SCREENING FOR EPIGENETIC INHIBITORS OF OSTEOSARCOMA METASTASIS by IAN BAYLES Submitted in partial fulfillment of requirements for the degree of Doctor of Philosophy Department of Genetics and Genome Sciences CASE WESTERN RESERVE UNIVERSITY May 2020 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Ian Bayles Candidate for the degree of Doctor of Philosophy* Committee chair Ahmad Khalil Committee Member Peter Scacheri Committee Member Edward Greenfield Committee Member Fulai Jin Date of Defense November 22nd, 2019 *We also certify that written approval has been obtained for any proprietary material contained therein. 2 Table of Contents AcKnowledgements .................................................................................................... 7 Abstract ........................................................................................................................ 9 Chapter 1: BacKground and Significance ............................................................ 11 Osteosarcoma................................................................................................................... 11 Osteosarcoma epidemiology ........................................................................................ 11 Classification .................................................................................................................. 12 Localization ..................................................................................................................... 13 Genetic Heterogeneity/Pathogenesis ........................................................................... 13 Therapeutic Strategies for Osteosarcoma ................................................................... 15 Cancer Metastasis............................................................................................................ 17 Genetic evolution of metastasis .................................................................................... 18 Epigenetic contribution to metastasis ........................................................................... 19 Epigenetic targeting compounds in cancer treatment ............................................. 21 Epigenetic Writers .......................................................................................................... 22 Epigenetic Erasers ......................................................................................................... 24 Epigenetic Readers........................................................................................................ 25 CDKs and their Inhibitors .............................................................................................. 26 Chapter 2. Ex vivo screen identifies CDK12 as a metastatic vulnerability in osteosarcoma ............................................................................................................ 29 Abstract.............................................................................................................................. 30 Introduction ....................................................................................................................... 31 Results ............................................................................................................................... 33 Ex vivo screening identifies CDK inhibitors as potent suppressors of metastatic OS outgrowth ........................................................................................................................ 33 CDK12 inhibitors reduce metastatic cell outgrowth in the lung microenvironment .. 35 CDK12 knockout reduces lung metastatic outgrowth in vivo ..................................... 36 CDK12 inhibition is broadly effective against multiple human OS cell lines in vitro . 37 E9 impairs transcription elongation in OS cells ........................................................... 39 CDK12 inhibition affects transcription elongation and RNA processing in OS cells 42 3 MYC levels correlate with sensitivity to E9 ex vivo. .................................................... 43 Discussion ......................................................................................................................... 44 Methods ............................................................................................................................. 48 Chapter 3: Discussion and Future Directions .................................................. 104 Summary .......................................................................................................................... 104 The effects of the tumor microenvironment in compound screening ................. 107 Using the PuMA technique to study lung metastasis in other cancers .............. 109 CDK Inhibitors in Osteosarcoma and Other Cancers ............................................. 109 CDK12 inhibition as a therapeutic option in osteosarcoma .................................. 112 Combining CDK12 inhibitors with other therapeutic options in cancer ............. 113 Effects of TP53 mutational status on CDK12 inhibition ......................................... 117 Efficacy of CDK12 inhibitors in vivo........................................................................... 118 Effects of CDK12 inhibition in osteosarcoma transcription. ................................. 119 Implications of CDK12 inhibition from CDK12 loss in other cancers .................. 121 Bibliography ............................................................................................................ 125 4 List of Tables Supplemental Table 2-1: Compounds used in Screens…………………………85 Supplemental Table 2-2: Top 100 GSEA terms………………………………….94 5 List of Figures Figure 1-1: Overview of epigenetic inhibitors in cancer treatment. ....................23 Figure 2-1: An ex vivo screen identifies compounds that inhibit the growth of metastatic OS......................................................................................................66 Supplemental Figure 2-1: Parallel in vitro screen data .....................................67 Figure 2-2: CDK inhibitors reduce metastatic cell outgrowth in the lung microenvironment ................................................................................................68 Supplemental Figure 2-2.1: CDK expression across tumor samples ................70 Supplemental Figure 2-2.2: Protein expression of CDK12 after CRISPR knockout ..............................................................................................................71 Figure 2-3: THZ531 and E9 show broad activity against OS cell line models. ...72 Supplemental Figure 2-3: Cell line data after CDK12 inhibitor treatment .........74 Figure 2-4: E9 treatment impairs transcription elongation in OS cells. ...............76 Supplemental Figure 2-4: E9 ChIP-seq in MG63.3 Cells ..................................78 Figure 2-5: E9 affects transcript levels in a gene-length and expression- dependent manner. .............................................................................................79 Supplemental Figure 2-5: E9 impairs transcription in MG63.3 cells ..................81 Figure 2-6: High levels of MYC correlate with insensitivity of OS cells to E9 treatment ex vivo .................................................................................................84 Figure 3-1: Bliss Synergy Score of THZ531 and Olaparib ...Error! BooKmarK not defined. 6 AcKnowledgements I want to start off by thanking Dr. Peter Scacheri, my Ph.D. thesis advisor and mentor. His guidance and wisdom over the last five and half years has helped mold me into the scientist I am today. He reminded me that while science can be tedious and frustrating as experiments inevitably fail, as long as you keep the end goal in mind and celebrate your successes, it will all be worth it in the end. His excitement over every result of data (“This is AWESOME!”) helped drive me to put my all into my work and I couldn’t be more thankful for his support and guidance. I want to thank my wife and partner, the future-doctor Kathleen Plona for all of her help and support and for dealing with me squirreled away in my office staring at my computer screen for nights on end. I truly appreciate her support and cannot wait to begin our future together as scientists. I want to thank you my lab mates for their help and support, especially Zach and Cindy. They are the foundations of the current Scacheri lab and I hope Pete realizes how incredible they are to the graduate students in the lab. I want to thank my committee members, Dr. Ahmad Khalil, Dr. Edward Greenfield, and Dr. Fulai Jin for their help and input over the years, and for helping to guide this thesis to where it is today. 7 I want to thank my family, my mother, father, and sister, for their help and inspiration during this process. I lastly want to thank my fellow BSTP classmates and whiskey drinking partners newly minted doctors, Tristan de Jesus and Douglas Oswald. Thank you for helping to keep me grounded and sane during our respective PhDs and for sharing your science with me. 8 Screening for Epigenetic Inhibitors of Osteosarcoma Metastasis by IAN BAYLES Abstract Osteosarcoma is a the most common cancer of the bone, known for its notoriously complex and heterogeneous genotypes and high propensity to metastasize to the lung. When distal metastatic lesions present in patients, mortality rates increase significantly. Indeed, this is the case