Vertical combination strategies endowed with anti-tumor synergism to target MAPK activation in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) ranks as the 4th/5th leading cause of cancer death worldwide. KRAS is mutated (mut) in 90% of human PDAC and results in the constitutive activation of multiple signaling cascades (MAPK, PI3K, NFκB, etc). Hitting a single point along the RAF/MEK/ERK cascade disrupts intra-pathway negative feedback loops, thereby causing paradoxical pathway activation and functional resistance. Thus, combining agents simultaneously inhibiting RAF and MEK represents a potential strategy to synergistically inhibit tumor growth and delay resistance. Molecular and functional effects of single and combined MEK (trametinib, T), BRAF (dabrafenib, D), and RAF (using the pan-RAF inhibitor RAF265, R) inhibition were dissected by WB and conservative isobologram analysis to assess functional synergism. In HPAFII cells (but not in the BRAF-mut M14 melanoma) D and R induced the formation of BRAF/CRAF complexes, as assessed by immunoprecipitation. In a panel of PDAC cell lines selective BRAF inhibition with D induced hyperphosphorylation of MEK, ERK, and p90RSK (paradox effect) and the combination of D+T suppressed cell growth with highly synergistic effects in 6/9 cell lines tested (CI 0.05-0.8); conversely, R did not induce paradox MAPK activation and did not result in growth inhibitory synergism when combined with T. Specific proteins and their phosphorylation states were also analyzed using Kinexus Antibody Microarray and preliminary results show that the different treatments cause upregulation of EGFR family, wnt/ß-catenin and c-KIT signaling, suggesting potential new strategies for both “horizontal” and “vertical” combinations of agents to achieve synergistic PDAC killing. Overall, our data indicate that, in appropriate cellular contexts,vertical RAF/MEK inhibition-based combination strategies exert highly synergistic antitumor effects.