Investigation of the Role of Cbl-B in Leukemogenesis and Migration

INVESTIGATION OF THE ROLE OF CBL-B IN LEUKEMOGENESIS AND MIGRATION

Karla Michelle Badger-Brown

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Medical Biophysics University of Toronto

Investigation of the Role of CBL-B in Leukemogenesis and Migration Doctor of Philosophy, 2009 Karla Michelle Badger-Brown Department of Medical Biophysics University of Toronto

ABSTRACT CBL proteins are E3 ubiquitin ligases and adaptor proteins. The mammalian homologs – CBL, CBL-B and CBL-3 show broad tissue expression; accordingly, the CBL proteins play roles in multiple cell types. We have investigated the function of the CBL-B protein in hematopoietic cells and fibroblasts. The causative agent of chronic myeloid leukemia (CML) is BCR-ABL. This oncogenic fusion down-modulates CBL-B protein levels, suggesting that CBL-B regulates either the development or progression of CML. To assess the involvement of CBL-B in CML, bone marrow transduction and transplantation (BMT) studies were performed. Recipients of BCR-ABL-infected CBL-B(-/-) cells succumbed to a CML-like myeloproliferative disease with a longer latency than the wild-type recipients. Peripheral blood white blood cell numbers were reduced, as were splenic weights. Yet despite the reduced leukemic burden, granulocyte numbers were amplified throughout the animals. As well, CBL- B(-/-) bone marrow (BM) cells possessed defective BM homing capabilities. From these results we concluded that CBL-B negatively regulates granulopoiesis and that prolonged latency in our CBL-B(-/-) BMT animals was a function of perturbed homing. To develop an in vitro model to study CBL-B function we established mouse embryonic fibroblasts (MEFs) deficient in CBL-B expression. Transduction of the wild-type and CBL-B-deficient MEFs with BCR-ABL did not confer transformation; nevertheless, the role of CBL-B in fibroblasts was evaluated. The CBL-B(-/-) MEFs showed enhanced chemotactic migration toward serum in both Transwell migration and time-lapse video microscopy studies. The biochemical response to serum was extensively evaluated leading to the development of a model. We predict that CBL-B deficiency either: (a) augments GRB2-associated binding protein 2 (GAB2) phosphorylation leading to enhanced extracellular signal-regulated kinase (ERK) and protein kinase B (PKB / Akt) signaling, or (b) alleviates negative control of Vav3 resulting in stimulation of Rho effectors. In either case, our results reveal a negative regulatory role for CBL-B in fibroblast migration. The two studies detailed herein expand our knowledge of CBL-B function. They strongly suggest that CBL-B can modulate granulocyte proliferation and point toward a role for CBL-B in the motility of numerous cell types.

ACKNOWLEDGMENTS My PhD endeavor was driven by a desire for independent scientific- and self- discovery. I was searching for an opportunity to steer my own course and follow a road less traveled. While the experiences I take from this journey are my own, I cannot pretend that I was not significantly aided along the way. This road has been pebbled by many friends, colleagues and family that have provided useful experimental and personal guidance. As a newcomer from Manitoba I would have expected Toronto to be a difficult city to navigate, and yet I felt immediately welcomed into the Department of Medical Biophysics. From the “2001 Freshmen” to the Drowned Rats and the editorial board of Hypothesis, each experience granted me new friendships. Many a merry meeting was imbibed with not only beverage but scientific discourse. There is no doubt that I will happily cultivate these friendships for many years to come. As a PhD student I had the fortunate opportunity to exist as a true member of not only one, but two great labs. Thanks so much to the Dick lab members for sharing your input, reagents and a cozy Isolab corner; you guys all deserve a huge thanks for helping me evaluate my science from a stem cell point of view and entertaining me in the process. And to the “Barber Babes” and “Boys” - I can’t thank you enough for useful advice, help in the rough patches, and again, a healthy dose of fun. I particularly want to thank Hani and Terri. It is with great regard that I value your scientific intellect and drive. As two strong women, there is no doubt in my mind that you will find great success in your future. Which brings be to the Barber boss – Dwayne. Thanks for being an excellent supervisor. From the moment I stepped into your office for my initial interview I knew that our relationship would work. You have always shown confidence in me, providing the independence that I so desperately craved. As well, you have continuously challenged me to strive for greater success. From my experience under your tutelage I really feel that if I work hard enough, no task is insurmountable. Which brings me to “family”. Thanks first to my family – Mum, Ron, Holly and Julie. It’s been difficult being so far away from home, but I thank you for your support of my dreams. A special thank- you goes out to my role model - Mum. You were “single-Mum-extrodinaire”! I can’t tell you how much I appreciated your encouragement then, and now, as I near graduation. To my other family, Covell and Mary, you have grown to be not only my “in-laws”, but also my second set of parents. Thanks goes to Mary for recognizing those moments when a warm hug and delicious Sunday dinner would be appreciated. And thanks to Covell - my “great self-development over these years” is definitely partly a function of your useful guidance and advice. Membership in the Brown family would not have existed without my dearest Nicholas. You have been an integral part of this PhD process. Thanks so much for taking the time to truly appreciate my science and for your unwavering support in trying times. Moreover, it is from you Nicholas, that I have learned compassion and love, and for that, I am truly blessed.

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TABLE OF CONTENTS

ABSTRACT...... ii

ACKNOWLEDGMENTS ...... iii

TABLE OF CONTENTS...... iv

LIST OF TABLES ...... vi

LIST OF FIGURES ...... vii

LIST OF ABBREVIATIONS...... ix

LIST OF APPENDICES...... xvi

CHAPTER 1. INTRODUCTION ...... 1 1.1. Hematopoiesis...... 2 1.1.1. Hematopoiesis during ontogenesis...... 2 1.1.2. Adult hematopoiesis ...... 2 1.1.3. Differentiation of the HSC into mature blood cells ...... 2 1.1.4. Hematopoietic Growth Factors ...... 6 1.1.5. Methods to identify hematopoietic cells ...... 6 1.1.6. Summary ...... 8 1.2. Chronic Myeloid Leukemia...... 10 1.2.1. BCR-ABL is the causative agent of CML...... 10 1.2.2. Molecular mechanisms of BCR-ABL-mediated transformation...... 12 1.2.3. Biological mechanisms of BCR-ABL-mediated in vitro transformation...... 20 1.2.4. Modeling CML in animals ...... 30 1.2.5. Using the BMT assay to identify key pathways of BCR-ABL-mediated leukemogenesis..... 33 1.2.6. Therapies for CML...... 37 1.2.7. Summary ...... 43 1.3. The CBL family of proteins...... 44 1.3.1. The domain structure of CBL proteins...... 44 1.3.2. CBL proteins mediate ubiquitination ...... 47 1.3.3. CBL proteins function as adaptors ...... 51 1.3.4. CBL protein-mediated interactions that regulate ERK, PKB and Rho signaling ...... 51 1.3.5. The role of CBL proteins in cellular migration...... 56 1.3.6. The role of CBL proteins in cellular adhesion ...... 56 1.3.7. In vivo functions of CBL proteins...... 57

1.3.8. CBL family members and human disease...... 59 1.3.9. Summary ...... 59

THESIS STATEMENT ...... 60

CHAPTER 2. CBL-B DEFICIENCY DELAYS BCR-ABL-MEDIATED LEUKEMOGENESIS.. 61 2.1. Abstract...... 62 2.2. Introduction...... 63 2.3. Materials and Methods ...... 64 2.4. Results...... 70 2.5. Discussion...... 88

CHAPTER 3. CBL-B IS A CRITICAL REGULATOR OF MOUSE EMBRYONIC FIBROBLAST MOTILITY...... 93 3.1. Abstract...... 94 3.2. Introduction...... 95 3.3. Materials and Methods ...... 96 3.4. Results...... 100 3.5. Discussion...... 118

CHAPTER 4. DISCUSSION AND FUTURE DIRECTIONS ...... 123 4.1. Discussion...... 124 4.2. Future directions to delineate the role of CBL-B in hematopoietic cells ...... 125 4.3. Future directions to delineate the role of CBL-B in fibroblasts ...... 141 4.4. Major scientific concepts emerging from this thesis...... 147 4.5. Closing Remarks...... 150

CHAPTER 5. REFERENCES ...... 152

LIST OF TABLES

Table 1-1. Hematopoietic cytokines...... 7 Table 1-2. Alternative names for chemical inhibitors of proteins in the BCR-ABL signaling pathway... 28 Table 1-3. The results of BMT assays that determined the essential domains and residues of BCR-ABL required for MPD development...... Error! Bookmark not defined. Table 1-4. Altered leukemogenesis when donor cells with genetic deletions of key effectors are used in BCR-ABLp210 BMTs...... 36 Table 1-5. Transcription factors that activate early myeloid genes...... 38 Table 4-1. Exogenous introduction of constructs that will alter protein activation...... 144

LIST OF FIGURES Figure 1-1. Hematopoietic stem cells reside in the bone marrow...... 3 Figure 1-2. Differentiation of the HSC into all of the hematopoietic lineages...... 5 Figure 1-3. Cell surface markers of hematopoietic stem cells...... 9 Figure 1-4. A chromosomal translocation results in the BCR-ABL fusion...... 11 Figure 1-5. Domain structure of BCR-ABL...... 13 Figure 1-6. Autoinhibition of ABL...... 15 Figure 1-7. Oligomerization as a means of BCR-ABL activation...... 16 Figure 1-8. BCR-ABL signaling mechanisms...... 18 Figure 1-9. BCR-ABL stimulates three oncogenic processes...... 21 Figure 1-10. Potential mechanisms of BCR-ABL-mediated regulation of adhesion...... 23 Figure 1-11. The physiological processes involved in migration of a cell across sub-stratum...... 25 Figure 1-12. Chemical inhibitors that target components of the BCR-ABL signaling pathway...... 27 Figure 1-13. Signaling pathways proposed to play a role in the inhibition of apoptosis by BCR-ABL.... 29 Figure 1-14. Bone marrow transduction and transplantation protocol...... 32 Figure 1-15. Transcription factors operate at multiple stages of differentiation to control myelopoiesis.39 Figure 1-16. Timeline of CML treatments...... 40 Figure 1-17. Imatinib binding to BCR-ABL...... 42 Figure 1-18. Domain structure of CBL proteins...... 45 Figure 1-19. The ubiquitination pathway...... 48 Figure 1-20. Different forms of ubiquitin modification...... 49 Figure 1-21. CBL functions as an adaptor in associations with multiple substrates...... 52 Figure 1-22. CBL proteins control Rho family activation...... 55

Figure 2-1. BCR-ABL expression results in constitutive tyrosine phosphorylation of CBL and CBL-B proteins, and a reduction in CBL-B protein levels...... 71 Figure 2-2. Transplant recipients of CBL-B deficient cells show delayed latency of BCR-ABL-mediated leukemogenesis...... 72 Figure 2-3. Myeloproliferative disease in the majority of CBL-B(-/-) BCR-ABL BMT recipients was confirmed by the peripheral blood differential and histopathology...... 74 Figure 2-4. CBL-B(-/-) MPD animals display enhanced Gr-1+ and Mac-1+ percentages in the spleen. .. 79 Figure 2-5. Recipients of BCR-ABL-expressing wild-type or CBL-B knock-out cells show similar protein tyrosine phosphorylation and activation profiles...... 81 Figure 2-6. CBL-B(-/-) cells do not effectively home to the BM...... 85 Figure 2-7. BCR-ABL-induced disease is oligoclonal...... 87

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Figure 2-8. CBL-B(-/-) BCR-ABL BMT recipients have poor homing to the BM but enhanced granulocyte infiltration into the lungs...... 91

Figure 3-1. CBL-B deletion in mouse embryonic fibroblasts does not alter cell growth...... 101 Figure 3-2. CBL-B expression modifies wound closure rates...... 103 Figure 3-4. CBL-B expression reduces Vav3 phosphorylation and total Vav3 protein levels...... 108 Figure 3-5. Rac activity is unaffected by CBL-B expression...... 110 Figure 3-6. CBL-B expression does not alter phosphorylation of PAK1/2/3, p38-MAPK or SAPK...... 111 Figure 3-7. CBL-B deficiency leads to enhanced phosphorylation of ERK1/2 and MEK1/2 proteins. .. 113 Figure 3-8. Activation of the PI3’K-PKB pathway is impaired by CBL-B expression, while inhibition of PI3’K activity perturbs MEF chemotaxis...... 114 Figure 3-9. CBL-B negatively regulates GAB2 phosphorylation...... 117 Figure 3-10. A proposed mechanism for the CBL-B-mediated regulation of serum-dependent migration...... 122

Figure 4-1. Comparison of the colony formation ability of CBL-B WT and KO cells after BCR-ABL infection of BMCs...... 126 Figure 4-2. CBL-B co-immunoprecipitates tyrosine-phosphorylated proteins of 55, 60 and 210 kDa in BCR-ABL-expressing cells...... 131 Figure 4-3. CBL-B-mediated ubiquitination of Vav may negatively regulate Rac activity and block neutrophil proliferation...... 133 Figure 4-4. Motility of wild-type and CBL-B(-/-) BMCs is enhanced by SDF-1a treatment...... 135 Figure 4-5. Comparison of the lymphoid transformation ability between CBL-B WT and KO BMCs after BCR-ABL infection...... 139 Figure 4-6. Schematic representation of the three human TEL/JAK2 fusion gene variants that have been cloned...... 142

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LIST OF ABBREVIATIONS

1o Primary 2o Secondary 4H Four-helix 5-FU 5-Fluorouracil ABL Abelson AGM Aorta-gonad-mesonephros ALL Acute lymphoblastic leukemia AML Acute myeloid leukemia APC Allophycocyanin ARE Adenylate uridylate-rich element ASO Antisense oligonucleotide ATP Adenosine Triphosphate AU Adenylate uridylate aUPD Acquired uniparental disomy B Basophil B-ALL B-cell acute lymphoblastic leukemia β-ME β-mercaptoethanol

Bcl-xL Basal cell lymphoma-extra large BCR breakpoint cluster region BFU-E Blast-forming unit-erythroid BM Bone marrow BMC Bone marrow cell BMT Bone marrow transduction and transplantation BSA Bovine serum albumin c-ABL Cellular Abelson C-C Coiled-coiled C/EBP CCAAT/enhancer binding protein CA Constitutively active CAM Cell adhesion media CAP CBL-associated protein CBF Core binding factor CBL Casitas B-lineage lymphoma CDC42 Cell division cycle 42 protein

CFU Colony-forming unit CFU-E Colony-forming unit-erythroid CIN85 CBL-interacting protein of 85 kDa CLP Common lymphoid progenitor CML Chronic myeloid leukemia CMML Chronic myelomonocytic leukemia CMP Common myeloid progenitor

CO2 Carbon dioxide COOH C-terminus Cool (Pix) Cloned out of library CSF Colony-stimulating factor CXCL12 (SDF-1α) CXC chemokine ligand 12 CXCR4 CXC chemokine receptor 4 ddH2O Double-distilled water DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl sulfoxide DN Dominant negative DNA Deoxyribonucleic acid DTT Dithiothreitol EAE Experimental autoimmune encephalomyelitis EDTA Ethylene diamine tetraacetic acid EGFR Epidermal growth factor receptor ELISA Enzyme-linked immunosorbent assay EMH Extramedullary hematopoiesis

Eo Eosinophil EPO Erythropoietin ERK Extracellular signal-regulated kinase FACS Fluorescence activated cell sorting FAK Focal adhesion kinase FAP Focal adhesion protein FCS Fetal calf serum FDCP-1 Factor-dependent cell progenitor -1 FL Flt3 ligand FSC Forward scatter FTI Farnesyl transferase inhibitor

x g Gram G-CSF Granulocyte colony-stimulating factor GAB2 GRB2-associated binding protein 2 GAP Guanosine triphosphatase-activating protein GDP Guanosine diphosphate GEF Guanine nucleotide exchange factor GFP Green fluorescent protein GM-CSF Granulocyte-macrophage colony-stimulating factor GMP Granulocyte and macrophage progenitor GRB2 Growth factor receptor-bound protein 2 GST Glutathione S transferase GTP Guanosine triphosphate GTPase Guanosine triphosphatase Gy Gray H&E Hematoxylin and eosin HECT Homologous to E6-AP carboxyl terminus HLA Human leukocyte antigen HNRPK Heterogeneous nuclear ribonucleoprotein K HRP Horseradish peroxidase HSC Hematopoietic stem cell HSC/P Hematopoietic stem cells and progenitors HSCT Hematopoietic stem cell transplantation hSpry2 Human Sprouty 2 IB Immunoblotting ICAM-1 Inter-cellular adhesion molecule-1 ICSBP Interferon Consensus Sequence Binding Protein ID Immunodepletion IF Intrafemoral IFN-α Interferon-alpha IL Interleukin IMDM Iscove’s modified Dulbecco’s media IP Immmunoprecipitation IRES Internal ribosomal entry site IV Intravenous(ly) JAK Janus kinase

JNK Jun N-terminal kinase K Lysine kDa KiloDalton KO Knock-out KSL c-kit+/ Sca-1+/Lin- LFA-1 Leukocyte factor antigen-1 Lin Lineage LRP1 Lipoprotein receptor-related protein 1 LT-HSC Long-term hematopoietic stem cell LTR Long terminal repeat LY LY294002 LZ Leucine zipper M Monocyte MAPK Mitogen-activated protein kinase MDM2 Murine double mutant 2 MEF Mouse embryonic fibroblast MEK MAPK/ERK kinase MEP Megakaryocyte and erythrocyte progenitor mg Milligram MGM MEF generation medium mL Millilitre MMM MEF maintenance medium MPD Myeloproliferative disease MPP Multi-potential progenitor MSCV Murine stem cell virus mTOR Mammalian target of rapamycin

Na3VO4 Sodium orthovanadate

Na4P2O7 Sodium pyrophosphate NaF Sodium fluoride NES Nuclear export signal NF-κΒ Nuclear factor of kappa light chain enhancer in B cells

NH2 N-terminus NLS Nuclear localization signals OB Osteoblast OC Osteoclast

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OCI Ontario Cancer Institute P-loop Phosphate-binding loop PAK p21-activated kinase PB Peripheral blood PBD Protein-binding domain PBS Phosphate-buffered saline PCR Polymerase chain reaction PE Phycoerythrin Ph Philadelphia chromosome PH Pleckstrin homology PI Propidium iodide PI3'K Phosphatidyl-inositol 3’ kinase

PIP2 Phosphatidyl-inositol-4,5-diphosphate

PIP3 Phosphatidyl-inositol-3,4,5-triphosphate Pix (Cool) PAK-interactive exchange factor PKB (AKT) Protein kinase B PLCγ Phospholipase C gamma PLGF Platelet-derived growth factor PMSF Phenylmethyl sulfonyl fluoride PTK Protein tyrosine kinase PTP1B Protein tyrosine phosphatase 1B pTyr Phospho-tyrosine PVDF polyvinylidene fluoride Q-RT-PCR Quantitative reverse-transcriptase polymerase chain reaction R Arginine RAC Ras-related C3 botulinum toxin substrate RBC Red blood cell RING Really interesting new gene rm Recombinant murine RNA Ribonucleic acid RNAi Ribonucleic acid interference RT-PCR Reverse-transcriptase polymerase chain reaction SAPK Stress-activated protein kinase Sca-1 Stem cell antigen-1 SCF Skp1/Cullin/F box

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SCF Stem cell factor SCID Severe combined immuno-deficient SDF-1α (CXCL12) Stromal cell-derived growth factor 1 alpha SDS Sodium dodecyl sulfate SDS-PAGE SDS – polyacrylamide gel electrophoresis SH Src homology SHP1 SH2-containing phosphatase 1 SHP2 (PTPN11) SH2-containing phosphatase 2 shRNA Short hairpin ribonucleic acid siRNA Small interfering RNA SLAM Signaling lymphocyte activation molecule Sli-1 Suppressor of lineage defect 1 SNP Single nucleotide polymorphism SOS Son of Sevenless Spi1 (PU.1) Spleen focus forming proviral integration oncogene SSC Side scatter SSM Serum starvation medium ST-HSC Short-term hematopoietic stem cell STAT Signal transducer and activator of transcription TBS Tris-buffered saline TBST Tris-buffered saline with Tween-20 TCR T cell receptor TF Transcription factor TK Tyrosine kinase TKB Tyrosine kinase-binding TKI Tyrosine kinase inhibitor TLVM Time-lapse video microscopy TPO Thrombopoietin Ub Ubiquitin UBA Ubiquitin-associated UbcH7 Ubiquitin-conjugating enzyme H7 UTR Untranslated region VSV-G Vesicular stomatitis virus G WBC White blood cell WT Wild-type

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Y Tyrosine

LIST OF APPENDICES

APPENDIX 1. BCR-ABL FAILS TO INDUCE STABLE TRANSFORMATION OF MOUSE EMBRYONIC FIBROBLASTS...... 190 A.1. Abstract...... 191 A.2. Introduction...... 192 A.3. Materials and Methods...... 193 A.4. Results...... 194 A.5. Discussion...... 202 A.6. References...... 204

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CHAPTER 1

INTRODUCTION

1 2 1.1. Hematopoiesis

1.1.1. Hematopoiesis during ontogenesis Hematopoiesis is the formation and development of blood cells. During human ontogenesis, hematopoiesis begins in the yolk sac where yolk-sac stem cells differentiate into primitive erythroid cells.1,2 In the early stages of gestation, an intra-embryonic region called the aorta-gonad-mesonephros (AGM) also emerges as a major site of hematopoietic stem cell (HSC) generation. Around weeks five to six, HSC begin to enter the circulation and migrate from the yolk sac or AGM to the fetal liver (and other hematopoietic organs like the spleen and thymus). Hematopoiesis continues in the liver until approximately seven months of gestation at which time the stem cells begin to migrate to the bone marrow. By birth, the bone marrow is the major site of hematopoiesis. In mice, embryonic hematopoiesis proceeds in a similar manner, but with a shorter time scale and a smaller contribution from the bone marrow.3 HSCs accumulate in the yolk sac and the AGM until around embryogenesis day 11 (E11) when the HSCs migrate to the fetal liver. This organ is the major site of hematopoiesis in mouse embryos and contributes to hematopoiesis until two weeks post- parturition. Not until E15-E16 do HSC numbers decrease in the fetal liver, possibly due to seeding of the spleen and bone marrow. Only at birth does the bone marrow become the major site of murine adult hematopoiesis.

1.1.2. Adult hematopoiesis In mammals, adult hematopoiesis is carried out in the bone marrow microenvironment, composed of two spatially distinct regions called the endosteal and vascular niches (Figure 1-1). The localization of HSCs within these compartments influences the commitment of these cells toward quiescence, proliferation or differentiation. The endosteum contains bone-forming osteoblasts, bone- resorbing osteoclasts and the nearby stromal cells. Hematopoietic stem cells, and other primitive hematopoietic cells, lie close to the osteoblasts that line the endosteal surface,4 while more committed progenitors that develop from the HSCs, display a looser connection with the osteoblasts.5 Quiescent HSCs are primarily found within this region of the bone marrow.6 In contrast, hematopoietic cell proliferation, differentiation and maturation are proposed to occur in the region surrounding the vascular endothelial cells.7,8 From this location, the mature cells can easily egress through the sinusoidal wall into the periphery where they carry out their inherent functions.

1.1.3. Differentiation of the HSC into mature blood cells The initial building block of hematopoiesis is the HSC. This cell can either self-renew or differentiate into all of the cellular blood elements, giving it the designation of a pluripotent cell. Normally, homeostatic levels of stem cells are maintained within the bone marrow. However, HSC expansion and

Figure 1-1. Hematopoietic stem cells reside in the bone marrow. Quiescent stem cells lie within the endosteal niche in close proximity to osteoblasts (OBs), osteoclasts (OCs) and stromal cells that line the bone surface. When HSC proliferate or differentiate, they migrate to the vascular niche. Mature hematopoietic cells enter the sinusoidal vessel through spaces between endothelial cells, and move from this location to populate the periphery. Adapted from ref. (9).

4 differentiation may occur when the local concentration of growth factors is altered. For example, patients undergoing HSC transplantation (HSCT) are usually exposed to a myelo-ablative treatment (like irradiation or chemotherapy) that kills differentiated cells, followed by transplantation with HSCs that home to the bone marrow. The myelo-ablation enhances production of growth factors that promote expansion of HSCs and reconstitution of the hematopoietic system.9 HSCT contributes many more HSCs than are normally found in the bone marrow – usually only 1 in 104 cells bears this classification. Therefore, in healthy individuals with a constant turnover of differentiated cells, the mechanism regulating HSC cell division and differentiation must be more complex than a single cell division producing two daughter cells. Indeed, eight key cell types are formed from a single HSC, highlighting the necessity for a complex, branching network of hematopoiesis. Hematopoietic stem cells initiate this multi-armed hematopoietic pathway through the primary generation of two cell types that form the lymphoid and myeloid compartments (Figure 1-2).10,11 The direct downstream lymphoid progenitors that develop from a stem cell are called common lymphoid progenitors (CLPs). The CLP matures into either pre-B or pre-T cells. In a series of further steps, the pre-B cells terminally differentiate into B cells in the bone marrow. Meanwhile, immature T cells migrate to the thymus where they are referred to as thymocytes. These thymocytes undergo a series of maturation and selection events before release into the bloodstream as T cells. B and T cells are also referred to as lymphocytes, and in general, these cells perform specialized roles in acquired immunity. The entire production of cells in the lymphoid arm is termed lymphopoiesis. Many more fully differentiated cell types with distinct functional roles are created during myeloid hematopoiesis (called myelopoiesis). Firstly, a common myeloid progenitor (CMP) is formed from the hematopoietic stem cell. The CMP undergoes a further bifurcated division into either progenitors of the erythroid and megakaryocytic lineages (called MEPs) or granulocyte and macrophage progenitors (GMPs). The production of fully differentiated erythroid cells, i.e., red blood cells (RBCs) or erythrocytes, is called erythropoiesis. For an MEP to become an RBC, a blast-forming unit-erythroid (BFU-E) cell first matures from the MEP. The BFU-E becomes a colony-forming unit-erythroid (CFU- E) cell that divides through multiple immature erythroid divisions in which the cells are termed erythroblasts. In the final stages of erythroid development reticulocytes are formed following nuclei extrusion. These cells are released into the periphery where they mature into functional RBCs with the full complement of hemoglobin for transport of oxygen. The MEP is also the basic cell from which megakaryocytes are formed during thrombocytopoiesis. Megakaryocytes function to produce platelets for blood clotting. The granulocyte and macrophage progenitor can mature into four functionally- and morphologically- distinct cell types through lineage-specific CFUs and blast stages. In one pathway, monocytes are formed and exit the bone marrow to circulate in the blood. When monocytes enter into tissues they mature into macrophages - their purpose being to phagocytose exogenous particles, digest

Figure 1-2. Differentiation of the HSC into all of the hematopoietic lineages. The HSC forms common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs). The CLP forms both the B cell and T cell lineages through multiple differentiation steps that include blast stages (not depicted, but represented by the blue shading). The CMP differentiates into macrophage/erythroid progenitor (MEP) cells and granuloocyte/macrophage progenitors (GMPs). Through multiple blast forming unit (BFU) and colony-forming unit (CFU) stages, the MEP forms erythrocytes and megakaryocytes (Meg). Likewise, the GMP further differentiates through multiple CFU and blasts stages into the basophil (B), neutrophil, and eosinophil (Eo) granulocytes; and the monocytes (M).

6 and present antigens to T cells, and secrete cytotoxic and inflammatory-activating cytokines. The granulocytes - basophils, eosinophils and neutrophils - are also produced from the GMP. The basophils are generated in response to allergens, while eosinophils and neutrophils both function as phagocytes. Neutrophils are highly motile and will migrate in response to chemotactic factors to sites of inflammation. This brief introduction into the multiple cell types formed during hematopoiesis emphasizes the complexity of the system, and reveals the importance of growth factors that must regulate the differentiation decisions.

1.1.4. Hematopoietic Growth Factors The direction of HSC differentiation, as well as hematopoietic cell survival, proliferation, maturation and functional activation, are tightly controlled through the action of a family of small glycoproteins called cytokines that bind to their cognate receptors on hematopoietic cell surfaces. Two types of cytokines are important for hematopoiesis: the colony-stimulating factors (CSFs) and the interleukins (ILs). (Chemokines also belong to this family but are important for chemotactic migration and so will be discussed in more detail in Section 1.2.4.) Colony-stimulating factors were so named for their ability, in early hematopoietic studies, to produce distinct colony types when bone marrow cells were cultured in semi-solid agar. The name interleukin refers to the ability of specific cytokines to communicate (inter-) with white blood cells (-leukin for leukocyte). Cytokines interact with multiple cell surface receptors, are produced in many tissues and organs, and exist in variable structural forms. Some of the important hematopoietic cytokines and their relative functions are included in Table 1-1. While it was originally hypothesized that specific cytokines would be identified for each hematopoietic lineage, it later became clear that some cytokines have overlapping functions. For example, IL-3 is important for the survival and differentiation of all myeloid lineages. In contrast, thrombopoietin (TPO) is specific to megakaryocyte maturation. Cytokine production occurs in multiple organs: stem cell factor (SCF) is generated by bone marrow stromal cells while erythropoietin (EPO) is produced by the kidneys and TPO is made in the liver. Cytokines like EPO exclusively made in a secreted form, but SCF exists as both a soluble growth factor, and as a membrane-bound cytokine produced through alternative splicing and the absence of proteolytic cleaveage.12,13 These descriptions of function, localization, and form, highlight the complexity of the cytokines that control the proliferation and maturation of the hematopoietic cell types.

1.1.5. Methods to identify hematopoietic cells In early studies, hematopoietic cell types were distinguished on the basis of the colony type formed from a progenitor (in colony formation assays) or the histological phenotype after cell staining

7 Table 1-1. Hematopoietic cytokines.

Cytokine Receptor Function in hematopoiesis (ligand)

Also known as Steel factor or KIT ligand; stimulates the Stem cell factor (SCF) c-kit survival, proliferation and differentiation of stem cells and early hematopoietic progenitors.14

Granulocyte/Macrophage Stimulates granulocytic and/or macrophage survival and GM-CSFR CSF* (GM-CSF) differentiation.15

Stimulates the proliferation of HSCsφ and uncommitted Flt3 ligand (FL) Flt3 progenitors.16

Also called multi-CSF; IL-3 is involved in growth, Interleukin-3 (IL-3) IL-3R proliferation and survival of hematopoietic progenitors and the differentiation of myeloid cells.17

Granulocyte-CSF G-CSFR Induces granulocyte generation.18

Erythropoietin (EPO) EPO-R Promotes erythroid differentiation.19

Also termed CSF-1; stimulates macrophage colony Macrophage-CSF M-CSF formation.20,21

IL-5 IL-5R Induces eosinophil production.22

IL-11 IL-11R Promotes megakaryopoiesis.23

Enhances proliferation and maturation of the Thrombopoietin (TPO) Mpl megakaryocyte lineage,24,25 and promotes survival of HSCs.26

Also called T cell proliferation factor (TCPF); required for IL-2 IL-2R T cell proliferation.27

Influences the differentiation of predominantly B cells, but IL-6 IL-6R also T cells, macrophages and megakaryocytes.28

Important for B cell maturation and T lymphocyte IL-7 IL-7R growth.29,30

* Colony stimulating factor φ Hematopoietic stem cell

8 with dyes. Unfortunately, neither one of these techniques was useful for the analysis of live progenitors: in the first technique the “starting material” was depleted; and in the second, the dyes were cytotoxic. To circumvent this problem, a technique was developed by which fluorescently tagged antibodies to cell- surface antigens or receptors were used to specifically label cell populations that were then identified with fluorescence-activated cell sorting (FACS) machinery. Cell surface markers have been generated for each of the major hematopoietic lineages. For example, in mice, granulocytes can be detected with the Gr-1 and Mac-1 antibodies. Likewise, erythroid cells bear the Ter-119 marker, B cells are B220-positive (B220+), and T cells can be detected with a Thy- 1.2 antibody. Lineage-specific markers have been combined in a lineage (Lin) cocktail that is useful for the enrichment of hematopoietic stem cells. As many of the HSC cell-surface markers are present on progenitor cells, these FACS markers are often used in combination. Human HSCs are commonly detected as CD34+, CD38- and Lin-. The typical cell surface phenotype of a murine HSC is stem cell antigen-1-positive (Sca-1+), c-kit+, and Lin-.31,32 As such, these mouse HSCs are often referred to as KSL cells. Several other HSC markers have been identified, including the receptor tyrosine kinase Flk-2 and Thy1.1.31,33 These markers help to distinguish HSCs with long-term, multilineage repopulating capacity (LT-HSC) from those with short-term repopulating capability (ST-HSC) (Figure 1-3). Some additional markers of HSCs include: a combination of the signaling lymphocyte activation molecules (SLAMs),34 rhodamine and Hoechst dyes,35 and CD133.36 Before the advent of FACS technology, researchers wishing to isolate mouse HSCs from the bone marrow discovered a “quick-and-dirty” method of purification. They utilized the knowledge that HSCs divide infrequently, only around every 30-60 days.37,38 By administering a classic chemotherapeutic drug called 5-fluorouracil (5-FU), that specifically targets actively dividing cells and initiates their apoptosis, scientists were able to enrich for quiescent HSCs in murine bone marrow.39 This technique is still employed today.

1.1.6. Summary In review, hematopoiesis describes HSC self-renewal and differentiation into lymphoid and myeloid cells. During morphogenesis, hematopoiesis begins in the yolk sac and AGM, but eventually moves to the bone marrow at birth, where differentiation selectively occurs in the vascular niche of the bone marrow. Scientists can detect and sort different hematopoietic cell types through antibodies to cell- surface markers and FACS technology. The precise binding of cytokines to their cognate receptors maintains homeostatic hematopoiesis in healthy individuals.

Figure 1-3. Cell surface markers of hematopoietic stem cells. Early hematopoieitic progenitors can be divided into long-term (LT-HSC) and short-term (ST-HSC) hematopoieitic cells and multi-potential progenitors (MPPs) based on the combinatorial expression of five cell surface markers: Lin, c-kit, Sca-1, Thy1.1 and Flk-2. Adapted from ref. (40).

10 1.2. Chronic Myeloid Leukemia Steady-state levels of each hematopoietic cell lineage are maintained in normal, healthy individuals. This balance is upset during times of stress or illness. Uncontrolled hematopoiesis due to cancer, visualized in the blood or bone marrow as cellular expansion, is called leukemia. This disease exists in several forms depending upon the hematopoietic lineage affected and the ability of leukemic cells to mature into fully differentiated cell types. Chronic myeloid leukemia (CML) is characterized by the expansion of the myeloid lineage, with normal maturation of these cells.41 The median age-of-onset for CML is 53 years and patients present with symptoms of weight loss, malaise and splenomegaly. Peripheral blood analysis shows an elevated white blood cell (WBC) count and an increase in mature and immature myeloid cells, as well as erythroid forms and platelets. Without treatment, CML inevitably progresses over a period of three to five years in an accelerated phase toward blast crisis. In CML blast crisis the leukemic cells fail to mature, and greater than 20% of the bone marrow cells (BMCs) are blasts.42 The prognosis for these blast crisis patients is very poor, and most succumb to their leukemia within a few months. While the progression of this disease involves the acquisition of additional genetic lesions,43 a single chromosomal abnormality is associated with the chronic phase of CML.

1.2.1. BCR-ABL is the causative agent of CML A minute chromosome, termed the Philadelphia chromosome (Ph) for the city in which it was discovered, is commonly identified in the leukemic cells of CML patients.44 This chromosomal abnormality results from a reciprocal translocation between chromosomes 9 and 22,45 and generates a novel BCR-ABL fusion gene through exchange of breakpoint cluster region (bcr) sequences on chromosome 22 with those of the c-abl gene on chromosome 9.46,47 The specific DNA breakpoints in the t(9;22) translocation have been characterized. Three prominent variations exist that encode proteins of differing molecular weights. The translocation breakpoint most frequently associated with CML shows fusion of sequences just upstream of abl exon a2, with either exon 13 or exon 14 of the major breakpoint cluster region of the bcr gene (M-bcr; Figure 1-4). The resulting e13a2 and e14a2 translocations are transcribed and translated into proteins of 210 kiloDaltons (kDa) in size.48,49 Also observed are fusions of the same abl region with the minor bcr region (m-bcr) at bcr exon 1.50,51 The resulting protein from this e1a2 fusion has a molecular weight of 185-190 kDa (referred to for the remainder of this thesis as the 190-kDa protein).52-54 A third translocation variant between bcr exon 19 (in the m-bcr region) and abl creates an e19a2 translocation55 that yields a protein of 230 kDa.56 Each of these BCR-ABL proteins –referred to as p190, p210 and p230 – is associated with a particular leukemia.58 The p190 protein is found in two-thirds of patients with Ph-positive acute lymphoblastic leukemias (ALLs) and in rare cases of CML. The p230 isoform is associated with chronic

Figure 1-4. A chromosomal translocation results in the BCR-ABL fusion. Breakpoints occurring within abl and bcr sequences produce four common variants: e1a2 that creates the p190 protein; e13a2 or e14a2 that gives a 210 kDa fusion protein; and the p230 protein, formed from an e19a2 translocation. Adapted from ref. (57).

12 neutrophilic leukemia,59 a more benign disease than CML. While the p210 isoform is observed in one- third of Ph-positive ALLs, it is most prominently associated with CML disease, more so than the other two BCR-ABL fusions. For this reason, the p210 form has been used in the majority of all further investigations into the functional consequence of BCR-ABL expression and the mechanism of action, as well as in rational drug design. The prevalence of the t(9;22) translocations in these diseases led to the hypothesis that BCR- ABL was directly responsible for cellular transformation and leukemogenesis. Cellular transformation is characterized in adherent cell lines as the loss of adhesion dependence (i.e., the formation of colonies in semi-solid culture media) and the loss of contact-inhibited growth. BCR-ABL transforms Rat-1 fibroblasts and a small subset of the murine NIH 3T3 fibroblast population.60-62 In suspension cell cultures, transformed cells proliferate in the absence of growth factors and may form foci in liquid culture. BCR-ABL also relieves the growth factor dependence of many hematopoietic cell lines including IL-3-dependent Ba/F3 cells.63 These in vitro studies showed that BCR-ABL is capable of inducing cellular transformation. BCR-ABL was also verified as the causative agent of leukemogenesis, through animal modeling studies: Mice transplanted with BCR-ABL(p210)-transduced BMCs succumbed, after a short latency, to a myeloproliferative disease (MPD) that resembled human CML.64,65 Based on these conclusive observations, the causative agent of CML was verified as BCR-ABL.

