Abstract 5769: A novel, high-throughput approach to discovering drug-resistance genes identifies CRAF, CrkL, and TPL2/COT as drivers of resistance to BRAFV600E inhibition

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Activating mutations in the BRAF kinase are found in 50-70% of melanomas and have nominated BRAF, along with downstream components of the MAP kinase pathway, as therapeutic targets. Until recently, MAPK pathway inhibitors have shown poor efficacy in clinical trials, leading many to doubt the therapeutic potential of BRAF/MAPK inhibition. However, the recent clinical success of the BRAFV600E-specific kinase inhibitor PLX4032 has validated MAPK pathway inhibition as a viable treatment strategy. Despite initial success, most targeted therapeutics will eventually succumb to acquired resistance. It is therefore imperative that we identify drivers of targeted drug resistance in a rapid, systematic manner that identifies parallel drug targets, ultimately facilitating an effective long-term treatment strategy. Accordingly, we report here a novel, high-throughput approach to systematically identify kinases capable of driving resistance to clinically efficacious, BRAFV600E-specific kinase inhibitors. We screened an open reading frame (ORF) expression library consisting of ∼600 kinases and kinase-related ORFs for their ability to drive resistance to PLX4720 in BRAFV600E-mutant cell lines. Parallel analytical approaches and subsequent validation identified RAF1 (CRAF), CRKL (CrkL) and MAP3K8 (TPL2/COT) as the strongest candidate resistance genes. CRAF and TPL2/COT are both MAP3 kinases that directly phosphorylate and activate MEK, whereas CrkL is an adapter protein that is amplified and phosphorylated in melanoma. CRAF, CrkL and TPL2/COT expressing cells proliferate and show constitutive MAPK pathway activation at concentrations of PLX4720 that are 10-fold over IG50. While CRAF-expressing cells show a dose-dependent increase in MAPK activation when treated with PLX4720, MAPK pathway activation is weakly dampened in CrkL and TPL2/COT-expressing cells upon PLX4720 treatment, suggesting that CrkL and TPL2/COT drive resistance largely, but not exclusively, through MAPK pathway activation. Accordingly, CRAF, CrkL and TPL2/COT segregate into two categories when queried for their ability to drive resistance to a panel of MAPK pathway inhibitors: CRAF mediates resistance only to BRAFV600E-specific inhibitors, while CrkL and TPL2/COT drive resistance to BRAFV600E-specific inhibitors and modify sensitivity to allosteric inhibitors of MEK kinases. Combinatorial treatment with BRAFV600E inhibitors and MEK inhibitors effectively inhibits the proliferation of cells expressing CRAF, CrkL and TPL2/COT, suggesting that combinatorial strategies may address acquired resistance in the clinic. These data highlight the utility and broad applicability of rapid, ORF-based methods of uncovering drivers of targeted resistance and identify CRAF, CrkL and TPL2/COT as putative resistance alleles to BRAFV600E-specific inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5769.