Abstract 3706: Oncogenic RAF mutants that signal as functional dimers are resistant to current RAF inhibitors but sensitive to a novel inhibitor of RAF dimer kinase activity

Abstract

Abstract Dysregulation of ERK signaling is a common event in malignancy. Receptor activation of RAS leads to dimerization and activation of RAF kinase, which in turn activates ERK. Physiologic activation of the pathway is limited by ERK dependent feedback inhibition of RAS activation and other components of the pathway. Oncogenic mutations and translocations of RAF family members have been identified in many human tumors and serve as drivers of tumor growth. We now show that these mutants are active despite feedback inhibition of RAS and that this is associated with dysregulated ERK output. All translocations and almost all of the RAF mutants tested so far form RAS-independent constitutive dimers, the activity of which is dependent on their dimerization. Mutations at one site, V600E and V600K allow RAF to function as a monomer in cells with low RAS activity and as a RAS-dependent dimer in cells with adequate RAS activity. In tumors expressing any of these mutants, RAF/MEK/ERK activity is elevated despite feedback inhibition of RAS. Currently available RAF inhibitors bind to one protomer of the RAF dimer and cause negative cooperativity of binding to the second site. This is sometimes associated with transactivation of the unbound protomer. These inhibitors cannot be used to inhibit ERK signaling in tumors in which ERK is driven by RAF-dimers (those with mutant RAS, NF1 loss, atypical RAF mutations, RAF translocations.) In contrast, in tumors with V600E BRAF mutations, ERK causes strong feedback inhibition of RAS and V600E BRAF exists as a monomer that is inhibited by these drugs. Acquired resistance to RAF inhibitors usually results from a molecular lesion that causes RAS-dependent or independent dimerization of V600E (NRAS mutation, V600E BRAF amplification, V600E aberrantly spliced isoform.) Current RAF inhibitors inhibit V600E BRAF dimers at concentrations 10-100 fold greater than those required to inhibit the corresponding monomer in cells. We take this as a provisional measure of the degree of negative cooperativity of binding to the second site. We have now identified inhibitors that inhibit V600E dimers at concentrations close (1-3 fold higher) than those required to inhibit the monomer. They do not cause dissociation of dimers and their inhibition is abolished by gatekeeper mutations. Phenotypically, therefore, binding of these compounds to dimers is associated with only minimal negative cooperativity. These drugs inhibit ERK signaling and the proliferation of tumor cells with atypical BRAF mutations and translocations as well as V600E melanoma models with acquired resistance to Vemurafenib. However, they are much less active in cancer cells with KRAS mutation or receptor-dependent activation of RAF. Selective inhibitors of subsets of RAF dimers may allow effective ERK inhibition of large subset of tumors without inhibiting signaling in normal cells. Citation Format: Zhan Yao, Merna Torres, Min Wei, Aphrothiti Hanrahan, Omar Abdel-Wahab, David Solit, Lusong Luo, Poulikos Poulikakos, Neal Rosen. Oncogenic RAF mutants that signal as functional dimers are resistant to current RAF inhibitors but sensitive to a novel inhibitor of RAF dimer kinase activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3706. doi:10.1158/1538-7445.AM2014-3706