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Targeted Degradation of the Myc Oncogene Using Pp2a

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Targeted Degradation of the Myc Oncogene Using Pp2a TARGETED DEGRADATION OF THE MYC ONCOGENE USING PP2A- B56ALPHA SELECTIVE SMALL MOLECULE MODULATORS OF PROTEIN PHOSPHATASE 2A AS A THERAPEUTIC STRATEGY FOR TREATING MYC- DRIVEN CANCERS BY CAROLINE C. FARRINGTON Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor: Dr. Goutham Narla, M.D., Ph.D. Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY May 2020 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Caroline Farrington candidate for the degree of Pharmacology * Committee Chair Dr. Ruth Keri, Ph.D Committee Member and Thesis Advisor Dr. Goutham Narla, M.D., Ph.D Committee Member Dr. Amar Desai Ph.D Committee Member Dr. Marvin Nieman Committee Member Dr. David Wald M.D., Ph.D Date of Defense January 17, 2020 *We also certify that written approval has been obtained for any proprietary material contained therein. Dedication “To laugh often and much; to win the respect of intelligent people and the affection of children; to earn the appreciation of honest critics and endure the betrayal of false friends; to appreciate beauty; to find the beauty in others; to leave the world a bit better whether by a healthy child, a garden patch, or a redeemed social condition; to know that one life has breathed easier because you lived here. This is to have succeeded.” - Ralph Waldo Emerson This work is primarily dedicated to my mother who raised me with the somewhat disillusioned mother’s belief that I have more talent in my pinky than others have in their whole body and, more importantly, that I have a lot to give back to others and share with the world. It is in her memory that I found my way to biomedical research and she is smiling ear to ear watching my success wherever she is now. This work is also dedicated to my father, who persistently taught me that said talent was useless without hard work. Lastly, this work is dedicated to my husband Angus whose love has buoyed me in a wild sea. Table of Contents Table of Contents List of Tables 5 List of Figures 6 Acknowledgments 8 Abstract 10 Chapter 1: Introduction and Background 12 1.1 Introduction to Phosphatases 13 1.2 Structure and stoichiometry of the PP2A Holoenzyme 13 1.2.1 Structural contributions to PP2A activity 14 1.2.2 Subunit stoichiometry and activity 16 1.3 Dysregulation of PP2A in human disease 16 1.4 Mechanisms of PP2A inactivation in cancer 18 1.4.1 PP2A subunits and cancer: Mutation, deletion, inactivation, 19 and aberrant expression 1.4.2 Post-translational modifications critical to PP2A activity 24 1.4.3. Endogenous inhibitors of PP2A 26 1.4.4 Exogenous inhibitors of PP2A 28 1.5 Approaches to activate PP2A in cancer 29 1.5.1 Inhibition of PP2A inhibitors 28 1.5.2 Promethylating agents 31 1.5.3 PME-1 inhibitors 31 1.5.4 Agents with undefined mechanism of action 32 2 1.5.5 Small Molecule Activators of PP2A 32 1.6 The oncogene MYC 35 1.6.1 MYC background 33 1.6.2 Regulation of the MYC gene 36 1.6.3 Structure and regulation of MYC protein 37 1.6.4 Approaches to target MYC at the transcriptional level 39 1.6.5 Inhibition of MYC protein and activity 40 1.7 Statement of Purpose 43 Chapter 2: Small molecule activation of PP2A for the treatment of MYC driven cancers 55 2.1 Abstract 56 2.2 Introduction 56 2.3 Results 60 2.3.1 SMAPs inhibit tumor growth in c-MYC driven Burkitt’s Lymphoma 60 2.3.2 SMAPs decrease tumor burden and c-MYC expression in a KRAS model of Non-Small Cell Lung Cancer 61 2.3.3 SMAPs inhibit tumor growth in c-MYC expressing xenograft models of TNBC 63 2.3.4 SMAP treatment results in proteasome mediated MYC degradation 65 2.3.5 SMAPs inhibit the transcription of c-MYC target genes 67 2.4 Discussion 69 2.5 Experimental Procedures 71 2.6 Acknowledgements 77 Chapter 3: Summary of Discoveries and Future Directions 98 3.1 Summary 99 3 3.2 SMAPs for the management of N-MYC and L-MYC driven cancer 99 3.3 SMAPs, MYC and CIP2A 103 3.4 Understanding vulnerabilities in PP2A inactivated/MYC driven cancers 105 3.5 Status of the PP2A- B56a Holoenzyme 107 3.6 Understanding mechanisms of resistance to SMAPs in MYC driven cancers 107 References 111 4 List of Tables Table 1.1 PP2A subunit alterations in cancer 48 Table 2.1: c-MYC target genes used to assess c-MYC transcriptional activity and corresponding