Because amplification of mutant BRAF in AR cells caused hyperactivation of MEK and resistance to AZD6244, we hypothesized that inhibiting excess BRAF activity might restore sensitivity to AZD6244. To test this hypothesis, we treated parental and AR cells with increasing concentrations of AZD6244 or the BRAF inhibitor AZ628, alone or in combination. Although AR cells were resistant to treatment with either compound alone, they were highly sensitive to the combination. In fact, the IC50s for the combination treatment in AR cellswere similar to the IC50sofeither inhibitor alone in parental cells. Moreover, parental COLO201 cells engineered to overexpress V600E BRAF were resistant to AZD6244 and AZ628, but were sensitive to the combination. The combination ofAZD6244 andAZ628 also inhibited the parental cell linesmore potently than did either treatment alone, suggesting that combinatorial targeting of the MAPK pathway may be an advantageous strategy in BRAF mutant tumors, even in the absence of BRAF gene amplification. Combined MEK and BRAF inhibition also more potently decrease ERK phosphorylation in parental and AR cells, and again, astrong correlation between BIM induction and the absolute amount of phospho-ERK was observed. Consistent with these findings, we observed that the combination of AZD6244 and AZ628 enhanced the apoptotic response in parental and AR cells (Fig. 4C). At a concentration of 100 nM, either AZD6244 or AZ628 alone was sufficient to cause marked apoptosis in COLO201 cells. In contrast, at this same concentration, neither AZD6244 nor AZ628 alone caused a substantial increase in apoptosis in COLO201-AR cells. However, when these agents were combined at 100 nM each, we observed an increase in apoptosis in the AR cells that was equivalent to that induced by either agent alone in parental COLO201 cells. Similarly, in parental COLO201 cells, the combination of AZD6244 and AZ628 induced considerably more apoptosis than equal concentrations of either agent alone. In fact, the combination of 10 nM AZD6244 and 10 nM AZ628 induced nearly as much apoptosis as 100 nM of either agent alone. Collectively, these findings suggest that the combination of BRAF and MEK inhibition can not only overcome the resistance caused by BRAF amplification but also potentially enhance antitumor efficacy against BRAF-mutant tumors in general and allow for lower effective doses of each drug, regardless of amplification status.