Abstract
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 MAPKs, 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 MAPKs and the PI3K pathway, as well as increased NRAS expression, which, contrary to previous reports, was not associated with 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.
Highlights
The response to vemurafenib in V600E BRAFϩve melanoma is short lived due to acquisition of vemurafenib resistance
The results indicate a mechanism of acquired vemurafenib resistance in V600E BRAF؉ve melanoma cells that involves increased activation of all three human MAPKs and the PI3K pathway, as well as increased NRAS expression, which, contrary to previous reports, was not associated with mutations in the NRAS gene
The present study is a contribution to these efforts, and the results provide important novel insight into the complexity of the vemurafenib resistance phenotype in melanoma and advance the basis upon which such resistance may be overcome clinically
Summary
The response to vemurafenib in V600E BRAFϩve melanoma is short lived due to acquisition of vemurafenib resistance. The activated BRAF 3 MEK 3 ERK pathway, resulting from the V600E BRAF, has been shown to drive melanoma cell proliferation and is implicated in poor therapeutic outcome in melanoma [5,6,7] This strong relationship between the activated ERK pathway, increased melanoma cell growth, and the V600E BRAF mutation has led to significant efforts to target this pathway in the treatment of melanoma [8, 9], and in 2011, vemurafenib was approved by the United States Food and Drug Administration for advanced melanoma therapy. Other studies have shown that melanoma cells become dependent on the MAPK pathway in the setting of vemurafenib resistance and that intermittent dosing strategies may combat this dependence and delay the development of resistance to vemurafenib [13] Despite these advances, the mechanisms underlying acquired resistance to BRAF-targeted therapy remain not fully understood, making it difficult to design more effective strategies to overcome it clinically. These cell lines provide a model and platform for further mechanistic investigations and for testing alternative novel therapeutic strategies for overcoming acquired vemurafenib resistance in melanoma
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