Mechanistic insights into bacillus Calmette-Guérin (BCG) sensitivity in non-muscle invasive bladder cancer (NMIBC) with ERCC2 alterations.

813 Background: Alterations in the nucleotide excision repair (NER) gene ERCC2 are common in bladder cancer (BC) and confer increased tumor mutational burden (TMB) and neoantigen load. Clinically, ERCC2- altered NMIBC tumors are associated with a favorable response to BCG. Because BCG can generate DNA-damaging reactive oxygen species (ROS) which are repaired by the NER pathway, we sought to determine whether differential response to ROS can explain the sensitibity of ERCC2 -altered NMIBC to BCG. To this end, we treated ERCC2 -altered and wild-type (WT) BC cell lines with hydrogen peroxide (H2O2) to assess the effect of free radical-induced DNA damage on cell death. Methods: The previously described BC cell line KE183 ( ERCC2 -altered KU-19-19 via CRISPR/Cas9) and parental cells (WT KU-19-19) were treated for 48 hours with graded concentrations of H2O2 using serial dilutions by two with a maximum dose of 500uM. A mono-tetrazolium salt (MTT) cell viability assay was performed following treatment and dose-response curves were generated to calculate half-maximal inhibitory concentrations (IC50) across experimental replicates (n=8). Results: The parental (WT) cell line exhibited an IC50 of 39.6 ± 3.3 uM H2O2 while the ERCC2 -altered KE183 cell line demonstrated an IC50 of 43.5 ± 7.2 uM H2O2, indicating comparable levels of cell death in response to oxidative stress. Despite the established increased sensitivity of ERCC2 -altered tumors to intravesical BCG, these results reveal no measurable difference in cytotoxicity following exposure to ROS. Conclusions: These findings suggest that the enhanced BCG sensitivity observed in ERCC2 -altered NMIBC is independent of free radical-induced DNA damage. Instead, BCG sensitivity associated with ERCC2 alterations likely reflects immunogenic mechanisms, such as increased TMB and neoantigen presentation, rather than differences in ROS-mediated DNA damage. Further investigation into ERCC2 alterations as a biomarker of the immunologic effects, rather than oxidative stress, of BCG treatment sensitivity in NMIBC are warranted.