Quantifying replication stress in cancer without proliferation confounding

Replication stress (RS) is a major driver of genomic instability and cancer development, caused by impaired DNA replication that can lead to chromosomal instability (CIN). Although RS is mechanistically linked to CIN, its relationship with cellular proliferation is complex. Depending on the context, RS can either promote or suppress cell growth. Existing RS gene expression signatures overlook this complexity, relying on the overexpression of oncogenes such as MYC, which introduces a proliferation bias. To disentangle genuine RS from confounding cell cycle and proliferation transcription profiles, we developed and validated a novel gene expression signature that accurately predicts RS independently of oncogene activity. This signature captures RS-related transcriptional changes across diverse cellular contexts, enabling a more robust and proliferation-independent measure of RS in both experimental and clinical samples. Applying our signature to patient data, we discovered a link between RS and the non-homologous end-joining (NHEJ) DNA repair pathway. Specifically, we observed that MSH2 and MSH6—core components of mismatch repair—are associated with elevated RS and may indicate a shift toward NHEJ-mediated repair under stress conditions. Our study provides a refined approach to quantify RS and sheds light on its broader impact on DNA repair network dynamics.