Purpose: Targeting RAF for antitumor therapy in RAS-mutant tumors holds promise. Herein, we describe in more detail novel characteristics in the type II RAF inhibitor, LXH254.

Experimental design: LXH254 was profiled in biochemical, in vitro, plus vivo assays, including analyzing individuals activities in the drug in the large panel of cancer-derived cell lines plus a comprehensive number of in vivo models. Furthermore, activity of LXH254 was assessed in cells where different groups of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF.

Results: We describe an unpredicted paralog selectivity of LXH254, which has the ability to potently hinder BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and NRAS mutants, but had only modest activity in KRAS mutants. In RAS-mutant lines, insufficient ARAF, while not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated capacity LXH254 needed both kinase function and dimerization. Greater concentrations of LXH254 were required to hinder signaling in RAS-mutant cells expressing only ARAF in compliance with BRAF or CRAF. In addition, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in the manner similar to dabrafenib. Finally, in vivo, LXH254 drove complete regressions of isogenic variants of RAS-mutant cells missing ARAF expression, while parental lines were only modestly sensitive.

Conclusions: LXH254 can be a novel RAF inhibitor, which has the ability to hinder dimerized BRAF and CRAF, additionally to monomeric BRAF, while largely sparing ARAF.