Abstract 1627: Switching cell fate from senescence to apoptosis by the combination of a p53 corrector with the MDM2 antagonist ASTX295

Abstract Background: ASTX295 is a potent MDM2 antagonist designed to have a shorter half-life (t1/2 4-6 hours in plasma), which leads to an improved safety profile (bone-marrow sparing), making it more amenable to combinations compared to other MDM2 antagonists. The recent success of targeting TP53:Y220C mutant cancers with a p53 corrector molecule holds great promise for treatment of all Y220C TP53 mutant cancers. However, for effective therapy, a high level of p53 signalling will be required to overcome the elevated apoptotic thresholds seen in some cancer cells with co-mutations in pro-survival signalling pathways. We rationalised that this could be achieved by removing the MDM2-driven negative feedback loop that is triggered following Y220C correction. Materials and Methods: Corrected p53 signalling was determined in two TP53:Y220C mutant pancreatic cancer cell lines with additional MAPK mutations: BxPC3 (BRAF:L485-P490del) and T3M-4 (KRAS:Q61H). Two small-molecule TP53:Y220C correctors were tested alone and in combination with ASTX295 using real-time microscopy, western blotting, and qPCR. Markers of p53 signalling (at the protein and mRNA level), senescence (β-galactosidase), apoptosis (Annexin V), and DNA damage (phospho-H2A.X) were measured after treatment with compounds. A mouse xenograft model was established in male CB17 SCID mice inoculated subcutaneously with BxPC3 cells, allowing PK/PD and efficacy evaluation. Results: The combination of ASTX295 and the Y220C corrector led to increased cell death in both BxPC3 and T3M-4 cell lines in vitro, with elevated p53 signalling (p21), apoptosis (PUMA), and DNA damage (phospho-H2A.X) when MDM2 is antagonised. The KRAS mutant line, T3M-4, was less sensitive to the combination, but could be driven to apoptosis at higher concentrations of the corrector. PK/PD evaluation in BxPC3-xenograft-bearing mice demonstrated prolonged p53 signalling following combination treatment up to 16 h post dose, with a subsequent reduction at 24 h consistent with the ASTX295 exposure profile. Conclusions: We have shown that MDM2 antagonism leads to sustained corrected p53 signalling over time, which drives cell death, even in tumor cells harbouring co-mutations conferring higher apoptotic resistance. Indeed, our study provides proof of concept that efficacy could be achieved in multiple tumor types in which the p53 pathway can be targeted therapeutically through combination with ASTX295, which would drive and maintain p53 signalling levels above the apoptotic threshold. Citation Format: George Ward, Judit Espana-Agusti, Keisha Hearn, Mark Wade, Lynsey Fazal, Hugh Walton, Andrea Biondo, John Lyons, Martin Sims, Simone Jueliger, Jessica Brothwood, Andrew Woodhead, Louise Walsh, Tomoko Smyth, Nicola Wilsher, Maria Ahn. Switching cell fate from senescence to apoptosis by the combination of a p53 corrector with the MDM2 antagonist ASTX295 [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 1627.