Antitumor synergy of AOH1996 and next-generation caPCNA Inhibitors in combination with targeted and chemotherapeutic agents

Background: AOH1996 is a small molecule that selectively targets the cancer-associated isoform of proliferating cell nuclear antigen (caPCNA), disrupting DNA replication and repair, and promoting DNA strand breaks through inhibition of effective resolution of transcription-replication conflicts. Building on its promising preclinical efficacy, we are also developing and testing next-generation caPCNA inhibitors with improved potency and druglikeness. We have developed two new inhibitors that exhibit up to 10-fold higher cellular potency relative to the parent compound, and an inhibitor with similar potency as the parent compound but 2.4-fold more soluble. Given PCNA's diverse role in processes that cancer cells depend upon for survival, e.g., coordinating DNA synthesis, damage tolerance, cell cycle control, chromatin assembly, and stress response signaling, it is hard to predict what other anticancer drugs would combine productively with AOH1996 and next-generation inhibitors. To that end, we screened a panel of 179 anticancer drugs with AOH1996 and three next-generation inhibitors to identify potential combinations that exhibit synergy. Methods: A systematic combination drug screen was performed using AOH1996 and next-generation inhibitors and a drug panel of 179 different anticancer drugs. Synergy evaluation: We performed initial screens using the human neuroblastoma cell line SK-N-AS. We assessed the synergy of the combinations using the Chou-Talalay method for drug combination analysis. For favorable drug combinations, we tested additional cell lines including cancer cell types where the drug identified would be used clinically in a treatment regimen. Mechanistic studies: We analyzed cell cycle effects and apoptosis by flow cytometry. We also assessed DNA damage and checkpoint activation by using immunoblotting and immunofluorescence to measure gammaH2AX, cleaved PARP, and phospho-Chk1. Results: Both AOH1996 and its next-generation analogs demonstrated synergy with some DNA-damaging and repair inhibiting agents such as cisplatin. Tyrosine kinase inhibitors (TKIs) in particular EGFR TKIs synergized well with AOH1996 and next-generation inhibitors. Inhibitors of the mitochondrial apoptosis pathway (e.g., venetoclax) also synergized as well. Conclusions: AOH1996 and its next-generation analogs exhibit potent synergistic activity with targeted therapies and DNA repair inhibitors, supporting PCNA inhibition as a promising strategy to enhance therapeutic efficacy and overcome resistance. These findings provide a preclinical rationale for clinical development of AOH1996 combination regimens, as well as continued optimization of caPCNA-targeting analogs with superior potency and tolerability.