Abstract
Diffuse midline glioma (DMG), remains a universally fatal brain cancer defined by H3K27M oncohistone mutations in H3F3A, HIST1H3B, or HIST1H3C. These alterations drive global hypomethylation and widespread transcriptional dysregulation. Through CRISPR-Cas9 loss-of-function screens across 38 patient-derived models, we identified consistent, mutation-independent dependencies on PIK3CA and MTOR, confirming PI3K/mTOR signaling as a core therapeutic vulnerability in DMG. However, systemic pan-PI3K inhibition (e.g., paxalisib) induces hyperglycemia and hyperinsulinemia, undermining treatment efficacy in vivo. To mitigate this, we developed multimodal strategies that combined clinically relevant PI3K/mTOR inhibitors (paxalisib, GCT-007, everolimus) with the antihyperglycemic agent metformin, which restored glucose homeostasis, suppressed insulin receptor activity, and extended survival in DMG xenograft models. Phosphoproteomic profiling identified therapy-induced activation of calcium-dependent PKC signaling, prompting combination with the brain-penetrant PKC inhibitor enzastaurin, further extending survival. Notably, GCT-007, a selective, brain-penetrant PIK3CA inhibitor, synergized with enzastaurin to achieve long-term survival in immunocompetent mice. Mechanistically, PI3K–PKC inhibition promoted differentiation from OPC-like to OC-like states, upregulating JAK/STAT signaling, MHC-II expression, and inducing features of demyelination. Spatial multiomics confirmed CD3+ and CD8+ T cell infiltration and PD-L1 expression on myeloid populations and HLA-DRA on DMG cells, indicating immune remodeling. This tumor microenvironmental shift mirrors neuroinflammatory responses observed in multiple sclerosis, where CD8+ T cell-driven demyelination occurs. Importantly, GCT-007 + enzastaurin regimens sensitized tumors to immune checkpoint inhibitors (ICIs), producing durable responses and long-term survival in immune-competent models that historically show resistance to radiotherapy and small-molecule inhibitors. Together, our findings define a multimodal, precision treatment strategy that targets core DMG genetic dependencies, rewires tumor cell identity, circumvents systemic resistance, and primes an immunologically “cold” tumor for immune engagement and clinical translation. We are currently designing an early-phase clinical trial that incorporates sequential small-molecule therapies and ICIs delivered via focused ultrasound, aiming to provide patients with a meaningful and durable survival benefit.