled receptor, family C, group 5, member C GPRC5C 2.041 Hairy/enhancer-of-split related with YRPW motif-like HEYL 2.041 RAB37, member RAS oncogene family RAB37 2.043 Malignant fibrous histiocytoma amplified sequence 1 Mfhas1 2.046 Zinc finger, SWIM-type containing 6 ZSWIM6 2.047 Adenylate cyclase 2 (brain) ADCY2 2.048 Carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2 CHST2 2.049 Interferon gamma receptor 2 (interferon gamma transducer 1) IFNGR2 2.049 ADP-ribosylation factor-like 5B ARL5B 2.051 Mitochondrial ribosomal protein L12 MRPL12 2.052 Transmembrane protein 49 TMEM49 2.054 Coenzyme Q5 homolog, methyltransferase (S. cerevisiae) COQ5 2.067 Zinc finger protein 642 ZNF642 2.069 Ubiquitin-conjugating enzyme E2N (UBC13 homolog, yeast) UBE2N 2.069 Small proline-rich protein 1B (cornifin) SPRR1B 2.070 Interferon-induced protein with tetratricopeptide repeats 2 IFIT2 2.074 Ras association (RalGDS/AF-6) and pleckstrin homology domains 1 RAPH1 2.074 Golgi phosphoprotein 4 GOLPH4 2.075 Integrator complex subunit 6 INTS6 2.075 Phosphodiesterase 4D interacting protein (myomegalin) PDE4DIP 2.077 Coagulation factor II (thrombin) receptor-like 3 F2RL3 2.079 Chloride intracellular channel 5 CLIC5 2.084 Angiopoietin-like 2 ANGPTL2 2.090 Non-metastatic cells 5, protein expressed in (nucleoside-diphosphate kinase) NME5 2.090 Fibroblast growth factor 12 FGF12 2.096 Rab15 effector protein REP15 2.097 Erythrocyte membrane protein band 4.1-like 3 EPB41L3 2.100 Cold shock domain containing E1, RNA-binding CSDE1 2.101 GTP binding protein overexpressed in skeletal muscle GEM 2.103 Proprotein convertase subtilisin/kexin type 6 PCSK6 2.103 Mal, T-cell differentiation protein 2 MAL2 2.105 Zona pellucida binding protein ZPBP 2.105 Growth arrest and DNA-damage-inducible, beta GADD45B 2.120 Nuclear factor of activated T-cells 5, tonicity-responsive NFAT5 2.123 Heparin-binding EGF-like growth factor HBEGF 2.124 SERTA domain containing 1 SERTAD1 2.124 Protease, serine, 23 PRSS23 2.124 Tyrosinase (oculocutaneous albinism IA) TYR 2.130 Adhesion molecule with Ig-like domain 2 AMIGO2 2.131 TSC22 domain family, member 1 TSC22D1 2.132 Chemokine (C-C motif) ligand 23 CCL23 2.135 STAM binding protein-like 1 STAMBPL1 2.140 Mannose receptor, C type 1 MRC1 2.140 Laminin, alpha 1 LAMA1 2.142 Zic family member 2 (odd-paired homolog, Drosophila) ZIC2 2.144 Early growth response 2 (Krox-20 homolog, Drosophila) EGR2 2.146 G protein-coupled receptor 137C GPR137C 2.155 Rab15 effector protein REP15 2.155 Aquaporin 4 AQP4 2.156 Major histocompatibility complex, class I, C HLA-C 2.159 PiggyBac transposable element derived 5 PGBD5 2.165 Acid phosphatase 5, tartrate resistant ACP5 2.175 G protein-coupled receptor 65 GPR65 2.182

Appendices 302 CASP2 and RIPK1 domain containing adaptor with death domain CRADD 2.183 Ventricular zone expressed PH domain homolog 1 (zebrafish) VEPH1 2.185 Interleukin 11 IL11 2.187 Lactoperoxidase LPO 2.192 Angiotensin II receptor, type 1 AGTR1 2.196 Transmembrane protein 16B TMEM16B 2.208 CD44 molecule (Indian blood group) CD44 2.208 Thyrotropin-releasing hormone degrading enzyme TRHDE 2.209 CD2 molecule CD2 2.217 Granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine esterase 3) GZMA 2.221 Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G APOBEC3G 2.227 Ankyrin repeat domain 57 ANKRD57 2.236 G protein-coupled receptor 65 GPR65 2.239 Zinc finger protein 513 ZNF513 2.240 Natural killer cell group 7 sequence NKG7 2.242 Chemokine (C-C motif) ligand 2 CCL2 2.243 Thrombomodulin THBD 2.246 Glucose 6 phosphatase, catalytic, 3 G6PC3 2.249 Coatomer protein complex, subunit epsilon COPE 2.252 START domain containing 4, sterol regulated STARD4 2.258 Transducin-like enhancer of split 3 (E(sp1) homolog, Drosophila) TLE3 2.261 ADAM metallopeptidase domain 8 ADAM8 2.262 Cartilage intermediate layer protein, nucleotide pyrophosphohydrolase CILP 2.278 AT hook, DNA binding motif, containing 1 AHDC1 2.281 BCL2-associated athanogene 4 BAG4 2.283 Ribosomal protein S28 RPS28 2.299 G protein-coupled receptor 68 GPR68 2.304 Relaxin 1 RLN1 2.318 TNF receptor-associated factor 3 TRAF3 2.337 Thrombomodulin THBD 2.339 Insulin-like growth factor 1 receptor IGF1R 2.351 Transducin-like enhancer of split 3 (E(sp1) homolog, Drosophila) TLE3 2.356 Steroid-5-alpha-reductase, alpha polypeptide 2 (3-oxo-5 alpha-steroid delta 4- SRD5A2 2.362 dehydrogenase alpha 2) RER1 retention in endoplasmic reticulum 1 homolog (S. cerevisiae) RER1 2.364 Tissue factor pathway inhibitor 2 TFPI2 2.365 COMM domain containing 3 COMMD3 2.372 Coiled-coil domain containing 62 CCDC62 2.374 Hemoglobin, zeta HBZ 2.378 Leucine rich repeat containing 6 LRRC6 2.384 Amplified in breast cancer 1 ABC1 2.399 Gremlin 1, cysteine knot superfamily, homolog (Xenopus laevis) GREM1 2.401 Nephroblastoma overexpressed gene NOV 2.402 Keratin 23 (histone deacetylase inducible) KRT23 2.413 EF-hand domain family, member D1 EFHD1 2.436 Latent transforming growth factor beta binding protein 2 LTBP2 2.437 Small proline-rich protein 2A SPRR2A 2.445 UDP glucuronosyltransferase 2 family, polypeptide B17 UGT2B17 2.447 Prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) PTGS1 2.462 Gamma-glutamyltransferase 3 GGT3 2.463 Lymphocyte cytosolic protein 1 Lcp1 2.469 TNF receptor-associated factor 1 TRAF1 2.473 Ribosomal protein S28 RPS28 2.497 Carbohydrate (chondroitin) synthase 1 CHSY1 2.505 BTB and CNC homology 1, basic leucine zipper transcription factor 2 BACH2 2.510

Appendices 303 Tumor necrosis factor receptor superfamily, member 12A TNFRSF12A 2.518 Golgi autoantigen, golgin subfamily a, 1 GOLGA1 2.520 Sciellin SCEL 2.525 AXIN1 up-regulated 1 AXUD1 2.536 Transmembrane and coiled-coil domains 2 TMCO2 2.537 Interleukin 11 IL11 2.538 Neuroblastoma breakpoint family, member 1 NBPF1 2.546 Cystatin F (leukocystatin) CST7 2.557 Ankylosis, progressive homolog (mouse) ANKH 2.587 Guanidinoacetate N-methyltransferase GAMT 2.590 Pygopus homolog 1 (Drosophila) PYGO1 2.591 Replication factor C (activator 1) 2, 40kDa RFC2 2.609 Early growth response 3 EGR3 2.618 Neuroglobin NGB 2.621 NADH dehydrogenase (ubiquinone) flavoprotein 3, 10kDa NDUFV3 2.622 Spermatogenesis and oogenesis specific basic helix-loop-helix 2 SOHLH2 2.639 Lipase, endothelial LIPG 2.682 TNF receptor-associated factor 3 TRAF3 2.685 Transketolase-like 1 TKTL1 2.690 ATP/GTP binding protein 1 AGTPBP1 2.691 5'-nucleotidase, ecto (CD73) NT5E 2.709 Tetratricopeptide repeat domain 27 TTC27 2.723 Discs, large (Drosophila) homolog-associated protein 4 DLGAP4 2.725 UDP-glucose ceramide glucosyltransferase-like 2 UGCGL2 2.739 Wingless-type MMTV integration site family, member 7A WNT7A 2.751 HCG1776047 hCG_1776047 2.787 AT-binding transcription factor 1 ATBF1 2.797 SH2 domain protein 2A SH2D2A 2.806 Claudin 18 CLDN18 2.812 Chemokine (C-X-C motif) receptor 4 Cxcr4 2.849 Early growth response 1 EGR1 2.856 Ankylosis, progressive homolog (mouse) ANKH 2.870 Potassium inwardly-rectifying channel, subfamily J, member 2 KCNJ2 2.875 Polymerase (RNA) III (DNA directed) polypeptide H (22.9kD) POLR3H 2.878 Centrobin, centrosomal BRCA2 interacting protein CNTROB 2.882 BMP2 inducible kinase BMP2K 2.888 RAS, dexamethasone-induced 1 RASD1 2.922 P antigen family, member 1 (prostate associated) PAGE1 2.924 Pleckstrin homology-like domain, family A, member 1 PHLDA1 2.927 Citron (rho-interacting, serine/threonine kinase 21) CIT 2.930 Ankylosis, progressive homolog (mouse) ANKH 2.949 Cytotoxic and regulatory T cell molecule CRTAM 3.004 Rho GTPase activating protein 9 ARHGAP9 3.013 Neurotrophic tyrosine kinase, receptor, type 2 NTRK2 3.051 Chemokine (C-C motif) ligand 4-like 1 CCL4L1 3.064 Transmembrane protein 158 TMEM158 3.091 Interleukin 1, beta IL1B 3.096 Ubiquitination factor E4A (UFD2 homolog, yeast) UBE4A 3.131 SH3 domain and tetratricopeptide repeats 2 SH3TC2 3.136 Macrophage erythroblast attacher MAEA 3.139 Phosphatidylinositol 4-kinase type II PI4KII 3.161 Ankylosis, progressive homolog (mouse) ANKH 3.176 Pleckstrin homology domain containing, family F (with FYVE domain) member 1 PLEKHF1 3.202 Gremlin 1, cysteine knot superfamily, homolog (Xenopus laevis) GREM1 3.208

Appendices 304 5'-nucleotidase, ecto (CD73) NT5E 3.220 TNF receptor-associated factor 7 TRAF7 3.264 Granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1) GZMB 3.291 Colony stimulating factor 1 receptor, formerly McDonough feline sarcoma viral (v-fms) CSF1R 3.292 oncogene homolog ATPase, Class VI, type 11A ATP11A 3.305 Latrophilin 3 LPHN3 3.308 Dermokine DMKN 3.325 CD93 molecule CD93 3.348 Acidic (leucine-rich) nuclear phosphoprotein 32 family, member E ANP32E 3.349 Integrin-binding sialoprotein (bone sialoprotein, bone sialoprotein II) IBSP 3.367 Sperm mitochondria-associated cysteine-rich protein SMCP 3.401 UDP glucuronosyltransferase 2 family, polypeptide B17 UGT2B17 3.418 Erythrocyte membrane protein band 4.1 (elliptocytosis 1, RH-linked) EPB41 3.422 Calcium binding and coiled-coil domain 2 CALCOCO2 3.425 MAP6 domain containing 1 MAP6D1 3.446 Transmembrane protein 158 TMEM158 3.458 Clathrin, light chain (Lca) CLTA 3.476 ADAM metallopeptidase domain 19 (meltrin beta) ADAM19 3.485 Solute carrier family 35, member E1 SLC35E1 3.488 Gap junction protein, beta 2, 26kDa GJB2 3.489 Cyclin A1 CCNA1 3.526 Potassium large conductance calcium-activated channel, subfamily M, beta member 1 KCNMB1 3.535 Neural cell adhesion molecule 1 NCAM1 3.619 Tetratricopeptide repeat domain 17 TTC17 3.661 IL2-inducible T-cell kinase ITK 3.668 Ankyrin repeat and SOCS box-containing 1 ASB1 3.682 Forkhead box A3 FOXA3 3.704 Hemoglobin, beta HBB 3.757 Transcription factor EC TFEC 3.788 FK506 binding protein 6, 36kDa FKBP6 3.795 Chemokine (C-C motif) ligand 3-like 3 CCL3L3 3.840 Galactosylceramidase GALC 3.860 BCL2-related protein A1 BCL2A1 3.879 Protein phosphatase 1, regulatory (inhibitor) subunit 9B PPP1R9B 3.929 Cell growth regulator with EF-hand domain 1 CGREF1 3.998 Inositol 1,4,5-triphosphate receptor, type 2 ITPR2 4.084 Regulator of G-protein signalling 5 RGS5 4.129 Membrane associated guanylate kinase, WW and PDZ domain containing 1 MAGI1 4.198 Forkhead box P1 FOXP1 4.226 ATPase, Class V, type 10A ATP10A 4.239 Endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, 2 EDG2 4.244 Paf1, RNA polymerase II associated factor, homolog (S. cerevisiae) PAF1 4.259 FXYD domain containing ion transport regulator 2 FXYD2 4.305 Mitochondrial ribosomal protein S28 MRPS28 4.318 Ras-related associated with diabetes RRAD 4.477 Ecotropic viral integration site 2A EVI2A 4.499 Phosphoglucomutase 2-like 1 PGM2L1 4.657 Tenascin N TNN 4.689 Cyclin A1 CCNA1 4.699 Nerve growth factor receptor (TNFRSF16) associated protein 1 NGFRAP1 4.787 Arrestin, beta 2 ARRB2 4.901 Dual specificity phosphatase 5 DUSP5 5.355 Alpha-2-HS-glycoprotein AHSG 5.423 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 10 GALNT10 5.447 (GalNAc-T10) Appendices 305 Hect domain and RLD 4 HERC4 5.926 Cyclin-dependent kinase-like 1 (CDC2-related kinase) CDKL1 5.977 RNA binding motif protein 25 RBM25 6.146 Calcitonin gene-related peptide-receptor component protein RCP9 6.215 Heterogeneous nuclear ribonucleoprotein U (scaffold attachment factor A) HNRPU 6.292 TIP41, TOR signalling pathway regulator-like (S. cerevisiae) TIPRL 6.338 RAN binding protein 3 RANBP3 6.384 Ubiquitin-conjugating enzyme E2I (UBC9 homolog, yeast) UBE2I 6.443 Peroxiredoxin 6 PRDX6 6.522 MAP/microtubule affinity-regulating kinase 2 MARK2 6.647 Prohibitin PHB 6.941 SFRS protein kinase 3 SRPK3 7.103 Numb homolog (Drosophila) NUMB 7.140 NK2 transcription factor related, locus 8 (Drosophila) NKX2-8 7.968 NIMA (never in mitosis gene a)-related kinase 3 NEK3 8.074 Usher syndrome 1C (autosomal recessive, severe) USH1C 8.568 Sine oculis homeobox homolog 6 (Drosophila) SIX6 9.046 Potassium channel tetramerisation domain containing 9 KCTD9 9.073 Developmentally regulated GTP binding protein 1 DRG1 9.250 Growth hormone regulated TBC protein 1 GRTP1 9.519 Piwi-like 1 (Drosophila) PIWIL1 9.540 Malic enzyme 1, NADP(+)-dependent, cytosolic ME1 10.624 Oxysterol binding protein-like 10 OSBPL10 10.695 Ribosomal protein L28 RPL28 10.847 Protein-L-isoaspartate (D-aspartate) O-methyltransferase domain containing 1 PCMTD1 11.217 PWP1 homolog (S. cerevisiae) PWP1 13.723 Regulator of G-protein signalling 5 RGS5 13.751 Hexokinase 1 HK1 14.535 Pleckstrin homology-like domain, family A, member 1 PHLDA1 14.571 ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide 1 ATP5C1 14.993 Keratin 34 KRT34 15.623 Asparagine-linked glycosylation 6 homolog (S. cerevisiae, alpha-1,3-glucosyltransferase) ALG6 16.889 Melanoma inhibitory activity family, member 3 MIA3 17.325 Formyl peptide receptor-like 1 FPRL1 17.864 Schlafen family member 11 SLFN11 20.253 SNAP-associated protein SNAPAP 20.553 Nucleolar and coiled-body phosphoprotein 1 NOLC1 22.561 Chemokine (C-X-C motif) ligand 14 CXCL14 24.181 Integral membrane protein 2C ITM2C 25.002 Serine peptidase inhibitor, Kazal type 1 SPINK1 29.735 Sterol-C4-methyl oxidase-like SC4MOL 31.007 Interleukin 1 receptor-like 1 IL1RL1 34.141 ADAM metallopeptidase domain 12 (meltrin alpha) ADAM12 34.905 Abhydrolase domain containing 6 ABHD6 35.965 HLA complex P5 HCP5 37.617 Decapping enzyme, scavenger DCPS 39.870 Casein kinase 1, gamma 2 CSNK1G2 41.849 Twist homolog 1 (acrocephalosyndactyly 3; Saethre-Chotzen syndrome) (Drosophila) TWIST1 44.466 Protamine 1 PRM1 44.717 FAT tumor suppressor homolog 1 (Drosophila) FAT 46.684 Zinc finger protein 37 homolog (mouse) ZFP37 50.962 Attractin ATRN 54.592 ATP-binding cassette, sub-family C (CFTR/MRP), member 9 ABCC9 75.720 CDC-like kinase 1 CLK1 96.863

Appendices 306

Table F.2. Genes changed by greater than 2-fold in ALL-3 xenograft cells after 6 hrs. Gene Name Gene ID 6hrs Paralemmin PALM 0.014 Transmembrane protein 186 TMEM186 0.017 Protein tyrosine phosphatase-like (proline instead of catalytic arginine), member b PTPLB 0.019 Regulator of G-protein signalling 12 RGS12 0.021 SEC14-like 2 (S. cerevisiae) SEC14L2 0.025 Ecotropic viral integration site 2A EVI2A 0.026 HLA complex P5 HCP5 0.027 Dihydrolipoamide branched chain transacylase E2 DBT 0.027 CDC-like kinase 1 CLK1 0.027 Myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) MX1 0.030 WAP four-disulfide core domain 2 WFDC2 0.038 Arrestin, beta 2 ARRB2 0.041 Salvador homolog 1 (Drosophila) SAV1 0.042 Lysyl oxidase-like 1 LOXL1 0.058 Transducer of ERBB2, 2 TOB2 0.068 Echinoderm microtubule associated protein like 1 EML1 0.071 Solute carrier family 25 (mitochondrial carrier; Graves disease autoantigen), member 16 SLC25A16 0.072 BCL2-associated athanogene 4 BAG4 0.080 Peroxiredoxin 1 PRDX1 0.091 Calcium binding tyrosine-(Y)-phosphorylation regulated (fibrousheathin 2) CABYR 0.093 Tetratricopeptide repeat domain 4 TTC4 0.103 Sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1 SPOCK1 0.113 Usher syndrome 1C (autosomal recessive, severe) USH1C 0.126 Transmembrane and coiled-coil domains 2 TMCO2 0.130 DNA (cytosine-5-)-methyltransferase 3 alpha DNMT3A 0.131 ATPase, H+ transporting, lysosomal 42kDa, V1 subunit C2 ATP6V1C2 0.135 Plakophilin 4 PKP4 0.140 LAG1 homolog, ceramide synthase 5 (S. cerevisiae) LASS5 0.141 Complement component 4 binding protein, beta C4BPB 0.141 Protein-O-mannosyltransferase 1 POMT1 0.143 Phosphatidylinositol 3,4,5-trisphosphate-dependent RAC exchanger 1 PREX1 0.150 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 PLOD2 0.152 Transmembrane protein 47 TMEM47 0.157 Synaptotagmin-like 1 SYTL1 0.159 Fc fragment of IgG, low affinity IIIa, receptor (CD16a) FCGR3A 0.176 Membrane-spanning 4-domains, subfamily A, member 5 MS4A5 0.176 Disabled homolog 2, mitogen-responsive phosphoprotein (Drosophila) DAB2 0.177 Neurotrophic tyrosine kinase, receptor, type 2 NTRK2 0.183 Prion protein 2 (dublet) PRND 0.187 Ankyrin repeat domain 18A ANKRD18A 0.193 Zinc finger protein 167 ZNF167 0.193 P antigen family, member 1 (prostate associated) PAGE1 0.200 Potassium inwardly-rectifying channel, subfamily J, member 8 KCNJ8 0.202 Ring finger protein 138 RNF138 0.203 Pygopus homolog 1 (Drosophila) PYGO1 0.204 Heat shock transcription factor family member 5 HSF5 0.204 Cyclin M2 CNNM2 0.205 Microtubule associated serine/threonine kinase family member 4 MAST4 0.207 Embryonal Fyn-associated substrate EFS 0.211 B-cell CLL/lymphoma 11B (zinc finger protein) BCL11B 0.219 Solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 SLC7A2 0.222

Appendices 307 Eukaryotic translation initiation factor 4A, isoform 2 EIF4A2 0.224 Cytochrome P450, family 3, subfamily A, polypeptide 4 CYP3A4 0.225 Mal, T-cell differentiation protein 2 MAL2 0.225 Paternally expressed 3 PEG3 0.230 Pyrimidinergic receptor P2Y, G-protein coupled, 6 P2RY6 0.231 Ankyrin repeat domain 38 ANKRD38 0.238 Chloride channel, calcium activated, family member 1 CLCA1 0.243 Nedd4 family interacting protein 2 NDFIP2 0.245 Grainyhead-like 1 (Drosophila) GRHL1 0.246 Major histocompatibility complex, class I, C HLA-C 0.247 Leukemia inhibitory factor receptor alpha LIFR 0.250 Proline rich Gla (G-carboxyglutamic acid) 1 PRRG1 0.250 Junctophilin 1 JPH1 0.251 MCM3 minichromosome maintenance deficient 3 (S. cerevisiae) MCM3 0.251 Forkhead box F2 FOXF2 0.254 Osteoglycin OGN 0.256 Sodium channel, voltage-gated, type II, alpha subunit SCN2A 0.258 Zinc finger, FYVE domain containing 20 ZFYVE20 0.263 Kinesin family member 5C KIF5C 0.267 Kinesin family member 26B KIF26B 0.270 Eukaryotic translation initiation factor 5A2 EIF5A2 0.274 Zinc finger protein 300 ZNF300 0.274 Chromodomain helicase DNA binding protein 5 CHD5 0.274 CAMSAP1L Calmodulin regulated spectrin-associated protein 1-like 1 0.274 1 Sine oculis homeobox homolog 6 (Drosophila) SIX6 0.275 Down syndrome critical region gene 8 DSCR8 0.277 MOB1, Mps One Binder kinase activator-like 1B (yeast) MOBK1B 0.277 Carbonic anhydrase VIII CA8 0.278 Myosin, light chain 9, regulatory MYL9 0.278 ST7 overlapping transcript 4 (non-coding RNA) ST7OT4 0.278 Leucine rich repeat containing 61 LRRC61 0.279 Protocadherin 17 PCDH17 0.280 InaD-like (Drosophila) INADL 0.280 Sodium channel, voltage-gated, type III, alpha subunit SCN3A 0.282 Tu translation elongation factor, mitochondrial TUFM 0.282 Calcyclin binding protein CACYBP 0.284 Bassoon (presynaptic cytomatrix protein) BSN 0.285 Homeobox B4 HOXB4 0.286 Zinc finger protein 532 ZNF532 0.289 Rho GTPase-activating protein RICS 0.290 Trichorhinophalangeal syndrome I TRPS1 0.292 RAS protein activator like 1 (GAP1 like) RASAL1 0.293 Rh blood group, CcEe antigens RHCE 0.294 RAB23, member RAS oncogene family RAB23 0.294 Heat shock 27kDa protein 3 HSPB3 0.295 Dystrobrevin, alpha DTNA 0.296 Microsomal glutathione S-transferase 1 MGST1 0.299 Protein phosphatase 4, regulatory subunit 1 PPP4R1 0.300 Calnexin Canx 0.301 WW and C2 domain containing 2 WWC2 0.302 Lymphocyte antigen 6 complex, locus K LY6K 0.302 CDC14 cell division cycle 14 homolog B (S. cerevisiae) CDC14B 0.305 Abhydrolase domain containing 6 ABHD6 0.306 Heat shock 70kDa protein 4-like HSPA4L 0.306

Appendices 308 Zinc finger protein 238 ZNF238 0.309 GRB2-associated binding protein 1 GAB1 0.310 Protocadherin 17 PCDH17 0.311 Family with sequence similarity 129, member A FAM129A 0.312 Acyltransferase like 1 AYTL1 0.312 Pregnancy-associated plasma protein A, pappalysin 1 PAPPA 0.313 Calcium/calmodulin-dependent protein kinase IV CAMK4 0.313 WAP four-disulfide core domain 5 WFDC5 0.313 Claudin 1 CLDN1 0.314 Collagen, type XI, alpha 1 COL11A1 0.314 Immunoglobulin superfamily, member 10 IGSF10 0.314 SAM domain, SH3 domain and nuclear localization signals 1 SAMSN1 0.314 Fibroblast growth factor 12 FGF12 0.316 Collagen, type IV, alpha 3 (Goodpasture antigen) COL4A3 0.316 IKAROS family zinc finger 1 (Ikaros) IKZF1 0.317 Hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 7 HSD3B7 0.317 WNT1 inducible signaling pathway protein 1 WISP1 0.321 G protein-coupled receptor 126 GPR126 0.321 Lipase, member I LIPI 0.323 Protein phosphatase 2 (formerly 2A), regulatory subunit B'', alpha PPP2R3A 0.324 Desmoplakin DSP 0.324 Two transmembrane domain family member A TTMA 0.324 Bone morphogenetic protein receptor, type IA BMPR1A 0.325 Chemokine (C-C motif) receptor-like 1 CCRL1 0.327 Embryonal Fyn-associated substrate EFS 0.327 ClpB caseinolytic peptidase B homolog (E. coli) CLPB 0.328 Pregnancy-associated plasma protein A, pappalysin 1 PAPPA 0.329 Multiple EGF-like-domains 10 MEGF10 0.331 G protein-coupled receptor 161 GPR161 0.332 Ubiquitin specific peptidase 2 USP2 0.332 Nuclear casein kinase and cyclin-dependent kinase substrate 1 NUCKS1 0.334 Solute carrier family 38, member 4 SLC38A4 0.334 Fibronectin leucine rich transmembrane protein 3 FLRT3 0.335 Scavenger receptor class B, member 1 SCARB1 0.337 Solute carrier family 6, member 15 SLC6A15 0.337 Growth hormone regulated TBC protein 1 GRTP1 0.338 Chondroitin polymerizing factor CHPF 0.339 OTX2 0.340 Leucine rich repeat neuronal 1 LRRN1 0.341 Galanin GAL 0.341 Amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 4 ALS2CR4 0.342 Sterile alpha motif and leucine zipper containing kinase AZK ZAK 0.345 RNA binding motif protein 6 RBM6 0.345 Golgi autoantigen, golgin subfamily a, 1 GOLGA1 0.348 RP5- SAM domain containing 1 0.350 875H10.1 Urothelial cancer associated 1 UCA1 0.350 LY6/PLAUR domain containing 6 LYPD6 0.351 TSPY-like 6 TSPYL6 0.352 GPI deacylase PGAP1 0.353 EPH receptor A2 EPHA2 0.354 Olfactomedin 1 OLFM1 0.355 Insulin-like growth factor 2 (somatomedin A) IGF2 0.355 Wolfram syndrome 1 (wolframin) WFS1 0.355 Collagen, type XI, alpha 1 COL11A1 0.357

Appendices 309 Cyclin M2 CNNM2 0.357 V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog KIT 0.358 AT rich interactive domain 2 (ARID, RFX-like) ARID2 0.358 Protocadherin alpha subfamily C, 2 PCDHAC2 0.359 Gonadotropin-releasing hormone 1 (luteinizing-releasing hormone) GNRH1 0.360 Doublecortin and CaM kinase-like 2 DCAMKL2 0.361 Synaptojanin 2 binding protein SYNJ2BP 0.362 Developmentally regulated GTP binding protein 1 DRG1 0.362 Fucosyltransferase 1 (galactoside 2-alpha-L-fucosyltransferase, H blood group) FUT1 0.363 Progestin and adipoQ receptor family member III PAQR3 0.365 Serine/threonine kinase 33 STK33 0.365 Beaded filament structural protein 1, filensin BFSP1 0.367 5-hydroxytryptamine (serotonin) receptor 2B HTR2B 0.367 Complement factor H CFH 0.367 Ring finger protein 157 RNF157 0.367 Pelota homolog (Drosophila) PELO 0.368 Olfactory receptor, family 2, subfamily A, member 7 OR2A7 0.371 G antigen 10 GAGE10 0.372 Myosin, light chain 1, alkali; skeletal, fast MYL1 0.372 Maternally expressed 3 MEG3 0.373 Sarcoglycan, delta (35kDa dystrophin-associated glycoprotein) SGCD 0.374 Pregnancy-associated plasma protein A, pappalysin 1 PAPPA 0.375 Nucleoporin 93kDa NUP93 0.375 Kelch-like 3 (Drosophila) KLHL3 0.375 Protein phosphatase 1, regulatory (inhibitor) subunit 9A PPP1R9A 0.375 Dachshund homolog 1 (Drosophila) DACH1 0.376 Phosphodiesterase 4D interacting protein (myomegalin) PDE4DIP 0.377 Cordon-bleu homolog (mouse) COBL 0.377 Filamin A interacting protein 1 FILIP1 0.378 GTPase activating Rap/RanGAP domain-like 3 GARNL3 0.379 Thy-1 cell surface antigen THY1 0.379 Cytokine receptor-like factor 1 CRLF1 0.379 Transmembrane protein 30B TMEM30B 0.380 Proline rich Gla (G-carboxyglutamic acid) 4 (transmembrane) PRRG4 0.382 Transforming growth factor, beta 3 TGFB3 0.382 Transmembrane phosphatase with tensin homology TPTE 0.383 Disabled homolog 1 (Drosophila) DAB1 0.383 Titin TTN 0.383 Protein phosphatase 2 (formerly 2A), regulatory subunit B'', alpha PPP2R3A 0.384 Bromodomain adjacent to zinc finger domain, 2B BAZ2B 0.385 Orthopedia homeobox OTP 0.385 Tetraspanin 12 TSPAN12 0.385 Family with sequence similarity 24, member B FAM24B 0.385 Seizure related 6 homolog (mouse)-like 2 SEZ6L2 0.385 Integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor) ITGA2 0.386 ADP-ribosyltransferase 4 (Dombrock blood group) ART4 0.386 Makorin, ring finger protein, 2 Mkrn2 0.386 ADAM metallopeptidase domain 12 (meltrin alpha) ADAM12 0.386 ADAM metallopeptidase domain 19 (meltrin beta) ADAM19 0.388 Neurofilament, heavy polypeptide 200kDa NEFH 0.388 CDC14 cell division cycle 14 homolog B (S. cerevisiae) CDC14B 0.388 Potassium channel tetramerisation domain containing 7 KCTD7 0.389 Notch homolog 3 (Drosophila) NOTCH3 0.390 Fibronectin leucine rich transmembrane protein 2 FLRT2 0.390

