TP53 Mutations within T Cells Induce T-Cell Exhaustion and Functional Impairment in TP53 Mutant AML

TP53 mutations in AML are present in 30% of R/R AML patients and are associated with poor prognosis and resistance to therapy. Earlier experiments have revealed markers of immune dysfunction in the bone marrow microenvironment of TP53m AML. However, what the role of TP53 mutations within immune cells remain largely unknown.

Single-cell cytokine secretion analysis (IsoPlexisR) revealed that T cells from the bone marrow (BM) of AML patients with TP53 mutations exhibited significant functional defects compared to those from TP53 wild-type AML patients. Analysis of a pooled scRNAseq dataset from 24 AML patients and CyTOF data from 38 AML patients demonstrated increased exhaustion in both T-cells and NK cells in patients with TP53 mutations versus those with TP53 wild type. Simultaneous single-cell DNA and surface antigen analysis using Tapestri (MissionBio) enabled direct phenotype-genotype correlations, revealing that 34% (range 8% to 91%, n=4) of CD4+ and 39% (range 5% to 90%, n=4) of CD8+ BM-resident T cells carried monoallelic TP53 mutations. These mutations were identical to those found in the leukemic blasts of the same patients. Digital-droplet PCR (ddPCR) with specific TP53 mutation primers confirmed the presence of TP53 mutations in highly pure (>99%) FACS-sorted T cells, detecting 36% of patients carried somatic mutations in T-cells. Notably, these patients did not have Li-Fraumeni syndrome. Further phenotypic profiling revealed that TP53-mutant T cells exhibited higher proliferation, but lower adhesion compared to TP53 wild-type T cells. TP53 mutations significantly alter the immune landscape in AML patients by enhancing the proliferative and activation profiles of T and NK cells while impairing T cell functionality. Therefore, we hypothesize that monoallelic TP53 mutations impair normal p53 function in T cells, which regulates the cell cycle and proliferation through a dominant-negative effect, subsequently inducing T-cell exhaustion.

To test our hypothesis, we engineered CD123-targeted CAR-T cells to specifically target AML cells overexpressing the mutant p53-Y220C using a lentiviral vector. Overexpressed mutant p53 is expected to exert a dominant-negative effect on wild-type p53. Control CAR-T cells were generated without mutant p53 overexpression, serving as baseline comparison. Using high-parametric CyTOF analysis, we compared the proteomic landscape of p53-mutant (p53mt) CAR-T cells versus control CAR-T cells. Results revealed that p53mt CAR-T cells exhibited increased expression of exhaustion markers, including PD-1, LAG3, TIGIT, TIM3, and CD39, a marker regulating T cell activation. In subsequent re-challenge experiments in vitro, we added AML cells to the culture of TP53-mutant and TP53-wild-type (TP53-wt) CD123-CAR-T cells every two days until day 14. The CyTOF-monitored exhaustion score was 49% in CD4 and 70% in CD8 of TP53-mut CAR-T cells, while it was 38% in CD4 and 57% in CD8 of TP53-wt CAR-T cells. These results strongly suggest that TP53 mutations expedite the exhaustion process in T cells. To further assess the functional impact, we examined the killing capacity of TP53mt CAR-T cells against Molm13 cells using the IncuCyte Live-Cell Analysis System. p53mt CAR-T cells exhibited a significant reduction in cytotoxicity compared to control CAR-T cells. This result aligns with the observation that TP53-mut CD123 CAR-T cells exhibited impaired cytokine secretion (TNF-α, IFN-γ, and IL-2) when exposed to AML cells. Additionally, Timelapse Imaging Microscopy in Nanowell Grids (TIMING) corroborated these findings, demonstrating a delayed cytotoxic response of p53mt CAR-T cells against target cells. To investigate the in vivo impact of mutant p53 on CAR-T cell functionality, we generated a patient-derived xenograft (PDX) mouse model using venetoclax-resistant AML patient cells. Following engraftment, we treated the mice with p53mt CAR-T cells, control CAR-T cells, or empty vector T cells. Bioluminescence imaging (BLI) revealed a significantly higher tumor burden in mice treated with p53mt CAR-T cells compared to other groups. Additionally, these mice exhibited significantly worse survival.

In summary, we discovered TP53 mutations in T-cells from patients with TP53 mutant AML. And TP53 mutations in T cells induce T-cell exhaustion and impair their anti-tumor efficacy, underscoring the need for targeted therapeutic strategies.

Battula:Daiichi Sankyo Company, Ltd: Research Funding; InCyte Corporation: Research Funding; Y-mAbs Therapeutics: Research Funding; CytoMed Therapeutics: Research Funding; Inspirna: Research Funding; FATE Therapeutics: Research Funding. Varadarajan:CellChorus: Other: Co-Founder; AuraVax Therapeutics: Other: Co-Founder. Issa:Kura Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: consultancy/ad board fees, Research Funding; Merck: Research Funding; Syndax Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: consultancy/ad board fees, Research Funding; Astex: Research Funding; NuProbe: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: consultancy/ad board fees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: consultancy/ad board fees, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: consultancy/ad board fees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: consultancy/ad board fees; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: consultancy/ad board fees; Celgene: Research Funding. Andreeff:Kintor Pharmaceutical: Research Funding; Boehringer-Ingelheim: Honoraria; Syndax: Honoraria, Research Funding; Oxford Biomedical: Research Funding; Ona: Honoraria; Glycomimetics: Honoraria; Paraza: Honoraria; Aptose: Honoraria; Daiichi-Sankyo: Research Funding; Sellas: Honoraria, Research Funding; Oncolyze: Current holder of stock options in a privately-held company; Roivant: Honoraria; Chimerix: Current holder of stock options in a privately-held company; Ellipses: Research Funding; SentiBio: Current holder of stock options in a privately-held company, Honoraria, Research Funding; Eterna: Current holder of stock options in a privately-held company, Honoraria, Research Funding.