Abstract Chimeric Antigen Receptor (CAR) T-cell therapy has shown limited efficacy in pediatric brain tumors due to challenges such as T-cell exhaustion, poor persistence, and the immunosuppressive tumor microenvironment (TME). DNA methylation, a critical epigenetic mechanism regulating T-cell differentiation, memory formation, and effector function, offers a promising therapeutic target to enhance CAR T-cell antitumor activity by silencing inhibitory pathways. This study aimed to determine whether DNA methyltransferase inhibitors (DNMTis) during CAR T-cell manufacturing enhance persistence, cytotoxicity, and antitumor efficacy in preclinical models of medulloblastoma, glioma, osteosarcoma, and ovarian cancer. Murine and human T cells were transduced with B7-H3 CAR or control CAR constructs and treated with DNMTis, including 5-AZA, GSK3685032, or Decitabine, during transduction. Functional assays evaluated cytotoxicity, persistence, and expansion. Memory phenotypes and exhaustion markers were evaluated by FACS, while therapeutic efficacy was assessed using in vivo tumor models. DNMTi treatment markedly enhanced murine CAR T-cell proliferation, persistence, and functionality. DNMTi-treated CAR T cells maintained cytolytic activity through 12 repeated stimulation cycles, whereas untreated cells exhibited functional decline after 7 cycles. Although initial cytotoxic activity was comparable, DNMTi-treated cells demonstrated significantly greater tumor-lytic capacity during repeated stimulations. Additionally, DNMTi-treated CAR T cells displayed increased proportions of memory-like phenotypes, sustained cytokine secretion, and reduced expression of exhaustion markers. In human models, DNMTi-treated B7-H3 CAR T cells mirrored these functional improvements, demonstrating superior persistence, cytotoxicity, and memory phenotype formation across medulloblastoma and glioma models. Early mechanistic studies revealed changes in memory T cell phenotypes and epigenetic remodeling of exhaustion markers in modified CAR T cells during successive stimulations. Epigenetic modulation with DNMTis during CAR T-cell manufacturing enhances persistence, functionality and antitumor efficacy. This approach addresses intrinsic and TME-associated barriers, highlighting as a strategy to optimize CAR T-cell therapy for pediatric brain tumors and solid malignancies.
Ghorayeb et al. (Fri,) studied this question.