High-grade gliomas are the leading cause of brain tumor-related death, underscoring the urgent need for a deeper understanding of high-grade glioma pathobiology and novel avenues for therapy. We have recently discovered that neuronal activity robustly promotes high-grade glioma growth and that a synaptic molecule called neuroligin-3 is a crucial activity-regulated mechanism for glioma growth. Activity-regulated cleavage and release of neuroligin-3 from synapses, mediated by the protease ADAM10, is required for glioma growth, although it is not yet clear what mediates this striking dependency. Further, we have found that a subset of xenografted gliomas evolve in vivo to circumvent neuroligin-3 dependency over a period of 6 months in the context of a neuroligin-3 deficient brain microenvironment. In the present proposal, we seek to leverage single cell genomics together with patient-derived glioblastoma orthotopic xenografts and immunocompetent murine glioblastoma allografts in neuroligin-3 knockout or wild type mice to dissect neuroligin-3 signaling within the intact glioma ecosystem. Using a similar strategy, we will also uncover the mechanisms by which some xenografted gliomas circumvent neuroligin-3 dependency, findings that will inform not only neuron-glioma interactions but also fundamental mechanisms of glioma progression. Finally, we will perform preclinical efficacy and safety testing of ADAM10 inhibition to block neuroligin-3 release into the tumor microenvironment in an effort to provide sufficient preclinical evidence to bring this novel therapeutic strategy to a clinical trial for adult high-grade gliomas. This future trial will complement our Pediatric Brain Tumor Consortium-sponsored phase 1 clinical trial of ADAM10 inhibition for pediatric high grade glioma. Taken together, the proposed experiments will elucidate fundamental mechanisms of glioma growth and progression and advance a promising new therapeutic approach for these lethal brain cancers.