STEM-27. TRACING THE ORIGINS OF H3.3K27M MUTANT DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas (DMGs) are universally fatal pediatric brain tumours defined by their midline localization, early onset, and distinct mutational landscape. The H3.3K27M mutation is found in approximately 80% of DMGs, marking it as a key initiating event. However, H3.3K27M alone does not induce brain tumour formation in experimental models, suggesting that its oncogenic potential may be linked to specific developmental windows and susceptible precursor populations. Identifying the cellular states that are vulnerable to transformation is critical to understanding DMG initiation. To establish a reference framework for these vulnerable states, we generated a single-cell RNA sequencing atlas of normal hindbrain development. Using a Sox2-eGFP reporter mouse and FACS-based enrichment of Sox2+ precursors, we profiled over 120,000 cells across ten developmental stages, capturing critical transitions in neurogenesis and gliogenesis. To reveal regional differences, we integrated the atlas with a similarly generated forebrain atlas, providing a comprehensive map of neurodevelopmental trajectories where DMG mutations might exert their effects. Building on this developmental reference, we utilized inducible mouse models to introduce H3.3K27M mutations in precursors and employ lineage tracing to track their fate over time. When combined with loss of Trp53 and overexpression of PDGFRa, alterations prevalent in DMG, mice develop tumours that resemble the human disease. H3.3K27M-driven tumours consistently localize to the pons, in contrast to H3 wild-type tumours, which occur more variably across the forebrain and hindbrain. Tracking these pontine tumours using serial MRI revealed that tumours arise well before symptom onset and exhibit growth dynamics that vary by location and timing. Earlier-arising tumours are more aggressive and tend to occur in posterior locations. Together, our integrated approach combining lineage tracing, tumour modelling, and a developmental atlas provides a powerful platform to dissect the early events in DMG pathogenesis and identify developmental vulnerabilities that may be targeted for early intervention.