Translational mechanisms of stem cell fate regulation in epidermal oncogene tolerance Yi Cai A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2019 Reading Committee: Slobodan Beronja, Chair Adam Geballe Andrew C. Hsieh Program Authorized to Offer Degree: Molecular and Cellular Biology v © Copyright 2019 Yi Cai v University of Washington Abstract Translational mechanisms of stem cell fate regulation in epidermal oncogene tolerance Yi Cai Chair of the Supervisory Committee: Slobodan Beronja, Associate Member Division of Human Biology, Fred Hutchinson Cancer Research Center Morphologically and functionally normal human skin carries a surprisingly high burden of oncogenic lesions, suggesting that the skin has exceptional capacity to tolerate oncogene hyperactivity. To date, we have made significant progress in understanding how homeostatic adult epidermis suppresses the expansion of individual clones derived from somatic mutations. However, the mechanisms behind oncogene tolerance during epidermal development, a time of significant physiological tissue expansion, has not been explored. This is exemplified in RASopathies, where patients rarely develop epidermal defects or cancers despite germline mutations in the RAS/MAPK signaling pathway. Here, we find that oncogenic RAS-induced progenitor cell hyperproliferation is coordinated with differentiation to restrain aberrant growth and preserve epidermal development. We uncover a novel translation mechanism mediated by initiation factor eIF2B5 that co-regulates RAS proliferation and differentiation, resulting in hyperplastic but controlled growth. Using in vivo ribosome profiling, we reveal that eIF2B5 v selectively regulates the translation of a substantial subset of the oncogenic RAS translatome. Furthermore, by coupling ribosome profiling with genetic screening, we provide direct functional evidence that RAS-induced differentiation is driven by eIF2B5-mediated translation of ubiquitination genes. We reveal ubiquitin ligase FBXO32 as a promoter of epidermal differentiation with no effect on proliferation, thus restraining RAS-driven pathological growth and tumorigenesis. Our study challenges the accepted view that oncogenic translation is inherently tumor promoting and unveils how it directly steers cell fate to mediate epidermal oncogene tolerance. v Acknowledgements I would like to thank my doctoral advisor, Slobodan Beronja, for giving me the freedom to grow as a scientist. His encouragement, support, and enthusiasm expanded my critical thinking and curiosity. Thanks to the past and present members of the Beronja lab--Madeline Sandoval, Zhe Ying, Megan Kufeld, Kamya Rajaram, Julie Rytlewski, Na Ge, for making the lab a warm and intellectually stimulating community. I would also like to thank Andrew Hsieh and members of the Hsieh lab, especially Sonali Arora, Yiting Lim, Yuzhen Liu, and Alexandre Germanos, for being wonderful collaborators and sharing their expertise on all things translational. I would like to thank the rest of my thesis committee--Adam Geballe, Julie Overbaugh, and Hannele Ruohola- Baker--for insightful suggestions, constructive criticisms, and guidance. Finally, I thank my family and friends for their generous and unwavering encouragement. v Table of Contents List of Figures................................................................................................................................viii List of Tables....................................................................................................................................x Chapter 1 – Introduction.................................................................................................................1 1.1 Stem cell dynamics in epidermal growth………………………………………………………………..…1 1.2 Epidermal tolerance to oncogene activation……………………………….………………….………..4 1.3 Regulation of epidermal progenitor cell behavior………………………………………………………7 Chapter 2 – Materials and Methods………………………………………………………………………………………....12 Chapter 3 – Oncogenic RAS activation alters stem cell dynamics to preserve epidermal development………..