New tools to study the role of RAS/CRAF interaction in RAS-driven lung cancer.

The RAF/MAPK pathway is a major RAS effector pathway implicated in RAS oncogenic properties, regulating a diversity of cellular processes. As RAF kinases (A-, B-, CRAF) are thought to be attractive therapeutic targets for RAS-driven cancers, pan-RAF inhibitors are currently under development. However, preclinical studies focusing on targeting only the CRAF kinase by genetic inactivation revealed effective inhibition of tumor development without inducing significant systemic toxicities in a model of KRAS-driven lung tumor initiation. Recently, Sanclemente et al. further demonstrated that the complete ablation of CRAF in established advanced lung tumors (KrasG12V/Trp53) triggers sustainable tumor regression. Although this approach validates CRAF as a powerful therapeutic target for KRAS-driven lung cancer, specific CRAF inhibitors are not available yet. It was described more than 20 years ago that RAS GTPases strongly interact with CRAF kinase through its Ras Binding domain. This interaction is required to activate CRAF. Many efforts are being made to try to disrupt this interaction with small compounds, but currently none of those drugs are potent enough for further testing in vivo. Surprisingly, this therapeutic approach has never been proven to be efficient in vivo. Using CRISPR/Cas9 technology, we engineered a new mouse model in which the Ras binding domain of CRAF is constitutively mutated to completely abolish its interaction with RAS GTPases (CRAFR89L). We also engineered two murine lung cancer cell lines (KrasG12D/Trp53) that are heterozygous for this mutation (CRAFR89L/KO). Preliminary data confirm that CRAF interaction with RAS is minimally involved in 2D cell proliferation, as well as KRAS-driven MAPK pathway activation, but important for anchorage-independent cell growth. Citation Format: Romain Baer, Ian Rosewell, David Hancock, Miriam Molina-Arcas, Julian Downward. New tools to study the role of RAS/CRAF interaction in RAS-driven lung cancer [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 B19.