LAUSR

Significance RAF inhibitors unexpectedly induce ERK activation in normal and oncogenic RAS tumor cells, making them unsuitable for treating RAS-driven cancers. The precise mechanism of this paradox is not fully understood but is believed to be RAS dependent. In this study, we discovered that classical RAS proteins are not essential for RAF inhibitor-induced ERK activation in H/N/KRAS-less mouse embryonic fibroblasts. We further showed that the MRAS/SHOC2 complex is required for the classical RAS-independent paradoxical ERK activation. Our findings provide new insights into the mechanism of paradoxical ERK activation by RAF inhibitors, and they have important therapeutic implications for developing effective RAF inhibitors. RAF inhibitors unexpectedly induce ERK signaling in normal and tumor cells with elevated RAS activity. Paradoxical activation is believed to be RAS dependent. In this study, we showed that LY3009120, a pan-RAF inhibitor, can unexpectedly cause paradoxical ERK activation in KRASG12C-dependent lung cancer cell lines, when KRAS is inhibited by ARS1620, a KRASG12C inhibitor. Using H/N/KRAS-less mouse embryonic fibroblasts, we discovered that classical RAS proteins are not essential for RAF inhibitor-induced paradoxical ERK signaling. In their absence, RAF inhibitors can induce ERK phosphorylation, ERK target gene transcription, and cell proliferation. We further showed that the MRAS/SHOC2 complex is required for this process. This study highlights the complexity of the allosteric RAF regulation by RAF inhibitors, and the importance of other RAS-related proteins in this process.