CRAF-mediated inactivation of epigenetic repressor complexes promotes KRAS-driven tumorigenesis.

Oncogenic mutations in KRAS serve as the initiating event in many human cancers, most notably pancreatic and lung cancers. While KRAS can signal through multiple downstream effector pathways, the MAPK pathway functions as one of the most critical downstream effectors promoting tumorigenesis. Despite its prominent role, inhibitors targeting the MAPK pathway have limited efficacy in KRAS mutant tumors. In the case of BRAF inhibitors, paradoxical activation is observed in KRAS-mutant tumors, while MEK and ERK inhibitors disable negative feedback loops resulting in pathway reactivation. Combinations of MEK and ERK inhibitors have been explored clinically, but a safe combination dose has not yet been established. Given this, identifying novel vulnerabilities in KRAS mutant tumors represents a significant unmet clinical need. Hence, we analyzed the dependency of KRAS-mutant tumors in the Achilles dataset and found a significant subset of KRAS and NRAS-mutant tumors that appear to depend on CRAF. Importantly, KRAS-mutant tumors were not dependent on BRAF or ARAF, the other 2 RAF isoforms. This is consistent with the studies in KRAS-mutant lung GEMMs, where CRAF and not BRAF depletion has been shown to reduce tumor incidence, suggesting that CRAF serves as the main signaling hub downstream of mutant KRAS. In this work, we investigate the kinase-dependent and -independent roles of CRAF in KRAS-mutant tumors. We demonstrate that CRAF depletion in KRAS-mutant human cancer cells inhibits colony formation on soft agar assay and mediates tumor growth inhibition in mouse xenograft models. CRAF loss resulted in activation of cell death and cell cycle arrest pathways without any alterations to downstream MAPK signaling. Interestingly, this suppression of growth upon CRAF loss in KRAS mutant cancer cells was rescued by expression of kinase-dead variants of CRAF, suggesting CRAF kinase function is not required to promote KRAS-driven tumorigenesis. In KRAS-mutant tumor cells, we observe that CRAF preferentially heterodimerizes with ARAF and upon treatment with a MEK inhibitor, the dimer preference on CRAF switches to BRAF, resulting in elevated CRAF kinase activity. To explore molecular determinants of CRAF kinase independent functions, we performed an enrichment screen to identify effectors whose loss confers a growth advantage in the CRAF-null KRAS mutant cells. We identified a class of epigenetic repressor complexes that were strongly depleted in the CRAF-null KRAS mutant cells and are in the process of validating these hits. Further, to dissect CRAF kinase-dependent versus -independent functions, we are establishing a CRAF gene signature using KRAS-mutant cancer cells and also, identifying CRAF-interacting partners that promote CRAF kinase-independent functions. Thus far, this study elucidates novel kinase-independent roles of cRAF that could be harnessed to effectively target KRAS-mutant human cancers. Citation Format: Avinashnarayan Venkatanarayan, Christiaan Klijn, Shiva Malek. CRAF-mediated inactivation of epigenetic repressor complexes promotes KRAS-driven tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B14.