Appendices 310 PR domain containing 10 PRDM10 0.390 Phosphorylase, glycogen; liver (Hers disease, glycogen storage disease type VI) PYGL 0.391 Zinc finger protein 135 ZNF135 0.391 Ribonucleoprotein, PTB-binding 2 RAVER2 0.392 Proprotein convertase subtilisin/kexin type 1 PCSK1 0.393 Rho family GTPase 2 RND2 0.393 Synaptopodin 2 SYNPO2 0.393 Frizzled homolog 1 (Drosophila) FZD1 0.394 TEA domain family member 1 (SV40 transcriptional enhancer factor) TEAD1 0.395 Phospholipase A2 receptor 1, 180kDa PLA2R1 0.396 Zinc finger protein 793 ZNF793 0.396 Mesothelin MSLN 0.397 Jagged 2 JAG2 0.397 Growth factor receptor-bound protein 10 GRB10 0.397 Protease, serine, 12 (neurotrypsin, motopsin) PRSS12 0.398 Insulin-like growth factor 1 receptor IGF1R 0.398 Variable charge, X-linked 3A VCX3A 0.398 Histocompatibility (minor) HA-1 HMHA1 0.398 Solute carrier family 19 (thiamine transporter), member 2 SLC19A2 0.399 Proline/arginine-rich end leucine-rich repeat protein PRELP 0.399 Microtubule-associated protein 9 MAP9 0.400 Acetyl-Coenzyme A carboxylase alpha ACACA 0.401 Coiled-coil domain containing 80 CCDC80 0.402 Neuroblastoma breakpoint family, member 1 NBPF1 0.402 COBL-like 1 COBLL1 0.402 SLC2A4 regulator SLC2A4RG 0.402 Transmembrane protein 47 TMEM47 0.402 Protein kinase (cAMP-dependent, catalytic) inhibitor alpha PKIA 0.402 LINE-1 type transposase domain containing 1 L1TD1 0.403 Developmental pluripotency associated 4 DPPA4 0.403 overlapping transcript (non-coding RNA) SOX2OT 0.404 Solute carrier family 36 (proton/amino acid symporter), member 3 SLC36A3 0.404 Numb homolog (Drosophila) NUMB 0.404 Neuronal guanine nucleotide exchange factor NGEF 0.405 Double C2-like domains, alpha DOC2A 0.406 LRP16 protein LRP16 0.406 DKFZP564O DKFZP564O0823 protein 0.406 0823 Ribonuclease III, nuclear RNASEN 0.406 Neurobeachin NBEA 0.406 ST6 beta-galactosamide alpha-2,6-sialyltranferase 2 ST6GAL2 0.407 Family with sequence similarity 111, member B FAM111B 0.407 Follistatin FST 0.408 Coiled-coil domain containing 113 CCDC113 0.408 SET and MYND domain containing 1 SMYD1 0.408 Plasminogen activator, tissue PLAT 0.409 Reversion-inducing-cysteine-rich protein with kazal motifs RECK 0.409 Musculin (activated B-cell factor-1) MSC 0.410 Serine hydroxymethyltransferase 2 (mitochondrial) SHMT2 0.410 Tumor necrosis factor receptor superfamily, member 17 TNFRSF17 0.411 TAF7-like RNA polymerase II, TATA box binding protein (TBP)-associated factor, 50kDa TAF7L 0.412 Elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 4 ELOVL4 0.412 Coiled-coil domain containing 4 CCDC4 0.412 VAMP (vesicle-associated membrane protein)-associated protein A, 33kDa VAPA 0.415 Transcription elongation regulator 1-like TCERG1L 0.415

Appendices 311 RAS protein activator like 2 RASAL2 0.415 Kelch-like 29 (Drosophila) KLHL29 0.416 Palmdelphin PALMD 0.416 Zinc finger protein 649 ZNF649 0.417 Titin Ttn 0.417 Period homolog 3 (Drosophila) PER3 0.417 Ubiquitination factor E4B (UFD2 homolog, yeast) UBE4B 0.419 Wingless-type MMTV integration site family, member 7A WNT7A 0.419 Reversion-inducing-cysteine-rich protein with kazal motifs RECK 0.420 Sp6 transcription factor SP6 0.420 Anterior gradient homolog 3 (Xenopus laevis) AGR3 0.420 Zinc finger protein 333 ZNF333 0.421 SLIT-ROBO Rho GTPase activating protein 1 SRGAP1 0.421 Phosphodiesterase 8B PDE8B 0.421 V-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) ERBB3 0.422 Teashirt family zinc finger 2 TSHZ2 0.422 Seizure related 6 homolog (mouse)-like 2 SEZ6L2 0.422 Mevalonate (diphospho) decarboxylase MVD 0.422 Tripartite motif-containing 5 TRIM5 0.422 Ras association (RalGDS/AF-6) and pleckstrin homology domains 1 RAPH1 0.424 G protein-coupled receptor 126 GPR126 0.425 Macrophage expressed gene 1 MPEG1 0.425 Amphiregulin (schwannoma-derived growth factor) AREG 0.425 Bestrophin 3 BEST3 0.425 Delta/notch-like EGF repeat containing DNER 0.425 Phosphotyrosine interaction domain containing 1 PID1 0.425 Family with sequence similarity 46, member A FAM46A 0.426 Tumor protein D52-like 3 TPD52L3 0.426 Chloride channel 5 (nephrolithiasis 2, X-linked, Dent disease) CLCN5 0.426 Kynureninase (L-kynurenine hydrolase) KYNU 0.427 Transmembrane channel-like 4 TMC4 0.427 Centrosomal protein 76kDa CEP76 0.428 N-acetylneuraminate pyruvate lyase (dihydrodipicolinate synthase) NPL 0.428 Ankyrin repeat domain 57 ANKRD57 0.428 Trophoblast-derived noncoding RNA TncRNA 0.428 Solute carrier family 37 (glycerol-6-phosphate transporter), member 4 SLC37A4 0.428 F-box protein 2 FBXO2 0.429 Somatostatin receptor 1 SSTR1 0.429 Leucine rich repeat containing 3B LRRC3B 0.429 RAN binding protein 17 RANBP17 0.429 Protocadherin beta 7 PCDHB7 0.430 CD36 molecule (thrombospondin receptor) CD36 0.430 Membrane protein, palmitoylated 7 (MAGUK p55 subfamily member 7) MPP7 0.430 Adipocyte-specific adhesion molecule ASAM 0.431 Hormonally upregulated Neu-associated kinase HUNK 0.431 Laminin, beta 2 (laminin S) LAMB2 0.431 Aminolevulinate, delta-, synthase 2 (sideroblastic/hypochromic anemia) ALAS2 0.431 Fibroblast growth factor 12 FGF12 0.431 Solute carrier family 31 (copper transporters), member 1 SLC31A1 0.432 Solute carrier family 2 (facilitated glucose transporter), member 13 SLC2A13 0.433 Transmembrane protein 56 TMEM56 0.433 Odz, odd Oz/ten-m homolog 3 (Drosophila) ODZ3 0.433 Hepatocyte nuclear factor 4, gamma HNF4G 0.435 Ets variant gene 1 ETV1 0.435

Appendices 312 Rho-related BTB domain containing 2 RHOBTB2 0.436 Aminoacylase 1 ACY1 0.437 Receptor interacting protein kinase 5 RIPK5 0.437 Bone morphogenetic protein 6 BMP6 0.437 Ubiquitin specific peptidase 53 USP53 0.438 Arylsulfatase family, member K ARSK 0.438 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, SMARCD3 0.439 member 3 RAB23, member RAS oncogene family RAB23 0.439 Synaptotagmin XII SYT12 0.439 Caldesmon 1 CALD1 0.440 Ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative function) ENPP4 0.440 Zinc finger protein 273 ZNF273 0.440 Solute carrier family 36 (proton/amino acid symporter), member 3 SLC36A3 0.440 Cholinergic receptor, nicotinic, alpha 5 CHRNA5 0.441 PDZ domain containing 2 PDZD2 0.441 Erythrocyte membrane protein band 4.1 like 4A EPB41L4A 0.442 Popeye domain containing 3 POPDC3 0.442 Wingless-type MMTV integration site family, member 5A WNT5A 0.443 Kinase D-interacting substance of 220 kDa KIDINS220 0.443 Islet cell autoantigen 1, 69kDa ICA1 0.443 TBC1 domain family, member 15 TBC1D15 0.444 Peripheral myelin protein 22 PMP22 0.444 CD97 molecule CD97 0.444 Chloride intracellular channel 2 CLIC2 0.444 Synuclein, alpha interacting protein (synphilin) SNCAIP 0.446 Oculocutaneous albinism II (pink-eye dilution homolog, mouse) OCA2 0.446 Contactin 3 ( associated) CNTN3 0.446 Protein kinase D3 PRKD3 0.446 Ring finger protein 128 RNF128 0.446 Actin binding LIM protein 1 ABLIM1 0.447 Solute carrier family 41, member 2 SLC41A2 0.447 Ring finger protein 32 RNF32 0.448 Sorbin and SH3 domain containing 2 SORBS2 0.449 CAMP responsive element binding protein 5 CREB5 0.449 Dihydrofolate reductase DHFR 0.450 Rho guanine nucleotide exchange factor (GEF) 10 ARHGEF10 0.450 Splicing factor, arginine/serine-rich 10 (transformer 2 homolog, Drosophila) SFRS10 0.450 Cholecystokinin B receptor CCKBR 0.450 Cholecystokinin CCK 0.450 CD80 molecule CD80 0.451 Microfibrillar associated protein 5 MFAP5 0.452 Dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex) DLAT 0.452 Nedd4 family interacting protein 2 NDFIP2 0.453 EPH receptor A7 EPHA7 0.453 Rho GTPase-activating protein RICS 0.454 Delta/notch-like EGF repeat containing DNER 0.454 Zinc finger protein 83 ZNF83 0.455 Neuropeptide Y NPY 0.456 Ring finger protein 121 RNF121 0.456 Aldo-keto reductase family 1, member C2 (dihydrodiol dehydrogenase 2; bile acid binding AKR1C2 0.456 protein; 3-alpha hydroxysteroid dehydrogenase, type III) 5-hydroxytryptamine (serotonin) receptor 2B HTR2B 0.457 LON peptidase N-terminal domain and ring finger 2 LONRF2 0.457 Protein tyrosine phosphatase, receptor type, Q PTPRQ 0.457 Synapse associated protein 1, SAP47 homolog (Drosophila) SYAP1 0.458 Appendices 313 TSPY-like 5 TSPYL5 0.458 Transcription factor 7-like 1 (T-cell specific, HMG-box) TCF7L1 0.458 Podoplanin PDPN 0.459 CXXC finger 4 CXXC4 0.459 Leukotriene B4 12-hydroxydehydrogenase LTB4DH 0.459 Flavin containing monooxygenase 1 FMO1 0.459 Solute carrier family 38, member 1 SLC38A1 0.460 Killer cell lectin-like receptor subfamily C, member 2 KLRC2 0.460 Tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2 TANC2 0.460 EH domain binding protein 1 EHBP1 0.460 Prostaglandin E receptor 3 (subtype EP3) PTGER3 0.460 Elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 2 ELOVL2 0.460 Gamma-aminobutyric acid (GABA) A receptor, beta 3 GABRB3 0.461 Sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) and short SEMA4F 0.461 cytoplasmic domain, (semaphorin) 4F Alcohol dehydrogenase, iron containing, 1 ADHFE1 0.461 V-erb-a erythroblastic leukemia viral oncogene homolog 4 (avian) ERBB4 0.462 Transmembrane protein 37 TMEM37 0.462 Farnesyltransferase, CAAX box, alpha FNTA 0.463 Nestin NES 0.464 MAS-related GPR, member F MRGPRF 0.464 Ring finger protein 32 RNF32 0.464 Ventricular zone expressed PH domain homolog 1 (zebrafish) VEPH1 0.464 SH3-domain GRB2-like (endophilin) interacting protein 1 SGIP1 0.464 RNA binding protein, autoantigenic (hnRNP-associated with lethal yellow homolog (mouse)) RALY 0.465 BUB3 budding uninhibited by benzimidazoles 3 homolog (yeast) BUB3 0.465 Membrane associated guanylate kinase, WW and PDZ domain containing 1 MAGI1 0.465 Glucuronic acid epimerase GLCE 0.466 Uveal autoantigen with coiled-coil domains and ankyrin repeats UACA 0.466 Neuroblastoma breakpoint family, member 1 NBPF1 0.466 THAP domain containing 10 THAP10 0.466 Sorting nexin 7 SNX7 0.466 Pelota homolog (Drosophila) PELO 0.466 Melanoma antigen family C, 1 MAGEC1 0.467 Zinc finger protein 662 ZNF662 0.467 Pseudouridylate synthase 7 homolog (S. cerevisiae) PUS7 0.467 Pleiotropic regulator 1 (PRL1 homolog, Arabidopsis) PLRG1 0.468 Alpha-kinase 3 ALPK3 0.468 Keratin, hair, basic, 5 KRTHB5 0.468 Netrin 4 NTN4 0.468 Kininogen 1 KNG1 0.469 G protein-coupled receptor 177 GPR177 0.469 Lyrm7 homolog (mouse) LYRM7 0.469 Cytochrome P450, family 2, subfamily U, polypeptide 1 CYP2U1 0.469 Zic family member 2 (odd-paired homolog, Drosophila) ZIC2 0.470 Metaxin 3 MTX3 0.470 Kaptin (actin binding protein) KPTN 0.471 NUAK family, SNF1-like kinase, 2 NUAK2 0.472 Rho GTPase activating protein 28 ARHGAP28 0.472 OCIA domain containing 2 OCIAD2 0.472 Seizure related 6 homolog (mouse)-like 2 SEZ6L2 0.472 Dynein, axonemal, heavy chain 5 DNAH5 0.472 Interleukin 15 IL15 0.472 CAP-GLY domain containing linker protein family, member 4 CLIP4 0.473 Chromobox homolog 5 (HP1 alpha homolog, Drosophila) CBX5 0.473

Appendices 314 Phosphatidylinositol transfer protein, beta PITPNB 0.474 Synaptopodin 2 SYNPO2 0.474 Hemoglobin, zeta HBZ 0.474 Retinoic acid induced 14 RAI14 0.474 Discoidin, CUB and LCCL domain containing 2 DCBLD2 0.475 Carboxypeptidase A6 CPA6 0.475 SAM and SH3 domain containing 1 SASH1 0.475 LIM and cysteine-rich domains 1 LMCD1 0.475 Synaptotagmin binding, cytoplasmic RNA interacting protein SYNCRIP 0.476 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 12 GALNT12 0.476 (GalNAc-T12) PERP, TP53 apoptosis effector PERP 0.476 Basonuclin 2 BNC2 0.476 Kinesin family member 21A KIF21A 0.476 Family with sequence similarity 114, member A1 FAM114A1 0.477 DiGeorge syndrome critical region gene 8 DGCR8 0.477 Phosphodiesterase 10A PDE10A 0.477 Ovo-like 1(Drosophila) OVOL1 0.477 Neuronal growth regulator 1 NEGR1 0.478 NIPA-like domain containing 3 NPAL3 0.478 ADAM metallopeptidase with thrombospondin type 1 motif, 5 (aggrecanase-2) ADAMTS5 0.478 Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 SVEP1 0.478 Sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6D SEMA6D 0.478 Coiled-coil domain containing 113 CCDC113 0.478 Protein kinase (cAMP-dependent, catalytic) inhibitor beta PKIB 0.479 IQ motif containing GTPase activating protein 1 IQGAP1 0.479 DENN/MADD domain containing 2C DENND2C 0.480 Spindlin family, member 3 SPIN3 0.480 C-type lectin domain family 2, member L CLEC2L 0.480 Membrane associated guanylate kinase, WW and PDZ domain containing 3 MAGI3 0.481 Multiple PDZ domain protein MPDZ 0.481 Prostate androgen-regulated transcript 1 PART1 0.482 BAT2 domain containing 1 BAT2D1 0.482 WW domain containing oxidoreductase WWOX 0.482 Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A SEMA3A 0.483 Solute carrier family 24 (sodium/potassium/calcium exchanger), member 6 SLC24A6 0.483 Dolichyl-diphosphooligosaccharide-protein glycosyltransferase DDOST 0.484 Odz, odd Oz/ten-m homolog 2 (Drosophila) ODZ2 0.485 Roundabout, axon guidance receptor, homolog 1 (Drosophila) ROBO1 0.485 Tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) TNFRSF11B 0.485 Sorting nexin 19 SNX19 0.485 Formyl peptide receptor-like 1 FPRL1 0.485 Ras protein-specific guanine nucleotide-releasing factor 2 RASGRF2 0.485 Family with sequence similarity 83, member A FAM83A 0.486 BTB and CNC homology 1, basic leucine zipper transcription factor 2 BACH2 0.486 Fibronectin leucine rich transmembrane protein 3 FLRT3 0.486 Membrane-spanning 4-domains, subfamily A, member 1 MS4A1 0.486 Prostaglandin E synthase PTGES 0.487 BCL2-associated athanogene 2 BAG2 0.487 CAMSAP1L Calmodulin regulated spectrin-associated protein 1-like 1 0.487 1 MAX interactor 1 MXI1 0.488 G protein-coupled receptor 162 GPR162 0.488 TRM5 tRNA methyltransferase 5 homolog (S. cerevisiae) TRMT5 0.488 Protease, serine, 21 (testisin) PRSS21 0.488 Myelin basic protein MBP 0.488 Appendices 315 SH3-domain GRB2-like (endophilin) interacting protein 1 SGIP1 0.489 transcription factor 1 0.489 RNA binding motif protein 35A RBM35A 0.489 F-box and leucine-rich repeat protein 7 FBXL7 0.489 Abhydrolase domain containing 5 ABHD5 0.490 Lipoma HMGIC fusion partner LHFP 0.490 PDZ and LIM domain 4 PDLIM4 0.490 GINS complex subunit 2 (Psf2 homolog) GINS2 0.490 Tyrosinase-related protein 1 TYRP1 0.490 Zinc fingers and 1 ZHX1 0.490 G protein-coupled receptor 98 GPR98 0.491 Prospero-related homeobox 1 PROX1 0.492 Fibronectin 1 FN1 0.492 Nudix (nucleoside diphosphate linked moiety X)-type motif 14 NUDT14 0.492 Acyltransferase like 1 AYTL1 0.492 Coiled-coil domain containing 90A CCDC90A 0.493 Paternally expressed 10 PEG10 0.493 Folliculin interacting protein 1 FNIP1 0.493 Solute carrier family 16, member 7 (monocarboxylic acid transporter 2) SLC16A7 0.493 ATP-binding cassette, sub-family A (ABC1), member 8 ABCA8 0.494 Leucine-rich repeat-containing G protein-coupled receptor 5 LGR5 0.494 AT hook, DNA binding motif, containing 1 AHDC1 0.495 Protein phosphatase 1, regulatory (inhibitor) subunit 1A PPP1R1A 0.495 Cannabinoid receptor 1 (brain) CNR1 0.495 Endothelial differentiation, sphingolipid G-protein-coupled receptor, 3 EDG3 0.495 Homeobox D9 HOXD9 0.496 Erythrocyte membrane protein band 4.1-like 3 EPB41L3 0.496 Dysbindin (dystrobrevin binding protein 1) domain containing 2 DBNDD2 0.496 Sterile alpha motif domain containing 3 SAMD3 0.496 UDP-glucose ceramide glucosyltransferase-like 2 UGCGL2 0.497 Urotensin 2 UTS2 0.497 Inhibitor of DNA binding 3, dominant negative helix-loop-helix protein ID3 0.497 Usher syndrome 2A (autosomal recessive, mild) USH2A 0.497 NDRG family member 2 NDRG2 0.497 Zinc finger, FYVE domain containing 20 ZFYVE20 0.497 TBC1 domain family, member 15 TBC1D15 0.498 Neurobeachin NBEA 0.498 Paternally expressed 3 PEG3 0.498 GM2 ganglioside activator GM2A 0.498 La ribonucleoprotein domain family, member 6 LARP6 0.499 Guanine nucleotide binding protein (G protein), gamma 12 GNG12 0.499 Protein kinase N3 PKN3 0.499 Coiled-coil domain containing 110 CCDC110 0.500 TBC1 domain family, member 4 TBC1D4 2.001 Interleukin 11 receptor, alpha chain 1 Il11ra1 2.004 Striatin, calmodulin binding protein 4 STRN4 2.005 G1 to S phase transition 2 GSPT2 2.005 Phospholipase C, beta 1 (phosphoinositide-specific) PLCB1 2.009 Major histocompatibility complex, class I, F HLA-F 2.010 Ubiquitin specific peptidase 18 USP18 2.015 Immediate early response 2 IER2 2.017 Nucleolar and coiled-body phosphoprotein 1 NOLC1 2.020 subfamily 0, group B, member 1 NR0B1 2.021 CD1c molecule CD1C 2.022

Appendices 316 Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha NFKBIA 2.024 Dual specificity phosphatase 6 DUSP6 2.025 Sorting nexin 4 SNX4 2.026 Serpin peptidase inhibitor, clade B (ovalbumin), member 9 SERPINB9 2.028 Heat shock protein 90kDa alpha (cytosolic), class B member 1 HSP90AB1 2.029 Leucine rich repeat containing 6 LRRC6 2.034 Transient receptor potential cation channel, subfamily A, member 1 TRPA1 2.036 Jun B proto-oncogene JUNB 2.037 Exportin 5 XPO5 2.038 Lysosomal-associated membrane protein 3 LAMP3 2.039 Coiled-coil domain containing 81 CCDC81 2.042 Potassium voltage-gated channel, shaker-related subfamily, member 5 KCNA5 2.043 Glutathione peroxidase 4 (phospholipid hydroperoxidase) GPX4 2.048 LRP16 protein LRP16 2.049 Catenin (cadherin-associated protein), alpha 1, 102kDa CTNNA1 2.051 Acyl-CoA synthetase long-chain family member 5 ACSL5 2.051 Ribosomal protein S28 RPS28 2.052 Phospholipase A2, group IVA (cytosolic, calcium-dependent) PLA2G4A 2.058 F-box and WD repeat domain containing 8 FBXW8 2.068 SMAD family member 3 SMAD3 2.071 Hematopoietic cell signal transducer HCST 2.074 Regulator of G-protein signalling 1 RGS1 2.082 SMAD family member 1 SMAD1 2.084 Arylsulfatase G ARSG 2.086 Hemoglobin, beta HBB 2.086 Hippocalcin-like 1 HPCAL1 2.091 Low density lipoprotein receptor-related protein 3 LRP3 2.092 Inhibin, beta A (activin A, activin AB alpha polypeptide) INHBA 2.093 Tumor necrosis factor receptor superfamily, member 4 TNFRSF4 2.099 Guanine nucleotide binding protein (G protein), gamma 11 GNG11 2.111 Roundabout, axon guidance receptor, homolog 2 (Drosophila) ROBO2 2.118 LIM domain only 2 (rhombotin-like 1) LMO2 2.119 Phosphatidic acid phosphatase type 2B PPAP2B 2.121 Major histocompatibility complex, class II, DR beta 4 HLA-DRB4 2.127 Malic enzyme 1, NADP(+)-dependent, cytosolic ME1 2.138 Tubulin, beta 3 TUBB3 2.139 Homer homolog 2 (Drosophila) HOMER2 2.139 Ephrin-B1 EFNB1 2.139 Pleckstrin homology domain containing, family F (with FYVE domain) member 1 PLEKHF1 2.141 Rho GTPase activating protein 27 ARHGAP27 2.146 Integrin, alpha X (complement component 3 receptor 4 subunit) ITGAX 2.148 Potassium inwardly-rectifying channel, subfamily J, member 2 KCNJ2 2.149 Thromboxane A synthase 1 (platelet, cytochrome P450, family 5, subfamily A) TBXAS1 2.150 Kelch-like 2, Mayven (Drosophila) KLHL2 2.151 Fibrinogen beta chain FGB 2.162 Calmin (calponin-like, transmembrane) CLMN 2.167 GTP cyclohydrolase 1 (dopa-responsive dystonia) GCH1 2.168 Regulator of G-protein signalling 13 RGS13 2.171 Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 1 LILRB1 2.172 Aquaporin 4 AQP4 2.173 Ribonuclease P 40kDa subunit RPP40 2.184 Tensin 1 TNS1 2.187 Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 1 LILRB1 2.190 Thrombomodulin THBD 2.196

Appendices 317 Phosphoribosyl pyrophosphate synthetase 2 PRPS2 2.196 Leukocyte immunoglobulin-like receptor, subfamily A (with TM domain), member 4 LILRA4 2.196 Mitogen-activated protein kinase kinase kinase 4 MAP3K4 2.196 Inositol 1,4,5-triphosphate receptor, type 1 ITPR1 2.197 CASP2 and RIPK1 domain containing adaptor with death domain CRADD 2.199 NGFI-A binding protein 2 (EGR1 binding protein 2) NAB2 2.203 Activin A receptor, type I ACVR1 2.203 Bridging integrator 2 BIN2 2.207 Claudin 1 CLDN1 2.208 Pleckstrin PLEK 2.209 Cystatin F (leukocystatin) CST7 2.223 Tumor necrosis factor (ligand) superfamily, member 11 TNFSF11 2.225 Docking protein 2, 56kDa DOK2 2.235 Pyruvate dehydrogenase kinase, isozyme 3 PDK3 2.237 Phosphofructokinase, platelet PFKP 2.239 Integrin-binding sialoprotein (bone sialoprotein, bone sialoprotein II) IBSP 2.249 CD93 molecule CD93 2.262 Basic leucine zipper transcription factor, ATF-like BATF 2.263 Transmembrane protein 49 TMEM49 2.275 Aspartate beta-hydroxylase domain containing 2 ASPHD2 2.277 Interleukin 6 receptor IL6R 2.277 G protein-coupled receptor 65 GPR65 2.279 RCSD domain containing 1 RCSD1 2.280 V- musculoaponeurotic fibrosarcoma oncogene homolog G (avian) MAFG 2.285 SMAD family member 3 SMAD3 2.290 Striatin, calmodulin binding protein 4 STRN4 2.302 5'-nucleotidase, ecto (CD73) NT5E 2.310 Purinergic receptor P2Y, G-protein coupled, 1 P2RY1 2.318 Lymphocyte antigen 6 complex, locus D LY6D 2.331 Ribosomal protein L28 RPL28 2.342 CD34 molecule CD34 2.346 Myeloid differentiation primary response gene (88) MYD88 2.346 Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G APOBEC3G 2.353 Keratin 23 (histone deacetylase inducible) KRT23 2.356 Lactoperoxidase LPO 2.357 Prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) PTGS1 2.369 Solute carrier family 25 (mitochondrial carrier, Aralar), member 12 SLC25A12 2.385 Granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine esterase 3) GZMA 2.394 Uroplakin 3A UPK3A 2.399 Peptidylprolyl isomerase F (cyclophilin F) PPIF 2.403 Calpain 5 CAPN5 2.409 Glucosidase, beta; acid (includes glucosylceramidase) GBA 2.418 CD52 molecule CD52 2.426 Matrix metallopeptidase 16 (membrane-inserted) MMP16 2.427 CD93 molecule CD93 2.440 Granulysin GNLY 2.441 Monocyte to macrophage differentiation-associated 2 MMD2 2.445 Peroxisome biogenesis factor 26 PEX26 2.451 Group-specific component (vitamin D binding protein) GC 2.452 ADP-ribosylation factor-like 4C ARL4C 2.455 Tumor necrosis factor (ligand) superfamily, member 13b TNFSF13B 2.477 Ficolin (collagen/fibrinogen domain containing) 1 FCN1 2.486 Heparin-binding EGF-like growth factor HBEGF 2.507 ADAM metallopeptidase domain 8 ADAM8 2.527

Appendices 318 Runt-related transcription factor 2 RUNX2 2.542 Enolase 1, (alpha) ENO1 2.559 Cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) Cdkn2c 2.570 Metallothionein 1 Mt1 2.584 Forkhead box F2 FOXF2 2.584 Early growth response 2 (Krox-20 homolog, Drosophila) EGR2 2.586 Cellular retinoic acid binding protein 2 CRABP2 2.607 Metastasis suppressor 1 MTSS1 2.626 Solute carrier family 27 (fatty acid transporter), member 2 SLC27A2 2.633 Docking protein 2, 56kDa DOK2 2.653 Natural killer cell group 7 sequence NKG7 2.653 Mitochondrial ribosomal protein L12 MRPL12 2.660 Coenzyme A synthase COASY 2.685 FCH and double SH3 domains 1 FCHSD1 2.697 Microtubule associated monoxygenase, calponin and LIM domain containing 2 MICAL2 2.698 G protein-coupled receptor, family C, group 5, member C GPRC5C 2.699 CD1c molecule CD1C 2.701 Acid phosphatase, prostate ACPP 2.704 Metadherin MTDH 2.708 Protein phosphatase 2, regulatory subunit B', alpha isoform PPP2R5A 2.746 5'-nucleotidase, ecto (CD73) NT5E 2.751 Granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1) GZMB 2.757 Solute carrier family 27 (fatty acid transporter), member 2 SLC27A2 2.783 RNA binding motif protein 25 RBM25 2.809 Major histocompatibility complex, class II, DQ beta 1 HLA-DQB1 2.816 Cyclin-dependent kinase inhibitor 3 (CDK2-associated dual specificity phosphatase) CDKN3 2.830 Nedd4 binding protein 1 N4BP1 2.840 ATP-binding cassette, sub-family C (CFTR/MRP), member 9 ABCC9 2.842 Eukaryotic translation initiation factor 4 gamma, 3 EIF4G3 2.843 Platelet-derived growth factor receptor, beta polypeptide PDGFRB 2.877 Glycogen synthase kinase 3 beta GSK3B 2.882 Sterol-C4-methyl oxidase-like SC4MOL 2.901 Ubiquitin-conjugating enzyme E2D 4 (putative) UBE2D4 2.916 Ficolin (collagen/fibrinogen domain containing) 1 FCN1 2.941 Gremlin 1, cysteine knot superfamily, homolog (Xenopus laevis) GREM1 2.948 Luteinizing hormone beta polypeptide LHB 2.956 Pregnancy specific beta-1-glycoprotein 10 PSG10 2.989 NIMA (never in mitosis gene a)-related kinase 3 NEK3 2.990 Chemokine (C-X-C motif) ligand 13 (B-cell chemoattractant) CXCL13 3.103 Coenzyme Q5 homolog, methyltransferase (S. cerevisiae) COQ5 3.163 Chemokine (C-C motif) ligand 2 CCL2 3.178 Junctophilin 2 JPH2 3.202 Claudin 3 CLDN3 3.208 ATPase, Class I, type 8B, member 3 ATP8B3 3.219 MAP/microtubule affinity-regulating kinase 2 MARK2 3.227 TNF receptor-associated factor 1 TRAF1 3.249 Transmembrane protein 158 TMEM158 3.275 Kallikrein-related peptidase 8 KLK8 3.309 BCL2-related protein A1 BCL2A1 3.318 Interleukin 17B IL17B 3.324 Phosphatidylinositol 4-kinase, catalytic, beta polypeptide PIK4CB 3.358 Fibroblast growth factor 1 (acidic) FGF1 3.360 CD163 molecule-like 1 CD163L1 3.408 CD1a molecule CD1A 3.444

Appendices 319 Distal-less homeobox 4 DLX4 3.466 Colony stimulating factor 1 receptor, formerly McDonough feline sarcoma viral (v-fms) CSF1R 3.536 oncogene homolog Ubiquilin 1 UBQLN1 3.546 Thimet oligopeptidase 1 THOP1 3.644 responder (tazarotene induced) 3 RARRES3 3.650 Asparaginyl-tRNA synthetase 2, mitochondrial (putative) NARS2 3.660 Coagulation factor VIII-associated (intronic transcript) 1 F8A1 3.758 Cadherin-like 22 CDH22 3.818 Nuclear transcription factor Y, beta NFYB 3.838 TP53 activated protein 1 TP53AP1 3.948 Transmembrane protein 158 TMEM158 3.962 Ubiquitin-conjugating enzyme E2N (UBC13 homolog, yeast) UBE2N 3.998 Isocitrate dehydrogenase 3 (NAD+) beta IDH3B 4.072 Myosin XVIIIA MYO18A 4.143 ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide 1 ATP5C1 4.205 Cyclin-dependent kinase 5 CDK5 4.283 Meis homeobox 3 pseudogene 1 MEIS3P1 4.604 Cyclin A1 CCNA1 4.657 Nipped-B homolog (Drosophila) NIPBL 4.750 RAB37, member RAS oncogene family RAB37 4.874 Early growth response 1 EGR1 4.886 Gap junction protein, alpha 3, 46kDa GJA3 4.929 ATP-binding cassette, sub-family A (ABC1), member 6 ABCA6 4.945 SAR1 gene homolog B (S. cerevisiae) SAR1B 5.051 Cystatin F (leukocystatin) CST7 5.057 COP9 constitutive photomorphogenic homolog subunit 2 (Arabidopsis) COPS2 5.313 Prohibitin PHB 5.362 Potassium large conductance calcium-activated channel, subfamily M, beta member 1 KCNMB1 5.685 Tyrosine kinase, non-receptor, 1 TNK1 5.702 Dual specificity phosphatase 5 DUSP5 5.893 Thioredoxin domain containing 13 TXNDC13 5.904 Ubiquitin specific peptidase 25 USP25 5.963 SH2 domain protein 2A SH2D2A 6.048 Growth hormone receptor GHR 6.162 E2F transcription factor 4, p107/p130-binding 6.166 TNF receptor-associated factor 7 TRAF7 6.171 Cyclin A1 CCNA1 6.360 Superkiller viralicidic activity 2-like 2 (S. cerevisiae) SKIV2L2 6.502 RAB35, member RAS oncogene family RAB35 6.695 Peroxiredoxin 6 PRDX6 6.761 Mitogen-activated protein kinase kinase 7 MAP2K7 6.990 Zinc finger protein 662 ZNF662 7.636 Amyloid beta precursor protein (cytoplasmic tail) binding protein 2 APPBP2 8.179 Membrane-spanning 4-domains, subfamily A, member 6A MS4A6A 8.185 Glutamate receptor, ionotropic, N-methyl D-aspartate 2A GRIN2A 9.266 Histone cluster 1, H2bk HIST1H2BK 10.268 MAP6 domain containing 1 MAP6D1 12.555 Mab-21-like 1 (C. elegans) MAB21L1 14.217 RNA binding motif protein 10 RBM10 30.216 Ankyrin repeat and sterile alpha motif domain containing 1A ANKS1A 34.726 Interleukin 1 receptor-like 1 IL1RL1 41.176

