Mechanisms of Resistance to MAPK Pathway Inhibition in RAS-Mutant Cancers

Abstract

The RAS family of genes are among the most frequently mutated genes in human cancers, including nearly all pancreatic cancers, ~40% of colorectal cancers, and ~30% of lung cancers. Although most RAS-mutant cancers depend on RAS signaling for proliferation and survival, direct RAS inhibitors have not yet been developed for clinical use. An alternative approach to treating RAS-mutant cancers is to inhibit RAS effector pathways, such as the RAS-RAF-MEK-ERK (MAPK) pathway. However, while some patients with RAS-mutant cancers benefit clinically from MAPK pathway inhibition (MAPKi), most do not respond to this regimen. Moreover, of the patients who do respond, nearly all progress to secondary therapeutic resistance. In this work, we applied systematic gain- and loss-of-function (GOF and LOF) screening approaches to identify modifiers of MAPK pathway dependence. Using RAS- or BRAF-mutant pancreatic and lung cancer cell lines, we performed a genome scale open reading frame (ORF) screen and six genome scale CRISPR-Cas9 knockout screens to investigate mechanisms of resistance to MEK or BRAF inhibition. We found that the most potent GOF mediators of resistance were overexpression of components of the RTK-RAS-MAPK pathway, which restored MAPK signaling. Contrastingly, the majority of LOF events that mediated resistance to MAPKi were not direct regulators or effectors of the MAPK pathway. From our LOF screens, we identified KEAP1 and CIC as generalizable modulators of resistance to MAPKi in RAS-mutant cancers. We found that KEAP1 deletion mediates resistance through mechanisms orthogonal to the MAPK pathway, such as reducing oxidative stress and promoting anabolic metabolism. Conversely, CIC loss promotes resistance by partially restoring the transcriptional pathway downstream of ERK. Understanding mechanisms of intrinsic resistance can enable the identification of predictive biomarkers that improve patient selection for targeted therapy. In addition, identifying mechanisms of acquired resistance can inform the development of novel agents or combination therapy strategies. Our studies highlight the ability of systematic and comprehensive in vitro functional screens to identify clinically relevant mediators of resistance and to provide novel insights into well-studied pathways. While we selected specific genes for detailed mechanistic studies, other genes that were identified from our screens may contribute additional biological insights.