1.2.2. Molecular mechanisms of BCR-ABL-mediated transformation The human bcr and abl genes, that are juxtaposed in the bcr-abl fusion, encode proteins with functional roles in normal cells. The BCR protein has a mass of approximately 160 kDa and is primarily found primarily in the cytoplasm.66-68 Its domain structure consists of an N-terminal coiled-coiled (C-C) domain, a dbl-like sequence and pleckstrin homology (PH) domain, and a RAC-GAP domain at the C- terminus (Figure 1-5). The C-C domain allows for dimerization of the protein.69 The dbl-like and PH domains are associated with guanine nucleotide exchange factor (GEF) activity for Rho proteins,70 and the RAC-GAP domain encodes a guanosine triphosphatase-activating protein (GAP) for the small guanosine triphosphate (GTP)-binding protein, p21-RAC.71 The ABL protein is 145 kDa in size,72 and is derived from c-abl – the cellular homologue of the Abelson murine leukemia virus. Two ABL isoforms exist – Type 1a and Type 1b – generated through 73,74 alternative splicing of the first exon. The Type 1b isoform contains a sequence at the NH2 terminus for myristoylation.75 Near the N-terminus, ABL also contains a protein tyrosine kinase (TK) domain,72,76 as well as Src homology -2 (SH2) and SH3 domains for protein-protein interactions.75,77-81 The central portion of the protein contains many proline-rich sequences for binding SH3-containing proteins.82 Closer to the C-terminus, ABL has three nuclear localization signals (NLS),83,84 and sequences that confer DNA-binding property on the protein.85,86 Located at the extreme 3’end of the protein are binding

Figure 1-5. Domain structure of BCR-ABL.

The BCR and ABL protein structures consist of several domains. BCR contains an N-terminal (NH2) coiled-coil (CC) domain, an interior dbl-like sequence and pleckstrin homology (PH) domain, and a RAC-GAP domain at the C-terminus (COOH). ABL contains N-terminal SH3, SH2 and tyrosine kinase (TK) domains. Further to the C-terminus are several proline-rich sequences (P) followed by three nuclear localization signals (NLS), three DNA-binding domains (DNA), G- and F-actin binding domains and a putative nuclear export signal (NES). In the p210 BCR-ABL fusion protein (formed from the breakpoints indicated by the arrows) all ABL domains are retained but the BCR RAC-GAP domain is lost. Adapted from ref. (57).

14 domains for F-actin and G-actin,87,88 as well as a putative nuclear export signal (NES).89,90 Due to these C-terminal localization signals and binding domains for cytoplasmic and nuclear structures, the ABL protein can be found both in the cytoplasm and in the nucleus. In response to DNA damage, ABL functions as a mediator of cell cycle arrest,91-93 apoptosis,94,95 and DNA repair.96 As well, ABL functions as an intermediate messenger in integrin signaling.97,98 The kinase activity of c-ABL is tightly controlled in normal cells. It was originally thought that the SH3 domain regulated ABL activity through the recruitment of inhibitory proteins.99-101 It is now believed that a cis-acting mechanism evokes an auto-inhibited conformational state (Figure 1-6). The SH3102 and SH2103 domains pack against ABL to hold it in an auto-inhibited state, while the N-terminal “cap” region with the associated myristoylated sequence forms an intramolecular regulatory engagement with the kinase domain of ABL.104,105 Based on this regulation, ABL is likely either activated by phosphorylation of residues within the catalytic domain and the release of intramolecular SH3-SH2 interactions106 or association of tyrosine phosphorylated ligands with the SH2 domain and displacement of the inhibitory myristoylation clamp.105 Moreover, Hantschel et al. suggest that myristate disruption by a fatty acid protein could disrupt the regulation by the SH2 domain.105 In any case, ABL activity is disrupted by fusion to BCR.107 The coiled-coil sequence of BCR allows for oligomerization of BCR-ABL proteins (Figure 1-7), leading to intermolecular autophosphorylation and constitutive tyrosine kinase activity.108 The BCR-ABL fusion contains elevated tyrosine kinase activity compared to c-ABL.72,109,110 As well, BCR-ABL is excluded from the nucleus and is entirely cytoplasmic.111 These combined features of strong kinase activity and cytoplasmic localization allow for the stimulation of a myriad of signaling pathways that mediate the oncogenic capacity of BCR-ABL. In a review by Van Etten,112 he suggested that “it is not an exaggeration to say that nearly every known cell signaling pathway has been shown to be affected by BCR-ABL in one publication or another.” As a thorough review of each of these pathways is beyond the scope of this introduction, the major mechanisms of BCR-ABL-mediated transformation will be discussed, with an emphasis on those pathways that intersect with the Casitas B-lineage lymphoma (CBL) family of proteins. BCR-ABL signal transduction begins with autophosphorylation. The initial phosphorylation event is proposed to occur at tyrosine 1294 (Y1294) in the activation loop of the ABL catalytic domain, followed by secondary phosphorylations at multiple tyrosine residues including Y177 (Figure 1-7).108 This tyrosine is highly phosphorylated on BCR-ABL and is the site of a high affinity association with the SH2 domain of growth factor receptor-bound protein 2 (GRB2; Figure 1-8).112-114 Some evidence suggests that association of the GRB2 adaptor protein at Y177 propagates a signal to Ras,113 most likely through binding with the son of Sevenless (SOS) guanine nucleotide exchange factor, either directly or indirectly through Shc.114 Moreover, GRB2 can recruit GRB2-associated binding protein 2 (GAB2) to BCR-ABL.115 Therefore, signals emanating from BCR-ABL can also be transferred through GAB2 and

Figure 1-6. Autoinhibition of ABL. ABL activity is inhibited by a “sandwich” of the SH3 and SH2 domains, as well as the N-terminal “cap” and myristate (blue) association with the SH2- and catalytic (Cat.)- domain, respectively. ABL is activated following phosphorylation of tyrosine 412 and 245 or ligand binding to the SH2 and SH3 domains and subsequent myristate release. Adapted from ref. (105).

16 Figure 1-7. Oligomerization as a means of BCR-ABL activation. This figure depicts only the Bcr sequences and the SH3, SH2 and TK domains of the ABL kinase. The monomeric BCR-ABL is structurally defined by interactions between the SH3 domain and the linker region between the SH2 domain and catalytic domains. When two monomers oligomerize at the coiled- coil (C-C) domain of BCR, a primary phosphorylation event occurs (in green) at Y1294. A secondary phosphorylation follows at Y1127, creating an active conformation of the kinase that allows for tertiary phosphorylations at residues like Y177. Adapted from ref. (108).

Figure 1-7

Figure 1-8. BCR-ABL signaling mechanisms. A plethora of signaling proteins are activated downstream of BCR-ABL. Shown here are those proteins and pathways with important roles in BCR-ABL-mediated adhesion, migration, proliferation and survival. Phosphorylated Y177 binds GRB2, which co-ordinates signals along the ERK1/2 cascade or to PI3’K and PKB. One of the components of this pathway, Ras, is thought to control the activation of the transcription factor NF-κB, while ERK1/2 may control Myc activity. Additionally, a signaling complex which associates with ABL, and contains proteins like CrkL and CBL, can activate PI3’K signaling to PKB. STATs are likely directly recruited to BCR-ABL. Finally, CBL-B expression is down-regulated by BCR-ABL which may result in modulation of signals to Vav, the Rho family and proteins, and finally of SAPK, p38-MAPK, ERK1/2 and NF-κB.

19 SH2-containing phosphatase 2 (SHP2; also known as PTPN11) to Ras.116 Regardless of the mechanism of Ras activation, the most prominent signaling pathway stimulated by Ras is the mitogen-activated protein kinase (MAPK) cascade that involves downstream signaling to Raf followed by activation of MAPK kinases 1 and 2 (MEK1/2) and extracellular-regulated kinases 1 and 2 (ERK1/2). (This cascade is herein referred to as the ERK pathway.) In support of this mechanistic pathway, the kinase activity of each of these MAPK proteins is upregulated in BCR-ABL-expressing cells.117,118 Moreover, signals emanating from Y177 are important for transformation113,119: both GRB2 and Ras mutants impair transformation.120,121 In addition to the interactions that occur at Y177, a multitude of proteins form complexes with the ABL region of BCR-ABL. One of the most prominently tyrosine phoshorylated proteins in BCR- ABL-expressing cells is CrkL,122-124 which binds to the proline-rich region in ABL. CrkL can also interact with the fusion protein through CBL,125,126 which itself binds directly to the ABL-SH2 domain through a phosphorylated tyrosine or indirectly in a phosphotyrosine-independent association with GRB2.127,128 CBL and CrkL coordinate complex formation with proteins like p62-Dok (Dok-1) and paxillin,129,130 plus Crk and phosphatidyl-inositol 3’ kinase (PI3’K).125 The interaction of CBL with PI3’K is mediated through association with the p85 subunit of PI3’K: the N- and C-terminal SH2 domains plus the SH3 domain of p85-PI3’K can interact with CBL. This CBL/PI3’K interaction may stimulate PI3’K as its activity is increased 14-fold in CBL immunoprecipitates from BCR-ABL transformed cells.131 Active PI3’K propagates signals to downstream targets like protein kinase B (PKB; also known as AKT), which stimulates anti-apoptotic processes to promote BCR-ABL-mediated transformation.132 The importance of CBL in BCR-ABL signaling is demonstrated by reduced colony formation in the absence of CBL.133 CBL-B, a homolog of CBL, is also phosphorylated by BCR-ABL.134 CBL-B and BCR-ABL can co-immunoprecipitate, but do so in a fashion distinct from CBL, as neither PI3’K or CrkL are significantly associated with CBL-B. Therefore, to date, the mechanism of BCR-ABL-mediated regulation of CBL-B is undefined. In BCR-ABL-transformed cells, CBL-B binds to Vav.134 This protein interacts with BCR-ABL and can be activated by the kinase.135 Vav is a GEF for the Rho family, which includes cell division cycle 42 protein (CDC42), Ras-related C3 botulinum toxin substrate (Rac) and Rho itself.136-138 Through a plethora of effectors, Rho proteins activate MAPK cascades (ERK, p38-MAPK and stress-activated protein kinase [SAPK]/Jun N-terminal kinase [JNK]) plus PI3’K and nuclear factor of kappa light chain enhancer in B cells (NF-κB).139 Vav deficiency reduces bone marrow transformation.135 Additionally, Rac is required for growth factor-independent proliferation of BCR-ABL cells,140 and the activation of one of its potential downstream effectors, SAPK, is essential for transformation.141,142 Of interest, BCR- ABL down-modulates CBL-B expression,134 possibly to control its E3 ubiquitin ligase activity (as

20 detailed in Section 1.3) that may negatively regulate Vav signaling to Rho family proteins and their downstream effectors. The signal transducer and activator of transcription (STAT) proteins are also constitutively activated in BCR-ABL-expressing cells.143-147 STAT1, STAT3 and STAT5 are all targets of BCR-ABL. As BCR-ABL mutants with deletion of the SH3 and SH2 domains do not show constitutive activation of STAT5 it is postulated that STAT proteins are directly recruited to BCR-ABL whereupon they are 148 phosphorylated. The anti-apoptotic protein, basal cell lymphoma-extra large (Bcl-xL), may be a downstream target of STAT activation,149 although this pathway is controversial. In any case, STAT proteins are key contributors to BCR-ABL-mediated transformation.150 BCR-ABL expression also augments the protein levels of the transcription factor Myc.151 Recent evidence suggests that through ERK1/2 regulation, BCR-ABL increases the expression and activation of heterogeneous nuclear ribonucleoprotein K (HNRPK), which in turn enhances Myc expression at the level of translation.151 Myc activity is also essential for BCR-ABL-mediated transformation.152 The activity of another transcription factor, NF-κB, is enhanced by BCR-ABL.153 As noted above, NF-κB activation may occur downstream of Rho proteins,139 but most studies suggest that signals downstream of Ras regulate NF-κB.154,155 Similarly to Myc, NF-κB activity is necessary for transformation from BCR-ABL.155 This brief discussion highlights the complexity of BCR-ABL signaling in terms of the sheer magnitude of the signaling proteins involved. Additionally, while the pathways presented in this introduction were more-or-less described in exclusion, crosstalk exists between the cascades. For example, while GAB2 was described as an upstream element of the ERK pathway, this protein is a prominent activator of PI3’K signaling in multiple cell systems including those transformed by BCR- ABL.115 On the whole, BCR-ABL initiates multiple, interconnected signaling cascades that culminate in oncogenesis.

1.2.3. Biological mechanisms of BCR-ABL-mediated in vitro transformation The discussion above described signaling proteins that are phosphorylated and or activated by BCR-ABL. These signals can be grouped into three biological mechanisms that are implicated in in vitro cellular transformation and in vivo leukemia development. These mechanisms include (1) altered adhesion, (2) constitutive mitogenic signaling and (3) inhibited apoptosis (Figure 1-9). The following sections describe the results of cell culture studies that determined the signaling pathways and proteins involved in each mechanism. BMT data that revealed the important in vivo proteins in each mechanism are discussed in Section 1.2.5. Growth and survival of many cell types is dependent on cell adhesion to sub-stratum. In the bone marrow, the extracellular matrix of attachment is produced by stromal cells, which include

Figure 1-9. BCR-ABL stimulates three oncogenic processes. By altering the adhesive properties of cells, activating mitogenic pathways and inhibiting apoptosis, BCR-ABL induces in vitro cell transformation and in vivo leukemogenesis. Adapted from ref. (57).

22 adipocytes, endothelial cells, fibroblasts, and macrophages. In cell culture systems this environment is often mimicked with matrix proteins including fibronectin, collagen, vitronectin or laminin. Integrins are the primary cell surface proteins that link the extracellular matrix to the intracellular cytoskeleton – of which actin is a major component. Integrins signal through cytoskeletal proteins like focal adhesion kinase (FAK), tensin and paxillin at specialized cell surface structures called Focal Adhesions. “Inside- out” signaling through integrins and focal adhesion proteins (FAPs) controls the adhesion of cells to the extracellular matrix (Figure 1-10).

BCR-ABL modifies cell adhesion Multiple studies have investigated the possibility that BCR-ABL modifies cell adhesion as a means of initiating transformation. There is conflicting evidence as to whether BCR-ABL promotes or perturbs adhesion. Studies with primitive CML progenitors show diminished adhesion to fibronectin and stromal layers as compared to normal progenitors.156,157 However, BCR-ABL-expressing hematopoietic cell lines and CML progenitors display increased adhesion to fibronectin.158-160 These phenotypic differences may be due to the use of alternative cell types. In either case, it is evident that BCR-ABL alters cell adhesion. The exact signaling pathway responsible for the altered adhesion of BCR-ABL cells has not been identified, but probably involves a combination of signals relayed through BCR-ABL association with actin, modification of integrin expression, and activation of focal adhesion proteins. BCR-ABL uses its C-terminal actin-binding domain to localize with actin filaments in punctate aggregates.87,107,161 Deletion of the ABD reduces adhesion to fibronectin,162 abrogates the transformation of Rat-1 fibroblasts, and impairs the BCR-ABL-mediated IL-3-independence of Ba/F3 cells.87 These data suggest that the localization of BCR-ABL on actin filaments may facilitate kinase interaction with membrane receptors like integrins or with associated focal adhesion proteins (Figure 1-10).

β1-integrin is the primary cell surface protein involved in binding to fibronectin, and the protein expression of this integrin is upregulated by BCR-ABL.159,162,163 Expression of the α5 hetero-dimer chains is also enhanced by BCR-ABL, suggesting that α5β1 is the major integrin involved in BCR-ABL- mediated adhesion.

The mechanism of α5β1-integrin regulation has not been identified except that BCR-ABL phosphorylates several focal adhesion proteins including paxillin, vinculin, FAK, talin, and tensin.161 Additional signals may be relayed through the ERK cascade,165 but multiple lines of evidence suggest that the CBL and CrkL proteins play prominent roles (Figure 1-10). First, and as mentioned above, CBL and CrkL are prominent substrates of BCR-ABL,122,124,166,167 and show associations with p130CAS, FAK, paxillin and talin.166,168 Therefore, CBL and CrkL could potentially act as adaptor proteins in the coordination of FAP phosphorylation by BCR-ABL. Second, CrkL over-expression leads to enhanced

Figure 1-10. Potential mechanisms of BCR-ABL-mediated regulation of adhesion. Integrins link the extracellular matrix to the intracellular cytoskeleton and focal adhesion proteins (FAPs; e.g., talin, tensin, vinculin, paxillin and FAK) that reside at the cell surface. The primary integrin involved in BCR-ABL adhesion mechanisms is α5β1. β1-integrin ligation studies have identified the CBL and CrkL adaptors as important mediators of “outside-in” signaling. Likewise, these proteins (and those of the ERK pathway) may modulate signals from BCR-ABL to the FAPs and integrins. As well, BCR-ABL association with F-actin may effect “inside-out” signals that regulate adhesion processes. Adapted from ref. (164).

169 adhesion of cells on fibronectin. Third, CBL and CrkL are key mediators of β1-integrin signaling. 170 These two proteins form a complex after β1-integrin ligation, and CBL displays enhanced tyrosine 171,172 phosphorylation after (a) cell adhesion to fibronectin, or (b) β1-integrin ligation. CBL also forms interactions with Src and PI3’K after integrin ligation.172 If we assume that the same pathways are operating in both the “outside-in” and “inside-out” mechanisms, then CBL and CrkL would be likely candidates for adhesion signaling from BCR-ABL. Combined, these data imply that BCR-ABL regulates cell adhesion through modification of integrin expression or function through a pathway involving F- actin, FAPs, CrkL and CBL proteins.

BCR-ABL modifies cell migration Changes in cell adhesion may also regulate the motility of cells, because adhesion formation and de-adhesion are required at the leading and trailing ends, respectively, of a motile cell (Figure 1-11). In all cell systems tested in vitro – primary CML cells, CD34+ cells transduced with BCR-ABL, and hematopoietic and fibroblastic cell lines transformed by BCR-ABL – the spontaneous motility was enhanced when the oncogene was expressed.173-175 BCR-ABL cells also respond to chemotactic stimuli, including stromal cell-derived growth factor 1 alpha (SDF-1α), but generally with reduced migration.174,176,177 Most of the major pathways stimulated by BCR-ABL have been implicated in migration. Involvement of the ERK pathway has been demonstrated in experiments that show reduced spontaneous migration of BCR-ABL Y177F cell.115 Moreover, GAB2(-/-) BCR-ABL cells show impaired migration toward SDF-1α. For this reason, PKB signaling may also be required for BCR-ABL-dependent migration. The Rho family of proteins is also implicated in the motility of CML cells. The role of this family in in vivo migration toward SDF-1α has been extensively studied by Williams and colleagues (and will be detailed in Section 1.2.5). Ba/F3 BCR-ABL cells show Vav and RAC1 regulation of spontaneous motility and RhoA-dependent amoeboid movement.178 Support for the upstream regulation of Vav and RAC by CBL-B is provided by experiments that show reduced spontaneous motility of BCR- ABL-expressing hematopoietic cells that over-express CBL-B.134 These data indicate that CBL proteins modulate both BCR-ABL-dependent adhesion and migration processes. In summary, it is evident that the BCR-ABL kinase can transform cells through modulation of key signaling proteins that play roles in adhesion and migration.

BCR-ABL promotes mitogenesis Mitogenesis is a second biological mechanism that is activated by BCR-ABL in the process of cellular transformation. Inducible expression of BCR-ABL in quiescent cells (those in the Go phase) promotes entry into S phase.118 The fusion protein stimulates the cell cycle by maintaining cyclin

Figure 1-11. The physiological processes involved in migration of a cell across sub-stratum. In order for a cell to initiate forward movement a series of coordinated events must occur. When an extracellular cue is received, the actin filaments become polarized and lamellipodia or filopodia protrude from the leading edge. Cell-substratum contacts form at focal adhesion complexes. The process of locomotion is completed when rear adhesions detach and the cell body contracts. Adapted from ref. (179).

26 activation, even in the absence of serum or growth factors. This mechanism may account for the observation that BCR-ABL induces growth factor independence of cells. 63 However, BCR-ABL does not appear to operate at a level greater than normal mitogenic signaling as Ba/F3 BCR-ABL cells in IL- 3-supplemented media expand at an equivalent rate to the parental cells.63 Additionally, the presence of BCR-ABL in a cell population that does not automatically promote mitogenesis as BCR-ABL is detectable in the quiescent stem cell population of CML patients.180 The upstream elements that control cell cycle entry in BCR-ABL cells are similar to those operating in normal cells. As implied by their name, the ERK and SAPK mitogen-activated protein kinase pathways control proliferation in BCR-ABL cells. Growth is impaired in BCR-ABL expressing cells treated with chemical inhibitors of Ras,181 MEK,182 and RAC (Figure 1-12; Table 1-2)183; plus peptide inhibitors of GRB2.184 As well, the introduction of dominant negative (DN) proteins of GRB2,120 ERK,182 RAC,140 and the SAPK effector c-Jun,141 blocks cell proliferation and transformation mediated by BCR-ABL. Incubation of Ph+ cells with GRB2 antisense oligonucleotides (ASOs) also reduces proliferation.185 Thus, experiments with MAPK inhibitors, dominant-negatives and antisense oligonucleotides demonstrate the importance of these pathways in BCR-ABL-dependent mitogenesis. Some additional BCR-ABL-stimulated proliferative signals have been identified. STAT5 has pleiotropic physiologic functions, one of which is proliferation. Expression of DN STAT5 impairs proliferation of BCR-ABL cells.186 Experimental evidence also implicates the PKB pathway in the growth of BCR-ABL cells: ASOs to PI3’K block CML cell growth.187 As for adhesion, CBL proteins may play roles in BCR-ABL-mediated proliferation as both CBL and CBL-B are predicted to exist upstream of the MAPK and PKB signaling cascades. These data suggest that there are multiple pathways that trigger mitogenesis of BCR-ABL cells.

BCR-ABL protects cells from apoptosis Cellular proliferation not only depends on mitogenic signals, but also on the activation of pathways that protect BCR-ABL cells from apoptosis. Several groups have shown survival of BCR- ABL cells after growth factor withdrawal or treatment with genotoxic agents – conditions that would induce death in normal cells.188 The major pathways involved in impaired apoptosis include those downstream of Y177 in BCR-ABL, plus the PI3’K and STAT5 proteins (Figure 1-13). Evidence for Y177 anti-apoptotic signals in BCR-ABL cells is provided by data that shows impaired survival of Y177F mutants,189 induction of cell death by DN Ras,190 and decreased viability when either DN Raf or PI3’K inhibitors abolish the negative regulation of the pro-apoptotic protein Bad.191 BCR-ABL may also control cell survival through PKB-dependent phosphorylation of murine double mutant 2 (MDM2): BCR-ABL cells treated with ASOs to MDM2 show increased apoptosis and p53 inhibition with small interfering RNA (siRNA) reduces cell survival after drug treatment.192,193 Additionally, STAT5

Figure 1-12. Chemical inhibitors that target components of the BCR-ABL signaling pathway. Several common inhibitors are in use either in vitro or in the clinic. When multiple drugs are available for each cellular target, they have been indicated.

28 Table 1-2. Alternative names for chemical inhibitors of proteins in the BCR-ABL signaling pathway.

Generic Name Chemical Code Trade Name

Imatinib STI571 Gleevec

Nilotinib AMN107 Tasigna

Dasatinib MBS354825 Sprycel

Lonafarnib SCH66336

Tipifarnib R115777 Zarnestra

Figure 1-13. Signaling pathways proposed to play a role in the inhibition of apoptosis by BCR- ABL. Several groups have shown a role for signals downstream of GRB2 and Y177 in the negative regulation of the pro-apoptotic factor Bad. Alternatively, Bad may be regulated through the PI3’K/PKB pathway. PKB also stimulates MDM2, which may be a mechanism of controlling p53 activation. Signals relayed from BCR-ABL to either the PI3’K or ERK pathways may be transferred through CBL proteins. Finally,

STAT activity downstream of BCR-ABL may stimulate the Bcl-xL anti-apoptotic factor.

activation plays an important role in BCR-ABL-mediated survival as DN STAT5 reduces Bcl-xL protein expression and enhances the apoptotic fraction of cells.149,194 Again, CBL proteins may assist BCR-ABL in the control of these survival mechanisms due their upstream location in these signaling pathways. The experimental results described above implicate the mechanisms of altered adhesion, enhanced mitogenesis, and impaired apoptosis in in vitro cellular transformation. Continued experimentation in both cell lines and in murine models will determine which regulators are essential for BCR-ABL-mediated leukemogenesis.

1.2.4. Modeling CML in animals The work described above utilized in vitro cell systems or ex vivo CML samples for BCR-ABL- mediated transformation analyses. These models serve useful purposes in that the cell lines can be quickly propagated and display characteristic features of transformation (e.g., factor-independent growth and anchorage-independent growth). However, based on the fact that in vivo cellular interactions can influence disease development, the generation of leukemia is more accurately modeled in an animal- based system. As such, four methods have been designed that study the BCR-ABL oncogene either in the context of a manipulated murine genome or after injection of BCR-ABL-expressing cells into mice. These systems include tumor formation in nude (athymic) mice; plus transgenic, knock-in, and retroviral transduction models. Tumor formation in nude mice was used to test the ability of transformed cells to expand in an in vivo context. By this assay BCR-ABL was determined to be tumorigenic, as subcutaneous injection of BCR-ABL-expressing hematopoietic cells led to tumor formation in mice and eventual death in the animals.63 In an extension of this model; with the use of DNs, ASOs and super-repressor mutants; the Bcl-2,195 GRB2,120 NF-κB,155 and protein tyrosine phosphatase 1 B (PTP1B)196 substrates were identified as important regulators of BCR-ABL-mediated tumor formation in nude mice. Despite the useful knowledge gained through this model, a major caveat still existed – the fact that amplification of cells was occurring in a non-native environment, i.e., subcutaneous layers versus bone marrow. Therefore, investigators created additional models to study hematopoietic cells in their native bone marrow microenvironment. Three murine systems were investigated for their ability to accurately model BCR-ABL disease within the bone marrow. Several transgenic and knock-in models were generated utilizing the BCR- ABL p210 isoform but in most cases the resulting animals either succumbed to embryonic lethality or diseases other than CML197-200; only a single transgenic model of p210 driven by the tec promoter can induce CML in the progeny of founder animals and after a long latency of eight to 12 months.201 More consistently, the bone marrow transduction and transplantation (BMT) assay elicits an MPD that mimics

31 human CML,64,65 with a short latency of approximately four weeks.202,203 The BMT experimental approach is detailed below (and in Figure 1-14). In order to generate CML, the transduction conditions must be carefully controlled. BCR-ABL must infect HSCs – otherwise, alternative diseases will develop.204 To enrich for a BMC population with a major HSC component, two methods have been employed; some groups perform FACS sorting of HSCs but significant HSC enrichment is more easily performed with 5-FU intraperitoneal injection four to six day prior to BMC isolation. The proper infection of 5-FU-enriched cells yields CML from all isoforms of BCR-ABL, while non-5-FU treated donor marrow induces a mixture of CML, B cell ALL (B-ALL) and macrophage disease.205 Harvested BMCs are transduced with an appropriate vector in a cytokine cocktail that maintains HSC survival. The vector utilized by most groups is a derivative of the murine stem cell virus (MSCV) in which expression of the internal sequences is driven by long terminal repeat (LTR) promoters.202 This vector was originally designed to transduce embryonic stem cells,206 but also works effectively for the transduction of HSCs.202,207 The cytokine cocktail includes IL-3, IL-6 and SCF.202,208 Before transduction, the cells are “pre-stimulated” overnight in this cocktail to induce entry into cell cycle209; cycling is required for incorporation of the retroviral DNA into the genomic DNA and efficient transduction.210,211 Furthermore, most groups use two rounds of infections to enhance the number of infected cells. A combination of these techniques should give successful infection of HSCs. The vectors now currently in use are also bicistronic. Protein products of both the inserted sequence (e.g., BCR-ABL) and a fluorescent marker (e.g., GFP) are produced in tandem as a result of an internal ribosomal entry site (IRES) inserted between the two gene sequences. This system facilitates the detection of transduced cells by flow cytometric evaluation of GFP fluorescence.202,207 If the cells show good expression of GFP, then lethally irradiated mice are intravenously injected (through the tail vein) with the transduced cells. The gamma-irradiation is an effective conditioning regime; all rapidly proliferating cells are killed and the resulting hematologic-deficient environment is easily repopulated by the transplanted leukemic cells. The dose of irradiation is also important, with a single nine Gray (Gy) dose being typical for most transplants. Once cells are injected, they travel through the periphery and eventually home and engraft in the bone marrow of the animal. Cells may also home to other organs, including the spleen and liver. Sometimes, cells lodge within the lungs of the recipient mouse, as this is a “first-pass” organ. Homing depends upon multiple forces; however, the homing of normal cells is most prominently influenced by SDF-1α (also known as CXCL12). This chemokine is produced by the BM stromal cells and is a potent chemo-attractant for both mouse and human hematopoietic progenitors.212 These primitive cells migrate toward SDF-1α because they consistently express the cognate CXC chemokine receptor 4 (CXCR4) on their cell surface.213

Figure 1-14. Bone marrow transduction and transplantation protocol. The retroviral vector encoding a bicistronic messages of BCR-ABL and green fluorescent protein (GFP) is used to transfect producer cell line and create infectious viral particles. This virus is used to transduce donor BMCs. The transduced cells that express BCR-ABL protein are intravenously injected into a lethally-irradiated wild-type recipient and the animal is monitored for leukemia development.

33 While this mechanism of homing may be employed in normal cells, BCR-ABL cells respond poorly to SDF-1α in vitro.174,176,177 Likewise, BCR-ABL cells display impaired homing to SDF-1α- injected spleens.176 This homing defect is not due to reduced CXCR4 expression, as CML cells display similar levels of the receptor on the cell surface as normal cells.177 Nevertheless, alternative homing mechanisms must be at play, because BCR-ABL cells are able to expand in the mouse BM to initiate leukemia. Based on studies showing reduced adhesion, migration and leukemic growth in severe combined immuno-deficient (SCID) mice, it is likely that signals emanating from the SH3 domain of BCR-ABL (and involved in expression of the α2 integrin) are required for homing.214 Moreover, the Rac protein,140 and the cell surface receptor CD44,215 are required for homing of BCR-ABL expressing cells. In any case, it is clear that alternative homing mechanisms must be efficiently operating as BCR- ABL cells still home to and expand in the BM microenvironment. Growth of the leukemic clone beyond the BM capacity leads to extravasation of cells into the peripheral blood and their movement to other organs including the spleen, liver and lungs. During this process the animals lose weight, display enlarged abdomens due to splenic expansion, and become moribund. Infiltration of cells into the lungs and the resulting pulmonary hemorrhage is the ultimate cause of death in these animals. BCR-ABL BMTs in a BALB/c background give rise to a full-blown MPD in three to four weeks.202 Several characteristic features are visualized upon histopathological work-up. As described above, there is evidence of extramedullary hematopoiesis in the spleen and liver: the organs are enlarged and normal splenic architecture is disrupted. The differential count is elevated and the peripheral blood (PB) is predominated by granulocytes. Both BCR-ABL-infected and non-infected myeloid cells are expanded in these mice due to excessive growth factor production (IL-3 and GM-CSF) by the leukemic cells.207 BCR-ABL is visualized at both the DNA (by Southern blotting) and protein (by immunoblotting) level. The outward disease phenotype and the results of laboratory tests in the BMT are highly similar to the findings in a CML patient, and therefore, the BMT serves as a useful model for determining the essential signaling pathways required for leukemogenesis.

1.2.5. Using the BMT assay to identify key pathways of BCR-ABL-mediated leukemogenesis As mentioned briefly in Section 1.2.3., leukemogenesis is the ultimate in vivo consequence of biological signaling from BCR-ABL. As demonstrated above, the BMT model serves as a useful mimic of human CML. To identify the key signaling proteins and pathways in BCR-ABL leukemogenesis, the donor BMCs for BMT have either: (a) been infected with BCR-ABL constructs containing mutations of key residues or domains, or (b) been derived from gene knock-out animals. These studies have identified key pathways that control proliferation, apoptotis, myeloid differentiation and homing of leukemic cells. Essential signaling pathways have been identified through transplantation experiments that utilize mutant constructs of the BCR-ABL protein. Through these BMTs, investigators have shown that Y177, the SH2 domain and the coiled-coil region are essential for development of an MPD (Table 1-3).

34 Table 1-3. Results of BMT assays that determined the essential domains and residues of BCR-ABL required for MPD development.

BCR-ABL construct Phenotype

KDa (K1176R) No disease207

ΔCC T-cell leukemia/lymphoma203,225

ΔCC/ΔSH3 MPD or non-fatal MPD >>b T cell leukemia/lymphoma225

Y177F Non-fatal MPD >> T-cell leukemia/abdominal lymphoma225; T-cell leukemia/lymphoma203; or B and T lymphoid leukemias226

BCR1-222-ABL Mixed MPD and B-ALL203

ΔSH2 Long-latency MPD or B-lymphoid leukemia227; B-lymphoid leukemia >> MPD228

SH2 R1053K or B-lymphoid leukemia227; or R1057Kc B-lymphoid leukemia >> MPD228 a KD, kinase dead. b >>, transitioned to. c Point mutations of the SH2 domain.

35 Additionally, the kinase activity of BCR-ABL is required for disease development. However, the SH3 domain of BCR-ABL appears to be dispensable. Nevertheless, MPD can be restored in most of the BMT recipients (71%) if the SH3 is deleted along with the CC domain. Combined, the results of these mutations studies implied that proteins downstream of Grb2 and those interacting with the SH2 domain may be involved in BCR-ABL-mediated leukemogenesis. Therefore, investigators initiated studies with donor cells either deficient (or containing mutations) in BCR-ABL target substrates proposed (by in vitro studies) to play essential roles downstream of BCR-ABL. A list of those substrates utilized in BMT experiments with the p210 isoform of BCR-ABL is detailed in Table 1-4. By these analyses the loss or deletion of most substrates led to either an impairment of abrogation of MPD. The strongest phenotype was observed with GAB2(-/-) donor cells in which all recipients succumbed to a T-cell lymphoma,216 i.e., GAB2 is required for BCR- ABL-mediated myeloid disease. Yet in one study, genetic deletion of Dok-1 from donor cells, led to a decrease in MPD latency, indicating that Dok-1 is a negative regulator of BCR-ABL signaling.217 Moreover, deletion of both Dok1 and Dok2 accelerates transformation to blasts crisis in transgenic Tec- BCR-ABL mice.218,219 Dinulescu et al. showed that genetic deletion of CBL from donor cells in BCR- ABL transplants has no effect on disease development, as the disease latency for MPD are similar in both WT and KO backgrounds.133 The authors concluded that CBL was dispensable for BCR-ABL disease (reasoning that lengthened latency in a single recipient mouse was due to poor tail vein injections). They suggested that CBL-B – the other CBL family member with significant hematopoietic tissue expression220 – may have compensated for the loss of CBL. Therefore, this study implied that BMTs with CBL-B-deficient cells might reveal a role for this family member in BCR-ABL-mediated disease.

Granulopoiesis regulators identified with the BMT model One of the characteristic features of chronic phase CML patients is the massive cellular expansion in the myeloid lineage. In the last eight years, the BMT has been used to test those proteins that regulate granulopoiesis, and as a result, may play prominent roles in BCR-ABL mediated leukemogenesis. Granulopoiesis may potentially arise from the autocrine production by BCR-ABL of IL-3, G- CSF and GM-CSF. CD34+ cells from chronic phase CML patients display enhanced levels of IL-3 and G-CSF mRNA,221 and several groups have detected the IL-3 and GM-CSF cytokines in the conditioned media of BCR-ABL expressing cell lines.222-224 Additionally, the BCR-ABL BMT recipients display similar characteristics to mice transplanted with BMCs that have been retrovirally transduced (or engineered) to express IL-3 or G-CSF and transgenic mice expressing G-CSF. 234-237 Moreover, Zhang and Ren have demonstrated that mice with CML-like disease induced by BCR-ABL have enhanced concentrations of IL-3 and GM-CSF in the sera and increased gene expression of these cytokines in BMCs.207

36 Table 1-4. Altered leukemogenesis when donor cells with genetic deletions of key effectors are used in BCR-ABLp210 BMTs.

Donor cells Phenotype

GAB2(-/-) T-cell lymphoma216

Attenuation of MPD or B-lymphoid N17 H-Rasa leukemia/lymphoma229

p85 (PI3’K) mutantb MPD with longer latency230

PKB K179Mc Impaired MPD132

STAT5a/bΔN/ΔN MPD, but more with B-ALL than WT231

STAT5a(-/-) ALL or CML-ALL or CML232

STAT5a/bnull/null d Mice survived for six months without disease233

Dok-1(-/-) MPD with shorter latency217

Rac1Δ/Δ/Rac2(-/-) Significant delay of MPD183

CBL(-/-) MPD133

a Dominant-negative construct. b Multiple mutations in the SH3 and SH2 domains of p85. c Dominant-negative kinase-dead mutant. d These transplanted cells were infected with the BCR-ABLp190 construct.

37 These observations suggested that IL-3 could be mediating the disease phenotype. To test this hypothesis, Li et al. performed BCR-ABL BMTs with either IL-3(-/-) or GM-CSF(-/-) donor cells.238 These animals still succumbed to an MPD indicating that BCR-ABL may be affecting IL-3 or GM-CSF signaling by means distinct from cytokine secretion. As Wong et al. observed enhanced IL-3R cell surface expression in BCR-ABL-induced hematopoietic progenitors, they hypothesized that impaired IL- 3R expression may perturb MPD development.239 Through BMTs using IL-3Rβc/β double knock-out cells, it was found that the IL-3R chains were dispensable for MPD. These studies eliminated IL-3 signaling as a key component in BCR-ABL-mediated leukemogenesis, but did not rule out the involvement of G-CSFR and GM-CSFR signaling. The expression of each of these myeloid receptors (as well as many others; see Table 1-5) is controlled by the action of two transcription factors: PU.1 (also known as spleen focus-forming proviral integration oncogene [Spi1]) and CCAAT/enhancer binding protein alpha (C/EBPα). PU.1 and C/EBPα control myeloid cell fate decisions at multiple stages of granulopoiesis (Figure 1-15). BCR- ABL can potentially promote transcription factor binding and myeloid gene transcription through the ERK and SAPK pathways that control PU.1 and C/EBP phosphorylation, associated-factor binding and translation regulation.240 As C/EBPα acts on many myeloid regulators and the C/EBPα(-/-) mouse lacks peripheral blood neutrophils, it is plausible that this transcription factor is required for CML development. When BCR-ABL transplant experiments were performed with C/EBPα(-/-) donor fetal liver cells, the recipient disease transformed to an erythroleukemia.241 These data suggested that C/EBPα played a role in the myeloproliferative phenotype but was not essential for disease generation as a whole. Knock-out mouse models exist in which genetic deletion alone induces a leukemia that closely resembles human CML. The deletion of both Dok1 and Dok2,218,219 or the loss of at least one copy of interferon consensus sequence binding protein (ISCBP),242 leads to a CML-like syndrome with all three phases of disease development. These mouse models indicate that Dok proteins and ICSBP are critical regulators of myeloid neoplasia, and suggest that these proteins may be important components of BCR- ABL-mediated disease. The aforementioned mouse models defined key pathways and signaling molecules that are required for disease development and myelopoiesis. These studies have aided investigators in pinpointing essential substrates for targeted therapies. In addition, the BMT has served as a useful model for the testing of inhibitors before clinical use, and as a result, will continue to operate as a powerful tool in the future.