Appendices 320 Table F.3. Genes changed by greater than 2-fold in ALL-3 xenograft cells after 24 hrs. Gene Name Gene ID 24 hrs Membrane-spanning 4-domains, subfamily A, member 5 MS4A5 0.004 Phosphoinositide-3-kinase, regulatory subunit 3 (p55, gamma) PIK3R3 0.016 RAS-like, family 10, member A RASL10A 0.021 Poly(A) polymerase alpha PAPOLA 0.022 Myosin XVIIIA MYO18A 0.044 RAB6A, member RAS oncogene family RAB6A 0.050 Caudal type homeobox transcription factor 2 CDX2 0.058 Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole PAICS 0.060 succinocarboxamide synthetase Ankyrin repeat and sterile alpha motif domain containing 1B ANKS1B 0.061 SEC14-like 1 (S. cerevisiae) SEC14L1 0.083 Nucleolar and spindle associated protein 1 NUSAP1 0.086 Pregnancy specific beta-1-glycoprotein 4 PSG4 0.095 Ring finger protein 138 RNF138 0.107 Protein phosphatase 2, regulatory subunit B', alpha isoform PPP2R5A 0.107 ATPase, H+ transporting, lysosomal 42kDa, V1 subunit C2 ATP6V1C2 0.118 Mediator of RNA polymerase II transcription, subunit 28 homolog (S. cerevisiae) MED28 0.128 NIMA (never in mitosis gene a)-related kinase 3 NEK3 0.131 MAP6 domain containing 1 MAP6D1 0.133 CD96 molecule CD96 0.143 Cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) Cdkn2c 0.145 Numb homolog (Drosophila) NUMB 0.147 Fc fragment of IgG, low affinity IIIa, receptor (CD16a) FCGR3A 0.147 LIM and senescent cell antigen-like domains 3 LIMS3 0.151 ATP-binding cassette, sub-family C (CFTR/MRP), member 9 ABCC9 0.152 Ribonuclease P 40kDa subunit RPP40 0.153 Paired-like homeodomain transcription factor 1 PITX1 0.174 Solute carrier family 25 (mitochondrial carrier; Graves disease autoantigen), member 16 SLC25A16 0.184 Isocitrate dehydrogenase 3 (NAD+) beta IDH3B 0.188 Usher syndrome 1C (autosomal recessive, severe) USH1C 0.193 Peroxisome proliferator-activated receptor alpha PPARA 0.196 Galactosylceramidase GALC 0.199 Selenocysteine lyase SCLY 0.201 Nipped-B homolog (Drosophila) NIPBL 0.204 Structural maintenance of chromosomes 1A SMC1A 0.206 Interleukin 6 receptor IL6R 0.208 Pleckstrin homology domain containing, family H (with MyTH4 domain) member 2 PLEKHH2 0.214 Ecotropic viral integration site 2A EVI2A 0.220 Nucleolar protein 4 NOL4 0.221 GULP, engulfment adaptor PTB domain containing 1 GULP1 0.223 Pannexin 3 PANX3 0.224 Ubiquitin specific peptidase 53 USP53 0.225 Ubiquitin associated protein 1 UBAP1 0.230 TNF receptor-associated factor 2 TRAF2 0.230 Arrestin, beta 2 ARRB2 0.230 Thyrotropin-releasing hormone TRH 0.231 Cyclin A1 CCNA1 0.235 Protein-O-mannosyltransferase 1 POMT1 0.245 Tripartite motif-containing 26 TRIM26 0.249 Pyruvate dehydrogenase (lipoamide) alpha 2 PDHA2 0.249 Pleckstrin PLEK 0.253 Breast carcinoma amplified sequence 3 BCAS3 0.254 Prolactin receptor PRLR 0.255

Appendices 321 Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 1 CITED1 0.256 Acyl-Coenzyme A oxidase 2, branched chain ACOX2 0.257 SAM domain and HD domain, 1 Samhd1 0.259 RAB37, member RAS oncogene family RAB37 0.260 MADS box transcription enhancer factor 2, polypeptide C (myocyte enhancer factor 2C) MEF2C 0.264 Zic family member 1 (odd-paired homolog, Drosophila) ZIC1 0.266 Ubiquitin specific peptidase 31 USP31 0.270 General transcription factor IIIC, polypeptide 2, beta 110kDa GTF3C2 0.271 Histidine triad nucleotide binding protein 3 HINT3 0.272 Pregnancy-associated plasma protein A, pappalysin 1 PAPPA 0.272 Proteasome (prosome, macropain) 26S subunit, non-ATPase, 14 PSMD14 0.274 Ubiquitin-activating enzyme E1-domain containing 1 UBE1DC1 0.275 Echinoderm microtubule associated protein like 1 EML1 0.277 Immunoglobulin lambda-like polypeptide 1 IGLL1 0.277 Colony stimulating factor 2 (granulocyte-macrophage) CSF2 0.278 Contactin 6 CNTN6 0.282 Basal cell adhesion molecule (Lutheran blood group) BCAM 0.283 1-acylglycerol-3-phosphate O-acyltransferase 2 (lysophosphatidic acid acyltransferase, beta) AGPAT2 0.284 Natural killer cell group 7 sequence NKG7 0.287 Tumor necrosis factor (ligand) superfamily, member 13b TNFSF13B 0.289 Similar to 2010300C02Rik protein MGC42367 0.292 Amyloid beta precursor protein (cytoplasmic tail) binding protein 2 APPBP2 0.293 YME1-like 1 (S. cerevisiae) YME1L1 0.293 Group-specific component (vitamin D binding protein) GC 0.294 Interleukin 15 IL15 0.296 Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 1 LILRB1 0.296 Ras responsive element binding protein 1 RREB1 0.296 Cystatin F (leukocystatin) CST7 0.298 Selectin L (lymphocyte adhesion molecule 1) SELL 0.299 Sine oculis homeobox homolog 6 (Drosophila) SIX6 0.301 Runt-related transcription factor 2 RUNX2 0.301 Cbl-interacting protein Sts-1 STS-1 0.304 T cell receptor alpha locus TRA@ 0.307 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6, 14kDa NDUFA6 0.307 Stonin 2 STON2 0.312 Transcobalamin I (vitamin B12 binding protein, R binder family) TCN1 0.312 Synaptotagmin XVII SYT17 0.313 /X (CCAAT-binding transcription factor) NFIX 0.315 TCR gamma alternate reading frame protein TARP 0.316 Solute carrier family 23 (nucleobase transporters), member 1 SLC23A1 0.316 Protein phosphatase 1, catalytic subunit, beta isoform PPP1CB 0.318 Eukaryotic translation initiation factor 4A, isoform 2 EIF4A2 0.319 Killer cell lectin-like receptor subfamily K, member 1 Klrk1 0.321 Ubiquitin-like, containing PHD and RING finger domains, 1 UHRF1 0.322 Translocase of outer mitochondrial membrane 70 homolog A (S. cerevisiae) TOMM70A 0.322 Insulinoma-associated 1 INSM1 0.323 NEFA-interacting nuclear protein NIP30 NIP30 0.325 Frizzled homolog 1 (Drosophila) FZD1 0.326 Deltex 4 homolog (Drosophila) DTX4 0.327 Heparan sulfate 6-O-sulfotransferase 1 HS6ST1 0.329 Regulator of chromosome condensation (RCC1) and BTB (POZ) domain containing protein 1 RCBTB1 0.330 Polycystic kidney and hepatic disease 1 (autosomal recessive) PKHD1 0.331 Transferrin TF 0.332 Protein tyrosine phosphatase, non-receptor type 1 PTPN1 0.333

Appendices 322 E2F transcription factor 4, p107/p130-binding E2F4 0.334 Cystatin F (leukocystatin) CST7 0.337 Tropomyosin 1 (alpha) TPM1 0.340 Tumor necrosis factor (ligand) superfamily, member 10 TNFSF10 0.342 Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMP-N-acetylneuraminate CMAH 0.343 monooxygenase) Solute carrier family 22 (organic anion/cation transporter), member 11 SLC22A11 0.346 Interleukin 1, beta IL1B 0.352 Protein tyrosine phosphatase, non-receptor type 4 (megakaryocyte) PTPN4 0.354 A kinase (PRKA) anchor protein 11 AKAP11 0.357 Wingless-type MMTV integration site family, member 11 WNT11 0.358 COMM domain containing 3 COMMD3 0.360 CD86 molecule CD86 0.361 Phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, type III PIP5K3 0.361 POU domain, class 6, transcription factor 1 POU6F1 0.361 associated protein 2 THRAP2 0.364 Immunoglobulin heavy constant gamma 1 (G1m marker) IGHG1 0.365 Toll-like receptor 3 TLR3 0.366 Major histocompatibility complex, class II, DO alpha HLA-DOA 0.366 Ankyrin repeat domain 15 ANKRD15 0.366 G protein-coupled receptor 85 GPR85 0.367 Interferon regulatory factor 4 IRF4 0.368 Aspartate beta-hydroxylase ASPH 0.371 ATP-binding cassette, sub-family C (CFTR/MRP), member 2 ABCC2 0.372 Eyes absent homolog 2 (Drosophila) EYA2 0.372 Purinergic receptor P2Y, G-protein coupled, 11 P2RY11 0.372 ATPase, Ca++ transporting, plasma membrane 4 ATP2B4 0.372 Bone morphogenetic protein 7 (osteogenic protein 1) BMP7 0.375 Major histocompatibility complex, class II, DQ beta 1 HLA-DQB1 0.377 Calpain 3, (p94) CAPN3 0.377 Papillary renal cell carcinoma (translocation-associated) PRCC 0.377 Cysteine/tyrosine-rich 1 CYYR1 0.378 Thyrotropin-releasing hormone TRH 0.378 Colony stimulating factor 1 receptor, formerly McDonough feline sarcoma viral (v-fms) CSF1R 0.383 oncogene homolog Myosin X MYO10 0.383 Secretogranin II (chromogranin C) SCG2 0.384 Zinc finger E-box binding homeobox 2 ZEB2 0.384 Mucosal vascular addressin cell adhesion molecule 1 MADCAM1 0.385 Scm-like with four mbt domains 2 SFMBT2 0.385 Docking protein 2, 56kDa DOK2 0.386 Neurotrophic tyrosine kinase, receptor, type 2 NTRK2 0.387 beta Cbfb 0.389 Solute carrier family 6 (neurotransmitter transporter, dopamine), member 3 SLC6A3 0.390 Basic leucine zipper and W2 domains 1 BZW1 0.390 Paired box gene 1 PAX1 0.392 Forkhead box F2 FOXF2 0.392 STEAP family member 3 STEAP3 0.393 Transcription factor 12 (HTF4, helix-loop-helix transcription factors 4) TCF12 0.396 Protein kinase C, epsilon PRKCE 0.400 Tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase 2 TNKS2 0.400 Solute carrier family 35, member B1 SLC35B1 0.400 Nucleobindin 2 NUCB2 0.401 Hemoglobin, zeta HBZ 0.401 Pleiotropic regulator 1 (PRL1 homolog, Arabidopsis) PLRG1 0.403

Appendices 323 Calcium channel, voltage-dependent, alpha 2/delta subunit 2 CACNA2D2 0.404 Runt-related transcription factor 2 RUNX2 0.404 Integrin, beta 8 ITGB8 0.405 Solute carrier family 25 (mitochondrial carrier, Aralar), member 12 SLC25A12 0.405 Coiled-coil domain containing 81 CCDC81 0.407 Bestrophin 3 BEST3 0.408 Sarcoglycan, epsilon SGCE 0.411 Solute carrier family 23 (nucleobase transporters), member 1 SLC23A1 0.411 Proline-rich protein HaeIII subfamily 1 PRH1 0.414 Acyl-CoA synthetase long-chain family member 3 ACSL3 0.414 Integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) ITGA4 0.414 Immunoglobulin lambda locus IGL@ 0.414 N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 3 NDST3 0.415 Indoleamine-pyrrole 2,3 dioxygenase INDO 0.416 Dynein, axonemal, heavy chain 7 DNAH7 0.417 Coiled-coil domain containing 69 CCDC69 0.419 Guanylate binding protein 2, interferon-inducible GBP2 0.419 Chemokine (C-C motif) receptor 7 CCR7 0.419 Ribosomal protein S28 RPS28 0.419 Tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase 2 TNKS2 0.420 Phosphodiesterase 5A, cGMP-specific PDE5A 0.420 Solute carrier family 15 (H+/peptide transporter), member 2 SLC15A2 0.421 Insulin INS 0.421 PI-3-kinase-related kinase SMG-1 SMG1 0.421 Synovial sarcoma, X breakpoint 4 SSX4 0.422 CD24 molecule CD24 0.426 Armadillo repeat containing, X-linked 6 ARMCX6 0.426 Isocitrate dehydrogenase 1 (NADP+), soluble Idh1 0.427 RAS guanyl releasing protein 1 (calcium and DAG-regulated) RASGRP1 0.427 Heat shock 22kDa protein 8 HSPB8 0.429 Melanoma antigen family D, 4 MAGED4 0.431 RAB, member of RAS oncogene family-like 2B RABL2B 0.432 Ankyrin repeat domain 28 ANKRD28 0.432 ATPase, Cu++ transporting, alpha polypeptide (Menkes syndrome) ATP7A 0.432 Phosphodiesterase 6G, cGMP-specific, rod, gamma PDE6G 0.432 Lymphoid-restricted membrane protein LRMP 0.433 TCDD-inducible poly(ADP-ribose) polymerase TIPARP 0.434 Signal transducer and activator of transcription 4 Stat4 0.435 Gremlin 1, cysteine knot superfamily, homolog (Xenopus laevis) GREM1 0.435 E74-like factor 1 (ets domain transcription factor) ELF1 0.436 ADAM metallopeptidase with thrombospondin type 1 motif, 1 ADAMTS1 0.436 Prolactin PRL 0.437 Tetratricopeptide repeat domain 4 TTC4 0.438 Eukaryotic translation initiation factor 4E family member 3 EIF4E3 0.438 Gremlin 1, cysteine knot superfamily, homolog (Xenopus laevis) GREM1 0.438 AKT1 substrate 1 (proline-rich) AKT1S1 0.439 Neutrophil cytosolic factor 2 (65kDa, chronic granulomatous disease, autosomal 2) NCF2 0.440 A disintegrin and metallopeptidase domain 19 (meltrin beta) Adam19 0.440 Nuclear receptor subfamily 2, group F, member 2 NR2F2 0.441 Microfibrillar-associated protein 3 MFAP3 0.441 Zinc finger protein 800 ZNF800 0.442 Lemur tyrosine kinase 2 LMTK2 0.443 Denticleless homolog (Drosophila) DTL 0.443 Yamaguchi sarcoma viral (v-yes) oncogene homolog 1 Yes1 0.445

Appendices 324 DOT1-like, histone H3 methyltransferase (S. cerevisiae) DOT1L 0.445 Sortilin-related receptor, L(DLR class) A repeats-containing SORL1 0.447 Heparan sulfate (glucosamine) 3-O-sulfotransferase 3B1 HS3ST3B1 0.448 Zinc finger and BTB domain containing 33 ZBTB33 0.448 Ets variant gene 6 (TEL oncogene) ETV6 0.448 TFIIA-alpha/beta-like factor ALF 0.448 XIAP associated factor-1 XAF1 0.449 Ubiquitin protein ligase E3B UBE3B 0.451 Neighbor of BRCA1 gene 1 NBR1 0.451 Serum/glucocorticoid regulated kinase 2 SGK2 0.451 RNA binding motif protein 5 RBM5 0.451 Colony stimulating factor 1 (macrophage) Csf1 0.452 FERM domain containing 4B FRMD4B 0.452 Survival of motor neuron 2, centromeric SMN2 0.452 Lipin 2 LPIN2 0.454 BTB (POZ) domain containing 16 BTBD16 0.454 Hexokinase 1 HK1 0.454 Mannosidase, beta A, lysosomal MANBA 0.454 Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta NFKBIZ 0.454 Nuclear receptor subfamily 5, group A, member 2 NR5A2 0.455 Histone cluster 1, H4i HIST1H4I 0.455 Pleiomorphic adenoma gene 1 PLAG1 0.456 Integrin-binding sialoprotein (bone sialoprotein, bone sialoprotein II) IBSP 0.457 Phospholipase A2, group IVB (cytosolic) PLA2G4B 0.457 Yippee-like 5 (Drosophila) YPEL5 0.458 CCR4-NOT transcription complex, subunit 6-like CNOT6L 0.458 V-abl Abelson murine leukemia viral oncogene homolog 1 ABL1 0.459 Teashirt family zinc finger 2 TSHZ2 0.459 Nuclear receptor interacting protein 1 NRIP1 0.460 WD repeat and SOCS box-containing 1 WSB1 0.460 Nuclear receptor co-repressor 1 Ncor1 0.461 Heterogeneous nuclear ribonucleoprotein H1 (H) HNRPH1 0.462 TATA element modulatory factor 1 TMF1 0.462 PTPRF interacting protein, binding protein 1 (liprin beta 1) PPFIBP1 0.463 Transcription factor Dp-1 TFDP1 0.463 Myosin, heavy chain 9, non-muscle MYH9 0.464 Dynamin 3 DNM3 0.464 Regulator of G-protein signalling 13 RGS13 0.464 Cellular retinoic acid binding protein 2 CRABP2 0.465 Tetratricopeptide repeat domain 27 TTC27 0.465 Tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase 2 TNKS2 0.465 Proteasome (prosome, macropain) 26S subunit, non-ATPase, 10 PSMD10 0.465 Guanylate cyclase 1, soluble, alpha 3 GUCY1A3 0.466 Fucosyltransferase 1 (galactoside 2-alpha-L-fucosyltransferase, H blood group) FUT1 0.466 MADS box transcription enhancer factor 2, polypeptide A (myocyte enhancer factor 2A) MEF2A 0.467 Potassium inwardly-rectifying channel, subfamily J, member 15 KCNJ15 0.467 IBR domain containing 3 IBRDC3 0.467 Oral-facial-digital syndrome 1 OFD1 0.468 Protocadherin 9 PCDH9 0.468 Dom-3 homolog Z (C. elegans) DOM3Z 0.468 Solute carrier family 17 (sodium phosphate), member 1 SLC17A1 0.469 ADAM metallopeptidase with thrombospondin type 1 motif, 1 ADAMTS1 0.469 Leukocyte-associated immunoglobulin-like receptor 2 LAIR2 0.469 Solute carrier family 6, member 16 SLC6A16 0.470

Appendices 325 Peroxisomal membrane protein 3, 35kDa (Zellweger syndrome) PXMP3 0.471 Receptor-interacting serine-threonine kinase 3 RIPK3 0.471 Mab-21-like 1 (C. elegans) MAB21L1 0.471 Peptidylglycine alpha-amidating monooxygenase COOH-terminal interactor PAMCI 0.472 Calmin (calponin-like, transmembrane) CLMN 0.472 Connector enhancer of kinase suppressor of Ras 1 CNKSR1 0.473 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 PLOD2 0.474 Menage a trois homolog 1, cyclin H assembly factor (Xenopus laevis) MNAT1 0.474 Histone cluster 1, H4e HIST1H4E 0.475 Diacylglycerol kinase, epsilon 64kDa DGKE 0.475 SH2 domain containing 3A SH2D3A 0.475 Heat shock protein 90kDa alpha (cytosolic), class B member 1 HSP90AB1 0.477 Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 SVEP1 0.477 Tripartite motif-containing 34 TRIM34 0.477 Interferon, gamma-inducible protein 16 IFI16 0.477 Placenta-specific 8 PLAC8 0.478 Oral-facial-digital syndrome 1 OFD1 0.478 Stromal interaction molecule 2 STIM2 0.479 Metallothionein-like 5, testis-specific (tesmin) MTL5 0.479 Ropporin 1-like ROPN1L 0.479 Serine peptidase inhibitor, Kazal type 1 SPINK1 0.479 Protocadherin 9 PCDH9 0.480 Regulator of chromosome condensation (RCC1) and BTB (POZ) domain containing protein 1 RCBTB1 0.480 COP9 constitutive photomorphogenic homolog subunit 7A (Arabidopsis) COPS7A 0.480 H2A histone family, member J H2AFJ 0.480 Cullin-associated and neddylation-dissociated 2 (putative) CAND2 0.481 CD200 molecule CD200 0.481 Fibroblast growth factor 1 (acidic) FGF1 0.482 Spectrin, alpha, erythrocytic 1 (elliptocytosis 2) SPTA1 0.482 Prion protein Prnp 0.482 Phospholipase C, beta 1 (phosphoinositide-specific) PLCB1 0.483 Dapper, antagonist of beta-catenin, homolog 1 (Xenopus laevis) DACT1 0.483 Zinc finger, CCHC domain containing 2 ZCCHC2 0.483 Suppressor of Ty 3 homolog (S. cerevisiae) SUPT3H 0.483 Golgi phosphoprotein 2 GOLPH2 0.484 Dehydrogenase/reductase (SDR family) member 3 DHRS3 0.484 Death inducer-obliterator 1 DIDO1 0.485 Ubiquitin-like, containing PHD and RING finger domains, 1 UHRF1 0.486 Titin TTN 0.486 Upstream transcription factor 2, c-fos interacting USF2 0.486 Signal-induced proliferation-associated 1 like 1 SIPA1L1 0.486 Retinoic acid receptor responder (tazarotene induced) 3 RARRES3 0.486 Casein kinase 1, delta CSNK1D 0.487 Golgi phosphoprotein 2 GOLPH2 0.487 Ring finger protein 13 RNF13 0.487 Brain specific protein CGI-38 0.487 Erythrocyte membrane protein band 4.9 (dematin) EPB49 0.488 G protein-coupled receptor, family C, group 5, member A GPRC5A 0.489 Pre-B lymphocyte gene 3 VPREB3 0.489 Ankyrin repeat domain 31 ANKRD31 0.489 RANBP2-like and GRIP domain containing 2 RGPD2 0.489 Carbonic anhydrase XIV CA14 0.489 Cylindromatosis (turban tumor syndrome) CYLD 0.489 CD163 molecule-like 1 CD163L1 0.490

Appendices 326 Tudor domain containing 7 TDRD7 0.491 Lipase, hepatic LIPC 0.491 Olfactory receptor, family 2, subfamily A, member 7 OR2A7 0.491 Prion protein 2 (dublet) PRND 0.492 Interferon-induced protein with tetratricopeptide repeats 2 IFIT2 0.492 Src-like-adaptor SLA 0.493 EPH receptor B2 EPHB2 0.494 Neurexin 3 NRXN3 0.494 Casein kinase 1, gamma 1 CSNK1G1 0.494 B-box and SPRY domain containing BSPRY 0.494 Armadillo repeat containing, X-linked 4 ARMCX4 0.495 Cas-Br-M (murine) ecotropic retroviral transforming sequence b CBLB 0.495 T-cell lymphoma invasion and metastasis 2 TIAM2 0.495 Mitogen-activated protein kinase kinase kinase 1 MAP3K1 0.495 Splicing factor, arginine/serine-rich 5 SFRS5 0.496 Ankyrin repeat and sterile alpha motif domain containing 6 ANKS6 0.496 Vav 3 oncogene VAV3 0.496 Low density lipoprotein receptor-related protein 3 LRP3 0.496 Transmembrane protein 163 TMEM163 0.497 Down syndrome critical region gene 1 DSCR1 0.498 Cysteine and glycine-rich protein 2 CSRP2 0.498 Interleukin 2 receptor, gamma (severe combined immunodeficiency) IL2RG 0.498 Solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system Xag), SLC1A1 0.498 member 1 Ribosomal protein L41 RPL41 0.498 Microtubule associated monoxygenase, calponin and LIM domain containing 1 MICAL1 0.499 Cyclin A1 CCNA1 0.499 Tumor necrosis factor (ligand) superfamily, member 13 TNFSF13 0.499 Nucleosome assembly protein 1-like 4 Nap1l4 0.499 DIRAS family, GTP-binding RAS-like 3 DIRAS3 0.499 Calcium channel, voltage-dependent, alpha 2/delta subunit 2 CACNA2D2 0.499 Interleukin 23, alpha subunit p19 IL23A 0.499 Arrestin, beta 1 ARRB1 0.499 Chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) CXCL1 0.500 Pregnancy-associated plasma protein A, pappalysin 1 PAPPA 2.000 Phytanoyl-CoA 2-hydroxylase interacting protein-like PHYHIPL 2.002 ATPase, Na+/K+ transporting, alpha 2 (+) polypeptide ATP1A2 2.002 UV radiation resistance associated gene UVRAG 2.003 Gap junction protein, alpha 3, 46kDa GJA3 2.005 Membrane-spanning 4-domains, subfamily A, member 1 MS4A1 2.009 Vasohibin 2 VASH2 2.011 Proline rich Gla (G-carboxyglutamic acid) 1 PRRG1 2.014 Guanylate cyclase 1, soluble, alpha 3 GUCY1A3 2.016 F-box protein 2 FBXO2 2.017 Conserved nuclear protein NHN1 NHN1 2.018 Mediterranean fever MEFV 2.019 Elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 4 ELOVL4 2.021 Retinol dehydrogenase 10 (all-trans) RDH10 2.022 Rhabdoid tumor deletion region gene 1 RTDR1 2.022 Protocadherin alpha subfamily C, 2 PCDHAC2 2.023 Interleukin 1 receptor, type I IL1R1 2.023 Lactamase, beta 2 LACTB2 2.028 Sodium channel, voltage-gated, type III, alpha subunit SCN3A 2.028 Zinc finger protein 202 ZNF202 2.029 Membrane associated guanylate kinase, WW and PDZ domain containing 3 MAGI3 2.030

Appendices 327 Zinc finger and BTB domain containing 38 ZBTB38 2.031 Gremlin 2, cysteine knot superfamily, homolog (Xenopus laevis) GREM2 2.032 Leukemia inhibitory factor receptor alpha LIFR 2.032 Rho GTPase activating protein 28 ARHGAP28 2.033 Myeloid leukemia factor 1 MLF1 2.036 Schwannomin interacting protein 1 SCHIP1 2.039 SERTA domain containing 4 SERTAD4 2.040 Tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase 2 TNKS2 2.042 Small nuclear RNA activating complex, polypeptide 4, 190kDa SNAPC4 2.048 CAP-GLY domain containing linker protein 2 CLIP2 2.053 Tumor protein -like TP73L 2.056 Fibronectin type III domain containing 1 FNDC1 2.069 F-box protein 2 FBXO2 2.072 Biphenyl hydrolase-like (serine hydrolase; breast epithelial mucin-associated antigen) BPHL 2.073 Ets homologous factor EHF 2.075 Solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 SLC7A2 2.080 Signal sequence receptor, delta (translocon-associated protein delta) SSR4 2.088 Deleted in liver cancer 1 DLC1 2.089 Spondin 1, extracellular matrix protein SPON1 2.089 UDP-glucose ceramide glucosyltransferase-like 2 UGCGL2 2.091 Inhibitor of DNA binding 3, dominant negative helix-loop-helix protein ID3 2.093 Caveolin 2 CAV2 2.096 Brain-specific angiogenesis inhibitor 3 BAI3 2.096 FLYWCH-type zinc finger 1 FLYWCH1 2.097 Paternally expressed 3 PEG3 2.097 Solute carrier family 22 (organic anion/cation transporter), member 10 SLC22A10 2.098 Ataxia telangiectasia mutated (includes complementation groups A, C and D) ATM 2.098 Glutamate receptor, metabotropic 7 GRM7 2.102 Storkhead box 1 STOX1 2.102 Eukaryotic translation initiation factor 4 gamma, 1 EIF4G1 2.103 Immunoglobulin heavy constant gamma 1 (G1m marker) IGHG1 2.104 Leucine rich repeat containing 51 LRRC51 2.105 S100 calcium binding protein A2 S100A2 2.106 Dickkopf homolog 3 (Xenopus laevis) DKK3 2.110 Dual specificity phosphatase 13 DUSP13 2.111 Bromodomain adjacent to zinc finger domain, 2B BAZ2B 2.112 Prospero-related homeobox 1 PROX1 2.112 CD93 molecule CD93 2.114 Intersectin 1 (SH3 domain protein) ITSN1 2.117 Dickkopf homolog 2 (Xenopus laevis) DKK2 2.119 Armadillo repeat containing 2 ARMC2 2.121 EF-hand calcium binding domain 2 EFCAB2 2.124 Slit homolog 3 (Drosophila) SLIT3 2.124 Ribonuclease, RNase A family, 11 (non-active) RNASE11 2.125 Acyltransferase like 1 AYTL1 2.127 Solute carrier family 2 (facilitated glucose transporter), member 9 SLC2A9 2.127 Neural cell adhesion molecule 1 NCAM1 2.129 Synuclein, alpha interacting protein (synphilin) SNCAIP 2.129 Discs, large homolog 2, chapsyn-110 (Drosophila) DLG2 2.134 Anterior gradient homolog 2 (Xenopus laevis) AGR2 2.136 Nitric oxide synthase 1 (neuronal) adaptor protein NOS1AP 2.137 Inhibitor of DNA binding 4, dominant negative helix-loop-helix protein ID4 2.137 Kelch repeat and BTB (POZ) domain containing 3 KBTBD3 2.138 Keratin 7 KRT7 2.140

Appendices 328 Alpha-kinase 2 ALPK2 2.140 Hook homolog 3 (Drosophila) HOOK3 2.141 Sterile alpha motif domain containing 9-like SAMD9L 2.144 Bestrophin 3 BEST3 2.145 Carbamoyl-phosphate synthetase 1, mitochondrial CPS1 2.146 Aquaporin 6, kidney specific AQP6 2.147 Son of sevenless homolog 1 (Drosophila) SOS1 2.152 WAP four-disulfide core domain 1 WFDC1 2.159 Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 4 LILRB4 2.161 Phosphodiesterase 9A PDE9A 2.161 Down syndrome critical region gene 8 DSCR8 2.161 Tubulin tyrosine ligase-like family, member 2 TTLL2 2.162 GM2 ganglioside activator GM2A 2.166 Unc-13 homolog C (C. elegans) UNC13C 2.174 Crumbs homolog 1 (Drosophila) CRB1 2.174 V-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian) MAF 2.175 Phospholipase A2, group XIIA PLA2G12A 2.177 Myelin transcription factor 1-like MYT1L 2.177 Sine oculis homeobox homolog 1 (Drosophila) SIX1 2.178 Protein tyrosine phosphatase, receptor type, C PTPRC 2.179 G protein-coupled receptor 68 GPR68 2.184 HEAT repeat containing 4 HEATR4 2.184 Cysteine-rich, angiogenic inducer, 61 CYR61 2.188 Centromere protein J CENPJ 2.188 InaD-like (Drosophila) INADL 2.190 Serologically defined colon cancer antigen 1 SDCCAG1 2.191 Sarcoglycan, delta (35kDa dystrophin-associated glycoprotein) SGCD 2.193 Chemokine (C-C motif) ligand 23 CCL23 2.199 ERO1-like beta (S. cerevisiae) ERO1LB 2.203 Tolloid-like 2 TLL2 2.203 Nestin NES 2.209 Zinc finger, MYM-type 2 ZMYM2 2.216 Tubulin folding cofactor A TBCA 2.218 Delta-like 1 homolog (Drosophila) DLK1 2.220 Fibronectin leucine rich transmembrane protein 2 FLRT2 2.229 TBC1 domain family, member 8B (with GRAM domain) TBC1D8B 2.230 Neurofilament, heavy polypeptide 200kDa NEFH 2.230 Zinc finger protein 71 ZNF71 2.237 SRY (sex determining region Y)-box 4 SOX4 2.238 Gamma-aminobutyric acid (GABA) A receptor, alpha 1 GABRA1 2.250 Methylthioadenosine phosphorylase MTAP 2.257 Germ cell associated 1 GSG1 2.257 Phosphatidylinositol transfer protein, beta PITPNB 2.262 Zinc finger protein 708 ZNF708 2.263 Ankyrin repeat domain 45 ANKRD45 2.266 WW and C2 domain containing 2 WWC2 2.269 Brain protein 44-like BRP44L 2.276 R-spondin 3 homolog (Xenopus laevis) RSPO3 2.277 Musculin (activated B-cell factor-1) MSC 2.278 2'-5'-oligoadenylate synthetase 2, 69/71kDa OAS2 2.278 Aminolevulinate, delta-, synthase 2 (sideroblastic/hypochromic anemia) ALAS2 2.283 Pregnancy-associated plasma protein A, pappalysin 1 PAPPA 2.285 Structural maintenance of chromosomes 6 SMC6 2.290 PALM2- PALM2-AKAP2 protein 2.298 AKAP2

