Atypical BRAF Research Articles

Abstract A41: 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 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, Torres N Merna, Wei Min, Hanrahan Aphrothiti, Abdel-Wahab Omar, Solit David, Luo Lusong, Poulikakos Poulikos, Rosen Neal. 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 AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A41. doi: 10.1158/1557-3125.RASONC14-A41

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 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

Identification of a Novel ComplexBRAFMutation Associated With Major Clinical Response to Vemurafenib in a Patient With Metastatic Melanoma

There is an increasing interest in BRAF V600 mutations in melanomas and their associated sensitivity to vemurafenib, a BRAF inhibitor. However, physicians cannot find information in the literature about vemurafenib response for rare and/or atypical BRAF mutations. We describe the identification of a novel complex BRAF mutation associated with major clinical response to vemurafenib in a patient with metastatic melanoma. Using a pyrosequencing method, we determined that the tumor positive for mutated BRAF, uncovering a novel c.1799_1803delinsAT; p.V600-K601>D variant. We uncovered this atypical BRAF mutation with 2 different sequencing methods, both in the primary lesion and in 1 metastasis. The patient was immediately treated with vemurafenib as monotherapy and achieved a prolonged (5.5-month) positive response. We analyzed the consequences of the BRAF V600-K601>D mutation in terms of amino acids. We referred to the published data and databases to screen chemical properties of well-known BRAF V600 mutations and other complex BRAF mutations to find common features of activated BRAF mutations. Importantly, we highlighted that both the site of the mutation and the involved amino acids are important to predict vemurafenib response. Our conclusion is that complex BRAF mutation surrounding codon 600 could also be sensitive to BRAF inhibitors.