Abstract 604: Second site rescue mutants as tools to uncover reactivation mechanisms of p53 cancer mutants.

Abstract TP53 is an important tumor suppressor and is the most frequently mutated gene in human cancer. A large fraction of cancers with mutated p53 carry missense mutations, so they express full-length but nonfunctional p53. Therefore, reactivating mutated p53 to a wildtype-like conformation has long been a major therapeutic goal. However, the mechanisms underlying p53 reactivation are still poorly understood. To dissect this mechanism, this study employs a genetic strategy in which a second amino acid is mutated in addition to the G245S mutation found in cancer. This second mutation induces structural changes to compensate for the defect caused by p53 cancer mutations. Two second site rescue mutations were used in this study: N239Y, known to stabilize p53, and T123P, located within the transient L1/S3 pocket implicated in small molecule binding. When expressed alongside the cancer mutation G245S, both second site rescue mutations restored tumor suppressive properties, including suppressed growth, activated canonical p53 downstream targets, and restored DNA binding both in vitro and in cells. Differential scanning fluorimetry confirmed that N239Y increased folding stability of the G245S mutant. However, despite rescuing p53 activity, T123P further destabilized the structural stability of the G245S mutant. These findings demonstrate that p53 reactivation can occur through distinct and mechanistically divergent pathways, including those independent of global thermostabilization.Our results provide a genetic framework to classify reactivation mechanisms of p53 cancer mutants and define gene-expression signatures associated with each mechanism. Rather than being a direct treatment, this framework can help guide the identification and development of future small molecule p53 reactivators. Citation Format: Fiona Law, Zane Norman, Mark Villamil, Peter Kaiser. Second site rescue mutants as tools to uncover reactivation mechanisms of p53 cancer mutants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 604.