Targeting the PI3K pathway to overcome resistance to BRAF or MEK inhibition in BRAF-mutant melanoma

Abstract

BRAF is mutated in approximately 50% of melanomas. Development of BRAF and MEK inhibitors have improved outcomes for patients with BRAF-mutant melanoma, but their efficacy is limited by both intrinsic and acquired resistance. A growing body of evidence suggests that activation of the PI3K pathway mediates resistance to these agents, providing a strong rationale for combination therapy in melanoma. A panel of BRAF-mutant metastatic melanoma cell lines were tested in cell viability assays for sensitivity to inhibitors of BRAF (vemurafenib), MEK (selumetinib), PI3K isoforms (A66, TGX 221, CAL 101, AS 252424), pan-PI3k (ZSTK 474), pan-PI3K/mTOR (BEZ 235), mTORC1 (rapamycin) and mTORC1/2 (KU 0063794). Basal expression of relevant proteins and effects of selected inhibitors on protein expression were assessed using western blotting. Cell viability assays and western blotting were also used to evaluate combination therapy with inhibitors of both the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signalling pathways. Results showed that five of nine (56%) cell lines tested were resistant to both vemurafenib and selumetinib, with failure to achieve 50% reduction in cell viability at concentrations ≤10 μM despite >80% inhibition of pERK across all cell lines. Increased activation of the PI3K pathway, as indicated by a high pAKT : AKT ratio, was predictive of resistance. Good single-agent activity was observed with ZSTK 474, KU 0063794 and BEZ 235 in cell viability assays, while selective inhibition of PI3K isoforms or mTORC1 alone had limited activity. Combination of ZSTK 474 + selumetinib resulted in synergistic reduction of cell viability in most cell lines, whereas synergy for BEZ 235 + selumetinib tended to be restricted to cell lines that were at least moderately sensitive to selumetinib alone. Combination of ZSTK 474 or BEZ 235 with vemurafenib tended to be less synergistic than the selumetinib combinations. Despite the lower synergy observed, BEZ 235 + selumetinib was consistently associated with the greatest reductions in cell viability across all cell lines. Overall, these results suggest that resistance to BRAF or MEK inhibition in BRAF-mutant melanoma is at least partly mediated by activation of the PI3K pathway and can be overcome by combined inhibition of the RAS/RAF/MEK/ERK and PI3K pathways.