As the Wnt/β-catenin signaling pathway is linked to melanoma pathogenesis and to patient survival, we conducted a kinome siRNA screen in melanoma cells to expand our understanding of kinases that regulate this pathway, and to illuminate potential therapeutic directions. We found that BRAF signaling, which is constitutively activated in many melanomas by the BRAFV600E mutation, negatively regulates Wnt/β-catenin signaling in human melanoma cells. As inhibitors of BRAFV600E show promise in ongoing clinical trials we investigated whether altering Wnt/β-catenin signaling might enhance the efficacy of the BRAFV600E inhibitor, PLX4720. Surprisingly, endogenous β-catenin is required for PLX4720 to induce apoptosis in melanoma cells, while activation of Wnt/β-catenin signaling strongly synergizes with PLX4720 to decrease tumor growth in vivo and to increase apoptosis in vitro. This synergistic enhancement of apoptosis correlates with a reduction in the abundance of a β-catenin antagonist, AXIN1. In support of the hypothesis that AXIN1 is a mediator rather than a marker of apoptosis, melanoma cell lines that are resistant to apoptosis after treatment with a BRAFV600E inhibitor become susceptible, and undergo apoptosis, when AXIN1 is reduced by siRNA. These findings point to a role for Wnt/β-catenin signaling and AXIN1 in regulating the efficacy of inhibitors of BRAFV600E, and may stimulate consideration of potential combination therapies and biomarkers for use in conjunction with targeted BRAF therapy.

T he limitations of revolutionary new mutation-specific inhibitors of BRAFV600E include the universal recurrence seen in melanoma patients treated with this novel class of drugs. Recently, our lab showed that simultaneous activation of the Wnt/β-catenin signaling pathway and targeted inhibition of BRAF V600E by PLX4720 synergistically induces apoptosis across a spectrum of BRAF V600E melanoma cell lines. As a follow-up to that study, treatment of BRAF-mutant and NRAS-mutant melanoma lines with WNT3A and the MEK inhibitor AZD6244 also induces apoptosis. The susceptibility of BRAF-mutant lines and NRAS-mutant lines to apoptosis correlates with negative regulation of Wnt/β-catenin signaling by ERK/ MAPK signaling and dynamic decreases in abundance of the downstream scaffolding protein, AXIN1. Apoptosisresistant NRAS-mutant lines can sensitize to AZD6244 by pretreatment with AXIN1 siRNA, similar to what we previously reported in BRAF-mutant cell lines. Taken together, these findings indicate that NRAS-mutant melanoma share with BRAF-mutant melanoma the potential to regulate apoptosis upon MEK inhibition through WNT3A and dynamic regulation of cellular AXIN1. Understanding the cellular context that makes melanoma cells susceptible to this combination treatment will contribute to the study and development of novel therapeutic combinations that may lead to more durable responses.