1.2.6. Therapies for CML Since the first description of CML in 1845, the therapeutic management of this disease has changed dramatically (Figure 1-16).243 CML was originally treated with Fowler’s solution of 1% arsenic trioxide.

38 Table 1-5. Transcription factors that activate early myeloid genes.a

Gene Transcription factors

Myb-induced myeloid protein -1 (Mim-1) C/EBPs, c-Mybb

Myeloperoxidase (MPO) C/EBPα, PU.1, CBFc, c-Myb

C/EBP , PU.1, CBF, c-Myb, Sp1 Neutrophil elastase α IL-3R C/EBPα, PU.1, CBF

G-CSFR C/EBPα, PU.1

GM-CSFR C/EBPα, PU.1

Lysozyme PU.1

c-Fes PU.1, Sp1

a Adapted from ref. (240). b c-Myb, cellular homolog of the myeloblastosis viral oncogene. c CBF, core binding factor.

Figure 1-15. Transcription factors operate at multiple stages of differentiation to control myelopoiesis. Adapted from ref. (240).

Figure 1-16. Timeline of CML treatments. CML was first described in 1845. Since then, multiple treatment modalities have been tested in the clinic. Arsenic trioxide was first used followed by radiotherapy directed toward the spleen. In the middle of the 20th century chemotherapy treatments (busulfan and hydroxyurea) were introduced. Hematopoietic stem cell transplantation (HSCT) was established in 1968 and still exists as the only curative therapy for CML. Within the last twenty years interferon and imatinib have been tested in the clinic and are both relatively effective treatments for chronic phase CML patients. Adapted from ref. (243).

41 Spleen irradiation became the common treatment in the latter part of the 19th century until chemotherapy (busulfan and hydroxyurea) was implemented in 1953. The first curative option for CML, the hematopoietic stem cell transplantation (HSCT), was established in 1968. However, widespread use of this treatment is hindered by the need for human leukocyte antigen (HLA)-matched donors and the high risk of complication, especially in patients over 40 years of age. As the median age-of-onset for CML is 53 years,41 HSCT is not a viable option for the majority of patients. Therefore, alternative strategies have been developed. In 1983, physicians began administering interferon-alpha (IFN-α) to CML patients. IFN-α promotes cytoreduction of CML cells by inducing the proliferation of multiple cell types involved in immunosurveillance.244 IFN-α has proved to be a very effective treatment strategy, but one that only works in a minority of CML patients.245,246 Therefore, IFN-α is another treatment that is useful but not a successful strategy for the management of all CML patients. During this time, scientists were investigating the idea of creating targeted inhibitors of BCR- ABL; therefore, blocking proliferation of leukemic cells and sparing normal healthy cells. By the mid- 1990s, Druker and colleagues discovered the tyrosine kinase inhibitor (TKI) imatinib (also known as imatinib mesylate, STI571, CGP57148 and Gleevec [Table 1-2]) that specifically targets the BCR-ABL kinase by binding in the ATP pocket (Figure 1-17).247 The oral imatinib drug formation successfully passed phase I,248 phase II,249 and phase III250 trials for chronic phase patients - showing good tolerability; the production of hematologic, cytogenetic and molecular remission; and an enhanced patient survival advantage over IFN-α.251,252 Based on the results of these trials, imatinib has been accepted as the first-line therapy for chronic phase CML patients.42 Unfortunately, imatinib also has associated deficiencies. This TKI must be taken for the lifetime of the patient, as even those patients who achieve a complete molecular response (characterized as the absence of BCR-ABL mRNA transcripts in nested PCRs) cannot discontinue therapy due to eventual relapse.253,254 Another drawback of imatinib is resistance, due sometimes to BCR-ABL amplification but most often results as a consequence of BCR-ABL mutants that emerge as a result of treatment.255,256 The mutants impair imatinib binding to the kinase and the patients have sub-optimal or failed responses. Over 100 BCR-ABL mutations have been detected in imatinib-resistant patients and through in vitro mutagenesis studies.257 To overcome the resistance associated with the BCR-ABL mutants, several second-generation TKIs have been developed. When tested in the laboratory, nilotinib and dasatinib show greater efficacy toward imatinib-resistant mutations.258,259 As a result, clinical trials are currently underway in which these 2nd-generation compounds are being tested in imatinib-resistant chronic phase patients.260-262 Unfortunately, these second-generation inhibitors are still ineffective against patients with the threonine to isoleucine mutation at residue 315 (T315I).

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Figure 1-17. Imatinib binding to BCR-ABL. This three-dimensional ribbon diagram depicts the binding of imatinib (orange) within the adenosine triphophate (ATP)-binding site of BCR-ABL (green). The location of the activation loop is shown (magenta) as is the phosphate-binding loop (P-loop; yellow). The TKI-resistant mutant T315I is located within the P-loop. This figure was originally published in Blood. Quintas-Cardama A, Cortes J. Molecular biology of BCR-ABL1-positive chronic myeloid leukemia. 2009;113:1619-1630. © the American Society of Hematology.257

43 As a result, investigation into alternative CML therapies is ongoing. One strategy is the use of combination therapy – in which imatinib is given in combination with an inhibitor of one of the important downstream signaling pathways. Several combination approaches are in the testing stages. Inhibitors of the PKB pathway may be useful for combination therapy. When CML cells or murine MPD models are treated with rapamycin, an inhibitor that targets the mammalian target of rapamycin (mTOR) effector of PKB, in combination with imatinib, there is a synergistic decrease in proliferation.263 Additionally, inhibitors of Ras pathways show some promise as CML therapies. Farnesyl transferase inhibitors (FTIs) have been developed (lonafarnib and tipifarnib) that block farnesylation of the Ras protein, leading to altered localization of the protein and reduced downstream activation. In vitro, lonafarnib inhibits the proliferation of BCR-ABL- expressing cells, including those that are imatinib-resistant.181,264 Moreover, lonafarnib given as oral gavage to BCR-ABL BMT mice effectively abrogates leukemia.181 Based on these successes, dose- finding trials for a combination of lonafarnib and imatinib were initiated and tolerable doses were identified.265 As CBL proteins exist upstream of both Ras and PKB in BCR-ABL signaling, inhibitors that target CBL proteins may be useful for a combination approach. These unique strategies may prove to be efficacious as therapies for all CML patients, and therefore warrant further investigation.

1.2.7. Summary CML results from uncontrolled hematopoiesis of the myeloid lineage. The causative agent of CML is the bcr-abl gene, which is formed by the t(9;22) translocation. The constitutive tyrosine kinase activity of the BCR-ABL fusion protein results in the activation of multiple signaling pathways that control cell adhesion, migration, proliferation and survival. Cellular transformation by BCR-ABL is observed in cell lines, and the human disease and can be effectively recapitulated in BMT studies in mice. The BMT model has been used to determine the essential pathways for leukemogenesis and to test protein inhibitors for use in human CML patients. A continued study of the mechanism through which BCR-ABL elicits disease will ultimately result in even better treatment modalities for patients with CML.

44 1.3. The CBL family of proteins In the early 1970s, scientists working out of Lake Casitas, California, discovered wild mice afflicted with pre- and pro-B-cell lymphomas.266,267 The oncogenic agent was later identified as the Cas NS-1 retrovirus, generated through recombination of the Cas-Br-M virus with the cellular oncogene aptly named v-CBL, for Casitas B-lineage lymphoma.268 In further studies it was discovered that v-Cbl was a truncated form of the full-length protein – CBL (Figure 1-18A).269 Since then, two additional mammalian homologs have been isolated, CBL-B and CBL-3 (or CBL-C),270-272 as well as multiple orthologues in lower organisms. The best characterized of these non-mammalian CBL proteins are found in Drosophila melanogaster, D-CBL(long) and D-CBL(short), and Caenorhabditis elegans (suppressor of lineage defect – 1 or Sli-1), but CBL proteins have been described in other worms, sea squirts, insects, amphibians, fish and birds.273 However, plants and single-cell organisms like yeast, do not possess CBL orthologues, and therefore, the CBL gene probably represents an evolutionary divergence of multi-cell eukaryotes.

1.3.1. The domain structure of CBL proteins The essential nature of CBL proteins in higher organisms is emphasized by the relatively unaltered domain structure throughout the course of evolution (Figure 1-18A). The highly homologous N-terminal region contains a tyrosine kinase-binding (TKB) domain, followed by a short linker sequence that joins the TKB domain to a RING (Really Interesting New Gene) finger motif. Variation exists within the length and amino acid sequence of the C-terminal half, but each CBL protein contains multiple tyrosine residues and a proline-rich region. CBL, CBL-B and D-CBL(long) also possess a combined ubiquitin-associated (UBA) / leucine zipper (LZ) domain at the C terminus. The TKB domain is comprised of a four-helix bundle (4H), a calcium-binding EF hand, and a variant SH2 domain (Figure 1-18B).274 While its name describes the fact that this region facilitates binding of CBL to multiple protein tyrosine kinases (PTKs), the association of CBL with adaptor molecules can also occur within the TKB domain. However, it is believed that the strongest associations of CBL proteins with their binding partners is not mediated through this region. Instead, it has been suggested that the TKB is required for the correct alignment of target substrates during ubiquitination.275 Indeed, this domain is essential for ubiquitination.276 The important functional role of the TKB domain in CBL proteins is evidenced by the evolutionary conservation of this region; in fact, CBL and CBL-B share greater than 80% amino acid identity in their TKB domains. Joining the TKB domain and RING finger is a short intervening region that is also required for the E3 ubiquitin ligase activity of CBL.277 The linker sequence contains two essential tyrosines (Y371 and Y368) for CBL ligase activity. When Y371 is replaced with phenylalanine, CBL E3 activity is perturbed,278 while substitution of Y371 with glutamate promotes ligase activity.279 Cellular

letters to nature

Figure 1-18. Domain structure of CBL proteins. (A) The CBL and CBL-B homologs each contain a tyrosine kinase-binding (TKB) domain near the N- terminus, followed by a RING finger motif and a proline-rich region (Pro-rich). A ubiquitin-associated region is found at the C-terminus. Scattered throughout each protein are multiple tyrosine- (closed circles) and serine- (open circles) phosphorylation sites. The CBL-3 homolog, oncogenic v-CBL, and the many of the other orthologs illustrated are missing domains found in the longer forms. The long version of Drosophila protein (D-CBL(long)) is the only ortholog in that species that retains all CBL domains. Adapted from ref. (280). (B) A ribbon diagram of the TKB domain of CBL, which contains a four-helix bundle (yellow) at the amino-terminus followed by an EF-hand (green) and an SH2 domain (blue). The EF-hand co-ordinates a calcium ion (red). Reprinted by permission from Macmillan Publishers Ltd: Nature, 398(6722):84-90, copyright 1999.274

Figure 1 Cbl domain structure and sequence comparisons. a, Ribbon diagram of Residues that are identical in at least three of the sequences are shaded yellow. unliganded Cbl-N. The N-terminal 4H domain is coloured yellow, the EF-hand Secondary-structure elements are shown above the sequence and are coloured domain green, and the SH2 domain blue. Secondary-structure elements are as in a and b. Black squares indicate residues that coordinate calcium. Red labelled aA±aD in the 4H domain and by established conventions for the EF-hand circles mark residues that interact with the bound ZAP-70 peptide. d, Structure- and SH2 domains. The bound Ca2+ ion is indicated by a red sphere. Arginine 294 is based sequence alignment of Cbl and Lck23 SH2 domains. Seventy structurally universally conserved in SH2 domains and participates in phosphotyrosine equivalent residues are shaded yellow; a-carbons of these seventy residues coordination. b, Diagram of c-Cbl domain structure. The Cbl-N region and superimpose with an r.m.s.d. of 1.47 AÊ . The secondary-structure elements that are adjacent RING ®nger domain are conserved in all Cbl homologues. The present in Lck and other SH2 domains, but not in the Cbl SH2 domain, are C-terminal region, which contains proline-rich segments and tyrosine phos- indicated by open boxes. e, Superposition of the Cbl SH2 domain (blue) with the phorylation sites, is more variable and is completely absent in D-Cbl. A putative Lck SH2 domain (yellow). The structural elements that are absent in the Cbl leucine zipper has been found near the C terminus of Cbl. c, Aligned sequences domain are red. of the Cbl-N portion of human c-Cbl, human Cbl-b, Drosophila D-Cbl, and Sli-1.

© 1999 Macmillan Magazines Ltd NATURE | VOL 398 | 4 MARCH 1999 | www.nature.com 85 46 transformation occurs if these tyrosines are deleted by either single point mutations or the loss of 17 amino acids from the linker region (as is found in the 70Z/3 mutant).277,281 The RING finger of CBL proteins contains the E3 activity.282 This region is approximately 40 to 100 amino acids in length and contains a conserved sequence of eight cysteines and several histidine residues that co-ordinates two zinc ions.283 This sequence also binds to ubiquitin E2 enzymes as characterized in structural evidence showing the association of CBL with the ubiquitin-conjugating enzyme H7 (UbcH7).275 Sequences in the C-terminal region allow for interactions with SH3- and SH2-containing proteins. SH3 binding is mediated by proline-rich sequences; CBL contains 15 potential contacts, CBL-B has 17, and CBL-3 has five, while D-CBL does not possess any sequences for SH3 domain interactions. The C- terminal region also contains multiple tyrosine residues – in fact, CBL has 22 tyrosine residues. Tyrosine phosphorylation of CBL can be accomplished by the Syk kinase; as well as the Fyn, Yes, Src and Lyn members of the Src kinase family.284-286 Phosphorylation of CBL tyrosine residues creates docking sites for multiple SH2-containing proteins including Vav1 and PI3’K.287-289 Additionally, CBL can be phosphorylated on serine and threonine amino acids leading to its association with the 14-3-3 protein or attenuation of its tyrosine phosphorylation.290,291 At the extreme C-terminal end of CBL is found a LZ motif superimposed with a UBA domain. As such, the respective functions of dimerization and ubiquitin binding are associated with CBL proteins: homo- and hetero-dimers can form between the CBL proteins,292,293 and the UBA domain can facilitate an interaction with ubiquitin.294 The importance of UBA-mediated dimerization differs between CBL and CBL-B: CBL dimerization is essential for adaptor protein binding and tyrosine phosphorylation but deletion of the CBL-B UBA domain does not alter epidermal growth factor receptor (EGFR) binding and ubiquitination.278,293 CBL and CBL-B also display differences in their UBA-mediated ubiquitin binding ability: only the CBL-B isoform has this property. A recent crystal structure has revealed that structural differences account for the distinct ubiquitin binding attributes: CBL-B requires association with ubiquitin for dimerization while tight CBL dimers can form in the absence of ubiquitin.295,296 Therefore, elucidation of the distinct functional role of the UBA/LZ motif in each CBL isoform still requires further experimentation. This description of CBL domain structure highlights how each domain plays an essential role in mediating protein interactions. The multifaceted structure of CBL proteins potentiates their function as E3 ubiquitin ligases and adaptor proteins. To perform these functions, in multiple cell types, it is estimated that CBL proteins interact with over 150 substrates. As such, a description of each interaction would be beyond the scope of this introduction. Instead, the description that follows will summarize a few of the key CBL associations and the functional consequence of CBL expression.

47 1.3.2. CBL proteins mediate ubiquitination CBL-mediated interactions play prominent roles in ubiquitination – a three-step sequential process whereby a 76 amino acid ubiquitin molecule is transferred to a substrate protein.297,298 To initiate the sequence, an E1 activating enzyme forms a thiol ester bond between its active site cysteine residue and the C-terminal glycine on ubiquitin (Figure 1-19). Next, the E2 conjugating enzyme accepts ubiquitin from E1 at its own active site through trans-esterification. Finally, through binding of the E3 ubiquitin protein ligase to both the substrate protein and the E2, the E3 promotes the formation of an isopeptide bond between the C-terminal glycine of ubiquitin and either: (a) the ε-amino group of a lysine residue on the substrate (as depicted in Figure 1-19), (b) the N-termini of substrate proteins (although this event happens infrequently) or (c) the lysine of another ubiquitin molecule already attached to the substrate. It has been suggested that a fourth enzyme, the E4 poly-ubiquitin chain conjugating factor, may facilitate this ubiquitin chain extension. The precise synchronization of each of these steps allows for ubiquitination of proteins. Three different ubiquitin modifications exist. In one ubiquitination modification, termed monoubiquitination (Figure 1-20), a single ubiquitin molecule is attached to a substrate and regulates its transcription, DNA repair, histone function, endocytosis or membrane trafficking.299,300 Second, monoubiquitination at multiple substrate sites can occur, and is most prominently observed in receptor tyrosine kinase endocytosis and trafficking to the lysosomes.301 In a third mechanism, poly-ubiquitin chains are formed on internal lysines within ubiquitin. Seven lysines exist within ubiquitin - K6, K11, K27, K29, K33, K48 and K63 – five of which (K6, K11, K29, K48 and K63) can function as a linkage for polyubiquitination chains. K48-linked poly-ubiquitin chains generally commit proteins to degradation in the 26S proteasome.302 Meanwhile, K6 or K63 poly-ubiquitin chain formation alters substrate activation, stability, sub-cellular localization, or its interaction with other proteins. Ubiquitin ligation to a substrate is specified by the E3 ligase. Membership in the E3 family usually depends on the presence of a homologous to E6-AP carboxyl terminus (HECT) or RING finger domain in the protein sequence.303,304 E3s can be single or multi-subunit in nature. Multi-subunit E3s, for example the Skp1/Cullin/F box (SCF) protein, require at least three proteins to perform the E3 role: a RING finger protein that has binds the E2 (Rbx1 for SCF), a cullin family protein that binds the RING (Cul1 for SCF), and another adaptor that links the culling to the substrate (Skp1 for SCF). Meanwhile, single subunit E3s can coordinate the transfer of ubiquitin from the E2 to the substrate without the help of additional proteins. E3 ligases can be regulated through: (a) auto-ubiquitination, (b) the action of de- ubiquitinating enzymes, or (c) post-translational modifications such as phosphorylation, oxidation, sumoylation, acetylation, or neddylation.298 CBL proteins belong to the E3 family based on the presence of a RING finger and their ability to transfer ubiquitin from an E2 to a substrate.278,282,305 The CBL family members are examples of single subunit E3s, having the ability to bind to both the substrate and the E2 conjugating enzyme. Most

Figure 1-19. The ubiquitination pathway. ATP-activated free ubiquitin (Ub) binds to the carboxyl terminus of the E1 enzyme through a thiol ester bond. The E2 conjugating enzyme then displaces E1 from Ub through trans-esterification. Next, the E3 ubiquitin ligase, which in this case is shown with the substrate (S) already attached, binds to the E2. Finally, for RING type E3’s, the Ub is transferred directly to the ε-amino group of a lysine residue on the substrate. Adapted from ref. (298).

Figure 1-20. Different forms of ubiquitin modification. Ubiquitin (Ub) attaches to proteins at lysine (K) residues in one of three ways. In mono-ubiquitination, a single Ub molecules attaches to a protein. In multi-ubiquitination, several Ub moieties are attached at individual lysines throughtout the substrate. Poly-ubiquitination is characterized by a single polymeric chain of Ub associated at one lysine. The most common biological function associated with each form of ubiquitination is indicated on the right. Adapted from ref. (306).

50 importantly, CBL proteins can select substrates for ubiquitination, contributing to either degradation in the lysosomal and proteasomal pathways or modification of protein localization. In the following paragraphs, examples of each of these mechanisms will be presented. EGFR trafficking is one of the most well studied examples of how CBL-mediated ubiquitination can target proteins for lysosomal degradation.307-310 CBL proteins (CBL and CBL-B) are recruited to the EGFR both indirectly through GRB2,311,312 or directly through binding of the CBL TKB domain to phosphorylated tyrosines in the EGFR.278,313 Association of CBL with the EGFR contributes to formation of a multi-subunit protein complex that is required for internalization.280 Several studies have debated the role of CBL-mediated ubiquitination in receptor endocytosis, but recent data from Huang et al. suggests that ubiquitination is not essential for internalization.314 Rather, a prominent role for CBL is visualized at later stages of lysosomal sorting,309,315 where CBL-mediated ubiquitination of the EGFR triggers entry into late endosomes with subsequent lysosomal degradation.316 In the absence of ubiquitination by CBL, the EGFR is recycled.317 CBL proteins can also ubiquitinate substrates for trafficking to the proteasome, as evidenced by studies with the non-receptor protein tyrosine kinase Syk. CBL and Syk interact primarily through binding of the TKB of CBL with a phosphorylated tyrosine residue in the linker region between the two SH2 domains of Syk.318,319 Using its RING finger activity, CBL directs the down-regulation of activated Syk through ubiquitination and degradation in the proteasome.320-322 Syk also directs the tyrosine phosphorylation of CBL,284 which may stimulate its E3 activity. Therefore, the interaction between CBL and Syk may be one in which Syk directs its own proteasomal degradation through association with and phosphorylation of CBL. In a third regulatory role, CBL-mediated ubiquitination does not affect protein stability. An example of this function occurs in T cells with the CBL-B isoform. CBL-B interacts with the p85-subunit of PI3’K, through association of its proline-rich region with the SH3 domain of p85, and initiates ubiquitination of the p85-PI3’K subunit.323 This interaction does not cause down-regulation of the PI3’K complex. Instead, it sequesters PI3’K away from the CD28 docking protein at the plasma membrane and impairs PI3’K activity.324 This is an interesting alternative mechanism of CBL protein-mediated ubiquitination that does not lead to proteasomal or lysosomal degradation. As CBL E3s bind a plethora of substrates, one can imagine that without regulation of CBL ligase activity, cellular signaling would run amuck. Thankfully, CBL proteins, like other E3s, are subject to regulatory control. CBL proteins themselves can be degraded by ubiquitination machinery, either through auto-ubiquitination,308,325,326 or as targets of other ubiquitin ligase such as NEDD4 and Itch.327 Phosphorylation of CBL promotes interactions with SH2- and SH3-domain containing proteins; yet both cortactin and Src homology 2 containing phosphatase 1 (SHP1) control CBL phosphorylation.328,329 Finally, other proteins block interactions of CBL with potential substrates, as is observed in EGFR signaling with Sprouty,330 and Cdc42.331

51 1.3.3. CBL proteins function as adaptors CBL family members not only bind to proteins during ubiquitination. Due to the multiple tyrosine, serine and threonine phosphorylation sites throughout the protein; the proline-rich motif; and the N-terminal SH2 domain; CBL proteins can also act as adaptors. To simplify this discussion we have included a diagram of some of the CBL interactors that are modulated by CBL adaptor function (Figure 1-21). As less is known about the associations formed with CBL-B, we have not included its specific interactions in this diagram. Needless to say, the high amino acid similarity between CBL and CBL-B suggests that both proteins bind many of the same signaling proteins. Yet, there are some dissimilarities. As discussed above, CBL-B associates with PI3’K through its proline-rich motif and ubiquitinates the p85 subunit.323 In contrast, CBL function as an adaptor in this context, associating with PI3’K through an interaction of the phosphorylated tyrosine residue 731 with the SH2 domain of p85.288,289 Moreover, CBL associates with 14-3-3 proteins, while CBL-B does not – likely due to the fact that CBL-B is missing a second RSXSXP consensus sequence.290 In conclusion, there are multiple interactions mediated through CBL domain- or phosphorylated residue- binding that co-ordinate signals to downstream substrates independent of ubiquitination.

1.3.4. CBL protein-mediated interactions that regulate ERK, PKB and Rho signaling The adaptor and E3 ubiquitin ligase functions of CBL proteins allow them to regulate many signaling pathways. Our review will focus on those CBL interactions that control the ERK, PKB and Rho signaling pathways. In each case, examples will be given of protein activation and the upstream interaction with CBL proteins that modifies these pathways.

CBL proteins and ERK signaling CBL-mediated regulation of ERK pathways is quite complex and is highly context specific. In some cell types ERK phosphorylation is unaffected by changes in the level of CBL protein expression.332- 334 In those cell types that are responsive to CBL levels, ERK activation is predominantly elevated in the absence of CBL proteins,335-338 suggesting a negative regulation of ERK phosphorylation. In general, CBL proteins control ERK activation through ubiquitination and down-regulation of cell surface receptors or kinases that co-ordinate downstream signaling events. For example, CBL may directly associate with and ubiquitinate the EGFR to reduce ERK activation.339,340 In T cells, CBL contacts and ubiquitinates the TCR-CD3 receptor indirectly through ZAP-70.341 Control of CBL-mediated receptor ubiquitination and ERK activation is achieved by several regulatory proteins, such as human Sprouty 2 (hSpry2),339 CDC42,331 and low density lipoprotein receptor-related protein 1 (LRP1).342 As observed in EGFR signaling with hSpry2, this protein intercepts CBL-mediated ubiquitination of the receptor, preventing endocytosis and sustaining ERK signaling.339 In sum, it is clear that no single

Figure 1-21. CBL functions as an adaptor in associations with multiple substrates. The numerous domains and phosphorylated residues of CBL co-ordinate associations with several proteins. In the TKB domain CBL can bind APS,343 and tubulin.344 The proline-rich region co-ordinates associations with Grb2,345,346 CBL-associated protein (CAP),347 and Nck.348 A phospho-serine (open circles) motif in CBL (RSXSXP) mediates binding to 14-3-3.290,349 Three tyrosine residues (closed circles) in the C-terminal region are important for CBL adaptor binding to BCR-ABL,127 the Crk family of proteins,350-352 the p85 subunit of PI3’K,288,289 and the CBL-interacting protein of 85 kDa (CIN85) that is constitutively bound to endophilin.353

53 mechanism of CBL-B-mediated ERK regulation is defined and that the local concentrations of adaptor and regulatory proteins can greatly influence ERK activation.

CBL proteins and PKB signaling The downstream signals from CBL proteins to PKB are more clearly identified, likely due to the direct association of CBL proteins with PI3’K. CBL association with PI3’K appears to up-regulate or sustain PKB activation, while CBL-B acts as a negative regulator of this pathway. The direct association of CBL with the p85-PI3’K subunit can occur (a) through association of its SH3 domain with the proline-rich sequences of CBL,131 or (b) through binding of the p85-SH2 domain to CBL consensus sequence pYXXP at tyrosine 731.288 It is believed that the SH3 association is constitutive and that phosphorylation of CBL allows for p85-SH2 domain association and enhanced interaction of the two proteins as has been observed after stimulation of the TCR,354 B-cell antigen receptor,355 and EGFR356; or following BCR-ABL transformation of hematopoietic cell lines.125 The association of p85 and CBL is postulated to enhance PKB activation. In v-Src expressing fibroblasts depletion of Shp2 reduces CBL and p85 association and impairs PKB activation.357 It is hypothesized that Shp2 is required for PKB activation by functioning as an adaptor between p85 and CBL or through regulation of CBL phosphorylation at Y731. In another example, Src inhibition in EGF-stimulated cells leads to reduced PKB activity.358 Again, it is presumed that Src impairs PI3’K and CBL association as it is known that Src proteins phosphorylate CBL at the site important for PI3’K binding.288 CBL-B appears to regulate the PKB pathway in a distinct manner. CBL-B does not bind to p85 through a conserved tyrosine residue; Instead, the proline-rich region of CBL-B co-ordinates association with the SH3 domain of p85.323 In T cells, CBL-B facilitates the ubiquitination of p85-PI3’K, and in a proteolysis-independent mechanism excludes PI3’K from its site of activity at the membrane.324 In support of a role for CBL-B in the negative regulation of the PI3’K/PKB pathway, CBL-B(-/-) T cells show hyper-activation of PKB.359 Furthermore, CBL-B over-expression decreases the amplitude and duration of PKB signaling in EGFR-expressing 32D cells.312 While exceptions exist,360 for the most part these studies suggest that CBL expression enhances PKB activity while CBL-B acts as a negative regulator of this pathway.

CBL proteins and Rho signaling CBL proteins can also control the activation of the Rho signaling pathway that plays a central role in cell migration and adhesion (Section 1.2.3). There are three mechanisms by which CBL proteins can control this pathway: (1) through the negative regulation of GEFs that activate Rho proteins, (2) through the sequestration of GEFs required for activity of the Rho effector PAK proteins, or (3) through cross-talk following regulation of PI3’K.

54 The most prominent GEF family to be identified as binding partners for CBL proteins is the Vav family. There are three mammalian Vav family members: Vav1,361 Vav2,362,363 and Vav3.137 Vav1 is predominantly expressed in hematopoietic cells while Vav2 and Vav3 are more broadly expressed. CBL associates with Vav proteins through a phospho-tyrosine interaction between Y699/700 of CBL and the SH2 domain of Vav.287,364,365 CBL-B mediates association with Vav through the proline-rich region of CBL-B binding to the SH3-SH2-SH3 region of Vav.220 The association of CBL and Vav proteins regulates the activity of the GEF. CBL protein expression negatively regulates Vav phosphorylation in T cells,332,364,366,367 and B cells.368 Modulation of Vav phosphorylation may be achieved through one of two mechanisms (Figure 1-22). In the first case, CBL may ubiquitinate Vav leading to its degradation. Enhanced ubiquitination of Vav is visualized in wild-type T cell lines as compared to cell lines with depletion of CBL.364 Moreover, CBL-B co- immunoprecipitates mono-ubiquitinated Vav in BCR-ABL-expressing cells.134 An alternative degradation-independent mechanism of Vav regulation has been proposed by Fang and colleagues.324 They suggest that CBL-B-mediated reduction in PI3’K activity (as described above) could reduce the 369 production of PIP3 – a phosphoinositide that enhances Vav phosphorylation. This theory would imply that CBL, as a predominantly positive regulator of the PI3’K pathway, could act in an opposing fashion to the ubiquitination method and increase Vav activity. Regardless of the mode of regulation it is evident that Vav phosphorylation is controlled by CBL family member expression. As phosphorylation of Vav proteins is required for its GEF activity toward Rac, CDC42 and Rho136-138; the negative regulation of phospho-Vav could potentially control Rho signaling. A direct substrate for Rac1 and CDC42 is the p21-activated kinase (PAK) protein.370 The cloned out of library (Cool) (also known as PAK-interactive exchange factor (Pix)) family can complex with PAK proteins and regulate their activity.371 As well, the Cool/Pix proteins can act as GEFs for Rac.372 Both CBL and CBL-B compete with PAK for binding to Cool proteins.373,374 Although the functional consequence of CBL protein-mediated competition is not yet clearly defined, these data suggest that CBL proteins may also employ the Cool/Pix GEFs to regulate Rho signaling. In a final proposed method, CBL proteins may control Rho activation through PI3’K regulation. As the lipid products of PI3’K bind Rac1 and promote GDP release,375 there is a possibility that the CBL protein-mediated regulation of PI3’K could alter Rho signaling events. Yet despite these numerous studies investigating direct binding partners of Rho proteins, there are limited reports that directly measure Rho family activity following modulation of CBL expression. One study in v-Abl transformed fibroblasts that over-express CBL shows increased activation of the RhoA and Rac1 GTPases.376 As well, truncated CBL that acts as a dominant negative causes increased Rac activation in response to PDGFR stimulation.377 Nevertheless, these studies suggest that CBL proteins can regulate the Rho GTPase pathways possibly through modulation of GEF or PI3’K activity.

Figure 1-22. CBL proteins control Rho family activation. CBL proteins may control Rho activation through one of three pathways. CBL-mediated ubiquitination of Vav may promote degradation, leading to reduced availability of this GEF for Rho stimulation. PI3’K may be positively controlled by CBL association or negatively regulated by CBL-B. The availability of the PIP3 product may control Vav or Rac activity. Finally, the competitive association of CBL proteins with Cool/Pix family members may affect either Rho family or PAK activity.

56 1.3.5. The role of CBL proteins in cellular migration Our discussion above indicated that CBL proteins modulate the activation of ERK, PKB and Rho pathways. These cascades have all been shown to mediate CBL protein-dependent migration of cells. CBL proteins control both spontaneous motility and the migration of cells toward multiple chemokines in positive and negative regulatory manners. CBL proteins are positive regulators of cell motility in breast cancer and T cell lines,378,379 fibroblasts,380 osteoclasts,381 and CD34+ hematopoietic progenitors.382 In the breast cancer cell lines, introduction of a mutant CBL construct (CBL N480), which effectively ubiquitinates the EGFR without regulation by CDC42 and Cool/Pix proteins, leads to decreased migration toward fetal calf serum (FCS) and reduced Rac1 and CDC42 activation.378 The introduction of either CBL or CBL-B siRNA into Jurkat T cells causes a decrease in chemotaxis toward SDF-1α.379 While a pathway of migratory response has not been defined, tyrosine phosphorylation of CBL proteins is enhanced after SDF-1α treatment, as is association of CBL with CrkL, 14-3-3β, and PI3’K.383 In v-Abl transformed NIH 3T3 cells, migration in the absence of exogenous growth factors, or in the presence of 10% serum and PDGF, is enhanced by CBL over-expression.380 In this system migration is dependent upon the E3 ubiquitin ligase activity of CBL and the activation of Rac1 and PI3’K. These data suggest that cellular chemotactic movement can be controlled in a positive manner by CBL proteins through mechanisms prominently involving the PKB and Rho family pathways. CBL proteins can also play negative regulatory roles in the cell motility. CBL(-/-) MEFs show enhanced migration toward platelet-derived growth factor-BB (PDGF-BB) in a dose-dependent manner,384 and over-expression of CBL impairs CXCL8-induced chemotaxis of a pre-B cell line, possibly through a mechanism involving PKB.385 Ba/F3 cells expressing BCR-ABLp210 that are stably transfected with CBL-B display reduced spontaneous migration as compared to control-transfected cells.134 The reduced motility of these cell types to CBL modulation may be a function of altered cellular environments or chemotactic stimuli. Further mechanistic research should clarify the observed variability in CBL protein-mediated migratory responses.

1.3.6. The role of CBL proteins in cellular adhesion As described earlier in Section 1.2.3, migration and adhesion are interconnected processes given the need for modification of adhesive contacts during cell movement. Therefore, it is not surprising that CBL protein expression both promotes and inhibits cell adhesion. Yet the signaling pathways modulated by CBL proteins to control adhesion are quite similar; CrkL and PI3’K are key players in CBL-mediated adhesion. Fibroblast adhesion is enhanced when CBL is over-expressed, with both CrkL and PI3’K playing a role.386 Teckchandani and colleagues propose that increased adhesion is due to enhanced fibronectin

57 deposition, that requires activation of RhoA, Rac1 and possibly Rap1,376 which are likely regulated upstream through recruitment of PI3’K, CrkL and Vav to CBL.386 In contrast, multiple other cell types show upregulation of adhesion when CBL or CBL-B are depleted. CBL-B(-/-) bone marrow-derived mononuclear phagocytes display increased adhesion to endothelial cells or inter-cellular adhesion molecule 1 (ICAM-1) following leukocyte function-associated antigen-1 (LFA-1) activation,387 and CBL-B-deficient T cells show enhanced adhesion to ICAM-1 through reduced ubiquitination of CrkL and enhanced association between CrkL, C3G, and Rap1.388 As well, CBL(-/-) thymocytes have increased adhesion following integrin activation.389 These cells also utilize the CrkL-C3G-Rap1 pathway to modulate adhesion. Continued studies in other cell types should reveal the significance of the PI3’K and CrkL-C3G-Rap1 pathways in cellular adhesion modulated by CBL proteins.

1.3.7. In vivo functions of CBL proteins To assess the role of CBL family members in vivo, genetic deletion in murine models was performed. Whole animal single knock-outs were created for each of the three mammalian CBL isoforms and an attempt was made to generate CBL/CBL-B double knock-out mice – but this genetic deletion was embryonic lethal.390 Additionally, the role of CBL and CBL-B expression in lymphocyte functions has been studied in conditional knock-outs that deplete CBL and CBL-B in either the T- or B- cell lineages. Moreover, the function of individual CBL domains has been studied with transgenic mice containing loss-of-function mutations. This section details each of these animal models. CBL-deficient mice are healthy and fertile.337,391 Naramura and colleagues did not visualize any gross abnormalities in their CBL(-/-) animals other than a mild splenomegaly, but Murphy et al. described a severe splenomegaly in their animals (characterized by fibrosis and significant EMH), lymphadenopathy from increased T and B cell numbers, and a slight thickening of mammary fat pads. Nevertheless, both groups confirmed an altered T cell phenotype. The total number of thymocytes is comparable between CBL-deficient and wild-type mice, but several cell surface receptors – such as those responsible for thymocyte positive selection (T cell receptor [TCR], CD5 and CD69) – are up-regulated in CBL(-/-) animals, leading to enhanced positive selection of CD4+ thymocytes. Immunoblotting experiments of antigen-stimulated thymocytes show that CBL-deficiency causes hyper-phosphorylation of multiple substrates, including ZAP-70 and ERK1/2. On the other hand, PI3’K and PLCγ1 phosphorylations are reduced. Signaling in the peripheral T cells contrasts sharply with that in the thymocytes: the absence of CBL reduces both the phosphorylation of peripheral CD4+ T cells and their proliferation. These CBL animal models suggest an important role for CBL in T cell proliferation. Young CBL-B(-/-) animals are similar to CBL knockout animals in that they are healthy and fertile, with only slight enlargement in the spleens and lymph nodes.332,392 However, after six months of age, these mice develop a spontaneous autoimmune disorder characterized by the infiltration of

58 lymphocytes into multiple organs and the resulting parenchymal damage. Milder effects are seen in CBL-B heterozygotes, suggesting some responses from the loss of a single copy of CBL-B. Additionally, CBL-B(-/-) animals are highly susceptible to experimental autoimmune encephalomyelitis (EAE). The autoimmunity is not due to a breakdown of self-tolerance, but rather a decrease in the activation threshold of the peripheral lymphocytes. CBL-B deficiency in T cells relieves the necessity for CD28-costimulation of the TCR in proliferation; plus, the loss of CBL-B enhances IL-2 production, Vav1 phosphorylation and its associated GEF activity. The older animals also display some progressive alterations in facial features and increased osteoclastogenesis. As described briefly, CBL-3 expression is not hematopoietic, but instead restricted to epithelial cells. The CBL-3-deficient animals bare no obvious defects: they are viable, healthy and fertile.393 The conditional knock-out animals noted above were generated through the use of the Cre-loxP system. B-cell specific ablation of both CBL and CBL-B was achieved when CBL floxed/CBL-B(-/-) mice were crossed to CD19-Cre transgenics.368 At birth these mice are normal and fertile, yet by 10 months of age one-half of the DKOs become moribund with systemic lupus erythematosus-like disease, possibly due to impaired B cell anergy to self antigen. BCR signaling in the DKO mice shows enhanced phosphorylation of the Syk, Vav and phospholipase C gamma 2 (PLCγ2) proteins but attenuation of BLNK phosphorylation. Dual CBL and CBL-B deletion in the T cell lineage was achieved when the same CBL floxed/CBL-B(-/-) mice were crossed to Lck-Cre transgenics.390 These mice are normal until three weeks of age at which time they become increasingly moribund. By 12-16 weeks of age the mice develop symptoms suggestive of autoimmunity, with vascular thickening and EMH. DKO T cells are hyperactive and driven to develop into effector-memory cells. As well, these cells show impaired TCR down- modulation, probably as a result of defective intracellular vesicle sorting, which leads to sustained ERK phosphorylation. Two CBL knock-in transgenics with loss-of-function mutations have been created to assess the functional importance of the TKB394 and RING finger domains.334 Glycine 304 in the TKB domain is the binding site for ZAP-70, yet the G304E knock-in mouse does not show any alterations in ZAP-70 activity. Similarly to the CBL(-/-) animals, cell surface expression of CD5 and CD69 is enhanced; meanwhile, CD3ε expression is reduced. The thymocytes of these mice show constitutive activation of Rac. The RING finger mutant mice were generated through targeted introduction of a cysteine to alanine mutation at residue 379. The homozygous mutants show reduced viability, with most embryos dying in utero. The surviving homozygous and heterozygous mutant mice show a large increase in CD5 and CD69 expression. Thymic loss occurs as the animals age and is due to enhanced susceptibility of the

59 mutant thymocytes to CD3-induced death. These mice also show enhanced activation of PKB and PLCγ1, likely through increased association of p85-PI3’K with the mutant CBL. These CBL protein animal studies define the important roles of CBL proteins in the lymphoid lineage. Moreover, they provide useful models for continued investigations of CBL proteins function in vivo.