Appendices 329 Transient receptor potential cation channel, subfamily M, member 1 TRPM1 2.304 B-cell linker BLNK 2.304 Receptor-interacting serine-threonine kinase 4 RIPK4 2.305 Gamma-aminobutyric acid (GABA) A receptor, alpha 2 GABRA2 2.306 Retinol binding protein 1, cellular RBP1 2.314 Cat eye syndrome chromosome region, candidate 8 CECR8 2.328 4-hydroxyphenylpyruvate dioxygenase HPD 2.331 LOH11CR2 Loss of heterozygosity, 11, chromosomal region 2, gene A 2.342 A LON peptidase N-terminal domain and ring finger 2 LONRF2 2.345 Golgi autoantigen, golgin subfamily a, 1 GOLGA1 2.350 WAP four-disulfide core domain 2 WFDC2 2.372 Two transmembrane domain family member A TTMA 2.386 Prostaglandin I2 (prostacyclin) receptor (IP) PTGIR 2.387 SAM and SH3 domain containing 1 SASH1 2.387 TEA domain family member 1 (SV40 transcriptional enhancer factor) TEAD1 2.389 ICEBERG caspase-1 inhibitor ICEBERG 2.390 Flavin containing monooxygenase 1 FMO1 2.391 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 3 ST8SIA3 2.395 Cyclin-dependent kinase-like 1 (CDC2-related kinase) CDKL1 2.406 Sodium channel, voltage-gated, type II, alpha subunit SCN2A 2.408 MCM2 minichromosome maintenance deficient 2, mitotin (S. cerevisiae) MCM2 2.409 RAB39B, member RAS oncogene family RAB39B 2.414 Nuclear casein kinase and cyclin-dependent kinase substrate 1 NUCKS1 2.422 Phosphorylase kinase, alpha 1 (muscle) PHKA1 2.432 Fibrinogen-like 2 FGL2 2.432 Farnesyltransferase, CAAX box, alpha FNTA 2.435 Potassium channel, subfamily U, member 1 KCNU1 2.440 DnaJ homology subfamily A member 5 DNAJA5 2.444 Lysyl oxidase-like 1 LOXL1 2.471 Protocadherin 17 PCDH17 2.477 Cysteine sulfinic acid decarboxylase CSAD 2.485 TEA domain family member 1 (SV40 transcriptional enhancer factor) TEAD1 2.491 Malic enzyme 3, NADP(+)-dependent, mitochondrial ME3 2.501 Sterile alpha motif domain containing 13 SAMD13 2.502 Polymerase I and transcript release factor PTRF 2.507 BCL2-associated athanogene 2 BAG2 2.512 FRAS1 related extracellular matrix 1 FREM1 2.514 Gamma-aminobutyric acid (GABA) A receptor, alpha 1 GABRA1 2.514 Cytokine receptor-like factor 1 CRLF1 2.523 TMDC II TMDCII 2.527 Zinc finger protein, multitype 2 ZFPM2 2.529 TBC1 domain family, member 15 TBC1D15 2.534 Amphiregulin (schwannoma-derived growth factor) AREG 2.537 Tu translation elongation factor, mitochondrial TUFM 2.540 Ribosomal protein S6 kinase, polypeptide 1 Rps6kb1 2.540 Latrophilin 3 LPHN3 2.543 B-cell CLL/lymphoma 11B (zinc finger protein) BCL11B 2.569 NK2 transcription factor related, locus 8 (Drosophila) NKX2-8 2.577 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 PLOD2 2.580 TSPY-like 6 TSPYL6 2.586 Zinc finger protein 135 ZNF135 2.592 Microtubule associated serine/threonine kinase family member 4 MAST4 2.596 ADP-ribosylation factor-like 13B ARL13B 2.604 Speckle-type POZ protein SPOP 2.616

Appendices 330 Asparagine-linked glycosylation 9 homolog (S. cerevisiae, alpha- 1,2-mannosyltransferase) ALG9 2.617 CD52 molecule CD52 2.644 Galanin GAL 2.649 G protein-coupled receptor 158 GPR158 2.650 Glycophorin E GYPE 2.651 Glutamate receptor, ionotropic, AMPA 2 GRIA2 2.665 Transmembrane phosphatase with tensin homology TPTE 2.665 Multiple EGF-like-domains 10 MEGF10 2.674 Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2-like MTHFD2L 2.674 Small nucleolar RNA host gene (non-protein coding) 6 SNHG6 2.689 Cytokine-like 1 CYTL1 2.691 G antigen 7B GAGE7B 2.707 Malic enzyme 1, NADP(+)-dependent, cytosolic ME1 2.712 Musculin (activated B-cell factor-1) MSC 2.715 Cysteine-rich secretory protein 2 CRISP2 2.719 Myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog,Drosophila);translocated to 4 MLLT4 2.720 ATPase, Na+/K+ transporting, alpha 3 polypeptide ATP1A3 2.732 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 5 GALNT5 2.733 (GalNAc-T5) Fibrous sheath interacting protein 1 FSIP1 2.733 Sperm antigen with calponin homology and coiled-coil domains 1 SPECC1 2.741 Integrin, beta 8 ITGB8 2.743 Integrin, alpha 8 ITGA8 2.758 Cytotoxic T-lymphocyte-associated protein 4 CTLA4 2.758 Acetyl-Coenzyme A carboxylase alpha ACACA 2.766 Microsomal glutathione S-transferase 1 MGST1 2.773 Mov10l1, Moloney leukemia virus 10-like 1, homolog (mouse) MOV10L1 2.777 Hairy/enhancer-of-split related with YRPW motif-like HEYL 2.796 Tyrosine kinase, non-receptor, 1 TNK1 2.816 Transmembrane protein 182 TMEM182 2.824 Folliculin FLCN 2.845 Sarcolipin SLN 2.852 SET and MYND domain containing 1 SMYD1 2.852 Spermatogenesis and centriole associated 1 SPATC1 2.853 Mitogen-activated protein kinase 8 MAPK8 2.861 ST7 overlapping transcript 4 (non-coding RNA) ST7OT4 2.863 COBL-like 1 COBLL1 2.881 WW and C2 domain containing 2 WWC2 2.887 Potassium channel tetramerisation domain containing 1 KCTD1 2.935 Tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) TNFRSF11B 2.943 Ankyrin repeat domain 38 ANKRD38 2.951 Sorting nexin 9 SNX9 2.956 Synaptopodin 2 SYNPO2 2.960 Immunoglobulin heavy constant gamma 1 (G1m marker) IGHG1 2.977 Growth hormone receptor GHR 2.977 DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 DDX4 2.981 Major histocompatibility complex, class II, DR beta 4 HLA-DRB4 2.988 Contactin 4 CNTN4 2.995 BUB3 budding uninhibited by benzimidazoles 3 homolog (yeast) BUB3 2.999 Neuroblastoma breakpoint family, member 1 NBPF1 3.003 Zinc finger with KRAB and SCAN domains 1 ZKSCAN1 3.005 BolA homolog 1 (E. coli) BOLA1 3.011 Ubiquitously transcribed tetratricopeptide repeat gene, Y-linked UTY 3.025 Malic enzyme 1, NADP(+)-dependent, cytosolic ME1 3.028 Kelch-like 3 (Drosophila) KLHL3 3.049

Appendices 331 Paraspeckle component 1 PSPC1 3.051 Integral membrane protein 2C ITM2C 3.057 Armadillo repeat containing, X-linked 5 ARMCX5 3.075 Coiled-coil domain containing 113 CCDC113 3.077 Four and a half LIM domains 5 FHL5 3.079 Solute carrier family 36 (proton/amino acid symporter), member 3 SLC36A3 3.084 DnaJ homology subfamily A member 5 DNAJA5 3.086 Actin filament associated protein 1-like 1 AFAP1L1 3.088 Target of myb1-like 2 (chicken) TOM1L2 3.096 Heat shock transcription factor family member 5 HSF5 3.103 Malic enzyme 1, NADP(+)-dependent, cytosolic ME1 3.110 Sterile alpha motif and leucine zipper containing kinase AZK ZAK 3.115 Testicular soluble adenylyl cyclase SAC 3.118 Delta/notch-like EGF repeat containing DNER 3.127 CD209 molecule CD209 3.132 Polycystic kidney disease 2-like 1 PKD2L1 3.141 HESX homeobox 1 HESX1 3.154 Plasticity related gene 1 LPPR4 3.168 B-cell CLL/lymphoma 8 BCL8 3.178 Amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 4 ALS2CR4 3.204 Desmoglein 1 DSG1 3.207 Transmembrane protein 30B TMEM30B 3.229 Ectonucleoside triphosphate diphosphohydrolase 3 ENTPD3 3.238 Makorin, ring finger protein, 2 Mkrn2 3.267 LIM domain only 3 (rhombotin-like 2) LMO3 3.274 G protein-coupled receptor 126 GPR126 3.299 Neuroligin 4, X-linked NLGN4X 3.315 Desmoplakin DSP 3.343 Tripartite motif-containing 36 TRIM36 3.361 Double C2-like domains, alpha DOC2A 3.404 Protein phosphatase 4, regulatory subunit 1 PPP4R1 3.405 Serpin peptidase inhibitor, clade B (ovalbumin), member 3 SERPINB3 3.410 ST6 beta-galactosamide alpha-2,6-sialyltranferase 2 ST6GAL2 3.411 Ribosomal protein L28 RPL28 3.436 Chloride intracellular channel 6 CLIC6 3.439 GPI deacylase PGAP1 3.441 Transmembrane and coiled-coil domains 2 TMCO2 3.443 Retinal degeneration 3 RD3 3.453 Coiled-coil domain containing 62 CCDC62 3.477 RAB35, member RAS oncogene family RAB35 3.478 Teashirt family zinc finger 2 TSHZ2 3.479 Keratin 19 KRT19 3.488 Tetratricopeptide repeat domain 18 TTC18 3.577 Placenta-specific 7 PLAC7 3.596 Fatty acid binding protein 4, adipocyte FABP4 3.605 Endothelial differentiation, sphingolipid G-protein-coupled receptor, 3 EDG3 3.611 Spastic paraplegia 7, paraplegin (pure and complicated autosomal recessive) SPG7 3.614 Phosphoglycerate kinase 1 PGK1 3.658 Single stranded DNA binding protein 4 SSBP4 3.674 CAMSAP1L Calmodulin regulated spectrin-associated protein 1-like 1 3.682 1 Phytoceramidase, alkaline PHCA 3.692 ROD1 regulator of differentiation 1 (S. pombe) ROD1 3.735 Distal-less homeobox 4 DLX4 3.748 Matrix metallopeptidase 16 (membrane-inserted) MMP16 3.758

Appendices 332 HCG23177 hCG_23177 3.768 TAF7-like RNA polymerase II, TATA box binding protein (TBP)-associated factor, 50kDa TAF7L 3.787 Dipeptidyl-peptidase 10 DPP10 3.790 Dpy-19-like 2 (C. elegans) DPY19L2 3.816 GTPase activating protein (SH3 domain) binding protein 1 G3BP1 3.851 TRNA splicing endonuclease 54 homolog (S. cerevisiae) TSEN54 3.927 Scinderin SCIN 3.938 Protein phosphatase 1H (PP2C domain containing) PPM1H 3.980 Menage a trois homolog 1, cyclin H assembly factor (Xenopus laevis) MNAT1 3.993 Uridine phosphorylase 2 UPP2 4.000 Aquaporin 9 AQP9 4.020 Progestin and adipoQ receptor family member III PAQR3 4.032 Secretory leukocyte peptidase inhibitor SLPI 4.048 Osteopetrosis associated transmembrane protein 1 OSTM1 4.073 Kinesin family member 21A KIF21A 4.076 Serpin peptidase inhibitor, clade B (ovalbumin), member 4 SERPINB4 4.085 Sarcoglycan, beta (43kDa dystrophin-associated glycoprotein) SGCB 4.090 Serine PI Kazal type 5-like 3 SPINK5L3 4.117 RAB3B, member RAS oncogene family RAB3B 4.144 Cell adhesion molecule 1 CADM1 4.169 MON1 homolog A (yeast) MON1A 4.179 RAB23, member RAS oncogene family RAB23 4.220 Erythrocyte membrane protein band 4.1 like 4A EPB41L4A 4.222 Potassium voltage-gated channel, shaker-related subfamily, member 1 (episodic ataxia with KCNA1 4.249 myokymia) WNT1 inducible signaling pathway protein 1 WISP1 4.330 Hemoglobin, beta HBB 4.428 Membrane-spanning 4-domains, subfamily A, member 6A MS4A6A 4.484 Trinucleotide repeat containing 6C TNRC6C 4.499 Claudin 1 CLDN1 4.503 RAN binding protein 17 RANBP17 4.504 Bassoon (presynaptic cytomatrix protein) BSN 4.505 Grancalcin, EF-hand calcium binding protein GCA 4.519 Coiled-coil domain containing 101 CCDC101 4.520 LYR motif containing 5 LYRM5 4.588 Ring finger protein 32 RNF32 4.592 Supervillin SVIL 4.738 Collagen, type XII, alpha 1 COL12A1 4.818 Potassium inwardly-rectifying channel, subfamily J, member 8 KCNJ8 4.920 Interleukin 24 IL24 5.055 Sex comb on midleg-like 2 (Drosophila) SCML2 5.166 Prokineticin receptor 1 PROKR1 5.183 Potassium channel tetramerisation domain containing 7 KCTD7 5.204 Lactamase, beta 2 LACTB2 5.245 Prohibitin PHB 5.285 Ras association (RalGDS/AF-6) and pleckstrin homology domains 1 RAPH1 5.355 Leucine rich repeat containing 3B LRRC3B 5.389 Transmembrane protease, serine 12 TMPRSS12 5.520 Suppressor of variegation 3-9 homolog 2 (Drosophila) SUV39H2 5.562 NUF2, NDC80 kinetochore complex component, homolog (S. cerevisiae) NUF2 5.583 MADS box transcription enhancer factor 2, polypeptide D (myocyte enhancer factor 2D) MEF2D 5.644 Mannosidase, alpha, class 1A, member 1 MAN1A1 5.730 Sestrin 1 SESN1 5.902 AT rich interactive domain 2 (ARID, RFX-like) ARID2 5.977 Erythrocyte membrane protein band 4.1 (elliptocytosis 1, RH-linked) EPB41 6.213

Appendices 333 Ras and Rab interactor 3 RIN3 6.220 Lipase, member I LIPI 6.228 Solute carrier family 35, member E1 SLC35E1 6.500 Abelson helper integration site 1 AHI1 6.582 Leucine rich repeat neuronal 1 LRRN1 6.708 Solute carrier family 38, member 4 SLC38A4 6.860 Peroxidasin homolog (Drosophila)-like PXDNL 7.132 Microtubule-associated protein 9 MAP9 7.336 FK506 binding protein 14, 22 kDa FKBP14 7.591 Membrane-spanning 4-domains, subfamily A, member 7 MS4A7 7.864 Trafficking protein particle complex 6B TRAPPC6B 7.878 Nedd4 family interacting protein 2 NDFIP2 8.012 Zinc finger E-box binding homeobox 1 ZEB1 8.058 Thrombospondin, type I, domain containing 3 THSD3 8.235 Variable charge, X-linked 3A VCX3A 8.411 Supervillin SVIL 8.700 Cyclin M2 CNNM2 9.357 ATPase, Class V, type 10B ATP10B 9.553 NADPH oxidase 4 NOX4 9.957 Grainyhead-like 1 (Drosophila) GRHL1 10.730 Vesicle transport through interaction with t-SNAREs homolog 1A (yeast) VTI1A 11.270 Roundabout, axon guidance receptor, homolog 2 (Drosophila) ROBO2 11.860 G protein-coupled receptor 126 GPR126 11.924 Glutamate receptor, metabotropic 3 GRM3 12.044 Homeobox B9 HOXB9 12.755 Synaptopodin 2 SYNPO2 14.399 Collagen, type XI, alpha 1 COL11A1 14.907 Zic family member 3 heterotaxy 1 (odd-paired homolog, Drosophila) ZIC3 15.119 Angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 ACE2 16.211 RAB27B, member RAS oncogene family RAB27B 18.190 HFM1, ATP-dependent DNA helicase homolog (S. cerevisiae) HFM1 19.852 SH3 and cysteine rich domain STAC 22.133 Nedd4 family interacting protein 2 NDFIP2 22.733 Mal, T-cell differentiation protein 2 MAL2 24.055 DnaJ (Hsp40) homolog, subfamily A, member 1 DNAJA1 25.648 Sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6D SEMA6D 25.852 SAC3 domain containing 1 SAC3D1 27.912 FYVE, RhoGEF and PH domain containing 2 FGD2 30.769 CDK5 regulatory subunit associated protein 2 CDK5RAP2 30.955 Interleukin 17B IL17B 31.202 One cut domain, family member 2 ONECUT2 31.837 Spermatogenic leucine zipper 1 SPZ1 34.436 Tubulin, delta 1 TUBD1 66.140 Matrix metallopeptidase 27 MMP27 87.248 Anaphase promoting complex subunit 2 ANAPC2 95.406 Integrin-binding sialoprotein (bone sialoprotein, bone sialoprotein II) IBSP 152.94 Collagen, type XXI, alpha 1 COL21A1 191.58 Metastasis associated 1 family, member 3 MTA3 363.84

Appendices 334  

APPENDIX G

G.1 Genes Two-Fold Differentially Regulated by FL in ALL-17

Xenograft Cells

Table G.1. Genes changed by greater than 2-fold in ALL-17 xenograft cells after 2 hrs. Gene Name Gene ID 2 hrs WD repeat domain 22 WDR22 0.012 Prokineticin receptor 1 PROKR1 0.016 SET domain containing 5 SETD5 0.024 Peroxiredoxin 1 PRDX1 0.026 Neuroblastoma, suppression of tumorigenicity 1 NBL1 0.030 Protease, serine, 23 PRSS23 0.030 RAS-like, family 10, member A RASL10A 0.035 Beta-site APP-cleaving enzyme 1 BACE1 0.036 Solute carrier family 31 (copper transporters), member 1 SLC31A1 0.038 Protein phosphatase 4, regulatory subunit 1 PPP4R1 0.055 Solute carrier family 35, member E1 SLC35E1 0.067 Phosphoserine phosphatase PSPH 0.070 Nedd4 family interacting protein 2 NDFIP2 0.071 Nipped-B homolog (Drosophila) NIPBL 0.095 Arrestin, beta 2 ARRB2 0.113 Solute carrier family 22 (organic cation transporter), member 16 SLC22A16 0.131 Hairy/enhancer-of-split related with YRPW motif 2 HEY2 0.149 Ankyrin repeat domain 31 ANKRD31 0.151 Fas apoptotic inhibitory molecule FAIM 0.156 RAB9B, member RAS oncogene family RAB9B 0.160 ATPase, Class V, type 10A ATP10A 0.163 Superkiller viralicidic activity 2-like 2 (S. cerevisiae) SKIV2L2 0.163 NK2 transcription factor related, locus 8 (Drosophila) NKX2-8 0.172 Zinc finger protein 3 ZNF3 0.173 Sarcolipin SLN 0.181 Sine oculis homeobox homolog 6 (Drosophila) SIX6 0.190 Attractin ATRN 0.201 Pleckstrin homology domain containing, family A member 5 PLEKHA5 0.210 Zinc finger protein 649 ZNF649 0.211 Melanoma antigen family A, 4 MAGEA4 0.211 ADAM-like, decysin 1 ADAMDEC1 0.213 Mannose-binding lectin (protein A) 1, pseudogene 1 MBL1P1 0.221 Makorin, ring finger protein, 2 Mkrn2 0.225

Appendices 335 Dynein, axonemal, light intermediate chain 1 DNALI1 0.228 Erythroid associated factor ERAF 0.229 Chemokine (C-X-C motif) ligand 14 CXCL14 0.236 Par-3 partitioning defective 3 homolog (C. elegans) PARD3 0.246 TRNA phosphotransferase 1 TRPT1 0.271 Protogenin homolog (Gallus gallus) PRTG 0.271 Thyroid transcription factor 1 TITF1 0.274 Proteasome (prosome, macropain) 26S subunit, non-ATPase, 14 PSMD14 0.279 Peptidase D PEPD 0.290 G protein-coupled receptor 177 GPR177 0.303 Cyclin-dependent kinase-like 1 (CDC2-related kinase) CDKL1 0.308 WAP four-disulfide core domain 2 WFDC2 0.309 Sterile alpha motif domain containing 4A SAMD4A 0.314 Integrin-binding sialoprotein (bone sialoprotein, bone sialoprotein II) IBSP 0.317 Protein tyrosine phosphatase-like (proline instead of catalytic arginine), member b PTPLB 0.319 Zinc finger E-box binding homeobox 1 ZEB1 0.319 RAD9 homolog A (S. pombe) RAD9A 0.323 HLA-B associated transcript 2 BAT2 0.323 F-box and leucine-rich repeat protein 7 FBXL7 0.324 Met proto-oncogene (hepatocyte growth factor receptor) MET 0.329 Neuron derived neurotrophic factor NENF 0.334 Developmentally regulated GTP binding protein 1 DRG1 0.337 Phytanoyl-CoA 2-hydroxylase interacting protein-like PHYHIPL 0.343 Neurexophilin 4 NXPH4 0.344 Glycosylphosphatidylinositol specific phospholipase D1 GPLD1 0.345 Paralemmin PALM 0.352 Coiled-coil domain containing 90A CCDC90A 0.353 Salvador homolog 1 (Drosophila) SAV1 0.354 Rho GTPase activating protein 5 ARHGAP5 0.368 SH2 domain containing 5 SH2D5 0.371 Double C2-like domains, alpha DOC2A 0.374 Interleukin 17B IL17B 0.378 Coenzyme Q10 homolog A (S. cerevisiae) COQ10A 0.391 Eukaryotic translation initiation factor 4A, isoform 2 EIF4A2 0.391 Small nucleolar RNA host gene (non-protein coding) 6 SNHG6 0.399 Hairy/enhancer-of-split related with YRPW motif-like HEYL 0.405 RAB6A, member RAS oncogene family RAB6A 0.417 SAC3 domain containing 1 SAC3D1 0.419 Kinesin family member 21A KIF21A 0.425 RAB35, member RAS oncogene family RAB35 0.426 MCM2 minichromosome maintenance deficient 2, mitotin (S. cerevisiae) MCM2 0.426 Pleiotropic regulator 1 (PRL1 homolog, Arabidopsis) PLRG1 0.428 Bestrophin 3 BEST3 0.434 LIM domain only 3 (rhombotin-like 2) LMO3 0.435 Vesicle transport through interaction with t-SNAREs homolog 1A (yeast) VTI1A 0.437 Ribosomal protein S4, Y-linked 1 RPS4Y1 0.445 Usher syndrome 2A (autosomal recessive, mild) USH2A 0.446 TIA1 cytotoxic granule-associated RNA binding protein TIA1 0.449 Tetratricopeptide repeat domain 27 TTC27 0.450 Fibronectin leucine rich transmembrane protein 2 FLRT2 0.457 Echinoderm microtubule associated protein like 1 EML1 0.457 Mitogen-activated protein kinase kinase kinase 4 MAP3K4 0.469 DIP2 disco-interacting protein 2 homolog C (Drosophila) DIP2C 0.471 Nestin NES 0.474

Appendices 336 Down syndrome critical region gene 8 DSCR8 0.478 Nebulette NEBL 0.478 Ring finger protein 13 RNF13 0.479 Molybdenum cofactor synthesis 1 MOCS1 0.482 Phosphodiesterase 4D interacting protein (myomegalin) PDE4DIP 0.483 Claudin 18 CLDN18 0.485 SET and MYND domain containing 1 SMYD1 0.487 Prolactin receptor PRLR 0.487 Neurofilament, heavy polypeptide 200kDa NEFH 0.490 ICEBERG caspase-1 inhibitor ICEBERG 2.007 Serpin peptidase inhibitor, clade B (ovalbumin), member 3 SERPINB3 2.008 Ankylosis, progressive homolog (mouse) ANKH 2.014 DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 DDX4 2.020 Desmoglein 1 DSG1 2.028 Collagen, type XII, alpha 1 COL12A1 2.029 Natural killer cell group 7 sequence NKG7 2.034 Protein tyrosine phosphatase, receptor type, O PTPRO 2.035 Inositol(myo)-1(or 4)-monophosphatase 1 IMPA1 2.036 Solute carrier family 20 (phosphate transporter), member 2 SLC20A2 2.039 Tenascin C (hexabrachion) TNC 2.041 Zinc finger protein 662 ZNF662 2.043 Interleukin 18 receptor 1 IL18R1 2.047 Protein phosphatase 1, regulatory (inhibitor) subunit 9B PPP1R9B 2.048 SUMO1/sentrin specific peptidase 5 SENP5 2.059 Transmembrane protein 56 TMEM56 2.059 FRAS1 related extracellular matrix 1 FREM1 2.061 Hephaestin HEPH 2.062 Mannosyl (alpha-1,6-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase MGAT2 2.065 Potassium intermediate/small conductance calcium-activated channel, subfamily N, KCNN2 2.067 member 2 Fukuyama type congenital muscular dystrophy (fukutin) FCMD 2.069 Transmembrane emp24 protein transport domain containing 8 TMED8 2.075 Acyltransferase like 1 AYTL1 2.075 Flavin containing monooxygenase 1 FMO1 2.077 Ubiquitin specific peptidase 9, X-linked USP9X 2.078 Histone deacetylase 2 HDAC2 2.091 Guanine nucleotide binding protein (G protein), alpha 15 (Gq class) GNA15 2.094 BIC transcript BIC 2.102 Developmental pluripotency associated 4 DPPA4 2.103 Elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 2 ELOVL2 2.107 Leukocyte specific transcript 1 LST1 2.108 RNA (guanine-9-) methyltransferase domain containing 3 RG9MTD3 2.112 Transcription factor CP2-like 1 TFCP2L1 2.117 Collagen, type XXI, alpha 1 COL21A1 2.126 Empty spiracles homeobox 2 EMX2 2.126 ADP-ribosylation factor 4 ARF4 2.131 Tyrosine kinase, non-receptor, 1 TNK1 2.133 Membrane-bound transcription factor peptidase, site 1 MBTPS1 2.134 GM2 ganglioside activator GM2A 2.135 Family with sequence similarity 116, member B FAM116B 2.142 Calmodulin regulated spectrin-associated protein 1-like 1 CAMSAP1L1 2.145 HECT, UBA and WWE domain containing 1 HUWE1 2.148 Glucosamine-6-phosphate deaminase 2 GNPDA2 2.148 Motile sperm domain containing 2 MOSPD2 2.155 Family with sequence similarity 19 (chemokine (C-C motif)-like), member A4 FAM19A4 2.158

Appendices 337 TAF7-like RNA polymerase II, TATA box binding protein (TBP)-associated factor, TAF7L 2.158 50kDa Solute carrier family 30 (zinc transporter), member 1 SLC30A1 2.158 Single stranded DNA binding protein 4 SSBP4 2.159 Fibronectin 1 FN1 2.161 Septin 4 SEPT4 2.167 SAR1 gene homolog B (S. cerevisiae) SAR1B 2.181 Potassium voltage-gated channel, shaker-related subfamily, member 1 (episodic KCNA1 2.187 ataxia with myokymia) Tetratricopeptide repeat domain 18 TTC18 2.187 Ret finger protein-like 3 antisense RFPL3S 2.190 Epididymal sperm binding protein 1 ELSPBP1 2.192 Tripartite motif-containing 36 TRIM36 2.197 Opioid growth factor receptor OGFR 2.202 Papillary renal cell carcinoma (translocation-associated) PRCC 2.204 Membrane-spanning 4-domains, subfamily A, member 7 MS4A7 2.210 Alanyl (membrane) aminopeptidase (aminopeptidase N, aminopeptidase M, ANPEP 2.211 microsomal aminopeptidase, CD13, p150) Nucleoporin 85kDa NUP85 2.212 Ubiquitin-like 7 (bone marrow stromal cell-derived) UBL7 2.216 Mannose receptor, C type 2 MRC2 2.223 Carbohydrate sulfotransferase 10 CHST10 2.231 Hemoglobin, beta HBB 2.233 Dual specificity phosphatase 10 DUSP10 2.235 Transmembrane and coiled-coil domains 2 TMCO2 2.240 Vascular cell adhesion molecule 1 VCAM1 2.242 Phytanoyl-CoA 2-hydroxylase interacting protein-like PHYHIPL 2.255 Bardet-Biedl syndrome 4 BBS4 2.267 Zinc finger protein 354B ZNF354B 2.279 Runt-related transcription factor 1 (acute myeloid leukemia 1; aml1 oncogene) RUNX1 2.288 GULP, engulfment adaptor PTB domain containing 1 GULP1 2.292 Sestrin 1 SESN1 2.293 Neurotrophic tyrosine kinase, receptor, type 2 NTRK2 2.300 Potassium large conductance calcium-activated channel, subfamily M, alpha KCNMA1 2.306 member 1 Solute carrier family 22 (organic cation transporter), member 2 SLC22A2 2.308 Jub, ajuba homolog (Xenopus laevis) JUB 2.318 TMDC II TMDCII 2.324 G protein-coupled receptor 126 GPR126 2.330 Neurotrophic tyrosine kinase, receptor, type 2 NTRK2 2.336 Cystatin E/M CST6 2.347 Inhibitor of growth family, member 3 ING3 2.348 Inducible T-cell co-stimulator ligand ICOSLG 2.350 Destrin (actin depolymerizing factor) DSTN 2.359 Pseudouridylate synthase 1 PUS1 2.362 Ubiquitin fusion degradation 1 like (yeast) UFD1L 2.366 Calmodulin regulated spectrin-associated protein 1-like 1 CAMSAP1L1 2.377 Zinc finger, SWIM-type containing 6 ZSWIM6 2.381 Ras protein-specific guanine nucleotide-releasing factor 2 RASGRF2 2.382 Hippocalcin-like 1 HPCAL1 2.383 ST3 beta-galactoside alpha-2,3-sialyltransferase 5 ST3GAL5 2.383 Thrombospondin, type I, domain containing 7A THSD7A 2.384 Lix1 homolog (mouse) LIX1 2.385 Son of sevenless homolog 1 (Drosophila) SOS1 2.391 Armadillo repeat containing, X-linked 5 ARMCX5 2.396 V-maf musculoaponeurotic fibrosarcoma oncogene homolog G (avian) MAFG 2.397 Suppressor of variegation 3-9 homolog 2 (Drosophila) SUV39H2 2.399

