LXH254, a Potent and Selective ARAF-Sparing Inhibitor of BRAF and CRAF for the Treatment of MAPK-Driven Tumors

Purpose: Targeting RAF for antitumor therapy in RAS-mutant tumors holds promise. Herein, we describe in detail novel properties of the type H RAF inhibitor, LXH254. Experimental Design: LXH254 was profiled in biochemical, in vitro, and in vivo assays, including examining the activities of the drug in a large panel of cancer-derived cell lines and a comprehensive set of in vivo models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF. Results: We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit 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, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Finally, in vivo, LXH254 drove complete regressions of isogenic variants of RAS-mutant cells lacking ARAF expression, while parental lines were only modestly sensitive. Conclusions: LXH254 is a novel RAF inhibitor, which is able to inhibit dimerized BRAF and CRAF, as well as monomeric BRAF, while largely sparing ARAF.

Automated summary

LXH254, a novel RAF inhibitor, demonstrates potent inhibition of BRAF and CRAF while sparing ARAF. Loss of ARAF, but not BRAF or CRAF, sensitizes RAS-mutant cells to LXH254, which requires both kinase function and dimerization. In cells expressing only ARAF, LXH254 leads to paradoxical activation of MAPK signaling similar to dabrafenib.