University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2018-02-13 RUNX1 Is an Oncogenic Transcription Factor that Regulates MYB and MYC Enhancer Activity in T-ALL AHyun Choi University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Cancer Biology Commons Repository Citation Choi A. (2018). RUNX1 Is an Oncogenic Transcription Factor that Regulates MYB and MYC Enhancer Activity in T-ALL. GSBS Dissertations and Theses. https://doi.org/10.13028/M20D6J. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/957 Creative Commons License This work is licensed under a Creative Commons Attribution 4.0 License. This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. RUNX1 IS AN ONCOGENIC TRANSCRIPTION FACTOR THAT REGULATES MYB AND MYC ENHANCER ACTIVITY IN T-ALL A Dissertation Presented By AHYUN CHOI Submitted to the Faculties of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY FEBRUARY 13, 2018 CANCER BIOLOGY ii RUNX1 IS AN ONCOGENIC TRANSCRIPTION FACTOR THAT REGULATES MYB AND MYC ENHANCER ACTIVITY IN T-ALL A Dissertation Presented By AHYUN CHOI This work was undertaken in the Graduate School of Biomedical Sciences Cancer Biology Under the mentorship of Michelle Kelliher, Ph.D., Thesis Advisor The signatures of the Dissertation Defense Committee signify completion and approval as to style and content of the Dissertation Lucio H. Castilla, Ph.D., Member of Committee Glen Raffel, MD., Ph.D., Member of Committee Thomas Fazzio, Ph.D., Member of Committee Alan B. Cantor, M.D., Ph.D., External Member of Committee Merav Socolovsky, MBBS., Ph.D., Chair of Committee Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences February 13, 2018 iii ACKNOWLEDGEMENTS These works could not be done without support and help from lots of people. First of all, I would like to thank my mentor, Dr. Michelle Kelliher, for her support and guidance. I have learned from her a way of thinking and handling the science. I also thank the lab member of the Kelliher lab, past and present, for their help and making the enjoyable lab life. Their advice and achievement have inspired me and kept me moving forward. I appreciate the advice and support from my committees, Dr. Lucio Castilla, Dr. Merav Socolovsky, Dr. Glen Raffel, and Dr. Thomas Fazzio. Additionally, I thank Dr. Alan Cantor for serving as an external examiner of my dissertation defense. I also appreciate Dr. John Pulikkan in the Castilla's lab for his friendship and guidance. I would like to thank my friends and family. Their support and advice help me going through this long journey. I also really enjoyed the adventures I have had with friends for years. Lastly, I cannot appreciate enough the understanding and support of my parents and husband, Hwanjong. They make me not to get lost, follow what I want, and smile every day. Thank you. iv ABSTRACT RUNX1, a transcription factor required for hematopoiesis and lymphocyte differentiation, is one of the most commonly targeted genes in hematopoietic malignancies. Mutations in the RUNX1 gene are associated with a poor prognosis in a subset of T cell acute lymphoblastic leukemia (T-ALL) and RUNX1 has been proposed as a tumor suppressor in TLX1/3-transformed human T-ALL. Recent ChIP-seq studies in human T-ALL cell lines demonstrated that a large portion of TAL1- and NOTCH1- bound regions contain RUNX binding sites in promoter or enhancer regions, suggesting oncogenic roles for RUNX1 in T-ALL. To interrogate RUNX1 functions in leukemogenesis, we depleted RUNX1 in a T-ALL mouse model and in human T-ALL cell lines. We found that RUNX1 is required for the maintenance of mouse T-ALL growth in vivo and the survival of human T-ALL cell lines in vitro. In addition, inhibition of the RUNX1 activity with a small molecule inhibitor impairs the growth of human T-ALL cell lines and primary patient samples. RUNX1 depletion reduces the expression of a subset of TAL1- and NOTCH1- regulated genes including the MYB and MYC oncogenes, respectively. We demonstrate that RUNX1 regulates transcription factor binding and acetylation of H3K27 at the Myb and Myc enhancer loci. These studies provide genetic and pharmacological evidences that RUNX1 supports T-ALL cell survival and suggest RUNX1 inhibitor as a therapeutic strategy in T-ALL treatment. v TABLE OF CONTENTS Signature Page ii Acknowledgements iii Abstract iv Table of Contents v List of Tables vii List of Figures viii Copyrighted Materials Produced by the Author xi Chapter I: Introduction 1 Chapter II: RUNX1/ and/or RUNX3 is required