Molecular therapies targeting the EGFR/MAPK signaling pathway have markedly improved treatment outcomes for colorectal cancer (CRC). However, the emergence of secondary resistance—whether through pre-existing factors or adaptive mechanisms—remains a major challenge in achieving long-term therapeutic success. The DNA damage response (DDR) plays a pivotal role in enabling cancer cells to cope with therapy-induced genotoxic stress. In CRC, dysregulation of the DDR pathways contributes to the survival of tumor cells despite DNA damage, suggesting DDR as a potential therapeutic target in the context of secondary resistance.

We generated a series of models with acquired resistance to anti-EGFR based targeted therapies, referred to as the ARes platform. To assess the impact of secondary resistance on cancer cells' dependence on DDR pathways for survival, we performed a drug screening, by exposing parental and resistant models to a range of DDR inhibitors (DDRi). In vivo validation of these findings was performed using xenograft mouse models. Mechanistic studies were conducted to evaluate DNA damage levels, repair capacity, cell cycle dynamics, and replication stress in selected cell pairs. These studies employed a range of techniques, including western blotting, comet assay, immunofluorescence for foci analysis, cytofluorimetry, and fiber assay, to provide comprehensive insights into WEE1i efficacy.

Pharmacological screening of the ARes platform with several DDRi revealed WEE1 as the most promising therapeutic target. In vivo experiments using xenograft models demonstrated that CRCs with acquired resistance to anti-EGFR therapies exhibited sustained or even enhanced sensitivity to WEE1i, consistent with in vitro observations. Mechanistic investigations showed that resistant cells maintain constitutive activation of the MAPK pathway and present heightened basal levels of DNA damage. In the same models, elevated RAD51 expression mediates tolerance to DNA damage and replication stress. However, we found that WEE1i treatment accelerates G2/M entry and disrupts RAD51 activity, leading to higher DNA damage accumulation and consequent cell death. Remarkably, these effects were observed even at sublethal concentrations of WEE1i, suggesting potential for synergistic strategies when combined with conventional chemotherapeutic agents.

Our results highlight the therapeutic potential of WEE1 inhibition in CRC patients who have developed acquired resistance to EGFR-targeted therapies. These findings provide a preclinical evidence for future clinical investigations into the use of WEE1 inhibitors as part of combination therapies to overcome secondary resistance in CRC.