Abstract LB-114: RAF inhibitor LXH254 effectively inhibits B-and-CRAF, but not ARAF
Authors: Monaco Kelli-Ann , Delach Scott , Fordjour Paul , Parikh Salonee , Feng Yun , Jaskelioff Mariela , Stuart Darrin , Caponigro Giordano 
Published: August 2019

RAS mutant tumors have proven largely refractory to inhibitors of the MAPK pathway in clinic. In contrast, selective inhibitors of BRAFV600E but not wild type RAF have been successfully developed in BRAFV600E mutant melanoma, NSCLC, and thyroid cancers. Success of these drugs results largely from the therapeutic index imparted by lack of activity against wild RAFs; however, this selectivity also prevents their utility in indications beyond BRAFV600E disease, such as RAS-mutant tumors. In contrast, MEK1/2 inhibitors, while effective pre-clinically in RAS mutant models, have proven ineffective against RAS mutant tumors due at least in part to the poor therapeutic index of these molecules that results from pathway inhibition in normal cells. Thus, to inhibit effectively MAPK pathway signaling in tumors, inhibitors with improved tolerability via differential pathway inhibition in tumor vs normal tissues are required. Recent data in mice conditionally lacking different RAF paralogs suggest that inhibitors biased towards CRAF might retain anti-tumor properties, with improved tolerability. LXH254 is a RAF inhibitor with high selectivity and nanomolar potency for BRAF and CRAF. Profiling of LXH254 activity in Ras mutant lines lacking either ARAF, BRAF or CRAF by CRISPR-Cas9 modification revealed that loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. This increased sensitivity to LXH254 from ARAF knockout was rescued by over-expression of wild-type ARAF but not by either ARAF that harbored mutations that impaired kinase activity (K336M, D447A) or its ability to dimerize (R362H). Comparing the effect of LXH254 on MAPK pathway signaling in RAS mutant cells engineered to express only one of the three RAF paralogs indicated that significantly higher concentrations of LXH254 are required to inhibit MAPK signaling in cells expressing only ARAF. Furthermore, at low concentrations of LXH254, cells expressing only ARAF displayed paradoxical activation of MAPK signaling in a manner similar to the BRAFV600E selective inhibitor dabrafenib. Thus, both LXH254 and dabrafenib inhibit mutationally activated monomeric BRAF, and promote/stabilize RAS-dependent dimerization of WT RAFs. However, because dabrafenib is only able to effectively inhibit one of the two RAF protomers in each dimer it paradoxically activates MAPK signaling in a RAS-dependent manner. In contrast, LXH254 retains activity against dimerized B-and C-RAF (but not ARAF) thereby theoretically extending its utility beyond BRAF mutant disease with better tolerability relative to, e.g. MEK1/2 inhibitors.

Citation Format: Kelli-Ann Monaco, Scott Delach, Paul Fordjour, Salonee Parikh, Yun Feng, Mariela Jaskelioff, Darrin Stuart, Giordano Caponigro. RAF inhibitor LXH254 effectively inhibits B-and-CRAF, but not ARAF [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-114.