1.3.8. CBL family members and human disease As evidenced by the discussion above, CBL proteins are essential modulators of signaling events, and as such, it would be expected that these proteins have important roles in human disease. It is postulated that CBL-B could play a role in the development or progression of multiple sclerosis, based on the knowledge that EAE is a good model of demyelinating disease,395 and the evidence that CBL-B deficiency enhances susceptibility to EAE.332 Yokoi and colleagues discovered CBL-B as a major susceptibility gene for diabetes mellitus in the rat.396 This same group recently identified CBL-B mutations in human diabetic subjects.397 In the last two years, CBL and CBL-B mutations have been characterized in a number of hematopoietic diseases, particularly the myeloproliferative neoplasms. In 2007, two papers were published that identified CBL and CBL-B RNA splice mutations, missense mutations and nucleotide insertions in several AML patients.398,399 Recently, single nucleotide polymorphism (SNP) and alleles- specific copy number screens have identified 11q acquired uniparental disomy (aUPD) or deletion in patients with BCR-ABL-negative CMLs, myelofibrosis, secondary AML, myelodysplastic syndromes and MDS/MPD diseases such as chronic myelomonocytic leukemia (CMML).400-402 CBL mutations are prominently associated with the 11q aUPD/deletions. Initial studies suggest that these mutations may confer shorter survival.401 Overall, these data point toward an important role for CBL proteins in multiple human diseases and will undoubtedly be the focus of many studies in the years to come.

1.3.9. Summary CBL proteins have been discovered in multiple organisms with three mammalian members: CBL, CBL-B and CBL-3. The multi-domain structure of these proteins allows them to function as both adaptor proteins and E3 ubiquitin ligases. As such, the CBL proteins regulate numerous cell-signaling mechanisms including the ERK, PKB and Rho family cascades, which control multiple biologic functions such as migration and adhesion. Mouse models with genetic deletion of the mammalian CBL proteins have expanded our knowledge of CBL function. The propensity of the CBL-B(-/-) mice to develop EAE suggests that CBL-B may play a role in multiple sclerosis. Furthermore, recent evidence indicates that CBL proteins may be involved in the development of diabetes and myeloid neoplasms.

60 THESIS STATEMENT CBL proteins are E3 ubiquitin ligases and adaptor proteins. There are three mammalian homologs: CBL, CBL-B and CBL-3. CBL and CBL-B have been well characterized in lymphocytes and the ubiquitin ligase function of CBL has been thoroughly investigated in epidermal growth factor receptor (EGFR) signaling. Moreover, CBL and CBL-B are upstream regulators of several guanine nucleotide exchange factors (GEFs) that control the activation of the Rho family of cytoskeletal regulators. As such, CBL proteins have been investigated for their role in modulation of migration, although a detailed signaling mechanism is not yet defined. As CBL and CBL-B knock-out mice show defects in T cell proliferation and activation,332,337,391,392 T cell receptor (TCR) signaling has been extensively studied. Yet little is known about the role of CBL proteins in myeloid cells. Recent evidence indicates that CBL proteins are mutated in some acute myeloid leukemias (AMLs) and other myeloid malignancies.398-400 As well, CBL is highly phosphorylated in cell lines expressing BCR-ABL134,281 – the causative agent of chronic myeloid leukemia (CML). Yet, bone marrow transduction and transplantation (BMT) experiments indicate that CBL is dispensable for CML development.133 As CBL-B is the other CBL family member with significant hematopoietic tissue expression,220 this homolog may compensate for the loss of CBL and may be a necessary component of CML disease development. CBL proteins modulate the motility of several cell types, although the data collected on CBL far outweighs our knowledge of CBL-B-dependent migration. While CBL and CBL-B have significant interactome overlap, the modulation of several key substrates varies between the proteins; for example, CBL predominantly promotes activation of phosphatidyl-inositol 3’ kinase (PI3’K), while CBL-B negatively regulates this protein. As such, it is important that migratory responses and signaling pathways are also studied in the context of CBL-B.

Based on our review of the literature the following HYPOTHESIS was developed:

CBL-B is an essential regulator of chronic myeloid leukemogenesis and cellular motility.

To test this hypothesis the following aims were investigated.

AIM 1: Assess the contribution of CBL-B toward BCR-ABL-mediated CML development in a murine BMT assay.

AIM 2: Utilize mouse embryonic fibroblasts (MEFs) deficient in CBL-B to study the potential regulation of motility and the biochemical pathways modulating this function.

CHAPTER 2

CBL-B DEFICIENCY DELAYS BCR-ABL-MEDIATED LEUKEMOGENESIS

A version of this chapter was submitted to Blood on March 3, 2009:

Badger-Brown KM, Bailey ML, Penninger JM, Barber DL. CBL-B is required for leukemogenesis mediated by BCR-ABL through negative regulation of bone marrow homing.

61 62 2.1. Abstract BCR-ABL induces chronic myeloid leukemia (CML) through the aberrant regulation of multiple signaling substrates. Previous research has shown that BCR-ABL mediates down-modulation of CBL-B protein levels. A murine bone marrow transplantation (BMT) study was performed to assess the contribution of CBL-B to BCR-ABL-induced disease. The predominant phenotype in the CBL-B(-/-) recipients was a CML-like myeloproliferative disease (MPD) similar to that observed in the wild-type animals, but with a longer latency, diminished circulating leukocyte numbers, and reduced spleen weights. Despite the decreased leukemic burden in comparison to their wild-type counter-parts, the CBL- B(-/-) animals displayed enhanced numbers of Gr-1+/Mac-1+ spleen cells and neutrophilia. All diseases were oligoclonal and no significant differences were observed in Abl kinase activity, global tyrosine phosphorylation, and PKB or ERK activation. Based on prior evidence of CBL-B-dependent motility toward serum-derived growth factor 1 alpha (SDF-1α) we hypothesized that CBL-B deficiency might impair bone marrow (BM) localization during transplantation. Our homing experiments showed reduced migration of CBL-B(-/-) cells to the bone marrow. We propose that BCR-ABL promotes granulopoiesis through negative regulation of CBL-B and that CBL-B deficiency diminishes homing of leukemic cells to the bone marrow, perturbing the proliferation of BCR-ABL-expressing malignant clones during CML development.

63 2.2. Introduction BCR-ABL is an oncogene formed by the reciprocal translocation of breakpoint cluster region (bcr) sequences on chromosome 22 with the c-abl gene on chromosome 9.45-47 The t(9;22) translocation is detected cytogenetically as the Philadelphia chromosome (Ph),44 and is harbored by the majority of patients with chronic myeloid leukemia (CML) and a small percentage of pediatric patients with acute lymphoblastic leukemia.41 BCR-ABL translocation variants encode proteins of 190 kDa,49,52,53 210 kDa,48and 230 kDa56 in size as determined by the breakpoints within the bcr gene; however, CML is most prominently associated with the p210 isoform.41 CML is characterized by a massive expansion of myeloid cells and their premature release from the BM microenvironment into the peripheral blood. The deregulated proliferation of these leukemic cells is due to the ability of BCR-ABL to confer factor-independent growth and protection from apoptosis,63,188,224,403,404 while the retention of cells within the bone marrow is dependent on the interplay between the adhesive and migratory forces. CML cells show inefficient adhesion to BM stroma and the extracellular matrix component fibronectin.156,157 As well, CD34+ CML cells display enhanced spontaneous motility.173,174 Combined, these alterations in cellular proliferation, apoptosis, adhesion and migration contribute to the pathogenesis of CML. BCR-ABL-mediated transformation has been extensively studied in cell lines but the pathogenesis of the human hematopoietic malignancy is more accurately reproduced in the murine BMT model. In this assay, BCR-ABL-transduced cells are intravenously injected into lethally irradiated recipient animals. These cells home to the BM niche where they reconstitute the entire hematopoietic compartment. The BCR-ABL-expressing cells display a growth advantage, and within approximately three weeks, the BMT recipients succumb to a fatal myeloproliferative disease (MPD) that resembles human CML..64,65 The oncogenic capacity of BCR-ABL results from deregulated tyrosine kinase activity,72,107 and enhanced activity relative to c-Abl.72,109,110 BCR-ABL constitutively activates several cell signaling cascades, including the Ras/ERK,113,118 PI3’K,187 Jak/STAT,143-146 SAPK/JNK,141 and Rac pathways.140 Transfer of signals to these pathways results from complex formation between BCR-ABL and its substrates. Of these, the Grb2 and Shc113,114; phosphatidylinositol-3 kinase (PI-3K)125,405,406; and CrkL,123 Crk and CBL proteins,125-127 are key signaling intermediates. CBL is prominently tyrosine phosphorylated by BCR-ABL.281 The CBL family was identified from the initial finding of a v-CBL retrovirus that caused B-cell lymphomas and myeloid leukemias in mice.268 The family consists of three mammalian members - CBL, CBL-B and CBL-3. Single gene deletion of the CBL family members in mice is not essential for development as all mice are viable and healthy.332,337,391,392 However, the CBL- and CBL-B-deficient mice exhibit defects in T cell development and activation, respectively.

64 The CBL proteins act as adaptor molecules, E3 ubiquitin ligases and transmitters of multiple phosphorylation signals that culminate in the activation of many cellular pathways.407 CBL family members also control cellular motility of hematopoietic cells. For example, both CBL and CBL-B positively regulate SDF-1α-stimulated motility of Jurkat T cells.379,383 As SDF-1α is a major chemokine known to regulate homing of cells to the BM, CBL proteins may critically regulate this key step in BM transplantation. CBL expression is required for efficient BCR-ABL-dependent in vitro cell transformation,133 but leukemia latency and phenotype is similar between recipients of wild-type and CBL knock-out BCR- ABL-transduced bone marrow cells (BMCs). These results suggest that other proteins can compensate in vivo for the loss of CBL. As CBL-B shows significant hematopoietic tissue expression,220 this CBL family member could play an essential role downstream of BCR-ABL. Interestingly, CBL-B protein levels are reduced by BCR-ABL expression, and the introduction of CBL-B into BCR-ABL-expressing cells augments spontaneous motility.134 In this study, we utilized the murine BMT assay to assess the role of CBL-B in the development of BCR-ABL-induced leukemia. In comparison to the wild-type BCR-ABL transplant animals, the recipients of CBL-B(-/-) BCR-ABL BMCs succumbed to a longer latency MPD, with a reduced leukemic burden at morbidity but enhanced granulopoiesis. Impaired engraftment in the BM suggested CBL-B-dependent regulation of motility that could account for the prolonged survival of the transplant animals receiving CBL-B-deficient BCR-ABL-expressing leukemic cells.

2.3. Materials and Methods 2.3.1. DNA Constructs The pcDNA3-TEL-JAK2(5-19) construct used for in vitro experiments was described previously,408 while the pGD-210 (BCR-ABL) construct was kindly provided by R. van Etten (Medford, MA). The Mig210 construct, generously provided by M. Carroll (Philadelphia, PA), was used to express the p210 isoform of BCR-ABL in BMCs that has been previously demonstrated to efficiently generate MPD in mice.202 The vesicular stomatitis virus G (VSV-G) expression plasmid was courteously supplied by G. Sauvageau (Montreal, QC). Both MSCV-IRES-EGFP (MIEV) and the vector expressing Gag and Pol sequences (SV-ψ--env-) were generously provided by J. E. Dick (Toronto, ON).

2.3.2. Cell Culture Ba/F3 cells expressing the pcDNA3, pcDNA3-TEL-JAK2(5-19) or pGD-210 (BCR-ABL) constructs were generated by H. Kim via retroviral transduction. All Ba/F3 cell lines s were maintained in RPMI Complete media (RPMI 1640 plus antibiotics containing 10% (v/v) Fetal Calf Serum (FCS; HyClone, Thermo Fisher Scientific, Inc., Waltham, MA) and 50 µM β-mercaptoethanol (β-ME; Fisher)

65 supplemented with 100 pg/mL of interleukin-3 (IL-3; R & D Systems, Minneapolis, MN). For clonal maintenance, Ba/F3 pcDNA3 AND Ba/F3 pcDNA3-TEL-JAK2(5-19) cells were cultured in RPMI Complete media with the addition of 100 pg/mL IL-3 and 1 mg/ml Geneticin (Invitrogen, Carlsbad, CA). Ba/F3 BCR-ABL cells were grown in Ba/F3 Complete media supplemented with 100 pg/mL IL-3, 1 mg/ml Geneticin and 1 µg/ml of Imatinib (Novartis, Basel, Switzerland) dissolved in dimethyl sulfoxide (DMSO; Fisher). For experimentation all cell lines were expanded in WEHI IL-3-conditioned media (RPMI Complete media supplemented with 10 – 15% culture supernatant from WEHI-3 cells). For production of the virus used in BMT, 293T cells were expanded in Iscove’s Modified Dulbecco’s Media (IMDM) plus antibiotics supplemented with 10% FCS.

2.3.3. Animals Studies were approved by the Animal Care Committee at the Ontario Cancer Institute (OCI), University Health Network, Toronto, ON. The animals used for BMTs were back-crossed in our laboratory onto the BALB/c line to a minimum of six generations (F6). CBL-B-heterozygous matings produced both wild-type and CBL-B(-/-) donor animals. All animals were housed in the OCI Animal Research Centre in micro-isolator cages and maintained with autoclaved chow and acidified water. BALB/c recipient mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Genomic DNA was isolated from all animals by Proteinase K digestion of tail clippings and phenol/chloroform extraction. DNA was resuspended in double-distilled water (ddH20) and genotypes were determined through PCR amplification of CBL-B WT or mutant sequences as described previously.392 Primer sequences are as follows: the sense-strand WT (5’- CATCTCAGTGTTTGAATTTG-3’) and mutant (5’-TTCCTCCCACTCATGATCTATAG-3’); the antisense strand (5’-GGAAAAATATTAGTTACAACTGG-3’). WT and mutant reactions were performed ++ separately. Reaction mixes contained 1x PCR Buffer (minus Mg ), 2 mM MgCl2, 0.2 mM dNTP mix, 2.5 U recombinant Taq DNA Polymerase, 0.5 µM of one sense primer and 0.5 µM of the anti-sense primer. All components used in reaction mixes were obtained from Invitrogen (Carlsbad, CA). PCR conditions included an initial denaturation step at 93oC for 2 minutes followed by 40 cycles of: denaturation at 94oC for 30 seconds, annealing at 52oC for 30 seconds and extension at 72oC for 30 seconds. A final extension step at 72oC ran 7 minutes.

2.3.4. Generation of retroviral stocks Calcium phosphate transient co-transfections (CalPhos Mammalian Transfection Kit, Clontech, Mountain View, CA) of 293T cells (5 x 106) were performed with 10 µg of retrovirus, 10 µg of the SV- ψ--env- packaging construct and 3.5 µg of the VSV-G envelope vector. During virus production, 293T

66 cells were grown in IMDM media minus antibiotics supplemented with 10% FCS. Medium was changed at 16 hours post-transfection, followed by supernatant collection at 40 and 64 hours. Harvested virus was immediately filtered (0.5 µM filter) and concentrated by ultracentrifugation at 53,000 g for 90 minutes at 4oC. Viral pellets were re-suspended in a final volume 1/333 times the initial volume (Mig210) or 1/167 times the initial volume (for MIEV), ) and frozen, and on dry ice. Viral stocks were stored at –80oC. For estimation of the retroviral titer, 3 x 106 Ba/F3 cells were transduced in a single well of a 6- well suspension dish with 375 µL retroviral supernatant in a total volume of 3 mL WEHI IL-3- conditioned media supplemented with 3 µg/mL Polybrene® (1,5-dimethyl-1,5-diazaundecamethylene polymethobromide, hexadimethrine bromide; Sigma-Aldrich, St. Louis, MO). Infection media was replaced with 6 mL fresh WEHI at 24 hours. Cells were collected 48 hours post-infection and analyzed by flow cytometry for the expression of GFP. The relative viral titer in colony forming units (CFU)/mL was calculated as the percentage of GFP-positive (GFP+) cells multiplied by the number of cells infected (3 x 106) and divided by the volume of supernatant applied to these cells in milliliters (0.375 mL). All retroviral stocks were used at titers of 2 x 105 CFU/mL for BMC transduction.

2.3.5. Bone marrow transduction and transplantation Donor and recipient mice were used at six- to eight-weeks of age. Donor mice were primed with a 200 mg/kg 5-fluorouracil (5-FU; Sigma-Aldrich) intraperitoneal injection four days prior to BM 205 harvest. Animals were sacrificed by carbon dioxide (CO2) asphyxiation followed by cervical dislocation. BMCs were collected by flushing of the femurs and tibias with a Pre-stimulation Cocktail of High Glucose (H21) Dulbecco’s modified Eagle’s medium (DMEM) plus antibiotics that included 10% FCS, 5% WEHI IL-3-conditioned media, 6 ng/mL rmIL-3, 10 ng/mL recombinant murine interleukin-6 (rmIL-6), and 50 ng/mL recombinant murine stem cell factor (rmSCF). All cytokines were purchased from R & D Systems, Inc. (Minneapolis, MN). Cells were plated, without lysis of erythrocytes, into adherent-cell culture dishes at a concentration of 1 x 106 white blood cells (WBCs)/mL of Pre- stimulation Cocktail. Twenty-four hours post-plating, trypsinized BMCs at a concentration of 1 x 106 WBCs/mL, were subjected to two rounds of infection (24 hours each) in Pre-stimulation Cocktail containing 3 µg/mL Polybrene® and virus at a final infectious dose of 2 x 105 CFUs/mL. Transduced BMCs were collected, washed once in phosphate-buffered saline (PBS) and re-suspended to a concentration of 1.2 x 106 WBC/mL in PBS. Following lethal irradiation (9 Gy) each mouse received 5 x 105 WBCs through tail vein injection.

2.3.6. Analysis of diseased mice After transplantation, recipient mice were monitored every three days for signs of disease as evidenced by cachexia, weight loss and splenomegaly. Periodic saphenous vein bleeds were performed

67 and peripheral blood (PB) WBC counts were obtained. Pre-moribund animals were sacrificed by CO2 asphyxiation and hematopoietic tissues were harvested. Spleen and liver weights were measured. For histopathological examination, PB smears and BM cytospins were stained with May-Grünwald and Giemsa (both from EMD Chemicals Inc., Gibbstown, NJ). Spleen and liver single-cell suspensions were created by manual force through wire mesh and erythrocytes were lysed with ACK (pH 7.3; 155 mM ammonium chloride, 100 µM disodium ethylene diamine tetraacetic acid (EDTA) and 10 mM potassium bicarbonate). Cell suspensions were used for flow cytometry analysis, extraction of protein lysates for immunoblotting, and preparation of genomic DNA for Southern blotting.

2.3.7. Antibodies The monoclonal anti-phosphotyrosine antibody (4G10) and the anti-ERK1/2 rabbit polyclonal antibody (06-182) were purchased from Millipore (Billerica, MA). The monoclonal phospho-ERK1/2 (E-4) and the rabbit polyclonal Cbl (C-15) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Both of the CBL-B antibodies used for immunoprecipitation (H-454) and immunoblotting (G-1) were obtained from Santa Cruz. The mouse monoclonal antibody to Tubulin (DM1A) was procured from EMD Chemicals, Inc. (Gibbstown, NJ). Mouse monoclonal c-Abl (8E9) was obtained from BD Biosciences (San Jose, CA). Both polyclonal PKB antibodies for detection of phosphorylation at serine 473 (9271) and total PKB levels (9272) were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Secondary antibodies, either horseradish peroxidase (HRP)-labeled Protein A or HRP-linked mouse immunoglobulin G (IgG; from sheep), were obtained from GE Healthcare (Piscataway, NJ). All flow cytometry antibodies were acquired from BD Biosciences including: Phycoerythrin (PE)-conjugated anti-mouse Thy-1.2 (53-2.1), CD4 (H129.19), TER-119, B220 (RA3-6B2), Sca-1 (D7), and Gr-1 (RB6-8C5); and PerCP-Cy5.5-conjugated rat monoclonal CD8a (53-6.7) and CD11b/Mac-1 (M1/70).

2.3.8. Immunoprecipitation and Immunoblotting For biochemical studies in Ba/F3 cells, cytokine deprivation was initiated through triplicate washings with 10 mM HEPES (pH 7.4)/Hanks balanced salts, followed by incubation in RPMI Complete media for four hours at 37oC. Cells were stimulated for 10 minutes at 37oC in pre-warmed RPMI Complete media either in the presence or absence of 100 pg/mL IL-3. The cells were washed once in 10 mM HEPES (pH 7.4)/Hanks balanced salts containing 10 mM sodium pyrophosphate (Na4P2O7), 10 mM sodium fluoride (NaF), 10 mM EDTA and 1 mM sodium orthovanadate (Na3VO4), followed by complete lysis of Ba/F3 cells achieved through brief vortexing of 1 x 107 pelleted cells in 1 mL of ice-cold Lysis

Buffer (1 % (v/v) Triton X-100, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM Na4P2O7, 10 mM

NaF, 10 mM EDTA, 1 mM Na3VO4, 1 mM phenylmethyl sulfonyl fluoride (PMSF) and 1x Complete

68 Protease Inhibitor Cocktail (Roche, Mannheim, Germany)). After a five-minute incubation on ice, cell membranes were pelleted by a 10,000 g centrifugation at 4oC for 5 minutes. Protein concentration in the cell lysate was determined by the Bradford Colorimetric Assay (Bio-Rad, Hercules, CA) and detected at 595 nm. Immunoprecipitations with 1 – 2 mg of protein, 50 µL of Protein A Sepharose CL-4B beads (GE Healthcare) and 8 µg of antibody were incubated overnight at 4oC. For Cbl immunodepletion experiments, the beads were precipitated by centrifugation and the remaining protein suspension was subjected to a second round of overnight immunoprecipitation with the anti-CBL-B (H-121) antibody. Beads were washed three times with IP Wash Buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1 %

(v/v) Triton X-100, 10 mM Na4P2O7, 10 mM NaF, 10 mM EDTA and 1 mM Na3VO4), dried with a Hamilton syringe, and re-suspended in 50 µL of 1x Sample Buffer (69 mM Tris-HCl (pH6.8), 11% (v/v) glycerol, 2.2% (w/v) sodium dodecyl sulfate (SDS) and 0.02% (w/v) bromophenol blue) containing 50 mM dithiothreitol (DTT). For analysis of Ba/F3 cell lysates, each sample was equated to 100 mg of protein and an equivalent volume of 2 x Sample Buffer supplemented with 100 mM DTT was added. Directly before resolution by SDS-polyacrylamide gel electrophoresis (PAGE), all samples were boiled for five minutes at 95oC to release the immune complexes. Proteins samples were separated by Laemmli electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes for immunoblotting . Protein lysates from mouse tissue were prepared by the following protocol. Erythrocyte-free cell suspensions were washed once with PBS, and resuspended at a concentration of 1 x 107 cells/mL in PBS. An equal volume of 2x Sample Buffer plus 100 mM DTT was added and samples were immediately boiled at 95oC for five minutes. Lysates were cleared by centrifugation before SDS-PAGE and transfer to PVDF. For most immunoblot experiments, membranes were blocked for one hour with 2.5% (w/v) bovine serum albumin (BSA; Sigma-Aldrich) in Tris-buffered saline (TBS; 50 mM Tris-HCl (pH 8.0), 150 mM NaCl). The primary antibody was then added at an optimal concentration in TBS supplemented with 0.1% (v/v) Tween-20 (TBST) for a one-hour incubation. The membrane was subsequently washed four times with TBST, and the relevant HRP-conjugated secondary antibody was applied at the correct concentration in TBST for 30 minutes. Finally, the membrane was washed four more times in TBST before visualization with Western LightningTM Chemiluminescence Reagent Plus (ECL; PerkinElmer, Waltham, MA) and autoradiographic HyperfilmTM (GE Healthcare). Reprobing of the blots was achieved through stripping of the membranes for 30 minutes at 50°C in 62.5 mM Tris-HCl (pH 6.8), 2% (v/v) SDS and 0.1 M β-ME. Membranes were rinsed twice in TBST before blocking. For phospho-PKB IB, blocking was performed for one hour with 5% (w/v) skim milk powder dissolved in TBST. A 1:1000 dilution of primary antibody in 1% BSA/TBST was applied to the membrane overnight at 4oC. After washing the membrane six times, incubation with a 1:2000 dilution of

69 HRP-conjugated Protein A in 2% milk/TBST was performed for one hour. A final wash-cycle of six times was carried out and the membrane was visualized with ECL. BCR-ABL protein was detected by anti-ABL IB. Membranes were blocked for one hour in 5% milk/TBST. Incubation with a 1:250 dilution of primary antibody in 5% milk/TBST was allowed to proceed overnight at 4oC, followed by six washes. A one-hour incubation of a 1:2000 dilution of HRP- linked anti-mouse in 5% milk/TBST was followed by six final washes before ECL detection. Membranes probed for phospho-ERK1/2 were blocked first for one hour in 5% milk/TBST and then incubated overnight at 4oC with a 1:400 dilution of the primary antibody in 5% milk/TBST. The membrane was washed three times, incubated in HRP-linked sheep anti-mouse at a 1:5000 dilution in 5% milk/TBST, followed by a final three washes before ECL detection. For total ERK1/2 reprobing, membranes were blocked in 3% milk/TBST for one hour, and incubated with a rabbit polyclonal antibody (0.25 µg/ml in 3% milk/TBST) overnight at 4oC. After two washes, membranes were incubated with HRP-conjugated Protein A secondary antibody (1:5000 in TBST) for one hour, washed four times and developed with ECL. For detection of equivalent loading, an anti-Tubulin IB was performed. Membranes were blocked in 5% milk/TBST for one hour followed by two quick rinses with TBST. The primary antibody was applied at a 1:400 dilution in 2.5% BSA/TBST overnight at 4oC. The membranes were then rinsed three times, incubated 30 minutes with a 1:5000 dilution of HRP-linked anti-mouse in 2.5% BSA/TBST, then rinsed three times more before detection by ECL.

2.3.9. Flow cytometry Cells obtained from the peripheral blood, BM and spleen were analyzed by flow cytometry. All cells were blocked with anti-mouse CD16/CD32 (2.4G2; BD Biosciences) and stained with a combination of fluorescently labeled primary antibodies as described above. Measurements were made on a FACSCalibur machine (BD) and data was analyzed with FlowJo software (Ashland, OR).

2.3.10. Homing assay BMC harvested from wild-type and CBL-B(-/-) mice were ACK-treated and then labeled with the red fluorescent dye PKH26 (Sigma-Aldrich) according to the manufacturer’s instructions. Greater than 99% of the cells were efficiently labeled. Five million viable PKH26-labeled cells were transplanted by lateral tail vein injection into each lethally irradiated recipient BALB/c mouse. Separate recipients were used for each of the CBL-B -WT or -KO donor cells, while control animals were injected with PBS alone. Three hours after transplantation, BM and spleen cells were retrieved and red blood cells were lysed with ACK. Cells were washed once with PBS and the percent of PKH26+ cells in each tissue was measured by flow cytometry.

70 2.3.11. Southern blotting Genomic DNA from ACK-treated spleen or liver cell suspensions of diseased mice was obtained by Proteinase K digestion followed by phenol/chloroform extraction. To detect the provirus, 15 µg of genomic DNA was digested with XbaI, separated on a 1 % agarose gel, and then transferred to a HybondTM-XL membrane (GE Healthcare). The blot was hybridized with a radioactively labeled probe derived from a 1.1 kb HindIII fragment of the IRES-GFP sequences in the Mig210 retroviral vector. The washed membrane was exposed to x-ray film. For detection of the number of proviral integrants, 15 µg of genomic DNA was digested with the enzyme EcoRI, which cuts 5’ to the IRES-GFP gene sequences in the vector, and generates a specific fragment determined by the first EcoRI site encountered in the cellular sequences flanking the 3’ LTR. Agarose gel separation and radioactive probing were performed as for the XbaI digestion.

2.4. Results 2.4.1. CBL-B protein is specifically decreased in BCR-ABL-positive cells. CBL-B was previously shown to co-immunoprecipitate with the BCR-ABL oncogene.134 As well, CBL-B protein levels are significantly reduced in the presence of multiple translocation fusion kinases, including BCR-ABL, TEL-ABL, TEL-JAK2, and TEL-PDGFR. In our laboratory we consistently observed reduced expression of CBL-B in the cell line expressing BCR-ABL, but the Ba/F3 TEL-JAK2 cells showed equivalent CBL-B expression to the Ba/F3 control (Figure 2-1). Lysates from a Ba/F3 cell line were included as a negative control. This result suggested that CBL-B may be distinctly regulated by the BCR-ABL kinase.

2.4.2. CBL-B-deficiency lengthens the latency of leukemogenesis. As a result of our findings in Figure 2-1 we initiated murine BMT experiments to clearly determine the role of CBL-B in the development of BCR-ABL-induced leukemia. VSV-G-pseudotyped retroviral supernatant, for both the control (MIEV) and BCR-ABL (Mig210) vectors, was generated in 293T cells and concentrated by ultracentrifugation. High titer virus (at least 5 x 105 colony forming units per mL) was efficiently generated as determined by GFP expression of transduced Ba/F3 cells. As wild- type and CBL-B(-/-) BMCs from 5-fluorouracil (5-FU)-treated donors showed comparable levels of infection (Figure 2-2A) the subsequent injection into lethally-irradiated syngeneic recipient mice was executed. Recipient animals were monitored weekly for signs of cachexia and elevated peripheral WBC counts. Moribund animals were promptly euthanized and histopathological examination was performed. Within three weeks of transplantation, all animals receiving wild-type BCR-ABL-transduced BMCs succumbed to a fatal MPD that closely resembled human CML (Figure 2-2B). These mice displayed elevated WBC counts (18-242 x 106 /mL) with a differential shift toward mature neutrophils (Figure 2-3A and 2-3C). Enhanced numbers of nucleated erythroid cells were also observed. The BM

Figure 2-1. BCR-ABL expression results in constitutive tyrosine phosphorylation of CBL and CBL-B proteins, and a reduction in CBL-B protein levels. Ba/F3, Ba/F3 TEL-JAK2(5-19) and Ba/F3 BCR-ABL-p210 cell lysates were probed by immunoblotting (IB) for tyrosine-phosphorylated proteins with a phospho-tyrosine (pTyr) antibody (upper panel). Total CBL and CBL-B proteins were detected upon reprobing with CBL and CBL-B antibodies, respectively (lower panels).

72 Figure 2-2. Transplant recipients of CBL-B deficient cells show delayed latency of BCR-ABL- mediated leukemogenesis. (A) Transduction efficiency was compared between wild-type and CBL-B(-/-) BMCs. Mice were treated with 5-FU (200 mg/kg, intraperitoneally) four days prior to BMC harvest. The cells were pre-stimulated overnight in a cocktail of growth factors, then transduced by 2 rounds of co-culture with MIEV. The cells were washed, fixed, and analyzed after 48 hours of infection for GFP expression (a marker of infection efficiency). (B) Kaplan-Meier survival analysis of BMT recipients from 5-FU-treated donors transduced with control MIEV or Mig210 virus. The number of recipients are shown in parentheses. BCR-ABL curves were generated from at least three independent experiments. The disease phenotype is indicated by the shading: CML-like MPD (closed), lymphoid disease (open).

Figure 2-2

74 Figure 2-3. Myeloproliferative disease in the majority of CBL-B(-/-) BCR-ABL BMT recipients was confirmed by the peripheral blood differential and histopathology. (A) Peripheral blood was obtained at morbidity. To determine counts of WBCs only, peripheral blood was diluted with Turk’s reagent that lyses erythrocytes and stains WBCs for microscopic visualization. All values were reported as the number of WBCs per milliliter (mL) of blood. Bars represent the median. (B) Extramedullary hematopoiesis was observed in the spleens of BCR-ABL BMT animals. This tissue was weighed during dissection and compared to the total body weight at morbidity. Bars represent the median. (C) Differentials were recorded for multiple animals (sample size of n = 5 for CBL-B(-/-); n = 4 for wild-type) through counts of 200 WBC in randomly-selected sections of the PB smear. (D) Histopathological analysis of PB and BM was visualized by microscopic evaluation of slides stained with May-Grünwald and Giemsa.

Figure 2-3

76 was hypercellular due to the expansion of maturing granulocytes or fully differentiated neutrophils (Figure 2-3D). Extramedullary hematopoiesis (EMH) was evidenced by splenomegaly (0.2-0.9 g) and hepatomegaly (1.5-3 g) (Table 2-1, Figure 2-3B), and the expansion of GFP-positive cells in these organs (data not shown). Again, immature myeloid cells had infiltrated both of these organs. A high number of immature erythroid cells were also observed in the spleens. The wild-type BCR-ABL-infected recipients showed signs of pulmonary hemorrhage, the probable cause of death. While 100% of the recipients of wild-type BCR-ABL-infected recipients died within 21 days of transplantation, the CBL-B(-/-) BCR-ABL recipients exhibited a prolonged survival (Figure 2-2B; median = 27 days, mean = 28 days; P = 0.0106 as determined by the log-rank test). The majority (90%) of these animals displayed an MPD with similar disease pathology to their wild-type counterparts, showing BM myeloproliferation, EMH and pulmonary hemorrhage. The PB counts and spleen weights showed tighter distributions of the values in these CBL-B(-/-) animals, 43 – 127 x 106 cells/mL and 0.3 – 0.5 g, respectively (Figure 2-3A and 2-3B). However, when these wild-type and CBL-B(-/-) populations were compared for variance of the means by a two-tailed Mann-Whitney U test, there was no significant difference between the two populations at P < 0.05 (P = 0.1416 for PB counts and P = 0.0735 for spleen weights). The differentials were also distinct, with the CBL-B(-/-) BMT animals displaying a larger expansion of granulocytes (compared to the wild-type mice) that overwhelmed the lymphocyte population (Figure 2-3C). While the wild-type BCR-ABL transplants showed a highly homogeneous pathology, the CBL- B(-/-) BCR-ABL BMTs did not give 100% penetrance to MPD (Figure 2B): a single animal receiving the CBL-B(-/-) BCR-ABL marrow was diagnosed with a lymphoid leukemia characterized by mild leukocytosis (25 x 106 cells/mL) and a PB differential predominated by blasts (Figure 2-3B). Control mice receiving vector-infected (MIEV) cells did not display any signs of disease development, and remained healthy until they were euthanized at the end of the 118-day study (Figure 2- 2B). These animals had normal WBC counts (10-20 x 106 /mL), composed primarily of lymphocytes (Figure 2-3B), and typical spleen and liver weights.

2.4.3. CBL-B(-/-) BCR-ABL-positive MPD animals have enhanced numbers of Gr-1+ and Mac-1+ spleen cells. Immunophenotyping was used to dissect cell types that might be alternatively regulated in the diseased tissues of the BMT mice. For this experiment we utilized spleen cells as this organ yielded a sufficiently large number of cells for multiple analyses and was of keen interest given the differing spleen weights of the diseased mice from the two CBL-B genotypes. The proliferation of BCR-ABL-positive (BCR-ABL+) cells was measured by flow cytometry enumeration of GFP+ cells. (As GFP and BCR-ABL are encoded in a bicistronic message, GFP expression can act as a surrogate marker for BCR-ABL.) Those mice suffering from an MPD had

78 significant splenic extramedullary hematopoiesis, as approximately 20% of the cells were GFP+ (Figure 2-4A). In contrast, the vector control-infected mice showed minimal infiltration of GFP+ cells. The percentages of Gr-1+/Mac-1+ granulocytes and TER-119+ erythroid cells were considerably increased in these diseased animals. The Gr-1+ and Mac-1+ GFP-negative cells were also amplified, possibly through enhanced cytokine production from BCR-ABL+ cells.207 As well, Gr-1 displayed a wide range of expression, indicative of the expansion of both mature and immature myeloid cells in the spleen. This contrasts with Gr-1 expression in the peripheral blood, where most cells had very high levels of Gr- 1 expression and were therefore more differentiated (data not shown). While myeloid cells were amplified in the spleens of BCR-ABL BMTs from both CBL-B genotypes the percentages varied: CBL-B deficient recipients showed enhanced percentages of Gr-1+ and Mac-1+ percentages as compared to their wild-type counterparts, with a statistically significantly increase in Mac-1+ cells (Figure 2-4B; T test, P = 0.05). These data are akin to the observations in the peripheral blood, where CBL-B deficiency enhanced granulopoiesis.

