Abstract LB-096: A gain-of-function screen identifies drivers of drug resistance in BRAFV600E melanoma cell lines

Abstract

Abstract Melanoma is the deadliest form of skin cancer, and until recently patients had few treatment options upon diagnosis. Fortunately, this lack of treatment options changed with the discovery of vemurafenib, a highly selective kinase inhibitor that specifically targets the BRAFV600E mutant protein, found in ~50% of all melanoma cases. Initially, the kinase inhibitor provided complete or partial response to over 50% of patients and prolonged progression-free survival. However, the majority of patients relapse once tumors acquire resistance to vemurafenib. Some of the genetic resistance mechanisms are known, including BRAF amplification, expression of truncated BRAF, and RAS mutation. However, a significant portion of vemurafenib resistance cases is unexplained. While drug resistance can be delayed by combining vemurafenib with cobimetinib, a MEK inhibitor, mechanisms of resistance to this drug combination are not well characterized. We performed a gain-of-function screen to identify drivers of drug resistance to vemurafenib or vemurafenib combined with cobimetinib. Briefly, A375 melanoma cells were engineered to express the hyperactive transposase SB100. These cells were then subsequently transfected with the mutagenic T2Onc3 transposon to produce a population of mutagenized A375 cells. Three weeks of vemurafenib treatment selected for cells with the acquired ability to grow in the presence of drug. Candidate driver mutations were then identified by profiling the sites of transposon insertion in drug-resistant cells to find genes that were recurrently over-expressed by transposon insertions. This led to the discovery of five genes that act as drivers of vemurafenib resistance when overexpressed in the human melanoma cell line A375. Candidate genes were then validated in multiple melanoma cell lines by assessing cell growth and viability in varying concentrations of vemurafenib. An analogous screen was performed in A375 to identify drivers of resistance to the combination of vemurafenib and cobimetinib. In addition, we are assessing the diversity of drug resistance mechanisms by performing mutagenesis in three additional human melanoma cell lines. The characterization of the novel drug resistance pathways identified here will improve treatment outcomes for patients with BRAFV600E mutant melanoma. Citation Format: Charlotte R. Feddersen, Jacob L. Schillo, Hayley R. Vaughn, Andrew P. Voigt, Eliot Y. Zhu, Lexy S. Wadsworth, Christopher S. Stipp, Adam J. Dupuy. A gain-of-function screen identifies drivers of drug resistance in BRAFV600E melanoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-096.