Abstract
Diffuse midline glioma (DMG) is a universally fatal high-grade glioma. Its immunologically cold tumor immune microenvironment (TIME) presents a major barrier to durable anti-tumor responses. Dordaviprone (ONC201) is a brain-penetrant DRD2 antagonist and ClpP agonist that disrupts mitochondrial complex II (CII) activity; currently under clinical evaluation for DMG. In melanoma, CII inhibition has been shown to enhance MHC presentation and T cell–mediated killing. Here, we investigated the immunomodulatory effects of dordaviprone in DMG mouse models, patient tumor tissues, and blood samples. Immunocompetent DMG-PPK mice (Pdgfra(D842V), Trp53(DN), H3f3a(K27M) mutations) were treated with dordaviprone (125 mg/kg, twice weekly) for 1 week (acute) or 3 weeks (chronic) and compared to naïve, sham (orthotopic surgery without tumor engraftment), and vehicle-treated controls. Murine tumors were analyzed by single-cell multiome (scRNA-seq and scATAC-seq) and spatial transcriptomics. Murine blood and bone marrow (BM) and human blood samples were profiled by scRNA-seq and flow cytometry. In models, dordaviprone promoted microglial activation and macrophage infiltration within the TIME. Spatial transcriptomics identified increased expression of the MHC-I subunit B2m in tumor cells and tumor-infiltrating Cd8+ T cells following dordaviprone treatment. Flow cytometry of murine and human peripheral blood confirmed CD4+ and CD8+ lymphopenia at baseline. Dordaviprone increased circulating lymphocytes in models (log(2) fold change=1.03; patient data pending), reversing DMG-induced CD3+ T cell sequestration in the BM. Immunohistochemistry confirmed elevated levels of B2M (p=0.0281) and tumor-infiltrating lymphocytes (TILs), including CD45+ (p=0.0275) and CD3+ T cells (p=0.0103) in dordaviprone-treated mice. Despite enhanced TIL recruitment, dordaviprone did not improve survival (n=10), likely due to persistent immunosuppressive myeloid activity, i.e. Havcr2 (TIM-3), Cd74, Cd274 (PD-L1). This study demonstrates that DMG tumors contribute to systemic lymphopenia, partially reversed by dordaviprone. While supporting its immunomodulatory effects, including enhanced antigen presentation and TIL recruitment, our findings highlight future combination strategies to leverage dordaviprone-induced immunoreactive TIME.