…....................................................................................................................27 3.1 HrasG12V activation induces epidermal overgrowth ....................................................27 3.2 HrasG12V activation alters stem cell dynamics to limit epidermal overgrowth ............28 Summary...........................................................................................................................31 Chapter 4 -- eIF2B5 coordinates progenitor cell behaviors to mediate oncogene tolerance……….37 4.1 eIF2B5 mediates HrasG12V-induced translation upregulation......................................37 4.2 HrasG12V progenitor cell behaviors and tissue growth are dependent on eIF2B5......41 4.3 eIF2B5 mediates the translation of a specific subset of oncogenic mRNAs .................42 Summary…........................................................................................................................45 Chapter 5 – eIF2B5-regulated FBXO32 drives HrasG12V differentiation to restrain tumorigenesis…….63 5.1 -- Oncogenic RAS-induced differentiation is driven by eIF2B5-regulated ubiquitination...63 vi 5.2 eIF2B5-regulated FBXO32 drives RAS-induced differentiation ...................................65 5.3 FBXO32 drives differentiation to restrain RAS tumor initiation and growth................67 5.4 Concurrent manipulation of progenitor cell behaviors downstream of eIF2B5..........68 Summary………………………………………………………………………………………………………………………70 Chapter 6 – Discussion……………………………………............................................................................87 6.1. Coordinated progenitor cell behaviors mediate RAS oncogene tolerance..................87 6.2 eIF2B5 coordinates RAS progenitor cell behaviors to regulate oncogenic growth......89 6.3 eIF2B5-driven differentiation restrains RAS tumorigenesis…………………………………..…91 6.4 Translation dysregulation as a therapeutic target……………………………………………………93 References ...................................................................................................................................96 Appendix A – Translation screen to identify essential oncogenic growth genes……………………....108 Appendix B – In vivo ribosome profiling results……………………………………………………………………….116 vii Figures 1.1 Epidermal stem cell models...................................................................................................11 3.1 Oncogene activation can be induced in mouse epidermis………………......................................32 3.2 HrasG12V induces epidermal expansion……………………………………………………………....................34 3.3 HrasG12V induces compensatory progenitor cell differentiation…………………..........................35 4.1 HrasG12V progenitor cell behaviors are regulated by the translation machinery......................46 4.2 HrasG12V translation upregulation is mediated by eIF2B5........................................................48 4.3 Cultured primary keratinocytes phenocopy in vivo epidermal basal cells...............................50 4.4 HrasG12V regulation of eIF2B5..................................................................................................51 4.5 HrasG12V progenitor cell behaviors and tissue growth are dependent on eIF2B5....................52 4.6 In vivo basal cell ribosome profiling........................................................................................54 4.7 In vivo ribosome profiling generates high quality, reproducible libraries................................55 4.8 eIF2B5 regulates translation of a specific subset of oncogenic mRNAs...................................56 4.9 HrasG12V and eIF2B5 mRNA specific translation is associated with a functional guanine-rich cis-regulatory element..................................................................................................................58 5.1 eIF2B5 regulates oncogenic translation of distinct gene sets that uniquely promote proliferation or differentiation……………………………………………………………………………………………......71 5.2 FBXO32 is translationally regulated by eIF2B5 .......................................................................73 5.3 FBXO32 drives RAS-induced differentiation to suppress clonal expansion…………………………..74 5.4 FBXO32 ubiquitin ligase function drives RAS-induced differentiation to restrain clonal expansion......................................................................................................................................76 5.5 FBXO32 is necessary and sufficient to restrain RAS tumor initaiton and growth…………………..78 viii 5.6 HrasG12V growth inhibition through simultaneous targeting of proliferation and differentiation mechanisms…………………………………………………………………………………………………………………………….80 6.1 Model of translational regulation in epidermal oncogene tolerance……………………………………95 ix Tables Table 2.1 shRNA and primer sequences used in this study............................................................26 Table 4.1 Regulators of HrasG12V progenitor proliferation within translation machinery..............59 Table 4.2 Regulators of HrasG12V progenitor renewal within translation machinery.....................62 Table 5.1 Regulators of HrasG12V progenitor proliferation within eIF2B5-regulated oncogenic translatome..................................................................................................................................82 Table 5.2