Highlights d Loss of SFPQ/PSF causes apoptosis in BRAF V600E -driven cancer cells d BRAF-mutant cells depend on SFPQ to maintain faithful DNA replication d SFPQ depletion upon BRAF V600E induction causes R-loop formation d Inhibition of ATR kinase Chk1 sensitizes BRAF V600E cells to replication stress

Expression of the epidermal growth factor ligands amphiregulin (AREG) and epiregulin (EREG) is positively correlated with a response to EGFR‐targeted therapies in colorectal cancer. Gene‐body methylation sites, which show a strong inverse correlation with AREG and EREG gene expression, were identified in cell lines using targeted 454 FLX‐bisulfite sequencing and SIRPH analyses for AREG/EREG promoters and intragenic CpGs. Upon treatment of colorectal cancer cells with 5‐aza‐2′‐desoxycytidine, methylation decreases at specific intragenic CpGs accompanied by upregulation of AREG and EREG gene expression. The same AREG gene‐body methylation was also found in human colorectal cancer samples and is independent of KRAS and NRAS mutations. Methylation is specifically decreased in the tumor epithelial compartment as compared to stromal tissue and normal epithelium. Investigation of a promoter/enhancer function of the AREG exon 2 region revealed a potential promoter function in reverse orientation. Retrospective comparison of the predictive power of AREG gene‐body methylation versus AREG gene expression using samples from colorectal cancer patients treated with anti‐EGFR inhibitors with complete clinical follow‐up revealed that AREG expression is superior to AREG gene methylation. AREG and EREG genes undergo a complex regulation involving both intragenic methylation and promoter‐dependent control.

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.