AOH1996, a cancer-associated PCNA inhibitor, restores platinum sensitivity in urothelial carcinoma: In vitro and in vivo study

Urothelial carcinoma (UC) frequently develops resistance to platinum-based chemotherapy, resulting in limited treatment efficacy and dismal clinical outcomes. Proliferating cell nuclear antigen (PCNA) is a key regulator of DNA replication and repair, and cancer-associated PCNA (caPCNA), a structurally altered form selectively expressed in malignant cells, has emerged as a promising therapeutic target. AOH1996 is a first-in-class small-molecule inhibitor that selectively binds caPCNA and disrupts PCNA-dependent survival pathways. In this study, we investigated the antitumor activity of AOH1996 in UC and its ability to overcome resistance to cisplatin-based chemotherapy. Cisplatin-sensitive (T24, BFTC905) and cisplatin-resistant (T24/R) UC cell lines were treated with AOH1996 alone or in combination with cisplatin or gemcitabine. AOH1996 significantly reduced cell viability and clonogenic survival in both sensitive and resistant cells, induced apoptosis as evidenced by increased Annexin V-positive fractions and cleavage of caspase-3 and PARP, and caused cell-cycle arrest with accumulation of cells in the S/G2 phase. Mechanistically, AOH1996 downregulated PCNA-dependent DNA repair signaling and enhanced DNA damage, as shown by increased gammaH2AX expression. Combination index analysis demonstrated synergistic interactions between AOH1996 and cisplatin or gemcitabine, particularly in cisplatin-resistant T24/R cells. In a xenograft mouse model of UC, AOH1996 significantly inhibited tumor growth and further potentiated the antitumor effect of cisplatin without causing overt systemic toxicity or significant body weight loss. Together, these findings provide preclinical evidence that targeting caPCNA with AOH1996 exerts robust antitumor activity and restores chemosensitivity in UC, supporting caPCNA inhibition as a promising therapeutic strategy for patients with platinum-resistant disease.