Abstract B014: Phenotypic CRISPR genome-wide screening for the discovery of genetic regulators of allele-specific mutant KRAS

Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most frequently mutated oncogene detected in 30% of all human cancers. Drugs that can impede the function of oncogenic KRAS offer exceptional therapeutic value. However, direct targeting KRAS is challenging due to its protein structure that lacks deep binding pockets for small-molecule inhibitors. Despite the recent success in targeting the G12C allele, targeted therapy for another hotspot mutant – G12V mutant KRAS has not been described. Here, we utilize CRISPR-Cas9 based genome-wide knockout screens and leverage a high-throughput microfluidic cell sorting platform to identify genes that regulate mutant KRAS harboring G12V mutation. The screens were conducted in two colorectal cancer cell lines HT29 and SW480, expressing wild-type KRAS and homozygous G12V KRAS, respectively. We have identified a list of genes whose loss-of-function reduces wild-type or mutant G12V KRAS protein expression. Our top gene identified from the mutant KRAS screen - denoted as selective-mutant KRAS modulator (SMKM) has shown promising results in selectively decreasing G12V KRAS protein expression. Through multiple assays including live cell imaging, western blot, flow cytometry, and proteomic analysis in SMKM knockout cells and SMKM inhibitor-treated cells. We found highly selective reduction in G12V KRAS protein expression. More importantly, genetically or pharmacologically inhibiting SMKM has demonstrated anti-tumor activity. We conducted cell viability assays including CellTiter-glo, MTT, and clonogenic assay. We treated a panel of G12V KRAS mutant and wild-type KRAS cell lines with SMKM inhibitor and found significantly reduced cell viability in G12V KRAS mutant cell lines across colorectal and lung cancer. To further demonstrate the downstream impact on mutant KRAS signaling, secondary assays including western blot and Lumit immunoassay were conducted. We observe a reduction in the aberrant phospho-ERK and phospho-AKT signaling in SMKM inhibitor treated cells. Our in vivo efficacy trial using heterozygous G12V KRAS lung cancer NCI-H441 xenograft model has shown SMKM inhibition significantly reduces tumor growth and increases the survival rate. Together, these data indicate the feasibility of selectively targeting G12V KRAS with SMKM inhibition. Lastly, our results provide a first-in-class small molecule oral inhibitor based selective protein clearance as an effective anti-G12V mutant KRAS therapeutic strategy. Citation