Targeting MAD2L2-dependent translesion synthesis impairs DNA damage tolerance and enhances cellular response to cisplatin.

Translesion synthesis (TLS) is a DNA damage tolerance pathway that enables cells to replicate across damaged DNA, thereby promoting cell survival under genotoxic stress, however, contributing to genomic instability and therapy resistance. C#3 is a small molecule that disrupts the interaction between MAD2L2 and Rev1, a key complex required for DNA polymerase ζ-mediated TLS. Here, we further investigated the impact of C#3 on MAD2L2-dependent TLS activity and cellular responses to DNA damage. Consistent with our previous findings, C#3 enhanced cellular sensitivity to cisplatin in multiple cancer cell lines and increased DNA damage signaling following treatment. Using a non-replicating plasmid assay, we demonstrate that C#3 impairs lesion bypass across DNA adducts in mammalian cells. Disruption of the MAD2L2-Rev1 axis was further supported by reduced formation of MAD2L2-Rev1 complexes, as assessed by proximity ligation assay. Functionally, cells exposed to C#3 during recovery from cisplatin treatment exhibited persistent γH2AX signaling, consistent with delayed resolution of replication-associated DNA damage. In-vivo, combined treatment with C#3 and cisplatin reduced tumor growth in syngeneic melanoma and triple-negative breast cancer mouse models compared with either treatment alone. Together, these findings demonstrate that pharmacological disruption of the MAD2L2-Rev1 axis impairs TLS associated DNA damage tolerance, enhances cellular responses to cisplatin-induced DNA damage, and suppresses tumor growth in-vivo. These results support targeting MAD2L2-dependent TLS as a potential strategy for improving the efficacy of DNA damaging chemotherapy.