Abstract
To unravel vulnerabilities of KRAS-mutant CRC cells, a shRNA-based screen specifically inhibiting MAPK pathway components and targets was performed in CaCo2 cells harboring conditional oncogenic KRASG12V. The custom-designed shRNA library comprised 121 selected genes, which were previously identified to be strongly regulated in response to MEK inhibition. The screen showed that CaCo2 cells expressing KRASG12V were sensitive to the suppression of the DNA replication licensing factor minichromosome maintenance complex component 7 (MCM7), whereas KRASwt CaCo2 cells were largely resistant to MCM7 suppression. Similar results were obtained in an isogenic DLD-1 cell culture model. Knockdown of MCM7 in a KRAS-mutant background led to replication stress as indicated by increased nuclear RPA focalization. Further investigation showed a significant increase in mitotic cells after simultaneous MCM7 knockdown and KRASG12V expression. The increased percentage of mitotic cells coincided with strongly increased DNA damage in mitosis. Taken together, the accumulation of DNA damage in mitotic cells is due to replication stress that remained unresolved, which results in mitotic catastrophe and cell death. In summary, the data show a vulnerability of KRAS-mutant cells towards suppression of MCM7 and suggest that inhibiting DNA replication licensing might be a viable strategy to target KRAS-mutant cancers.
Highlights
RAS genes constitute the most commonly mutated oncogenes in human malignancies and serve as drivers of cellular transformation and tumor maintenance[1]
We demonstrate that the suppression of maintenance complex component 7 (MCM7) is synthetic lethal in KRAS mutated colorectal cancer cells, which rely on high MCM7 levels
We find that endogenous or weak ectopic expression of mutant KRAS did not cause detectable replication stress by itself, suggesting that cells harboring endogenous mutant KRAS levels have adapted to oncogene-induced stress by activation of DNA damage repair[50]
Summary
RAS genes constitute the most commonly mutated oncogenes in human malignancies and serve as drivers of cellular transformation and tumor maintenance[1]. Even though RAS genes were the first oncogenes to be discovered, no targeted therapy for KRAS, NRAS, or HRAS mutant cancers has made its way to clinical application. This failure was due to the high affinity of RAS proteins for the cofactor GTP, rendering its displacement by competing drugs inefficient, and due to mutation, which occurs in a small subset of KRAS-mutant cancer patients, were identified and further developed[3,4]. RAS proteins activate downstream signaling pathways via different effectors including the RAF proteins, RAL-GDS, and PIK3CA among others.
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