Despite intensive study, no drugs in clinical use specifically target KRAS-mutant tumors. Uncharacterized feedback mechanisms and parallel pathways have stymied the treatment of KRAS-mutant tumors with Raf and PI3K inhibitors, and the KRas protein itself does not easily accommodate binding of small-molecule inhibitors. These challenges demand more systematic and quantitative characterization of the physical and genetic relationships between Ras regulators and effectors. Thus we used tandem affinity purification of Kras, Hras and Nras, their activated alleles and key proteins with known regulatory (GEFs, GAPs) or effector (Raf, RalGDS) roles in both 293 cells and A549 NSCLC cells to generate a high-confidence protein-protein interaction (PPI) network. This map of 220 proteins and 1,400 physical interactions was used to design an sgRNA library with 10 guides/gene. This library was screened in Cas9-expressing A549 cells and grown for 14 days before analysis for dropout or enhanced representation of sgRNAs. Approximately 120 genes showed positive or negative growth effects. PPIs and genetic interactions (GIs) were cross-referenced with public PPI data and TCGA patient data to assemble a combined physical PPI and genetic map informed by cancer mutations. This map suggests many hypotheses for PPIs critical for growth control. This set was used to construct a sgRNA library covering 120 genes of probable relevance to the Ras pathway with ~60 “safe harbor” control sgRNAs. This library was screened in a two-cassette sgRNA system testing 14K pairwise genetic effects to identify quantitative changes in growth in A549 and H23 NSCLC lines. This screen showed >100 genetic interactions, which in conjunction with PPIs identify coupling between the Raf/MEK/ERK kinase, Ral and Rap GTPase, RNA processing and cell adhesion pathways. The screen identified new candidate effector pathways for cell adhesion, RNA processing, Rap GTPase regulation and protein processing, including the RADIL, RGL and RIN Kras effectors. Validation focused on using the synthetic lethal interactions observed in the sgRNA screen to predict drug combinations showing drug synergy in A549 and H23 cells. Using 11-point dose titrations and isobologram analysis of drug combinations, we see strong synergy among PI3 kinase, Raf and Erk inhibitors in these cells. Using the recently described Kras G12C inhibitor, expressed in H23 cells, we have validated that sgRNA deletion of the the key Kras effector for specific pathway,s including cell adhesion (RADIL), growth signaling (RAF) and endocytosis/ macropinocytosis (RIN), is affected and that use of the Kras inhibitor ARS-853 shows much-reduced effects on specific Kras effector pathways in cells deleted for these effectors. These systematic data underscore the limitations of our current understanding of Kras-driven cancers, revealing new genetic vulnerabilities and target candidates.

Citation Format: Peter K. Jackson. A combined protein-protein interaction and genetic interaction map defines new and critical Kras effectors in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1313.