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.

为了揭示KRAS突变CRC细胞的脆弱性，在包含条件致癌KRAS ^G12V的CaCo2细胞中进行了基于shRNA的筛选，该筛选特异性抑制MAPK途径的成分和靶标。定制设计的shRNA文库包含121个选定的基因，这些基因先前已被确定对MEK抑制有强烈的调节作用。屏幕显示，表达KRAS ^G12V的CaCo2细胞对DNA复制许可因子微染色体维持复合物7（MCM7）的抑制敏感，而KRAS ^wtCaCo2细胞在很大程度上抵抗MCM7抑制。在同基因的DLD-1细胞培养模型中获得了相似的结果。如核RPA聚焦增加所示，在KRAS突变背景下敲低MCM7导致复制应激。进一步的研究表明，同时MCM7敲低和KRAS ^G12V后有丝分裂细胞显着增加表达。有丝分裂细胞百分比的增加与有丝分裂中DNA损伤的强烈增加相吻合。综上所述，有丝分裂细胞中DNA损伤的积累是由于复制应力仍未解决，导致了有丝分裂灾难和细胞死亡。总之，数据显示出KRAS突变细胞容易受到MCM7抑制的抑制，并表明抑制DNA复制许可可能是靶向KRAS突变癌症的可行策略。