Preclinical evidence shows that hitting a single point along the RAF/MEK/ERK cascade disrupts intra-pathway negative feedback loop, may cause paradoxical pathway activation and may lead to functional resistance. Thus, the “vertical” combination of agents simultaneously inhibiting RAF and MEK has been proposed as a strategy to synergistically inhibit tumor growth and delay resistance. Experimental procedures: Molecular and functional effects of single and combined MEK (using trametinib, T), BRAF (using dabrafenib, D), and RAF (using the pan-RAF inhibitor, RAF265, R) inhibition were dissected by WB analysis and conservative isobologram analysis to assess functional synergism, using a fixed dose-ratio experimental design. Summary data: We examined the in vitro molecular and functional consequences of D and T, alone or in combination, in a panel of different human BRAFV600E melanoma cell lines; both drugs inhibited cell growth and inactivated the MAPK pathway, but little or no synergistic growth inhibition was observed with their combination (CI: 0.7 - 1.3x106). Conversely, combined D+T suppressed malignant growth with highly synergistic effects in KRAS-mutant lung (2/5 cell lines tested) and pancreatic (4/6 cell lines tested) cancer models (CI: 0.1 - 0.7), in which selective BRAF inhibition induced hyperphosphorylation of MEK, ERK, and p90RSK (paradox effect). At concentrations inhibiting both BRAF and CRAF, R did not induce paradox MAPK activation and did not result in growth inhibitory synergism when combined with T. To define the role of RAS gene status in determining sensitivity/resistance to single and combined RAF and MEK blockade, we analyzed two isogenic tumor cell line models: H1299 expressing individual codon 12 KRAS mutants and isogenic HCT116 clones differing for the presence of a homo or heterozygous G13D KRAS mutation. Conversely, in lung cancer models driven by either EGFR mutations (HCC827, H1650) or HER-2 overexpression (Calu-3), selective BRAF inhibition also induced a paradox MAPK activation, which could be blocked using a reversible EGFR/HER-2 inhibitor (Lapatinib); in this context, combination (D+T) resulted in a non-synergic growth inhibitory effects. Conclusions: Overall, our data indicate that, in appropriate cellular contexts, vertical RAF/MEK inhibition-based combination strategies exert highly synergistic antitumor effects across different cancer models.