Abstract
A high percentage of patients with BRAF(V600E) mutant melanomas respond to the selective RAF inhibitor vemurafenib (RG7204, PLX4032) but resistance eventually emerges. To better understand the mechanisms of resistance, we used chronic selection to establish BRAF(V600E) melanoma clones with acquired resistance to vemurafenib. These clones retained the V600E mutation and no second-site mutations were identified in the BRAF coding sequence. Further characterization showed that vemurafenib was not able to inhibit extracellular signal-regulated kinase phosphorylation, suggesting pathway reactivation. Importantly, resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed a rare activating mutation in KRAS, resulting in a K117N change in the KRAS protein. Elevated levels of CRAF and phosphorylated AKT were also observed. In addition, combination treatment with vemurafenib and either a MAP/ERK kinase (MEK) inhibitor or an AKT inhibitor synergistically inhibited proliferation of resistant cells. These findings suggest that resistance to BRAF(V600E) inhibition could occur through several mechanisms, including elevated RAS-GTP levels and increased levels of AKT phosphorylation. Together, our data implicate reactivation of the RAS/RAF pathway by upstream signaling activation as a key mechanism of acquired resistance to vemurafenib, in support of clinical studies in which combination therapy with other targeted agents are being strategized to combat resistance.
Highlights
The BRAF oncogene is mutated in approximately 8% of all human tumors; the prevalence is much higher in melanoma, where a mutation is documented in more than 50% of all melanoma [1]
All cell lines were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 10% of heat-inactivated FBS (HI-FBS; GIBCO/BRL) and 2 mmol/L L-glutamine (GIBCO/BRL)
The A375 melanoma cell line is driven by the BRAFV600E oncogene and is exquisitely sensitive to proliferation inhibition
Summary
The BRAF oncogene is mutated in approximately 8% of all human tumors; the prevalence is much higher in melanoma, where a mutation is documented in more than 50% of all melanoma [1]. Other tumor types with a substantial incidence of mutated BRAF include papillary thyroid (30%– 70%), ovarian (15%–30%), and colorectal cancers In melanoma cells BRAFV600E causes deregulated proliferation by overcom-. Authors' Affiliations: 1Discovery Oncology, 2Pharma Development, 3Translational Research Sciences, Hoffmann-La Roche Inc., Nutley, New Jersey; 4Roche NimbleGen, Madison, Wisconsin; and 5Plexxikon Inc., Berkeley, California. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Acquisition of the BRAFV600E mutation seems to be an early event in melanoma development with a high percentage of premalignant melanocytic nevi found to harbor the mutation [5]
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