DEFINING THE MECHANISM OF ACTION OF BROMODOMAIN AND EXTRATERMINAL INHIBITORS IN TRIPLE-NEGATIVE BREAST CANCERS By JENNIFER MICHELE BRANCATO Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Ruth A. Keri, Ph.D. Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY May, 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Jennifer Michele Brancato candidate for the degree of Doctor of Philosophy*. Youwei Zhang, Ph.D. (Committee Chair) Ruth Keri, Ph.D. (Committee Member) Goutham Narla, M.D., Ph.D. (Committee Member) Peter Scacheri, Ph.D. (Committee Member) Analisa DiFeo, Ph.D. (Committee Member) William Schiemann, Ph.D. (Committee Member) Date of Defense February 2, 2018 *We also certify that written approval has been obtained for any proprietary material contained therein. 1 DEDICATION I dedicate this work to my family. First, to my parents who have always pushed me to dream big and work hard to achieve my goals. I would not be where I am today without your love and support. Also, to my husband and my son. I know the last several years were not always easy, especially when I had to focus on my work instead of spending time with you. Your patience and encouragement helped to carry me through this process. I love you. 2 TABLE OF CONTENTS List of Tables ...................................................................................................... 6 List of Figures ..................................................................................................... 7 Acknowledgements .......................................................................................... 10 ABSTRACT ........................................................................................................ 12 Chapter 1: Introduction .................................................................................... 14 1.1 Overview of breast cancer ................................................................. 15 1.1.1 Breast cancer subtypes ............................................................ 16 1.1.1.1 Receptor status ................................................................. 17 1.1.1.2 Intrinsic subtypes of breast cancer .................................... 21 1.1.1.3 Additional breast cancer classification systems ................ 26 1.1.2 Therapeutic options for the treatment of breast cancer............. 30 1.1.3 Resistance to cytotoxic chemotherapy ...................................... 37 1.1.3.1 Taxane-specific resistance mechanisms ........................... 39 1.1.3.2 Anthracycline-specific resistance mechanisms ................. 40 1.2 Basics of transcriptional control of genes ........................................... 41 1.2.1 Transcriptional regulatory elements .......................................... 41 1.2.2 RNA polymerase ....................................................................... 45 1.2.3 Other transcription-related proteins........................................... 46 1.2.4 Transcription initiation, elongation, and termination .................. 50 1.3 Targeting the epigenome in cancer ................................................... 52 1.3.1 Chromatin structure .................................................................. 53 1.3.2 DNA methylation ....................................................................... 54 1.3.3 Histone modifications ................................................................ 57 1.4 Targeting Bromodomain and extraterminal proteins in breast cancer 61 1.4.1 BET protein structure and function ........................................... 61 1.4.1.1 BET protein structure ........................................................ 61 1.4.1.2 BET proteins and transcription ......................................... 63 3 1.4.1.3 BET proteins and the cell cycle ......................................... 64 1.4.1.4 BET proteins and inflammation ......................................... 65 1.4.1.5 BET proteins and development ......................................... 66 1.4.1.6 BRDT and spermatogenesis ............................................. 66 1.4.2 BET proteins in cancer .............................................................. 66 1.4.2.1 BET proteins and super-enhancers ................................... 66 1.4.2.2 Expression of the BET family in breast tumors and cell lines ................................................................................................................. 68 1.4.2.3 Essentiality of BET proteins in breast cancer .................... 69 1.4.3 Targeting BET proteins in breast cancer ................................... 70 1.4.3.1 BET inhibitor structure and selectivity ............................... 70 1.4.3.2 Impact of breast cancer cell growth and tumor formation .. 72 1.4.3.3 BET inhibitors and hypoxia ................................................ 76 1.4.3.4 BET inhibitors and angiogenesis ....................................... 77 1.4.3.5 BET inhibitors and cancer stem cells ................................ 79 1.4.3.6 BET inhibitors and metastasis ........................................... 81 1.4.3.7 BET inhibitors and metabolism .......................................... 85 1.4.3.8 Mechanism(s) of action of BET inhibitors in breast cancer 86 1.4.3.9 Resistance to BET inhibitors ............................................. 90 1.4.3.10 BET inhibitor adverse effects .......................................... 92 1.4.4 Alternative BET protein targeting mechanisms ......................... 94 1.4.4.1 Combination therapies ...................................................... 95 1.4.4.2 BET protein degraders .................................................... 106 1.4.4.3 Dual protein kinase/BET protein inhibitors ...................... 108 1.4.4.4 CDK7 inhibitors ............................................................... 109 1.4.5 Future perspectives and conclusions ...................................... 111 1.5 Mitosis .............................................................................................. 113 1.5.1 The cell cycle and cell cycle checkpoints ................................ 113 1.5.2 Cyclin-dependent kinases ....................................................... 116 4 1.5.3 The mitotic kinases ................................................................. 119 1.5.3.1 Polo-like kinases ............................................................. 121 1.5.3.2 Aurora kinases ................................................................ 122 1.5.4 Activity of mitotic kinases in M phase ...................................... 123 1.5.5 Targeting mitotic kinases in breast cancer .............................. 128 1.5.5.1 Polo-like kinase 1 ............................................................ 129 1.5.5.2 Aurora kinase A ............................................................... 130 1.5.5.3 Aurora kinase B ............................................................... 132 1.6 Statement of purpose ....................................................................... 133 Chapter 2: Bromodomain and extraterminal protein inhibition blocks growth of triple-negative breast cancers through the suppression of Aurora kinases ............................................................................................................ 158 2.1 Abstract ............................................................................................ 159 2.2 Introduction ...................................................................................... 159 2.3 Materials and methods ..................................................................... 161 2.4 Results ............................................................................................. 167 2.4.1 BET inhibition blocks growth of diverse TNBC cells without consistently down-regulating MYC ................................................... 167 2.4.2 Sustained BET inhibition induces apoptosis and senescence in TNBC cells ....................................................................................... 168 2.4.3 BET inhibition abrogates TNBC tumor growth ........................ 170 2.4.4 Aurora kinases are downstream targets of BETi ..................... 171 2.5 Discussion ....................................................................................... 173 Chapter 3: Mitotic vulnerability in triple-negative breast cancer associated with LIN9 is targetable with BET inhibitors .................................................. 191 3.1 Abstract ............................................................................................ 192 3.2 Introduction ...................................................................................... 192 3.3 Materials and methods ..................................................................... 195 3.4 Results ............................................................................................. 202 3.4.1 Sustained BET activity is necessary for normal progression through mitosis ................................................................................. 202 5 3.4.2 BET activity is necessary for sustained expression of cell-cycle- associated genes ............................................................................. 204 3.4.3 BET inhibitors fail to induce a luminal differentiation signature 205 3.4.4 BETi proteins directly modulate the mitotic transcriptional program ...........................................................................................