Abstract Background: Colorectal cancer (CRC) is the second most common cause of cancer deaths in the USA. Oxaliplatin containing regimens are generally considered first line therapy for fit patients, either in the form of FOLFIRINOX or NALIRIFOX. However, acquired resistance is inevitable and a significant percentage of patients treated with adjuvant FOLFOX (5-FU/leucovorin/oxaliplatin) fail to respond. Furthermore, oxaliplatin treatment has toxic side effects. Recent studies have shown that oxaliplatin kill cells by inducing stress to nucleolus. We and others have shown that oxaliplatin, but not cisplatin resistance in tumor cells is dependent upon the levels of DDB2, a DNA repair protein involved in damage recognition in chromatin during global genome nucleotide excision repair (GG-NER). However, the role of DDB2 in suppressing oxaliplatin induced nucleolar stress is not known. Therefore, defining the molecular roles of DDB2 in oxaliplatin response, to assess CRC response rates correlate with low DDB2 levels and the mechanisms of how DDB2 acts to remove these DNA lesions will fill an urgent gap in our knowledge. Methods: DDB2 protein levels were measured by western blot and IHC assay in CRC tissue microarrays. The IC50s for oxaliplatin and cisplatin was obtained by performing cell titer glo (CTG) cell viability and clonogenic assay. The accumulation of DDB2 in nucleolus after oxaliplatin treatment was imaged by confocal microscopy. We used our newly developed SMADNE assay to calculate DDB2 binding kinetics and equilibrium dissociation constant (KD). Results: We found that high DDB2 expressing CRC cell lines have higher IC50 for oxaliplatin and DDB2 KO cells are more sensitive to oxaliplatin than cisplatin therapy. Our IHC data suggests that DDB2 is differentially expressed in CRC patient’s tumor. Interestingly, single molecule analysis by C-trap, reveals that DDB2 binds to oxaliplatin and cisplatin adducts with similar affinities, indicating that while DDB2 recognizes cisplatin lesions loss of DDB2 does not increase cisplatin sensitivity. Confocal microscopic imaging demonstrates that oxaliplatin treatment dramatically facilitated DDB2 accumulation in nucleolus with mutual colocalization of NPM1 (a marker for nucleolar stress). The combination of DDB2 inhibitors (NAEi or CSN5i-3) and oxaliplatin kills HCT116 CRC cells synergistically. Conclusions: Our studies demonstrate that DDB2 is a potential biomarker for response to oxaliplatin and a new clinically targetable protein in the treatment of CRC. These studies will lay the foundation for clinical trials of combination therapy of Pevonedistat or CSN5i with oxaliplatin and 5FU. Citation Format: Vinod Kumar, Dennis Hsu, Orlando D. Schärer, Matthew A. Schaich, Katherine E. Helfrich, Simon C. Watkins, Bennett Van Houten. Defining the role of nucleolar stress in mediating DDB2 induced oxaliplatin resistance in colorectal cancer 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 3117.
Kumar et al. (Fri,) studied this question.