Appendices 338 Neuroblastoma breakpoint family, member 1 NBPF1 2.399 Vacuolar protein sorting 13 homolog A (S. cerevisiae) VPS13A 2.401 Zinc finger, FYVE domain containing 20 ZFYVE20 2.403 GTPase, IMAP family member 4 GIMAP4 2.412 FYN oncogene related to SRC, FGR, YES FYN 2.416 EF-hand calcium binding domain 2 EFCAB2 2.418 Glycophorin E GYPE 2.421 Placenta-specific 7 PLAC7 2.425 Sodium channel, voltage-gated, type II, alpha subunit SCN2A 2.428 Potassium inwardly-rectifying channel, subfamily J, member 8 KCNJ8 2.435 Procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), beta P4HB 2.438 polypeptide Forkhead box J2 FOXJ2 2.454 Sperm associated antigen 6 SPAG6 2.456 Tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase 2 TNKS2 2.465 Dickkopf homolog 3 (Xenopus laevis) DKK3 2.466 RP5- SAM domain containing 1 2.467 875H10.1 Phospholipase C, beta 4 PLCB4 2.469 Gamma-aminobutyric acid (GABA) A receptor, alpha 2 GABRA2 2.474 SUMO1/sentrin specific peptidase 7 SENP7 2.477 Methylmalonyl Coenzyme A mutase MUT 2.488 Ankyrin repeat domain 39 ANKRD39 2.494 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- GALNT5 2.499 acetylgalactosaminyltransferase 5 (GalNAc-T5) Membrane-spanning 4-domains, subfamily A, member 7 MS4A7 2.511 Paraspeckle component 1 PSPC1 2.515 Kelch-like 3 (Drosophila) KLHL3 2.516 Zinc finger protein 219 ZNF219 2.519 Caveolin 2 CAV2 2.532 Zinc finger protein 236 ZNF236 2.535 RAB3B, member RAS oncogene family RAB3B 2.535 Metastasis associated 1 family, member 3 hCG_1783907 2.536 Membrane-spanning 4-domains, subfamily A, member 5 MS4A5 2.542 Polybromo 1 PB1 2.548 Family with sequence similarity 44, member C FAM44C 2.548 Supervillin SVIL 2.550 Microtubule-associated protein 9 MAP9 2.559 SEC14-like 1 (S. cerevisiae) SEC14L1 2.576 Lipase, endothelial LIPG 2.580 Bardet-Biedl syndrome 12 BBS12 2.588 Transmembrane protein 182 TMEM182 2.591 Coiled-coil domain containing 91 CCDC91 2.601 CD1c molecule CD1C 2.605 Radical S-adenosyl methionine domain containing 1 RSAD1 2.613 LON peptidase N-terminal domain and ring finger 2 LONRF2 2.622 Acyl-CoA synthetase medium-chain family member 3 ACSM3 2.640 Transmembrane protein 30B TMEM30B 2.642 Solute carrier family 25, member 38 SLC25A38 2.643 CD93 molecule CD93 2.653 Protein tyrosine phosphatase, receptor type, U PTPRU 2.654 SEC14-like 2 (S. cerevisiae) SEC14L2 2.669 Tetratricopeptide repeat domain 7A TTC7A 2.682 Chemokine (C-C motif) ligand 3-like 3 CCL3L3 2.686 KH domain containing, RNA binding, signal transduction associated 3 KHDRBS3 2.687 Anaphase promoting complex subunit 2 ANAPC2 2.700

Appendices 339 Ankyrin repeat and sterile alpha motif domain containing 6 ANKS6 2.715 NUF2, NDC80 kinetochore complex component, homolog (S. cerevisiae) NUF2 2.731 Interleukin 23, alpha subunit p19 IL23A 2.753 Potassium large conductance calcium-activated channel, subfamily M, !member 2 KCNMB2 2.763 Hepatoma-derived growth factor (high-mobility group protein 1-like) HDGF 2.781 Basic helix-loop-helix domain containing, class B, 3 BHLHB3 2.797 Progestin and adipoQ receptor family member III PAQR3 2.799 Chemokine (C-C motif) ligand 28 CCL28 2.810 Chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) CXCL1 2.834 WW and C2 domain containing 2 WWC2 2.845 5',3'-nucleotidase, mitochondrial NT5M 2.865 Podocan PODN 2.866 Phosphoglycerate kinase 1 PGK1 2.917 Neurocalcin delta NCALD 2.941 Sterile alpha motif domain containing 9-like SAMD9L 2.970 Kelch-like 28 (Drosophila) KLHL28 2.977 Transmembrane channel-like 6 TMC6 2.980 Mitogen-activated protein kinase kinase 7 MAP2K7 3.039 Desmoglein 1 DSG1 3.057 Farnesyltransferase, CAAX box, alpha FNTA 3.062 SAM domain and HD domain, 1 Samhd1 3.062 Amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 16 ALS2CR16 3.078 Ankyrin repeat domain 29 ANKRD29 3.094 Surfeit 2 SURF2 3.098 Junctophilin 2 JPH2 3.147 Cyclic nucleotide gated channel beta 3 CNGB3 3.154 AT rich interactive domain 2 (ARID, RFX-like) ARID2 3.158 Mesoderm specific transcript homolog (mouse) MEST 3.162 Mindbomb homolog 2 (Drosophila) MIB2 3.163 Solute carrier family 25 (mitochondrial carrier, Aralar), member 12 SLC25A12 3.164 Transmembrane protein 45A TMEM45A 3.171 WW and C2 domain containing 2 WWC2 3.174 Nuclear cap binding protein subunit 2, 20kDa NCBP2 3.194 Angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 ACE2 3.204 TXK tyrosine kinase TXK 3.259 Spermatid perinuclear RNA binding protein STRBP 3.299 Synaptogyrin 1 SYNGR1 3.308 DnaJ homology subfamily A member 5 DNAJA5 3.368 Fibroblast growth factor 1 (acidic) FGF1 3.381 Solute carrier family 20 (phosphate transporter), member 1 SLC20A1 3.387 Ubiquitously transcribed tetratricopeptide repeat gene, Y-linked UTY 3.396 C-type lectin domain family 2, member B CLEC2B 3.416 Ras association (RalGDS/AF-6) domain family 5 RASSF5 3.419 Sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) SEMA6D 3.429 6D Actin filament associated protein 1-like 1 AFAP1L1 3.457 Zinc finger, X-linked, duplicated B ZXDB 3.467 Vaccinia related kinase 2 VRK2 3.471 Phosphoglucomutase 5 PGM5 3.479 Tetraspanin 12 TSPAN12 3.483 ATP-binding cassette, sub-family C (CFTR/MRP), member 2 ABCC2 3.529 Coiled-coil domain containing 3 CCDC3 3.561 Major histocompatibility complex, class II, DR beta 4 HLA-DRB4 3.571 Ephrin-B2 EFNB2 3.573 Zinc finger protein 117 ZNF117 3.573

Appendices 340 Nuclear casein kinase and cyclin-dependent kinase substrate 1 NUCKS1 3.590 Transforming growth factor beta regulator 1 TBRG1 3.643 Phytoceramidase, alkaline PHCA 3.737 Monocyte to macrophage differentiation-associated 2 MMD2 3.773 Phospholipase A2, group XIIA PLA2G12A 3.830 Non-metastatic cells 5, protein expressed in (nucleoside-diphosphate kinase) NME5 3.858 SEC24 related gene family, member C (S. cerevisiae) SEC24C 3.875 Target of myb1-like 2 (chicken) TOM1L2 3.885 Lactamase, beta 2 LACTB2 3.925 DOT1-like, histone H3 methyltransferase (S. cerevisiae) DOT1L 3.952 SEC63 homolog (S. cerevisiae) SEC63 3.988 Leucine rich repeat containing 8 family, member B LRRC8B 4.004 Zinc finger and BTB domain containing 10 ZBTB10 4.024 Interleukin 31 receptor A IL31RA 4.098 Collagen, type XI, alpha 1 COL11A1 4.103 AT hook, DNA binding motif, containing 1 AHDC1 4.110 ST6 beta-galactosamide alpha-2,6-sialyltranferase 2 ST6GAL2 4.146 Zinc finger protein, multitype 2 ZFPM2 4.174 Interleukin 24 IL24 4.192 Ribonuclease, RNase A family, 11 (non-active) RNASE11 4.198 Flavin containing monooxygenase 2 (non-functional) FMO2 4.214 Male-specific lethal-1 homolog MSL-1 4.221 MAP/microtubule affinity-regulating kinase 2 MARK2 4.223 Sterol-C4-methyl oxidase-like SC4MOL 4.241 Lysyl oxidase-like 1 LOXL1 4.285 Tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) TNFRSF11B 4.291 Solute carrier family 41, member 2 SLC41A2 4.313 Lectin, galactoside-binding, soluble, 1 (galectin 1) LGALS1 4.332 Sperm antigen with calponin homology and coiled-coil domains 1 SPECC1 4.392 CDC14 cell division cycle 14 homolog B (S. cerevisiae) CDC14B 4.408 C-type lectin domain family 14, member A CLEC14A 4.418 Cell growth regulator with EF-hand domain 1 CGREF1 4.432 Additional sex combs like 1 (Drosophila) ASXL1 4.472 Ras association (RalGDS/AF-6) and pleckstrin homology domains 1 RAPH1 4.566 Ring finger protein 180 RNF180 4.586 Heparan sulfate 6-O-sulfotransferase 2 HS6ST2 4.604 Nedd4 family interacting protein 2 NDFIP2 4.637 Endothelial differentiation, sphingolipid G-protein-coupled receptor, 3 EDG3 4.667 Myotubularin related protein 9 MTMR9 4.728 Mal, T-cell differentiation protein 2 MAL2 4.752 Matrix metallopeptidase 16 (membrane-inserted) MMP16 4.831 Claudin 11 (oligodendrocyte transmembrane protein) CLDN11 4.888 Aconitase 1, soluble ACO1 4.929 Ventricular zone expressed PH domain homolog 1 (zebrafish) VEPH1 4.965 Cholinergic receptor, nicotinic, alpha 5 CHRNA5 5.020 REST corepressor 3 RCOR3 5.032 Usher syndrome 1C (autosomal recessive, severe) USH1C 5.033 DENN/MADD domain containing 4C DENND4C 5.152 Integrator complex subunit 8 INTS8 5.158 Meis homeobox 1 MEIS1 5.159 Cytochrome P450, family 19, subfamily A, polypeptide 1 CYP19A1 5.392 Lipase, member I LIPI 5.590 Mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, MGAT4A 5.764 isozyme A Solute carrier family 38, member 4 SLC38A4 5.901

Appendices 341 Prohibitin PHB 5.964 RNA binding motif protein 9 RBM9 6.233 Leukotriene B4 receptor 2 LTB4R2 6.852 Biphenyl hydrolase-like (serine hydrolase; breast epithelial mucin-associated BPHL 6.965 antigen) Dynein, axonemal, heavy chain 7 DNAH7 6.978 Bromodomain adjacent to zinc finger domain, 2B BAZ2B 6.995 Spectrin, beta, non-erythrocytic 5 SPTBN5 7.047 Fibronectin leucine rich transmembrane protein 3 FLRT3 7.055 Clathrin, light chain (Lca) CLTA 7.083 Nucleoporin 50kDa NUP50 7.191 Transcription factor Dp-1 TFDP1 7.679 G patch domain containing 4 GPATCH4 8.235 Ecotropic viral integration site 2A EVI2A 8.325 Zinc finger protein, multitype 2 ZFPM2 8.576 Pregnancy specific beta-1-glycoprotein 10 PSG10 8.673 Src homology 2 domain containing E SHE 8.952 Myosin XVIIIA MYO18A 9.077 CLP1, cleavage and polyadenylation factor I subunit, homolog (S. cerevisiae) CLP1 9.278 Nucleolar and spindle associated protein 1 NUSAP1 9.447 Lymphocyte cytosolic protein 2 (SH2 domain containing leukocyte protein of 76kDa) LCP2 10.246 Protein phosphatase 2, regulatory subunit B', alpha isoform PPP2R5A 10.638 Yippee-like 2 (Drosophila) YPEL2 10.831 Zinc finger protein 542 ZNF542 12.678 Phospholipase A2, group IIA (platelets, synovial fluid) PLA2G2A 13.070 Membrane-spanning 4-domains, subfamily A, member 6A MS4A6A 13.197 Coiled-coil domain containing 62 CCDC62 13.652 Nuclear transcription factor Y, beta NFYB 14.323 Cytochrome P450, family 17, subfamily A, polypeptide 1 CYP17A1 14.806 Variable charge, X-linked 3A VCX3A 14.849 Calnexin Canx 16.316 Lethal giant larvae homolog 1 (Drosophila) LLGL1 16.808 Ribosomal protein L28 RPL28 18.562 CDC-like kinase 1 CLK1 20.155 Coiled-coil domain containing 101 CCDC101 20.308 Peroxisome proliferator-activated receptor alpha PPARA 21.120 Calpain 14 CAPN14 22.232 Cyclin M2 CNNM2 25.106 SH3 and cysteine rich domain STAC 27.493 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- GALNTL2 30.140 acetylgalactosaminyltransferase-like 2 Zinc finger, MYM-type 2 ZMYM2 32.029 Numb homolog (Drosophila) NUMB 32.311 Fibrinogen-like 2 FGL2 33.445 Beta-1,3-glucuronyltransferase 2 (glucuronosyltransferase S) B3GAT2 34.703 Stromal cell-derived factor 2 SDF2 35.183 Dihydrolipoamide branched chain transacylase E2 DBT 38.281 Osteopetrosis associated transmembrane protein 1 OSTM1 38.573 Cysteine-rich secretory protein 2 CRISP2 43.847 P antigen family, member 2 (prostate associated) PAGE2 44.247 Membrane-spanning 4-domains, subfamily A, member 6E MS4A6E 51.553 Beta-1,3-glucuronyltransferase 1 (glucuronosyltransferase P) B3GAT1 65.149 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 5 SERPINA5 76.437 ADAMTS-like 1 ADAMTSL1 82.345 Retinaldehyde binding protein 1 RLBP1 86.533 Klotho beta KLB 88.617 Appendices 342 Ankyrin repeat and sterile alpha motif domain containing 1B ANKS1B 115.49 Aryl hydrocarbon receptor AHR 123.25 Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), LILRB4 129.7 member 4 Neurotrophic tyrosine kinase, receptor, type 3 NTRK3 149.18 Mucin 2, oligomeric mucus/gel-forming MUC2 164.79 Regulator of G-protein signalling 18 RGS18 198.05 Interleukin 18 binding protein IL18BP 235.29

Table G.2. Genes changed by greater than 2-fold in ALL-17 xenograft cells after 6 hrs. Gene Name Gene ID 6 hrs Twist homolog 1 (acrocephalosyndactyly 3; Saethre-Chotzen syndrome) (Drosophila) TWIST1 0.001 Protocadherin 17 PCDH17 0.003 Endothelial differentiation, sphingolipid G-protein-coupled receptor, 3 EDG3 0.006 Carbonic anhydrase VI CA6 0.007 V-raf murine sarcoma 3611 viral oncogene homolog ARAF 0.015 Astrotactin 2 ASTN2 0.016 Complement factor H CFH 0.017 Eukaryotic translation initiation factor 4A, isoform 2 EIF4A2 0.018 Fibronectin leucine rich transmembrane protein 3 FLRT3 0.021 Prostaglandin E receptor 3 (subtype EP3) PTGER3 0.022 Zinc finger, HIT domain containing 2 Znhit2 0.025 Scavenger receptor class B, member 1 SCARB1 0.025 Ankyrin repeat domain 38 ANKRD38 0.027 G protein-coupled receptor 107 GPR107 0.027 Forkhead box F2 FOXF2 0.028 Transmembrane protein 177 TMEM177 0.030 EPB41L4 Erythrocyte membrane protein band 4.1 like 4A 0.030 A Family with sequence similarity 123A FAM123A 0.030 Zinc finger protein 238 ZNF238 0.032 Protocadherin alpha subfamily C, 2 PCDHAC2 0.033 RAB35, member RAS oncogene family RAB35 0.036 Cyclic nucleotide gated channel alpha 1 CNGA1 0.038 FAST kinase domains 2 FASTKD2 0.043 Solute carrier family 35, member F5 SLC35F5 0.052 Fibronectin 1 FN1 0.054 Filamin A interacting protein 1 FILIP1 0.061 Delta/notch-like EGF repeat containing DNER 0.063 Laminin, beta 2 (laminin S) LAMB2 0.066 Latrophilin 2 LPHN2 0.066 Membrane associated guanylate kinase, WW and PDZ domain containing 3 MAGI3 0.067 Cellular retinoic acid binding protein 2 CRABP2 0.068 Integrin, alpha 5 (fibronectin receptor, alpha polypeptide) ITGA5 0.070 AT rich interactive domain 2 (ARID, RFX-like) ARID2 0.071 Coiled-coil domain containing 110 CCDC110 0.072 LYR motif containing 5 LYRM5 0.074 Myosin VI MYO6 0.075 Roundabout, axon guidance receptor, homolog 1 (Drosophila) ROBO1 0.076 CAMSAP1 Calmodulin regulated spectrin-associated protein 1-like 1 0.076 L1 Dachshund homolog 1 (Drosophila) DACH1 0.076 Disabled homolog 2, mitogen-responsive phosphoprotein (Drosophila) DAB2 0.082 FLYWCH-type zinc finger 1 FLYWCH1 0.086

Appendices 343 Protein tyrosine phosphatase, receptor type, B PTPRB 0.087 Beta-1,3-glucuronyltransferase 1 (glucuronosyltransferase P) B3GAT1 0.087 Ribosomal protein S6 kinase, 90kDa, polypeptide 3 RPS6KA3 0.087 Carbonic anhydrase VIII CA8 0.088 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 3 B4GALT3 0.091 Sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1 SPOCK1 0.095 G protein-coupled receptor 177 GPR177 0.111 Ubiquitin-like 7 (bone marrow stromal cell-derived) UBL7 0.112 Ankyrin repeat and SOCS box-containing 1 ASB1 0.112 Zinc finger protein 662 ZNF662 0.115 Nucleolar and spindle associated protein 1 NUSAP1 0.115 Vanin 1 VNN1 0.120 Methylmalonic aciduria (cobalamin deficiency) cblA type MMAA 0.121 Protein phosphatase 2, regulatory subunit B', alpha isoform PPP2R5A 0.123 Serine PI Kazal type 5-like 3 SPINK5L3 0.124 RNA binding motif protein, Y-linked, family 1, member A1 RBMY1A1 0.127 Dual specificity phosphatase 5 Dusp5 0.128 RAN binding protein 3 RANBP3 0.132 Transmembrane and coiled-coil domains 2 TMCO2 0.133 Makorin, ring finger protein, 2 Mkrn2 0.138 Superkiller viralicidic activity 2-like 2 (S. cerevisiae) SKIV2L2 0.146 Retinoblastoma binding protein 5 RBBP5 0.150 Amyloid beta precursor protein (cytoplasmic tail) binding protein 2 APPBP2 0.153 Chemokine (C-C motif) ligand 3 CCL3 0.153 Folliculin FLCN 0.154 Coenzyme Q5 homolog, methyltransferase (S. cerevisiae) COQ5 0.157 TSPY-like 6 TSPYL6 0.157 Interleukin 1 receptor, type I IL1R1 0.158 SAM domain and HD domain, 1 Samhd1 0.159 CD209 molecule CD209 0.162 KRR1, small subunit (SSU) processome component, homolog (yeast) KRR1 0.162 Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 SVEP1 0.163 Basonuclin 2 BNC2 0.165 TRAPPC6 Trafficking protein particle complex 6B 0.166 B Solute carrier family 38, member 4 SLC38A4 0.169 Coiled-coil domain containing 113 CCDC113 0.171 Biphenyl hydrolase-like (serine hydrolase; breast epithelial mucin-associated antigen) BPHL 0.175 G protein-coupled receptor 126 GPR126 0.177 Hairy and enhancer of split 1, (Drosophila) HES1 0.177 Dihydrouridine synthase 4-like (S. cerevisiae) DUS4L 0.178 Glucosamine-phosphate N-acetyltransferase 1 GNPNAT1 0.179 Like-glycosyltransferase LARGE 0.180 Major histocompatibility complex, class I, C HLA-C 0.181 Myosin, light chain 9, regulatory MYL9 0.184 Non-metastatic cells 5, protein expressed in (nucleoside-diphosphate kinase) NME5 0.187 TEA domain family member 2 TEAD2 0.192 Piccolo (presynaptic cytomatrix protein) PCLO 0.194 Anterior gradient homolog 3 (Xenopus laevis) AGR3 0.198 Scavenger receptor class A, member 3 SCARA3 0.200 Ubiquitin family domain containing 1 UBFD1 0.202 Cytochrome P450, family 2, subfamily U, polypeptide 1 CYP2U1 0.204 Calpain 14 CAPN14 0.205 Plakophilin 4 PKP4 0.205 Farnesyltransferase, CAAX box, alpha FNTA 0.206

Appendices 344 FRAS1 related extracellular matrix 1 FREM1 0.206 Transmembrane protein 56 TMEM56 0.207 Solute carrier family 41, member 2 SLC41A2 0.211 Myosin, heavy chain 9, non-muscle MYH9 0.219 CD97 molecule CD97 0.223 Squalene epoxidase Sqle 0.226 Bardet-Biedl syndrome 12 BBS12 0.228 Proline rich 5 (renal) PRR5 0.228 Chemokine (C-X-C motif) ligand 9 Cxcl9 0.233 Cytokine receptor-like factor 1 CRLF1 0.246 Zinc finger protein-like 1 ZFPL1 0.246 Embryonal Fyn-associated substrate EFS 0.247 Cysteine and glycine-rich protein 1 CSRP1 0.251 Gamma-aminobutyric acid (GABA) A receptor, alpha 2 GABRA2 0.252 Hippocalcin like 4 HPCAL4 0.253 Flavin containing monooxygenase 1 FMO1 0.254 Cordon-bleu homolog (mouse) COBL 0.263 Transmembrane protein 37 TMEM37 0.263 Cyclin-dependent kinase-like 2 (CDC2-related kinase) CDKL2 0.267 Sphingomyelin phosphodiesterase, acid-like 3A SMPDL3A 0.269 Cysteine sulfinic acid decarboxylase CSAD 0.273 Peroxisomal membrane protein 3, 35kDa (Zellweger syndrome) PXMP3 0.274 EH domain binding protein 1 EHBP1 0.276 Desmoglein 1 DSG1 0.280 Complement component 4 binding protein, beta C4BPB 0.281 Squalene epoxidase SQLE 0.281 Single stranded DNA binding protein 4 SSBP4 0.284 Polycystic kidney disease 2-like 1 PKD2L1 0.285 IKAROS family zinc finger 1 (Ikaros) IKZF1 0.287 Ribosomal protein S28 RPS28 0.294 Sarcoglycan, delta (35kDa dystrophin-associated glycoprotein) SGCD 0.296 Junctophilin 2 JPH2 0.297 Dedicator of cytokinesis 5 DOCK5 0.299 Transcription factor CP2-like 1 TFCP2L1 0.300 Pannexin 3 PANX3 0.302 Macrophage erythroblast attacher MAEA 0.308 Keratin 7 KRT7 0.309 Transmembrane, prostate androgen induced RNA TMEPAI 0.313 GRB2-associated binding protein 1 GAB1 0.317 Claudin 1 CLDN1 0.322 Kinesin family member 5C KIF5C 0.324 RAP1 interacting factor homolog (yeast) RIF1 0.330 Family with sequence similarity 19 (chemokine (C-C motif)-like), member A4 FAM19A4 0.331 TNFRSF1 Tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) 0.331 1B SET domain containing (lysine methyltransferase) 8 SETD8 0.331 Parvin, alpha PARVA 0.333 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 PFKFB2 0.334 Midline 1 (Opitz/BBB syndrome) MID1 0.336 Mal, T-cell differentiation protein 2 MAL2 0.344 Structural maintenance of chromosomes 1A SMC1A 0.348 Chemokine (C-X3-C motif) receptor 1 CX3CR1 0.353 RAB6A, member RAS oncogene family RAB6A 0.354 Mitochondrial ribosomal protein S2 MRPS2 0.355 Tetraspanin 12 TSPAN12 0.356

Appendices 345 Frizzled homolog 1 (Drosophila) FZD1 0.356 Sterol-C4-methyl oxidase-like SC4MOL 0.356 SH3-binding domain kinase 1 SBK1 0.356 SEC14-like 2 (S. cerevisiae) SEC14L2 0.358 Interleukin 1 receptor accessory protein IL1RAP 0.360 Phosphoglycerate kinase 1 PGK1 0.361 CAMP responsive element binding protein 5 CREB5 0.364 RWD domain containing 2 RWDD2 0.364 Vestigial like 3 (Drosophila) VGLL3 0.365 Dynein, axonemal, heavy chain 5 DNAH5 0.367 Bone morphogenetic protein 6 BMP6 0.367 SAR1 gene homolog B (S. cerevisiae) SAR1B 0.368 Plasminogen activator, tissue PLAT 0.369 Transmembrane emp24 domain trafficking protein 2 TMED2 0.371 Sine oculis homeobox homolog 6 (Drosophila) SIX6 0.371 Transmembrane protein 16F TMEM16F 0.372 Double C2-like domains, alpha DOC2A 0.375 5-hydroxytryptamine (serotonin) receptor 2B HTR2B 0.379 GPI-anchored membrane protein 1 GPIAP1 0.383 Tyrosinase-related protein 1 TYRP1 0.384 Stabilin 2 STAB2 0.385 Serine palmitoyltransferase, long chain base subunit 2 SPTLC2 0.389 NADPH oxidase 4 NOX4 0.391 EPH receptor A2 EPHA2 0.391 Iduronate 2-sulfatase (Hunter syndrome) IDS 0.391 Calcitonin gene-related peptide-receptor component protein RCP9 0.391 Signal transducer and activator of transcription 4 Stat4 0.391 COBL-like 1 COBLL1 0.392 Calnexin Canx 0.392 Acyltransferase like 1 AYTL1 0.394 Claudin 1 CLDN1 0.394 MPHOSP M-phase phosphoprotein 1 0.398 H1 Hbs1-like (S. cerevisiae) Hbs1l 0.400 Osteopetrosis associated transmembrane protein 1 OSTM1 0.402 Tumor protein p73-like TP73L 0.403 Solute carrier family 35, member E1 SLC35E1 0.403 THAP domain containing 10 THAP10 0.405 Serine hydroxymethyltransferase 2 (mitochondrial) SHMT2 0.406 Guanylate binding protein 2, interferon-inducible GBP2 0.408 Myoglobin MB 0.410 Amyloid beta (A4) precursor-like protein 2 APLP2 0.410 Mitogen-activated protein kinase kinase 7 MAP2K7 0.412 Coiled-coil domain containing 109B Ccdc109b 0.413 Fukuyama type congenital muscular dystrophy (fukutin) FCMD 0.414 Chloride channel, calcium activated, family member 1 CLCA1 0.416 Olfactomedin 1 OLFM1 0.416 Ubiquitin-like, containing PHD and RING finger domains, 1 UHRF1 0.419 Peptidase D PEPD 0.419 Membrane-spanning 4-domains, subfamily A, member 7 MS4A7 0.419 Discs, large homolog 2, chapsyn-110 (Drosophila) DLG2 0.419 Delta/notch-like EGF repeat containing DNER 0.420 SPARC related modular calcium binding 2 SMOC2 0.422 Phospholipase A2 receptor 1, 180kDa PLA2R1 0.422 Colony stimulating factor 1 (macrophage) Csf1 0.423

Appendices 346 Ubiquitin-conjugating enzyme E2D 4 (putative) UBE2D4 0.425 Integrin beta 3 binding protein (beta3-endonexin) ITGB3BP 0.426 DNA fragmentation factor, 40kDa, beta polypeptide (caspase-activated DNase) DFFB 0.426 Developmental pluripotency associated 4 DPPA4 0.426 PMP22 claudin domain-containing protein PMP22CD 0.427 G antigen 7B GAGE7B 0.428 Death-associated protein DAP 0.430 Adaptor-related protein complex 1, gamma 2 subunit AP1G2 0.430 Ring finger protein 180 RNF180 0.432 Neuropeptide Y receptor Y1 NPY1R 0.433 Myeloperoxidase MPO 0.433 Protein tyrosine phosphatase, non-receptor type 13 (APO-1/CD95 (Fas)-associated PTPN13 0.434 phosphatase) Hormonally upregulated Neu-associated kinase HUNK 0.439 Placenta-specific 7 PLAC7 0.440 Zinc finger protein, multitype 2 ZFPM2 0.440 POU domain, class 6, transcription factor 2 POU6F2 0.440 Cytochrome P450, family 19, subfamily A, polypeptide 1 CYP19A1 0.441 Kallikrein-related peptidase 8 KLK8 0.441 Tubulin, delta 1 TUBD1 0.444 Potassium large conductance calcium-activated channel, subfamily M, beta member 4 KCNMB4 0.444 Guanylate binding protein 4 GBP4 0.446 Fas apoptotic inhibitory molecule FAIM 0.448 Insulin-like 6 INSL6 0.450 Sciellin SCEL 0.451 Checkpoint suppressor 1 CHES1 0.451 Peroxisome proliferator-activated receptor alpha PPARA 0.451 DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 DDX4 0.451 Galactosylceramidase GALC 0.451 DENN/MADD domain containing 2A DENND2A 0.452 Brain expressed, X-linked 1 BEX1 0.452 Interleukin 2 receptor, alpha chain Il2ra 0.453 Transient receptor potential cation channel, subfamily A, member 1 TRPA1 0.454 Small nuclear ribonucleoprotein polypeptide N SNRPN 0.454 Usher syndrome 1C (autosomal recessive, severe) USH1C 0.454 RNA binding motif protein 12B RBM12B 0.455 Phytoceramidase, alkaline PHCA 0.455 La ribonucleoprotein domain family, member 6 LARP6 0.458 Potassium large conductance calcium-activated channel, subfamily M, beta member 1 KCNMB1 0.459 Prokineticin receptor 1 PROKR1 0.462 Spondin 1, extracellular matrix protein SPON1 0.463 Membrane-spanning 4-domains, subfamily A, member 6E MS4A6E 0.463 Flavin containing monooxygenase 2 (non-functional) FMO2 0.463 Pleckstrin PLEK 0.463 WD repeat domain 78 WDR78 0.464 Par-3 partitioning defective 3 homolog (C. elegans) PARD3 0.466 DEP domain containing 6 DEPDC6 0.466 Pelota homolog (Drosophila) PELO 0.467 Tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase 2 TNKS2 0.468 Neuropilin (NRP) and tolloid (TLL)-like 2 NETO2 0.468 1-acylglycerol-3-phosphate O-acyltransferase 2 (lysophosphatidic acid acyltransferase, beta) AGPAT2 0.468 Ring finger protein 128 RNF128 0.469 Actin filament associated protein 1-like 1 AFAP1L1 0.471 SET and MYND domain containing 1 SMYD1 0.471 CDC14 cell division cycle 14 homolog B (S. cerevisiae) CDC14B 0.471

Appendices 347 COBL-like 1 COBLL1 0.474 Family with sequence similarity 123A FAM123A 0.474 Gap junction protein, chi 1, 31.9kDa GJC1 0.475 Roundabout, axon guidance receptor, homolog 2 (Drosophila) ROBO2 0.475 E2F transcription factor 1 E2F1 0.477 Zinc finger protein 323 ZNF323 0.477 ST6 beta-galactosamide alpha-2,6-sialyltranferase 2 ST6GAL2 0.480 Polybromo 1 PB1 0.481 Protein kinase (cAMP-dependent, catalytic) inhibitor alpha PKIA 0.481 Family with sequence similarity 87, member B FAM87B 0.482 Vesicle transport through interaction with t-SNAREs homolog 1A (yeast) VTI1A 0.483 THUMP domain containing 3 THUMPD3 0.484 Integrator complex subunit 1 INTS1 0.484 Desmoplakin DSP 0.485 Cylicin, basic protein of sperm head cytoskeleton 1 CYLC1 0.486 EGF-containing fibulin-like extracellular matrix protein 2 EFEMP2 0.487 TBC1 domain family, member 15 TBC1D15 0.487 HEG homolog 1 (zebrafish) HEG1 0.487 ATP-binding cassette, sub-family A (ABC1), member 5 ABCA5 0.488 ATP-binding cassette, sub-family A (ABC1), member 6 ABCA6 0.488 ATP-binding cassette, sub-family C (CFTR/MRP), member 9 ABCC9 0.490 Four and a half LIM domains 5 FHL5 0.491 Orthodenticle homeobox 2 OTX2 0.491 BTB (POZ) domain containing 7 BTBD7 0.492 Spindlin family, member 3 SPIN3 0.493 ATP-binding cassette, sub-family C (CFTR/MRP), member 9 ABCC9 0.493 RP5- SAM domain containing 1 0.493 875H10.1 Splicing factor 1 SF1 0.494 ABI gene family, member 3 ABI3 0.494 Small nuclear ribonucleoprotein polypeptide N SNRPN 0.494 V-abl Abelson murine leukemia viral oncogene homolog 1 ABL1 0.495 HLA- Major histocompatibility complex, class II, DR beta 4 0.496 DRB4 Zinc finger E-box binding homeobox 1 ZEB1 0.499 Angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 ACE2 0.499 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-like 2 GALNTL2 0.499 Acyl-CoA synthetase long-chain family member 5 ACSL5 0.499 Bone morphogenetic protein receptor, type IA BMPR1A 0.500 Zinc finger protein 3 ZNF3 2.003 ADAM metallopeptidase domain 19 (meltrin beta) ADAM19 2.008 Alcohol dehydrogenase IB (class I), beta polypeptide ADH1B 2.010 Rho guanine nucleotide exchange factor (GEF) 3 ARHGEF3 2.012 C-type lectin domain family 4, member E CLEC4E 2.013 Molybdenum cofactor synthesis 1 MOCS1 2.022 Ribosomal protein S9 Rps9 2.023 SERTA domain containing 4 SERTAD4 2.033 KIT ligand KITLG 2.033 CDV3 homolog (mouse) CDV3 2.034 WNT1 inducible signaling pathway protein 1 WISP1 2.036 Fibronectin leucine rich transmembrane protein 3 FLRT3 2.037 Integrin, beta 8 ITGB8 2.040 CDK5RAP CDK5 regulatory subunit associated protein 2 2.044 2 Myosin, heavy chain 11, smooth muscle MYH11 2.047 SMAD family member 6 SMAD6 2.052