2.4.4. The phospho-tyrosine profile of MPD mice is similar between wild-type and CBL-B(-/-) recipients. To test the prevalence of BCR-ABL protein throughout the diseased animals we derived cell lysates from both the splenic and hepatic compartments. Ba/F3 BCR-ABL cells were included as a positive control. Negative controls included both Ba/F3 lysates and protein from the spleen or liver cells of a control-infected BMT mouse. Loading was compared between lanes with an anti-ABL immunoblot and detection of the c-ABL protein was observed at 150 kDa. Anti-ABL immunoblots clearly showed expression of BCR-ABL at 210 kDa in the CBL-B(-/-) BCR-ABL mice, in both the liver and the spleen (Figure 2-5A, Lanes 7 and 8). While anti-ABL immunoblots did not detect BCR-ABL in the wild-type BCR-ABL samples (Lanes 2 and 4), its presence was inferred from the more sensitive phospho-tyrosine immunoblot where a phosphorylated band was visualized at the appropriate molecular weight in the BCR-ABL+ lanes and was absent from the control wild-type lanes. From the phospho-tyrosine immunoblot, BCR-ABL expression could also be confirmed in both the liver and spleen of wild-type-diseased animals. In agreement with the results of others,64 lower molecular weight bands were visualized between the 210 kDa BCR-ABL and c-ABL proteins, representative of degraded BCR-ABL formed from cleavage with endogenous proteases of the neutrophil compartment. Given that the BCR-ABL protein could be observed in both the wild-type and CBL-B(-/-) animals, we assessed the global phospho-tyrosine profiles of these diseased mice for differences that might explain the altered disease phenotypes. Similar proteins were tyrosine phosphorylated in each of the BCR-ABL BMT recipients (Figure 2-5A, and lower exposures). Therefore, this analysis was

79 Figure 2-4. CBL-B(-/-) MPD animals display enhanced Gr-1+ and Mac-1+ percentages in the spleen. Immunophenotypic analysis by flow cytometry was performed on splenic cell suspensions. (A) Histograms of green fluorescent protein (GFP) expression are shown, as well as two-parameter dot plot profiles for cells stained with phycoerythrin-labelled Gr-1 or TER-119, and PerCP-Cy5.5-conjugated Mac-1 antibodies. Representative examples from each BMT group are shown, including control (MIEV)- infected recipients and MPD animals from both the wild-type and CBL-B(-/-) backgrounds. (B) The average Gr-1 and Mac-1 staining from animals of each BMT group (n=2 for controls; n=4 for BCR-ABL animals) are depicted graphically. Error bars represent standard deviation, *, P = 0.05 by T test.

Figure 2-4

81 Figure 2-5. Recipients of BCR-ABL-expressing wild-type or CBL-B knock-out cells show similar protein tyrosine phosphorylation and activation profiles. (A) BCR-ABL protein expression and tyrosine phosphorylation was detected. Cells from mouse spleen and liver and cytokine-deprived Ba/F3 (B) cell lines were lysed in equal volumes of PBS and 2 x SDS loading buffer at a concentration of 107 cells/mL. Samples were resolved with 6% SDS-PAGE and tyrosine-phosphorylated proteins were detected by a pTyr immunoblot (upper panel). Reprobes for BCR-ABL and c-ABL were detected with an anti-ABL antibody (lower panel). (B) Spleen cell lysates from control-infected mice or BCR-ABL-BMT animals were probed with antibodies directed toward total BCR-ABL (anti-ABL) or CBL-B proteins, plus phosphorylation-specific antibodies for the PKB and ERK proteins. These blots were stripped and reprobed with total PKB, ERK or tubulin antibodies as loading controls. Phosphorylation levels were quantified using ImageJ software.

Figure 2-5

83 insufficient to derive altered signaling pathways that might contribute to the longer latenc CBL-B(-/-) BMT latency.

2.4.5. BCR-ABL protein expression is not enhanced by CBL-B deficiency. The results presented in Figure 2-5A suggested that BCR-ABL protein expression was stronger in CBL-B(-/-) cells as compared to the wild-type cells, even though our results from flow cytometry revealed similar percentages of GFP+ cells. To thoroughly investigate relative protein levels, lysates from equivalent numbers of spleen cells were obtained from multiple BCR-ABL and control BMT animals. Immunoblots were probed for expression of the BCR-ABL tyrosine kinase with an anti-ABL antibody. Our results showed ranges in BCR-ABL protein expression among the animals, with no visible trend among the CBL-B(-/-) cells (Figure 2-5B). Instead, the level of BCR-ABL protein correlated with GFP expression. For example, Mouse 21R had 49% GFP+ cells in the spleen and a strong expression of BCR-ABL. Meanwhile, Mouse 21RL had 14% of its splenic cells as GFP+ and concordantly showed weaker expression of BCR-ABL. As a result, phenotypic difference between the CBL-B MPD animals could not be explained by altered BCR-ABL protein expression.

2.4.6. CBL-B deficiency does not alter BCR-ABL-mediated PKB and ERK signaling. As our biochemical studies to address BCR-ABL expression and global tyrosine phosphorylation profiles did not show divergences to explain the differing disease pathogenesis of the wild-type and CBL-B(-/-) animals we investigated the relative activation levels of two key effectors of BCR-ABL transformation. We again chose lysates from spleen cells as this organ was highly infiltrated by granulocytes at the time of morbidity, with 20-50% of the cells expressing the Gr-1 and Mac-1 markers. For both protein kinase B (PKB) and extracellular-regulated kinase (ERK), the levels of protein phosphorylation were highly variable among recipients (Figure 2-5B). When protein phosphorylation was compared to total protein levels, there were no clear differences in the PKB and ERK activation profiles between the diseased CBL-B(-/-) and wild-type mice. The dissimilarity in total protein levels could be explained by the variability in myeloid content of the spleen cell lysates, as those animal whose spleens were predominated by Gr-1+/Mac-1+ cells (e.g., Mouse 21R with 51% Gr-1+/Mac-1+) produced more protein as compared to animals whose Gr-1+/Mac-1+ splenic content was lower (e.g., Mouse 21RL with 22% Gr-1+/Mac-1+). Therefore, from this analysis, we could not derive any changes in PKB and ERK phosphorylation between the CBL-B(-/-) and wild-type animals that would explain the altered leukemic phenotypes.

2.4.7. CBL-B knockout cells display decreased homing to the BM. Okabe et al. recently showed that Jurkat T cells treated with CBL-B-specific siRNA were less motile toward SDF-1α than untreated cells.379 As SDF-1α is the major chemokine that controls BMC

84 homing, we hypothesized that CBL-B(-/-) intravenously injected cells may be impaired in their homing to the BM microenvironment. The resulting reduction in cell numbers may account for the delayed latency and reduced leukemic burden. To test this hypothesis, we performed in vivo homing assays with the use of the red fluorescent marker PKH26 (Sigma) for labeling intravenously injected cells. BMCs were derived from wild-type and CBL-B(-/-) animals and labeled ex vivo with PKH26. Equivalent labeling of wild-type and CBL-B(-/-) cells was apparent (Figure 2-6A). Labeled cells were intravenously transplanted into irradiated recipients and allowed to migrate for three hours. Following this homing period, mice were sacrificed and cell suspensions were created from the BM and spleen. The percentages of homed cells were determined by flow cytometry and are presented in their raw form in Figure 2-6B or graphically as in Figure 2-6C and 2-6D. PKH26-stained cells were readily observed in those mice injected with the fluorophore-labeled cells, while no fluorescence was visualized in control mice injected with saline (data not shown). As depicted in Figure 2-6C, there was a statistically significant decrease in the ability of CBL-B(-/-) cells to home to the BM. Splenic homing was similar between recipients of wild-type and CBL-B knock-out cells (Figure 2-6D). These figures point to a positive role for CBL-B in regulating effective homing to the BM.

2.4.8. BCR-ABL DNA integrates into the BMC of CBL-B(-/-) BCR-ABL recipient mice in an oligoclonal fashion. As CBL-B(-/-) BCR-ABL BMT animals eventually succumbed to an MPD, we presumed that the infected cells contained BCR-ABL provirus. To confirm the presence of bcr-abl sequences in affected tissues, DNA was isolated from the spleens and livers of multiple animals. Southern blot analysis was performed on DNA digested with XbaI, which releases the provirus by cutting once within each of the 5’ and 3’ long-terminal repeats (LTRs). Proviral sequences were hybridized with a 32P-labelled ires-gfp probe. Bcr-abl provirus with an intact structure was detected in Ba/F3 cells infected with Mig210, as well as in the wild-type BCR-ABL and CBL-B(-/-) BCR-ABL recipient tissues (Figure 2-7A). Hybridization of the probe with the negative control sample was not observed as this DNA was derived from an MSCV-IRES-EGFP (MIEV)-infected transplant animal, and under our conditions, the ires-gfp probe could not hybridize with egfp sequences. The clonality of the leukemic animals was addressed in EcoRI digests of the same genomic DNA. EcoRI digestion creates one cut within the Mig210 proviral DNA, and another outside the proviral sequences. Southern blots were performed with the same ires-gfp probe. Enumeration of the provirus detected several distinct bands in each of the tissues of the wild-type and CBL-B(-/-) -BCR- ABL recipients, supporting the oligoclonal nature of the disease (Figure 2-7B). Three or four proviral integrants were observed in each animal, regardless of the genetic background of the injected cells. Therefore, the observed delay in leukemogenesis of the CBL-B(-/-) BCR-ABL BMT animals could not be attributed to a reduced number of infectious clones.

85 Figure 2-6. CBL-B(-/-) cells do not effectively home to the BM. (A) Donor BM cells from wild-type and CBL-B(-/-) animals, were labeled with PKH26, which fluoresces in the FL3 (red) channel. Viable cells were selected from forward scatter (FSC) versus side scatter (SSC) profiles and the percentage of fluorescently labeled cells was measured in dot plots of the FL3 channel. Control cells that were not subject to labeling had very little fluorescence in this channel, while 100% of the viable cells that were labeled with PKH26 successfully picked up the marker. (B) IV- injected cells were allowed to home for three hours at which time the BM and spleen were collected. Single cell suspensions of these tissues were measured by flow cytometry in the SSC and FL3 channels to detect homed cells. After controlling for the number of injected cells, the percentage of PKH26+ cells in each CBL-B background was averaged from four independent experiments, and graphed for each of the BM (C) and splenic (D) compartments. Error bars represent the standard deviation.

Figure 2-6

Figure 2-7. BCR-ABL-induced disease is oligoclonal. Southern blot analysis of genomic DNA isolated from murine spleen (S) and liver (L) or Ba/F3 cell lines. DNA was hybridized with a probe derived from the ires-gfp sequences of the Mig210 vector. (A) XbaI digestion, which demonstrates the structure of the provirus because it cuts once in each of the 5’ and 3’ LTRs. (B) EcoRI digestion, which demonstrates the integration pattern of the provirus. EcoRI cuts 5’ to the ires-gfp gene sequences in the vector and generates a specific fragment determined by the first EcoRI site encountered in cellular sequences flanking the 3’ LTR. The positive control is DNA from Ba/F3 Mig210 cells and the negative control is MIEV-infected liver.

88 2.5. Discussion Both CBL and CBL-B are highly phosphorylated in BCR-ABL-expressing cell lines,134,281 and CBL is a phosphorylated target in chronic phase CML cells.126 As such, it is probable that these proteins play important roles in BCR-ABL-mediated leukemogenesis. However, BMT studies showed that CBL is dispensable for CML disease development.133 Given that BCR-ABL expression down-regulates CBL- B,134 it is conceivable that this protein is the primary CBL family member involved in CML. To address the functional role of CBL-B downstream of BCR-ABL in an in vivo context we pursued murine BMT studies. Our results show that CBL-B(-/-) BCR-ABL-infected BMCs are less effective at inducing leukemia in mice as compared to animals receiving wild-type BCR-ABL-transduced cells. The CBL-B(- /-) BCR-ABL recipients have a delayed latency of disease development that is not due to ineffective generation of granulocytes, but rather an impaired ability of the CBL-B(-/-) cells to home to the BM niche. Our initial experiments contrast the previously published observations from Sattler et al. in which they showed that both the TEL-JAK2 and BCR-ABL fusion kinases could down-regulate the expression of CBL-B in Ba/F3 cells.134 While we observed a reduction in CBL-B protein levels in the presence of BCR-ABL, TEL-JAK2 did not decrease CBL-B expression below the endogenous level in Ba/F3 cells. We do not believe the reason for this discrepancy is attributable to a lower expression of TEL-JAK2 in our system, as prior to immunoblotting our cells were sorted to greater than 90% GFP positivity (a surrogate marker for TEL-JAK2), and the protein bands for TEL-JAK2(5-19) were easily observed in phospho-tyrosine immunoblots at the appropriate molecular weights of 73- and 77- kDa.408 Instead, we conclude that the TEL-JAK2(5-19) isoform used in our studies may be distinct from that utilized by Sattler and colleagues in their experiments. TEL-JAK2(5-19) is missing the JH2 domain found in both the TEL-JAK2(4-17) and TEL-JAK2(5-12) isoforms (schematically depicted in ref. 409). A specific association between CBL-B (or one of its upstream regulatory proteins) with the JH2 domain could potentially mediate the TEL-JAK2 isoform-specific modulation of CBL-B. Detailed biochemical studies between CBL-B and the various TEL-JAK2 isoforms may clarify these regulatory differences. The BCR-ABL oncogene significantly altered CBL-B protein levels in vitro, so our laboratory initiated BMT studies to assess the contribution of CBL-B in BCR-ABL-mediated MPD development. The wild-type recipients became moribund approximately three weeks post-transplant and succumbed to an MPD as displayed by elevated WBC counts, expansion of the neutrophil compartment, hypercellular BM, extramedullary hematopoiesis of the spleen and liver, and eventual death through pulmonary hemorrhage.202,205,207 The disease observed in the majority of the CBL-B(-/-) BMTs was also characterized as an MPD, but with key phenotypic differences from wild-type recipients. As an exception to the CBL-B(-/-) MPD, a single mouse expired from a lymphoid leukemia. This alternate disease phenotype suggested that: (a) CBL-B was required for BCR-ABL-mediated myeloid disease development, (b) the donor cells were transduced with low titer virus, or (c) the target cells for

89 transformation in this instance were not multipotent stem cells. We believe the latter possibility is most likely. The CBL-B(-/-) MPD was distinct in multiple respects. Compared to the wild-type MPD animals the CBL-B(-/-) recipients displayed (1) enhanced granulopoiesis in the peripheral blood and spleen, (2) reduced leukemic burden at the time of morbidity, and (3) an overall prolongation of survival. The first observed phenotypic difference was the more effective granulocyte production in the CBL-B(-/-) BCR-ABL BMTs. Differential counts showed enhanced numbers of neutrophils in these animals as compared to the wild-type recipeints, with a consequential decrease in the percentage of lymphoid cells. Additionally, flow cytometric spleen cell analyses showed elevated percentages of both GFP+ and GFP- granulocytes (Gr-1+/Mac-1+ cells). These data suggest that CBL-B is a negative regulator of myeloid maturation. Several groups initially argued that BCR-ABL controlled granulopoiesis through autocrine production of IL-3, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Granulopoiesis could be induced in cell cultures through treatment with IL-3 and G-CSF,410,411 or in animals through direct administration of these cytokines.412-414 Also, CD34+ cells isolated from chronic phase CML patients displayed enhanced levels of autocrine IL-3 and G-CSF production,221 and murine BMT studies of CML-like disease induced by BCR-ABL showed animals with elevated IL-3 and GM-CSF levels in the serum.207 But, BCR-ABL BMTs later showed that MPD development was unaltered by deletion of IL-3 or GM-CSF cytokines and IL-3 receptor chains,238,239 suggesting that BCR-ABL must control granulopoiesis through an alternative mechanism. Based on our findings in CBL-B(-/-) recipients we propose that BCR-ABL may actively reduce CBL-B expression to promote myeloid expansion. In support of our hypothesis, several recent studies suggest that CBL mutations co-operate in the pathogenesis of myeloid malignancies. Mono- or bi-allelic mutations of CBL proteins in regions that co-ordinate ubiquitination have been found in patients with acute myeloid leukemia (AML),398,399 myelodysplastic syndrome (MDS)/MPD, and chronic myelomonocytic leukemia (CMML).400 To confirm the role of CBL proteins in myeloid disease our future experiments will focus on the regulation of biological pathways that modulate the expression of myeloid-specific genes. The second phenotypic difference in the CBL-B(-/-) MPD animals was the presence of morbidity in the absence of significant leukemic burden (i.e., high PB WBC counts and massive splenic EMH). Several groups suggest that the ultimate cause of death in murine CML transplants is not the level of leukemic burden, but rather the infiltration of mature myeloid cells into the lungs and the consequential lung hemorrhage.205,415 Moreover, BMTs performed with the mutant Y177F BCR-ABL yield T-cell or B- cell lymphomas and leukemias and the absence of pulmonary involvement203,225,226 and suggest that a mere transformation of leukemic phenotype away from a myeloid disease spares the animals from lung manifestations. Therefore, we argue that the prominent neutrophil expansion in the CBL-B(-/-) MPD

90 animals led to increased infiltration of the lungs and morbidity in the absence of massive leukemic burden. The third, and most striking difference in our CBL-B(-/-) BCR-ABL animals, was the delayed latency of disease development. Southern and Western blots did not identify a plausible mechanism to explain this difference .The provirus was detected in spleen and liver samples from both genotypes – indicative of the massive infiltration of BCR-ABL-expressing cells. As well, the disease was oligoclonal in both CBL-B genotypes suggesting that BCR-ABL was sufficient for disease generation. Likewise, parallel results were observed in immunoblots. The phospho-tyrosine profiles of the diseased animals were identical and the level of BCR-ABL expression was similar. Furthermore, the intensity of PKB and ERK1/2 phosphorylations was reflective of the number of infiltrating leukemic cells in the splenic compartment and was not distinctly regulated by CBL-B. Since PKB and ERK activity were not influenced by CBL-B deletion we speculated that cell survival and proliferation, respectively, were not appreciably different between the wild-type and CBL- B(-/-) backgrounds and could not account for the delayed latency. In support of our hypothesis, our preliminary colony formation assays did not show any significant difference in the ability of CBL-B(-/-) cells to form myeloid colonies (data not shown). Additionally, the growth rate of BCR-ABL-expressing Ba/F3 cells is not altered by forced expression of CBL-B.134 Therefore, we predicted that CBL-B perturbed leukemogenesis through modulation of cellular migration to the bone marrow. Our in vivo homing assays confirmed that CBL-B-deficient cells were less capable of homing to the BM niche than the wild-type cells (Figure 6). We surmise that decreased homing of the CBL-B-deficient cells to the BM – the major site of hematopoiesis - impairs proliferation of the CML progenitors which consequently retards leukemic progression. Skorski et al. previously showed that impaired homing of hematopoietic cells transduced with SH3 deletion mutants (∆SH3) of BCR-ABL led to decreased tissue infiltration as a function of time and prolonged survival compared recipients of WT BCR-ABL cells.214 The ∆SH3-BCR- ABL protein was abnormally localized to the cytosol. As CBL-B can co-immunoprecipitate BCR- ABL,134 contains a proline-rich motif that could potentially interact with the BCR-ABL SH3 domain, and can alter cellular localization of the p85 subunit of PI3’K,324 we postulate that CBL-B may regulate BCR-ABL distribution. A link between the association of the BCR-ABL SH3 domain with CBL-B, cytoskeletal localization and efficient homing would be of great interest for further evaluation of transplant outcomes. The observed CBL-B-specific differences in BCR-ABL-mediated disease can be summarized in the proposed model (Figure 2-8). CBL-B(-/-) cells are less efficient at homing to the BM then their wild- type counterparts. The wild-type cells proliferate rapidly, while the decreased number of CBL(-/-) cells in this organ gives the impression of slower proliferation, even though the growth rates of wild-type and CBL-B(-/-) BMCs are comparable. As a result of increased engraftment, egress of wild-type cells proceeds at a faster rate than for the CBL-B(-/-) cells, leading to a hastened disease latency.

Figure 2-8. CBL-B(-/-) BCR-ABL BMT recipients have poor homing to the BM but enhanced granulocyte infiltration into the lungs. This model depicts the differences in BCR-ABL-mediated leukemogenesis between the wild-type CBL- B knock-out backgrounds. Wild-type recipients have good homing of BMCs to their niche, massive proliferation of multiple cell types that egress into the peripheral blood and eventually to the lungs to cause hemorrhaging. The CBL-B(-/-) BMTs have reduced homing to the BM leaving fewer cells to divide. These BCR-ABL-expressing cells have the propensity to become neutrophils, which efficiently induce death in the animals through invasion of the lungs.

92 Nevertheless, the BM niche in the CBL-B(-/-) BMTs continues to increase in cellularity and eventually leukemic cells migrate into the PB and to other organs. These extramedullary cells are primarily granulocytic in nature, due to the proposed proliferative stimulus provided by CBL-B deficiency. The abundance of neutrophils invading the lungs causes pulmonary hemorrhage and death even in the absence of massive extramedullary infiltration in these animals. We conclude that CBL-B functions in vivo as a negative regulator of BCR-ABL-mediated granulopoiesis. BCR-ABL may reduce CBL-B expression levels in an effort to enhance granulocyte proliferation, a characteristic feature of chronic phase CML. Additionally, our studies show an essential positive regulatory role for CBL-B in BMC homing, suggesting that inefficient homing of CBL-B(-/-) cells to the BM may account for the observed attenuation of disease development in our CBL-B-deficient BMT studies of BCR-ABL-mediated leukemogenesis.

CHAPTER 3

CBL-B IS A CRITICAL REGULATOR OF MOUSE EMBRYONIC FIBROBLAST MOTILITY

93 94 3.1. Abstract CBL proteins play critical roles in the modulation of the essential biological process of cell migration. In this report, we describe the creation of CBL-B-deficient mouse embryonic fibroblasts (MEFs) for motility studies. Using time-lapse video microscopy (TLVM), we observed a positive influence of CBL-B on spontaneous fibroblast migration. In contrast, after serum stimulation, CBL-B(-/- ) cells displayed enhanced chemotaxis compared to wild-type cells, as evidenced by both TLVM and Transwell migration assays. In the absence of CBL-B, lamellipodia were the principal leading edge protrusions as compared to the filopodia forward extension that predominated in CBL-B wild-type (WT) cells. CBL-B expression triggered reduced Vav3 tyrosine phosphorylation and total protein levels, without altering downstream signaling to the Rac1 guanosine triphosphatase (GTPase), p21-activated kinase (PAK), p38 mitogen-activated protein kinase (p38MAPK) and stress-activated protein kinase (SAPK / Jun N-terminal kinase (JNK)). Instead, the extracellular signal-regulated kinase (ERK) and protein kinase B (PKB / Akt) pathways showed significant regulation. ERK tyrosine phosphorylation was constitutively increased in CBL-B(-/-) cells, and the serum-stimulated phosphorylation of the upstream regulator of ERK, mitogen-activated protein kinase kinase (MEK), was augmented by CBL-B deficiency. Serine phosphorylation of PKB was enhanced in the absence of CBL-B and fibroblast treatment with LY294002 reduced cell motility. CBL-B expression impaired the serum-stimulated phosphorylation of GAB2, a known upstream regulator of PKB and ERK. These results demonstrate that CBL-B is an essential upstream element in pathways that control fibroblast locomotion.

95 3.2. Introduction Cell migration plays a vital role in many physiological and pathological processes, including embryogenesis, wound repair, immune responses and tumor cell metastases.179,416 Locomotion is composed of a series of co-ordinated events. Extracellular signals initiate a polarization of actin filaments within the cell, followed by protrusion of lamellipodia or filopodia at the leading edge and the subsequent formation of cell-substratum adhesions at the cell front. The opposing contractile force is exerted through detachment of rear adhesions and tail retraction, resulting in the forward movement of the cell body. This extremely orchestrated process involves a multitude of biochemical cascades. Phospholipase C gamma (PLCγ) may facilitate polarization of the cell417; while Cdc42, Rac and Rho GTPases enable the formation of filopodia, lamellipodia and focal adhesions, respectively.418-420 Contraction of the cell body is coordinated by Myosin and mitogen-activated protein kinase (MAPK) signaling proteins,421,422 and the release of rear adhesion contacts requires Calpain.423 Our understanding of this intricate mechanism is strengthened through the continued identification of additional intermediary substrates essential for locomotion. The CBL protein, a known E3 ubiquitin ligase and adaptor molecule,280,282 associates with and regulates many of the aforementioned proteins in response to chemotactic stimuli. The key role of CBL in motility is evidenced by its involvement in the locomotion of many cell types: macrophages,424 osteoclasts,381 T and B cell lines,379,385 breast cancer cell lines,425 and fibroblasts.426 CBL belongs to a family of three mammalian members that includes CBL, CBL-B,270 and CBL-3.271,272 CBL and CBL-B share a common domain structure of an N-terminal tyrosine-kinase-binding domain followed by a RING finger motif that mediates the E3 ubiquitin ligase activity of CBL. The C-terminus contains a proline- rich region and a sequence that displays homology to both the leucine zipper and ubiquitin associated (UBA) domains.280 While CBL and CBL-B are highly homologous, particularly within the TKB domain, dissimilarity exists within the quantity of serine and tyrosine residues in the C-terminal region and the affinity of the UBA domain for ubiquitinated proteins.294 These variations account for differences in their relative interactomes, raising the possibility that CBL and CBL-B may utilize alternative signaling pathways for the regulation of migration. Indeed, motility differences are observed after ligand binding to CXCR3 in CD34+ hematopoietic progenitors: CBL antisense induces migration while CBL-B antisense does not elicit a migratory effect.382 Alternatively, both CBL and CBL-B positively regulate the SDF-1α-stimulated motility of the Jurkat T cell line, and immunoprecipitations show the formation of similar protein complexes in these cells.379 These homolog-specific differences highlight the importance of continued investigation in additional cell systems as to the role of CBL-B in locomotion. Here we describe the generation of CBL-B-deficient MEFs for the study of migration. We observe a negative regulatory role for CBL-B in the chemotaxis of MEFs toward serum. This motility involves signals propagated along both the PKB and ERK pathways, possibly through a mechanism involving the upstream adaptor GAB2 and the cytoskeletal protein Paxillin.

96 3.3. Materials and Methods 3.3.1. Generation of mouse embryonic fibroblasts To create wild-type and CBL-B(-/-) mouse embryonic fibroblasts (MEFs), a mating was initiated between a male and female BALB/c CBL-B(+/-) pair. (The generation of BALB/c CBL-B(-/-) mice is described in Chapter 2.) The female was monitored daily for the presence of a vaginal plug, at which point it was assumed that coitus had taken place the evening prior (Day 0). At 13.5 days post-coitus, the female was euthanized and each embryo was harvested and processed separately to avoid cross- contamination of genetic material. After decapitation and removal of the dark red organs from the body (the liver and heart), the remaining “fibroblastic” tissue was dissociated into single cells through mincing and incubation with 0.05% Trypsin/EDTA (Invitrogen, Carlsbad, CA) for one hour at 37oC. Homogeneous cell suspensions were plated in MEF Generation Medium (MGM) composed of High Glucose (H21) Dulbecco’s Modified Eagle’s Medium (DMEM) plus antibiotics, supplemented with 10% Fetal Calf Serum (FCS; HyClone, Thermo Fisher Scientific, Inc., Waltham, MA), 1x non-essential amino acids (Invitrogen), 200 mM L-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (β-ME; Fisher) and 20 mM HEPES, pH 7.3 (Fisher). Cells from each embryo were passaged independently in MGM according to the specifications of Todaro and Green.427 At Passage 20, the MEFs were considered to be immortal and were subsequently maintained in a MEF Maintenance Medium (MMM) of DMEM (H21) containing antibiotics, 10% FCS and 0.1 mM β-ME. These “late passage” cells were frozen in DMEM with 50% FCS and 7.5% dimethyl sulfoxide (DMSO; Fisher), stored at -70oC, and used for all subsequent experiments. Genomic DNA for genotyping was isolated by phenol/chloroform extraction of concentrated MEF cell pellets. PCR analysis was performed as described in Chapter 2.

3.3.2. Proliferation assay Cell proliferation was recorded over 10 days. MEFs were seeded at a low density of 2 x 103 cells per cm2 in MMM. Growth was monitored daily by trypsinizing triplicate cultures and performing manual cell counts. Trypan blue was added to exclude dead cells. Values were recorded as the mean of three replicates ± standard deviation. Proliferation assays were repeated in duplicate.

3.3.3. Adhesion assay A 96-well plate was pre-coated overnight at 4oC with 1 μg/ml of human plasma fibronectin (Millipore, Billerica, MA). The following day, the wells were blocked with DMEM supplemented with 1% (w/v) bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO). During blocking, sub-confluent cultures of MEFs were harvested with trypsin, washed three times in Cell Adhesion Media (CAM; DMEM plus 0.2% (w/v) BSA) and re-suspended in CAM at equivalent concentrations. The wells were subsequently washed three times with CAM and loaded with 2.5 x 104 cells/well in a volume of 100 µL

97 of CAM. Adherence to fibronectin was allowed to proceed for 30 minutes at 37°C, at which time cell adhesion was tested through at least five washes with CAM. Washing was completed when less than 2% of the input cells remained in wells that were BSA-blocked but not coated with fibronectin. XTT Reagent (1 mg/mL Sodium 3,3'-428-Bis(4-Methoxy-6-Nitro)Benzene Sulfonic Acid Hydrate (XTT; Diagnostic Chemicals Ltd., Charlottetown, PE) in CAM plus 125 mM phenazine methosulfate (Sigma-Aldrich)) was added to the remaining adhered cells and the plates were incubated at 37°C for four hours. The formazan product was recorded at 450 nM on a plate reader. After subtraction of background cell binding to BSA only-coated wells, the percentage of adhered cells was determined by dividing the colorimetric response of the adhered cells by the initial cell input. The cell adhesion assay was repeated four times.

3.3.4. Migration assays For wound healing experiments, confluent MEF cultures were plated by seeding 5 x 106 cells/well in 24-well tissue culture plates six hours before initiation of the scratch assay. Subsequently, monolayers were scratched with a 200 µL plastic pipette tip and washed once in the same media to be used for chemotaxis. The plates were re-fed with either MMM (10% FCS) or with DMEM plus β-ME alone, and allowed to heal while live-cell images were acquired in 20-minute intervals for 12 hours on a Zeiss Axiovert 200M Inverted Fluorescent Microscope (Thornwood, NY) equipped with a Roper Scientific Coolsnap HQ camera (Photometrics; Tucson, AZ). The wound distance was measured over time from triplicate images and the mean was calculated. Results are representative of three independent experiments ± standard error. To assess directed migration, modified Boyden chamber experiments were initiated using uncoated Transwell migration chambers (24-well format, 8 µm pore size; Becton Dickinson (BD) Falcon, Franklin Lakes, NJ). The filters were placed into the lower chamber containing either 800 µL DMEM alone (to assess spontaneous motility) or DMEM with the addition of 10% FCS to analyze chemotactic responses. Filters were pre-incubated for at least 30 minutes before cells were added to the upper chamber. Sub-confluent MEFs were trypsinized from plates and washed three times in phosphate- buffered saline (PBS). Cell counts were performed, MEFs were resuspended in DMEM alone, and 5 x 104 cells were added to the upper Transwell chamber in a volume of 300 µL. The cells were allowed to migrate to the underside of the top chamber for four hours. Non-migrated cells on the upper side of the membrane were removed with a cotton swab, and migrated cells attached to the bottom surface of the membrane were fixed for 10 minutes in 25% (v/v) methanol, 75% (v/v) glacial acetic acid, followed by staining for one minute in 0.5 % (w/v) crystal violet in methanol. Membranes were washed in tap water and allowed to dry overnight before counting. In each experiment, a mean was calculated for three separate filter counts, where each filter count was the sum of three randomly selected 40x-objective fields. Each data point graphically represented is the mean of four experiments ± standard error. The Student’s t-test was used in statistical analyses.

3.3.5. Antibodies Sources of antibodies to tyrosine-phosphorylated proteins (4G10), CBL, CBL-B, Tubulin, phospho-ERK1/2, total ERK1/2, phospho-PKB and total PKB are described in Chapter 2. Total polyclonal PAK1 (αPAK, N-20), Vav (Vav1, C-14), and SAPK (C-17; also known as JNK), plus monoclonal GST (B-14) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal GAB2 (06-967) and Vav3 (07-465) antibodies were purchased from Millipore (Billerica, MA). Mouse anti-PI3’K was purchased from BD Transduction LaboratoriesTM. All other primary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA): polyclonal antibodies to detect phospho-GAB2 (3881), phospho-MEK1/2 (9121), total MEK1/2 (9122) and phospho-PAK1/2/3 (2605), plus monoclonal antibodies for detection of phospho-p38MAPK(28B10), total p38MAPK(5F11) and phospho-SAPK(G9). Secondary antibodies were described previously in Chapter 2.

3.3.6. Immunoprecipitation and immunoblotting FCS depletion of MEFs was initiated through triplicate washing with PBS, followed by incubation in Serum Starvation Media (SSM; DMEM supplemented with 1 mg/mL BSA and 0.1 mM β- ME) for 16 hours. Subsequently, cells were stimulated for 10 minutes at 37oC in pre-warmed DMEM plus β-ME either in the presence of absence of 10% FCS. In experiments designed to block proteasomal function, two hours prior to stimulation the SSM was replaced with fresh SSM containing 20 µM MG132 (EMD Chemicals, Inc., Gibbstown, NJ). For cell lysis of MEFs, the Lysis Buffer recipe, as well as the methods for protein quantification and separation, were identical to those outlined in Chapter 2 for Ba/F3 cells. However, lysis of these adherent cells was performed directly on the tissue culture plates by the addition of 1 mL of Lysis Buffer to eight 10-cm2 plates and collection with a rubber policeman. Most immunoblotting (IB) was performed according to the general protocol described in Chapter 2; nevertheless, some supplementary methods were required. Alternative immunoblotting strategies for detection of tubulin, phospho-ERK1/2, total ERK1/2, phospho-PKB and total PKB are described in detail in Chapter 2. For detection of total GAB2, membranes were blocked for 20 minutes with 3% (v/v) skim milk powder dissolved in Tris buffered saline (TBS; 50 mM Tris-HCl (pH 8.0), 150 mM NaCl) supplemented with 0.1% (v/v) Tween-20 (TBST). Overnight incubation at 4oC was performed with 1 µg/ml of primary polyclonal antibody in 3% milk/TBST. Membranes were washed twice and incubated with secondary horseradish peroxidase (HRP)-linked Protein A antibody at a 1:5000 dilution in 3% milk/TBST. The

99 membrane was washed a final four times and detected with Western LightningTM Chemiluminescence Reagent Plus (ECL; PerkinElmer, Waltham, MA). For p46 SAPK reprobing, membranes were blocked in 5% milk/TBST for one hour, and incubated with rabbit polyclonal antibody for one hour at a dilution of 1:1000 in 5% (w/v) BSA dissolved in TBST. After three washes, membranes were incubated with HRP-conjugated Protein A secondary antibody (1:2000 in 5% milk/TBST) for one hour, washed three times and visualized with ECL. Vav1 was detected with the Vav (C-14) antibody from Santa Cruz Biotechnology, Inc. Membranes were blocked in 2.5% BSA/TBST for one hour, rinsed twice in TBST, then incubated overnight at 4oC with a 1:200 dilution of polyclonal antibody in 2.5% BSA/TBST. Membranes were washed four times, then subjected to a one hour secondary antibody incubation of HRP-conjugated Protein A at a 1:5000 dilution in 2.5% BSA/TBST. Membranes were washed four times and protein was detected by ECL. For detection of phosphorylated forms of PAK1/2/3, the manufacturer’s instructions were followed with the exception that all wash steps were increased to four times rather than the recommended three. Immunoblotting with Vav3, phospho-MEK1/2, total MEK1/2 and phospho-GAB2 polyclonal antibodies, plus phospho-p38MAPK, total p38MAPK and phospho-SAPK monoclonal antibodies, was performed according to the manufacturer’s instructions.

3.3.7. Rac activation assay The activation of Rac was measured by the affinity of the guanosine triphosphate (GTP)-bound form of RAC for the protein-binding domain (PBD) of p21/Cdc42/Rac1-activated kinase 1 (PAK1).429 The pGEX2TK-PAK-CD plasmid was a generous gift from R. Rottapel (Toronto, ON). Similarly to the immunoblotting experiments described above, cells were serum-starved overnight and stimulated for 10 minutes with 10% FCS. Cell lysates were collected with a rubber policeman after direct addition of 1 mL of ice-cold RAC Lysis Buffer onto four 10-cm2 plates. Rac Lysis Buffer was composed of 50 mM HEPES (pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride, 1% (v/v) Triton X-100, 1 mM sodium orthovanadate, 10 mM sodium fluoride and 1x Complete Protease Inhibitor Cocktail (Roche, Mannheim, Germany). Cell lysates were clarified of cellular debris by centrifugation at maximum speed for 30 minutes and the protein concentration was determined by the Bradford colorimetric assay (Bio- Rad, Hercules, CA). One mg of cell lysate was incubated with 25 µg of the glutathione S transferase (GST)-fused PAK1 PBD bound to Glutathione SepharoseTM 4B beads (GE Healthcare, Piscataway, NJ). After 1.5 hours, beads were washed four times with RAC Lysis Buffer, resuspended in 50 µL of 1x Sample Buffer (69 mM Tris-HCl (pH6.8), 11% (v/v) glycerol, 2.2% (w/v) sodium dodecyl sulfate (SDS) and 0.02% (w/v) bromophenol blue) supplemented with 50 mM dithiothreitol (DTT), and separated by

100 SDS – polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the gel was transferred to a polyvinylidene fluoride membrane. GTP-bound Rac1 was identified in the pull-down reactions by immunoblotting for Rac1. The general protocol for immunoblotting was followed with the exception of six final washes rather than the usual four. Detection of Rac1 in whole cell lysates was used as a loading control, as was reprobing of the blot for levels of GST in the pull-downs. GST reprobes were performed according to the standard immunoblot protocol.

3.4. Results 3.4.1. The growth characteristics of mouse embryonic fibroblasts are unaltered by the genetic deletion of CBL-B. In order to generate wild-type and CBL-B(-/-) MEFs with genetically similar profiles, day 13.5 embryos were collected from a pregnant mouse of a CBL-B heterozygous mating. Nine immortalized cell lines were established according to the protocol of Todaro and Green,427 and CBL-B gene status was confirmed by PCR analysis of genomic DNA. Line 1 displayed genetic deletion of CBL-B (Figure 3- 1A) and the absence of CBL-B protein expression as confirmed by immunoblotting experiments (Figure 3-1B). Line 6 was used as the wild-type control for the remainder of the studies as it showed strong expression of CBL-B. The CBL-B(-/-) MEFs did not display any obvert signs of transformation. The growth of CBL-B knock-out cells in low-serum conditions was similar to that of the WT cells and proliferation rates in both 0.5%- and 10%- serum were comparable (Figure 3-1C). Contact-inhibited growth was maintained and no soft agar colonies were formed (data not shown). Our data indicates that CBL-B(-/-) MEFs can be effectively generated without alteration of their proliferation properties.

