Abstract PR-1: Acquired resistance to RAF inhibitors is mediated by splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.

Abstract

Abstract Tumors with mutant BRAF(V600E) are dependent on BRAF/MEK/ERK (ERK signaling) for their growth. ATP-competitive RAF inhibitors suppress ERK signaling in cells with mutant BRAF, but paradoxically enhance ERK signaling in cells with wild-type BRAF. We have shown previously the mechanistic basis of this phenomenon: active RAS promotes homo- and heterodimerization of RAF. When RAF exists in dimers, drug binding to one protomer transactivates the other unbound protomer. In BRAF(V600E) tumors, RAS is not activated, RAF is predominantly monomeric and ERK signaling is inhibited by the drug. These results predict that RAF inhibitors will be effective in tumors in which BRAF is mutated. Furthermore, because RAF inhibitors do not inhibit ERK signaling in other cells, the model predicts that they would have a higher therapeutic index and greater antitumor activity than MEK inhibitors. Confirming these predictions, RAF inhibitors have shown remarkable clinical activity in patients with melanomas that harbor mutant BRAF(V600E), however, resistance invariably develops. The mechanism of action of these drugs indicates that promotion of RAF dimerization (by lesions that activate RAS, or other means) could lead to drug resistance in mutant BRAF tumors. Indeed, RAS mutation has been reported as a mechanism of resistance in a subset of patients that progressed on the RAF inhibitor PLX4032 (vemurafenib). In order to identify novel mechanisms of resistance, we generated cell lines resistant to PLX4032. We found that a subset of cells resistant to PLX4032 express a 61kd splicing variant form of BRAF(V600E) that lacks exons 4–8, a region that encompasses the RAS-binding domain (p61BRAF(V600E)). p61BRAF(V600E) exhibits enhanced dimerization as compared to full length BRAF(V600E) in cells with low levels of RAS activation. In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signaling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to PLX4032, indicating that dimerization status of BRAF(V600E) determines the effects of the drug. We detected BRAF splicing variants in tumors collected at the time of progression on PLX4032, but not in matched pre-treatment tumors, supporting the clinical relevance of this mechanism and its potential utility as a therapeutic target. These data support the model that inhibition of ERK signaling by RAF inhibitors is dependent on levels of RAS-GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PR-1.