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UNLABELLED: The tumor-suppressor TP53 is the most frequently altered gene in cancer, and the Y220C hotspot, found in 1.8% of TP53-mutant tumors, creates a druggable cavity that destabilizes p53. Rezatapopt, a first-in-class, orally bioavailable reactivator of Y220C-mutant p53, has demonstrated promising initial efficacy in the phase 1/2 PYNNACLE trial. We report the first clinical mechanisms of resistance to this therapeutic class. Profiling of circulating tumor DNA, tumor biopsies, and rapid autopsy specimens upon rezatapopt progression revealed multiple heterogenous secondary TP53 alterations in cis with Y220C, including (i) DNA-binding domain mutations or frameshift/nonsense mutations that abolish transcriptional activity and (ii) mutations within the Y220C-binding surface predicted to hinder drug binding. Functional modeling confirmed that these double mutants eliminate p53 reactivation and target gene induction by rezatapopt. These findings establish a molecular framework for resistance to p53 Y220C reactivators and inform strategies to overcome resistance with next-generation agents.SIGNIFICANCE: This study illustrates how pan-cancer resistance to Y220C-mutant p53 reactivators emerges in patients, indicating that on-target acquired alterations can represent a major mechanism of clinical resistance. These insights establish a molecular basis for therapeutic failure and provide a framework for developing next-generation agents to overcome resistance. See related commentary by Liu and Gu, p. 620.