Abstract 5649: Potentiation of AKR1C3- and P53-dependent AST-3424 activity via PUMA-mediated degradation of Rad51

Abstract AST-3424 is a novel, highly tumor-selective prodrug activated by tumor-specific AKR1C3 to release AST 2660, a toxic bis-alkylating moiety that induces DNA alkylation and cross-linking. Currently undergoing evaluation in various clinical trials for solid and hematologic malignancies, AST-3424 has demonstrated promising efficacy in a Phase II study as a later-line monotherapy for advanced hepatocarcinoma, particularly in patients who have progressed after immunotherapy. Subgroup analysis suggests enhanced clinical benefit in patients with wild-type P53. This study elucidates the mechanism underlying AST-3424's enhanced therapeutic effect in cancer cells expressing high levels of AKR1C3 and wild-type P53. Co-treatment with AST-3424 and the P53 activator nutlin-3 significantly potentiated AST-3424's pharmacological activities, including in vitro cytotoxicity, reduced colony formation, apoptosis induction, DNA damage (γH2AX), and G2/M phase cell cycle arrest. Targeted deletion of P53 in P53 wild type cells markedly attenuated AST-3424 activities without affecting its AKR1C3 dependence, highlighting P53's pivotal role in cancer cell sensitivity to AST-3424. Given RAD51's crucial role in homologous recombination repair and genomic stability maintenance, we investigated its involvement in P53-dependent AST-3424 activity. Our findings reveal that AST-3424 activates the P53 signaling pathway while concurrently reducing RAD51 expression exclusively in P53 wild type cells. This P53-dependent reduction of RAD51 is mediated by upregulation of PUMA, which promotes RAD51 ubiquitination and diminishes its half-life. Importantly, the effects of AST-3424 on P53 signaling, RAD51, and PUMA were entirely dependent on AKR1C3, as evidenced by complete reversal of these effects upon treatment with the AKR1C3-specific inhibitor AST-3021. The AKR1C3 dependence confers tumor specificity to AST-3424's regulation of P53, RAD51, and PUMA, as AKR1C3 is highly expressed only in tumor cells. In conclusion, this study demonstrates that AST-3424's pharmacological activity is enhanced in cancer cells with high AKR1C3 expression and wild-type P53. We elucidate the mechanism of tumor-specific enhancement associated with P53-dependent RAD51 degradation, leading to decreased homology-directed DNA repair and synthetic lethality. These insights have significant implications for optimizing AST-3424's antitumor efficacy and potentially improving survival rates in patients with functional P53. Citation Format: Jianxin Duan, Tianyang Qi, Yizhi Wang, Xing Liu, Zhaoqiang Lu, Jibing Yu, Xiaohong Cai, Anrong Li, Don Jung, Chen Xun, Yanbin Xie, Fanying Meng, Shukui Qin. Potentiation of AKR1C3- and P53-dependent AST-3424 activity via PUMA-mediated degradation of Rad51 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5649.