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CRISPR Framework Helps Identify Combination Therapies for Optimal Targeting of KRAS Mutant

NEW YORK (GenomeWeb) – Researchers from the University of California, San Francisco used a genome-scale CRISPR interference (CRISPRi) functional genomics platform to systematically identify genetic interactions with a mutant form of KRAS called KRASG12C, and found combination therapies that target genes involved in its activity.

Inhibitors targeting KRASG12C are a promising new class of targeted cancer therapeutics, the authors noted in their study in Science Signaling yesterday. The inhibitors react with the mutant cysteine residue by binding covalently to the switch-II pocket that is present only in the inactive guanosine diphosphate (GDP)-bound form of KRASG12C, sparing the wild-type protein. In analyzing cellular models of KRASG12C-mutant lung and pancreatic cancer, the researchers found the presence of genes that were selectively essential in this oncogenic driver-limited cell state.

"Their loss enhanced cellular susceptibility to direct KRASG12C inhibition," the team wrote. "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."

In preclinical studies, an advanced-stage KRASG12C inhibitor called ARS-1620 has very specific anticancer activity against KRASG12C-mutant tumors with no observed dose-limiting toxicity in mice, the researchers noted. However, they added, it is likely that KRASG12C-dependent cancer cells will engage previously dispensable genes and pathways to maintain survival and proliferation.

The team hypothesized that such bypass pathways capable of sustaining cancer cell survival are likely to be distinct from synthetic lethal (SL) dependencies, and termed them collateral dependencies (CDs). Using a genome-wide CRISPRi platform they identified diverse mechanisms by which CDs influence KRASG12C-driven growth upon oncogene inactivation.

"This approach identified specialized roles of known RAS signaling components and highlights CDs involved in transcriptional regulation and other cellular processes outside the core RAS pathway," the authors wrote. "In a large panel of KRASG12C-driven cancer cells, the vast majority of CDs identified in our experiments are not SL, thus demonstrating that CDs are biologically distinct from SL dependencies."

The researchers then used pharmacology to analyze therapeutically targetable CDs and found that chemical inhibition of both known and unexpected RAS pathway genes resulted in KRASG12C inhibition. In investigating the mechanisms of these drug interactions, they further found that these combination therapies either directly cooperated with switch-II pocket inhibition to increase KRASG12C target engagement or independently blocked residual survival pathways.

"Both classes of combinations tested ultimately resulted in a deepened suppression of common signaling nodes, emphasizing the convergent nature of the oncogenic RAS signaling network," the team wrote. "Our study uncovered diverse mechanisms by which collateral pro-oncogenic signaling proteins sustain a mutant KRAS-dependent cancerous phenotype after acute chemical inhibition of KRASG12C."

Importantly, the researchers also noted that their study revealed that single-target inhibition of even the most well-validated oncogenic drivers can be limited by CDs that help sustain oncogenic phenotypes in the driver-limited state.

"The concept that a cancerous phenotype can be driven by the activity of a single oncogene is being revised given that, beyond BCR-ABL inhibitors in chronic myelogenous leukemia, targeted therapies have fallen short on their promise of promoting durable responses and cures for patients," they concluded. "We nominate CDs to be the underlying cellular processes that are engaged upon driver oncogene inhibition and limit the efficacy of targeted therapeutics."