Abstract The FDA approved RAF inhibitors vemurafenib and dabrafenib have elicited profound responses in melanoma patients with tumors harboring BRAFV600E mutation, but resistance limits their effectiveness. Furthermore, RAF inhibitors exhibit only modest efficacy in colorectal and thyroid BRAFV600E tumors and they are not effective in tumors harboring non-V600 BRAF mutations. While RAF/MEK inhibitor combinations demonstrate increased clinical efficacy, drug resistance develops as well, through similar mechanisms to those identified in RAF inhibitor monotherapy. This suggests that insufficient RAF inhibition limits treatment efficacy and indicates the need for more effective therapeutic approaches to potently and durably target RAF/ERK signaling. Although the unique biochemical properties of RAF inhibitors have been a topic of intensive investigation, a unified model of RAF inhibitor action has been lacking. Our biochemical and structural analysis in a panel of RAF inhibitors with different structural properties revealed that they exert their effects via the combined outcome of two distinct allosteric mechanisms. RAF priming and dimerization are the result of RAF/RAS-GTP interaction upon inhibitor binding to RAF and stabilization of the αC-helix in the IN (active) position. Resistance of the dimeric RAF to vemurafenib and dabrafenib is the result of stabilization of the αC-helix of the drug-bound protomer towards the OUT (inactive) position, a conformation that sterically prevents occupancy of both RAF protomers. Next generation RAF inhibitors that stabilize the αC-helix of RAF towards the IN position (αC-IN RAF inhibitors) allow occupancy of both RAF protomers and thus accomplish potent inhibition of the dimeric form of RAF. We further provide structural, biochemical and cell-based evidence that next generation αC-IN RAF inhibitors (such as MLN480, TAK632, LY3009120, AZ628 and others) will be more effective for the treatment of patients with tumors expressing non-V600 BRAF mutants and in BRAFV600E colorectal and thyroid tumors. However, αC-IN RAF inhibitors are predicted to have a low therapeutic window, since they inhibit dimeric wild-type BRAF also in normal cells. Together our findings provide a blueprint for the development of RAF inhibitors with defined biochemical properties and support therapeutic strategies in which relatively lower, non-toxic concentrations of αC-IN inhibitors will be administered in combination with currently approved RAF and MEK inhibitor regimens for the treatment of BRAFV600E melanoma, colorectal and thyroid cancers, as well as for tumors with BRAF mutations other than V600. Citation Format: Zoi Karoulia, Yang Wu, Tamer A. Ahmed, Qisheng Xin, Julien Bollard, Clemens Krepler, Xuewei Wu, Chao Zhang, Gideon Bollag, Meenhard Herlyn, James A. Fagin, Amaia Lujambio, Evripidis Gavathiotis, Poulikos I. Poulikakos. A unified model of RAF inhibitor action determines inhibitor activity in BRAF-dependent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1225. doi:10.1158/1538-7445.AM2017-1225