CONTRASTING ROLES OF C/EBPα AND NOTCH IN IONIZING RADIAITON- INDUCED MULTIPOTENT HEMATOPOIETIC PROGENITOR CELL SELF- RENEWAL DEFECTS by COURTNEY JO FLEENOR B.S., University of Iowa, 2008 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Immunology Program 2015 This thesis for the Doctor of Philosophy degree by Courtney Jo Fleenor has been approved for the Immunology Program by Philippa Marrack, Chair John Cambier Heide Ford James Hagman Jill Slansky James DeGregori, Advisor Date 12/31/14 ii Fleenor, Courtney Jo (Ph.D., Immunology) Contrasting Roles of C/EBPα and Notch in Ionizing Radiation-Induced Multipotent Hematopoietic Progenitor Cell Self-Renewal Thesis directed by Professor James V. DeGregori. ABSTRACT Ionizing radiation (IR) is associated with persistent panhematopoietic defects and increased risk of carcinogenesis, although the mechanism underlying these relationship remains largely unknown. The increasing exposure of humans to man-made sources of IR, primarily through medical procedures, exemplifies a critical need to understand how IR elicits adverse health affects. Mice exposed to IR develop a disease similar to human T-cell acute lymphoblastic lymphoma, of which over 50% are associated with activating Notch mutations. Furthermore, our lab has previously shown that the activated intracellular Notch mutant, ICN1, is selected for and promotes lymphomagenesis within previously irradiated mice. As pan-hematopoietic defects persist long after the exposure event, I hypothesized that IR reduces hematopoietic stem cell (HSC) function, increasing selective pressure for activating Notch mutations that repair or circumvent the IR-mediated defect. Consistent with this hypothesis, I observed that IR results in persistent, somatically heritable, cell-intrinsic reductions in multipotent hematopoietic progenitor cell (mHPC) self-renewal. mHPC from previously irradiated but homeostatically restored (IRP) mice exhibited reduced expansion and an iii increased propensity to differentiate in vitro, as well as persistent oxidative stress. RNA-seq revealed persistent alterations in gene expression resulting from IR, with decreased expression of HSC-associated genes, increased expression of myeloid differentiation-associated genes, including CEBPA, and a signature of antioxidant responses. Notch activation restored the expansion and reduced the precocious differentiation of IRP mHPC in vitro, as well as reversed a IR-induced gene expression changes. Loss of CEBPA expression is selected for within IRP HSC and mHPC pools, reverses IR-dependent precocious differentiation and restores self-renewal. Finally, Notch-mediated restoration of mHPC self-renewal prevents selection for loss of C/EBPα expression within IRP mHPC pools. We propose that in response to environmental insults, HSC initiate a program limiting their self-renewal, preventing the damaged HSC from contributing long-term to hematopoiesis. This “programmed mediocrity” is beneficial in the case of sporadic genotoxic insults, but promotes tumorigenesis when the entire HSC compartment is damaged, such as during total body irradiation, by reducing the fitness of the entire HSC pool and thereby increasing selective pressure for adaptive oncogenic mutations. The form and content of this abstract are approved. I recommend its publication. Approved: James DeGregori iv This dissertation is dedicated to my supportive and loving family: my parents, Jeff and Sharyl, my sisters, Stephanie and Kelsey, and my fiancé Chris. Without their love and support, I wouldn’t be the scientist or person I am today. v ACKNOWLEDGEMENTS I am forever in indebted to the many people who have provided advice, support, and encouragement throughout my graduate career. You all rock! I would like to thank my thesis committee, Pippa Marrack, John Cambier, Heide Ford, Jim Hagman, and Jill Slansky for all their support, advice, and encouragement. I would like to thank the Department of Immunology for their support throughout my graduate education. I would also like to thank members of the Cancer Research Institute Pre-doctoral Emphasis in Tumor Immunology Fellowship for providing funding for part of my graduate career. I would like to thank all the members of the flow core, sequencing core, EH&S, and animal facility and staff for their help with experiments. I would like to thank all the current and past members of the DeGregori lab for scientific discussion, experimental advice and technical help. Specifically, I want to thank Vadym Zaberezhnyy for help with all the bone marrow transplants and for being such a good sport whenever I was running behind schedule. I also want to thank Mark Gregory and Jen Salstrom for scientific discussions, for answering