Abstract LB289: Clinical resistance to Y220C-mutant p53 reactivators

Abstract Mutations in TP53 are observed across all tumor types and the vast majority are missense mutations occurring within its DNA binding domain (DBD), resulting in decreased target gene expression due to loss of DNA-binding affinity. Among them, the Y220C mutation is the ninth most frequent, accounting for 1.8% of all p53 mutations. Structural characterization of this substitution has revealed that it creates a small crevice in the p53 protein, compromising its thermal stability and impairing its function to effectively interact with DNA. This druggable cavity has led to the development of small molecules that selectively react with the mutant cysteine in Y220C to restore thermal stability to a comparable level as in the WT form of p53. This results in restoration of p53 functionality in tumor cells inducing cell death.Rezatapopt is a first-in-class orally bioavailable small molecule reactivator of Y220C-mutant p53 in clinical development for the treatment of patients with locally advanced or metastatic solid tumors harboring this mutation. This agent is currently under evaluation in the phase 1/2 PYNNACLE study. Preliminary analysis of phase 1 data has shown that rezatapopt displays clinical efficacy across multiple tumor types with a favorable safety profile.In this work, we describe the first mechanisms of resistance in patients treated with this novel class of Y220C-mutant p53 reactivators. In our patient cohort, we observe on target secondary mutations as a leading cause of drug resistance and disease progression. Interestingly, we identified three classes of acquired p53 alterations: 1) mutations affecting the DNA binding surface leading to known loss of p53 function, 2) mutations located in the Y220C binding surface which may seclude the crevice preventing drug binding, and 3) insertion/deletions downstream of Y220C resulting in nonfunctional protein products.Expression of these acquired alterations in cis with the Y220C mutation in NSCLC and HNSCC cell models confirmed their impact leading to resistance. Notably, these secondary alterations do not further compromise protein stability unless causing a shift in the reading frame. Furthermore, we observe a complete lack of p53 reactivation upon treatment of these p53 double mutant cells with rezatapopt, as target gene expression remains unaffected.This work provides the first insightful characterization of clinical acquired resistance to p53 Y220C reactivators. Future studies will elucidate whether more potent covalent Y220C reactivators manage to overcome the resistance mechanisms identified in this study. Citation Format: Ferran Fece de la Cruz, Andreas Varkaris, Parasvi S. Patel, Elijah W. Kushner, Alvin A. Morales-Giron, Bryanna L. Norden, Sara Ehnstrom, Jakob M. Riedl, Haley Barnes, Allison M. Kehlmann, Jessica L. Hopkins, Keelan Z. Guiley, Kevan M. Shokat, Aparna R. Parikh, Ryan B. Corcoran. Clinical resistance to Y220C-mutant p53 reactivators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB289.