Melanoma whole-exome sequencing identifies (V600E)B-RAF amplification-mediated acquired B-RAF inhibitor resistance

Abstract

The development of acquired drug resistance hampers the long-term success of B-RAF inhibitor therapy for melanoma patients. Here we show (V600E)B-RAF copy-number gain as a mechanism of acquired B-RAF inhibitor resistance in 4 out of 20 (20%) patients treated with B-RAF inhibitor. In cell lines, (V600E)B-RAF overexpression and knockdown conferred B-RAF inhibitor resistance and sensitivity, respectively. In (V600E)B-RAF amplification-driven (versus mutant N-RAS-driven) B-RAF inhibitor resistance, extracellular signal-regulated kinase reactivation is saturable, with higher doses of vemurafenib down-regulating phosho-extracellular signal-regulated kinase and re-sensitizing melanoma cells to B-RAF inhibitor. These two mechanisms of extracellular signal-regulated kinase reactivation are sensitive to the MEK1/2 inhibitor AZD6244/selumetinib or its combination with the B-RAF inhibitor vemurafenib. In contrast to mutant N-RAS-mediated (V600E)B-RAF bypass, which is sensitive to C-RAF knockdown, (V600E)B-RAF amplification-mediated resistance functions largely independently of C-RAF. Thus, alternative clinical strategies may potentially overcome distinct modes of extracellular signal-regulated kinase reactivation underlying acquired B-RAF inhibitor resistance in melanoma.

Conflict of interest statement

Competing financial interests: A.R. and R.S.L. are the authors of patent application under PCT Application Serial No. PCT/US11/061552 (Compositions and methods for detection and treatment of B-RAF inhibitor-resistant melanomas)

Figures

Figure 1. Exome sequencing identifies V600EB-RAF amplification as a candidate mechanism for BRAFi resistance

(a) Copy number variations (CNVs) called from whole exome sequence data on two triads of gDNAs using ExomeCNV and chromosome 7 as visualized by Circos (outer ring, genomic coordinates (Mbp); centromere, red; inner ring, log ratio values between baseline and disease progression (DP) samples’ average read depth per each capture interval; scale of axis for Pt #5 −5 to 5 and for Pt #8 −2.5 to 2.5). Two patients whose melanoma responded to and then progressed on vemurafenib. The genomic region coded orange represents the location of B-RAF (chr7:140,424,943–140,524,564), which shows an average log ratio value of 1.14 (2.2 fold gain; Pt #5) and 3.8 (12.8 fold gain; Pt #8). (b) B-RAF immunohistochemistry on paired tissues derived from the same patients as in a (scale bar = 50 μM) (c) Validation of V600EB-RAF copy number gain by gDNA qPCR (black and red by B-RAF primer set 1 and 2, respectively) and recurrence across distinct patients (positives highlighted in orange). PMN, peripheral mononuclear cells, and HDF, human dermal fibroblasts for diploid gDNAs. (d)B-RAF V600 mutant to WT ratio increases with disease progression or acquisition of B-RAFi resistance mediated by mutant B-RAF copy number gain. Chromatograms from Sanger sequencing for melanoma samples from patients who acquired B-RAFi resistance based on distinct molecular alterations: V600EB-RAF copy number gain, V600EB-RAF truncation, N-RAS mutation or RTK over-expression.

Figure 2. V600EB-RAF levels modulate melanoma sensitivity to vemurafenib

(a, b) Western blot of V600EB-RAFV600E and p-ERK, tubulin is used as a loading control. Growth curve of did not alter the pERK level in the absence of vemurafenib/PLX4032 but conferred growth resistance to the parental line, M395 P when exposed to indicated concentrations of PLX4032 for 72 h (relative to DMSO-treated controls; mean ± SEM, n = 5). Dashed line, 50% inhibition. (c, d) Transduction of shRNA to knockdown BRAFV600E in the drug-resistant sub-line, M395 R, did not alter the pERK level in the absence of PLX4032 but restored growth sensitivity to PLX4032 (72 h; mean ± SEM, n = 5). (e) Increasing (in M395 P) or decreasing (in M395 R) BRAFV600E levels decreased or increased pERK sensitivity to PLX4032 (0, 0.1, 1, 10 μM) treatments for 1 h, respectively.

Figure 3. Differential B-RAFi/MEKi sensitivities and C-RAF dependency

(a) Survival curves of B-RAFi acquired resistant sub-lines, with indicated mechanisms of resistance, to 72 h of B-RAFi (PLX4032) treatments, showcasing differential responses at the micro-molar drug range. Results are shown relative to DMSO-treated controls (mean ± SEM, n = 5; dashed line, 50% inhibition). (b) Survival curves of cell lines, engineered by viral transduction of M229 P to be B-RAFi resistant, to 72 h of B-RAFi (PLX4032) treatments, showcasing differential responses at the micro-molar drug range. Results are shown relative to DMSO-treated controls (mean ± SEM, n = 5). Expression of indicated viral expression constructs shown in Western blots. (c) Survival curves of B-RAFi acquired resistant sub-lines, with indicated mechanisms of resistance, to 72 h of MEKi (AZD6244) treatments, showcasing differential responses at the micro-molar drug range. Results are shown relative to DMSO-treated controls (mean ± SEM, n = 5). (d) Survival curves of cell lines (engineered by viral transduction of M229 P and M238 P to over-express V600EB-RAF rendering these parental cells resistant to B-RAFi) to 72 h of MEKi (AZD6244) treatments, showcasing differential responses at the micro-molar drug range. Pt55 R (double B-RAF and N-RAS mutant) is a short-term melanoma culture derived from a tumor which acquired PLX4032 (vemurafenib) resistance in a treated patient. Results are shown relative to DMSO-treated controls (mean ± SEM, n = 5). (e and f) Indicated cell lines were treated with constant ratios of PLX4032 and AZD6244 and survival measured after 72h. Relative synergies, expressed as log10 of CI values, are shown. (g) M249 (R4) and M395 R were seeded at single cell density and treated with indicated concentrations of PLX4032 and/or AZD6244. Inhibitors and media were replenished every two days, colonies visualized by crystal violet staining after 8 days of drug treatments, and quantified (% growth relative to cells treated with 1 μM PLX4032; representative of 2 experiments). Photographs representative of two independent experiments. (h) Survival curves of indicated cell lines after shScrambled or shC-RAF transduction (inset) and when treated with PLX4032 for 72 h. (i) Clonogenic assays of cell lines in e with 14 days (M249 R4) or 18 days (M395 R) of PLX4032 treatment. Results are representative of 2 experiments.

Figure 4. MAPK-reactivating mechanisms of acquired B-RAFi resistance and therapeutic implications

Distinct strategies to overcome acquired resistance driven by amplification of mutant B-RAF or mutations in N-RAS. Schematic of ERK-reactivating pathways (V600EB-RAF amplification indicated by stacked symbols, top; N-RAS mutation, bottom; mutant proteins in red and WT proteins in grey) and proposed strategies to restore B-RAFi sensitivity (increasing B-RAFi concentration or potency, top; switching B-RAFi to pan-RAFi, bottom). Alternatively, the combination of B-RAFi and MEKi are predicted to synergistically growth-inhibit melanomas with acquired resistance to B-RAFi monotherapy stemming from ERK reactivation.