Activating mutations in B-RAF and N-RAS occur in B60 and B15% of melanomas, respectively. The most common mutation in B-RAF is V600E, which activates B-RAF and the downstream MEK-ERK1/2 pathway. Thus, B-RAF V600E is a viable therapeutic target. PLX4720 is a selective inhibitor of mutant B-RAF and its analog, PLX4032, is currently undergoing clinical trials in melanoma. However, the effects of PLX4720 across the genotypic spectrum in melanoma remain unclear. Here, we describe that PLX4720 treatment rapidly induces hyperactivation of the MEK-ERK1/2 pathway in mutant N-RAS melanoma cells. Furthermore, we demonstrate that C-RAF is the major RAF isoform involved in this process. Importantly, PLX4720-induced hyperactivation of the MEK-ERK1/2 pathway promotes resistance to apoptosis in both non-invasive and invasive mutant N-RAS melanoma cells but does not enhance cell cycle properties. These findings underscore the need to genotypically stratify melanoma patients before enrollment on a mutant B-RAF inhibitor trial.

The recent RAF inhibitor trial with PLX4032/RG7204 in late-stage mutant B-RAF melanoma patients has been lauded as a success story for personalized cancer therapy since short-term clinical responses were observed in the majority patients. However, initial responses were followed by subsequent tumor re-growth and a subset of patients showed intrinsic resistance. Bi-directional translational efforts are now essential to determine the mechanisms underlying acquired/secondary and intrinsic resistance to RAF inhibitors.

Background: Reactivation of ERK1/2 frequently underlies acquired resistance to RAF inhibitors. Results: NRAS mutations are acquired during resistance to RAF inhibitors and promote CRAF and SHOC2-modulated ERK1/2 pathway re-activation. Conclusion: NRAS mutations in mutant BRAF cells alter RAF isoform and SHOC2 usage in the presence of RAF inhibitor. Significance: These studies delineate mechanisms mediating RAF inhibitor resistance in mutant BRAF cells.

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