Abstract Colorectal cancer (CRC) is one of the most common malignant tumors worldwide. Current treatment options for CRC include surgery, chemotherapy, and radiotherapy. Chemoresistance is a major factor contributing to poor prognosis in CRC patients. Oxaliplatin is recommended as a first-line chemotherapeutic agent for CRC, and overcoming oxaliplatin resistance holds great clinical significance. In this study, multi-omics cohort analysis using the ARGO database revealed for the first time that Replication Termination Factor 2 (RTF2) exhibits high-frequency mutations in CRC and shows elevated RNA and protein expression levels. We established 42 CRC organoids and performed sequencing and drug-sensitivity assays; RTF2 expression was markedly higher in the oxaliplatin-resistant organoid cohort. As a conserved replication-termination factor, RTF2 prevents DNA degradation during replication-fork stalling, maintains genome stability, and plays a critical role in replication-fork restart. Its high expression is significantly correlated with poor patient prognosis. We found that RTF2 is substantially upregulated in CRC cells compared with normal intestinal epithelial cells, and its expression is also elevated in oxaliplatin-resistant cell lines. Therefore, we generated RTF2-overexpression and RTF2-knockdown CRC cell models, as well as a subcutaneous xenograft nude-mouse model. Using the RTF2 inhibitor Aphidicolin in combination with oxaliplatin, we verified that targeting RTF2 enhances the cytotoxic effect of oxaliplatin on CRC cells. In recent years, increasing evidence has shown that many transcription factors exert biological functions through phase separation. RTF2, which contains an intrinsically disordered region (IDR), may also possess this property. We observed that fluorescently tagged RTF2 formed liquid-like droplets in the nucleus, suggesting potential phase separation, which was further confirmed by FRAP assays. Treatment of CRC cells with the phase-separation inhibitor 1,6-hexanediol reduced their oxaliplatin resistance. Results from comet assays and γH2AX immunofluorescence indicated that the mechanism by which RTF2 regulates resistance may be related to DNA double-strand break repair. Using DR-GFP and EJ5-GFP reporter systems, we found that 1,6-hexanediol significantly inhibited homologous recombination (HR) repair efficiency. These findings suggest that in CRC, RTF2 may enhance oxaliplatin resistance by undergoing phase separation and promoting HR-mediated DNA damage repair. In summary, our study reveals for the first time that RTF2 enhances oxaliplatin resistance in colorectal cancer by undergoing phase separation and promoting HR-mediated DNA repair. This discovery provides new mechanistic insights and potential therapeutic targets for overcoming chemoresistance and improving patient prognosis in CRC. Citation Format: Yiran Bie,Chong Chen,Haiqin Jiang,Zhizhong Xiong,Bin Zhong,Haoyang Xv,Yufeng Chen,Lei Lian,Xiaojian Wu,Peishan Hu. RTF2 drives oxaliplatin resistance in colorectal cancer by promoting phase separation-mediated homologous recombination repair abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3115.
Bie et al. (Fri,) studied this question.