Genomic and Functional Studies of Uveal Melanoma The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Place, Chelsea Schwartz. 2015. Genomic and Functional Studies of Uveal Melanoma. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:17464154 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Genomic and Functional Studies of Uveal Melanoma A dissertation presented by Chelsea Schwartz Place to The Division of Medical Sciences In partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biological Chemistry and Molecular Pharmacology Harvard University Cambridge, Massachusetts April 2015 © 2015 Chelsea Schwartz Place All rights reserved. Dissertation Advisor: Dr. Levi A. Garraway Chelsea Schwartz Place Genomic and Functional Studies of Uveal Melanoma Abstract Uveal melanoma (UM) is a rare form of melanoma that is lethal once metastatic. Primary tumors in the iris, ciliary body, and choroid of the eye metastasize in 50% of patients, despite effective treatment of the initial tumor. The majority of uveal melanomas harbor activating mutations in GNAQ or GNA11, which relay signaling to downstream effectors including protein kinase C (PKC) and the mitogen-activated protein kinase (MAPK) signaling pathway (RAF-MEK-ERK). Both PKC and MEK inhibitors are currently in clinical trials, however, MEK inhibition alone is insufficient to improve overall survival. These observations highlight a need to identify new drug targets for the design of novel therapies including combinations. To uncover novel UM biology and nominate strategies for combination therapy, genomic and functional genomic approaches were applied. Whole exome sequencing of primary and metastatic tumors identified somatic genetic alterations that drive tumorigenesis. Recurrent mutations in GNAQ, GNA11, BAP1, SF3B1, and EIF1AX were confirmed. Mutations in potential drivers of metastasis, SMARCA4 and IQGAP1, were also identified. Furthermore, the function of N-terminal tail mutations in the translation initiation factor EIF1AX was probed using loss of function studies to assess both viability and mRNA regulation at the level of translation. Upon EIF1AX knockdown, the efficiency of ribosomal protein translation was reduced in wild type, but not mutant cells. Deregulated translation may play an important role in UM tumorigenesis. To identify putative co-targets of MEK and PKC inhibitors, genome-scale RNA interference drug enhancer screens were performed. These screens nominated several novel iii genes and pathways for further study in UM. In particular, the mitochondrial folate pathway enzyme MTHFD2 was identified as a novel PKC inhibitor sensitizer. The strongest MEK inhibitor enhancer was the MAPK pathway member, BRAF. Indeed, targeting multiple nodes of the MAPK signaling pathway achieved stronger pathway suppression and synergistic effects. Co-inhibition of RAF/MEK or MEK/ERK may warrant clinical investigation in patients. Overall, these studies provide a foundation for our understanding of UM genomics and combination therapy opportunities. Several novel avenues for future study of UM biology and co- dependencies are uncovered. Translation of these findings into clinical studies will be of the utmost importance. iv TABLE OF CONTENTS Chapter 1. Introduction .............................................................................................................. 1 Clinical features of uveal melanoma ............................................................................. 2 Epidemiology ............................................................................................................... 2 Risk factors ................................................................................................................. 2 Diagnosis and standard of care .................................................................................. 3 Uveal melanoma biology ............................................................................................. 4 Prognostics ................................................................................................................. 4 The genetics of uveal melanoma ................................................................................... 5 Copy number alterations ............................................................................................. 5 Somatic and germline point mutations ........................................................................ 6 Prognostic value of genetic features ......................................................................... 10 Treatment strategies ..................................................................................................... 10 Targeting GNAQ/11-mutant tumors .......................................................................... 11 Other targeted therapy approaches .......................................................................... 14 Immunotherapy ......................................................................................................... 15 Precision medicine for uveal melanoma .................................................................... 17 Limitations to existing therapeutic strategies ............................................................ 17 Rationale for this work ............................................................................................... 18 Chapter 2. Systematic genomic characterization of primary and metastatic uveal melanoma ............................................................................................................................... 20 Attributions .................................................................................................................... 21 Abstract ......................................................................................................................... 22 Introduction ................................................................................................................... 22 Results ........................................................................................................................... 23 Discussion ..................................................................................................................... 56 Materials and Methods ................................................................................................. 58 Chapter 3. Systematic identification of targets for co-inhibition with MEK and protein kinase C inhibitors in uveal melanoma ................................................................................ 66 Attributions .................................................................................................................... 67 Abstract ......................................................................................................................... 68 Introduction ................................................................................................................... 68 v Results ........................................................................................................................... 70 Discussion ................................................................................................................... 101 Materials and Methods ............................................................................................... 104 Chapter 4. Conclusion and future directions ....................................................................... 109 Summary ...................................................................................................................... 110 Limitations of this study ........................................................................................... 111 Future directions for UM studies ............................................................................... 113 Genome characterization ........................................................................................ 113 Targeted therapy ..................................................................................................... 114 Mechanisms of resistance ....................................................................................... 117 Concluding Remarks .................................................................................................. 118 References .............................................................................................................................. 120 vi List of Figures Figure 1.1. Signaling downstream of mutated GNAQ or GNA11 (αQ/11) in UM ............................. 8 Figure 2.1. Somatic mutations in primary and metastatic uveal melanoma ............................... 24 Figure 2.2. Copy number profiles of 52 uveal melanomas ......................................................... 31 Figure 2.3. Trio 2 clinical timeline ............................................................................................... 33 Figure 2.4. EIF1AX-regulated growth and translation in uveal melanoma ................................. 36 Figure 2.5. EIF1AX sequence and protein expression across UM cell lines .............................