KRAS ^G12C inhibition produces a driver-limited state revealing collateral dependencies

Abstract

Inhibitors targeting KRASG12C, a mutant form of the guanosine triphosphatase (GTPase) KRAS, are a promising new class of oncogene-specific therapeutics for the treatment of tumors driven by the mutant protein. These inhibitors react with the mutant cysteine residue by binding covalently to the switch-II pocket (S-IIP) that is present only in the inactive guanosine diphosphate (GDP)–bound form of KRASG12C, sparing the wild-type protein. We used a genome-scale CRISPR interference (CRISPRi) functional genomics platform to systematically identify genetic interactions with a KRASG12C inhibitor in cellular models of KRASG12C mutant lung and pancreatic cancer. Our data revealed genes that were selectively essential in this oncogenic driver–limited cell state, meaning that their loss enhanced cellular susceptibility to direct KRASG12C inhibition. We termed such genes “collateral dependencies” (CDs) and identified two classes of combination therapies targeting these CDs that increased KRASG12C target engagement or blocked residual survival pathways in cells and in vivo. From our findings, we propose a framework for assessing genetic dependencies induced by oncogene inhibition.

Defining and targeting adaptations to oncogenic KRASG12Cinhibition using quantitative temporal proteomics

“…Finally, CDK4/6i and SHP2i worked in combination with KRASi in multiple 3D cultures supporting recent studies that tested its potential in combination with MEKi (Haines et al, 2018;Mainardi et al, 2018;Ruess et al, 2018). Indeed, in vivo pre-clinical evaluation of CDK4/6i in combination with KRASi showed promising results (Lou et al, 2019). Overall, our 26 approach was able to predict useful combinations with KRASi and drugs targeting multiple pathways that will benefit from further evaluation in pre-clinical models.…”

“…Next, we selected the 15 top up and downregulated pathways at 24 h and 7 days and analyzed their temporal response ( Figure S1D To assess phosphorylation-dependent signaling pathways after KRASi, we performed a global quantitative phosphoproteomic analysis identifying 21,048 phospho-peptides (Table S3). KRASi induced minor differences in the 1 h and 4 h phosphoproteome ( Figure S3A that is preferentially phosphorylated and activated in KRAS mutant cells, showed a decrease in phosphorylation upon KRAS inhibition (Lou et al, 2019;Vallejo et al, 2017). To identify the overall response pattern, we performed gene ontology (GO) enrichment analysis (biological processes) at 24 h, which revealed inhibition in DNA replication and cell cycle consistent with a cytostatic response ( Figure S3E-F).…”

“…We were further interested in correlating our proteome data to recent large scale efforts identifying KRAS collateral dependencies in the context of pharmacologic KRAS G12C inhibition. We compared our H358 KRASi proteome datasets (24 h and 7 days) to a recent CRISPR interference (CRISPRi) functional genomic screen with KRASi (Lou et al, 2019) ( Figure S4A-B). While this analysis showed poor correlation between datasets on a gene by protein basis, likely given the differential biological and technical approaches used, overall, both analyses identified dependencies in pathways such as cell cycle, cytoskeleton/cell adhesion and lipid biosynthesis (Lou et al, 2019).…”

“…We compared our H358 KRASi proteome datasets (24 h and 7 days) to a recent CRISPR interference (CRISPRi) functional genomic screen with KRASi (Lou et al, 2019) ( Figure S4A-B). While this analysis showed poor correlation between datasets on a gene by protein basis, likely given the differential biological and technical approaches used, overall, both analyses identified dependencies in pathways such as cell cycle, cytoskeleton/cell adhesion and lipid biosynthesis (Lou et al, 2019). This suggests that proteome analysis and combination CRISPR/Cas9 screens can provide complementary outputs towards a similar goal of identifying compensatory pathways to target for effective combination therapy.…”

Inhibition of both mutant and wild-type RAS-GTP in KRAS G12C colorectal cancer through cotreatment with G12C and EGFR inhibitors

“…Previously, it has been empirically shown that co-targeting EGFR along with a G12C inhibitor can result in increased inhibition of the KRAS G12C mutant in lung cancer and pancreatic cancer cell lines (12,13,(23)(24)(25)(26). We desired to test this combination in our colorectal cancer cells.…”