Targeting the DNA damage response and replication stress to overcome resistance to targeted therapies in colorectal cancer

Molecular therapies targeting the EGFR/MAPK pathway have improved clinical outcomes in colorectal cancer (CRC). However, primary and acquired resistance continue to limit durable responses. Oncogenic signaling activates stress response pathways that allow cancer cells to adapt to therapeutic pressure, while simultaneously generating exploitable vulnerabilities. Among these, defects in the DNA damage response (DDR) and replication stress (RS) pathways, which are essential for cancer cell survival, may represent promising therapeutic targets. This study aims to develop relevant preclinical models and identify new therapeutic strategies to overcome resistance by targeting DDR and RS mechanisms in colorectal cancer. We initially profiled 112 CRC cell lines to capture the genomic diversity of metastatic CRC and five patient-derived organoid models using a panel of DDR inhibitors (DDRis). Additionally, to investigate the role of DDR in acquired resistance, we developed a CRC platform model (“ARes platform”), comprising both 2D and 3D systems with secondary resistance to EGFR/MAPK inhibitors, and evaluated the response to DDRis. In the initial screening, we found that around 30% of CRC models, including those harboring KRAS or BRAF mutations, were sensitive to at least one RS-targeting DDRi. Acquisition of resistance to EGFR-targeted therapies led to either maintained or enhanced susceptibility to RS pathway inhibition. To define predictive biomarkers, we conducted molecular and functional assays in DDR sensitive and resistant models identified through the pharmacological screening and in parental versus secondary resistance models. A multimodal biomarker, incorporating phospho-RPA32 and RAD51 foci analysis, loss of ATM and RAD51C expression, and mutational signature profiling, emerged as a potential predictor of response to RS inhibitors. Increased DNA damage and replication stress emerged as common biomarkers of sensitivity to RS inhibitors in the acquired resistance setting. These findings support the targeting of replication stress, particularly through ATR and WEE1 inhibition, as a promising strategy to overcome both primary and secondary resistance in CRC. A composite biomarker approach may enable patient stratification and guide future clinical applications