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UNIVERSITY of CALIFORNIA, SAN DIEGO Characterization Of UNIVERSITY OF CALIFORNIA, SAN DIEGO Characterization of granulocyte-macrophage colony-stimulating factor signaling in RUNX1-ETO leukemogenesis A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by Stephanie Weng Committee in charge: Professor Dong-Er Zhang, Chair Professor Steven Dowdy Professor Gen-Sheng Feng Professor Mark Kamps Professor David Traver 2016 Copyright Stephanie Weng, 2016 All rights reserved. The Dissertation of Stephanie Weng is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2016 iii DEDICATION This dissertation is dedicated in loving memory to my mother, Linda Chien Weng. Thank you for being a continual source of inspiration in every aspect of my life. iv TABLE OF CONTENTS Signature page ............................................................................................................... iii Dedication ...................................................................................................................... iv Table of Contents ............................................................................................................ v List of Abbreviations ....................................................................................................... ix List of Figures ................................................................................................................. xi List of Tables ................................................................................................................ xiii Acknowledgements ...................................................................................................... xiv Vita .............................................................................................................................. xviii Abstract of the Dissertation ............................................................................................ xx Chapter 1: Introduction ................................................................................................... 1 1.1. Loss of a sex chromosome in hematological malignancies .................................. 2 1.2. Pseudoautosomal regions ................................................................................... 4 1.3. Pseudoautosomal region genes in the hematopoietic system .............................. 6 1.3.1. Tumor suppressors ....................................................................................... 7 1.3.2. Proto-oncogenes ..........................................................................................13 1.3.3. Context-dependent pseudoautosomal region genes ....................................16 1.3.4. Inactive pseudoautosomal region genes ......................................................20 1.3.5. Other less characterized pseudoautosomal region genes and regions .........21 1.4. Sex chromosome amplifications in cancer ..........................................................24 1.5. Discussion ..........................................................................................................24 1.6. Objectives of the dissertation ..............................................................................25 1.7. Acknowledgements .............................................................................................25 Chapter 2: Multiple GM-CSF-induced genes in RUNX1-ETO hematopoietic stem/progenitor cells function to inhibit self-renewal potential ........................................26 2.1. Introduction .........................................................................................................27 2.2. Results ...............................................................................................................29 2.2.1. GM-CSF induces a human myelopoiesis gene expression profile in RUNX1- ETO hematopoietic stem/progenitor cells ...............................................................29 2.2.2. GM-CSF promotes differentiation and reduces the long-term culture initiating cell frequency of primary human RUNX1-ETO hematopoietic stem/progenitor cells ..............................................................................................................................36 v 2.2.3. Characterization of the GM-CSF-induced gene expression profile in inhibiting self-renewal potential of RUNX1-ETO hematopoietic stem/progenitor cells ...........40 2.2.4. Validation and characterization of candidates from screen to identify GM- CSF-induced genes that reduce self-renewal potential of RUNX1-ETO hematopoietic stem/progenitor cells .......................................................................45 2.3. Discussion ..........................................................................................................50 2.4. Materials and Methods........................................................................................51 2.4.1. Plasmids ......................................................................................................51 2.4.2. Gene expression profiling.............................................................................52 2.4.3. Gene expression profiling data analysis .......................................................52 2.4.4. Quantitative reverse-transcription PCR (qRT-PCR)......................................53 2.4.5. CD34 + cell isolation, transduction, and culture .............................................56 2.4.6. Long-Term Culture-Initiating Cell (LTC-IC) assays .......................................56 2.4.7. Flow cytometry .............................................................................................57 2.4.8. Barcoded cDNA screen ................................................................................57 2.4.9. Cell culture ...................................................................................................60 2.4.10. Proliferation assays ....................................................................................60 2.4.11. Replating assays ........................................................................................60 2.4.12. Microscopy .................................................................................................60 2.4.13. Statistical analysis ......................................................................................61 2.5. Acknowledgements .............................................................................................61 Chapter 3: Attenuation of MYC reduces the leukemic potential of RUNX1-ETO cells ....63 3.1. Introduction .........................................................................................................64 3.2. Results ...............................................................................................................66 3.2.1 MYC-associated gene signatures are attenuated in GM-CSF-treated RUNX1- ETO hematopoietic stem/progenitor cells ...............................................................66 3.2.2 MXI1 expression in RUNX1-ETO hematopoietic stem/progenitor cells and t(8;21) cell lines reduces leukemic potential ...........................................................67 3.2.3 MYC knockdown reduces the leukemic potential of t(8;21) cell lines .............70 3.2.4 JQ1 treatment reduces the leukemic potential of RUNX1-ETO hematopoietic stem/progenitor cells and t(8;21) cell lines .............................................................73 3.2.5 MXI1 relieves the RUNX1-ETO-induced repression of RUNX1 target sites ...75 vi 3.3. Discussion ..........................................................................................................79 3.4. Materials and Methods........................................................................................81 3.4.1 Plasmids .......................................................................................................81 3.4.2 Cell culture ....................................................................................................81 3.4.3 Proliferation assays .......................................................................................81 3.4.4 Flow Cytometry .............................................................................................82 3.4.5 Colony forming assays ..................................................................................82 3.4.6 Western blot ..................................................................................................82 3.4.7 Quantitative reverse-transcription PCR (qRT-PCR) ......................................82 3.4.8 Luciferase assay ...........................................................................................84 3.4.9. Co-immunoprecipitation ...............................................................................84 3.4.10. Statistical analysis ......................................................................................85 3.5. Acknowledgements .............................................................................................85 Chapter 4: CSF2RA is post-transcriptionally downregulated in t(8;21) cell lines ............86 4.1. Introduction .........................................................................................................87 4.2. Results ...............................................................................................................89 4.2.1. CSF2RA is downregulated in t(8;21) cell lines ..............................................89
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