TMIC-97. Deciphering how DNA methylation programs in diffuse midline glioma influence CAR T cell efficacy in fully immune-competent murine models

Abstract Diffuse midline glioma (DMG) is a lethal pediatric brain tumor with no effective treatment options. A molecular hallmark of DMG is the histone H3K27 (H3K27M) mutation that dysregulates other epigenetic programs, including DNA methylation, which can further drive tumorigenesis and reprogramming of the tumor immune microenvironment (TIME). We investigated whether DNA methylation, through DNA methyltransferase 3a (Dnmt3a) knockout (KO), could reprogram the DMG TIME and synergize with B7-H3.CAR T-cell therapy for a more durable response. To achieve this, we used murine DMG cells established from in utero electroporation (IUE) DMG tumors, with or without Dnmt3a knockout (KO). Using flow cytometry and histology, we confirmed that Dnmt3a-KO DMG cells sustained B7-H3 expression, a brain tumor-specific CAR T cell target. In vitro, we showed that B7-H3.CAR T-cells could kill murine DMG cells with similar efficacy regardless of Dnmt3a status. Once implanted in vivo, we found that Dnmt3a-KO tumors proliferated faster compared to control-KO tumors, and had fewer immune infiltrates in both the primary IUE and secondary syngeneic models. Unexpectedly, when treated with murine B7-H3.CAR T-cells, we observed dose-dependent CAR T-induced neurotoxicity and brainstem inflammation in Control- and Dnmt3a-KO tumors that persisted for one week after T-cell administration. Our dose optimization studies indicate that the non-toxic dose was able to temporarily manage DMG tumors, but mice ultimately succumbed to the disease. When comparing the non-toxic dose between Control- and Dnmt3a-KO tumors, we noticed less severe weight loss in the Dnmt3a-KO group, suggesting better tolerance of B7-H3.CAR T treatment during the inflammation period. However, neither group improved overall survival. Transcriptomics analyses are ongoing to elucidate the immediate interaction between the DMG TIME and B7-H3.CAR T cells during the treatment. Together, our findings reveal that epigenetic programs modulate the DMG TIME and potentially influence B7-H3.CAR T therapeutic efficacy.