Abstract Pediatric gliomas present a significant challenge in neuro-oncology due to the immunosuppressive tumor microenvironment (TME) and limited efficacy of standard treatments. While chimeric antigen receptor (CAR) T cell therapy has revolutionized outcomes in hematologic cancers, its success in solid tumors is hindered by CAR T cell exhaustion and the hostile TME. A major barrier to sustained efficacy is the epigenetic regulation of exhaustion. Chronic antigen exposure increases DNA methylation, restricting critical effector gene expression and impairing CAR T cell function. This study evaluates the addition of DNA methyltransferase inhibitors (DNMTis) early during CAR T cell manufacturing to reprogram the DNA methylome and enhance anti-glioma activity. We hypothesize that pharmacologic modulation of DNA methylation can overcome premature exhaustion and the immunosuppressive challenges of the glioma microenvironment. To test this, we optimized transduction by incorporating DNMTi treatment of murine CAR T cells targeting B7-H3. In a comprehensive series of standard in-vitro repeat stimulation assays, DNMTi-treated CAR T cells showed extended persistence and superior anti-glioma activity compared to untreated controls. These cells expanded more robustly and maintained cytotoxicity through twelve rounds of stimulation, accompanied by sustained secretion of immune-stimulatory cytokines. To validate these findings, we performed in-vivo studies in immunocompetent mouse models bearing HGG and DIPG tumors, treated with B7H3- and GD2-targeting CAR T cells. DNMTi-treated CAR T cells improved tumor control and reshaped the immune landscape of the tumor microenvironment, indicating a broader immunomodulatory effect. This approach was also replicated in ovarian cancer models, where we observed survival benefits and tumor reduction. Additionally, this effect is being validated in human glioma models and with patient-derived samples. DNMTis represent a clinically accessible and readily implementable strategy for optimizing cell therapies in solid and brain tumors. These findings support further exploration of epigenetic modulation to advance next-generation immunotherapies for children with CNS malignancies.
VanNoy et al. (Tue,) studied this question.