Abstract 4609: Plixorafenib (plixo) synergizes with MEK inhibitors (MEKi) in MAPK pathway inhibition in BRAF V600 and non V600 alterations, with higher potency compared to early generation BRAFi and pan-RAFi
Authors: Tarcic Gabi , Cohen Limor , Shepherd Stacie Peacock , Levy Natalie Filippov , Billauer Hana , Birnbaum Lea , Gasasa Sara , Maor Roey , Abu-Liel Reham , Goldfarb Eden 
Published: March 2024

Abstract
The efficacy of BRAFi in V600 mutated tumors is established and requires effective inhibition of the MAPK pathway. However, the use approved BRAFi leads to paradoxical MAPK pathway activation associated with toxicities and to acquired resistance. Therefore, these inhibitors were combined with MEKi to mitigate toxicities, which has been shown to be more effective than monotherapy in some settings. Although effective, these combinations result in bothersome side effects, a high discontinuation rate, and risk of secondary malignancies, indicating a continued unmet need. Also, ~35% of all BRAF mutations occur outside the V600 codon, composed of a wide array of missense mutations, fusions, and in-frame deletions, which are not targeted by the earlier generation BRAFi. By design, plixo (FORE8394; PLX8394), a clinical stage BRAFi, binds the monomeric form of BRAF and inhibits its dimerization, while sparing the WT form, avoiding paradoxical MAPK pathway activation. We hypothesized that the combination of plixo with a MEKi will improve its efficacy and potency through maximal suppression of the MAPK pathway. Using ForeSight assay, a unique platform for testing MAPK signaling, we tested the efficacy of plixo and 4 MEKi (trametinib, cobimetinib, binimetinib and mirdametinib) across a cohort 18 BRAF alterations (V600E, 2 class II and 15 fusions) as single agents and in combinations. The combinations of plixo with the different MEKi were synergistic across all BRAF alterations tested. Furthermore, we compared the potency of a combination of plixo with binimetinib (bini) to that of another BRAFi (vemurafenib) or pan-RAFi’s (tovorafenib and lifirafenib) with bini in 2 class I, 3 class II and 7 BRAF fusions using the foresight assay. The IC:50 of BRAF V600E expressing cells treated with plixo + bini was 6nM compared with 57nM, 103nM and 190nM for the tovorafenib, vemurafenib, and lifirafenib combinations with bini respectively. A similar trend was shown in 9 of 11 additional alterations tested, indicating the potency of the combination of plixo + bini. Results were validated using western blot analysis and cell viability assays of both melanoma and CRC V600E mutated cell lines. Western blot correlated with ForeSight results both in single agents and combination treatment. Cells treated with plixo + bini showed complete growth inhibition while the same drug concentrations of vemurafenib + bini and tovorafenib + binimetinib showed no growth inhibition beyond the effect of bini single agent. These results indicate the improved efficacy of plixo + bini combination in inhibiting MAPK signaling and cancer cell proliferation. Overall results support that maximal suppression of the MAPK pathway with plixo is more potent in combination with a MEK inhibitor in BRAF V600 and non-V600 nonclinical models compared to BRAF or pan-RAF combinations.
Citation Format: Gabi Tarcic, Limor Cohen, Stacie Peacock Shepherd, Natalie Filippov Levy, Hana Billauer, Lea Birnbaum, Sara Gasasa, Roey Maor, Reham Abu-Liel, Eden Goldfarb. Plixorafenib (plixo) synergizes with MEK inhibitors (MEKi) in MAPK pathway inhibition in BRAF V600 and non V600 alterations, with higher potency compared to early generation BRAFi and pan-RAFi [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4609.