primer sequences for qRT-PCR 89 Table 2.2. Changes to c-MYC target genes in the Daudi cell line 90 Table 2.3 Changes to c-MYC target genes in the MDA-MB-231 cell line 91 5 List of Figures Figure 1.1 Organization of the PP2A Holoenzyme 45 Figure 1.2 Structures of the PP2A Core Enzyme and Holoenzyme 46 Figure 1.3 PP2A mutations in cancer 47 Figure 1.4 Post Translational Modifications of PP2A 49 Figure 1.5 Structure of PP2A in complex with PME-1 and LCMT 50 Figure 1.6 Approaches to activate PP2A 51 Figure 1.7 Mechanisms of c-MYC regulation exploited in cancer and approaches to target its expression and activity 52 Figure 1.8 Regulation of MYC protein 54 Figure 2.1 Structure of SMAP 1 and 2 79 Figure 2.2 SMAPs inhibit tumor growth and decrease c-MYC expression in a model of Burkitt’s Lymphoma. 80 Figure 2.3 Figure 2.4 SMAPs inhibit tumor growth and decrease c-MYC expression in KRAS driven NSCLC 81 Figure 2.4 SMAP treatment increases TUNEL staining and decrease c-MYC protein expression in NSCLC mouse models upon treatment with SMAPs 83 Figure 2.5 SMAPs inhibit tumor growth in models of triple negative breast cancer 84 Figure 2.6 SMAPs decrease c-MYC expression through a proteasome mediated mechanism and induce changes to c-MYC target genes 86 Figure 2.7 SMAPs do not induce changes to c-MYC mRNA in 6 Burkitts or breast cancer cell lines and inhibit c-MYC protein in breast cancer cell 88 Figure 2.8 Confirmation of MYC overexpression in Daudi cell line 92 Figure 2.9. SMAP inhibition of tumor growth and changes to c-MYC expression is abrogated by mutation to c-MYC phosphodegron 93 Figure 2.10 SMAP inhibition of tumor growth and changes to c-MYC expression is abrogated by mutation to c-MYC phosphodegron 95 Figure 2.11 Confirmation of c-MYC band and overexpression in Daudi tumor lysates 97 7 Acknowledgements 10 years ago I was a year into a post- baccalaureate pre-medical program, inspired to study medicine after taking care of my mom when she was sick and the interactions I had with her team of doctors. Yet, despite knowing I was on the right path, I was not sure a path through medical school was the way for me to find my way in this field. Enter Dr. Goutham Narla. Literally. He entered the restaurant where I was working while supporting myself in school and our encounter lead to an interview which lead to a summer internship and eventually a full time research assistant position in his lab at Mount Sinai Hospital in New York City. At the time I started in his lab I had barely finished a college level biology course, but I was hooked within a few weeks. A few years later I was applying to PhD programs. I interviewed at a handful of programs, well aware of the dogma to branch out and do my PhD with someone else, but all those experience did was tell me to trust my gut and do my PhD with Dr. Narla. Dr. Narla broadened my horizons and showed me what a career in biomedical research looked like. He taught me how to think critically, to think big picture while focusing on the small details, and perhaps most importantly,he demonstrated each day a passion for his work that I hope to embody in my career- essentially to never lose sight of why we do what we do. Thank you Goutham. I’d also like to thank my thesis committee for being supportive, providing insight and constructive criticism and teaching me how to receive feedback and think more deeply. Every meeting helped me become a better scientist and my work is better for it. I’d also like to thank both Goutham and my thesis committee, Dr. Keri, Dr. Nieman, and Dr. Wald and the pharmacology department for their patience and support. 8 My cancer diagnosis mid PhD truly disrupted my progress and my morale. In the months after chemo I was ready to give up multiple times on finishing my degree. But it was the support of these four that pushed me to persevere and it was their patience and understanding that allowed that to happen as I creeped into my 7th year of my PhD. I have lived away from home since I was 16. As a result, I have developed a rich group of friends that have become my chosen family. Francesca, Amanda, Caite, Regina, Tracey, Erin and Katie to name a few- strong women with incredible careers that have inspired and motivated me and didn’t think I was nuts when I decided to go back to school in my thirties. Your support got me here and helped me to the end.
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