RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E)

Abstract

Activated RAS promotes dimerization of members of the RAF kinase family. ATP-competitive RAF inhibitors activate ERK signalling by transactivating RAF dimers. In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity. This tumour-specific inhibition of ERK signalling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbour mutant BRAF(V600E). However, resistance invariably develops. Here, we identify a new resistance mechanism. We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61-kDa variant form of BRAF(V600E), p61BRAF(V600E), which lacks exons 4-8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) shows enhanced dimerization in cells with low levels of RAS activation, as compared to full-length BRAF(V600E). In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signalling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib. Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumours of six of nineteen patients with acquired resistance to vemurafenib. These data support the model that inhibition of ERK signalling 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.

Figures

Figure 1. Resistance to the RAF inhibitor vemurafenib (PLX4032) is associated with failure of the drug to inhibit ERK signaling

a. Vemurafenib IC50 curves (at 5 days) for the SKMEL-239 parental cell line and five vemurafenib-resistant clones. b. Effects of 2μM vemurafenib on ERK signaling in parental (Par) and resistant clones (C1-5). c. Western blot for components of the ERK and AKT signaling pathways in parental and resistant clones (2μM PLX4032/24 hours). d. Dose-response of pMEK and pERK downregulation at 1 hour to increasing concentrations of vemurafenib in parental and two representative resistant clones (C3 and C5). e. Graphic representation of the chemiluminescent signal intensities from 1d and IC50s for inhibition of MEK phosphorylation by vemurafenib in the parental and C3 and C5 clones.

Figure 2. A BRAF(V600E) variant that lacks exons 4-8 is resistant to the RAF inhibitor vemurafenib

a. PCR analysis of BRAF in cDNA from parental (P) and C3 cells. Sequencing of the 1.7kb product expressed in the C3 clones but not in parental cells revealed an in frame deletion of five exons (4-8) in cis with the V600E mutation. Abbreviations: CR1: Conserved Region 1, CR2: Conserved Region 2, CR3: Conserved Region 3, RBD: RAS-binding domain, CRD: Cystine-Rich Domain. b. Full length wild-type BRAF and the 1.7kb/61kd splice variant of BRAF(V600E) were expressed in 293H cells. The effect of vemurafenib (2μM for 1 hour) on ERK signaling in the presence of p61BRAF(V600E) was analyzed by western blot for pMEK and pERK. c. To compare levels of dimerization, 293H cells co-expressing FLAG tagged and V5-tagged p61BRAF(V600E), full length BRAF(V600E) and the corresponding dimerization-deficient mutants p61BRAF(V600E/R509H) and BRAF(V600E/R509H) were lysed followed by immunoprecipitation with FLAG antibody. Western blotting with V5 or FLAG antibodies was performed as indicated. d. Comparison of MEK/ERK activation and sensitivity of ERK signaling to vemurafenib (2μM for 1 hour) in 293H cells expressing either Flag-tagged BRAF(V600E) or the dimerization mutant Flag-tagged BRAF(V600E/R509H). e. Constructs expressing V5-tagged BRAF(V600E), p61BRAF(V600E) or the dimerization mutant p61BRAF(V600E/R509H) were transfected into 293H cells and treated with DMSO or 2μM vemurafenib for 1 hour.

Figure 3. Identification of splice variants of BRAF(V600E) in human tumors resistant to vemurafenib

a. PCR analysis of cDNA derived from tumor samples from patients treated with vemurafenib. In samples with only one band (full-length BRAF), both BRAF(V600E) and wild-type BRAF (1+2) were detected. In resistant tumor samples expressing shorter transcripts, the shorter transcript was a splicing variant of BRAF(V600E) (3, 4, 5). The figure shows samples from three patients with acquired resistance to PLX4032: baseline (B) and disease progression (DP) samples from patient 1 and post-treatment samples from patients 11 and 12. A tumor sample from a patient with de novo resistance to vemurafenib (patient 20) is also shown. The intermediate band in samples expressing splicing variants (Pts 1, 11, 12) is an artifact of the PCR reaction resulting from switching between two very similar templates. Representative Sanger sequencing traces showing the junction between exons 3 and 11 in the DP sample from patient 1 compared to the full-length transcript derived from the baseline pre-treatment sample. b. As in A, baseline (B) and disease progression (DP) samples from a patient with an exon 2-10 deletion. RNA/cDNA levels of the exon 2-10 deletion were determined by qPCR using an exon 1/11 junction primer. The data are shown as the average of duplicates and expressed as relative levels between patient-matched samples c. Exon organization of the splicing variants found in tumors from six patients that initially responded and then progressed on vemurafenib.