Histone H3K27M variants determine myeloid subset dependencies and immune reprogramming in diffuse midline glioma

Diffuse midline gliomas (DMGs) are highly aggressive, WHO grade 4 glial tumors that arise in midline central nervous system structures and are defined by K27M mutations in histone H3 genes. These K27M mutations shape intratumoral myeloid cell composition in DMG. In H3.1K27M DMGs, genetic ablation of monocyte recruitment reshapes the tumor microenvironment (TME) by reducing monocyte-derived macrophages (MDMs) and increasing microglia and neutrophil presence, with overall survival remaining unchanged, indicating compensatory myeloid remodeling is occurring. Here, by using CRISPR/Cas9-based genome editing, we generated a mouse model deficient for CCR1/CCR2/CCR3/CCR5 (Δ1235). Using this strain, we effectively abolished monocyte and MDM infiltration and reversed compensatory recruitment of CCR1+ neutrophils. Abolishing MDMs in tumors skewed remaining neutrophils and microglia toward a homeostatic state, reduced expression of immune checkpoint molecules on T cells, and extended the survival of H3.1K27M DMG-bearing mice. In contrast, H3.3K27M DMG showed independence from MDM recruitment, suggesting reliance on other TME-driven signaling. Last, H3.1K27M DMGs exhibited reduced microglia presence and a dose-dependent increase in MDM infiltration postirradiation. MDM depletion did not further enhance radiation efficacy, potentially due to compensatory recruitment of classical neutrophils. Collectively, these data reveal histone mutation–specific myeloid dependencies in DMG, highlighting MDM-independent mechanisms in H3.3K27M tumors and MDM-dependent pathways in H3.1K27M tumors.