Targeting the MAPK signaling pathway has transformed the treatment of metastatic melanoma. CRISPR-Cas9 genetic screens provide a genome-wide approach to uncover novel genetic dependencies that... Show moreTargeting the MAPK signaling pathway has transformed the treatment of metastatic melanoma. CRISPR-Cas9 genetic screens provide a genome-wide approach to uncover novel genetic dependencies that might serve as therapeutic targets. Here, we analyzed recently reported CRISPR-Cas9 screens comparing data from 28 melanoma cell lines and 313 cell lines of other tumor types in order to identify fitness genes related to melanoma. We found an average of 1,494 fitness genes in each melanoma cell line. We identified 33 genes, inactivation of which specifically reduced the fitness of melanoma. This set of tumor type-specific genes includes established melanoma fitness genes as well as many genes that have not previously been associated with melanoma growth. Several genes encode proteins that can be targeted using available inhibitors. We verified that genetic inactivation of DUSP4 and PPP2R2A reduces the proliferation of melanoma cells. DUSP4 encodes an inhibitor of ERK, suggesting that further activation of MAPK signaling activity through its loss is selectively deleterious to melanoma cells. Collectively, these data present a resource of genetic dependencies in melanoma that may be explored as potential therapeutic targets. Show less
The studies in this thesis explored several aspects of genetic dependencies in the development of familial and sporadic melanoma. CDKN2A is the most common high-penetrance susceptibility gene... Show moreThe studies in this thesis explored several aspects of genetic dependencies in the development of familial and sporadic melanoma. CDKN2A is the most common high-penetrance susceptibility gene responsible for up to 40% of melanoma families worldwide. Interestingly, more than half of germline variation in familial predisposition to melanoma remains to be determined. To identify novel high-penetrance melanoma susceptibility genes we applied Whole Exome Sequencing (WES) and co-segregation analysis in a Dutch melanoma family. We identified NEK11 as a candidate high-penetrance melanoma susceptibility gene and performed functional characterization in cancer cell lines to show loss-of-function (chapter 2). Our additional focus of investigation was a specific cohort of familial melanoma patients carrying a CDKN2A founder mutation, a 19-bp deletion known as the p16-Leiden mutation. Due to the variability in occurrence of pancreatic cancer (PC) and melanoma within familial melanoma families, we sought to examine genetic modifiers predicting the risk of PC and melanoma (chapter 3). In this specific cohort of familial melanoma patients, the timing of CDKN2A wild-type allele loss in melanoma development is unknown. We have applied a customized SNP-based digital PCR (dPCR) methodology to precisely quantify CDKN2A allelic imbalance depicting loss-of-heterozygosity (LOH) and attempted to deduce the order of genetic events based on absolute quantification of mutations and losses (CDKN2A LOH, BRAFV600E, TERT promoter, chromosome 9q LOH) (chapter 4). Finally, in addition to high-penetrance genes in familial melanoma, there are genes that are important fitness factors for cancer cell growth and may provide insight into the biology and progression of sporadic melanoma. The application of screening technologies has been successful in identifying genetic dependencies that could possibly be implemented as therapeutic targets in cancer. We have therefore analyzed Clustered Regular Interspaced Short Palindromic Repeats (CRISPR-Cas9) screening data to identify fitness genes in melanoma and used in-vitro systems to validate our findings (chapter 5). Combined, we hope to have uncovered novel genetic dependencies that could be used in the targeted treatment of sporadic as well as familial melanoma. Show less
