Abstract A095: The paradox-breaker BRAF inhibitor plixorafenib (FORE8394) efficiently inhibits non-V600 mutations and fusions
Authors: Cohen Limor , Filippov-Levy Natalie , Billauer Hana , Birnbaum Lea , Gasasa Sara , Maor Roey , Amrusi Yakir , Abu-Liel Reham , Goldfarb Eden , Tarcic Gabi 
Published: December 2023

Abstract
The use of several BRAF inhibitors to target BRAF V600 (Class I BRAF mutations) mutated tumors has shown clinical benefit in multiple types of cancer. Nonetheless, V600 mutations comprise about 40% of the BRAF-altered patient population, and the remainder of the BRAF mutated patients are in need of novel targeted therapies. Clinical trials aimed at evaluating the effect of these inhibitors in non-V600 patients, mostly in combination with a MEK inhibitor, have been performed with mixed results. However, the experimental sensitivity to a wide range of class 2 (RAS independent non-V600 including BRAF fusions) or class 3 (RAS dependent non-V600) alterations to different types of BRAF inhibitors has not been reported. Here we tested the efficacy of plixorafenib (a novel paradox braker BRAF inhibitor) compared with several BRAF inhibitors in BRAF V600 and non-V600 alterations. We implemented a high-throughput cell-based functional assay to profile the response of different BRAF variants to plixorafenib (paradox breaker), vemurafenib (a first-generation inhibitor) and tovorafenib (a pan-RAF inhibitor). This method quantifies MAPK signaling pathway activation using fluorescent imaging coupled with image analysis of cells expressing the mutated protein together with a fluorescently labeled ERK2 as a signaling pathway reporter. We also validated a subset of the findings using standard western blot and cell viability experiments. Comparing the response of 4 Class I, 26 Class II and 94 BRAF fusions to plixorafenib and vemurafenib Results showed that Plixorafenib was more potent in inhibiting the majority of BRAF alterations tested, i.e., a lower IC50 value was measured for plixorafenib over vemurafenib. Furthermore, of the 94 BRAF fusions proteins tested, over 75% showed at least a 50% inhibition over the concentrations tested, while less than 20% were inhibited by vemurafenib. A smaller cohort of 23 BRAF alterations was tested with the investigational pan-RAF inhibitor tovorafenib. The IC:50 values for the Class I and II BRAF alterations tested were higher with tovorafenib compared with plixorafenib, and 6 of 13 BRAF fusions tested were resistant to tovorafenib and sensitive to plixorafenib. In conclusion, we show here that plixorafenib inhibits a wide range of V600 and non-V600 BRAF alterations, more potently compared with first generation BRAFi (vemurafenib) and a pan-RAF inhibitor (tovorafenib). Importantly, this effect has the potential to overcome an unmet clinical need in cancers with non-V600 alterations.
Citation Format: Limor Cohen, Natalie Filippov-Levy, Hana Billauer, Lea Birnbaum, Sara Gasasa, Roey Maor, Yakir Amrusi, Reham Abu-Liel, Eden Goldfarb, Gabi Tarcic. The paradox-breaker BRAF inhibitor plixorafenib (FORE8394) efficiently inhibits non-V600 mutations and fusions [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A095.