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Protein Phosphatase Regulatory Protein 10 Cooperates with Neurofibromin Inactivation in Myeloid Leukemogenesis

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Protein Phosphatase Regulatory Protein 10 Cooperates with Neurofibromin Inactivation in Myeloid Leukemogenesis PROTEIN PHOSPHATASE REGULATORY PROTEIN 10 COOPERATES WITH NEUROFIBROMIN INACTIVATION IN MYELOID LEUKEMOGENESIS By ANGELA HADJIPANAYIS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1 © 2008 Angela Hadjipanayis 2 This work is dedicated to my extended family who never had the opportunity to obtain higher education. To my parents and husband for their loving support and belief in my abilities. To Camilynn I. Brannan whose research expertise led to the discovery of this locus. 3 ACKNOWLEDGMENTS First, I would like to thank my mentor Peggy Wallace for her indispensable support over the years, her encouragement, and her advice on how to become a better scientist. Second, I would like to thank my husband for taking a leap of faith in life and moving to Florida with me when he didn’t have a job at the time. Third, I would like to thank my parents for their love, support, and advice over the years. Fourth, I would like to thank my other committee members, Dr. Jim Resnick, Dr. Paul Oh, Dr. Stephen Hunger and Dr. Dan Driscoll for their valuable advice on the project. I also would like to thank Dr. Jennifer Embury for the tremendous effort on all the pathology of the mice. In addition, I would like to thank Dr. Kevin Shannon and Dr. Scott Kogan for their leukemia expertise and strategies in modeling leukemia. Lastly, I would like to thank the lab for their help on this project, their jokes, and all their personalities that made the days seem not as long. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 LIST OF ABBREVIATIONS ........................................................................................................11 ABSTRACT ...................................................................................................................................13 CHAPTER 1 INTRODUCTION ..................................................................................................................15 Neurofibromatosis Type 1 and JMML ...................................................................................15 Mouse Models of Nf1 .............................................................................................................18 Molecular and Biochemical Mechanisms Underlying Myeloid Leukemogenesis. ................19 Hyperactive Ras in JMML ......................................................................................................20 Nf1 Reconstitution Mouse Model ..........................................................................................21 Identification of Epi Loci in Nf1-Related Myeloid Leukemia ...............................................21 Viral Insertional Mutagenesis .................................................................................................23 MRPS18B ...............................................................................................................................24 PPP1R10 .................................................................................................................................24 PP1, a Serine/Threonine Protein Phosphatase ........................................................................25 2 MUTATIONAL ANALYSIS OF PPP1R10 IN LEUKEMIA ...............................................28 Introduction .............................................................................................................................28 Materials and Methods ...........................................................................................................29 Patient Samples ...............................................................................................................29 Polymerase Chain Reaction and Sequencing of PPP1R10 Exons ..................................29 Restriction Fragment Length Polymorphism for Intron 15 Loss of Heterozygosity .......29 Real-Time mRNA Quantification ...................................................................................30 Results .....................................................................................................................................31 Discussion ...............................................................................................................................32 3 IN VITRO ANALYSIS OF PPP1R10 AS A POTENTIAL ONCOGENE ............................41 Introduction .............................................................................................................................41 Materials and Methods ...........................................................................................................41 Cloning of Ppp1r10 in the MSCV Viral Vectors ............................................................41 Hematopoietic Cell Isolation and Retroviral Transduction .............................................42 Colony Formation Unit Granulocyte/Macrophage Colony Assay ..................................42 5 Serum Starvation of COS7 Cells .....................................................................................43 Western Blots ..................................................................................................................43 Results .....................................................................................................................................44 Discussion ...............................................................................................................................44 4 PPP1R10 COOPERATES WITH NF1 INACTIVATION TO CAUSE ACUTE MYELOMONOCYTIC LEUKEMIA IN A MOUSE MODEL .............................................50 Introduction .............................................................................................................................50 Materials and Methods ...........................................................................................................51 Ppp1r10+/- Mouse Construction .....................................................................................51 Breeding Ppp1r10 Mice ..................................................................................................51 Pathology/Sectioning/H and E Stain ...............................................................................51 Blood Smears and Manual Counts ..................................................................................52 Sudan Black Stain and MPO Stain ..................................................................................52 Flow Cytometry ...............................................................................................................53 Nucleic Acid Extraction ..................................................................................................53 Loss of Heterozygosity Analysis .....................................................................................53 Results .....................................................................................................................................54 Discussion ...............................................................................................................................58 5 DISCUSSION .........................................................................................................................89 APPENDIX A PRIMER LIST ........................................................................................................................94 B PFAFFL METHOD ................................................................................................................95 REFERENCES ..............................................................................................................................96 BIOGRAPHICAL SKETCH .......................................................................................................105 6 LIST OF TABLES Table page 2-1 NF1-JMML human patient samples and Intron 15 polymorphism LOH. .........................38 2-2 JMML human samples and Intron 15 polymorphism LOH. ..............................................39 2-3 mRNA Quantification of human leukemia RNA. ..............................................................40 4-1 Ppp1r10+/- aged mice necropsy data, M: E (2 to 8.25) ratios, and blast numbers............86 4-2 Nf1+/-Ppp1r10+/-aged mice necropsy data, M:E (2.33 to 13.25) ratios, and blast numbers. .............................................................................................................................87 4-3 Peripheral blood smear analysis of Nf1+/-Ppp1r10+/- (NF1PKO), Ppp1r10+/- (PKO), and Nf1+/-. .............................................................................................................88 4-4 Bone marrow analysis of Nf1+/-Ppp1r10+/- (NF1PKO), Ppp1r10+/- (PKO), and Nf1+/- mice. WNL signifies within normal limits. ............................................................88 7 LIST OF FIGURES Figure page 2-1 Human NF1-JMML analysis PPP1R10, Promoter Polymorphism (rs16867845). ............33 2-2 Human NF1-JMML analysis, PPP1R10 Exon 6 nonsense, S125X. ..................................33 2-3 Human NF1-JMML
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