3.4.2. Serum-dependent chemotactic migration is enhanced in the absence of CBL-B. After confirming a non-transformed phenotype in our MEFs, we sought to test the effect of CBL-B deficiency on serum-dependent motility, given the previously published report that the family member CBL enhances the migration of v-Abl-transformed NIH 3T3 toward serum.380 General cell movement was assessed through the closure of a wound in a cell confluent monolayer, while directed migration was quantified in Transwell experiments. In wound healing assays, cell monolayers were scratched with a pipette tip, washed, and media with or without serum was added to the MEF. Migration into the wound was visualized over a 12-hour period using parallel images collected by TLVM. In the absence of serum (media supplemented with 0.1% BSA only), CBL-B WT cells were quite motile, with a rate of wound closure greater than that of the CBL-B(-/-) cells (2.7 arbitrary units (au)/hour (h) versus 1.9 au/h; Figure 3-2A). The CBL-B-

101 Figure 3-1. CBL-B deletion in mouse embryonic fibroblasts does not alter cell growth. (A) Genetic deletion of CBL-B was confirmed through PCR analysis of genomic DNA from nine MEF lines. DNA was screened for the presence of the CBL-B gene with primers to detect either the WT or KO form of the gene. MEF Line 1 is CBL-B-deficient. MEF Lines 2, 4, 6 and 8 are WT for CBL-B, while MEF Lines 3, 5, 7 and 9 are heterozygous for the CBL-B gene. (B) Loss of CBL-B protein was assessed by immunoblotting MEF lysates with an antibody directed toward CBL-B. CBL protein levels were monitored after stripping the blot and reprobing with an anti-CBL-specific antibody. (C) and (D) To assess growth rates, wild-type and CBL-B(-/-) cell lines were plated in DMEM supplemented with FCS at low (0.5% FCS) and high (10% FCS) concentrations. Cell counts were performed with the trypan blue exclusion assay each day for four to eight days. The mean cell number of triplicate platings is graphically plotted ± standard deviation. The results are representative of at least two independent experiments.

102

Figure 3-1

103 Figure 3-2. CBL-B expression modifies wound closure rates. A monolayer of wild-type or CBL-B(-/-) MEFs was plated into 24-well plates six hours prior to the introduction of a scratch wound with a 200-µL pipette tip. The cultures were washed to remove scraped cells, and DMEM plus either 0.1% BSA (A) or 10% FCS (B) was added. Live cell imaging was performed with parallel differential interference contrast images recorded every 20 minutes for 12 hours (h). Representative images are shown of wounded MEFs at the initial time of wounding (0 h) plus at 6 and 12 h later. At least three independent experiments were performed, and in each set-up, at least three movies were recorded. The line graphs are representative of a single experiment and depict the wounded area (in arbitrary units; au) over time; with data points recorded every three hours and error bars representing the standard deviation. Bar charts display the rate of migration as calculated from the slope of the curves for all experiments combined, with standard error depicted in the error bars. (C) Representative images show the formation of lamellipodia in CBL-B(-/-) cells and filopodia in wild-type MEFs.

104

Figure 3-2

105 deficient MEFs displayed significant cell death after three hours in 0.1% BSA as evidenced by enhanced cell rounding and detachment from the tissue culture plates. This increase in cell death could explain the reduced spontaneous motility of these CBL-B(-/-) cells. In contrast to the limited movement observed in conditions of serum starvation, both wild-type and CBL-B-knock-out cells were quite responsive to serum. Again, differing velocities of movement were observed between the two genetic backgrounds. However, under conditions of serum stimulation the CBL-B-deficient cells were more motile. After 12 hours of observation, the CBL-B(-/-) cells completely achieved closure of the wound and reformed a tight monolayer (Figure 3-2B). Meanwhile the wild-type cells displayed slower movement into the wounded area (2.8 au/h versus the 4.2 au/h displayed by the CBL-B(-/-) MEFs) and were delayed in the formation of a monolayer. The enhanced motility of CBL-B(-/-) MEFs in these wound closure experiments can be directly attributed to an altered migration potential, as the proliferation rates between the CBL-B(-/-) and wild-type cells were comparable (as assessed previously in Figure 3-1C). Therefore, we conclude that the ability of cells to migrate in the absence of serum is dependent upon CBL-B expression while motility in the presence of serum is attenuated by the presence of CBL-B. Interestingly, the CBL-B(-/-) cells also displayed an altered morphology during serum-dependent migration. The wild-type cells moved in a manner characteristic of slow-moving fibroblasts, with the extension of filopodia-like protrusions at the leading edge (Figure 3-2C; as determined by visual analysis). The CBL-B(-/-) cells did not display such obvious protrusions, but rather moved with the extension of lamellipodia, an attribute most often visualized in more highly motile cells. These observations of enhanced velocity and modified morphology during serum-dependent migration of CBL- B-deficient MEFs suggest that CBL-B significantly alters the migration pathways in these cells. To investigate the possibility that the serum-dependent movement observed in the wound closure experiments was due to chemotactic responses, we initiated Transwell migration assays in which cells were allowed to migrate either spontaneously or toward a gradient of 10% serum. In contrast to observations of motility in the absence of serum as detected by TLVM, we did not observe any significant spontaneous Transwell motility (Figure 3-3A). However, CBL-B(-/-) cells were massively responsive to serum, with a 9.7-fold increase over spontaneous motility. As well, CBL-B-deficient cells showed a statistically significant 2.8-fold increase in motility over their WT counterparts (P < 0.01). These results confirm that CBL-B negatively regulates chemotactic signals induced by serum that control cell migration. As both the formation of adhesion contacts at the leading edge of the cell and the release of adhesion points at the trailing edge are important processes required for migration,430 we also assessed the MEFs for alterations in adhesion properties. Panning style assays were performed, where cells were monitored for their adherence to fibronectin after several washings. The adherent fraction was similar between the wild-type and CBL-B(-/-) MEFs (Figure 3-3B). Therefore, we conclude that while CBL-

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Figure 3-3. CBL-B-deficient fibroblasts display enhanced chemotaxis toward serum but no change in adhesion. (A) Wild-type and CBL-B(-/-) MEFs were applied to the upper chamber of Transwell migration chambers and allowed to migrate toward media either absent or present for 10% FCS. After four hours, the migrated cells on the underside of the membranes were counted after first removing the cells from the upper chambers and then staining the cells attached to the lower surface of the membranes with crystal violet. During microscopic examinations, values were calculated as the total number of cells migrating in three randomly-selected 40x-objective fields. Data represent the average of four independent experiments ± standard error. (B) CBL-B(-/-) and wild-type MEFs were subjected to a cell adhesion test in tissue culture plates coated with 1 µg/mL fibronectin. The graph represents the mean adhesion from four independent experiments ± standard error. In each experiment the average adhesion was calculated from recordings of four separate wells of a 96-well culture dish.

107 B expression significantly alters migration, CBL-B does not play a prominent role in controlling cellular adhesion.

3.4.3. CBL-B deficiency upregulates Vav3 protein levels and tyrosine phosphorylation. In our studies described above, serum-dependent chemotactic migration was the sole functional consequence of CBL-B deficiency. Therefore, we surmised that we could evaluate the downstream signaling events responsible for the altered motility by assessing total protein levels and phosphorylation responses after serum stimulation. Multiple immunoblotting experiments were initiated, focused on signaling molecules known to be responsible for cellular migration.ζ CBL-B deficiency leads to enhanced tyrosine phosphorylation of the Vav1/2 proteins in T cells.332,366 Tyrosine phosphorylation of Vav proteins stimulates their activity as guanine nucleotide exchange factors (GEFs) for the Rho/RAC family of cytoskeletal regulators.136-138 These data suggest that CBL-B could control cellular migration through the regulation of Vav phosphorylation. As the mammalian members of the Vav family show variable tissue expression, we initiated our studies by first determining which Vav homolog was expressed in our MEFs. Immunoblotting experiments with homolog-specific antibodies showed that while Vav1 was readily detected in spleen cells, expression of this protein was not observed in fibroblasts (data not shown). However, Vav3 expression was revealed within MEFs. Therefore, we continued our studies of CBL-B-regulated phosphorylation of Vav proteins by specifically studying Vav3. Immunoprecipitations were performed with an anti-Vav3 antibody followed by immunoblots to detect tyrosine-phosphorylated proteins. Preliminary evidence reveals that while tyrosine-phosphorylated Vav3 is visible in CBL-B-deficient cells, Vav3 tyrosine phosphorylation is undetectable in wild-type cells (Figure 3-4A). Treatment of these cells with MG132, to inhibit proteasomal degradation of proteins,431,432 did not restore Vav3 phosphorylation, suggesting that CBL-B either blocks Vav3 tyrosine phosphorylation or degrades phosphorylated-Vav3 through a proteasomal-independent mechanism. It is important to note that CBL-

ζ As detailed in the Appendix, these cell lines were originally developed for use in mechanistic studies evaluating the role of CBL-B in BCR-ABL signaling. As BCR-ABL expression was unable to confer transformation properties to the MEFs, those studies were discontinued. Many of the signaling molecules evaluated for transformation also play important roles in motility, and as such, signaling downstream of BCR-ABL was often evaluated alongside experiments to elucidate mechanisms of CBL-B- regulated motility. Consequently, many of the immunoblots presented in this section also depict protein expression and phosphorylation in the presence of BCR-ABL. The results presented here, in Chapter 3, will discuss the “control” cells only. The BCR-ABL-specific results have not been described in the narrative of this section but will be addressed in the Appendix.

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Figure 3-4. CBL-B expression reduces Vav3 phosphorylation and total Vav3 protein levels. Wild-type and CBL-B(-/-) MEF were serum starved overnight, then stimulated in the absence (-) or presence (+) of FCS. (A) Prior to protein extraction and serum stimulation, cells were pre-treated with either DMSO or the proteasomal inhibitor MG132. Immunoprecipitations (IPs) were performed overnight with an anti-Vav3 antibody and detection of tyrosine-phosphorylated proteins was achieved with a phospho-tyrosine (pTyr) immunoblot (IB; upper panel). Total Vav3 proteins were detected through reprobing with the anti-Vav3 antibody (lower panel). (B) Vav3 protein was detected in cell lysates with an anti-Vav3 antibody (upper panel). Equivalent loading was assessed with an anti-tubulin IB (lower panel).

109 B expression is not the only cellular mechanism of regulating Vav3 phosphorylation in these cells. Upon FCS stimulation, Vav3 phosphorylation is reduced in CBL-B(-/-) cells. The addition of MG132 to CBL- B KO cells enhances Vav3 phosphorylation, suggesting that alternative pathways regulate Vav3 proteasomal degradation. Our data present novel evidence that CBL-B negatively regulates the tyrosine phosphorylation of the Vav3 family member. As a direct interaction between Vav proteins and CBL-B has been observed,220 we hypothesized that CBL-B may also regulate the total protein level of Vav3 through its E3 ubiquitin ligase activity. We performed IBs to assess the total Vav3 protein levels in wild-type and CBL-B(-/-) protein lysates. Our IBs show a small, but reproducible, increase in Vav3 protein levels in the absence of CBL-B (Figure 3- 4B), suggesting that CBL-B not only regulates Vav3 phosphorylation in MEFs, but also downregulates total Vav3 protein expression. Phosphorylated Vav3 can stimulate the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) on Rac1, thus leading to its activation.137,433 Interestingly, Rac activation is important for the formation of lamellipodia418 – the morphological feature of our highly motile serum-stimulated CBL-B(-/-) MEFs. Therefore, we performed Rac activation assays to assess CBL-B-mediated regulation of GTP binding to Rac1. These experiments were performed multiple times but no consistent regulation of Rac was observed (Figure 3-5). Consequently, we conclude that enhanced Vav3 phosphorylation in CBL-B(-/-) cells does not translate into increased levels of active Rac. The p21-activated kinase (PAK) protein is the direct downstream substrate of Rac1.370 While phosphorylated Vav3 was unable to generate nucleotide exchange on RAC1, the stimulation of PAK activity could still be induced through an alternative mechanism: for example through Cdc42.370 Hence, we investigated PAK activity with IBs to detect phosphorylation of PAK1, PAK2 and PAK3 proteins. Our results show no difference in PAK1/2/3 serine phosphorylation between the wild-type and CBL-B(- /-) cells lines (Figure 3-6A). Similarly, the phosphorylation of p38-MAPK and SAPK proteins, known effectors of PAK activity,434,435 were unaffected by CBL-B deletion (Figures 3-6B and 3-6C, respectively). These results lead us to conclude that CBL-B-mediated regulation of Vav3 does not alter the Rac1 and PAK signaling pathways that control p38-MAPK and SAPK activation.

3.4.4. Serum-stimulated phosphorylation of proteins in the ERK signaling pathway is increased in CBL-B-deficient cells. As our efforts to link Vav3 activation to important downstream regulators of migration were unsuccessful, we turned our attention to additional pathways involved in serum-stimulated motility. A study from Krueger et al. demonstrated a requirement for ERK activation in serum-dependent motility of breast cancer cell lines.436 Interestingly, CBL-B acts as a negative regulator of ERK activity in multiple cell types.335,437 With this evidence in mind, we pursued immunoblotting studies to assess the phosphorylation of ERK1 and ERK2 in our MEFs after serum induction, and the potential regulation of

110

Figure 3-5. Rac activity is unaffected by CBL-B expression. CBL-B(-/-) and wild-type MEF were serum starved overnight then stimulated in the absence (-) or presence (+) of FCS. GST in vitro mixes were performed using a GST-PAK1 PBD construct. Precipitates were probed with an anti-Rac1 antibody to detect GTP-bound Rac1 protein (1st panel). Loading was compared by either probing the lysates for total Rac1 protein with an anti-Rac1 IB (2nd panel), or reprobing the pull-downs with an anti-GST antibody to detect the GST-PAK1 PBD construct (3rd panel). Two independent experiments are depicted.

111

Figure 3-6. CBL-B expression does not alter phosphorylation of PAK1/2/3, p38-MAPK or SAPK. Wild-type and CBL-B(-/-) MEFs were serum starved overnight, then stimulated in the absence (-) or presence (+) of FCS. (A) Lysates were probed with a phospho-specific antibody to detect serine phosphorylated PAK1/2/3 (upper panel). Phosphorylation levels were compared to total PAK1 protein through an anti-PAK1 reprobe (lower panel). (B) Phosphorylated p38-MAPK was detected by immunoblotting lysates with a phosphorylation-speciﬁc p38-MAPK antibody (upper panel). Total p38 was detected upon reprobing with an anti-p38-MAPK antibody (lower panel). (C) The phospho-specific SAPK antibody detected both the p46 and p54 isoforms of phosphorylated SAPK (upper panel). The blot was reprobed for total SAPK protein with an anti-SAPK antibody (lower panel).

112 phosphorylation by CBL-B. Our results showed enhanced ERK1/2 phosphorylation in CBL-B(-/-) cells, as compared to WT cells, after serum stimulation (Figure 3-7A). We also observed constitutive ERK1/2 phosphorylation of CBL-B-deficient cells even in the absence of serum stimulation. To extend our observations, we monitored the phosphorylation of MEK1 and MEK2, the upstream activators of ERK1/2.438,439 Here, we visualized enhanced serum-dependent MEK1/2 phosphorylation in cells lacking CBL-B (Figure 3-7B). However, MEK1/2 were not phosphorylated in the absence of serum. These data suggest that CBL-B controls ERK1/2 phosphorylation under conditions of both serum starvation and stimulation. The evidence indicates that ERK1/2 phosphorylation during serum stimulation may be regulated through a pathway involving MEK1/2 activation, while in the absence of serum, CBL-B may be modulating ERK1/2 phosphorylation through a pathway independent of MEK1/2.

3.4.5. CBL-B deficiency upregulates serum-induced protein kinase B phosphorylation. In addition to ERK signaling, activation of the PI3’K-PKB pathway has also been implicated in the regulation of migration,440 and CBL-B has been shown to ubiquitinate the p85 regulatory subunit of PI3’K.323 As ubiquitination often leads to degradation of the substrate, we initiated our studies of CBL- B-mediated regulation of the PI3’K-PKB pathway by assessing the total protein levels of p85-PI3’K. Our results show comparable levels of p85-PI3’K expression in both wild-type and CBL-B(-/-) cells in both the presence and absence of serum stimulation (Figure 3-8A). Therefore, we conclude that CBL-B is not affecting p85-PI3’K degradation. However, studies by Fang and Liu (Nat. Immunol., 2001) showed that CBL-B-dependent ubiquitination of p85-PI3’K in T cells did not lead to degradation of the protein, but rather to a modification of cellular localization.324 As situation at the plasma membrane is essential for PI3’K to effect its activity on phosphatidyl inositides, leading to the subsequent activation of PKB, CBL-B could still alter PKB activation in a degradation-independent mechanism. From immunoblotting studies with a phospho-specific antibody toward PKB, we observed an enhanced serum-dependent serine phosphorylation of PKB in the CBL-B(-/-) cells (Figure 3-8B). Therefore, we conclude that CBL-B negatively controls PKB phosphorylation through a mechanism that does not require degradation of p85- PI3’K. In an attempt to correlate our findings of strong PKB regulation by CBL-B with altered motility, we utilized the selective PI3’K inhibitor, LY294002.441 Transwell migration assays were performed in which CBL-B-deficient cells were allowed to migrate toward serum in either the presence or absence of increasing concentrations of LY294002. We observed a dose-dependent decrease in motility of these fibroblasts toward serum when LY294002 was added (Figure 3-8C). These data suggest that the PKB pathway is essential for CBL-B-mediated migration.

113

Figure 3-7. CBL-B deficiency leads to enhanced phosphorylation of ERK1/2 and MEK1/2 proteins. Wild-type and CBL-B(-/-) MEFs were serum starved overnight, then stimulated in the absence (-) or presence (+) of FCS. (A) An anti-phospho-MEK1/2 antibody was used to probe lysates for phosphorylated MEK1/2 (upper panel). Total MEK1/2 were revealed through immunoblotting with an anti-MEK1/2 antibody (lower panel). (B) Phosphorylated ERK1/2 were detected by immunoblotting lysates with an antibody specific for phosphorylated ERK1/2 (upper panel). Total ERK1/2 were detected upon reprobing with an anti-ERK1/2 antibody (lower panel).

114 Figure 3-8. Activation of the PI3’K-PKB pathway is impaired by CBL-B expression, while inhibition of PI3’K activity perturbs MEF chemotaxis. Wild-type and CBL-B(-/-) MEFs were serum starved overnight, then stimulated in the absence (-) or presence (+) of FCS. (A) An anti-PI3’K antibody was used to detect the p85-subunit of PI3’K (upper panel). A tubulin reprobe was performed as a loading control (lower panel). (B) Phosphorylated PKB proteins were detected in lysates using an anti-phospho-PKB antibody specific for the phosphorylation of serine 473 (upper panel). A total anti-PKB antibody was used for reprobing the membrane (lower panel). (C) CBL-B(-/-) MEFs were applied to the upper chamber of Transwell migration chambers and allowed to migrate toward media containing 10% FCS in the presence of increasing doses of LY294002 (LY) or the absence of LY but with an equivalent volume of DMSO to the bar depicted to the immediate right (DMSO). One sample did not contain either DMSO alone or LY (-). After four hours, migrated cells were detected by removing cells from the upper chambers and staining the attached cells on the lower surface of the membranes with crystal violet. During microscopic examinations, values were calculated as the total number of cells migrating in three randomly-selected 40x-objective fields. Data represent the average of triplicate wells ± standard deviation.

115

Figure 3-8

116 3.4.6. CBL-B may control ERK1/2 and PKB signaling through the regulation of GAB2. In an attempt to identify the direct binding partner of CBL-B in these MEFs, and in this way derive a signaling pathway for CBL-B-mediated regulation of migration, we performed immunoprecipitation experiments with antibodies directed toward CBL-B. As the CBL-B antibody non- specifically detects CBL, we first removed CBL protein from lysates with an overnight incubation with an anti-CBL antibody, and then performed an anti-CBL-B IP on the immunodepleted lysates (Figure 3- 9A). Immunodepletion was confirmed though the combined results of CBL and CBL-B IBs, whereby anti-CBL-B antibodies detected both of the CBL and CBL-B proteins at 120 kDa, while the anti-CBL antibody did not detect CBL protein in the immunodepleted lanes. Tyrosine-phosphorylated proteins were detected with an anti-phospho-tyrosine immunoblot (Figure 3-9A). Full-length tyrosine phosphorylated CBL-B was visualized at 120 kDa, and a 70-kDa tyrosine phosphorylated species was co-immunoprecipitated with CBL-B. Immunoblotting experiments with antibodies directed toward two candidate binding partners, Shp2 and Syk, did not detect the 70 kDa phospho-protein (data not shown). Interestingly, the CBL-B reprobe also detected a band at approximately 68 kDa. While the CBL-B antibody may have non-specifically bound to a lower molecular weight species, there is a strong possibility that the 68 kDa species is an unphosphorylated degradation product of CBL-B. As a result, our combined IP and anti-phospho-tyrosine IB studies were unsuccessful in identifying CBL-B interactors. While CBL-B may not associate with tyrosine-phosphorylated proteins, a more likely explanation is that CBL-B associates with many of the same proteins as CBL, and that immunodepletion of CBL from the lysates also depleted potential binding partners for CBL-B. As the reverse immunodepletion-immunoprecipitation is not possible due to the non-specificity of the CBL-B antibody, we proceeded in our elucidation of a mechanism via a candidate protein approach. In our search for upstream activators of the ERK1/2 and PKB signaling pathways, we considered the previous evidence from mast cells that shows downregulation of GAB2 expression and phosphorylation by CBL-B.437 As GAB2 overexpression can activate ERK1/2,442,443 and GAB2 association with p85-PI3’K enhances PI3’K activity downstream of multiple cytokine receptors,444-446 CBL-B could suppress both the ERK1/2 and PKB pathways through a potential negative regulation of GAB2. To investigate this hypothesis, we performed immunoblotting experiments to evaluate both the tyrosine phosphorylation and total protein levels of GAB2. The phosphorylation of GAB2 was indeed enhanced in the absence of CBL-B after serum stimulation (Figure 3-9B), while total GAB2 protein levels were similar between the wild-type and CBL-B(-/-) cells lines. Therefore, we conclude that CBL-B regulates GAB2 phosphorylation, but not expression, and in this way may modulate the activation of the ERK1/2 and PKB signaling pathways.

117

Figure 3-9. CBL-B negatively regulates GAB2 phosphorylation. Wild-type and CBL-B(-/-) MEFs were serum starved overnight, then stimulated in the absence (-) or presence (+) of FCS. (A) Primary overnight IPs were performed with an anti-CBL antibody, followed by secondary IPs on the remaining lysates with an anti-CBL-B antibody. Tyrosine-phosphorylated proteins were detected by phospho-tyrosine (pTyr) immunoblotting (top panel). CBL-B (2nd panel) and CBL (3rd panel) proteins were detected upon reprobing with the appropriate peptide-specific antibodies. (B) A phospho-specific anti-GAB2 antibody was used to detect phosphorylated GAB2 (upper panel). Total GAB2 levels were detected upon reprobing with an anti-GAB2 antibody (lower panel).

118 3.5. Discussion Recent evidence suggests that the CBL family of proteins play critical roles in cellular migration in a variety of cell types. Given the critical differences in protein interaction partners between the CBL and CBL-B homologs, it is essential to independently study the functional consequences of the expression of each homolog. Previous experiments by Teckchandani et al. have investigated the role of CBL in the migration of v-Abl-transformed NIH 3T3 fibroblasts.380 However, the influence of CBL-B expression on fibroblast migration has not been explored. In the present study we undertook experiments to elucidate the role of CBL-B in mouse embryonic fibroblast motility and the mechanistic consequences of CBL-B deletion in these cells. We show a CBL-B-dependent decrease in chemotactic locomotion toward serum. Our studies identify the ERK and PKB pathways as important purveyors of these serum-dependent migration signals. This study depicts the original derivation of CBL-B(-/-) MEFs with a paired wild-type line. We successfully created fibroblasts that were deficient for CBL-B at both the DNA and protein levels (Figures 3-1A and 1B) for use in subsequent studies. CBL-B did not alter the ability of these fibroblasts to proliferate in either low or high serum conditions (Figure 3-1C), nor did its deletion lead to transformation of these cells. The growth rates of CBL(-/-) MEFs were not previously analyzed,315,447 and therefore, could not be used for direct comparison. However, studies by Feschenko et al. show that over-expression of CBL in NIH 3T3 cells does not change the growth rates, anchorage dependence or contact inhibition of cells in media containing serum.386 These combined results suggest that mammalian CBL proteins do not play essential roles in fibroblast growth in response to serum. While CBL-B(-/-) MEFs did not display a serum-dependent alteration in growth potential, these cells did exhibit a modified migration phenotype. In the absence of serum, CBL-B(-/-) cells were reduced in their motility, as evidenced by TLVM. In the first three hours after serum withdrawal, CBL- B(-/-) cells displayed an altered rounded morphology that could be indicative of cell death or loss of adhesiveness. CBL-B has previously been shown to play a role in modulating each of these properties. CBL-B over-expression can protect MDA-MB-468 breast cancer cells from EGF-induced apoptosis,448 whereas CBL-B deletion can protect T helper 1 cells from CD3-stimulated apoptosis.449 The absence of CBL-B increases adhesion of both T cells and bone marrow-derived monuclear phagocytes,387,388 while lipid raft localization of CBL-B in mast cells enhances cell adhesion.450 As our studies do not show a significant effect of CBL-B deletion on MEF adhesion to fibronectin (Figure 3-3B), we conclude that CBL-B(-/-) MEF induce cell death in conditions of serum withdrawal. CBL-B was also involved in significant regulation of serum-dependent motility, as observed in both our TLVM and Transwell experiments. CBL-B was a strong negative regulator of serum chemotaxis. These results are in opposition to those of Teckchandani et al.380; they show enhanced

119 motility in 10% serum of v-Abl-transformed NIH 3T3 when CBL is over-expressed, while we show that complete deletion of CBL-B is advantageous to the serum motility of our fibroblasts. We initiated immunoblotting studies to elucidate a biochemical mechanism for the serum- induced migration differences. Our search for direct protein interaction partners of CBL-B yielded a single tyrosine-phosphorylated protein of 70 kDa (Figure 3-9A). However, we hypothesize that this protein is merely degraded CBL-B, as it was detected in anti-CBL-B IBs. Consistent with this theory, a previously published report revealed a 70 kDa protein through IB detection of immunoprecipitated CBL- B.220 Moreover, evidence exists for the ubiquitination of CBL-B and degradation in the proteasome and lysosome compartments.308 As an alternative to immunoprecipitations we assessed the phosphorylation and total protein levels of several known CBL-B target substrates. We indentified the GAB2 adaptor as a CBL-B- regulated protein. GAB2 phosphorylation was decreased in wild-type cells as compared to CBL-B(-/-) cells (Figure 3-9B). We believe GAB2 may act as a substrate for CBL-B-mediated ubiquitination as mast cell studies show decreased levels of phosphorylated GAB2 and multi-ubiquitination of total GAB2 when CBL-B is over-expressed.437 Down-modulation of GAB2 could have a major impact on migration signals propagated through both the ERK and PKB pathways. GAB2 siRNA impairs Shp2 association, MEK activation and EGF-induced motility of breast cancer cell lines,451 while GAB2 mutant constructs 452 reduce PI3’K activity and migration of β1-integrin cross-linked Ba/F3 cells. Based on our finding of GAB2 regulation, we were not surprised by our findings that both the ERK and PKB pathways were significantly affected by CBL-B expression. The activation of the ERK signaling cascade was elevated in the absence of CBL-B. MEK1/2 serine phosphorylation was increased after serum-stimulation and in the absence of CBL-B, while ERK1/2 proteins showed constitutively enhanced phosphorylation under CBL-B deficiency (Figure 3-7). ERK regulation by CBL-B is also observed in other cells types: ERK is more strongly phosphorylated in CBL-B(-/-) B cells after BCR- crosslinking,335 while over-expression of CBL-B impairs ERK phosphorylation in mast cells.437 From these results, we can conclude that CBL-B plays a negative regulatory role in the activation of the ERK pathway. The role of CBL in chemotaxis of fibroblasts toward serum has not been investigated. However, phosphorylation of ERK1/2 is required for platelet derived growth factor (PDGF)-mediated migration of CBL(-/-) MEFs.384 Therefore, we propose that CBL proteins commonly regulate the ERK pathway for the purpose of fibroblast migration. We were intrigued by that data that showed constitutive phosphorylation of ERK in the absence of a mimicked response from MEK. We conclude that either ERK phosphorylation is sustained in CBL-B(-/-) cells or that MAP kinase phosphatases are down-regulated in the absence of CBL-B. Future studies into this serum-independent phosphorylation of ERK will be of interest. The PKB pathway also displayed serum-dependent regulation by CBL-B. The serine phosphorylation of PKB was enhanced in the absence of CBL-B while levels of the upstream element,

120 PI3’K, remained unaltered by CBL-B expression (Figure 3-8). PI3’K activity was essential for motility, as treatment of the cells with LY294002 reduced motility. Similarly, CBL uses the PKB pathway for serum-dependent chemotaxis of fibroblasts. Over-expression of CBL enhances the migration of v-Abl- transformed NIH 3T3, a process also down-modulated by LY294002 and wortmannin treatment.380 These data suggest that CBL proteins exploit the same signaling pathway and yet initiate opposing forces. While we hypothesize that CBL-B regulates PI3’K through GAB2, it is also possible that CBL-B directly associates with the p85 subunit of PI3’K as has been observed previously in T cells.323 In this cell type CBL-B attaches to p85 via linkage of its proline-rich region with the SH3 domain of p85. This interaction leads to ubiquitination of p85 without degradation324; instead, CBL-B expression prevents the localization of p85 with the CD28 docking protein at the membrane – the site of PI3’K activity. Our results in wild-type fibroblasts are consistent with a degradation-independent mechanism of PI3’K downregulation, revealed as stable p85 levels and reduced PKB phosphorylation. Meanwhile, the direct PI3’K-CBL interaction - primarily mediated through binding of phospho-tyrosine residue 731 of CBL to the SH2 domain of p85 – results in PI3’K activation.288,289 In other words, CBL acts (in opposition to CBL-B) as a positive regulator of PKB. Through this brief analysis, we argue that CBL-B may also directly contact PI3’K. Moreover, based on the mechanism of the p85-CBL interactions it is not surprising that both CBL and CBL-B utilize the PKB signaling cascade for fibroblast chemotaxis. CBL-B-mediated regulation of PI3’K signaling may also affect other cellular pathways, including those modulated by the Rho/Rac GEF, Vav. Phosphorylation of Vav is enhanced in the 369 presence of phosphatidylinositol-3,4,5-trisphosphate (PIP3), the lipid product generated by the action of PI3’K on phosphatidylinositol-4,5-bisphosphate (PIP2). Our data shows an enhanced Vav3 phosphorylation in the absence of CBL-B (Figure 3-4A). These results are consistent with those observed in T cells, where CBL-B deficiency increased Vav phosphorylation.332,392 In addition, the impeded Vav3 phosphorylation of our wild-type MEF could not be restored by MG132 treatment. From these data, we conclude that CBL-B controls Vav3 through a proteasomal-independent mechanism. Two possible modes of regulation can be proposed. The first implicates PI3’K, where CBL-B sequestration of PI3’K away from its active site at the membrane, would leave PIP2 to predominate and inhibit Vav3 phosphorylation. A second model can also be put forward in which a direct interaction between CBL-B and Vav3 could mono-ubiquitinate Vav3 and facilitate its degradation through the lysosome. In support of our data, and of the theory of lysosomal-mediated degradation, Sattler et al. also failed to visualize increased Vav levels after MG132 treatment of BCR-ABL-transformed Ba/F3 cells.134 Nevertheless, we believe the first mechanism predominates in our fibroblasts, as total Vav3 levels are not severely diminished in wild-type cells (Figure 3-4B). In defense of this argument, Fang and colleagues could not demonstrate a direct ubiquitin transfer from CBL-B to Vav, despite numerous attempts.323 In contrast, CBL effectively ubiquitinates Vav, primarily through the direct association of Y699/Y700 (amino acid sequence YMTP) with the SH2 domain of Vav.287,364 CBL-B does not possess a

121 YMTP motif, and instead uses its proline-rich sequences to bind to Vav.220 Similar to the PI3’K mechanism described above, the method of CBL homolog binding to Vav may influence the downstream signaling events. The modified Vav phosphorylation was of interest as it indicated that the GEF activity was also affected.136 However, activation of the Vav primary substrate, Rac1,136 was unaltered in our fibroblasts (Figure 3-5). In fact, of the proposed pathways downstream of Vav that we analyzed – PAK,453 SAPK,136 and p38-MAPK454 – none were modulated by CBL-B-regulated phosphorylation of Vav (Figure 3-6). We reason that Vav3 may modify the activity of other target GTPases, including Cdc42 and RhoA.455 Taken together, we believe that CBL-B is a key regulator of cell motility and propose the mechanism detailed in Figure 3-10. CBL-B could degrade phosphorylated GAB2, resulting in decreased association with Shp2 and PI3’K proteins, and in this way reduce activation of both ERK and PKB. Alternatively, CBL-B could directly associate with p85-PI3’K to regulate is cellular distribution, and in this way, alter PKB signaling. Modulation of PI3’K may link to the observed reduction in phosphorylated Vav3 as a consequence of CBL-B expression. Modifications of the ERK and PKB pathways are of interest, as these two pathways are essential regulators of morphological changes in motility. ERK activation regulates the process of de-adhesion, allowing the rear cell body to retract during movement. As exemplified in fibroblasts, the MEK-specific inhibitor, PD98059, blocks the dissolution of focal adhesions and abrogates motility.456 Meanwhile, PKB localized to the leading edge of fibroblasts and establishes polarity by stabilizing microtubules.457,458 This evidence exposes a mechanism by which CBL-B can influence motility at both the front and rear of fibroblasts. Our study describes CBL-B as an important negative modulator of serum-dependent fibroblast motility. This knowledge furthers our understanding of wound repair, as fibroblast infiltration is a key component of this process. Precise regulation of fibrosis is essential, as too few fibroblasts at the site of injury can lead to chronic non-healing wounds, while too many fibroblasts may promote fibrotic disease. Therefore, continued investigation into the role of CBL proteins in fibroblast migration may aid in our understanding of these afflictions.

122

Figure 3-10. A proposed mechanism for the CBL-B-mediated regulation of serum-dependent migration.

CHAPTER 4

DISCUSSION AND FUTURE DIRECTIONS

123 124 4.1. Discussion Hematopoiesis describes the formation and development of blood cells. When this process goes astray, leukemia – the uncontrolled proliferation of blood elements, can result. CML represents a unique sub-type that is characterized by expansion of the myeloid lineage without a blockage in differentiation capacity. The causative agent of CML is a chromosomal translocation that results in the fusion of bcr and abl genes. The resulting protein fusion product, BCR-ABL, initiates a cascade of signaling events that induce factor-independent proliferation, reduced apoptosis and modified adhesion and migration properties. Fortunately, small molecule inhibitors were discovered that effectively block activation of the BCR-ABL kinase. Yet, as often occurs in biology, mutation of BCR-ABL has occurred such that the tyrosine kinase inhibitors (TKIs) are ineffective for the treatment of CML. Therefore, studies are still underway to identify those pathways that are prominently regulated by BCR-ABL and that may serve as useful targets for combination therapy with TKIs. CBL-B is a known adaptor protein and E3 ubiquitin ligase. Sattler and colleagues have previously shown that CBL-B mRNA and protein expression is down-modulated by BCR-ABL.134 We hypothesized that CBL-B may be an essential substrate of BCR-ABL. We initiated a murine BCR-ABL BMT with CBL-B(-/-) cells to determine the role of this protein in leukemogenesis (Chapter 2). We observed an MPD phenotype in these mice that was significantly delayed in latency as compared to recipients of wild-type BCR-ABL bone marrow. The CBL-B(-/-) recipients displayed elevated neutrophils numbers in the differential counts and enhanced Gr-1+ and Mac-1+ cells in the spleen. Homing analyses of the CBL-B WT and KO cells revealed a reduced ability of the CBL-B-deficient cells to home to the BM microenvironment. From these studies we concluded that the prolonged latency was a function of reduced BM homing of the CBL-B(-/-) cells. Homing is directed by chemokine-mediated migration. In an independent study of CBL-B- deficient MEF, we observed a reduced serum-dependent chemotactic migration in both Transwell migration and TLVM experiments as compared to control CBL-B WT cells. The signaling mechanisms responsible for the altered chemokinesis were elucidated. Immunoblotting studies identified enhanced GAB2 phosphorylation as a consequence of CBL-B-deficiency, and suggested that GAB2 may serve as a key intermediary whose function is specifically modulated by CBL-B during serum stimulation. As well, the serum-dependent activation of both the ERK and PKB pathways was significantly upregulated suggesting that these pathways may be critical mechanisms of CBL-B-mediated motility. Combined, our results indicate that CBL-B is an essential regulator of cellular motility in two different cell systems. In hematopoietic cells, CBL-B may intensify leukemogenesis by tempering the extravasation of cells during disease progression. As CBL-B controls fibroblast motility, normal fibrosis may be critically impaired resulting in either bleeding disorders or fibrotic disease. Continued investigations of the CBL-B-modulated motility in other cell types may reveal underlying disorders resulting from CBL-B deficiency.

125 Additionally, our BMT revealed that CBL-B was a negative regulator of granulopoiesis. We were initially surprised by these findings as the literature to date primarily describes CBL proteins as controllers of lymphocyte proliferation and activation. But multiple recent papers have identified CBL family mutations in diseases of the myeloid lineage.398-402 We believe that future studies within our laboratory, and the scientific community as a whole, will pinpoint the mechanisms through which CBL proteins influence myeloid diseases.

4.2. Future directions to delineate the role of CBL-B in hematopoietic cells

4.2.1. Immediate future directions with regard to the function of CBL-B in BCR-ABL-mediated leukemogenesis. Our results from Chapter 2 suggested that CBL-B influences BCR-ABL-mediated leukemogenesis by modulating the homing of BMCs in a BMT and granulocyte differentiation and production. However, as will any scientific project, supplementary experiments will provide additional support for our inferences. Those studies planned for the immediate future are detailed below. One area of keen interest was defined by the BMT results which showed increased percentages of granulocytes in the spleen of CBL-B(-/-) BCR-ABL recipients (Figure 2-5) without a compensatory increase in spleen mass (Figure 2-3A and Table 2-1). We hypothesized that the absence of a correlation was due to either: (a) the presence of granulocytes that were targeted for apoptosis (and therefore shrinking), or (b) other architectural distortions that were not visualized by flow cytometry. To rule out the first hypothesis, we will harvest spleen cells at sacrifice and test, by flow cytometry, for the presence of Annexin V- and propidium iodide (PI)- positivity. This analysis will measure cells in both the early (AnV+/PI-) and late (AnV+/PI+) stages of apoptosis. To investigate the second theory we will perfom comprehensive histopathological analysis of sectioned spleen tissue to visualize invading or expanding non-hematopoietic elements that could account for the altered spleen mass. Our model (in Figure 2-9) proposes that the delayed latency of CBL-B-deficient BCR-ABL BMTs is a function of perturbed homing and not of reduced growth rates. To confirm our assertion, the proliferation capacity of wild-type- and CBL-B(-/-)- BCR-ABL-infected BMCs will be compared. We have previously performed a single in vitro methylcellulose CFU assay. Unfortunately, our results were confounded by relatively minor enhancements of the proliferative potential in BCR-ABL-infected cultures (Figure 4-1). We hypothesize that colony formation may be enhanced with the use of commercially available reagents (e.g., MethoCult from Stem Cell Technologies). Moreover, proliferation as a result of BCR-ABL infection in a small proportion of BMCs may be masked by growth in cytokine of uninfected cells. Therefore, our future CFU experiments will utilize MethoCult media combined with quantification of only GFP+ (BCR-ABL-expressing) colonies. Nevertheless, we predict that BCR-ABL colony formation capacity will be equivalent in the CBL-B WT and KO cultures.

