Synergistic activity of vertical combinations of agents targeting multiple steps along the RAF/MEK/ERK cascade as a therapeutic strategy in human tumors.

e13572 Background: BRAF-selective kinase inhibitors have potent antitumor effects in mutant BRAF(V600E) tumors; however, in BRAF-wt cells, they paradoxically activate MEK/ERK. In addition, MEK blockade may induce compensatory signaling through both upstream pathway elements (RAF) and parallel pathways (PI3K/AKT/mTOR). Methods: We set out to define molecular and functional effects of single and combined BRAF (GSK2118436A, BRAF-I) and MEK (GSK1120212B, MEK-I) inhibition, using WB analysis to dissect signaling and a fixed dose-ratio experimental design to assess functional synergism by conservative isobologram analysis. Results: In A549 lung adenocarcinoma (KRAS G12S), BRAF-I (10 μM) induces hyperphosphorylation of CRAF, MEK, ERK, and p90RSK, while MEK-I (10 nM), alone or in combination with BRAF-I, potently offsets MAPK activation. Combined BRAF-I and MEK-I suppress malignant growth and survival at 72 h with highly synergistic effects in the A549 (lung, KRAS G12S), H1299 (lung, NRAS), HCT116 (colon, KRAS G1...