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The Function of the ASH1L Histone Methyltransferase in Cancer: a Chemical Biology Approach

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The Function of the ASH1L Histone Methyltransferase in Cancer: a Chemical Biology Approach The function of the ASH1L histone methyltransferase in cancer: A chemical biology approach by David Rogawski A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular and Cellular Pathology) in the University of Michigan 2016 Doctoral committee: Assistant Professor Jolanta E. Grembecka, Chair Assistant Professor Tomasz Cierpicki Associate Professor Yali Dou Associate Professor Ivan P. Maillard Associate Professor Ray C. Trievel When I heard the learn’d astronomer, When the proofs, the figures, were ranged in columns before me, When I was shown the charts and diagrams, to add, divide, and measure them, When I sitting heard the astronomer where he lectured with much applause in the lecture-room, How soon unaccountable I became tired and sick, Till rising and gliding out I wander’d off by myself, In the mystical moist night-air, and from time to time, Look’d up in perfect silence at the stars. —Walt Whitman Leaves of Grass For Maria ii Acknowledgements I would like to begin by thanking my high school and college teachers, who inspired in me a love for science, language, and art. A few teachers deserve special recognition: from Hammond High School (Columbia, MD), Bob Jenkins (World History), Hope Vasholz (English), and Stan Arnold (Calculus); and from Williams College, Tom Garrity (Math), Daniel Lynch (Biology), and the late Bill DeWitt (Biology). My honors thesis mentor Lois Banta at Williams College and my Masters thesis mentor Sigurd Wilbanks at the University of Otago (N.Z.) were essential role models for me as I embarked on a scientific career. I’ve been fortunate to work with many great collaborators in the Michigan Pathology Department. Ivan Maillard and Jennifer Chase provided the ΔSET mice, which were originally acquired from Gang Huang, Cincinnati Children’s Hospital, and Kenichi Nishioka, Saga University. Lili Chen and Andy Muntean provided the CALM-AF10 mouse bone marrow cells with doxycycline-inducible shRNA targeting Ash1l. Bo Zhou and Yali Dou helped with recombinant nucleosome reconstitution. Next I would like to thank Jola Grembecka and Tomek Cierpicki for creating a highly interdisciplinary lab environment where I could learn chemistry, biochemistry, and biology in one PhD thesis. Their lab is truly one-of-a-kind, and I am very fortunate to have had the opportunity to work there. Juliano Ndoj, who began as my undergraduate assistant, then became lab technician, and is now an important source of expertise in the lab, was instrumental to the success of this project. I thank fellow graduate students George Lund, Felicia Gray, and Jon Pollock, with whom I shared the ups and downs of lab research. I gained much from Hyoje iii Cho’s expertise in crystallography, Trupta Purohit patiently taught me cell culture, and Hongzhi Miao introduced me to flow cytometry. I was fortunate to work with several highly skilled synthetic chemists, without whom this project would not be possible, including Dmitry Borkin, Yu-Hsuan Kuo, Deanna Montgomery, John Zhuang, and Szymon Klossowski. Patricia Ernst at the University of Colorado Denver was a superb sabbatical mentor during the summer of 2015. Thank you to my thesis committee for their suggestions and advice over the years. I would like to thank my family and friends who have and continue to support me during my MD/PhD journey. Thanks to my friends in grad school, the MSTP, and the med school, with whom I’ve shared fun times and exciting adventures. I owe a colossal thanks to my mom Carol, step-dad Jim, dad Michael, and step-mom Julie for their love and support and all the opportunities they have given me. My sister Liz and brother Alex are simply the best siblings ever. Finally, thank you to my love and partner, Mary. You mean more to me than is appropriate to put on a PhD thesis acknowledgements page. Suffice to say that my PhD years will always feel special for having met you. Thank you for your support and all that you’ve done for me. iv Table of Contents Dedication ....................................................................................................................................... ii Acknowledgements ........................................................................................................................ iii List of Figures .............................................................................................................................. viii List of Tables ................................................................................................................................. ix Abstract ............................................................................................................................................x Chapter 1. H3K36 methyltransferases as cancer drug targets: rationale and perspectives for inhibitor development ......................................................................................................................1 Abstract ......................................................................................................................................1 Introduction ................................................................................................................................2 H3K36 methylation regulates diverse processes implicated in cancer ......................................4 Transcriptional regulation ....................................................................................................4 Alternative splicing ..............................................................................................................5 DNA repair...........................................................................................................................6 The reciprocal relationship between H3K36 and H3K27 methylation ................................7 H3K36 KMTases play important and varying roles in carcinogenesis .....................................8 NSD1: Activator of HOX genes in leukemia .....................................................................10 NSD2 (MMSET/WHSC1): An oncogenic driver in multiple myeloma ............................11 NSD3: Frequently overexpressed in diverse tumor types ..................................................13 ASH1L: HOX gene activator with emerging role in cancer ..............................................14 SETD2: An important tumor suppressor ...........................................................................15 Other H3K36 methyltransferases: SMYD2, SETMAR, and SETD3 ................................16 H3K36-specific SET domains: structural considerations for inhibitor development ..............17 Structural studies of NSD, ASH1L, and SETD2 SET domains demonstrate feasibility of inhibitor design ..............................................................................................................18 SMYD2 structures: U-shaped substrate binding................................................................21 Inhibitors of H3K36 methyltransferases ..................................................................................22 Challenges and opportunities in developing specific inhibitors of H3K36 KMTases ............24 Targeting the catalytic SET domain ..................................................................................24 Alternative druggable regions of H3K36 KMTase proteins ..............................................26 Conclusions and future perspectives ..................................................................................29 Chapter 2. Two loops undergoing concerted dynamics regulate activity of the ASH1L histone methyltransferase ...........................................................................................................................31 Abstract ....................................................................................................................................31 v Introduction ..............................................................................................................................32 Results ......................................................................................................................................34 Crystal structure of the ASH1L SET domain shows increased dynamics of the autoinhibitory and SET-I loops ..........................................................................................34 NMR studies reveal that the ASH1L active site is surrounded by two loops experiencing conformational dynamics .............................................................................36 ASH1L requires chromatin-interacting domains for robust enzymatic activity ................38 ASH1L SET-BAH requires native nucleosome substrate for optimal enzymatic activity................................................................................................................................41 Mutations of non-conserved residues in autoinhibitory loop have significant effects on ASH1L SET domain activity ........................................................................................41 NMR studies correlate the degree of structural perturbation to enzyme activity ..............43 Mutations affect structure and mobility of the ASH1L autoinhibitory loop .....................46 Structural insight into SET-I loop conformational dynamics ............................................49 Conformational exchange in the SET-I loop contributes to ASH1L activity ....................50 Discussion ................................................................................................................................53
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