Reactivating mutant p53 in T cells enhances anti-tumor activity in Acute Myeloid Leukemia

Abstract

Abstract Emerging evidence from our group suggests that mutant TP53 may play a critical role in T-cell exhaustion and the impairment of anti-tumor immunity, particularly in acute myeloid leukemias (AML). We have shown that structural alterations of the mutant p53 protein disrupt transcription-dependent and -independent regulatory functions of wild-type p53 in AML blasts, but TP53 mutations are also present in T and NK cells (Li,L et al. under review). Our data suggest that mutant p53 impairs T-cell functionality by upregulating exhaustion markers and reducing T-cell cytotoxicity, potentially contributing to the poor therapeutic outcomes characteristic of TP53 mutant AML. We further investigate the impact of mutant p53 on T-cell function and evaluate the efficacy of PC14586, a small molecule designed to restore wild-type p53 conformation and function of mutant p53-Y220C (Carter B et al., Blood 2025). The primary objectives are to determine whether PC14586 can correct the functional defects of TP53-Y220C mutant T cells and to assess its effects on TP53-Y220C mutant CAR-T cell functionality in both in vitro and in vivo models. We engineered CAR-T cells to co-express a chimeric antigen receptor (CAR) targeting AML-associated antigens and a mutant form of p53 (p53-Y220C mutation), thereby modeling the effects of p53 mutations commonly observed in AML on T-cell function. After confirming high transduction efficiency (>80% of primary T cells co-expressed both CAR and p53), we treated these TP53 mutant CAR-T cells with PC14586 and monitored mutant p53 protein expression over time. Treatment with PC14586 resulted in a time-dependent reduction of mutant p53 protein, with a 50% decrease in CD4 T cells and a 38% decrease in CD8 T cells at 72 hours, as confirmed by flow cytometry and western blot analysis. To further investigate the impact of p53 reactivation, we performed differential gene expression analysis comparing PC14586-treated and untreated TP53 mutant CAR-T cells. T cells treated with the p53 reactivator exhibited significantly decreased levels of exhaustion markers in both CD4 and CD8 subsets. Functional assessment using a real-time cell killing assay (InCuCyte) demonstrated that TP53 mutant CAR-T cells had significantly reduced anti-AML activity, which could be largely restored in vitro by PC14586. To evaluate in vivo efficacy of this approach, we employed a venetoclax-resistant patient-derived xenograft (PDX) mouse model of AML. NSG mice received a single intravenous dose of 3 × 10^6 T cells expressing both anti-CD123 CAR and mutant TP53, pre-treated with PC14586. Control groups received T cells expressing anti-CD123 CAR with or without mutant TP53, or empty vector-transduced T cells, administered one day after AML engraftment was established by the presence of circulating AML blasts. Notably, mice treated with PC14586-pre-treated TP53 mutant CAR-T cells exhibited reduced tumor burden and significantly prolonged survival compared to other groups. Conclusion: We recently reported TP53 mutations in T and NK cells from TP53 mutant AML patients (Li et al, ASH 2024). Our new findings establish reduced functionality of TP53 mutant T cells and enhanced anti-leukemia activity and improved therapeutic outcomes in vivo by pharmacological restoration of wild-type p53 by PC14586. A clinical trial of PC14586 (Rezatapopt, PMV Pharma) in p53-Y220C mutant AML is ongoing (Senapathi, J. et al., ASH 2025).