The development of acquired drug resistance hampers the long-term success of B-RAF inhibitor (B-RAFi) therapy for melanoma patients.,Here we show V600EB-RAF copy number gain as a mechanism of acquired B-RAFi resistance in four out of twenty (20%) patients treated with B-RAFi.,In cell lines, V600EB-RAF over-expression and knockdown conferred B-RAFi resistance and sensitivity, respectively.,In V600EB-RAF amplification-driven (vs. mutant N-RAS-driven) B-RAFi resistance, ERK reactivation is saturable, with higher doses of vemurafenib down-regulating pERK and re-sensitizing melanoma cells to B-RAFi.,These two mechanisms of ERK reactivation are sensitive to the MEK1/2 inhibitor AZD6244/selumetinib or its combination with the B-RAFi vemurafenib.,In contrast to mutant N-RAS-mediated V600EB-RAF bypass, which is sensitive to C-RAF knockdown, V600EB-RAF amplification-mediated resistance functions largely independently of C-RAF.,Thus, alternative clinical strategies may potentially overcome distinct modes of ERK reactivation underlying acquired B-RAFi resistance in melanoma.
The initial use of BRAF targeted therapeutics in clinical trials has demonstrated encouraging responses in melanoma patients, although a rise in drug-resistant cells capable of advancing malignant disease has been described.,The current study uses BRAFV600E expressing WM793 melanoma cells to derive data aimed at investigating the molecular determinant of cell invasion following treatment with clinical BRAF inhibitors.,Small-molecule inhibitors targeting BRAF reduced MEK1/2-ERK1/2 pathway activation and cell survival; yet, viable cell subpopulations persisted.,The residual cells exhibited an elongated cell shape, prominent actin stress fibers and retained the ability to invade 3-D dermal-like microenvironments.,BRAF inhibitor treatments were associated with reduced expression of RND3, an antagonist of RHOA activation, and elevated RHOA-dependent signaling.,Restoration of RND3 expression or RHOA knockdown attenuated the migratory ability of residual cells without affecting overall cell survival.,The invasive ability of BRAF inhibitor treated cells embedded in collagen gels was diminished following RND3 re-expression or RHOA depletion.,Conversely, melanoma cell movement in the absence of BRAF inhibition was unaffected by RND3 expression or RHOA depletion.,These data reveal a novel switch in the requirement for RND3 and RHOA in coordinating the movement of residual WM793 cells that are initially refractive to BRAF inhibitor therapy.,These results have important clinical implications because they suggest that combining BRAF inhibitors with therapies that target the invasion of drug-resistant cells could aid in controlling disease relapse.