Identification of pharmacological vulnerabilities in drug-resistant melanoma cells

Most BRAF-mutant melanoma tumors respond to BRAFi/MEKi combination therapy. Despite a strong initial response to these agents, most patients relapse within months or years. The goal of this dissertation is to identify pharmacologically tractable resistance mechanisms, and ultimately to prevent or reverse drug resistance in melanoma. We found that RhoA is activated in approximately half of BRAFi-resistant melanoma cells/tumors and demonstrated that inhibition of RhoA or disruption of actin polymerization resensitizes these cells to vemurafenib. The transcriptional profile of a subset of tumors in the TCGA dataset is similar to that of the BRAFi-resistant cells. Using gene expression-based drug response signatures we predicted that these tumors would be less sensitive to BRAF inhibitors and more sensitive to ROCK inhibitors. This finding is exciting since ROCK is a direct substrate of Rho, and we demonstrated that ROCK inhibition re-sensitizes BRAFi-resistant cells to vemurafenib. Rho-induced F-actin polymerization can modulate the activity of multiple transcriptional coactivators. Two of these transcriptional co-activators, MRTF-A and YAP1, are activated in BRAFi-resistant cells and inhibitors which disrupt these transcriptional processes re-sensitize BRAFi-resistant cells to vemurafenib. In chapter 3 we applied multiple high throughput approaches to identify pharmacological vulnerabilities of BRAFi-resistant melanoma cells. First, we leveraged the LINCS dataset to identify compounds which reverse a drug resistance gene signature. The most promising compound that we identified in this analysis was ibrutinib, which is clinically used as a BTK inhibitor. Interestingly, we found that ibrutinib does not reverse BRAFi resistance through BTK inhibition, but rather through the polypharmacology of the compound. The differentially expressed genes in ibrutinib-treated cells are enriched in YAP1 target genes, which suggests that ibrutinib may be modulating vemurafenib resistance by altering YAP1 activation. Consistent with this hypothesis, treatment with ibrutinib prevents the nuclear accumulation of YAP1. In chapter 4 we sought to identify compounds which selectively killed vemurafenibresistant melanoma cells. To this end we screened a well-annotated drug repurposing library which contains approximately 2,000 FDA-approved drugs, clinical inhibitors, and tool compounds. We found that BRAFi-resistant cells are more sensitive to inhibitors that disrupt mitosis, such as AURKi, PLKi, Chk1/2i, and compounds which disrupt kinesin and tubulin polymerization. The fate of the resistant cells upon drug treatment was nuclear fragmentation and death. But interestingly, a subset of the parental cells did not die upon drug treatment and instead underwent mitotic slippage and exited from mitosis, likely due to dysregulated Cyclin B1 degradation in the parental cells. This finding likely explains why the resistant cells are more sensitive to this class of inhibitors, and it suggests that disruption of mitosis may be a pharmacological vulnerability for melanoma cells/tumors that have developed resistance to BRAFi/MEKi therapy.