Abstract 4029: Neuronal activity-regulated mechanisms promoting growth of diffuse hemispheric glioma, H3G34-mutant

Abstract Neuronal activity robustly drives glioma progression, mediated via paracrine and synaptic mechanisms and offering novel therapeutic avenues for a disease with a devastating prognosis. While the activity-dependent regulation of diffuse midline glioma, H3K27-mutant (DMG-H3K27) and IDH-wildtype glioblastoma has been investigated thoroughly, the interaction of neurons with diffuse hemispheric glioma, H3G34-mutant (DHG-H3G34) remains to be elucidated. Recent transcriptomic analyses suggest a distinct cell-of-origin for DHG-H3G34, demonstrating these tumors primarily resemble early interneuron lineage cells. In the healthy context, neuronal progenitor cells are highly responsive to neuronal activity during development. We previously reported a proliferative response of patient-derived DHG-H3G34 cultures to active neurons in vitro, yet the key neuronal mechanisms governing the progression of DHG-H3G34 are unknown. Here, we explored the neuronal activity-dependent mechanisms that drive DHG-H3G34 growth. We found that activity-induced DHG-H3G34 proliferation in neuron-glioma co-culture is abrogated in the presence of a voltage-gated sodium channel blocker (tetrodotoxin) that prevents action potentials or an AMPA receptor inhibitor, confirming that activity-dependent mechanisms drive DHG-H3G34 malignant cell proliferation and raising the possibility of AMPAR-mediated synaptic mechanisms. Immuno-electron microscopy in two independent patient-derived models demonstrated multiple types of synaptic structures in DHG-H3G34 xenografts, including neuron-to-glioma synapses. Preliminary electrophysiological recordings of xenografted DHG-H3G34 cultures confirmed the presence of spontaneous and stimulation-evoked inward currents consistent with excitatory postsynaptic currents (EPSCs). Further optogenetic stimulation of both glutamatergic cortical projection neurons and GABAergic neurons promoted the proliferation of DHG-H3G34 xenografted cells within the stimulated circuits in vivo. Activity-regulated conditioned media harvested from optogenetically stimulated acute cortical slices induced DHG-H3G34 proliferation in monoculture, thus confirming paracrine signaling as one key mechanism in driving tumor growth. Subsequent proteomic analysis of active conditioned media indicated multiple novel proteins of interest including neuronal cell adhesion molecule (NrCAM) and neurofascin (NFASC). Both paracrine factors increased proliferation and migration in multiple DHG-H3G34 cell lines. Overall, these findings suggest that DHG-H3G34 gliomas integrate into neural circuits and leverage both conserved and tumor-type-specific activity-regulated mechanisms. Continued investigation into these activity-dependent mechanisms of glioma growth aims to identify potential therapeutic strategies for these lethal brain cancers. Citation Format: Kathryn R. Taylor, Samuel H. Wu, Richard Drexler, Gustavo A. Cruzeiro, Ilon Liu, Alexis E. Ivec, Lijun Ni, Carlos A. Biagi, Pamelyn J. Woo, Minhui Su, Youkyeong Gloria Byun, Mariella Filbin, Michelle Monje. Neuronal activity-regulated mechanisms promoting growth of diffuse hemispheric glioma, H3G34-mutant [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4029.