Appendices 348 Jub, ajuba homolog (Xenopus laevis) JUB 2.054 Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) CXCL12 2.057 B-cell CLL/lymphoma 11B (zinc finger protein) BCL11B 2.060 Mindbomb homolog 2 (Drosophila) MIB2 2.067 RAS protein activator like 2 RASAL2 2.073 Homeodomain interacting protein kinase 2 HIPK2 2.075 Integrin-binding sialoprotein (bone sialoprotein, bone sialoprotein II) IBSP 2.086 Zinc finger protein 300 ZNF300 2.094 Myeloblastosis oncogene Myb 2.102 SH3PXD2 SH3 and PX domains 2B 2.109 B Target of myb1-like 2 (chicken) TOM1L2 2.109 Latent transforming growth factor beta binding protein 1 LTBP1 2.111 Interferon induced transmembrane protein 5 IFITM5 2.113 Spectrin, beta, non-erythrocytic 5 SPTBN5 2.116 Echinoderm microtubule associated protein like 1 EML1 2.118 Melanoma associated antigen (mutated) 1-like 1 MUM1L1 2.123 Seizure related 6 homolog (mouse)-like 2 SEZ6L2 2.124 Solute carrier family 16, member 12 (monocarboxylic acid transporter 12) SLC16A12 2.133 Homeodomain-only protein HOP 2.134 Proline rich Gla (G-carboxyglutamic acid) 1 PRRG1 2.136 Myeloid cell nuclear differentiation antigen MNDA 2.141 Solute carrier family 36 (proton/amino acid symporter), member 3 SLC36A3 2.145 MGC1682 Esophageal cancer associated protein 2.155 4 FERM domain containing 4B FRMD4B 2.160 Solute carrier family 31 (copper transporters), member 1 SLC31A1 2.167 Ankyrin repeat domain 57 ANKRD57 2.169 Aldehyde dehydrogenase 7 family, member A1 ALDH7A1 2.170 Cytochrome P450, family 2, subfamily A, polypeptide 6 CYP2A6 2.175 Odz, odd Oz/ten-m homolog 3 (Drosophila) ODZ3 2.182 Spindlin family, member 3 SPIN3 2.183 KH domain containing, RNA binding, signal transduction associated 3 KHDRBS3 2.193 Filamin C, gamma (actin binding protein 280) FLNC 2.199 Natural killer cell group 7 sequence NKG7 2.201 Zinc finger, CCHC domain containing 10 ZCCHC10 2.203 Spermatogenic leucine zipper 1 SPZ1 2.206 Endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, 2 EDG2 2.211 V-erb-a erythroblastic leukemia viral oncogene homolog 4 (avian) ERBB4 2.213 Nestin NES 2.214 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1 B4GALT1 2.214 Cytochrome P450, family 46, subfamily A, polypeptide 1 CYP46A1 2.216 Cholinergic receptor, nicotinic, alpha 5 CHRNA5 2.219 Potassium channel tetramerisation domain containing 1 KCTD1 2.222 Ankyrin repeat domain 39 ANKRD39 2.223 Paternally expressed 3 PEG3 2.226 Iroquois homeobox protein 3 IRX3 2.228 GTPase activating protein (SH3 domain) binding protein 2 G3BP2 2.231 Transmembrane phosphatase with tensin homology TPTE 2.236 Solute carrier family 35, member F5 SLC35F5 2.237 Interleukin 15 IL15 2.242 Small nucleolar RNA host gene (non-protein coding) 6 SNHG6 2.243 Zinc finger, DHHC-type containing 13 ZDHHC13 2.247 Tubulointerstitial nephritis antigen TINAG 2.254 CCR4-NOT transcription complex, subunit 4 CNOT4 2.262 Serpin peptidase inhibitor, clade B (ovalbumin), member 3 SERPINB3 2.271 Appendices 349 Beta-1,3-glucuronyltransferase 2 (glucuronosyltransferase S) B3GAT2 2.276 Rh blood group, CcEe antigens RHCE 2.284 Heat shock transcription factor 2 binding protein HSF2BP 2.286 Cartilage paired-class homeoprotein 1 CART1 2.287 Attractin ATRN 2.289 Cytochrome P450, family 3, subfamily A, polypeptide 4 CYP3A4 2.295 G protein-coupled receptor 22 GPR22 2.297 CDC42BP CDC42 binding protein kinase alpha (DMPK-like) 2.308 A Solute carrier family 36 (proton/amino acid symporter), member 3 SLC36A3 2.313 Dipeptidyl-peptidase 10 DPP10 2.335 Solute carrier family 25 (mitochondrial carrier; Graves disease autoantigen), member 16 SLC25A16 2.337 Phosphorylase kinase, alpha 1 (muscle) PHKA1 2.343 G protein-coupled receptor 55 GPR55 2.352 Erythrocyte membrane protein band 4.1 (elliptocytosis 1, RH-linked) EPB41 2.367 Zinc finger protein 532 ZNF532 2.369 Gap junction protein, beta 2, 26kDa GJB2 2.370 Sestrin 1 SESN1 2.377 Ribonuclease, RNase A family, 11 (non-active) RNASE11 2.377 Multiple PDZ domain protein MPDZ 2.378 Synaptopodin 2 SYNPO2 2.381 Core-binding factor, runt domain, alpha subunit 2; translocated to, 2 CBFA2T2 2.386 Cyclin L2 CCNL2 2.399 Intraflagellar transport 122 homolog (Chlamydomonas) IFT122 2.401 RAS protein activator like 1 (GAP1 like) RASAL1 2.406 Guanidinoacetate N-methyltransferase GAMT 2.407 Interleukin 24 IL24 2.410 ClpB caseinolytic peptidase B homolog (E. coli) CLPB 2.422 Potassium inwardly-rectifying channel, subfamily J, member 8 KCNJ8 2.437 TATA element modulatory factor 1 TMF1 2.441 Rho-related BTB domain containing 3 RHOBTB3 2.443 RAB30, member RAS oncogene family RAB30 2.489 Chloride intracellular channel 6 CLIC6 2.495 Non-SMC element 4 homolog A (S. cerevisiae) NSMCE4A 2.496 Heparan sulfate (glucosamine) 3-O-sulfotransferase 4 HS3ST4 2.504 Dynein, axonemal, heavy chain 7 DNAH7 2.508 Zic family member 3 heterotaxy 1 (odd-paired homolog, Drosophila) ZIC3 2.508 HtrA serine peptidase 3 HTRA3 2.516 Ras and Rab interactor 3 RIN3 2.530 Bone morphogenetic protein 4 BMP4 2.547 Insulin-like growth factor binding protein-like 1 IGFBPL1 2.552 WW and C2 domain containing 2 WWC2 2.555 Low density lipoprotein-related protein 2 LRP2 2.576 Phosphatase and actin regulator 2 PHACTR2 2.583 Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 SVEP1 2.592 Iduronate 2-sulfatase (Hunter syndrome) IDS 2.600 P antigen family, member 2 (prostate associated) PAGE2 2.602 RABGGT Rab geranylgeranyltransferase, beta subunit 2.605 B Insulin-like growth factor 2 (somatomedin A) IGF2 2.615 Elastin microfibril interfacer 1 EMILIN1 2.634 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, SMARCD3 2.667 member 3 CCAAT/enhancer binding protein (C/EBP), beta CEBPB 2.696 PiggyBac transposable element derived 5 PGBD5 2.699 Phosphotyrosine interaction domain containing 1 PID1 2.700

Appendices 350 TAF7-like RNA polymerase II, TATA box binding protein (TBP)-associated factor, 50kDa TAF7L 2.700 Methylmalonyl Coenzyme A mutase MUT 2.710 Phosphoinositide-binding protein PIP3-E PIP3-E 2.722 TRNA splicing endonuclease 54 homolog (S. cerevisiae) TSEN54 2.740 Myosin binding protein C, fast type MYBPC2 2.756 Cannabinoid receptor 1 (brain) CNR1 2.785 Kelch-like 3 (Drosophila) KLHL3 2.808 Paternally expressed 10 PEG10 2.835 Transmembrane protein 67 TMEM67 2.836 Cartilage associated protein CRTAP 2.848 Transforming growth factor, beta 3 TGFB3 2.873 Intestine-specific homeobox ISX 2.889 Mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, isozyme A MGAT4A 2.928 Bassoon (presynaptic cytomatrix protein) BSN 2.929 Bestrophin 3 BEST3 2.947 Yes-associated protein 1, 65kDa YAP1 2.959 Pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor PCBD1 2.965 1 alpha (TCF1) Solute carrier family 13 (sodium-dependent dicarboxylate transporter), member 3 SLC13A3 3.036 Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2-like MTHFD2L 3.075 RAB3B, member RAS oncogene family RAB3B 3.105 Cysteine-rich secretory protein 3 CRISP3 3.115 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 PLOD2 3.120 Leukemia inhibitory factor receptor alpha LIFR 3.126 Proline synthetase co-transcribed homolog (bacterial) PROSC 3.139 Pregnancy-associated plasma protein A, pappalysin 1 PAPPA 3.154 Mitochondrial ribosomal protein S9 MRPS9 3.184 Membrane-spanning 4-domains, subfamily A, member 6A MS4A6A 3.248 Ring finger protein 111 RNF111 3.309 ADP-ribosyltransferase 4 (Dombrock blood group) ART4 3.312 F-box protein 28 FBXO28 3.331 Ras association (RalGDS/AF-6) and pleckstrin homology domains 1 RAPH1 3.332 Serine palmitoyltransferase, long chain base subunit 2 SPTLC2 3.341 Rho GTPase activating protein 28 ARHGAP28 3.368 Protease, serine, 21 (testisin) PRSS21 3.441 CD36 molecule (thrombospondin receptor) CD36 3.442 Src homology 2 domain containing E SHE 3.464 Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 SVEP1 3.480 Elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 4 ELOVL4 3.486 Collagen, type XI, alpha 1 COL11A1 3.522 Cat eye syndrome chromosome region, candidate 8 CECR8 3.648 Protocadherin alpha 6 PCDHA6 3.668 Hemoglobin, zeta HBZ 3.675 Transcription factor 3 (E2A immunoglobulin enhancer binding factors E12/E47) TCF3 3.874 Small proline-rich protein 2A SPRR2A 3.891 Lymphocyte antigen 9 LY9 4.010 GTPase, IMAP family member 6 GIMAP6 4.059 ERO1-like beta (S. cerevisiae) ERO1LB 4.095 Cyclin M2 CNNM2 4.123 Inducible T-cell co-stimulator ligand ICOSLG 4.156 Contactin 3 (plasmacytoma associated) CNTN3 4.182 YME1-like 1 (S. cerevisiae) YME1L1 4.225 Microtubule associated serine/threonine kinase family member 4 MAST4 4.439 Phospholipase A2, group IIA (platelets, synovial fluid) PLA2G2A 4.441 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 PFKFB4 4.469

Appendices 351 Inturned planar cell polarity effector homolog (Drosophila) INTU 4.524 Parathyroid hormone PTH 4.545 Slingshot homolog 1 (Drosophila) SSH1 4.687 Prostaglandin E receptor 3 (subtype EP3) PTGER3 4.814 Lipase, member I LIPI 4.939 V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog KIT 4.950 Cytochrome P450, family 26, subfamily A, polypeptide 1 CYP26A1 5.050 Phytanoyl-CoA 2-hydroxylase interacting protein-like PHYHIPL 5.113 Collagen, type XII, alpha 1 COL12A1 5.153 Insulin-like growth factor 1 receptor IGF1R 5.190 Dimethylglycine dehydrogenase DMGDH 5.569 V-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian) MAF 5.948 Sorting nexin 9 SNX9 6.158 Sorbin and SH3 domain containing 2 SORBS2 6.185 Protein tyrosine phosphatase type IVA, member 2 PTP4A2 6.411 Vacuolar protein sorting 29 homolog (S. cerevisiae) VPS29 6.461 Arrestin, beta 2 ARRB2 6.558 Hairy/enhancer-of-split related with YRPW motif-like HEYL 7.865 Neuron navigator 2 NAV2 8.010 Myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, MLLT4 8.045 4 Neurobeachin NBEA 9.247 AT hook, DNA binding motif, containing 1 AHDC1 10.811 X (inactive)-specific transcript XIST 11.253 Neurobeachin NBEA 12.349 Neuroblastoma breakpoint family, member 1 NBPF1 13.866 Ubiquitin specific peptidase 25 USP25 14.150 Neurocalcin delta NCALD 14.704 Fibroblast growth factor 12 FGF12 15.022 Neutrophil cytosolic factor 2 (65kDa, chronic granulomatous disease, autosomal 2) NCF2 15.578 Chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2) CXCL6 18.079 Tyrosylprotein sulfotransferase 1 TPST1 22.980 Nuclear receptor co-repressor 1 Ncor1 23.739 Dickkopf homolog 3 (Xenopus laevis) DKK3 24.467 Solute carrier family 37 (glycerol-6-phosphate transporter), member 4 SLC37A4 26.303 Nedd4 family interacting protein 2 NDFIP2 29.753 Family with sequence similarity 46, member A FAM46A 29.908 Regulator of G-protein signalling 20 RGS20 32.446 Interferon-induced protein with tetratricopeptide repeats 2 Ifit2 37.034 Solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 SLC7A2 38.743 Par-6 partitioning defective 6 homolog gamma (C. elegans) PARD6G 51.871 Anaphase promoting complex subunit 2 ANAPC2 72.437 N- downstream regulated gene 1 NDRG1 135.15 NIMA (never in mitosis gene a)-related kinase 3 NEK3 366.83 Opioid growth factor receptor OGFR 485.01

Table G.3. Genes changed by greater than 2-fold in ALL-17 xenograft cells after 24 hrs. Gene Name Gene ID 24 hrs Calcium channel, voltage-dependent, gamma subunit 4 CACNG4 0.006 Heat shock transcription factor family member 5 HSF5 0.013 Synaptopodin 2 SYNPO2 0.016 Eukaryotic translation initiation factor 4A, isoform 2 EIF4A2 0.021 IL2-inducible T-cell kinase ITK 0.025 Appendices 352 Bassoon (presynaptic cytomatrix protein) BSN 0.027 Membrane-spanning 4-domains, subfamily A, member 7 MS4A7 0.028 RAB6A, member RAS oncogene family RAB6A 0.030 Monocyte to macrophage differentiation-associated 2 MMD2 0.032 Interleukin 15 IL15 0.034 Phytoceramidase, alkaline PHCA 0.038 Placenta-specific 7 PLAC7 0.040 BCL2-associated athanogene 4 BAG4 0.047 Metallothionein 1 Mt1 0.057 Cysteine-rich secretory protein 2 CRISP2 0.064 P antigen family, member 1 (prostate associated) PAGE1 0.065 Usher syndrome 1C (autosomal recessive, severe) USH1C 0.076 Numb homolog (Drosophila) NUMB 0.077 Rho GTPase activating protein 5 ARHGAP5 0.086 Hepatitis B virus x interacting protein HBXIP 0.098 Membrane-spanning 4-domains, subfamily A, member 6A MS4A6A 0.116 Phospholipid scramblase 2 PLSCR2 0.116 Fibrinogen-like 2 FGL2 0.117 ATPase, Class V, type 10A ATP10A 0.123 Cordon-bleu homolog (mouse) COBL 0.137 Ras homolog gene family, member B RHOB 0.140 Dynein, axonemal, heavy chain 7 DNAH7 0.143 Calcium binding protein 1 (calbrain) CABP1 0.157 Structural maintenance of chromosomes 1A SMC1A 0.161 COP9 constitutive photomorphogenic homolog subunit 2 (Arabidopsis) COPS2 0.165 LIM domain only 3 (rhombotin-like 2) LMO3 0.166 Thyroid transcription factor 1 TITF1 0.172 Par-3 partitioning defective 3 homolog (C. elegans) PARD3 0.173 Aquaporin 9 AQP9 0.179 HLA- Major histocompatibility complex, class II, DR beta 1 0.188 DRB1 Microtubule-associated protein 9 MAP9 0.202 Zinc finger protein 318 ZNF318 0.215 Filamin C, gamma (actin binding protein 280) FLNC 0.217 Sulfiredoxin 1 homolog (S. cerevisiae) SRXN1 0.217 Kinesin family member 21A KIF21A 0.218 Alpha-kinase 2 ALPK2 0.227 Ring finger protein 144 Rnf144 0.227 Non-metastatic cells 5, protein expressed in (nucleoside-diphosphate kinase) NME5 0.229 Zinc finger, CCHC domain containing 5 ZCCHC5 0.229 Transketolase (Wernicke-Korsakoff syndrome) TKT 0.241 Sperm antigen with calponin homology and coiled-coil domains 1 SPECC1 0.241 Adenylate cyclase 7 ADCY7 0.242 RGM domain family, member B RGMB 0.242 Lactamase, beta 2 LACTB2 0.256 Cat eye syndrome chromosome region, candidate 8 CECR8 0.256 Prohibitin PHB 0.257 Sine oculis homeobox homolog 4 (Drosophila) SIX4 0.257 CD247 molecule CD247 0.258 Ankyrin repeat and sterile alpha motif domain containing 1A ANKS1A 0.258 Phosphodiesterase 1A, calmodulin-dependent PDE1A 0.258 Ecotropic viral integration site 2A EVI2A 0.259 MCM3 minichromosome maintenance deficient 3 (S. cerevisiae) MCM3 0.262 Beta-site APP-cleaving enzyme 1 BACE1 0.262 Histamine N-methyltransferase HNMT 0.264

Appendices 353 IQ motif containing H IQCH 0.274 RNA binding protein, autoantigenic (hnRNP-associated with lethal yellow homolog (mouse)) RALY 0.276 Coiled-coil domain containing 62 CCDC62 0.280 NADPH oxidase 4 NOX4 0.281 Cytochrome P450, family 19, subfamily A, polypeptide 1 CYP19A1 0.281 Ribonuclease, RNase A family, 11 (non-active) RNASE11 0.284 Nestin NES 0.285 Zinc finger protein 135 ZNF135 0.286 Ets variant gene 1 ETV1 0.288 RNA binding motif protein 6 RBM6 0.291 SEC24 related gene family, member C (S. cerevisiae) SEC24C 0.304 Sciellin SCEL 0.321 SERTA domain containing 4 SERTAD4 0.327 SET and MYND domain containing 1 SMYD1 0.327 Suppressor of variegation 3-9 homolog 2 (Drosophila) SUV39H2 0.327 P antigen family, member 2 (prostate associated) PAGE2 0.332 Neuroligin 4, X-linked NLGN4X 0.338 Pleckstrin homology domain containing, family B (evectins) member 1 PLEKHB1 0.338 Zinc finger, CCHC domain containing 7 ZCCHC7 0.339 Solute carrier family 36 (proton/amino acid symporter), member 3 SLC36A3 0.340 Single stranded DNA binding protein 4 SSBP4 0.341 SH3 domain binding glutamic acid-rich protein like 2 SH3BGRL2 0.344 ADAM metallopeptidase with thrombospondin type 1 motif, 5 (aggrecanase-2) ADAMTS5 0.344 Tyrosine kinase, non-receptor, 1 TNK1 0.346 Leukemia inhibitory factor receptor alpha LIFR 0.354 Hairy/enhancer-of-split related with YRPW motif-like HEYL 0.354 Anterior gradient homolog 3 (Xenopus laevis) AGR3 0.354 Developmentally regulated GTP binding protein 1 DRG1 0.355 Mov10l1, Moloney leukemia virus 10-like 1, homolog (mouse) MOV10L1 0.359 GM2 ganglioside activator GM2A 0.361 Acyl-Coenzyme A oxidase 2, branched chain ACOX2 0.362 Proteasome (prosome, macropain) 26S subunit, non-ATPase, 14 PSMD14 0.363 Coenzyme Q4 homolog (S. cerevisiae) COQ4 0.363 Coiled-coil domain containing 90A CCDC90A 0.368 WAP four-disulfide core domain 1 WFDC1 0.369 SRY (sex determining region Y)-box 4 SOX4 0.372 Solute carrier family 6, member 15 SLC6A15 0.372 Prenylcysteine oxidase 1 PCYOX1 0.374 HCG23177 hCG_23177 0.374 Sterile alpha motif domain containing 14 SAMD14 0.374 Leucine rich repeat containing 8 family, member B LRRC8B 0.375 Histidine triad nucleotide binding protein 3 HINT3 0.377 Major histocompatibility complex, class I, C HLA-C 0.379 Empty spiracles homeobox 2 opposite strand EMX2OS 0.379 RAN binding protein 3 RANBP3 0.380 ST6 beta-galactosamide alpha-2,6-sialyltranferase 2 ST6GAL2 0.381 Dipeptidyl-peptidase 4 (CD26, adenosine deaminase complexing protein 2) DPP4 0.386 Putative neuronal cell adhesion molecule PUNC 0.388 Elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 2 ELOVL2 0.390 Interferon-induced protein with tetratricopeptide repeats 2 Ifit2 0.390 Endothelial differentiation, sphingolipid G-protein-coupled receptor, 3 EDG3 0.393 Matrix metallopeptidase 27 MMP27 0.394 Nebulette NEBL 0.394 Zinc finger protein 229 ZNF229 0.394

Appendices 354 Discs, large homolog 2, chapsyn-110 (Drosophila) DLG2 0.395 Glutathione S-transferase A4 GSTA4 0.395 Chemokine (C-C motif) receptor-like 2 CCRL2 0.398 Cytochrome P450, family 27, subfamily B, polypeptide 1 CYP27B1 0.402 Death inducer-obliterator 1 DIDO1 0.402 Amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 4 ALS2CR4 0.402 Solute carrier family 35, member F5 SLC35F5 0.404 Low density lipoprotein receptor-related protein 4 LRP4 0.405 Aldehyde dehydrogenase 5 family, member A1 (succinate-semialdehyde dehydrogenase) ALDH5A1 0.408 Integrin, beta 8 ITGB8 0.408 ADAMDEC ADAM-like, decysin 1 0.408 1 CD24 molecule CD24 0.412 Neurocalcin delta NCALD 0.414 Calpastatin CAST 0.415 Ankyrin repeat domain 45 ANKRD45 0.415 Solute carrier family 35, member E1 SLC35E1 0.419 ADAM metallopeptidase with thrombospondin type 1 motif, 18 ADAMTS18 0.420 Fas (TNF receptor superfamily, member 6) FAS 0.422 Zic family member 2 (odd-paired homolog, Drosophila) ZIC2 0.424 Doublesex and mab-3 related transcription factor 3 DMRT3 0.424 CUB and Sushi multiple domains 1 CSMD1 0.425 Interleukin 1 receptor, type I IL1R1 0.426 WAP four-disulfide core domain 2 WFDC2 0.427 Caspase 12 CASP12 0.429 Cortexin 1 CTXN1 0.429 Molybdenum cofactor synthesis 1 MOCS1 0.431 Microsomal glutathione S-transferase 1 MGST1 0.431 Retinoblastoma binding protein 5 RBBP5 0.432 Elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 4 ELOVL4 0.432 Integrin-binding sialoprotein (bone sialoprotein, bone sialoprotein II) IBSP 0.432 Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 4 LILRB4 0.433 Ras and Rab interactor 3 RIN3 0.433 Carbonic anhydrase XIII CA13 0.433 Cysteine-rich, angiogenic inducer, 61 CYR61 0.434 Microtubule-associated protein 6 MAP6 0.436 Dpy-19-like 2 (C. elegans) DPY19L2 0.437 Phospholipase C, beta 4 PLCB4 0.439 Dual specificity phosphatase 12 DUSP12 0.439 Neuroepithelial cell transforming gene 1 NET1 0.441 Sestrin 1 SESN1 0.441 Neurogenic differentiation 2 NEUROD2 0.441 KH domain containing, RNA binding, signal transduction associated 3 KHDRBS3 0.442 Sodium channel, voltage-gated, type II, alpha subunit SCN2A 0.442 Dom-3 homolog Z (C. elegans) DOM3Z 0.443 T-cell leukemia/lymphoma 1A TCL1A 0.444 NEFA-interacting nuclear protein NIP30 NIP30 0.445 Dimethylarginine dimethylaminohydrolase 1 DDAH1 0.446 Paternally expressed 3 PEG3 0.447 Interleukin 31 receptor A IL31RA 0.448 Caveolin 2 CAV2 0.452 Secretory leukocyte peptidase inhibitor SLPI 0.452 Collagen, type VIII, alpha 1 COL8A1 0.454 Glyoxylate reductase/hydroxypyruvate reductase GRHPR 0.454 Membrane associated guanylate kinase, WW and PDZ domain containing 3 MAGI3 0.454

Appendices 355 Malic enzyme 1, NADP(+)-dependent, cytosolic ME1 0.455 Ribosomal protein S4, Y-linked 1 RPS4Y1 0.457 EF-hand domain family, member D1 EFHD1 0.459 Myeloperoxidase MPO 0.459 Flavin containing monooxygenase 1 FMO1 0.460 Bestrophin 3 BEST3 0.461 Phospholipase A2, group IIA (platelets, synovial fluid) PLA2G2A 0.462 Histidyl-tRNA synthetase 2, mitochondrial (putative) HARS2 0.465 Interleukin 2 receptor, alpha chain Il2ra 0.466 Stonin 2 STON2 0.466 Six transmembrane epithelial antigen of the prostate 1 STEAP1 0.466 Integrin, alpha 8 ITGA8 0.466 Superkiller viralicidic activity 2-like 2 (S. cerevisiae) SKIV2L2 0.466 Dual specificity phosphatase 13 DUSP13 0.467 Down syndrome critical region gene 8 DSCR8 0.467 Sterile alpha motif domain containing 3 SAMD3 0.467 Bestrophin 3 BEST3 0.467 Hermansky-Pudlak syndrome 4 HPS4 0.468 Peroxisomal biogenesis factor 11 gamma PEX11G 0.468 BTB and CNC homology 1, basic leucine zipper transcription factor 2 BACH2 0.468 Phenylalanine hydroxylase PAH 0.469 F-box protein 2 FBXO2 0.470 ATPase family, AAA domain containing 1 ATAD1 0.470 Potassium voltage-gated channel, subfamily H (eag-related), member 8 KCNH8 0.473 DIP2 disco-interacting protein 2 homolog C (Drosophila) DIP2C 0.474 Glycosyltransferase 25 domain containing 2 GLT25D2 0.475 Spastic paraplegia 7, paraplegin (pure and complicated autosomal recessive) SPG7 0.475 Protein phosphatase, EF-hand calcium binding domain 1 PPEF1 0.477 Allantoicase ALLC 0.477 V- myeloblastosis viral oncogene homolog (avian) MYB 0.479 Thrombospondin, type I, domain containing 7A THSD7A 0.481 Casein kinase 1, epsilon CSNK1E 0.481 Nuclear receptor subfamily 2, group F, member 2 NR2F2 0.482 Cytokine receptor-like factor 1 CRLF1 0.482 LON peptidase N-terminal domain and ring finger 2 LONRF2 0.483 Carbohydrate (chondroitin 4) sulfotransferase 12 CHST12 0.483 Sarcoglycan, beta (43kDa dystrophin-associated glycoprotein) SGCB 0.483 BTB and CNC homology 1, basic leucine zipper transcription factor 2 BACH2 0.484 Rho GTPase activating protein 9 ARHGAP9 0.484 Cysteine-rich, angiogenic inducer, 61 CYR61 0.485 Cytotoxic T-lymphocyte-associated protein 4 CTLA4 0.486 Interleukin 17B IL17B 0.488 Lysyl oxidase-like 1 LOXL1 0.488 Fibulin 5 FBLN5 0.489 Fc fragment of IgG, low affinity IIIa, receptor (CD16a) FCGR3A 0.489 Rho family GTPase 3 RND3 0.491 Transcription factor AP-2 beta (activating enhancer binding protein 2 beta) TFAP2B 0.491 Protocadherin alpha 6 PCDHA6 0.491 Pleckstrin homology domain containing, family H (with MyTH4 domain) member 1 PLEKHH1 0.492 Spondin 1, extracellular matrix protein SPON1 0.493 Prolactin receptor PRLR 0.493 G antigen 7B GAGE7B 0.493 Zinc finger protein 473 ZNF473 0.493 Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMP-N-acetylneuraminate CMAH 0.493 monooxygenase)

Appendices 356 Serum amyloid A-like 1 SAAL1 0.493 Nucleolar and spindle associated protein 1 NUSAP1 0.494 ATG4 autophagy related 4 homolog C (S. cerevisiae) ATG4C 0.494 Retinol dehydrogenase 13 (all-trans/9-cis) RDH13 0.495 Lipid phosphate phosphatase-related protein type 2 LPPR2 0.496 Small proline-rich protein 2A SPRR2A 0.496 Platelet derived growth factor D PDGFD 0.496 Carbonic anhydrase XIV CA14 0.497 Interleukin 7 receptor IL7R 0.498 Vanin 1 VNN1 0.498 Microtubule associated monoxygenase, calponin and LIM domain containing 3 MICAL3 0.498 Protein tyrosine phosphatase, receptor type, F PTPRF 0.499 Aldehyde dehydrogenase 1 family, member B1 ALDH1B1 0.499 DKFZP77 PRO0845 0.500 9L1068 G protein-coupled receptor 34 GPR34 2.002 ATP-binding cassette, sub-family B (MDR/TAP), member 1B Abcb1b 2.002 Transmembrane protein 163 TMEM163 2.006 C-type lectin domain family 14, member A CLEC14A 2.007 Myelin expression factor 2 MYEF2 2.079 HLA- Major histocompatibility complex, class II, DR beta 4 2.084 DRB4 Thrombospondin, type I, domain containing 3 THSD3 2.089 Purinergic receptor P2Y, G-protein coupled, 14 P2RY14 2.094 Protein tyrosine phosphatase, receptor type, O PTPRO 2.099 Complement component 3a receptor 1 C3AR1 2.100 Arylsulfatase B ARSB 2.112 RIMS binding protein 2 RIMBP2 2.118 Myelin transcription factor 1-like MYT1L 2.123 Kinase suppressor of ras 2 KSR2 2.127 Natural killer cell group 7 sequence NKG7 2.127 Torsin A interacting protein 1 TOR1AIP1 2.149 Prion protein Prnp 2.154 Integrin, alpha X (complement component 3 receptor 4 subunit) ITGAX 2.167 Isocitrate dehydrogenase 1 (NADP+), soluble Idh1 2.179 5'-nucleotidase, cytosolic III NT5C3 2.185 Tripartite motif-containing 36 TRIM36 2.199 Zinc finger, MYM-type 6 ZMYM6 2.199 ATPase, Class I, type 8B, member 3 ATP8B3 2.201 RAS guanyl releasing protein 2 (calcium and DAG-regulated) RASGRP2 2.203 ADAM metallopeptidase domain 8 ADAM8 2.206 GPI deacylase PGAP1 2.211 Trafficking protein particle complex 5 TRAPPC5 2.224 Inducible T-cell co-stimulator ligand ICOSLG 2.227 PWP1 homolog (S. cerevisiae) PWP1 2.227 Heparan sulfate (glucosamine) 3-O-sulfotransferase 1 HS3ST1 2.227 Phosphoinositide-binding protein PIP3-E PIP3-E 2.227 CD53 molecule CD53 2.233 Glycerol-3-phosphate dehydrogenase 2 (mitochondrial) GPD2 2.242 Family with sequence similarity 84, member A FAM84A 2.242 Phosphoinositide-binding protein PIP3-E PIP3-E 2.243 Nedd4 family interacting protein 1 NDFIP1 2.247 Phosphoinositide-3-kinase, class 2, gamma polypeptide PIK3C2G 2.254 Brain-specific angiogenesis inhibitor 3 BAI3 2.285 Chemokine (C-C motif) receptor 5 CCR5 2.291 Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 1 LILRB1 2.312 Appendices 357 TSC22 domain family, member 1 Tsc22d1 2.324 Grainyhead-like 1 (Drosophila) GRHL1 2.352 Colony stimulating factor 1 receptor, formerly McDonough feline sarcoma viral (v-fms) CSF1R 2.375 oncogene homolog Cellular retinoic acid binding protein 2 CRABP2 2.379 NIMA (never in mitosis gene a)-related kinase 3 NEK3 2.408 Transducer of ERBB2, 2 TOB2 2.419 Protease, serine, 23 PRSS23 2.462 Potassium inwardly-rectifying channel, subfamily J, member 2 KCNJ2 2.490 Zinc finger E-box binding homeobox 1 ZEB1 2.504 Prokineticin receptor 1 PROKR1 2.534 ANKRD18 Ankyrin repeat domain 18A 2.543 A Pleckstrin homology-like domain, family A, member 1 PHLDA1 2.584 Zinc finger, X-linked, duplicated B ZXDB 2.614 Gap junction protein, alpha 5, 40kDa GJA5 2.619 CD1c molecule CD1C 2.635 SAM domain and HD domain, 1 Samhd1 2.642 WW and C2 domain containing 2 WWC2 2.643 Interleukin 23, alpha subunit p19 IL23A 2.653 Cellular retinoic acid binding protein 2 CRABP2 2.653 SAC3 domain containing 1 SAC3D1 2.675 Protein tyrosine phosphatase, non-receptor type 9 Ptpn9 2.734 Chemokine (C-X-C motif) ligand 9 Cxcl9 2.749 Calpain 14 CAPN14 2.800 Myoglobin MB 2.832 Nuclear transcription factor Y, beta NFYB 2.834 TRAPPC6 Trafficking protein particle complex 6B 2.840 B Core binding factor beta Cbfb 2.857 Sine oculis homeobox homolog 6 (Drosophila) SIX6 2.917 V-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian) MAF 2.962 Sarcolipin SLN 2.972 Protein tyrosine phosphatase-like (proline instead of catalytic arginine), member b PTPLB 2.976 Arginine-rich, mutated in early stage tumors Armet 3.001 Collagen, type XI, alpha 1 COL11A1 3.029 Zinc finger protein 212 ZNF212 3.058 Solute carrier family 9 (sodium/hydrogen exchanger), isoform 3 regulator 1 Slc9a3r1 3.157 Zinc finger protein 91 ZNF91 3.181 Lethal giant larvae homolog 1 (Drosophila) LLGL1 3.255 Echinoderm microtubule associated protein like 3 EML3 3.278 Ryanodine receptor 3 RYR3 3.384 Inducible T-cell co-stimulator ligand ICOSLG 3.422 Coiled-coil domain containing 80 CCDC80 3.463 Wingless-type MMTV integration site family, member 7A WNT7A 3.466 V-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian) MAF 3.499 Yip1 domain family, member 5 YIPF5 3.525 GTPase, IMAP family member 6 GIMAP6 3.595 V-maf musculoaponeurotic fibrosarcoma oncogene homolog G (avian) MAFG 3.767 Usher syndrome 2A (autosomal recessive, mild) USH2A 3.768 Gap junction protein, beta 2, 26kDa GJB2 4.030 Pleckstrin homology domain containing, family A member 5 PLEKHA5 4.039 Ras association (RalGDS/AF-6) domain family 5 RASSF5 4.076 ANKRD13 Ankyrin repeat domain 13C 4.114 C Yip1 domain family, member 5 YIPF5 4.314 Protein phosphatase 2C, magnesium-dependent, catalytic subunit PPM2C 4.545

