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Reduced replication origin licensing selectively kills KRAS-mutant colorectal cancer cells via mitotic catastrophe.

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 KRAS^G12V. 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 KRAS^G12V were sensitive to the suppression of the DNA replication licensing factor minichromosome maintenance complex component 7 (MCM7), whereas KRAS^wt 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 KRAS^G12V 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.

SUBMITTER: Gastl B

PROVIDER: S-EPMC7330027 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Reduced replication origin licensing selectively kills KRAS-mutant colorectal cancer cells via mitotic catastrophe.

Gastl Bastian B Klotz-Noack Kathleen K Klinger Bertram B Ispasanie Sylvia S Salib Krenoula Hani Fouad KHF Zuber Johannes J Mamlouk Soulafa S Bublitz Natalie N Blüthgen Nils N Horst David D Morkel Markus M Schäfer Reinhold R Sers Christine C

Cell death & disease 20200701 7

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 KRAS<sup>G12V</sup>. 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 KRAS<sup>G12V</sup> were sensitive to the suppression of the DNA replication licensin ...[more]

PMID: 32612138

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Project description:BackgroundSotorasib is the first KRASG12C inhibitor approved by the US Food and Drug Administration for treating KRASG12C-mutant non-small-cell lung cancer (NSCLC). Clinical trials on the therapeutic use of sotorasib for cancer have reported promising results. However, KRASG12C-mutant cancers can acquire resistance to sotorasib after treatment. We incidentally discovered that sotorasib-resistant (SR) cancer cells are addicted to this inhibitor. In this study, we investigated the mechanisms underlying sotorasib addiction.MethodsSotorasib-resistant cells were established using KRASG12C-mutant pancreatic cancer and NSCLC cell lines. Cell viability in the presence or absence of sotorasib and in combination with multiple inhibitors was assessed through proliferation assay and annexin V/propidium iodide (PI) flow cytometry assays. The mechanisms underlying drug addiction were elucidated through 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, immunofluorescence staining, time-lapse microscopy, and comet assay. Furthermore, a subcutaneous xenograft model was used to demonstrate sotorasib addiction in vivo.ResultsIn the absence of sotorasib, the sotorasib-resistant cells underwent p21Waf1/Cip1-mediated cell cycle arrest and caspase-dependent apoptosis. Sotorasib withdrawal resulted in robust activation of mitogen-activated protein kinase (MAPK) pathway, inducing severe DNA damage and replication stress, which activated the DNA damage response (DDR) pathway. Persistent MAPK pathway hyperactivation with DDR exhaustion led to premature mitotic entry and aberrant mitosis, followed by micronucleus and nucleoplasmic bridge formation. Pharmacologic activation of the MAPK pathway with a type I BRAF inhibitor could further enhance the effects of sotorasib withdrawal on sotorasib-resistant cancer cells both in vitro and in vivo.ConclusionsWe elucidated the mechanisms underlying the sotorasib addiction of cancer cells. Sotorasib addiction appears to be mediated through MAPK pathway hyperactivity, DNA damage, replication stress, and mitotic catastrophe. Moreover, we devised a therapeutic strategy involving a type I BRAF inhibitor to strengthen the effects of sotorasib addiction; this strategy may provide clinical benefit for patients with cancer.

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Reduced replication origin licensing selectively kills KRAS-mutant colorectal cancer cells via mitotic catastrophe.

Publications

Reduced replication origin licensing selectively kills KRAS-mutant colorectal cancer cells via mitotic catastrophe.

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