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SSStttooonnnyyy BBBrrrooooookkk UUUnnniiivvveeerrrsssiiitttyyy The official electronic file of this thesis or dissertation is maintained by the University Libraries on behalf of The Graduate School at Stony Brook University. ©©© AAAllllll RRRiiiggghhhtttsss RRReeessseeerrrvvveeeddd bbbyyy AAAuuuttthhhooorrr... Mechanistic evaluation of NSD3 in the pathogenesis of acute myeloid leukemia A Dissertation Presented by Chen Shen to The Graduate School in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Molecular and Cellular Biology Stony Brook University May 2016 Stony Brook University The Graduate School Chen Shen We, the dissertation committee for the above candidate for the Doctor of Philosophy degree, hereby recommend acceptance of this dissertation. Dr. Christopher R. Vakoc– Dissertation Advisor Associate Professor, Cold Spring Harbor Laboratory Dr. Alea A. Mills - Chairperson of Defense Professor, Cold Spring Harbor Laboratory Dr. Adrian R. Krainer Professor, Cold Spring Harbor Laboratory Dr. Lloyd C. Trotman Associate Professor, Cold Spring Harbor Laboratory Dr. Ed Luk Assistant Professor, Department of Biochemistry and Cell Biology, Stony Brook University Dr. Christopher M. Hammell Assistant Professor, Cold Spring Harbor Laboratory This dissertation is accepted by the Graduate School Charles Taber Dean of the Graduate School ii Abstract of the Dissertation Mechanistic evaluation of NSD3 in the pathogenesis of acute myeloid leukemia by Chen Shen Doctor of Philosophy in Molecular and Cellular Biology Stony Brook University 2016 The bromodomain and extra-terminal (BET) protein BRD4 is a validated drug target in hematological malignancies, owing to its essential role in sustaining oncogenic transcriptional programs. To gain insight into the cancer-relevant mechanistic function of BRD4, I have investigated its mechanism of transcriptional activation in the MLL-fusion subtype of acute myeloid leukemia (AML) with experimental approaches including small hairpin RNA (shRNA) knockdown, clustered regularly-interspaced short palindromic repeats (CRISPR)-Cas9 knockout, biochemistry, RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq). In this study, I demonstrate that the AML maintenance function of BRD4 requires its interaction with NSD3, which belongs to a subfamily of H3K36 methyltransferases. Unexpectedly, AML cells were found to only require a short isoform of NSD3 that lacks the methyltransferase domain. I show that NSD3-short is an adaptor protein that sustains leukemia by linking BRD4 to the CHD8 chromatin remodeler, by utilizing a Pro-Trp-Trp-Pro (PWWP) module, and by employing an acidic transactivation domain. Phenotypic and transcriptional effects of genetic targeting of NSD3 or CHD8 mimic the effects of BRD4 inhibition. Furthermore, BRD4, NSD3, and CHD8 colocalize across the AML genome and are each released from super-enhancer regions upon chemical inhibition of BET bromodomains. These findings suggest that BET inhibitors exert therapeutic effects in leukemia by evicting BRD4- NSD3-CHD8 complexes from chromatin to suppress transcription. iii Dedication Page I sincerely dedicate this thesis to my beloved family, dear teachers and friends. iv Table of Contents List of Figures .............................................................................................................................. viii List of Tables .................................................................................................................................. x List of Abbreviations ..................................................................................................................... xi Acknowledgments........................................................................................................................ xiii Chapter 1: Introduction ................................................................................................................... 1 1.1 Acute myeloid leukemia ....................................................................................................... 1 1.1.1 Epidemiology and symptoms of acute myeloid leukemia ............................................. 1 1.1.2 Genetics of AML ........................................................................................................... 2 1.1.3 Classifications and diagnosis of AML ........................................................................... 2 1.1.4 Current therapies for AML patients ............................................................................... 3 1.2 BRD4 and BET inhibitors ..................................................................................................... 6 1.2.1 Basic mechanism of BRD4 functions ............................................................................ 6 1.2.2 Direct BRD4 interaction with transcription factors ....................................................... 8 1.2.3 BET inhibitors .............................................................................................................. 11 1.2.4 Therapeutic targeting of BRD4 with small molecules ................................................. 12 1.3 A uncharacterized NSD family protein NSD3.................................................................... 21 Chapter 2: A Short Isoform of NSD3 Lacking Catalytic Function Is Essential in Acute Myeloid Leukemia....................................................................................................................................... 24 2.1 NSD3 is required for AML cell proliferation ..................................................................... 26 2.2 NSD3 is required for maintaining the undifferentiated state of AML cells ....................... 30 2.3 c-Myc is an essential target gene of NSD3 in AML maintenance ...................................... 30 2.4 NSD3 regulates a similar global gene profile with BRD4 in AML .................................... 33 2.5 A short isoform of NSD3 is essential in AML ................................................................... 35 Chapter 3: NSD3-short Binds Directly to the BRD4 ET Domain ................................................ 39 3.1 NSD3 is an ET-domain associated protein ......................................................................... 39 3.2 NSD3-short 100-263 is a BRD4 interacting domain .......................................................... 41 3.3 NSD3-short 100-263 Binds BRD4 ET-domain directly ..................................................... 44 3.4 Dissociation of NSD3 from BRD4 impairs ET domain functions ..................................... 46 v Chapter 4: NSD3-short Is an Adaptor Protein that Links BRD4 to the CHD8 Chromatin Remodeling Enzyme ..................................................................................................................... 50 4.1 CHD8 is required for AML cell proliferation ..................................................................... 50 4.2 CHD8 is required for maintaining the undifferentiated state of AML cells ....................... 56 4.3 CHD8 regulates a similar global gene profile with NSD3 and BRD4 in AML ................. 58 4.4 NSD3-short bridges BRD4 to the CHD8 chromatin remodeler ......................................... 60 Chapter 5: BRD4 Recruits NSD3 and CHD8 to Super-Enhancer Regions at Oncogene Loci ..... 62 5.1 BRD4, NSD3, and CHD8 colocalize at active promoters and enhancers across the AML genome ...................................................................................................................................... 62 5.2 BRD4 recruits NSD3 and CHD8 to the Myc +1.7 Mb super-enhancer region ................... 66 Chapter 6: NSD3-short Uses Four Distinct Interaction Surfaces to Sustain AML Cell Proliferation .................................................................................................................................. 71 6.1 NSD3-short uses a PWWP reader module to sustain AML cell proliferation .................... 71 6.2 NSD3-short possesses an acidic transactivation domain .................................................... 76 6.3 CRISPR-Cas9 scanning of exons encoding NSD3-short in AML...................................... 78 Chapter 7: Conclusions and Perspectives ..................................................................................... 82 7.1 Summary ............................................................................................................................. 82 7.2 Discussions ......................................................................................................................... 84 7.2.1 BRD4 in AML maintenance ........................................................................................ 84 7.2.2 Functions of the PWWP domain in NSD3-short ......................................................... 86 7.2.3 Functions of NSD3-long .............................................................................................. 87 7.2.4 Interactions between NSD3 and BET family proteins other than BRD4 .................... 87 7.3 Perspectives and future directions ...................................................................................... 88 Chapter 8: Extended Materials and Methods ................................................................................ 91 8.1 Cell culture .......................................................................................................................... 91 8.2 Cell lines and plasmids