Abstract LB-428: RAS-independent dimerization of BRAF(V600E) splicing variants promotes resistance to RAF inhibitors
Authors: Poulikakos Poulikos I. , Persaud Yogindra , Janakiraman Manickam , Kong Xiangju , Ng Charles , Moriceau Gatien , Shi Hubing , Atefi Mohammad , Titz Bjoern , Gabay May Tal , Salton Maayan , Dahlman Kimberly B. , Tadi Madhavi , Wargo Jennifer A. , Flaherty Keith T. , Kelley Mark C. , Misteli Tom , Chapman Paul B. , Sosman Jeffrey A. , Graeber Thomas G. , Ribas Antoni , Lo Roger S. , Rosen Neal , Solit David B. 
Published: August 2012

Abstract
Over 50% of melanomas harbor activating mutations in BRAF, most commonly BRAF(V600E). Profound clinical activity has been observed with RAF inhibitors, including vemurafenib, which is now FDA approved for the treatment of patients with advanced melanomas whose tumors harbor a BRAF(V600E) mutation. RAF inhibitors affect ERK signaling in a mutation-specific manner: they inhibit ERK signaling in cells with BRAF(V600E), but paradoxically activate ERK signaling in cells with wild-type BRAF. We recently elucidated the mechanism of this phenomenon: activation of RAS promotes the dimerization of members of the RAF family. At non-saturating concentrations, binding of an ATP-competitive RAF inhibitor to one member of the dimer inhibits it, while also causing its transition to the activated state. This is associated with the allosteric transactivation of the other, non-drug bound member of the dimer. This leads to an overall increase in RAF specific activity and induction of ERK signaling. In BRAF(V600E) melanomas, RAS-GTP levels are low, BRAF(V600E) is found primarily as a monomer and RAF inhibitors effectively inhibit active BRAF(V600E) monomers. This model of RAF transactivation by RAF inhibitors predicts that any molecular lesion that enhances RAF dimerization will promote resistance to RAF inhibitors. To investigate mechanisms of acquired resistance to RAF inhibitors, we treated a sensitive BRAF(V600E) expressing melanoma cell line (SKME239) with the RAF inhibitor vemurafenib for 8 weeks and selected for resistant clones. We found that a subset of cells resistant to vemurafenib expressed a splicing variant form of BRAF(V600E) that lacked exons 4-8, a region that encompasses the RAS-binding domain. This form of BRAF(V600E) had a size of approximately 61KD (p61BRAF(V600E)). p61BRAF(V600E) exhibited enhanced dimerization as compared to full length BRAF(V600E) in cells with low levels of RAS-GTP. Ectopic expression of p61BRAF(V600E) conferred resistance to the RAF inhibitor. Moreover, a mutation that disrupts dimerization of p61BRAF(V600E) restored its sensitivity to vemurafenib. In tumors from patients that relapsed on vemurafenib we identified various splicing variants of BRAF(V600E), all of them lacking the RAS-binding domain (6/19). Disease progression samples obtained from a mutually exclusive subset (4/19) of the same group of patients harbored activating mutation in NRAS. We report the first RAF-inhibitor resistance mechanism that involves a structural change in BRAF and the first kinase inhibitor resistance mechanism that involves expression of aberrant splicing variants of the drug target. Tumors resistant to RAF inhibitors resulting from increased RAF dimerization retain sensitivity to inhibitors of downstream effectors of RAF such as MEK. Therefore, MEK inhibitors, if used in combination with RAF inhibitors, may delay or prevent the onset of this mechanism of resistance.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-428. doi:1538-7445.AM2012-LB-428