126

Figure 4-1. Comparison of the colony formation ability of CBL-B WT and KO cells after BCR- ABL infection of BMCs. Wild-type and CBL-B(-/-) donor BMCs were derived from 5-FU-treated mice. The cells were infected with BCR-ABL as per the methods described in Chapter 2 and then plated into methylcellulose media containing either low (0.1 ng/mL) or high (10 ng/mL) concentrations of IL-3, plus SCF and IL-6. Colonies were counted nine days after plating. Results are the mean of four independent cultures and error bars represent standard deviation.

127 As with other BMT studies, our CBL-B(-/-) BCR-ABL transplant recipients died as a result of cellular lodging in the lungs and resulting pulmonary hemorrhage. We believe that the CBL-B(-/-) recipients died not as a consequence of enhanced leukemic burden but as a result of greater numbers of granulocytes homing to and lodging in the lungs at an accelerated rate compared to the wild-type recipients. We plan to investigate this theory by sacrificing wild-type and CBL-B(-/-) BMT animals at serial time points, fixing and staining the lungs with H&E, and examination of stained sections for infiltrating cell elements. Original papers that defined the BCR-ABL BMT technology included serial passage studies to confirm clonality and provide proof that the disease was indeed a malignancy.202 As the system has been successfully employed for the last 10 years, investigators today rarely proceed with this step. Nevertheless, we will perform two assays to verify transplantability. We previously performed day 12 CFU-spleen (CFU-S) assays by transferring leukemic spleen cells from a primary (1o) recipient to a lethally irradiated secondary (2o) recipient. Spleen colonies formed in the 2o recipients of both our CBL- B WT and KO BCR-ABL transplants (data not shown). However, these colonies have not yet been assessed for clonality, and therefore, will be a focus of our future studies. We will also transplant BMCs, spleen cells, or a combination of the two, via IV injection into 2o recipients. We predict that our results will be equivalent to those observed previously by other groups in that most 2o recipients will succumb to an MPD, while others will display evidence of T- or B- cell leukemias and lymphomas.202,205 The above-defined experiments will complete our study evaluating the ability of CBL-B, in a murine model, to control leukemogenesis by BCR-ABL. Out of this study emerged new roles for CBL- B in granulopoiesis, homing, and potentially diseases caused by other oncogenes. The following section will discuss those expanded investigational avenues that will be pursued in future initiatives.

4.2.2. Broader investigational avenues stemming from this project.

Is CBL-B a negative regulator of BCR-ABL-mediated granulopoiesis? Our BMT studies revealed that the absence of CBL-B in BCR-ABL transplants led to enhanced numbers of neutrophils (and reduced lymphocyte numbers) in the peripheral blood, and an increase in the percentage of Gr-1+ and Mac-1+ cells in the spleen. These data led us to conclude that CBL-B may negatively regulate the granulopoiesis that is observed in CML cells. To investigate this hypothesis, further investigations will be performed: (a) on the BMT animals, (b) in CBL-B-deficient BCR-ABL-expressing cell lines, or (c) in CBL-B-deficient BCR-ABL-expressing BMCs. In the mice, we will perform similar experiments to those of Zhang and Ren when they assessed the production of IL-3 and GM-CSF in their BMT mice.207 Through the use of enzyme-linked immunosorbent assays (ELISAs) we will compare the serum concentrations of these myeloid cytokines between the two CBL-B donor backgrounds. Additionally, we will extract RNA from whole BM samples

128 (an organ highly infiltrated by BCR-ABL-expressing cells), and measure, via reverse transcriptase polymerase chain reaction (RT-PCR), the gene expression of these myeloid cytokines in BMCs. The expression of other myeloid markers (as indicated in Table 1-4) will also be evaluated. We predict that if CBL-B blocks granulopoiesis, then the CBL-B(-/-) BCR-ABL transplant animals will have enhanced myeloid cytokine concentrations in the serum and elevated myeloid cytokine transcript numbers in their BMCs. There are many other useful experiments for determination of CBL-B as a granulopoiesis regulator that cannot be tested from tissue samples of the BMT mice. For example, it would be of interest to perform immunoblots on tissue lysates with antibodies directed toward myeloid-specific proteins. However, these experiments cannot be performed because of two reasons. Firstly, insufficient amounts of protein lysate are obtained from the largely GFP+ BM. Secondly, the splenic compartment, that houses an abundance of cells for protein extraction, cannot be sorted for GFP-positivity due to biohazard issues (i.e., VSV-G-pseudotyped cells). Therefore, in vitro experiments with non-hazardous biologic samples will be useful for additional analyses. For these in vitro studies we will perform RNA-interference (RNAi) knock-down of CBL-B in a cell line into which BCR-ABL has been previously introduced. Small interfering RNA (siRNA) duplexes to CBL-B have been effectively used in 293Ts, BT20s, and adipocytes.313,387,459 For these experiments we will either utilize Ba/F3-BCR-ABL or FDCP-1 BCR-ABL cells. We believe that CBL-B RNAi will work in Ba/F3 cells based on promising results in our laboratory with siRNA and short hairpin RNA (shRNA) targeted toward the SH2B protein (Javadi et al., unpublished results). Forty-eight to 72 hours after the introduction of siRNA, Ba/F3 BCR-ABL cells will be collected and RNA or protein will be extracted. RT-PCR experiments will be performed on cDNA obtained from the RNA samples, and gene expression of myeloid transcription factors and response genes (described in Table 1-4) will be compared between the CBL-B siRNA-treated cells and the untreated cells (or those into which scrambled siRNA was introduced). (Note: From this point forward the untreated and scrambled siRNA-treated cells will be referred to as the “control” cells.) The protein expression of these myeloid genes will also be assessed. Furthermore, the phosphorylation status of PKB and ERK can be assessed with this system. We believe that if CBL-B negatively regulates granulopoiesis, our siRNA experiments will show enhanced RNA and protein expression of myeloid targets in those Ba/F3-BCR- ABL cells with CBL-B knock-down. As Ba/F3 BCR-ABL cells do not secrete IL-3, and the conditioned media from these cells cannot support the growth of Ba/F3,63 we cannot utilize this cell line for studies of secretion or growth. However, the factor-dependent cell progenitor – 1 (FDCP-1) murine myeloid cell line has been shown to secrete IL-3 when BCR-ABL is introduced,223,224 and therefore, will be used for these studies. We will test the ability CBL-B siRNA-treated FDCP-1 BCR-ABL cells to secrete IL-3 (through ELISA measurements) and compare the IL-3 concentration to that observed in the control cells. We will also

129 measure the ability of these cells to grow (via an XTT assay) in media that does not contain IL-3, and the capacity for the conditioned media from these cells to induce growth of FDCP-1 cells. Moreover, we will apply IL-3 neutralizing antibodies to the CBL-B siRNA-treated cells and record the resulting growth. We hypothesize that knock-down of CBL-B will increase secretion of IL-3, leading to a enhanced growth of FDCP-1 BCR-ABL cells, and FDCP-1 cells to which the conditioned media is applied. Additionally, we imagine that the anti-IL-3 antibodies will be less effective in controlling the growth of the FDCP-1 BCR-ABL cells with CBL-B knock-down. Our final assay to assess the role of CBL-B in BCR-ABL-mediated granulopoiesis, will monitor the ability of CBL-B to regulate colony formation in the presence of BCR-ABL. CBL-B WT and KO BMCs will be infected with BCR-ABL or a control retrovirus. Again, for these assays, we will use the bicistronic vector that expresses GFP so that transduced cells can be selected through sorting. We believe selection (via fluorescence microscopy) of the GFP-positive colonies is essential, based on previous studies that showed no enhancement of growth in cell populations where a percentage of BCR- ABL cells were transduced (i.e., Five – 10% GFP+; Figure 4-1). GFP-positive cells will be plated in methylcellulose culture media with appropriate concentrations of IL-3, SCF, G-CSF and GM-CSF. CBL- B deficiency may lead to a change in the number of clonal outgrowths, but in our assessment of granulopoiesis, we are uniquely interested in the colony type that emerges. We expect that CBL-B deficiency in BCR-ABL-expressing BMCs will lead to an increase in the number of CFU-GEMM or CFU-GM. Our CBL-B(-/-) BCR-ABL BMC recipients showed a reproducible but relatively minor enhancement of granulopoiesis in the peripheral blood. This minimal effect may be due to compensation by CBL. This family member compensation was also suggested by Dinulescu et al. to explain the absence of a change in MPD phenotype and latency in their CBL(-/-) BCR-ABL BMTs.133 Yet, we would argue that the CBL(-/-) BMTs also showed some trend toward an altered phenotype. Their results demonstrated a single mouse without evidence of disease at 50 days post-transplantation and the peripheral blood WBC counts were significantly enhanced in the CBL(-/-) transplants (76 x 103/µL) versus the WT BCR-ABL recipients (13 x 103/µL). This data points toward a role for CBL in CML development. As a result, we conjecture that a transplant of BCR-ABL-infected CBL/CBL-B DKO cells would reveal an even greater granulopoietic phenotype; and would therefore be an interesting pursuit. As the CBL/CBL-B DKO animals die in utero390 we will either use fetal liver cells for transduction and transplantation or cells from Mx1-Cre CBL-floxed/CBL-B(-/-) mice with deletion of the CBL genes in only the hematopoietic lineage. Support for this “combinatorial” study is provided by experiments from the Dok family of proteins. Dok-1(-/-) animals show increased ERK activation but no obvert defects in hematopoiesis.460 Yet, when both Dok-1 and Dok-2 are deleted in DKOs, the mice develop a spontaneous MPD evidenced by increased splenic weight; enhanced numbers of Gr-1+/Mac-1+ cells in the BM, spleen and the

130 peripheral blood; a greater percentage of neutrophils and monocytes in the differential count; and elevated overall leukocyte numbers.218,219 These data from the Dok DKOs backs the idea that, in some instances, deletion of multiple family members may reveal an underlying hematopoietic defect. In conclusion, our data reveal an underlying role for CBL-B in negatively regulating granulopoiesis during BCR-ABL-mediated disease development. We hypothesize that CBL-B may down-modulate components of the PKB and ERK pathways and impair transcription of myeloid genes. Careful examination of the CBL(-/-) transplants suggests that CBL may also play a role in granulopoiesis, and warrants BMTs with CBL and CBL-B DKO donor cells. Importantly, these data suggest that the down-regulation of CBL-B expression may be an active process by which BCR-ABL promotes myeloid differentiation over hematopoiesis in other lineages.

What are the downstream protein components of BCR-ABL signaling that may be influenced by CBL- B? In our BMT studies, we derived protein from spleen cell lysates and measured the phosphorylation of PKB and ERK. Our results were complicated by the presence of non-transduced cell lysate in our samples. Unfortunately, as described above, we cannot sort these samples for GFP- positivity given the biohazard nature of the samples. Therefore, analyses of downstream signaling components must be assessed by alternative means. We believe the Ba/F3-BCR-ABL cell line will serve as a useful model for investigations of signaling. Initial immunoprecipitation studies have been performed in the Ba/F3 and Ba/F3-BCR-ABL cell lines. Using the same protocol as for the MEFs (Chapter 3), we were able to specifically co- immunoprecipitate CBL-B interactors by first depleting the cells of CBL, and then performing CBL-B immunoprecipitations. Contrary to the results from the MEF studies in our experiments with Ba/F3 cells we observe the co-immunoprecipitation of multiple tyrosine-phosphorylated proteins with CBL-B (Figure 4-2). A prominent band is visible in both the CBL and CBL-B immunoprecipitations at 210 kDa, which is most likely BCR-ABL given its known molecular weight (MW) and the previous evidence that both CBL and CBL-B can co-immunoprecipitate the oncogene.127,128,134 Reprobes of the immunoblots with anti-ABL antibodies will confirm this band as BCR-ABL. Similar to Sattler and colleagues, we also observed tyrosine-phosphorylated proteins with MWs of 55- and 60- kDa. We have yet to identify these proteins, but we suspect that the 60-kDa protein is Dok-1 given its respective alignment with the CBL immunoprecipitations and the known interaction of this protein with CBL in BCR-ABL cells.129 Moreover, we suspect that the lower tyrosine phosphorylated band may be the 52- kDa isoform of Shc. In studies by Ren et al. Shc was identified in a multimeric complex with CBL and p85-PI3’K, GAB2, and GRB2.461 Further immunoblots will confirm the identity of these two phospho- proteins.

131

Figure 4-2. CBL-B co-immunoprecipitates tyrosine-phosphorylated proteins of 55, 60 and 210 kDa in BCR-ABL-expressing cells. Ba/F3 vector-infected or BCR-ABL-expressing cells were starved of IL-3 for six hours and then stimulated in the absence (-) or presence (+) of IL-3. Primary overnight IPs were performed with an anti-CBL antibody (CBL IP), followed by secondary IPs on the remaining immunodepleted (ID) lysates with an anti-CBL-B antibody (CBL ID/CBL IP). Tyrosine-phosphorylated proteins were detected by phospho-tyrosine (pTyr) immunoblotting (top panel). CBL (2nd panel), CBL-B (3rd panel) and GRB2 (4th panel) proteins were detected upon reprobing with the appropriate peptide-specific antibodies.

132 Interestingly, GRB2 was identified as an interacting partner with CBL-B either in the presence or absence of BCR-ABL. We are unsurprised by this result based on the evidence that the CBL homolog can bind to GRB2 in BCR-ABL cells.128 This experiment has been performed in duplicate, and in both instances the association of CBL-B with GRB2 was enhanced in the presence of BCR-ABL. We postulate that BCR-ABL expression initiates CBL-B phosphorylation, which enhances the association of CBL-B with the SH2 domains of GRB2. This result of a CBL-B/GRB2 co-immunoprecipitate may simply identify a mechanism by which CBL-B interacts with BCR-ABL or it may indicate that CBL-B may propagate downstream signaling through interactions with those proteins bound to GRB2, such as GAB2. Our data presented in Chapter 3 indicates that CBL-B can control the level of GAB2 tyrosine phosphorylation in fibroblasts. We postulate that CBL-B may also modulate the phosphorylation of GAB2 (and its downstream substrates, e.g., PKB and ERK) in hematopoietic cells expressing BCR-ABL. To investigate these phosphorylations we will utilize the Ba/F3 BCR-ABL CBL-B siRNA system and compare the level of phosphorylation in “control” and CBL-B knock-down cells. As GRB2 is known to associate with GAB2, and E3 ubiquitin ligases can indirectly ubiquitinate substrates,462 we hypothesize that CBL-B will down-modulate GAB2 phosphorylation through ubiquitination. In support of our theory, CBL-B has previously been shown to ubiquitinate GAB2 in in mast cells.437 Ubiquitination of phospho-GAB2 would also likely decrease the phosphorylation of its downstream substrates. Nevertheless, recent data indicates that the GAB2(-/-) BCR-ABL BMT recipients fail to develop CML-like disease.216 As our CBL-B(-/-) BMT experimental animals eventually succumbed to the MPD, we are not confident that the predominant substrate regulated by CBL-B is GAB2. Moreover, we cannot ignore the evidence from Sattler and colleagues that shows CBL-B association with ubiquitinated Vav in BCR-ABL-transformed cell lines.134 We believe CBL-B directly targets Vav for ubiquitination, leading to down-modulation or differential localization. Therefore, we will assess the total level of Vav protein and its ability to be phosphorylated. As Vav is predominantly a GEF for Rac in BCR-ABL cells,135,178 we will perform Rac activation assays (as described in Chapter 3) to determine if Vav regulation impairs Rac activation. We postulate that in CBL-B-depleted cells, RAC activity will be enhanced (Figure 4-3). Our theory is feasible given the fact that BCR-ABL down-regulates CBL-B,134 and that Rac is hyper- activated in chronic phase CML cells.183 Additionally, in our BMT experiments, the CBL-B(-/-) BCR- ABL recipients had enhanced neutrophil counts, and would theoretically have high Rac activity given the proposed hypothesis of CBL-B-mediated down-regulation of Vav. Similarly, but in the reverse mechanism, the Rac1/2 DKO BCR-ABL BMT animals showed a decreased neutrophil percentage in the differential count.183 These studies all provide support for a mechanism of signaling through CBL-B to Vav and Rac. There are many potential substrates of Rho proteins.139 We will assess the activation of ERK, PKB, SAPK, p38-MAPK, and NF-κB in the BaF3 siRNA system through immunoblotting with

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Figure 4-3. CBL-B-mediated ubiquitination of Vav may negatively regulate Rac activity and block neutrophil proliferation. In this model, activity or protein level is directly represented by the font and shape size. Under normal conditions (center panel) we postulate that CBL-B will ubiquitinate Vav, resulting in limited activation of Rac, and control of neutrophil proliferation. In CBL-B-deficient cells (left panel) we argue that Vav ubiquitination will be decreased. As a result, Rac activation will be dramatically enhanced, as will the proliferation of neutrophils. While evidence does not exist for a circumstance where CBL-B is over- expressed, we believe the results of this experiment would closely mimic a situation where Rac activity would be drastically reduced (right panel). Thomas et al. have shown that in the absence of Rac activity neutrophils numbers are greatly decreased.183

134 phospho-specific antibodies. We believe the phosphorylation of each of these proteins will be enhanced by CBL-B knock-down, potentially through a mechanisms involving Rac. The results of these in vitro signaling studies will provide support for continued functional studies, including the role of CBL-B in homing of BMCs.

How does CBL-B control the homing of bone marrow cells? Our results from Chapter 3 indicate that CBL-B is a negative regulator of serum-dependent chemotaxis of MEFs, and may control migration through pathways involving PKB and ERK. Meanwhile, the perturbed BM homing of CBL-B(-/-) BMCs (as described in Figure 2-6) would indicate, that in the context of the mouse and in hematopoietic cells, CBL-B is a positive regulator of migration. Combined, these results lead us to conclude that different migratory signaling mechanisms are at play in the BMCs, based on either altered protein environment or chemo-attractant. As described in the Introduction, BMCs prominently home to the chemokine SDF-1α that is produced by the BM stromal cells. Previous studies have investigated the role of CBL proteins in the in vitro migration of Jurkat T cells toward SDF-1α. SiRNA toward either CBL or CBL-B can effectively reduce SDF-1α-mediated migration.379 Moreover, this chemokine enhances CBL and CBL-B phosphorylation and their association with a plethora of signaling proteins, including PI3’K.383 However, ERK phosphorylation, while being enhanced by SDF-1α, is not altered by expression of the CBL proteins.379 Based on these migration studies, we believe that the homing of the CBL-B(-/-) BMCs is regulated by SDF-1α in a similar manner to the Jurkat T cells. To investigate this hypothesis, we will inject mouse spleens with SDF-1α and monitor homing of CBL-B WT and KO cells. Should homing of CBL-B(-/-) cells to the splenic compartment be severely reduced, as compared to wild-type cells, we would conjecture that homing of the BMCs was dependent upon SDF-1α. Similar to studies performed above in Jurkat T cells, we will initiate TLVM experiments to monitor the chemokinetic movement of CBL-B-deficient BMCs in media supplemented with SDF-1α. We predict that CBL-B(-/-) BMCs will be less motile in the presence of SDF-1α. Our initial experiment shows enhanced cell movement with SDF-1α treatment (Figure 4-4), but given the preliminary nature of this data we cannot make any conclusions with regard to the influence of CBL-B expression on chemokinesis. We will repeat this experiment and also expand upon this assay with the use of cell separation chambers to measure directed migration. Furthermore, we will assess the signaling pathways downstream of CBL-B that control chemotaxis toward SDF-1α. BMCs will be harvested from mice of both CBL-B genotypes, sorted (in the presence of serum but the absence of cytokine) for Lin- cells, and then stimulated with SDF-1α. Subsequent to stimulation the cells will be lysed, protein will be harvested and IBs will be performed to

135

Figure 4-4. Motility of wild-type and CBL-B(-/-) BMCs is enhanced by SDF-1α treatment. BMCs were derived from 5-FU-treated CBL-B WT and KO animals and sorted for Lin- c-kit+ cells. Sorted cells were applied to fibronectin-coated dishes in either the presence (plus) or absence (minus) of 100 ng/mL SDF-1α. Migration was observed by TLVM with images acquired every 20 seconds for 4 minutes. The time 0 image was pseudocolored as red, while the image at 4 minutes was pseudocolored green. By overlapping the two images we can observe migration as independent red and green cells versus the absence of migration as depicted by a yellow cell. These figures are representative of two replicates of an independent experiment.

136 assess potential target substrates of CBL-B activity. To uniquely identify important signaling pathways in SDF-1α-mediated motility, inhibitors could be added during the sorting step. We hypothesize that CBL-B-deficient cells will show decreased activation of migration targets such as Rac, given the proposed signaling mechanism described above. We will need to specifically monitor both the Rac1 and Rac2 isoforms, as these proteins play alternate roles in hematopoietic stem cell and progenitor (HSC/P) homing – Rac2 deficiency slightly enhances homing to the BM while Rac1(-/-) cells are impaired in their homing to the BM.463 Therefore, we suspect that Rac2 is specifically regulated by CBL-B. These studies of SDF-1α-mediated migration will delineate the biological signaling mechanisms responsible for the decreased homing of our CBL-B(-/-) BMCs.

Does CBL-B control the homing of BCR-ABL-expressing bone marrow cells? In our BMT studies we hypothesized that the delayed latency of leukemia development in the CBL-B(-/-) BCR-ABL transplants was a function of reduced homing to the bone marrow, based on the results described above and detailed in Figure 2-6. To substantiate our claim, we will perform intrafemoral (IF) injections of the BCR-ABL-infected cells. We hypothesize that this altered step in the transplant will either lead to an equivalent disease latency between donor backgrounds, or possibly even a shortened latency in the CBL-B(-/-) BCR-ABL transplants given the hypothesized role of CBL-B in granulopoiesis as described above. Using TLVM to monitor the motility of BCR-ABL cells toward SDF-1α may not be a useful experiment given the results of several studies that suggest that BCR-ABL+ cells are impaired in their response to SDF-1α174,176,177; rather, signals mediated through CD44,215 or downstream of Rac,140 may be important mediators of homing in BCR-ABL+ cells. Therefore, we will utilize the wild-type and CBL-B knock-out BCR-ABL-infected BMCs in two key experiments. We will assess Rac activation and will determine the CD44 cell surface expression level through flow cytometric evaluation of cells labeled with fluorescent anti-CD44 antibody. We will also perform homing assays with CBL-B WT and KO cells that are BCR-ABL-infected (as determined by GFP expression). We expect that the CBL-B-deficient BCR-ABL cells will home less effectively than the WT BCR-ABL cells, given our results from the Chapter 2 homing experiments with BMCs. Yet, based on the proposed CBL-B/Vav/Rac model and the previous evidence that Vav activity is required for motility of BCR-ABL Ba/F3 cells and that Rac activity negatively regulates the homing of BCR-ABL-expressing 32D cells,140,178 one may argue that CBL-B deficiency may enhance homing of BCR-ABL-infected cells. However, it is important to note that these previous experiments were not performed with primary cells. Additionally, CBL-B may increase the activity of Rac2, which as described earlier, is a negative regulator of homing. Furthermore, CBL-B may utilize other pathways to modulate cellular migration.

137 In a final assay, we will perform immunofluorescence assays to detect the cellular localization of BCR-ABL in CBL-B-deficient cells. Skorski et al. have shown that BCR-ABL localization at the cytoskeleton is important for cell homing.214 Therefore, we postulate that in the absence of CBL-B, BCR- ABL will occupy a cellular location distinct from the cytoskeleton. Combined these studies of CBL-B-mediated modulation of homing in general, and the role of CBL-B in this unique step of the BCR-ABL BMT will substantially expand our limited knowledge of the mechanism by which BCR-ABL cells home to the BM microenvironment.

How does BCR-ABL regulate CBL-B protein expression? Sattler et al. indicate that CBL-B protein expression is decreased in cells that express BCR-ABL, i.e., MO7e-BCR-ABL and K562 cells untreated with imatinib.134 These data correlate with our findings that BCR-ABL expression decreases CBL-B protein levels in Ba/F3 cells (Figure 2-1). From these data, one could hypothesize that BCR-ABL controls CBL-B protein stability. However, the same published paper reports additional data that suggests that CBL-B is regulated at the level of mRNA transcription. When the BCR-ABL-expressing cell lines were treated with imatinib, CBL-B mRNA levels increased. Combined, these data indicate that BCR-ABL elicits signals that culminate in down-modulation of CBL- B gene expression. We will investigate the mechanism used by BCR-ABL to either block CBL-B transcription or perturb mRNA stability. Our initial assay will define the nature of BCR-ABL-mediated downregulation. We will perform an mRNA decay assay in Ba/F3 BCR-ABL cells. We will first treat the cells with or without imatinib, and then block mRNA synthesis with the addition of actinomycin D. RNA will be extracted at multiple times points over a 24-hour period. (A detailed description of this procedure can be found in ref. 151). If CBL-B regulation does not occur at the level of mRNA turnover, then quantitative real-time reverse-transcription PCR (Q-RT-PCR) analysis will show comparable CBL-B mRNA decay in the presence or absence of imatinib treatment. Should CBL-B show differential decay plus and minus imatinib, then inhibitors to each of the major BCR-ABL signaling pathways (as detailed in Figure 1-12) will be added to the cell culture media and CBL-B protein and mRNA turnover will be analyzed. This method should determine those pathways that regulate CBL-B expression. There are several RNA sequence elements that regulate mRNA decay.464 For example, those mRNA products of genes with significant adenylate uridylate (AU)-rich elements (AREs) in their 3’- untranslated regions (UTRs) are often targets of rapid destabilization. A query of the ARE Database ARED-mRNA version 2.0 (http://rc.kfshrc.edu.sa/ARED) does not detect AREs in the 3’ UTR of CBL- B. Therefore, we postulate that BCR-ABL is down-regulating CBL-B at the level of transcription rather than mRNA turnover.

138 Transcription is predominantly controlled from the 5’UTR (or promoter) region of a gene. As no information is available for the CBL-B promoter, we will clone this region using the CLONTECH kit called GenomeWalker, which allows for easy cloning of short genomic sequences close to known sequences. Primers will be designed near the transcriptional start site of CBL-B. The cloned sequences will be inserted into the pGL3 Luciferase Reporter Vector (from Promega) for analysis of promoter activity. This construct will be transfected in Ba/F3 BCR-ABL cells to determine if indeed BCR-ABL regulates CBL-B transcription by modulating promoter activity. To gain some insight into CBL-B promoter regulation and the transcription factors (TFs) that bind in this region we will perform a bioinformatic analysis of the CBL-B promoters (approximately 200 to 1000 bases upstream of the transcriptional start site) in the human, rat and mouse genomes. We will perform an alignment of these promoter sequences to detect conserved elements that may represent transcription factor (TF) binding sites. From here we will proceed with the following laboratory experiments to identify TFs: (1) chromatin immunoprecipitation (ChIP), (2) luciferase assays, and (3) the electrophoretic mobility shift assay (EMSA) to confirm transcription factor binding. At this point we will either review the BCR-ABL literature for other studies that show regulation of these TFs by BCR-ABL or monitor mRNA and protein expression of these TFs in the presence or absence of BCR-ABL activity (i.e., plus or minus imatinib). From these studies we will be able to predict a pathway from BCR-ABL to promoter regulation of CBL-B.

Does CBL-B play a role in BCR-ABL-mediated lymphoid disease development? CBL-B protein expression is down-regulated in myeloid cell lines with active BCR-ABL protein.134 Therefore, our BMT studies focused on the ability of CBL-B to modulate MPD development. Yet, the same paper from Sattler and colleagues also showed a decrease in CBL-B protein levels in the BV173 line. These cells likely constitute a Ph+ ALL cell line based on the expression of the common ALL antigen.465 Yet, the role of CBL-B in lymphoid leukemogenesis downstream of BCR-ABL has not been addressed. Therefore, we will perform several in vitro and in vivo experiments to measure lymphoid transformation and leukemogenesis. The Whitlock-Witte B-lymphoid transformation assay is the prototypical cell culture experiment used to detect lymphoid transformation. Whitlock and Witte initially developed the conditions for long- term culture of B-lymphoid cells,466 The protocol was expanded in 1987 by McLaughlin et al. to monitor transformation by BCR-ABL, characterized by the loss of adhesion dependence of the lymphoid cells in culture.467 We will utilize this system to monitor BCR-ABL-mediated lymphoid transformation in the presence (CBL-B WT BMCs) and absence of CBL-B (CBL-B KO BMCs). We have performed this experiment a single time and have observed expansion of some CBL-B(-/-) BCR-ABL cultures after 21 days (Figure 4-5). As for the methylcellulose CFU assays with BCR-ABL, our observation of rapid transformation by BCR-ABL was likely hindered by a low percentage of infected (GFP+) cells. We will

139

Figure 4-5. Comparison of the lymphoid transformation ability between CBL-B WT and KO BMCs after BCR-ABL infection. Wild-type and CBL-B(-/-) donor BMCs were infected with BCR-ABL as per the methods described in Chapter 2 and then plated with untransduced BMCs for stromal support and in media for B-cell outgrowth to a total of 106 cells per well. Cultures were monitored daily and scored as positive when the number of non-adherent viable cells reached 106.

140 modify our protocol to include a sorting step for BCR-ABL+ cells. A continued investigation of CBL-B deficiency in BCR-ABL B-lymphoid cultures will elucidate the role of CBL-B in lymphoid transformation. In our next experiment we will perform a murine BMT specifically designed to initiate B-ALL downstream of BCR-ABL. Li and colleagues have shown that BCR-ABL transduction of non-5-FU- treated BMCs will produce a B-ALL.205 Therefore, we will derive donor cells from both wild-type and CBL-B(-/-) donors that are not injected with 5-FU. After BCR-ABL transduction, we will transplant these cells into recipient animals via intrafemoral injections (based on our newly derived data that CBL- B alters homing). We predict that CBL-B deficiency will result in an accelerated rate of lymphoid leukemogenesis. As for the myeloid disease described above, we will perform in vitro signaling studies in BV173s and determine the pathways that are modulated by CBL-B in the development of BCR-ABL- mediated lymphoid disease.

Is CBL-B an essential regulator of leukemogenesis caused by the p190 form of BCR-ABL? The reciprocal translocation between chromosomes 9 and 22 results in BCR-ABL gene fusions that encode proteins with molecular weights of 190,52-54 210,48,49 or 230 kDa.56 As p210 predominates in CML,58 this isoform has been predominantly utilized for mouse experiments investigating the role of BCR-ABL in myeloid disease. Yet, a small proportion of patients that succumb to CML harbor the p190 isoform. Therefore, we will also perform experiments to investigate the role of CBL-B in transformation and myeloid disease development caused by BCR-ABLp190. We will infect Ba/F3 cells with a retrovirus expressing the p190 isoform and assess CBL-B protein levels with immunoblots. Should CBL-B show alteration in expression, we will initiate in vitro clonogenic assays and in vivo BMTs with BCR-ABLp190- infected BMCs from 5-FU-treated wild-type and CBL-B knock-out animals. Again, intrafemoral injections will be used for transplantation of the BCR-ABL-transduced BMCs. We predict that CBL-B will also negatively regulate the MPD resulting from p190 isoform. As BCR-ABLp190 is the prominent isoform in two-thirds of all ALLs,58 the role of CBL-B in lymphoid disease development will also by investigated downstream of BCR-ABLp190. Unfortunately, a good murine model is unavailable for testing disease development from BCR- ABLp230. This isoform initiates CNL,59 but murine BMTs with either 5-FU-treated or untreated donor cells initiate MPD or predominantly B-ALL, respectively.205 As a result, our future experiments will not include an analysis of CBL-B in BCR-ABLp230-mediated leukemogenesis.

Is CBL-B a target of TEL-JAK2 fusions? In our preliminary signaling studies of Chapter 2, we performed IBs to measure CBL-B protein expression in the presence of known oncogenes. Our data revealed a BCR-ABL-dependent down- modulation of CBL-B, while the TEL-JAK2 TK fusion did not regulated CBL-B protein levels (Figure

141 2-1). Our results contradicted those of Sattler et al., in which they observed decreased CBL-B protein expression in the presence of three different TK fusions: TEL-JAK, BCR-ABL and TEL-PDGFR.134 We believe that the differing results may be a function of the TEL-JAK2 constructs utilized. In our studies, we employed Ba/F3s cells stably-expressing the TEL-JAK2(5-19) isoform. The fusion is one of three TEL-JAK2 translocations previously described in patients with either atypical CML or B/T-cell ALL (see Figure 4-6).468,469 Unfortunately, the studies by Sattler and colleagues did not describe the fusion used. We believe that their studies employed either the TEL-JAK2(5-12) or TEL-JAK2(4-17) fusions which contain a JH2 domain (as distinct from the TEL-JAK2(5-19) fusion). Therefore, this domain may differentially modulate the expression of CBL-B. To investigate the role of CBL-B downstream of TEL-JAK2 we will initiate studies will all three TEL-JAK2 fusions. We have previously cloned each of these isoforms into Ba/F3 cells and so a cell line model is readily available.408 We will first perform immunoblots to detect CBL-B expression downstream of each of the fusions. We predict that CBL-B protein levels will be reduced in the TEL- JAK2(4-17) and TEL-JAK2(5-12) lines, but not in the TEL-JAK2(5-19)-expressing cells. We will also perform methylcellulose CFU assays and BMTs to delineate the role of CBL-B in TEL-JAK2-mediated leukemias. Moreover, siRNA constructs to CBL-B will be introduced into these Ba/F3 TEL-JAK2 cell lines, and (as described above) we will monitor factor-independent proliferative potential and signal transduction in the absence of CBL-B expression. Given the overlapping pathways of TK fusion leukemias we hypothesize that CBL-B will modulate disease development and signaling in a similar fashion as for the BCR-ABL disease.

4.3. Future directions to delineate the role of CBL-B in fibroblasts

4.3.1. Immediate future directions to define the pathways regulating serum-dependent migration of fibroblasts in the absence of CBL-B. In the experiments detailed in Chapter 3 we found that CBL-B was a negative regulator of serum-dependent chemotaxis. From the results of our biochemical immunoblot studies in the presence and absence of serum we showed that PKB and ERK were important substrates of CBL-B. We hypothesized that these pathways were stimulated in the absence of CBL-B to effect the migration phenotype toward serum. To solidify our theory it is essential that we also monitor migration as a function of PKB or ERK activation and not only the phosphorylation events in the presence of serum. Therefore, we will perform Transwell migration assays with the addition of two types of exogenous agents: (1) inhibitors of the PKB and ERK pathways, and (2) WT, constitutively active (CA) and dominant-negative (DN) forms of PKB and MEK. Moreover, siRNA constructs to ERK or PKB could be utilized.

142

Figure 4-6. Schematic representation of the three human TEL/JAK2 fusion gene variants that have been cloned. TEL-JAK2(5-12) is the longest variant, containing the pointed (PNT) domain of TEL and two full-length domains of JAK2 – the JH1 kinase domain and the JH2 pseudokinase domain. TEL-JAK2(4-17) also contains both JAK2 kinase domains, but a shortened variant of the JH2 domain. The TEL-JAK2(5-19) isoform does not contain the JH2 domain but retains the JH1 domain. Adapted from ref. (408).

143 Our first experiment in this vein was previously described in Figure 3-6. In this assay, CBL-B(- /-) MEFs were stimulated to migrate toward serum in the presence of the PI3’K inhibitor LY294002. Our initial results showed that chemotaxis was impaired by PI3’K inhibition, suggesting that in the absence of CBL-B these fibroblasts utilize the PI3’K/PKB pathway to move toward serum. However, this assay was only performed a single time and needs to be repeated. Moreover, we will replicate this experiment in the presence of U0126 to inhibit MEK1/2. We expect that migration of CBL-B(-/-) MEFs will also be impaired by this inhibitor of the ERK pathway. To confirm the inhibitor studies we will be perform Transwell migration experiments following transient transfection with constructs that will either enhance (WT or CA) or inhibit (DN) PKB or MEK activity (Table 4-1). We expect that the WT and CA constructs will promote migration of CBL-B(-/-) cells while the DN constructs will block migration responses that result from CBL-B-deficiency. From these inhibitor and over-expression studies we will confirm or deny the involvement of the PKB and ERK pathways in CBL-B-deficient serum-dependent motility of MEFs.

4.3.2. Questions to be answered in the next phase of this project. The completion of the studies outlined in Section 4.3.1. will likely suggest that PKB and ERK are important downstream activators of CBL-B-deficient MEF chemotaxis toward serum. We will continue our studies by defining precise migration signaling pathways modulated by CBL-B.

What are the direct interaction partners of CBL-B? Our immunoprecipitation experiments to identify CBL-B interactors were not fruitful as we did not visualize any co-immunoprecipitated tyrosine-phosphorylated proteins. The inability to observe interacting proteins may be a function of the fact that: (a) CBL and CBL-B interact with the same proteins and that the CBL ID step removes all potential CBL-B binding partners, or (b) that CBL-B does not associate with tyrosine-phosphorylated proteins. Our next experiments will seek to identify either those potential proteins that bind to both CBL and CBL-B, or unphosphorylated CBL-B interactors. In the first experiment, we will transfect wild-type cells with siRNA directed toward CBL to remove expressed CBL protein. We will then perform CBL-B immunoprecipitations followed by phosphotyrosine immunoblots on membrane blots of the co-immunoprecipitated proteins. As the sequence similarity of CBL and CBL-B is high, leading to similar interactomes, we expect that this stategy to remove CBL will uncover CBL-B binding proteins. We are particularly interested in those associated proteins that may mediate the migratory phenotype. As CBL associates with several focal adhesion proteins such as paxillin and talin,166 we expect that CBL-B may also bind these FAPs. In the second assay proteins from CBL siRNA-treated wild-type MEFs will be co- immunoprecipitated with the anti-CBL-B antibody. The co-immunoprecipitated complex will be denatured and separated by SDS-PAGE and the gel will be stained for protein with silver stain. Distinct

144 Table 4-1. Exogenous introduction of constructs that will alter protein activation.

Wild-type Constitutively Active Dominant Negative

MEK MEK ΔN-S222D470 MEK K97M470 ERK1 ERK1 K72R471 ERK2 ERK2 K52R472 ERK1/ERK2 TEYEa PKB PKB E40K473 PKB K179M474 Rac1 Rac Q61L Rac N17 RhoA Rho Q63L Rho N19 CDC42 CDC42 Q61L CDC42 N17

a Glutamic acid substitutions at T183 and Y185; thought to act as a dominant negative to both ERK1 and ERK2.475

145 bands will be cut from the gel, trypsinized, diffused from the gel, and sent to a facility for identification by mass spectrometry. This process will detect CBL-B associated proteins that may not have been revealed in the phospho-tyrosine immunoblots. It will be of interest to study whether the CBL-B interactors that we distinguish by these two means can link to proteins detailed in Chapter 3 that were shown to be regulated by CBL-B. For example, we predict that GRB2 may bind to CBL-B in our MEFs, given the results described above in hematopoietic cells and the known association with CBL.128 Therefore, CBL-B may regulate GAB2 through the intermediary protein GRB2. In any event, these two studies will give us some indication of what pathways should be the focus of our future signaling experiments.

