A lung cancer-derived CRAF mutation to activate ERK pathway and to predict sensitivity to LY3009120 and trametinib.

Abstract

e23197 Background: During the Belgian FIELT II clinical study, two non-small cell lung cancers among 41 were found to harbor a CRAF mutation. One of these mutations (P207S) was previously found in fibrosarcoma and characterized as not activating the ERK pathway at higher levels compared to the wild-type CRAF. The other mutation (P261A) has never been reported in cancer but found in Noonan syndrome. CRAF mutations can be oncogenic thereby activation of the ERK pathway. Serine 259 is a negative regulatory site and its phosphorylation is essential for keeping the CRAF auto-inhibited. P261A CRAF prevents phosphorylation of S259 but its role in cancer and its possible response to small molecule inhibitors has yet to be uncovered. Methods: We generated recombinant CRAF expression vectors by site-directed mutagenesis and investigated the ERK pathway activation status induced by CRAF mutants, in HEK293T cells and BEAS-2B (lung epithelial) cells. We tested the effect of candidate inhibitors at a clinically relevant dose on ERK pathway activity in BEAS-2B cells expressing CRAF mutants. Results: Expression of P261A CRAF in both HEK293T and BEAS-2B cells leads to increased ERK pathway activation compared to wtCRAF, accompanied by decreased phosphorylation of S259. P207S CRAF induces ERK pathway activity at levels comparable to wtCRAF and we observed that co-expression of BRAF with P207S CRAF does not lead to increased ERK activity compared to wtCRAF/BRAF. RAF-inhibitors LY3009120 and AZD-628 suppress ERK pathway activity induced by P261A CRAF in BEAS-2B cells. LY3009120 showed stronger ERK-inhibitory effect compared to AZD-628. In contrast, Dabrafenib (RAF-inhibitor) treatment of mutant or wtCRAF expressing cells leads to paradoxical ERK activation. Combinatorial treatment of LY3009120 with Trametinib (MEK-inhibitor) leads to stronger ERK-inhibitory effects compared to either single agent treatment in cells expressing mutant CRAF. Conclusions: The P261A CRAF mutation is ERK pathway activating and predicts sensitivity to LY3009120 and Trametinib. CRAF inhibition should be clinically explored in lung cancers and perhaps other cancers with sensitizing CRAF mutations.