Abstract B25: Combined proteomic and genetic interaction mapping reveals new Ras pathway effectors and regulators
Authors: Kelly Marcus , Han Kyuho , Kostyrko Kaja , Mooney Nancie , Jeng Edwin , Demeter Janos , Sweet-Cordero Alejandro , Bassik Michael , Jackson Peter K. 
Published: June 2021

Despite intensive study, no drugs in clinical use specifically target KRAS-mutant tumors. Uncharacterized feedback pathways and unmapped compensatory pathways, including compensation among paralogs, hinder our ability to target Ras effector pathways, requiring a better catalogue of pathways upstream and downstream of Ras. We used tandem affinity purification of Kras, Hras and Nras, their activated alleles and key proteins with known regulatory (GEFs, GAPs) or effectors (Raf, RalGDS1, RIN1/2) in both 293 cells and A549 NSCLC cells to generate a high-confidence protein-protein interaction (PPI) network of 220 proteins showing 1,400 physical interactions. The network was used to design an sgRNA library (10 sgRNAs/gene) and screen Cas9-expressing A549 cells for strong growth dependencies. These data were then used to select 120 genes and construct a 2-gene tandem sgRNA library of highest relevance to the Ras pathway (with 60 control sgRNAs). This 2-gene sgRNA library was tested in A549 and H23 NSCLC lines for quantitative single and two gene-dependent quantitative changes in growth, showing 100s of strong synthetic lethals among 14K pairwise tests. These genetic interactions in conjunction with PPIs and TCGA data identify extensive coupling between Raf/MEK/ERK kinases, Ral and Rap GTPases, the Rap1GDS1 small GTPase controller, and RADIL cell adhesion pathways. The screen identified new candidate effector pathways for cell adhesion, Rap GTPase regulation, and protein processing, including new understudied Kras direct effectors RADIL, RGL1/2/3, and RIN1/2. Additional 20 x 20 custom libraries were screened in a broader panel of Kras-mutant versus other NSCLC lines. These screens revealed systematic Kras-dependent synthetic lethality among components of the MAP kinase pathway (ERK1/ERK2, ERK1/RAF1, MEK1/MEK2 etc.) and other interactions between the MAPK pathway and components of the Ral and Rap GTPase, RADIL cell adhesion pathways and RIN1-dependent macropinocytosis pathways. Using the recent Kras G12C inhibitor in H23 cells, we have validated that sgRNA knockouts of these Kras effector affect these specific, new pathways: cell adhesion via RADIL, growth signaling via Rap1GDS1 and RhoA, and macropinocytosis via the Rab5 GEF RIN1. Application of the Kras inhibitor ARS-853 shows much-reduced effects on specific Kras effector pathways in cells deleted for these specific effectors, showing these effectors are highly coupled to Kras. Our systematic data reveal new genetic vulnerabilities and target candidates with potential for new therapeutics.

Citation Format: Marcus Kelly, Kyuho Han, Kaja Kostyrko, Nancie Mooney, Edwin Jeng, Janos Demeter, Alejandro Sweet-Cordero, Michael Bassik, Peter K. Jackson. Combined proteomic and genetic interaction mapping reveals new Ras pathway effectors and regulators [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 B25.