Appendices 358 ATP-binding cassette, sub-family C (CFTR/MRP), member 9 ABCC9 4.610 IKAROS family zinc finger 1 (Ikaros) IKZF1 5.061 Calnexin Canx 5.304 Angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 ACE2 5.561 Potassium inwardly-rectifying channel, subfamily J, member 8 KCNJ8 5.602 Calcium binding tyrosine-(Y)-phosphorylation regulated (fibrousheathin 2) CABYR 6.789 Junctophilin 2 JPH2 7.076 Melanoma antigen family D, 4 MAGED4 7.193 Cyclin-dependent kinase inhibitor 3 (CDK2-associated dual specificity phosphatase) CDKN3 7.496 Cytochrome c oxidase subunit Vb COX5B 10.555 WNT1 inducible signaling pathway protein 1 WISP1 12.479 Syntaxin binding protein 6 (amisyn) STXBP6 13.061 Protein-O-mannosyltransferase 1 POMT1 18.175 Myosin XVIIIA MYO18A 18.932 Ubiquitin-conjugating enzyme E2N (UBC13 homolog, yeast) UBE2N 23.056 Zinc finger protein 3 ZNF3 25.994 TNF receptor-associated factor 7 TRAF7 27.204 HECT, UBA and WWE domain containing 1 HUWE1 40.653 Neuroblastoma, suppression of tumorigenicity 1 NBL1 47.533 Makorin, ring finger protein, 2 Mkrn2 48.151 Signal transducing adaptor molecule (SH3 domain and ITAM motif) 2 STAM2 49.861 Phosphatidylinositol 3,4,5-trisphosphate-dependent RAC exchanger 1 PREX1 67.608 Pleiotropic regulator 1 (PRL1 homolog, Arabidopsis) PLRG1 107.77 Aminoadipate aminotransferase AADAT 201.64

Appendices 359

 

APPENDIX H

H.1 Genes Two-Fold Differentially Regulated by FL in ALL-19

Xenograft Cells

Table H.1. Genes changed by greater than 2-fold in ALL-19 xenograft cells after 2 hrs. Gene Name Gene ID 2 hrs ATPase, Na+/K+ transporting, alpha 3 polypeptide ATP1A3 0.016 SRY (sex determining region Y)-box 1 SOX1 0.018 SAC3 domain containing 1 SAC3D1 0.025 Glutamate receptor, ionotropic, N-methyl D-aspartate 2A GRIN2A 0.031 Rho GTPase activating protein 23 ARHGAP23 0.034 START domain containing 5 STARD5 0.057 Ecotropic viral integration site 2A EVI2A 0.066 Mab-21-like 1 (C. elegans) MAB21L1 0.069 Ubiquitination factor E4A (UFD2 homolog, yeast) UBE4A 0.069 Pleckstrin PLEK 0.071 Cellular retinoic acid binding protein 2 CRABP2 0.083 Calcium binding tyrosine-(Y)-phosphorylation regulated (fibrousheathin 2) CABYR 0.094 Dual specificity phosphatase 12 DUSP12 0.105 MAP6 domain containing 1 MAP6D1 0.117 Sterol-C4-methyl oxidase-like SC4MOL 0.119 Guanidinoacetate N-methyltransferase GAMT 0.120 RNA binding motif protein 35B RBM35B 0.132 Nuclear casein kinase and cyclin-dependent kinase substrate 1 NUCKS1 0.149 Major histocompatibility complex, class II, DQ alpha 1 HLA-DQA1 0.168 SAM domain and HD domain, 1 Samhd1 0.177 Protein-O-mannosyltransferase 1 POMT1 0.187 Cholinergic receptor, nicotinic, alpha 4 CHRNA4 0.190 Protease, serine, 23 PRSS23 0.193 Esophageal cancer associated protein MGC16824 0.204 RAD9 homolog A (S. pombe) RAD9A 0.217 RAB37, member RAS oncogene family RAB37 0.227 Distal-less homeobox 4 DLX4 0.241 Fas apoptotic inhibitory molecule FAIM 0.243 Histone cluster 1, H3i HIST1H3I 0.247 Synaptopodin 2 SYNPO2 0.252 Lysyl oxidase-like 1 LOXL1 0.257 Protein phosphatase 1, regulatory (inhibitor) subunit 9B PPP1R9B 0.265

Appendices 361 Ankyrin repeat and sterile alpha motif domain containing 1A ANKS1A 0.267 Metallothionein 1 Mt1 0.275 Frizzled homolog 6 (Drosophila) FZD6 0.276 Sperm antigen with calponin homology and coiled-coil domains 1 SPECC1 0.281 Cytochrome P450, family 19, subfamily A, polypeptide 1 CYP19A1 0.284 WAP four-disulfide core domain 2 WFDC2 0.287 Mitogen-activated protein kinase kinase kinase 4 MAP3K4 0.287 Cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) Cdkn2c 0.291 WD repeat domain 20 WDR20 0.296 Zinc finger, CCHC domain containing 5 ZCCHC5 0.302 ATP-binding cassette, sub-family C (CFTR/MRP), member 9 ABCC9 0.309 Peroxidasin homolog (Drosophila)-like PXDNL 0.314 Interferon-induced protein with tetratricopeptide repeats 2 Ifit2 0.325 Keratin 19 KRT19 0.339 Thyroid transcription factor 1 TITF1 0.341 Growth hormone receptor GHR 0.341 Nuclear cap binding protein subunit 2, 20kDa NCBP2 0.344 WW and C2 domain containing 2 WWC2 0.349 Dual specificity phosphatase 5 Dusp5 0.349 Par-3 partitioning defective 3 homolog (C. elegans) PARD3 0.351 Lectin, galactoside-binding, soluble, 1 (galectin 1) LGALS1 0.352 CDC-like kinase 1 CLK1 0.354 Core binding factor beta Cbfb 0.356 Collagen, type IV, alpha 3 (Goodpasture antigen) COL4A3 0.360 Heat shock protein 90kDa alpha (cytosolic), class A member 1 Hsp90aa1 0.362 DEAH (Asp-Glu-Ala-His) box polypeptide 8 DHX8 0.365 TSC22 domain family, member 1 Tsc22d1 0.368 Ubiquitin specific peptidase 53 USP53 0.370 Eukaryotic translation initiation factor 4A, isoform 2 EIF4A2 0.372 Collagen, type XXI, alpha 1 COL21A1 0.377 Hemoglobin, beta HBB 0.379 FRAS1 related extracellular matrix 1 FREM1 0.382 Calcium channel, voltage-dependent, L type, alpha 1C subunit CACNA1C 0.382 Dipeptidyl-peptidase 4 (CD26, adenosine deaminase complexing protein 2) DPP4 0.387 Potassium voltage-gated channel, shaker-related subfamily, member 5 KCNA5 0.390 Disabled homolog 1 (Drosophila) DAB1 0.391 Solute carrier family 9 (sodium/hydrogen exchanger), isoform 3 regulator 1 Slc9a3r1 0.391 Brix domain containing 1 Bxdc1 0.391 Protein tyrosine phosphatase, non-receptor type 22 (lymphoid) Ptpn22 0.395 Chemokine (C motif) ligand 1 Xcl1 0.398 Melanoma inhibitory activity family, member 3 MIA3 0.399 DCMP deaminase DCTD 0.400 Prokineticin receptor 1 PROKR1 0.401 Pleckstrin homology domain containing, family A member 5 PLEKHA5 0.402 Calcium/calmodulin-dependent protein kinase IV Camk4 0.404 Epsin 2 EPN2 0.404 CAMP responsive element modulator Crem 0.408 Alanyl (membrane) aminopeptidase (aminopeptidase N, aminopeptidase M, ANPEP 0.410 microsomal aminopeptidase, CD13, p150) Fibronectin leucine rich transmembrane protein 2 FLRT2 0.411 Neurotensin receptor 2 NTSR2 0.411 Alcohol dehydrogenase, iron containing, 1 ADHFE1 0.413 Ubiquitously transcribed tetratricopeptide repeat gene, Y-linked UTY 0.422

Appendices 362 Nipped-B homolog (Drosophila) NIPBL 0.422 GTPase, IMAP family member 6 GIMAP6 0.424 Serine PI Kazal type 5-like 3 SPINK5L3 0.425 ADAM metallopeptidase domain 6 ADAM6 0.430 Teashirt family zinc finger 2 TSHZ2 0.432 Leucine-rich, glioma inactivated 1 LGI1 0.434 Tumor necrosis factor receptor superfamily, member 1b Tnfrsf1b 0.436 Leucine-rich repeat kinase 2 LRRK2 0.437 Heat shock protein 90kDa alpha (cytosolic), class B member 1 Hsp90ab1 0.438 Arginine-rich, mutated in early stage tumors Armet 0.438 Integrin-binding sialoprotein (bone sialoprotein, bone sialoprotein II) IBSP 0.440 Ankyrin repeat domain 13C ANKRD13C 0.441 BarH-like homeobox 1 BARX1 0.443 E2F transcription factor 4, p107/p130-binding E2F4 0.443 Leucine rich repeat containing 8 family, member B LRRC8B 0.445 Collagen, type XI, alpha 1 COL11A1 0.447 Asparaginyl-tRNA synthetase Nars 0.448 Hbs1-like (S. cerevisiae) Hbs1l 0.448 MCM2 minichromosome maintenance deficient 2, mitotin (S. cerevisiae) MCM2 0.449 HFM1, ATP-dependent DNA helicase homolog (S. cerevisiae) HFM1 0.449 Cyclin M2 CNNM2 0.450 Protein phosphatase 2, regulatory subunit B (B56), gamma isoform Ppp2r5c 0.451 Dpy-19-like 2 (C. elegans) DPY19L2 0.454 Leucine-rich, glioma inactivated 1 LGI1 0.455 Casein kinase 1, gamma 2 CSNK1G2 0.456 ER degradation enhancer, mannosidase alpha-like 3 EDEM3 0.457 Chemokine (C-C motif) ligand 5 Ccl5 0.457 Hydroxyacid oxidase (glycolate oxidase) 1 HAO1 0.458 SMAD family member 6 SMAD6 0.459 Signal transducer and activator of transcription 4 Stat4 0.459 Arginine-rich, mutated in early stage tumors Armet 0.460 Colony stimulating factor 1 (macrophage) Csf1 0.460 Ret finger protein-like 3 antisense RFPL3S 0.460 Cyclin A1 CCNA1 0.462 Phytoceramidase, alkaline PHCA 0.462 Syntrophin, basic 2 Sntb2 0.462 Cylicin, basic protein of sperm head cytoskeleton 1 CYLC1 0.464 Potassium large conductance calcium-activated channel, subfamily M, beta KCNMB1 0.464 member 1 BCL2-like 11 (apoptosis facilitator) BCL2L11 0.465 Golgi autoantigen, golgin subfamily a, 1 GOLGA1 0.466 Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 Dyrk2 0.467 Prolactin receptor PRLR 0.468 DNA (cytosine-5-)-methyltransferase 3 alpha DNMT3A 0.468 Membrane targeting (tandem) C2 domain containing 1 MTAC2D1 0.469 HCG23177 hCG_23177 0.470 Serine palmitoyltransferase, long chain base subunit 2 SPTLC2 0.470 Leukemia inhibitory factor receptor alpha LIFR 0.471 TNF receptor-associated factor 7 TRAF7 0.471 Similar to 2010300C02Rik protein MGC42367 0.471 Ectonucleoside triphosphate diphosphohydrolase 1 ENTPD1 0.472 LIM and cysteine-rich domains 1 LMCD1 0.475 1-acylglycerol-3-phosphate O-acyltransferase 2 (lysophosphatidic acid AGPAT2 0.476 acyltransferase, beta)

Appendices 363 SUMO/sentrin specific peptidase family member 8 SENP8 0.479 Calcium/calmodulin-dependent protein kinase (CaM kinase) II delta CAMK2D 0.480 Solute carrier family 25 (mitochondrial carrier, Aralar), member 12 SLC25A12 0.481 Interleukin 4 receptor, alpha Il4ra 0.487 Nuclear mitotic apparatus protein 1 Numa1 0.490 Calnexin Canx 0.490 Inducible T-cell co-stimulator ligand ICOSLG 0.490 ES cell associated transcript 8 ECAT8 0.490 Pleckstrin homology-like domain, family B, member 2 PHLDB2 0.491 BCL2/adenovirus E1B interacting protein 3-like Bnip3l 0.492 Similar to matrilin 2 precursor tcag7.216 0.493 Myosin VIIA and Rab interacting protein MYRIP 0.494 PRKC, apoptosis, WT1, regulator PAWR 0.495 Ribosomal protein S6 kinase, polypeptide 2 Rps6ka2 0.495 Pyrroline-5-carboxylate reductase 1 Pycr1 0.496 Apolipoprotein A-II APOA2 0.497 Metastasis associated 1 family, member 3 hCG_1783907 0.498 ADP-ribosylation factor-like 4C Arl4c 0.499 Transmembrane protein 182 TMEM182 0.499 RNA binding motif protein, Y-linked, family 1, member A1 RBMY1A1 0.499 DnaJ (Hsp40) homolog, subfamily B, member 14 DNAJB14 0.500 Protocadherin 17 PCDH17 2.002 ADAM-like, decysin 1 ADAMDEC1 2.003 LYR motif containing 5 LYRM5 2.004 G protein-coupled receptor 87 GPR87 2.024 Rho GTPase activating protein 28 ARHGAP28 2.033 Osteoglycin OGN 2.036 Ectonucleoside triphosphate diphosphohydrolase 3 ENTPD3 2.045 Dom-3 homolog Z (C. elegans) DOM3Z 2.047 Mortality factor 4 like 2 MORF4L2 2.050 Interferon regulatory factor 4 Irf4 2.057 Nucleolar and spindle associated protein 1 NUSAP1 2.087 Protein O-fucosyltransferase 2 POFUT2 2.089 SH2 domain containing 3A SH2D3A 2.089 Sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1 SPOCK1 2.094 Phosphodiesterase 4D interacting protein (myomegalin) PDE4DIP 2.098 Transient receptor potential cation channel, subfamily M, member 2 TRPM2 2.113 Cellular retinoic acid binding protein 2 CRABP2 2.117 Ankyrin repeat domain 38 ANKRD38 2.122 Pleiotropic regulator 1 (PRL1 homolog, Arabidopsis) PLRG1 2.148 Family with sequence similarity 44, member B FAM44B 2.158 Nestin NES 2.180 Protein tyrosine phosphatase-like A domain containing 2 PTPLAD2 2.203 Coiled-coil domain containing 90A CCDC90A 2.233 WD repeat and FYVE domain containing 3 WDFY3 2.247 FAT tumor suppressor homolog 1 (Drosophila) FAT 2.250 Zinc finger protein 3 ZNF3 2.259 Caveolin 1, caveolae protein, 22kDa CAV1 2.260 Tumor protein D52-like 3 TPD52L3 2.284 Son of sevenless homolog 1 (Drosophila) SOS1 2.296 Syntaxin binding protein 6 (amisyn) STXBP6 2.297 Arginine vasopressin-induced 1 AVPI1 2.321 Armadillo repeat containing, X-linked 2 ARMCX2 2.348

Appendices 364 Versican VCAN 2.399 Transmembrane channel-like 6 TMC6 2.401 Chemokine (C-X-C motif) ligand 13 (B-cell chemoattractant) CXCL13 2.427 Protein phosphatase 4, regulatory subunit 1 PPP4R1 2.435 Cell adhesion molecule with homology to L1CAM (close homolog of L1) CHL1 2.469 Protocadherin alpha 6 PCDHA6 2.483 Sodium channel, voltage-gated, type III, alpha subunit SCN3A 2.720 Plakophilin 4 PKP4 2.972 Bassoon (presynaptic cytomatrix protein) BSN 3.003 Protein tyrosine phosphatase-like (proline instead of catalytic arginine), member b PTPLB 3.022 Cysteine-rich, angiogenic inducer, 61 CYR61 3.031 Attractin ATRN 3.063 Ring finger protein 150 RNF150 3.110 Ventricular zone expressed PH domain homolog 1 (zebrafish) VEPH1 3.111 Wingless-type MMTV integration site family, member 5A WNT5A 3.155 HECT, UBA and WWE domain containing 1 HUWE1 3.266 Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) CXCL12 3.299 RAB6A, member RAS oncogene family RAB6A 3.317 Ankyrin repeat and SOCS box-containing 1 ASB1 3.328 Protein tyrosine phosphatase, receptor type, C-associated protein PTPRCAP 3.377 Chondroitin sulfate proteoglycan 4 CSPG4 3.427 Phospholipase A2, group IIA (platelets, synovial fluid) PLA2G2A 3.470 IKAROS family zinc finger 1 (Ikaros) IKZF1 3.624 Signal transducer and activator of transcription 3 (acute-phase response factor) STAT3 3.851 Myosin regulatory light chain MRLC2 MRLC2 4.799 4-hydroxyphenylpyruvate dioxygenase HPD 5.218 YTH domain containing 1 YTHDC1 5.360 Prohibitin PHB 5.614 Cyclin-dependent kinase-like 1 (CDC2-related kinase) CDKL1 5.797 Rho GTPase activating protein 12 ARHGAP12 5.818 Protein phosphatase 1, regulatory (inhibitor) subunit 3B PPP1R3B 6.096 Paralemmin PALM 6.680 Myosin XVIIIA MYO18A 6.703 V-raf murine sarcoma 3611 viral oncogene homolog ARAF 6.939 Ubiquitin-like 7 (bone marrow stromal cell-derived) UBL7 7.278 Zinc finger protein 649 ZNF649 7.682 Major histocompatibility complex, class II, DR beta 4 HLA-DRB4 8.147 Bromodomain adjacent to zinc finger domain, 2A BAZ2A 8.432 Amphiregulin (schwannoma-derived growth factor) AREG 10.013 MAP/microtubule affinity-regulating kinase 2 MARK2 11.558 Integrin beta 3 binding protein (beta3-endonexin) ITGB3BP 12.522 Transmembrane protein 186 TMEM186 12.832 Transcription factor 3 (E2A immunoglobulin enhancer binding factors E12/E47) TCF3 13.501 WD repeat domain 22 WDR22 13.675 SH2 domain containing 5 SH2D5 14.513 MON1 homolog B (yeast) MON1B 16.325 V-abl Abelson murine leukemia viral oncogene homolog 1 ABL1 22.985 Solute carrier family 22 (organic cation transporter), member 16 SLC22A16 29.230 Phosphoserine phosphatase PSPH 36.774 Solute carrier family 17 (sodium phosphate), member 1 SLC17A1 38.608 Methylmalonyl Coenzyme A mutase MUT 43.476

Appendices 365 Table H.2. Genes changed by greater than 2-fold in ALL-19 xenograft cells after 6 hrs. Gene Name Gene ID 6 hrs Hemoglobin, beta HBB 0.001 Cyclin M1 CNNM1 0.015 Zinc finger protein 3 ZNF3 0.015 SRY (sex determining region Y)-box 1 SOX1 0.025 Melanoma inhibitory activity family, member 3 MIA3 0.034 Bruno-like 4, RNA binding protein (Drosophila) BRUNOL4 0.076 Growth hormone receptor GHR 0.092 ADAMDE ADAM-like, decysin 1 0.099 C1 Solute carrier family 22 (organic cation transporter), member 16 SLC22A16 0.100 RNA binding motif protein 35B RBM35B 0.100 Thyroid transcription factor 1 TITF1 0.102 V-set and immunoglobulin domain containing 2 VSIG2 0.106 Bromodomain adjacent to zinc finger domain, 2A BAZ2A 0.114 Bassoon (presynaptic cytomatrix protein) BSN 0.126 Pleckstrin homology domain containing, family A member 5 PLEKHA5 0.145 Solute carrier family 25 (mitochondrial carrier, Aralar), member 12 SLC25A12 0.145 Septin 4 SEPT4 0.156 Arrestin, beta 2 ARRB2 0.158 Nipped-B homolog (Drosophila) NIPBL 0.158 Fas apoptotic inhibitory molecule FAIM 0.168 Amphiregulin (schwannoma-derived growth factor) AREG 0.190 Ubiquitin-like 7 (bone marrow stromal cell-derived) UBL7 0.205 Mitogen-activated protein kinase kinase 7 MAP2K7 0.220 Rap guanine nucleotide exchange factor (GEF) 1 RAPGEF1 0.224 YTH domain containing 1 YTHDC1 0.230 RAD9 homolog A (S. pombe) RAD9A 0.230 Cordon-bleu homolog (mouse) COBL 0.230 Sarcolipin SLN 0.236 Protein tyrosine phosphatase, receptor type, C-associated protein PTPRCAP 0.267 Coenzyme Q5 homolog, methyltransferase (S. cerevisiae) COQ5 0.268 Glutamate receptor, ionotropic, N-methyl D-aspartate 2A GRIN2A 0.269 Wingless-type MMTV integration site family, member 7A WNT7A 0.270 N-myc downstream regulated gene 1 NDRG1 0.272 Sterile alpha motif domain containing 13 SAMD13 0.285 Ankyrin repeat domain 39 ANKRD39 0.301 RAC/CDC42 exchange factor GEFT 0.303 Lymphocyte antigen 6 complex, locus D LY6D 0.310 Pleckstrin homology-like domain, family A, member 1 PHLDA1 0.310 Prion protein Prnp 0.313 Kinesin family member C1 KIFC1 0.326 Protein-O-mannosyltransferase 1 POMT1 0.332 Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 Slc3a2 0.333 Non-metastatic cells 5, protein expressed in (nucleoside-diphosphate kinase) NME5 0.337 SEC14-like 2 (S. cerevisiae) SEC14L2 0.337 Vascular cell adhesion molecule 1 VCAM1 0.346 Protein phosphatase 3 (formerly 2B), catalytic subunit, gamma isoform PPP3CC 0.355 Tumor protein D52-like 3 TPD52L3 0.355 Glucose 6 phosphatase, catalytic, 3 G6PC3 0.356 Phytanoyl-CoA 2-hydroxylase interacting protein-like PHYHIPL 0.356 GATA zinc finger domain containing 2A Gatad2a 0.356 Membrane-spanning 4-domains, subfamily A, member 5 MS4A5 0.359 Zinc finger, CCHC domain containing 14 ZCCHC14 0.360

Appendices 366 Hbs1-like (S. cerevisiae) Hbs1l 0.362 Phosphoinositide-binding protein PIP3-E PIP3-E 0.365 Potassium large conductance calcium-activated channel, subfamily M, beta member 4 KCNMB4 0.366 ATPase, Class I, type 8B, member 3 ATP8B3 0.370 Phosphoinositide-binding protein PIP3-E PIP3-E 0.371 Transforming growth factor beta regulator 1 TBRG1 0.373 Pregnancy upregulated non-ubiquitously expressed CaM kinase PNCK 0.380 Myosin, heavy chain 9, non-muscle MYH9 0.380 Ecotropic viral integration site 2A EVI2A 0.388 SAM domain and HD domain, 1 Samhd1 0.391 SAC3 domain containing 1 SAC3D1 0.398 Isoamyl acetate-hydrolyzing esterase 1 homolog (S. cerevisiae) IAH1 0.403 Interleukin 23, alpha subunit p19 IL23A 0.406 Signal transducing adaptor molecule (SH3 domain and ITAM motif) 2 STAM2 0.407 ATPase, Na+/K+ transporting, alpha 3 polypeptide ATP1A3 0.414 Protein tyrosine phosphatase, non-receptor type 22 (lymphoid) Ptpn22 0.415 Cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) Cdkn2c 0.420 Haloacid dehalogenase-like hydrolase domain containing 1A HDHD1A 0.422 Rh-associated glycoprotein RHAG 0.424 Amyloid beta precursor protein (cytoplasmic tail) binding protein 2 APPBP2 0.428 Colony stimulating factor 2 (granulocyte-macrophage) Csf2 0.430 Acyl-Coenzyme A dehydrogenase, long chain ACADL 0.440 Ubiquitin specific peptidase 9, X-linked USP9X 0.440 DEAH (Asp-Glu-Ala-His) box polypeptide 34 DHX34 0.441 Phosphatidylinositol 4-kinase, catalytic, beta polypeptide PIK4CB 0.441 Nuclear receptor co-repressor 1 Ncor1 0.441 TNFAIP3 interacting protein 1 TNIP1 0.448 Transducin (beta)-like 1X-linked TBL1X 0.448 Growth factor receptor-bound protein 10 GRB10 0.448 Peroxiredoxin 6 PRDX6 0.454 Mesoderm specific transcript homolog (mouse) MEST 0.455 Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member LILRB1 0.455 1 Syntaxin binding protein 6 (amisyn) STXBP6 0.456 Neutrophil cytosolic factor 2 (65kDa, chronic granulomatous disease, autosomal 2) NCF2 0.457 Placenta-specific 7 PLAC7 0.458 Zinc finger and BTB domain containing 48 ZBTB48 0.460 Makorin, ring finger protein, 2 Mkrn2 0.461 Killer cell lectin-like receptor subfamily K, member 1 Klrk1 0.464 Calnexin Canx 0.464 Colony stimulating factor 1 (macrophage) Csf1 0.465 Interleukin 4 receptor, alpha Il4ra 0.467 Topoisomerase (DNA) II beta Top2b 0.468 Semenogelin II SEMG2 0.469 Dehydrogenase/reductase (SDR family) member 9 DHRS9 0.471 Glycogen synthase kinase 3 beta GSK3B 0.473 Ribosomal protein L37 RPL37 0.474 Natural killer cell group 7 sequence NKG7 0.478 Ephrin-B2 EFNB2 0.478 Triggering receptor expressed on myeloid cells 1 TREM1 0.479 Chemokine (C-X-C motif) ligand 2 CXCL2 0.479 PWP1 homolog (S. cerevisiae) PWP1 0.481 Integrin-binding sialoprotein (bone sialoprotein, bone sialoprotein II) IBSP 0.483 Receptor (chemosensory) transporter protein 4 RTP4 0.487 Potassium inwardly-rectifying channel, subfamily J, member 2 KCNJ2 0.487

Appendices 367 Neurotrophic tyrosine kinase, receptor, type 2 NTRK2 0.490 Macrophage stimulating 1 (hepatocyte growth factor-like) MST1 0.490 Ubiquitin protein ligase E3 component n-recognin 1 UBR1 0.491 Chemokine (C-C motif) ligand 20 CCL20 0.493 Solute carrier family 7 (cationic amino acid transporter, y+ system), member 5 Slc7a5 0.493 Chemokine (C-C motif) ligand 5 CCL5 0.494 Alanyl-tRNA synthetase Aars 0.495 Transient receptor potential cation channel, subfamily M, member 1 TRPM1 0.496 Guanylate binding protein 2, interferon-inducible GBP2 0.497 Testis derived transcript (3 LIM domains) TES 0.497 Potassium voltage-gated channel, shaker-related subfamily, member 5 KCNA5 0.499 Uridine-cytidine kinase 1 Uck1 0.500 WAP four-disulfide core domain 1 WFDC1 2.003 Syntaxin binding protein 1 STXBP1 2.006 Adaptor-related protein complex 2, alpha 2 subunit AP2A2 2.007 CDC42BP CDC42 binding protein kinase alpha (DMPK-like) 2.014 A Fibrillin 1 FBN1 2.015 Dickkopf homolog 3 (Xenopus laevis) DKK3 2.019 Wingless-type MMTV integration site family, member 5A WNT5A 2.019 Teashirt family zinc finger 2 TSHZ2 2.034 Gamma-aminobutyric acid (GABA) A receptor, alpha 1 GABRA1 2.036 Transmembrane protein 56 TMEM56 2.036 Ring finger protein 157 RNF157 2.041 Hepatitis B virus x interacting protein HBXIP 2.042 Myosin VIIB MYO7B 2.044 Meis homeobox 1 MEIS1 2.044 Tetratricopeptide repeat domain 27 TTC27 2.053 Interleukin 31 receptor A IL31RA 2.054 Disabled homolog 1 (Drosophila) DAB1 2.058 RAB26, member RAS oncogene family RAB26 2.058 Low density lipoprotein-related protein 2 LRP2 2.061 ES cell associated transcript 8 ECAT8 2.061 Solute carrier family 2, (facilitated glucose transporter) member 8 SLC2A8 2.064 Dystonin DST 2.066 ADAMTSL ADAMTS-like 2 2.067 2 Mitogen-activated protein kinase 8 interacting protein 1 MAPK8IP1 2.067 Urotensin 2 UTS2 2.068 Carbonic anhydrase VB, mitochondrial CA5B 2.068 Aldehyde dehydrogenase 1 family, member A2 ALDH1A2 2.072 HFM1, ATP-dependent DNA helicase homolog (S. cerevisiae) HFM1 2.074 Ret finger protein-like 3 antisense RFPL3S 2.074 Caveolin 2 CAV2 2.076 Scavenger receptor class A, member 3 SCARA3 2.081 ADP-ribosylation factor-like 4C ARL4C 2.087 One cut domain, family member 2 ONECUT2 2.089 Phenylalanine hydroxylase PAH 2.093 Two transmembrane domain family member A TTMA 2.097 Amiloride-sensitive cation channel 1, neuronal (degenerin) ACCN1 2.100 Transferrin TF 2.102 Neural cell adhesion molecule 1 NCAM1 2.103 DNA (cytosine-5-)-methyltransferase 3 alpha DNMT3A 2.105 Nedd4 family interacting protein 2 NDFIP2 2.107 Zinc finger protein 483 ZNF483 2.108 Clusterin CLU 2.108 Appendices 368 Spindlin family, member 3 SPIN3 2.109 Major histocompatibility complex, class I, C HLA-C 2.110 Myosin, light chain 1, alkali; skeletal, fast MYL1 2.113 CD53 antigen Cd53 2.116 Leukemia inhibitory factor receptor alpha LIFR 2.117 Dynein, axonemal, heavy chain 5 DNAH5 2.126 Musculin (activated B-cell factor-1) MSC 2.126 Biphenyl hydrolase-like (serine hydrolase; breast epithelial mucin-associated antigen) BPHL 2.127 Collagen, type XI, alpha 1 COL11A1 2.133 BIC transcript BIC 2.136 Microtubule-associated protein tau MAPT 2.142 Phosphatidylinositol-specific phospholipase C, X domain containing 2 PLCXD2 2.143 Fibronectin leucine rich transmembrane protein 2 FLRT2 2.143 G protein-coupled receptor 162 GPR162 2.147 Protein O-fucosyltransferase 2 POFUT2 2.149 ICEBERG caspase-1 inhibitor ICEBERG 2.149 Calcium channel, voltage-dependent, gamma subunit 4 CACNG4 2.156 Islet cell autoantigen 1,69kDa-like ICA1L 2.163 MAP7 domain containing 2 MAP7D2 2.163 Pregnancy-associated plasma protein A, pappalysin 1 PAPPA 2.165 Exocyst complex component 2 EXOC2 2.175 Galanin GAL 2.175 Gap junction protein, beta 2, 26kDa GJB2 2.188 Ras association (RalGDS/AF-6) and pleckstrin homology domains 1 RAPH1 2.191 Discs, large homolog 2, chapsyn-110 (Drosophila) DLG2 2.194 EMI domain containing 1 EMID1 2.201 Kinesin family member 5A KIF5A 2.208 Orthopedia homeobox OTP 2.208 F-box and leucine-rich repeat protein 7 FBXL7 2.210 Latent transforming growth factor beta binding protein 1 LTBP1 2.212 Junctional adhesion molecule 3 JAM3 2.216 Spermatogenesis associated 22 SPATA22 2.218 Zinc finger protein-like 1 ZFPL1 2.218 Discs, large homolog 3 (neuroendocrine-dlg, Drosophila) DLG3 2.223 F-box protein 2 FBXO2 2.225 Erythrocyte membrane protein band 4.1-like 3 EPB41L3 2.229 Tripartite motif-containing 36 TRIM36 2.230 Ring finger protein 150 RNF150 2.231 Elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 4 ELOVL4 2.242 Fatty acid desaturase 1 FADS1 2.243 Ankyrin repeat domain 15 ANKRD15 2.249 X (inactive)-specific transcript XIST 2.258 Melanoma antigen family B, 2 MAGEB2 2.260 Cysteine-rich, angiogenic inducer, 61 CYR61 2.263 Folliculin FLCN 2.264 Solute carrier family 6, member 15 SLC6A15 2.267 Sperm associated antigen 6 SPAG6 2.272 FAM103A Family with sequence similarity 103, member A1 2.283 1 Melanoma antigen family A, 4 MAGEA4 2.289 Galanin GAL 2.294 LINE-1 type transposase domain containing 1 L1TD1 2.306 Glutamate receptor, ionotropic, AMPA 2 GRIA2 2.322 Pleckstrin homology domain containing, family B (evectins) member 1 PLEKHB1 2.323 Heart and neural crest derivatives expressed 2 HAND2 2.332