all my ridiculous questions sometimes pertaining to science, and for being both great friends and mentors. I’d like to thank Francesca Alvarez- Calderon, Matias Casas-Selvez, and Melanie Bui for being my partners in crime in and out of the lab. I would like to thank Matthew Divij, Kelly Higa, and Andrii Rozhok for their help on this project. vi Big thanks to all my family and friends for their unconditional love, support, and for helping keep me sane. Finally, I would like to thank my doctorate advisor, James DeGregori, for allowing me to pursue all my scientific hypotheses, no matter how wild. I can’t thank you enough for all your patience, advice, and encouragement over the years. vii TABLE OF CONTENTS CHAPTER I. INTRODUCTION 1 Ionizing Radiation 2 Types of Ionizing Radiation 2 Quantification of Radiation 4 Human Exposure to Ionizing Radiation 5 Hematopoiesis 9 The Role of Notch in Hematopoiesis 11 Regulation of Myeloid Differentiation by C/EBPα 15 Biological Effects of Ionizing Radiation 17 IR-induced DNA Damage 18 Induction of Reactive Oxygen Species by IR 21 Nrf2 Response to Oxidative Stress 21 ATM Response to Oxidative Stress 22 PKCδ Response to Oxidative Stress 23 Persistent Effects of IR on HSC Function 24 Adaptive Oncogenesis 26 II. MATERIALS AND METHODS 29 Mice 29 Flow Cytometry 29 Fluorescence Activated Cell Sorting 30 mHPC cultures 31 viii Bone Marrow Transplantation 32 Bone Marrow Transplantation of Cultured mHPC 32 Retrovirus and Lentivirus 33 Generation of Virus 33 Viral Transduction Efficiency 34 Viral Transduction of Murine Bone Marrow Cells 34 Knockdown Efficiency of shRNA Targets 34 RT-qPCR 35 Mathematical Modeling 36 Probability of Differentiation 36 LSK Fitness 38 ROS Detection 39 Chemical Compounds 39 RNA Sequencing 40 In Vivo All-Trans Retinoic Acid Treatment 42 Subcutaneous ATRA Pellets 42 Oral Administration of ATRA 43 In Vivo Analysis of ATRA Treatment on mHPC Fitness 43 In Vitro Analysis of mHPC Self-Renewal After In Vivo ATRA Treatment 44 Statistical Analysis 44 ix III. NOTCH ACTIVATION RESTORES IONIZING RADIATION- REDUCED MULTIPOTENT HEMATOPOIETIC PROGENITOR CELL SELF-RENEWAL 45 Introduction 45 Ionizing Radiation and Human Disease 45 Hematopoietic Stem and Progenitor Cells 46 Effect of IR Exposure on HSC Function 47 Results 48 Ionizing Radiation Exposure Results in a Long-Term Reduction in the Number of Phenotypic Hematopoietic Stem and Progenitor Cells 48 IRP BM Possesses Decreased Long-Term HSC Function In Vivo 52 IRP mHPC Exhibit Reduced Self-Renewal In Vitro 55 LShiK Population Represents the Most Primitive mHPC Population In Vitro 57 IRP LT-HSC Possess Reduced Expansion Potential In Vitro 58 IR Exposure Does Not Alter Cell Culture Initiating Frequency of LSK 59 IRP LSK Lose Long-Term Multipotent Reconstitution Potential More Quickly In Vitro 60 Exposure to X-rays or γ-rays Elicits the IRP mHPC Defect 61 IR Dose Threshold Required for Generation of IRP mHPC Expansion Defect 62 IRP mHPC Expansion Defect In Vitro is Cell Intrinsic 63 Activation of Notch Restores IRP mHPC Self-Renewal In Vitro 64 x IRP LSK Have Slightly Reduced Surface Expression of Notch2 67 IRP mHPC Increased Probability of Differentiation and Reduced Fitness are Restored by Notch Activation 68 Discussion 70 IV. INHIBITING IR-INDUCED PRECOCIOUS DIFFERENTIATION RESTORES SELF-RENEWAL OF PREVIOUSLY IRRADIATED MHPC 74 Introduction 74 Regulation of HSC Self-Renewal 75 Regulation of HSC Differentiation 75 Results 77 IR Exposure Causes Long-Term, Notch-Reversible Alterations in mHPC Gene Expression 77 IRP LSK Express Decreased Levels of HSC-Associated Genes 79 Effect of IR on Expression of Notch-Regulated Genes in mHPC 80 Monocyte Differentiation Programs are Enriched Within IRP mHPC 83 Inhibition of C/EBPα Restores IRP LSK Self-Renewal In Vitro 86 Inhibition of C/EBPα is Selected for Within IRP mHPC In Vivo 90 Notch Activation Prevents Selection for C/EBPα Inhibition Within LSK In Vitro 92 Discussion 95 V. IONIZING RADIATION CAUSES PERSISTENT OXIDATIVE STRESS IN HEMATOPOIETIC PROGENITOR CELLS 99 xi Introduction 99 Ionizing Radiation Induces Oxidative Stress 99 Cellular Responses to Oxidative Stress 100 Effect of Elevated ROS on mHPC Function 101 Results 102 IRP LSK Possess Increased Lipid Peroxidation 102 IRP LSK May Possess Elevated NADPH Oxidase Activity 104 IRP LSK Possess Increased Gene Expression Signature of Nrf2, a Master Regulator of Antioxidant Activity 106 Nrf2 Activation is Sufficient to Reduce LSK Expansion In Vitro 108 Nrf2 is Not Required for IR-Mediated Induction or Notch-Rescue of IRP LSK Expansion Defect 110 Inhibition of ATM Restores IRP LSK Expansion In Vitro 113 Inhibition of PKC Restores IRP LSK Expansion In Vitro 115 Vitamin E Supplementation