for T-ALL survival 50 Introduction 51 Results 52 Discussion 56 Methods and Materials 58 Figures 62 Chapter III: RUNX1 supports T-ALL survival by regulating Myb and Myc enhancer activity 76 Introduction 77 Results 78 Discussion 84 Methods and Materials 87 Figures 90 Chapter IV: Discussion 100 Appendix 122 vi Appendix I Evaluation of the efficacy of RUNX/CBFb inhibitor AI-12-126 on T-ALL progression in vivo 123 Appendix II RUNX1 regulates DNA accessibility in mouse T-ALL cells 128 Appendix III Repression of mTORC1 activity sensitizes T-ALL cells to ABT-263 treatment 139 Appendix IV Attempts to identify a compound that selectively kills ETP-ALL cells 148 Appendix V Identification of tumor suppressor(s) in chromosome 6q deleted region that cooperate with TAL1 to cause T-ALL 157 List of Primers 160 Reference 161 vii List of Tables Chapter II Table 2.1. Expression and mutation status in patient samples and human T- ALL cell lines. Table 2.2. Human T-ALL cell lines and primary patient samples are sensitive to AI-10-104 treatment. Table 2.3. shRNA clones used in the study Appendix Table A.II.1. Gene lists of functional annotations analyses Table A.IV.1. The list of compounds selected for the secondary screening Table A.IV.2. The list of 6 compounds identified in the secondary screening viii List of Figures Chapter I Figure 1.1. Thymocyte development Figure 1.2. The structure of RUNX proteins Chapter II Figure 2.1. RUNX1 is required for the maintenance of leukemic growth in vivo Figure 2.2. RUNX1 supports survival of mouse leukemic cells in vitro Figure 2.3. Cre activation has no significant effects on mouse T-ALL growth in vitro and RUNX3 protein expression in mouse leukemic cells Figure 2.4. RUNX1, but not RUNX3, is ubiquitously expressed in human T-ALL cell lines and primary patients T-ALL samples Figure 2.5. Knockdown of RUNX1, RUNX3, and CBFb results in apoptosis Figure 2.6. Treatment with a RUNX-CBFβ inhibitor impairs the growth of human T-ALL cell lines and primary pediatric T-ALL samples Figure 2.7. RUNX1/3-CBFb inhibitor is not detrimental to normal human hematopoietic stem and progenitor cells at low concentration Figure 2.8. Reduction of RUNX1 expression in TLX3-transformed T-ALL cell line induces cell apoptosis Chapter III Figure 3.1. RUNX1 regulates a subset of TAL1-, and NOTCH1-regulated genes ix Figure 3.2. RUNX1 knockdown in human T-ALL cell lines alters target the expression of target genes the expression of a subset of RUNX1-, TAL1-, and NOTCH1-regulated genes Figure 3.3. RUNX1 depletion derepresses CD4 cell surface expression on T- ALL cells Figure 3.4. RUNX1 is required for TAL1 binding to the Myb enhancers and for the retention of active chromatin marks Figure 3.5. RUNX1 is required for intracellular NOTCH1 binding and for chromatin accessibility at the N-Me Figure 3.6. Runx1 depletion has no effect on intracellular NOTCH1 binding to the Hes1 promoter or to gene desert regions Figure 3.7. RUNX1/3 binding to the oncogenic enhancers reflects their regulation on gene expression Figure 3.8. The reduced expression of RUNX1 or RUNX3 does not lead to increased expression of the other Figure 3.9. RUNX1 reduction interferes with the formation of loop between the MYB promoter and -94-kb and +14-kb enhancers Chapter VI Figure 4.1. Proposed model Appendix Figure A.I.1. AI-12-126 treatment does not suppress leukemia progression in mice transplanted with primary patient T-ALL sample x Figure A.I.2. Treatment of AI-12-126 does not inhibit leukemic growth in vivo Figure A.I.3. AI-12-126 treatment inhibits the growth of human T-ALL cell lines Figure A.II.1. Signals of ATAC-seq in Runx1 deleted mouse T-ALL cells and over-representative pathways Figure AII.2. The accessibility to the Myb and Myc enhancer regions is increased in Runx1-deleted cells. Figure A.III.1. Experimental strategy of used to determine the efficacy of combination treatment of AZD8055 and ATB-263 on leukemia progression in vivo Figure A.III.2. Treatment of AZD8055 and ABT-263 prolong survival of mice transplanted with primary T-ALL patient samples Figure A.III.3. Combination treatment of AZD8055 and ABT-263 inhibits leukemic burden in vivo Figure A.III.4. The in vivo growth of TALL-X-7 primary T-ALL patient sample is suppressed by treatment with ABT-263 Figure A.III.5. The expression of intracellular NOTCH1 and MCL-1 in primary T-ALL patient samples Figure A.IV.1. Secondary screening identifies compounds that are selectively effective against the Loucy ETP-ALL cell line Figure A.IV.2.