Appendices 369 V-maf musculoaponeurotic fibrosarcoma oncogene homolog G (avian) MAFG 2.337 RWD domain containing 2 RWDD2 2.339 Grainyhead-like 1 (Drosophila) GRHL1 2.345 Anaphase promoting complex subunit 2 ANAPC2 2.349 Cysteine-rich, angiogenic inducer, 61 CYR61 2.350 TEA domain family member 1 (SV40 transcriptional enhancer factor) TEAD1 2.360 Glutathione S-transferase A4 GSTA4 2.366 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1 B4GALT1 2.374 Pregnancy-associated plasma protein A, pappalysin 1 PAPPA 2.384 Heterogeneous nuclear ribonucleoprotein U (scaffold attachment factor A) HNRPU 2.386 Cell adhesion molecule 1 CADM1 2.395 Solute carrier family 36 (proton/amino acid symporter), member 3 SLC36A3 2.401 Prolactin receptor PRLR 2.406 Bestrophin 3 BEST3 2.407 Male-specific lethal 3-like 1 (Drosophila) MSL3L1 2.407 Usher syndrome 2A (autosomal recessive, mild) USH2A 2.410 Aspartate beta-hydroxylase ASPH 2.425 TEA domain family member 2 TEAD2 2.450 CUB and Sushi multiple domains 1 CSMD1 2.450 Integrin, alpha 5 (fibronectin receptor, alpha polypeptide) ITGA5 2.481 Protease, serine, 21 (testisin) PRSS21 2.486 Ring finger and CCCH-type zinc finger domains 2 RC3H2 2.498 Oxysterol binding protein-like 6 OSBPL6 2.503 G protein-coupled receptor 126 GPR126 2.506 TEA domain family member 1 (SV40 transcriptional enhancer factor) TEAD1 2.508 Alpha-kinase 3 ALPK3 2.512 Tetratricopeptide repeat domain 7A TTC7A 2.514 Vasohibin 2 VASH2 2.515 ADAM metallopeptidase with thrombospondin type 1 motif, 5 (aggrecanase-2) ADAMTS5 2.532 Dystrobrevin, alpha DTNA 2.535 Trafficking protein particle complex 6B TRAPPC6B 2.546 Nuclear cap binding protein subunit 2, 20kDa NCBP2 2.547 Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 SVEP1 2.571 Lipoma HMGIC fusion partner-like 1 LHFPL1 2.574 Synaptopodin 2 SYNPO2 2.575 Protein phosphatase 2, regulatory subunit B', alpha isoform PPP2R5A 2.576 Synaptopodin 2 SYNPO2 2.576 Plakophilin 4 PKP4 2.602 Melanoma antigen family A, 11 MAGEA11 2.605 Ribosomal protein L28 RPL28 2.612 Zinc finger CCCH-type containing 12C ZC3H12C 2.615 InaD-like (Drosophila) INADL 2.615 Follistatin FST 2.624 Heat shock 27kDa protein 3 HSPB3 2.630 Four and a half LIM domains 5 FHL5 2.630 Spermatogenic leucine zipper 1 SPZ1 2.632 Zinc finger protein 333 ZNF333 2.642 Ubiquitin specific peptidase 53 USP53 2.656 FRAS1 related extracellular matrix 1 FREM1 2.672 TIA1 cytotoxic granule-associated RNA binding protein TIA1 2.694 Vacuolar protein sorting 29 homolog (S. cerevisiae) VPS29 2.726 Disrupted in schizophrenia 1 DISC1 2.732 Galactosylceramidase GALC 2.733 CD93 molecule CD93 2.737

Appendices 370 Coiled-coil domain containing 90A CCDC90A 2.743 Fibulin 5 FBLN5 2.747 Alpha-kinase 2 ALPK2 2.749 Shugoshin-like 2 (S. pombe) SGOL2 2.772 SOX2 overlapping transcript (non-coding RNA) SOX2OT 2.784 Zinc finger protein 37 homolog (mouse) ZFP37 2.803 Leucine rich repeat neuronal 1 LRRN1 2.813 Nestin NES 2.829 G protein-coupled receptor 126 GPR126 2.835 KH domain containing, RNA binding, signal transduction associated 3 KHDRBS3 2.840 Cylicin, basic protein of sperm head cytoskeleton 1 CYLC1 2.886 Myosin XVIIIA MYO18A 2.908 Titin TTN 2.917 Pleiotropic regulator 1 (PRL1 homolog, Arabidopsis) PLRG1 2.931 Adipocyte-specific adhesion molecule ASAM 2.933 Collagen, type XI, alpha 1 COL11A1 2.947 Cytochrome P450, family 19, subfamily A, polypeptide 1 CYP19A1 2.955 Cyclin N-terminal domain containing 1 CNTD1 2.962 Coiled-coil domain containing 62 CCDC62 2.977 Family with sequence similarity 19 (chemokine (C-C motif)-like), member A4 FAM19A4 2.985 Cellular retinoic acid binding protein 2 CRABP2 3.007 Neurocalcin delta NCALD 3.009 Pleckstrin PLEK 3.016 ATPase, Na+/K+ transporting, beta 1 polypeptide ATP1B1 3.031 Contactin 3 (plasmacytoma associated) CNTN3 3.056 Hairy/enhancer-of-split related with YRPW motif-like HEYL 3.056 V-erb-a erythroblastic leukemia viral oncogene homolog 4 (avian) ERBB4 3.116 Sine oculis homeobox homolog 4 (Drosophila) SIX4 3.133 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 5 GALNT5 3.141 (GalNAc-T5) Potassium voltage-gated channel, Shal-related subfamily, member 2 KCND2 3.144 Calcium channel, voltage-dependent, gamma subunit 4 CACNG4 3.144 Ets variant gene 1 ETV1 3.216 Neurofilament, heavy polypeptide 200kDa NEFH 3.217 Glutamate receptor, ionotropic, AMPA 2 GRIA2 3.313 Zinc finger, X-linked, duplicated B ZXDB 3.346 Prokineticin receptor 1 PROKR1 3.382 SET and MYND domain containing 1 SMYD1 3.469 Zinc finger protein 229 ZNF229 3.501 Sestrin 1 SESN1 3.537 Sperm associated antigen 7 SPAG7 3.547 Microtubule-associated protein 9 MAP9 3.560 Membrane-spanning 4-domains, subfamily A, member 7 MS4A7 3.561 Stearoyl-CoA desaturase (delta-9-desaturase) SCD 3.596 Checkpoint suppressor 1 CHES1 3.656 Solute carrier family 35, member E1 SLC35E1 3.716 Attractin ATRN 3.747 Zinc finger protein 649 ZNF649 3.797 Kinesin family member 21A KIF21A 3.841 HLA- Major histocompatibility complex, class II, DQ alpha 1 3.850 DQA1 ER degradation enhancer, mannosidase alpha-like 3 EDEM3 3.995 Follistatin-like 4 FSTL4 4.080 Nestin NES 4.082 Stearoyl-CoA desaturase (delta-9-desaturase) SCD 4.111 Angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 ACE2 4.246 Appendices 371 LIM domain only 3 (rhombotin-like 2) LMO3 4.468 Interleukin 17B IL17B 4.547 Heat shock transcription factor family member 5 HSF5 4.720 Pyruvate kinase, liver and RBC PKLR 4.975 Alanyl (membrane) aminopeptidase (aminopeptidase N, aminopeptidase M, microsomal ANPEP 5.009 aminopeptidase, CD13, p150) Basic helix-loop-helix domain containing, class B, 3 BHLHB3 5.034 NIPA-like domain containing 1 NPAL1 5.038 Dual specificity phosphatase 12 DUSP12 5.319 Transcription factor CP2-like 1 TFCP2L1 5.343 Dynein, axonemal, heavy chain 7 DNAH7 5.693 Rho-associated, coiled-coil containing protein kinase 1 ROCK1 5.730 ATPase, Class V, type 10A ATP10A 5.934 Pregnancy specific beta-1-glycoprotein 10 PSG10 7.998 Plakophilin 4 PKP4 8.226 Lysyl oxidase-like 1 LOXL1 9.727 PMP22 claudin domain-containing protein PMP22CD 10.508 Transmembrane protein 186 TMEM186 16.032 Variable charge, X-linked 3A VCX3A 17.329 Zic family member 3 heterotaxy 1 (odd-paired homolog, Drosophila) ZIC3 21.659 Mediterranean fever MEFV 25.257 Leukocyte immunoglobulin-like receptor, subfamily B (with TM & ITIM domains), member 3 LILRB3 27.087 HLA- Major histocompatibility complex, class II, DR beta 4 30.054 DRB4 Eukaryotic translation initiation factor 4A, isoform 2 EIF4A2 32.401 Membrane-spanning 4-domains, subfamily A, member 6A MS4A6A 42.171 Echinoderm microtubule associated protein like 3 EML3 55.267

Table H.3. Genes changed by greater than 2-fold in ALL-19 xenograft cells after 24 hrs. Gene Name Gene ID 24 hrs PIN2-interacting protein 1 PINX1 0.002 Plakophilin 4 PKP4 0.013 Alanyl (membrane) aminopeptidase (aminopeptidase N, aminopeptidase M, microsomal ANPEP 0.020 aminopeptidase, CD13, p150) Acidic (leucine-rich) nuclear phosphoprotein 32 family, member E ANP32E 0.027 SERPINB Serpin peptidase inhibitor, clade B (ovalbumin), member 4 0.030 4 Acyl-Coenzyme A oxidase 2, branched chain ACOX2 0.040 Fas apoptotic inhibitory molecule FAIM 0.064 TRAPPC6 Trafficking protein particle complex 6B 0.098 B Membrane-spanning 4-domains, subfamily A, member 6A MS4A6A 0.099 PDZ and LIM domain 5 PDLIM5 0.101 Protein phosphatase 2, regulatory subunit B', alpha isoform PPP2R5A 0.102 Tumor protein D52-like 3 TPD52L3 0.103 Abhydrolase domain containing 6 ABHD6 0.105 V-maf musculoaponeurotic fibrosarcoma oncogene homolog G (avian) MAFG 0.108 Eukaryotic translation initiation factor 4A, isoform 2 EIF4A2 0.111 YTH domain containing 1 YTHDC1 0.118 RAB35, member RAS oncogene family RAB35 0.128 Signaling lymphocytic activation molecule family member 1 Slamf1 0.128 Melanoma antigen family A, 11 MAGEA11 0.137 V-raf murine sarcoma 3611 viral oncogene homolog ARAF 0.143 Enhancer of zeste homolog 2 (Drosophila) EZH2 0.146 Family with sequence similarity 19 (chemokine (C-C motif)-like), member A4 FAM19A4 0.148

Appendices 372 Calpain 14 CAPN14 0.159 Paf1, RNA polymerase II associated factor, homolog (S. cerevisiae) PAF1 0.160 Discs, large homolog 5 (Drosophila) DLG5 0.172 Potassium voltage-gated channel, subfamily H (eag-related), member 8 KCNH8 0.175 Mitogen-activated protein kinase kinase 7 MAP2K7 0.176 RAB6A, member RAS oncogene family RAB6A 0.179 IKAROS family zinc finger 1 (Ikaros) IKZF1 0.182 MAP/microtubule affinity-regulating kinase 2 MARK2 0.201 Endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, 2 EDG2 0.204 Interleukin 17B IL17B 0.204 Pleiotropic regulator 1 (PRL1 homolog, Arabidopsis) PLRG1 0.206 Empty spiracles homeobox 2 opposite strand EMX2OS 0.223 Phospholipase A2, group IIA (platelets, synovial fluid) PLA2G2A 0.228 Claudin 18 CLDN18 0.231 Adaptor-related protein complex 1, gamma 2 subunit AP1G2 0.262 Monooxygenase, DBH-like 1 MOXD1 0.265 Spermatogenesis associated, serine-rich 2 SPATS2 0.267 Membrane-spanning 4-domains, subfamily A, member 7 MS4A7 0.281 Tyrosine kinase, non-receptor, 1 TNK1 0.283 Usher syndrome 2A (autosomal recessive, mild) USH2A 0.284 Protein tyrosine phosphatase-like (proline instead of catalytic arginine), member b PTPLB 0.291 Pregnancy specific beta-1-glycoprotein 10 PSG10 0.294 FAT tumor suppressor homolog 1 (Drosophila) FAT 0.298 Angiopoietin-like 7 ANGPTL7 0.302 Sestrin 1 SESN1 0.302 RNA binding motif protein 35B RBM35B 0.308 Pleiomorphic adenoma gene-like 1 PLAGL1 0.316 Nestin NES 0.321 Cysteine-rich, angiogenic inducer, 61 CYR61 0.327 Epididymal sperm binding protein 1 ELSPBP1 0.331 Apolipoprotein C-IV APOC4 0.331 Neuroligin 4, X-linked NLGN4X 0.333 LIM domain only 3 (rhombotin-like 2) LMO3 0.344 TNFAIP3 interacting protein 1 TNIP1 0.348 DEAD (Asp-Glu-Ala-Asp) box polypeptide 59 DDX59 0.352 Phospholipid scramblase 2 PLSCR2 0.359 Thrombospondin, type I, domain containing 7B THSD7B 0.363 Putative neuronal cell adhesion molecule PUNC 0.371 Dynein, axonemal, heavy chain 7 DNAH7 0.371 Bromodomain adjacent to zinc finger domain, 2A BAZ2A 0.372 Anaphase promoting complex subunit 2 ANAPC2 0.374 Fibroblast growth factor 2 (basic) FGF2 0.374 RNA binding motif protein 35A RBM35A 0.375 Kinesin family member 21A KIF21A 0.376 Osteoglycin OGN 0.378 WAP four-disulfide core domain 1 WFDC1 0.380 YME1-like 1 (S. cerevisiae) YME1L1 0.381 Amine oxidase, copper containing 3 (vascular adhesion protein 1) AOC3 0.389 NIMA (never in mitosis gene a)-related kinase 3 NEK3 0.391 Protein kinase, interferon-inducible double stranded RNA dependent activator PRKRA 0.395 Coenzyme Q5 homolog, methyltransferase (S. cerevisiae) COQ5 0.404 ADAM metallopeptidase domain 19 (meltrin beta) ADAM19 0.407 ADP-ribosylation factor-like 4C ARL4C 0.410 Integrin, beta 8 ITGB8 0.413

Appendices 373 Molybdenum cofactor synthesis 1 MOCS1 0.414 Matrix metallopeptidase 11 (stromelysin 3) MMP11 0.414 Junctional adhesion molecule 3 JAM3 0.425 Family with sequence similarity 120A FAM120A 0.428 Ets variant gene 1 ETV1 0.430 Basal cell adhesion molecule (Lutheran blood group) BCAM 0.431 WAP four-disulfide core domain 2 WFDC2 0.444 Nuclear receptor coactivator 3 NCOA3 0.445 MutS homolog 4 (E. coli) MSH4 0.447 Anterior gradient homolog 3 (Xenopus laevis) AGR3 0.449 Phosphatidylinositol glycan anchor biosynthesis, class L PIGL 0.452 Calnexin Canx 0.461 SMAD family member 6 SMAD6 0.461 Myosin, heavy chain 9, non-muscle MYH9 0.462 DEAD (Asp-Glu-Ala-Asp) box polypeptide 43 DDX43 0.464 Septin 4 SEPT4 0.466 Kelch repeat and BTB (POZ) domain containing 6 KBTBD6 0.467 Lipoic acid synthetase LIAS 0.468 Isocitrate dehydrogenase 3 (NAD+) beta IDH3B 0.469 Junctophilin 2 JPH2 0.470 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105) NFKB1 0.470 Structural maintenance of chromosomes 1A SMC1A 0.472 Sodium channel, voltage-gated, type II, alpha subunit SCN2A 0.476 Carboxypeptidase B1 (tissue) CPB1 0.478 CD36 molecule (thrombospondin receptor) CD36 0.480 Keratin 6A KRT6A 0.480 TIA1 cytotoxic granule-associated RNA binding protein TIA1 0.486 Zinc finger protein 229 ZNF229 0.487 Immunoglobulin lambda-like polypeptide 1 IGLL1 0.488 Immunoglobulin superfamily containing leucine-rich repeat 2 ISLR2 0.489 Coiled-coil domain containing 80 CCDC80 0.490 Sine oculis homeobox homolog 6 (Drosophila) SIX6 0.492 Matrix metallopeptidase 16 (membrane-inserted) MMP16 0.496 FAM103A Family with sequence similarity 103, member A1 0.499 1 Myelin basic protein MBP 2.000 Hbs1-like (S. cerevisiae) Hbs1l 2.001 Transmembrane protein 27 TMEM27 2.003 Heat shock protein 90kDa alpha (cytosolic), class A member 1 Hsp90aa1 2.005 LRP2 binding protein LRP2BP 2.012 Myoglobin MB 2.019 Pleckstrin and Sec7 domain containing 4 PSD4 2.021 CD40 molecule, TNF receptor superfamily member 5 CD40 2.028 Yes-associated protein 1, 65kDa YAP1 2.030 SLIT and NTRK-like family, member 1 SLITRK1 2.030 G protein-coupled receptor 85 GPR85 2.032 Interleukin 24 IL24 2.035 Solute carrier family 22 (organic cation transporter), member 15 SLC22A15 2.036 Arylsulfatase D ARSD 2.037 Osteopetrosis associated transmembrane protein 1 OSTM1 2.041 FYVE, RhoGEF and PH domain containing 4 FGD4 2.041 Trafficking protein particle complex 5 TRAPPC5 2.044 Acid phosphatase, prostate ACPP 2.048 Proline-serine-threonine phosphatase interacting protein 1 PSTPIP1 2.050 CD93 molecule CD93 2.052

Appendices 374 Attractin ATRN 2.056 APOBEC3 Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3F 2.062 F WD repeat domain 1 WDR1 2.064 Guanylate binding protein 4 GBP4 2.066 Ankyrin repeat domain 12 ANKRD12 2.068 Malignant fibrous histiocytoma amplified sequence 1 Mfhas1 2.069 Myosin regulatory light chain interacting protein MYLIP 2.070 SGT1, suppressor of G2 allele of SKP1 like 1 (S. cerevisiae) SUGT1L1 2.073 Solute carrier family 27 (fatty acid transporter), member 3 SLC27A3 2.075 Potassium voltage-gated channel, shaker-related subfamily, member 5 KCNA5 2.081 Jumonji domain containing 2C JMJD2C 2.086 Phosphonoformate immuno-associated protein 5 PFAAP5 2.091 FRAS1 related extracellular matrix 1 FREM1 2.098 G protein-coupled receptor 55 GPR55 2.102 Lecithin-cholesterol acyltransferase LCAT 2.110 Zinc finger protein 518 ZNF518 2.110 Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation SPP1 2.114 1) Antagonist of mitotic exit network 1 homolog (S. cerevisiae) AMN1 2.119 Protein tyrosine phosphatase, receptor type, O PTPRO 2.120 Unkempt homolog (Drosophila)-like UNKL 2.120 Squalene epoxidase Sqle 2.125 DENN/MADD domain containing 3 DENND3 2.126 Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation SPP1 2.131 1) Platelet/endothelial cell adhesion molecule (CD31 antigen) PECAM1 2.133 YTH domain family 1 Ythdf1 2.136 AXIN1 up-regulated 1 AXUD1 2.137 Glypican 4 GPC4 2.138 Integrin, alpha L (antigen CD11A (p180), lymphocyte function-associated antigen 1; alpha ITGAL 2.138 polypeptide) Acidic repeat containing ACRC 2.147 Sphingomyelin phosphodiesterase, acid-like 3A SMPDL3A 2.150 Peroxidasin homolog (Drosophila)-like PXDNL 2.153 Fibronectin leucine rich transmembrane protein 2 FLRT2 2.158 Thymocyte selection-associated HMG box gene Tox 2.166 CD300a molecule CD300A 2.167 CD1c molecule CD1C 2.177 Solute carrier family 37 (glycerol-3-phosphate transporter), member 2 SLC37A2 2.182 Myosin, heavy chain 11, smooth muscle MYH11 2.193 Glutamate receptor, ionotropic, N-methyl D-aspartate 2A GRIN2A 2.196 Phosphoinositide-3-kinase, regulatory subunit 5, p101 PIK3R5 2.206 TSC22 domain family, member 1 Tsc22d1 2.211 RAB3B, member RAS oncogene family RAB3B 2.211 MGC4236 Similar to 2010300C02Rik protein 2.215 7 Single-stranded DNA binding protein 2 SSBP2 2.219 Inducible T-cell co-stimulator ligand ICOSLG 2.221 Family with sequence similarity 26, member B FAM26B 2.222 Myosin, heavy chain 8, skeletal muscle, perinatal MYH8 2.223 Sorting nexin 19 SNX19 2.243 Transmembrane protein 134 Tmem134 2.249 Gap junction protein, beta 2, 26kDa GJB2 2.253 Pyruvate dehydrogenase E1 alpha 1 Pdha1 2.272 IBR domain containing 2 IBRDC2 2.298 Selectin, platelet Selp 2.302

Appendices 375 Cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) Cdkn2c 2.314 CUG triplet repeat, RNA binding protein 2 CUGBP2 2.317 Solute carrier family 35, member F5 SLC35F5 2.321 Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 Dyrk2 2.322 Contactin 1 CNTN1 2.329 Mal, T-cell differentiation protein 2 MAL2 2.331 E530011L RIKEN cDNA E530011L22 gene 2.345 22Rik ADAMDE ADAM-like, decysin 1 2.345 C1 TH1-like (Drosophila) TH1L 2.346 Phosphatidylinositol glycan anchor biosynthesis, class C PIGC 2.368 CAP-GLY domain containing linker protein family, member 4 CLIP4 2.373 Cardiotrophin-like cytokine factor 1 CLCF1 2.377 Homeobox A11 HOXA11 2.377 Sine oculis homeobox homolog 4 (Drosophila) SIX4 2.382 CUG triplet repeat, RNA binding protein 2 CUGBP2 2.389 Sterile alpha motif domain containing 9-like SAMD9L 2.416 V-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian) MYCN 2.443 Carbonic anhydrase VI CA6 2.447 Protein tyrosine phosphatase, non-receptor type 9 Ptpn9 2.449 FERM domain containing 4B FRMD4B 2.454 POU domain, class 2, transcription factor 2 POU2F2 2.456 G protein-coupled receptor 68 GPR68 2.458 Epithelial stromal interaction 1 (breast) EPSTI1 2.471 Glucuronidase, beta pseudogene 1 GUSBP1 2.489 Signal transducer and activator of transcription 4 Stat4 2.491 Ribosomal protein S9 Rps9 2.500 Inositol 1,4,5-trisphosphate 3-kinase A ITPKA 2.505 Mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, isozyme A MGAT4A 2.533 Regulator of chromosome condensation (RCC1) and BTB (POZ) domain containing protein RCBTB2 2.535 2 BolA homolog 2 (E. coli) BOLA2 2.541 Solute carrier family 9 (sodium/hydrogen exchanger), member 5 SLC9A5 2.556 Translocase of outer mitochondrial membrane 20 homolog (yeast) TOMM20 2.565 Hippocalcin like 4 HPCAL4 2.594 Metastasis associated lung adenocarcinoma transcript 1 (non-coding RNA) MALAT1 2.630 Family with sequence similarity 129, member A FAM129A 2.659 Distal-less homeobox 4 DLX4 2.664 Calpain 5 CAPN5 2.714 Histidyl-tRNA synthetase HARS 2.720 Phosphoglucomutase 3 PGM3 2.751 Tetratricopeptide repeat domain 27 TTC27 2.752 Stathmin-like 3 STMN3 2.764 Zic family member 3 heterotaxy 1 (odd-paired homolog, Drosophila) ZIC3 2.776 Synapse associated protein 1, SAP47 homolog (Drosophila) SYAP1 2.784 Lymphocyte antigen 9 LY9 2.794 WW and C2 domain containing 2 WWC2 2.820 Ets homologous factor EHF 2.843 GABARAP GABA(A) receptor-associated protein like 1 2.851 L1 Cellular retinoic acid binding protein 2 CRABP2 2.856 Protein phosphatase 4, regulatory subunit 1 PPP4R1 2.862 Phosphodiesterase 6H, cGMP-specific, cone, gamma PDE6H 2.880 MutS homolog 3 (E. coli) MSH3 2.893 Family with sequence similarity 116, member B FAM116B 2.928 Interleukin 23, alpha subunit p19 IL23A 2.937

Appendices 376 Caspase 12 CASP12 2.951 Colony stimulating factor 1 (macrophage) Csf1 2.965 CD93 molecule CD93 2.992 Retinal degeneration 3 RD3 2.994 Chemokine (C-C motif) ligand 20 CCL20 3.055 Membrane-spanning 4-domains, subfamily A, member 7 MS4A7 3.063 AT rich interactive domain 2 (ARID, RFX-like) ARID2 3.072 BCL2-like 11 (apoptosis facilitator) BCL2L11 3.109 Histidine triad nucleotide binding protein 3 HINT3 3.110 Protein phosphatase 2C, magnesium-dependent, catalytic subunit PPM2C 3.120 Core binding factor beta Cbfb 3.134 Keratin 19 KRT19 3.177 Nuclear receptor co-repressor 1 Ncor1 3.230 Zinc finger protein 318 ZNF318 3.239 G protein-coupled receptor 64 GPR64 3.289 PCTAIRE protein kinase 3 PCTK3 3.308 Coiled-coil domain containing 109B Ccdc109b 3.310 Makorin, ring finger protein, 2 Mkrn2 3.313 Mitogen-activated protein kinase kinase kinase 4 MAP3K4 3.404 Methylmalonyl Coenzyme A mutase MUT 3.416 GTPase, IMAP family member 6 GIMAP6 3.453 Macrophage expressed gene 1 MPEG1 3.615 Chemokine (C-C motif) receptor 5 CCR5 3.633 G protein-coupled receptor 143 GPR143 3.668 Ankyrin repeat domain 39 ANKRD39 3.668 Myosin XVIIIA MYO18A 3.812 Cyclin-dependent kinase 5 CDK5 3.868 Neuroblastoma, suppression of tumorigenicity 1 NBL1 3.930 Mediterranean fever MEFV 3.933 Superkiller viralicidic activity 2-like 2 (S. cerevisiae) SKIV2L2 3.964 Prion protein Prnp 4.060 CD84 molecule CD84 4.114 SFT2 domain containing 3 SFT2D3 4.114 Galactosylceramidase GALC 4.141 CDC-like kinase 1 CLK1 4.286 Arrestin, beta 2 ARRB2 4.318 ROD1 regulator of differentiation 1 (S. pombe) ROD1 4.424 SAR1 gene homolog B (S. cerevisiae) SAR1B 4.489 Interleukin 31 receptor A IL31RA 4.525 Glycogenin 2 GYG2 4.579 Melanoma antigen family A, 4 MAGEA4 4.707 Urotensin 2 UTS2 4.847 CD1c molecule CD1C 4.918 Ras association (RalGDS/AF-6) domain family 5 RASSF5 5.081 Septin 11 SEPT11 5.275 Thymus high mobility group box protein TOX TOX 6.028 Coiled-coil domain containing 101 CCDC101 6.165 Attractin-like 1 ATRNL1 6.205 Beta-site APP-cleaving enzyme 1 BACE1 6.458 Thyroid transcription factor 1 TITF1 6.767 Solute carrier family 4, sodium bicarbonate cotransporter, member 7 SLC4A7 6.989 Amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein APBB1IP 7.140 Integrin-binding sialoprotein (bone sialoprotein, bone sialoprotein II) IBSP 7.200 MSX2 7.269

Appendices 377 Peroxiredoxin 6 PRDX6 7.775 Prostaglandin E receptor 2 (subtype EP2) Ptger2 8.696 Erythrocyte membrane protein band 4.1-like 3 EPB41L3 8.884 Heparan sulfate 6-O-sulfotransferase 2 HS6ST2 8.893 BCL2-associated athanogene 2 BAG2 9.338 Heat shock transcription factor family member 5 HSF5 10.553 Serine PI Kazal type 5-like 3 SPINK5L3 10.807 Solute carrier family 22 (organic cation transporter), member 16 SLC22A16 10.831 Sorting nexin 9 SNX9 10.871 Hairy/enhancer-of-split related with YRPW motif 2 HEY2 11.430 Nipped-B homolog (Drosophila) NIPBL 11.762 Transmembrane protein 186 TMEM186 12.047 Small nucleolar RNA host gene (non-protein coding) 6 SNHG6 18.242 SAC3 domain containing 1 SAC3D1 21.266 DCMP deaminase DCTD 21.524 Solute carrier family 25 (mitochondrial carrier, Aralar), member 12 SLC25A12 24.602 IQ motif and Sec7 domain 3 IQSEC3 27.865 Sperm associated antigen 7 SPAG7 29.568 Asparagine synthetase domain containing 1 ASNSD1 41.203 WD repeat domain 27 WDR27 43.330 Phosphoinositide-3-kinase, regulatory subunit 3 (p55, gamma) PIK3R3 71.325

Appendices 378  

APPENDIX I

I.1 HIF Chip

A ChIP assay was performed to assess the functional activity of HIF1. The PCR products of the VEGF promoter were assessed by capillary chromatography using a Bioanalyser (Agilent Technologies). The results are therefore displayed as an electronically generated gel, shown in Figure 5.25. The results show an increase in binding to the VEGF promoter region when cultured on MS5s at comparable levels to that measured to the control cells were cultures under low oxygen conditions.

3

2

1 Fold VEGF compared to ALL-3 only 0

-3 2 L S5 O M AL Low ith in -3 w 3 L ALL- AL Culture conditions

Figure I.1 ChIP assay of the HIF-1 binding capacity to the VEGF promoter. The graphed quantity equates to the amount of DNA 'pulled-down' compared to ALL 3 cells only, with the mock input subtracted from each sample. A 350 bp PCR product was amplified within the VEGF promoter region (-1386 to -1036 upstream from the start site) from the immunoprecipitated chromatin. Cells were grown ± MS5s under normal culturing conditions. Controls were cultured under low-oxygen conditions without MS5 stromal support.

Appendices 379