A study by researchers from Mass General Brigham has found how cancers become resistant to rezatapopt and other drugs designed to restore the tumor suppressor p53. The research, published in Cancer Discovery, showed that tumors evolve new mutations within the same TP53 gene in response to treatments which prevents it from reassuming its tumor suppressing role and uncovered ways to improve p53-targeted treatments using next-generation drugs or combination approaches.
“Our findings establish a molecular basis for why patients treated with rezatapopt may experience therapeutic failure and provide the first clinical evidence that on-target secondary TP53 mutations can lead to acquired resistance,” said first author Ferran Fece de la Cruz, PhD, an instructor with the Krantz Family Center for Cancer Research at the Mass General Brigham Cancer Institute. In addition, the work “galvanizes us to further investigate whether next-generation agents or combination therapies may overcome or delay the emergence of resistance.”
Mutations in TP53 are the most frequent genetic alterations in cancer, and the Y220C mutation creates a small cavity in the p53 protein that is not present in the wild-type form, which has been a fertile target for small molecule therapies. These agents, like rezatapopt, bind the mutant protein and restore tumor suppressor activity. Rezatapopt, an orally available Y220C reactivator, has shown initial efficacy across tumor types in the ongoing phase 1/2 PYNNACLE trial. But acquired resistance to this treatment led to the current study to find out how tumors develop therapeutic resistance.
For the research, the team examined blood and tumor samples from two patients with different metastatic solid tumors enrolled in PYNNACLE who initially responded to rezatapopt but later relapsed. Using ctDNA analysis, tumor biopsies, and, in one case, rapid autopsy specimens, they tracked tumor evolution during therapy. Tumor profiling uncovered multiple new TP53 alterations that arose during treatment, including nearly 100 de novo mutations in one patient. These secondary mutations were found in cis with the original Y220C alteration, indicating direct evolutionary pressure on the drug target itself.
The team then conducted functional studies to determine how these mutations affected drug response. The researchers expressed representative double mutants—Y220C plus a newly acquired alteration—in cultured cancer cells and assessed p53 activity and responsiveness to rezatapopt. The experiments showed two main classes of resistance. One class included DNA-binding domain mutations, frameshifts, or nonsense mutations that halt p53 transcriptional activity. “Functional modeling confirmed these double mutants eliminate p53 reactivation and target gene induction by rezatapopt,” the researchers wrote. Because these alterations remove p53 function entirely, they are likely to cause resistance to all targeted drugs using the same approach as rezatapopt.
The second class of resistance mutations clustered around the Y220C binding surface. Structural modeling indicated these changes reconfigure the druggable pocket and hinder rezatapopt binding without necessarily halting p53 activity. Some of these alterations “do not invariably abolish p53 activity, suggesting that resistance here may be drug-specific rather than universally applicable across all Y220C reactivators,” the researchers noted, providing an opportunity to develop new treatments with different profiles or binding chemistries that might retain activity against these tumors.
Prior research had established that the Y220C mutation creates a cryptic pocket adjacent to the DNA-binding domain of p53 and that small molecules could stabilize the mutant protein and restore function. But no prior research has shown that tumors would evolve additional TP53 mutations to evade these treatments. “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,” the researchers wrote.
The implications for clinical care include the potential need for molecular testing during treatment to monitor for resistance mutations and to guide therapy changes. Secondary mutations that disrupt drug binding but preserve p53 function could be addressed by switching to next-generation reactivators. By contrast, mutations that abolish p53 activity may require combination strategies targeting vulnerabilities associated with p53 loss.
The researchers noted that their analysis of this activity was derived from only two patients and that larger cohorts will be needed to define how often and how quickly these resistance mechanisms arise. Future studies will integrate patient-derived genomic data with functional assays to distinguish drug-specific escape mutations from other drivers of disease development. This research could help guide the design of next-generation p53-targeted therapies and trails of combination approaches aimed at extending the durability of responses in patients with TP53-mutant cancers.