Is GAB2 the upstream regulator of serum chemotaxis in CBL-B-deficient cells? From our studies in Chapter 3 we predicted that GAB2 was the upstream regulator of PKB and ERK activation in the MEFs, and that signals through this protein are responsible for the serum- dependent motility. To illustrate this pathway, we will infect the MEFs with GAB2 siRNA.451 Subsequent to infection we will perform Transwell migration assays and assess phosphorylation of MEK, ERK and PKB. We hypothesize that GAB2 inhibition will impair serum-dependent chemotaxis of our CBL-B(-/-) MEFs and that MEK, ERK and PKB phosphorylation will be reduced. These experiments will detail two potential signaling pathways for locomotion of our MEFs through CBL- B/GAB2/PKB and CBL-B/GAB2/MEK/ERK. Co-immunoprecipitation experiments with anti-GAB2 antibodies will define the intermediary proteins (such as PI3’K and SHP2) that may link GAB2 to the PKB and ERK cascades.

Does Vav3 play a role in CBL-B-dependent serum chemotaxis, and if so, what are the downstream targets that mediate these effects? Phosphorylated Vav acts as a GEF for the Rho family of cytoskeletal regulators.136-138 Therefore, we were encouraged by our findings of enhanced Vav3 tyrosine phosphorylation and total Vav3 proteins levels in our highly-motile CBL-B(-/-) MEFs. To complete our hypothesized link between Vav3 and motility it is essential that we monitor migration after impairment of Vav3 and identify the downstream signaling proteins that may be mediated locomotion. As for the GAB2 studies, we will monitor Transwell chemotaxis of our MEFs after RNAi- mediated knock-down of Vav3.476 We expect that reduced total Vav3 levels will decrease CBL-B(-/-) motility toward serum to a level comparable or less than observed with the wild-type cells. Moreover, we will determine the downstream target of Vav3, by performing GTPase activation assays after Vav3 siRNA treatment of the MEFs. As, our preliminary assays looking for Rac1 activation showed little regulation in the presence or absence of CBL-B (Figure 3-5) we predict that the substrate of Vav3 is either CDC42 or one of the Rho proteins. Following our elucidation of the correct GTPase we will use

146 inhibitors specific to the particular GTPase (Rac, SCH-51344; RhoA, C3 coenzyme or Y-27632), plus CA or DN constructs (Table 4-1) and monitor the resulting serum-dependent Transwell motility of wild- type and CBL-B(-/-) MEFs. We predict that signals downstream of one of these GTPases will contribute to the enhanced motility of the CBL-B KO cells and will be revealed through these experiments.

Does CBL-B control migration through ubiquitination of substrates? In our model depicted in Figure 3-10 we suggested three possible cascades downstream of CBL- B that could be controlled by its E3 ubiquitin ligase function. First, given the knowledge that CBL-B can ubiquitinate GAB2 in mast cells,437 we hypothesized that the decreased phosphorylation of GAB2 in CBL-B WT cells was a consequence of ubiquitination and degradation of phospho-GAB2. Second, as evidence suggests that CBL-B can bind ubiquitinated Vav,134 we predicted that the reduced total Vav3 and phosho-Vav3 protein was a function of CBL-B-mediated degradation. Third, we suggested that PI3’K activity may be modulated in the CBL-B WT MEFs by altered localization of ubiquitinated p85- PI3’K, which has previously been observed in T cells.324 Furthermore, we proposed that Vav3 phosphorylation may alternatively be reduced by the impaired localization of p85-PI3’K and the reduced 369 conversion of PIP2 to PIP3. Yet, to conclude that CBL-B ubiquitination activity is essential for the control of each of these pathways, and ultimately serum-dependent locomotion, several additional experiments are required. In our initial experiment we will perform Transwell migration assays with the MEFs in the presence of either MG132 (proteasomal inhibitor) or chloroquine (lysosomal inhibitor). Should these inhibitors impair the chemotactic migration of the CBL-B(-/-) MEFs we will proceed with further biochemical studies. Our preliminary results from Figure 3-4 suggest that CBL-B reduces tyrosine-phosphorylated Vav3 through a proteasomal-independent mechanism. We will repeat this assay, as well as add chloroquine to determine if CBL-B targets Vav3 to the lysosome. We will perform similar experiments in wild-type and CBL-B(-/-) MEFs in which the phosph-GAB2 antibody will be used to detect the level of this phosphorylated protein after serum-stimulation and inhibition of the proteasome and lysosome. These results will determine whether impaired ubiquitination in the CBL-B-deficient MEFs is responsible for the regulation of Vav3 or GAB2. Should neither one of these proteins show significant regulation through ubiquitination, we will then turn our attention to PI3’K. We specifically want to monitor the sub-cellular localization of the p85 subunit of PI3’K in wild-type and CBL-B(-/-) MEFs that are migrating toward serum. To do so we will initiate TLVM chemotactic assays as performed in Chapter 3. After several hours of migration we will fix the MEFs and then permeabilize and incubate with antibodies toward CBL-B, p85 and phalloidin (to detect changes in the actin cytoskeleton). We hypothesize that p85 will be localized in a sub-cellular location distinct from the cell membrane in wild-

147 type cells. While this assay will not determine the ubiquitination status of p85, it will confirm our suspicion that CBL-B control p85 localization. In conclusion, these proposed experiments will define the key signaling pathways and proteins that are responsible for CBL-B-mediated serum chemotaxis of MEFs.

4.4. Major scientific concepts emerging from this thesis. Several major concepts and ideas emerged from the work presented in this thesis. The utility of the BMT and cell line models was evaluated. Our experiments with CBL family members revealed that single proteins contained within in a family and bearing strong sequence similarity may have unique functional properties. Finally, our data supports a continued investigation into the use of combination therapy in the treatment of CML, and the role of CBL proteins in moderating granulopoiesis during myeloid disease development.

4.4.1. The BMT model should be continuously assessed and improved. The BMT is the prototypical model for chronic myeloid leukemogenesis in a murine host. As compared to cell line work, the BMT excels in its incorporation of environmental influences. Furthermore, of the four in vivo models – subcutaneous tumor formation, transgenics, knock-ins and retroviral transduction and transplantation – the transplantation system is the only version that consistently induces a CML-like disease within a suitably short time frame. As such, the BMT continues, and will continue, to be used by investigators to identify essential targets of BCR-ABL and to test emerging therapeutics. Yet, the classical BMT to produce MPD has several limitations. First, while the disease closely mimics human CML in the chronic phase, all mice succumb to their disease before progression to acute phase or blast crisis. Therefore, most scientists utilize the tec promoter p210BCR-ABL transgenic mice for studies of the acute disease. These mice acquire disease with CML characteristics at approximately six to eight months after birth and at one year their disease progresses to blast crisis.201 Second, almost all BMT recipients inevitably die of lung hemorrhaging resulting from infiltrating granulocytes. Human patients do not die of this complication. Third, the IV injection, homing and lodging steps of the BMT are foreign to the natural human occurrence of the disease and can be greatly affected by the phenotype of the donor cells and the recipient BM environment. Our BMT studies with CBL-B-deficient cells underlined the third caveat of this CML model. While one could easily jump to conclusions with regard to the ability of CBL-B(-/-) cells to induce proliferation of the BCR-ABL+ BMCs, without a proper analysis of the ability of the cells to home to the BM, the results could be confounded. Our homing experiments suggest that CBL-B impairs leukemic development not by interfering with cell proliferation but by reducing homing. We postulate that intrafemoral injections will reveal the true role of CBL-B in BCR-ABL disease development. We also

148 argue that the BMT model be re-evaluated, and that intrafemoral injections be included as an integral component of the technique. Through adoption of this step, leukemogenesis can be properly evaluated in BMT studies performed with donor cells known to harbor migration abnormalities.

4.4.2. There is still some utility in studying individual components of signaling cascades that are not causative agents of disease. The treatment approach to CML was drastically modified in the 1990s when imatinib was discovered. This agent specifically targets the BCR-ABL fusion kinase,247 blocking activation of downstream substrates and effectively halting proliferation of the leukemic clone. Yet, in patients resistance to the tyrosine kinase inhibitor (TKI) developed due to amplification of BCR-ABL or the emergence of BCR-ABL mutants such as T315I.255,256 This clinical picture revealed a caveat of single agent targeted therapy – that inhibitors designed to block activity of only the causative agent are immediately ineffective in situation of mutation, and resistance will inevitably follow. For this reason, we argue that combination therapy is a rational alternative to single target drugs. Hoover and colleagues have shown that the combination of SCH66336 (a farnesyl transferase inhibitor that predominantly impairs Ras activity) with imatinib decreases the growth of BCR-ABL+ cells that display imatinib resistance as a function of amplification or mutation.264 As well, rapamycin treatment (an inhibitor of the PKB effector mTOR) in combination with imatinib shows a synergistic decrease in the proliferation of CML cells.263 The design of these two treatment strategies was based upon evidence of ERK and PKB pathway activation downstream of BCR-ABL. Therefore, without basic research into the mechanisms of activation downstream of the causative agent (BCR-ABL) these valid, and possibly rewarding, approaches would not have been investigated. Moreover, it seems logical that inhibitors that specifically target the upstream regulators of both of these pathways (PKB and ERK) would be clever choices for combination therapy. As a result, by performing experiments that specifically detail the downstream signaling pathways of oncogenes, robust inhibitors for use in combination therapy can be developed. Based on this rationale, we pursued studies to expand upon our knowledge of CBL proteins in BCR-ABL-mediated disease. CBL is highly phosphorylated by BCR-ABL,281 and is known to act upstream of both the ERK and PKB pathways.125,128,131 Therefore, inhibitors to CBL would theoretically be suitable candidates for combination therapy. Yet the BMT studies with CBL-deficient donor cells revealed that CBL was dispensable for MPD development. As a result, we turned our attention to the related family member CBL-B. Again, our data indicates that CBL-B is not required for myeloid leukemogenesis initiated by BCR-ABL. While this approach did not yield an appropriate target for combination therapy, the experimental design was still rational. Recently, Van Etten and colleagues have identified a downstream BCR-ABL signaling component that may be a perfect candidate for combination therapy. Experimental evidence shows that

149 in BCR-ABL-expressing cells, GAB2 is an upstream regulator of both the ERK and PKB pathways,115 and all BMT recipients of BCR-ABL-infected GAB2(-/-) donor cells succumb to a T-cell lymphoma,216 suggesting that GAB2 is required for MPD development. This BMT phenotype is superior to the experiments with DN mutant donor cells of the ERK or PKB pathways alone where recipients still developed MPDs but with longer latencies (Table 1-4).132,229,230 From these results, GAB2 fits the criteria for an ideal target in combination therapy, and emphasizes the importance of continued investigation into the individual protein components of oncogenic signaling cascades.

4.4.3. Caution should be assumed when extending scientific findings to other protein family members. In this thesis we assessed the functional role of CBL-B in two distinct cell types: hematopoietic cells and fibroblasts. CBL-B belongs to the CBL protein family that contains three closely related mammalian member; CBL-B and CBL share greater than 80% amino acid identity in their tyrosine kinase binding domains. Our studies have shown that in one context – leukemic cells – these family members behave similarly, while in another context – fibroblasts – their roles are quite opposite. When we performed BMT studies of BCR-ABLp210-infected CBL-B(-/-) cells we observed a delayed latency of leukemia development. The results of Dinulescu et al. show that CBL contributes to in vitro transformation by BCR-ABL.133 Moreover, while this group argues that CBL is dispensable for leukemogenesis in the mouse; we counter that the prolonged latency in 20% of their recipients combined with enhanced WBC counts and spleen weights compared to control is suggestive of a negative regulator. Combining these studies, we propose that CBL and CBL-B function in a similar manner to control the leukemogenesis from BCR-ABL. In opposition to their roles in hematopoietic cells, CBL proteins do not appear to behave similarly in fibroblasts. In Chapter 3 we presented evidence that CBL-B deficient MEFs were enhanced in their migratory potential toward serum. Biochemical experiments showed increased PKB phosphorylation of CBL-B(-/-) in response to serum (as compared to WT controls) suggesting that the increased motility was a function of an augmented PI3’K/PKB pathway. When a comparison is made to fibroblasts with forced expression of CBL (i.e., enhanced CBL expression), the results are rather surprising: these cells display enhanced migration toward serum.380 Moreover, PI3’K activity is required for this motility. Were CBL and CBL-B to behave similarly in fibroblasts we would have predicted that the fibroblasts with forced expression of CBL would have shown reduced migration as compared to the cells transduced with vector control. In the discussion to Chapter 3 we eluded to a possible mechanism for the opposing roles, proposing that the manner in which CBL and CBL-B associate with the p85-subunit of PI3’K could alter response: CBL associates with PI3’K and stimulates its phosphorylation, while CBL-B binds to p85-PI3’K and sequesters the protein from its site of activation.

150 By this analysis it is clear that even though CBL and CBL-B are highly related and possess similar interactomes, their functional properties can be distinct in certain contexts. Therefore, we re- iterate our warning that all members of a particularly protein family should be thoroughly investigated before assumptions are made as to their functional roles.

4.4.4. Continued studies of CBL family members is critical to a greater understanding of myeloid disease. The Introduction to this thesis described several recent publications that identified CBL and CBL-B mutations in the development and progression of myeloproliferative neoplasms.398-402 Each of these mutations occurred either within the linker region or the RING domain – portions of the CBL protein required for the E3 ubiquitin ligase activity. Therefore, it is hypothesized that the mutant CBL proteins cannot co-ordinate receptor down-modulation and as a result, signaling is enhanced. Several studies support this argument. Caligiuri and colleagues observed reduced cellular proliferation of an AML cell line with RNA interference of the CBL mutant.398 Sargin et al. have demonstrated the importance of CBL in Flt3 signaling.399 Flt3 is commonly mutated through internal tandem duplication in AML. This group showed that CBL is required for the ubiquitination and degradation of FLT3 and that expression of the R420Q CBL RING finger mutant along with Flt3 led to cytokine-independent cell growth. This mutant was characterized in an AML patient without a Flt3 mutation; as such, CBL mutations may represent an alternative mechanism of myeloid disease development. Moreover, the introduction of the CBL mutations into NIH 3T3 cells can transform this cell line through possible activation of the PI3’K pathway.402 These data are of interest given our studies that show increased PKB activation in CBL-B(-/-) MEFs (Figure 3-8) and enhanced granulopoiesis in the absence of CBL-B and the presence of BCR-ABL (Chapter 2). Our findings concur with those of others that suggest that CBL proteins may co-operate with other oncogenes in leukemogenesis of myeloid origin and provide support for continued research into the role of CBL proteins in myeloid disease development and progression.

4.5. Closing Remarks In this body of work we have described the functions played by CBL-B – an E3 ubiquitin ligase and adaptor protein – in fibroblast and hematopoietic cells. We have shown that serum chemotaxis of fibroblasts is enhanced by CBL-B deficiency. We postulate that PKB and ERK activation are essential for the observed motility. As well, we have investigated the role of CBL-B in BCR-ABL-mediated leukemogenesis. Our results showed that CBL-B contributes to the MPD phenotype observed in BCR- ABL BMTs. We observed enhanced granulopoiesis in our CBL-B(-/-) BCR-ABL BMC recipients in both the peripheral blood and spleen, suggesting that CBL-B is a negative regulator of granulocyte proliferation. Moreover, we noticed impaired homing of CBL-B KO cells to the BM niche. Combining

151 these experiments, we propose that CBL-B utilizes its known E3 and adaptor functions to modify two key cellular functions: proliferation and migration. The observed consequence of CBL-B deletion is of utmost importance in human disease. As fibroblast migration is a key process in wound repair, we hypothesize that CBL-B will be revealed as a crucial regulator of chronic non-healing wounds and fibrotic disease. Furthermore, we suspect that BCR-ABL down-modulates CBL-B in CML cells to promote the characteristic feature of myeloid expansion. As CML is one of only a few leukemic diseases, we argue that an expanded analysis of CBL- B function in additional myeloid diseases is warranted. In sum, this thesis has underlined the significance of the CBL-B protein in two cell types and alludes to a role for CBL-B in multiple forms of human disease.

CHAPTER 5

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APPENDIX

BCR-ABL FAILS TO INDUCE STABLE TRANSFORMATION OF MOUSE EMBRYONIC FIBROBLASTS

190

191 A.1. Abstract Chronic myeloid leukemia results from BCR-ABL tyrosine kinase activity that stimulates multiple downstream signaling pathways. Murine bone marrow transplantation (BMT) studies conducted in our laboratory show impaired induction of the myeloproliferative phenotype in BCR-ABL BMT mice when CBL-B(-/-) donor bone marrow cells (BMCs) are used. These data suggest that pathways emanating from CBL-B may be required for a full leukemic phenotype. To investigate downstream signaling components we initiated experiments to generate wild-type and CBL-B(-/-) mouse embryonic fibroblasts (MEFs) that stably expressed the BCR-ABL oncogene. While a minor proliferative expansion was observed in the BCR-ABL-expressing cells in the absence of growth factor (GF), no other transformation phenotypes were observed. The BCR-ABL MEFs were not anchorage- independent as determined through adhesion, migration and soft agar colony formation assays. Furthermore, while BCR-ABL expression was observed, the tyrosine kinase (TK) activity of the fusion kinase was absent in these MEFs. From the results of our studies we conclude that BCR-ABL cannot confer stable transformation to newly derived mouse embryonic fibroblasts.

192 A.2. Introduction Chronic myeloid leukemia results from a translocation of sequences on chromosomes 9 and 22,1- 3 and the generation of the Philadelphia chromosome (Ph).4 The t(9;22) translocation juxtaposes breakpoint cluster region (bcr) sequences with those of the c-abl. Several translocation variants exist, and encode proteins of 185/190 kDa,5-7 210 kDa,8 and 230 kDa9 in size as determined by the breakpoints within the bcr gene; however, CML is most prominently associated with the p210 isoform.10 BCR-ABL is the causative agent of CML – instigating cellular transformation in vitro and leukemogenesis in mouse models. BCR-ABL is transforming to Rat-1 fibroblasts and a small subset of the mouse NIH 3T3 fibroblast population.11-13 Additionally, transient infection of newly-derived MEFs MEFs leads to soft agar growth.14 BCR-ABL also relieves the growth factor dependence of many hematopoietic cell lines including IL-3-dependent Ba/F3 cells.15 Furthermore, murine BMT of BCR- ABL(p210)-transduced BMCs leads to the development of a myeloproliferative disease that resembles human CML.16,17 Based on these conclusive observations in vitro and in vivo, the causative agent of CML has been verified as BCR-ABL. The oncogenic capacity of BCR-ABL results from deregulated TK activity.18,19 BCR-ABL constitutively activates several cell signaling cascades, including the Ras/ERK,20,21 PI3’K,22 Jak/STAT,23- 26 SAPK/JNK,27 and Rac pathways.28 Transfer of signals to these pathways results from complex formation between BCR-ABL and its substrates. Of these, the GRB2 and Shc20,29; phosphatidylinositol-3 kinase (PI-3K)30-32; and CrkL,33 Crk and CBL proteins,34-36 are key signaling intermediates. One of the most prominently tyrosine phosphorylated proteins in the BCR-ABL cascade is the CBL protein,37 that functions as an adaptor and E3 ubiquitin ligase,38,39 and belongs to a family consisting of three mammalian members - CBL, CBL-B and CBL-3. Single gene deletion of the CBL family members has been performed in mice. All animals are viable and healthy,40-43 but the CBL- and CBL-B-deficient mice exhibit defects in T cell development and activation, respectively. In BMT studies, CBL expression is not required for BCR-ABL-mediated disease development. We hypothesized that CBL-B could compensate for the loss of CBL in this in vivo model. BMT experiments performed in our laboratory (and detailed in Chapter 2) showed prolonged survival of CBL- B(-/-) BCR-ABL transplant recipients. To assess the signaling events that impaired leukemogenesis, we desired a cell line with stable expression of BCR-ABL in the absence of CBL-B. Encouraged by the results of Dinulescu et al.,14 which show the production of newly-derived MEFs transformed by transient infection of BCR-ABL, we initiated experiments to generate wild-type and CBL-B knock-out MEFs transduced by the p210 isoform of BCR-ABL. Our results show stable expression of BCR-ABL within these MEFs without the corresponding TK activity necessary to stimulate transformation.

193 A.3. Materials and Methods A.3.1. Generation of murine embryonic fibroblasts expressing BCR-ABL The derivation of those murine embryonic fibroblasts (MEFs) used in these experiments, as well as their culture conditions, was described previously in Chapter 3. Passage 20 MEFs were transfected by the Lipofectamine method with 4.4 µg of DNA in serum- free Dulbecco’s modified Eagle’s medium (DMEM). After five hours the transfection media was replaced with fresh DMEM containing 10% fetal calf serum FCS (FCS; HyClone, Thermo Fisher Scientific, Inc., Waltham, MA). Cells were expanded for three days before selection through fluorescence activated cell sorting (FACS) of green fluorescent protein (GFP)-positive cells to greater than 90%. Sorted cells were frozen in DMEM with 50% FCS and 7.5% dimethyl sulfoxide (DMSO; Fisher), stored at -70oC, and used for all subsequent experiments. Prior to experimentation, transfected MEFs were re-selected for the presence of the transgene through fluorescence activated cell sorting (FACS) of green fluorescent protein (GFP)-positive cells to greater than 90%. All cell lines were expanded in MEF Maintenance Medium (MMM) of Dulbecco’s modified Eagle’s medium (DMEM) H21 containing antibiotics, 10% FCS and 0.1 mM β-ME.

A.3.2. Proliferation assays Cell proliferation under low serum (0.5% FCS) was measured via two differing protocols: (1) the trypan blue exclusion assay, and (2) the XTT assay. In the first method, MEFs were seeded in triplicate at a low density of 5 x 103 cells per 35-mm2 plate in Low Serum Media (LSM; DMEM containing antibiotics, 0.5% FCS and 0.1 mM β-ME). Growth was monitored after four days of incubation at 37°C and measured at this time by trypsinizing the cultures and performing manual cell counts. Trypan blue was added to exclude dead cells. Values were recorded as the mean of three replicates ± standard deviation. In the second assay, MEFs (100/well) were added to a 96-well plate in a final volume of 100 µL MMM. The cells were allowed to adhere for two hours, at which time, the MEFs were washed twice with PBS and the media was replaced with LSM. The plate was incubated at 37°C for four days and then XTT Reagent (1 mg/mL Sodium 3,3'-44-Bis(4-Methoxy-6-Nitro)Benzene Sulfonic Acid Hydrate (XTT; Diagnostic Chemicals Ltd., Charlottetown, PE) and 125 mM phenazine methosulfate (Sigma- Aldrich, St. Louis, MO) was added. Cells were incubated for an additional four hours at 37°C prior to measuring the absorption of the soluble formazan reduction product at 450 nm. Values were recorded as the mean of four replicates ± standard deviation.

A.3.3. Soft agar assay Soft agar assays were performed in non-tissue culture-treated 35-mm2 plates that were prepared by first plating 1.5 mL of a 1:1 mixture of 2x DMEM H21 containing 20% FCS with 1.2% (w/v) DIFCO 194 noble agar (Becton Dickinson (BD) Falcon, Franklin Lakes, NJ). This layer was allowed to solidify for 20 to 30 minutes before adding a second layer containing 250 cells in a 2:1:1 mixture of media/FCS to agar to water. The soft agar cultures were incubated in a hydrated environment at 37oC for 14 days. The cells were fed once weekly with 100 µL of 1x DMEM H21 containing 10% FCS.

A.3.4. Foci formation assay MEFs were seeded at an initial concentration of 106 cells per 10-cm2 plate in MMM and grown for 21 days. Media was then aspirated and the plates were fixed for 10 minutes in 25% (v/v) methanol, 75% (v/v) glacial acetic acid, followed by staining for one minute in 0.5 % (w/v) crystal violet in methanol. The plates were washed in tap water and allowed to dry overnight.

A.3.5. Antibodies All primary antibodies used in immunoblots were previously described in Chapter 3 and the secondary antibodies were described in Chapter 2.

A.3.6. Immunoblotting These experiments were performed identically to those described in Chapter 3.

A.3.7. Adhesion assay The adhesion assay was performed in accordance with the protocol detailed in Chapter 3. The result for each individual assay was recorded as the mean of quadruplicate replicates. The data presented is the mean of five independent experiments ± standard error.

A.3.8. Migration assay Spontaneous migration of these MEFs was performed according to the Transwell migration protocol previously described in Chapter 3.

A.4. Results

A.4.1. BCR-ABL protein is expressed in CBL-B(+/+) and CBL-B(-/-) MEFs. In order to study the role of CBL-B in the regulation of cell signaling downstream of BCR-ABL, we transfected wild-type and CBL-B(-/-) MEFs with either a control retroviral vector (MIEV) or the p210 isoform of BCR-ABL (Mig210 or BCR-ABL). Transfected cells were collected by fluorescence activated cell sorting (FACS) of green fluorescent protein (GFP)-positive cells, and expression of BCR- ABL was analyzed by anti-ABL immunoblotting. As shown in Figure A-1, BCR-ABL protein was 195

Figure A-1. BCR-ABL is stably-expressed in wild-type and CBL-B(-/-) MEFs. Wild-type and CBL- B(-/-) MEFs were transfected with control retrovirus (vector) or one expressing BCR-ABL. Cells expressing the transgene were sorted to >90% GFP-positivity. These MEFs were serum starved overnight and then stimulated in the absence (-) or presence (+) of 10% FCS. Cell lysates were separated by SDS-PAGE and membranes were probed with an anti-ABL antibody to detect expression of BCR- ABL and confirm equal loading (as determined by c-ABL). 196 expressed in those cells transfected with the oncogene, but not in the control cells. This expression was stable throughout our studies. Additionally, the cells maintained GFP expression over several weeks of culture and any loss of GFP expression was comparable between the control- and BCR-ABL-infected cells. From these results we concluded that BCR-ABL expression was not toxic to the cells.

A.4.2. BCR-ABL relays a growth stimulus to wild-type MEFs under low serum conditions. Based on the findings that BCR-ABL expression was stable, we proceeded with an analysis of the growth of these cells. Previous experiments have demonstrated that BCR-ABL expression leads to growth of cells in factor-independent or low serum conditions.12,15 Therefore, to assess the transformation of our cells based on proliferative capacity, we plated wild-type cells that contained either the control or BCR-ABL constructs at low density in media containing 0.5% serum. Growth was evaluated after four days in culture by cell counts after trypan blue exclusion. The results shown in Figure A-2A showed a slight increase in growth of the BCR-ABL-expressing line over four days as compared to the input cell number. However, the variability of triplicate platings was quite high and suggested to us that our protocol required optimization. We hypothesized that the low serum conditions may have altered the adhesion of the MEFs to the tissue culture plate, resulting in read-outs that were more a measure of adhesion properties than cell growth. To overcome this variable, in the next experiment we plated cells in the presence of 10% FCS. These cells were allowed to adhere for two hours before the plates were washed and the media was changed to the low serum (0.5% FCS) conditions. We also chose to evaluate proliferation with an XTT assay to reduce the error that may be introduced during trypsinization and manual counting. In this analysis, we observed a statistically significant increase in the growth of the BCR-ABL+ WT MEFs over the control-infected cells (Figure A-2B; p < 0.05). These data indicated that BCR-ABL was able to induce proliferation of the MEF under low serum conditions and suggested that transformation may be occurring.

A.4.3. BCR-ABL fails to abrogate the anchorage requirement of wild-type MEFs. The traditional experiment used to measure transformation of adherent cell lines is the soft agar assay. As such, we assessed transformation according to the recognized protocols, but the growth of soft agar colonies was not observed. As a result, we concluded that BCR-ABL could not abrogate the anchorage requirement of these MEFs. To confirm that BCR-ABL was not able to confer anchorage-independence to these MEFs, we chose to evaluate cell adhesion with another method. Multiple studies have shown that BCR-ABL modifies cell adhesion to the extracellular matrix protein fibronectin.45-49 We performed our own anaylsis of the wild-type MEFs and their adherence to 1 µg/mL fibronectin after multiple wash steps. There 197

Figure A-2. BCR-ABL confers growth factor independence to wild-type MEFs. Wild-type MEFs, either present or absent for BCR-ABL, were subjected to assays to measure the proliferative index. (A) Cells were plated in 0.5% FCS. Cell proliferation was measured on Day 4 my manual counts following trypan blue exclusion. The mean cell count was recorded ± standard deviation. (B) Cells were plated in the presence of 10% FCS, allowed to adhere for two hours, and then washed and incubated four days in 0.5% FCS. XTT assays were performed in triplicate and the mean absorption values with standard deviation were graphed. 198 was no difference in the adhesive properties of those cells that stably-expressed BCR-ABL as compared to those that expressed the vector control (Figure A-3A). As two adhesion processes (adhesion formation at the leading edge of a cell, and de-adhesion at the trailing edge) are required for cellular motility, we also assessed the migration of our MEF populations. In vitro, BCR-ABL expression enhances the spontaneous cellular motility of multiple cell lines.50-52 Therefore, we assessed the motility of MEFs in Transwell migration experiments in which the cells were allowed to migrate spontaneously to the lower chamber. Very little spontaneous motility was observed after the four-hour migration period and migration was not enhanced by BCR-ABL expression (Figure A-3B). Combined, these results from soft agar assays, adhesion experiments on fibronectin, and Transwell migrations confirm that stable transduction of BCR-ABL does not confer anchorage independence to MEFs.

A.4.4. BCR-ABL has no associated TK activity in stable MEFs. As BCR-ABL expression was not transforming to our cells, we hypothesized that the protein may be expressed but not eliciting kinase activity. When the BCR-ABL TK is active, multiple downstream effectors are phosphorylated, even in the absence of exogenous growth stimuli. Therefore, we chose to initially measure kinase activity through the assessment of global tyrosine phosphorylation. In the wild-type MEFs, BCR-ABL expression did not lead to enhanced tyrosine phosphorylation of the proteins in the cell lysate (Figure A-4A). Additionally, we did not observed enhanced phosphorylation in the BCR-ABL-specific lanes at 210 kDa that would indicate that BCR-ABL was able to confer tyrosine phosphorylation to itself. Therefore, we hypothesized that while BCR-ABL was expressed in these MEFs, its expression was low or the kinase activity was inactive. To confirm our theory, we measured the tyrosine phosphorylation of two known effector proteins of BCR-ABL: extracellular-regulated kinase (ERK) and protein kinase B (PKB).21,53 Immunoblots were probed with phospho-specific antibodies, and results showed that BCR-ABL expression did not enhance the phosphorylation of either ERK1/2 or PKB (Figure A-4B). These data confirm that the TK activity of BCR-ABL is impaired in these MEFs. 199

Figure A-3. BCR-ABL does not alter adhesion of MEFs to fibronectin or spontaneous motility. Wild-type MEFs either present or absent for BCR-ABL were subjected to a cell adhesion test in tissue culture plates coated with 1 µg/mL fibronectin. The graph represents the mean adhesion from three independent experiments ± standard error. In each experiment the average adhesion was calculated from recordings of four separate wells of a 96-well culture dish. (B) Wild-type MEFs either present or absent for BCR-ABL were applied to the upper chamber of Transwell migration chambers and allowed to migrate toward media without FCS. After four hours, the migrated cells on the underside of the membranes were counted after first removing the cells from the upper chambers and then staining the cells attached to the lower surface of the membranes with crystal violet. During microscopic examinations, values were calculated as the total number of cells migrating in three randomly selected 40x-objective fields. Data represent the average of four independent experiments ± standard error. 200 Figure A-4. BCR-ABL expression does not confer constitutive global tyrosine phosphorylation or phosphorylation of ERK1/2 and PKB proteins. Wild-type MEFs, that were either present or absent for BCR-ABL, were serum starved overnight and then stimulated in the absence (-) or presence (+) of FCS. Cell lysates were separated by SDS-PAGE and membranes were probed with antibodies to detect phosphorylation events. (A) Tyrosine-phosphorylated proteins were detected with an anti-phospho- tyrosine (pTyr) antibody (upper panel). Total BCR-ABL and ABL proteins were detected upon reprobing with an anti-ABL antibody (lower panels). (B) Phosphorylated ERK1/2 were detected by 200mmunoblotting lysates with an antibody specific for phosphorylated ERK1/2 (first panel). Total ERK1/2 were detected upon reprobing with an anti-ERK1/2 antibody (second panel). Phosphorylated PKB proteins were detected in lysates using an anti-phospho-PKB antibody specific for the phosphorylation of serine 473 (third panel). A total anti-PKB antibody was used for reprobing the membrane (fourth panel).

201

Figure A-4 202 A.5. Discussion The CBL and CBL-B proteins are highly phosphorylated in BCR-ABL-expressing cell lines,37,54 yet in vivo, Dinulescu et al. argue that CBL is dispensable for CML development.14 Recent results from our laboratory demonstrating impaired myeloproliferative disease development in BCR-ABL BMTs (described in Chapter 2) suggest that CBL-B may be the dominant CBL homologue regulated in BCR- ABL-mediated CML development. To discern the mechanistic function of CBL-B in BCR-ABL- mediated leukemogenesis we initiated the generation of a cell line in which to perform our analyses. Our findings show that stable introduction of BCR-ABL in wild-type and CBL-B(-/-) cells induces a slight increase in growth but no other characteristic features of cellular transformation. Our biochemical examination predicts that the lack of transformation results from defunct BCR-ABL TK activity. We chose to create stable BCR-ABL-expressing cell lines by transfecting wild-type and CBL-B(-/- ) MEFs with a retroviral bicistronic vector that encodes for BCR-ABL and GFP. Transfected cells were selected by flow cytometric detection of GFP. The MEFs maintained BCR-ABL protein expression (Figure A-1) and showed only normal GFP drift. Our initial cell proliferation assays under low serum were hampered by reproducibility (Figure A- 2A). Given the known role of serum components, such as vitronectin,55 on cell attachment and adhesion, we altered our protocol to first include an adhesion step in 10% FCS. In this assay an increase in MEF growth was observed in the presence of BCR-ABL (Figure A-2B). Yet, proliferation proceeded at a rate 10-fold slower than that achieved by BCR-ABL cells in the presence of serum (data not shown). Based upon our findings in Figure A-4, which show the absence of global tyrosine phosphorylation or activation of known downstream substrates (ERK1/2 and PKB) we conclude that the BCR-ABL TK activity was damaged. As a result, we hypothesize that the minor growth enhancement observed in the MEFs resulted from a toxicity of the oncogene,11 rather than a growth signal provided by BCR-ABL. Assays to measure adhesion and migration were performed; stable BCR-ABL expression in the MEFs did not alter adhesion to fibronectin, spontaneous migration, or soft agar colony formation. These results align with those from two other groups,11,13 who failed to observe BCR-ABL-dependent transformation in the mouse NIH 3T3 fibroblast cell line. As Rat-1 fibroblasts can be transformed by BCR-ABL,11 our studies confirm that mouse fibroblasts are uniquely resistant to BCR-ABL transformation. There are several possible explanations for the failure of BCR-ABL to transform mouse fibroblasts, including: (1) an unsuitable signaling environment, (2) the inappropriate compartmentalization of the protein, and (3) poor expression strength. Several papers suggest that proteins are missing from NIH 3T3 cells that are required for transformation. Forced expression of the interleukin-3 receptor (IL-3R), along with BCR-ABL, leads to transformation of NIH 3T3s.56 This group suggests that signals emanating from IL-3R activate JAK2 and the GAB2/PI3’K cascade, while BCR- ABL stimulates the STAT5 protein. Only the combination of these signals results in transformation. 203 Further evidence for the involvement of IL-3 signaling is provided by data in which Rat-1 transformation by BCR-ABL is enhanced by v-myc expression.11 This finding is of interest, given the data that BCR- ABL induces Myc mRNA expression and protection from degradation through a pathway downstream of JAK2.57 Moreover, several groups detect IL-3 in the conditioned media of BCR-ABL-positive hematopoietic cells and it is suggested that this cytokine may contribute to autocrine growth.58-60 These studies all imply that the failed transformation of mouse fibroblasts by BCR-ABL may be due to reduced expression of proteins in the IL-3 signaling pathway. Secondly, failure of BCR-ABL to stably transform our MEFs may have been due to localization in a cellular compartment that was not conducive to transformation. Daley et al. found that attachment of a myristoylated Gag sequence to BCR-ABL could confer transformation to NIH 3T3,13 and Renshaw and colleagues state that a Gag-BCR-ABL construct can overcome the requirement for growth factors (GFs) of the P-3T3 line while BCR-ABL expression could not confer GF independence.12 These results imply that membrane localization of BCR-ABL is required for transformation of mouse fibroblasts. Finally, we hypothesize that our MEFs were resistant to transformation as a result of low BCR- ABL kinase activity. While we could detect BCR-ABL expression (Figure A-1) we were unable to observe noticeable TK activity (Figure A-4). It is known that the p185 isoform of BCR-ABL displays a 1.6-fold increase in TK activity over the p210 isoform,61 and the p185 construct can transform NIH 3T3.62 Therefore, we predict that transformation by BCR-ABL in our stable MEF lines may have resulted from low TK activity of the retroviral construct. Based on the results of our MEF experiments we suggest that further cell line studies of BCR- ABL and CBL-B utilize alternative strategies for depletion of CBL-B expression. RNA interference technology has been utilized successfully for the reduction of CBL-B in 293T cells, BT20s, and adipocytes.63-65 Based on promising results in our laboratory with siRNA and shRNA knock-down of proteins in the hematopoietic cell line Ba/F3 (Javadi et al., unpublished results), we believe CBL-B knock-down could be effectively achieved in Ba/F3 BCR-ABL-expressing cell lines. This model would serve as a useful tool in which to carry out experiments to discern the BCR-ABL signaling pathways that are differentially regulated in a CBL-B deficient environment.

204 A.6. References

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