MAP kinase hyper-activation and enhanced NRAS expression drive acquired vemurafenib resistance in V600E BRAF melanoma cells [Cell Biology]

July 25th, 2014 by Lidsky, M., Antoun, G., Speicher, P., Adams, B., Turley, R., Augustine, C., Tyler, D., Ali-Osman, F.

Although, targeting the V600E activating mutation in the BRAF gene, the most common genetic abnormality in melanoma, has shown clinical efficacy in melanoma patients, response is, invariably, short-lived. To better understand mechanisms underlying this acquisition of resistance to BRAF-targeted therapy in previously responsive melanomas, we induced vemurafenib-resistance in two V600E BRAF+ve melanoma cell lines, A375 and DM443, by serial in vitro vemurafenib exposure. The resulting, approximately, 10-fold more vemurafenib-resistant cell lines, A375rVem and D443rVem, had higher growth rates, and showed differential collateral resistance to cisplatin, melphalan and temozolomide. The acquisition of vemurafenib resistance was associated with significantly increased NRAS levels in A375rVem and D443rVem, increased activation of the prosurvival protein, AKT, and the MAP kinases, ERK, JNK, and P38, which correlated with decreased levels of the MAPK inhibitor protein, GSTP1. Despite the increased NRAS, whole exome sequencing showed no NRAS gene mutations. Inhibition of all three MAPKs and siRNA-mediated NRAS suppression, both reversed vemurafenib resistance significantly in A375rVem and DM443rVem. Together, the results indicate a mechanism of acquired vemurafenib resistance in V600E BRAF+ve melanoma cells that involves increased activation of all three human MAP kinases and the PI3K pathway, as well as increased NRAS expression, which, contrary to previous reports, was not associated mutations in the NRAS gene. The data highlight the complexity of the acquired vemurafenib resistance phenotype and the challenge of optimizing BRAF-targeted therapy in this disease. They also suggest that targeting the MAPKs and/or NRAS may provide a strategy to mitigate such resistance in V600E BRAF+ve melanoma.