Abstract
Diffuse Intrinsic Pontine Glioma (DIPG) is a universally fatal pediatric brain tumor with median survival of less than 12 months. While chimeric antigen receptor (CAR) T-cell therapy shows promise, its efficacy in DIPG is limited by a profoundly immunosuppressive tumor microenvironment (TME). MicroRNAs (miRs), small non-coding RNAs that regulate gene expression, play critical roles in both gliomagenesis and immune regulation. Given their expression in both tumor and immune cells, miRs represent a novel, underexplored strategy to modulate the TME and enhance immunotherapy. We hypothesize that targeting dysregulated miR expression in DIPG can reprogram the TME to support CAR T-cell activation and persistence.To investigate the role of miRs in DIPG, we performed small RNA sequencing on post-mortem DIPG tumors and normal brain tissue. Tumors showed significant dysregulation of multiple miRs, including upregulation of immune-regulatory miR-103a, miR-23c, and miR-361—associated with TGF-β signaling and T-cell exhaustion. Gene Set Enrichment Analysis of miR targets revealed enrichment in immunosuppressive and inflammatory pathways, consistent with a chronically inhibited TME. Bulk RNA sequencing of the same samples confirmed elevated expression of immunosuppressive genes (IL10, FOXP3, PD-L1, CTLA-4). Integrated pathway analysis identified miR–mRNA networks involved in immune evasion and impaired immune cell recruitment.To assess therapy-induced changes in miR expression, we treated glioma-bearing mice with B7-H3 CAR T cells, control CARs, or left them untreated. Tumors harvested at defined timepoints were profiled for miRs. Principal Component Analysis revealed distinct clustering in B7-H3 CAR–treated tumors. Differential expression analysis identified therapy-associated miRs (including miR-344, let-7d, miR-99b, and miR-30c) implicated macrophage polarization and T-cell dysfunction.Together, our data suggests that miRs contribute to DIPG immune suppression and are dynamically regulated by CAR T-cell therapy. Ongoing studies aim to define how targeting specific miRs can enhance CAR T-cell function, offering a new avenue to improve outcomes in DIPG.