Oncohistone inhibition reshapes tumor–microenvironment communication in Diffuse Midline Glioma (DMG)

Background

Diffuse midline glioma (DMG) is a lethal pediatric brain tumor driven by the H3K27M oncohistone, which disrupts epigenetic regulation and promotes tumor proliferation. While prior studies show that H3K27M is essential for tumor initiation, its role in established tumors, tumor microenvironment (TME) regulation, and therapeutic response remain unclear.

Methods

Here, we developed inducible and reversible H3.3K27M and H3.1K27M cell and mouse models to study oncohistone-dependent effects on tumor growth, recurrence, and the immune/stromal microenvironment. We generated a tetracycline-inducible PiggyBac-based oncohistone expression cassette in patient- and murine-derived models and validated inducible and reversible H3K27M expression.

Results

Re-expression of H3K27M in knockout cells induced morphological changes and suppressed astrocytic markers. Chromatin accessibility profiling revealed distinct states between ON, OFF, and OFF–ON groups, including PD1-mediated immunosuppressive mechanisms associated with H3K27M expression. Single-cell RNA sequencing demonstrated that the oncohistone reshapes the TME. H3K27M expression promotes tumor–neuron interactions, enhances neuronal excitability, excitatory/inhibitory imbalance, and synaptic connectivity that supports tumor proliferation. These effects are associated with increased glutamatergic signaling and enhanced tumor–neuron coupling through glutamate transport and receptor pathways, including EAAT1 ( SLC1A3 ) and AMPARs ( GRIA3 ). Conversely, H3K27M inhibition reduces neuronal excitation, disrupts tumor-associated signaling, and partially restores neuron–neuron and neuron–immune communications. These findings identify H3K27M as a key driver of excitatory neuron-to-tumor coupling and immunosuppression in DMG.

Conclusions

Overall, our findings demonstrate that H3K27M extensively reshapes TME in DMG and support direct oncohistone targeting as a potential therapeutic strategy, including potential CRISPR-based or small-molecule approaches for patients with H3K27M-mutant DMG.

Key Points

• We developed inducible and reversible H3K27M DMG models to investigate the role of H3K27M in the tumor microenvironment.

• H3K27M promotes tumor–neuron communication, while its inhibition disrupts these interactions, supporting H3K27M-targeted therapies for DMG.

Importance of Study

Diffuse midline glioma (DMG) remains one of the deadliest pediatric brain tumors, with limited effective treatment options and poor patient survival. Although the H3K27M oncohistone is recognized as a key driver of tumor initiation, its role in maintaining tumor progression and shaping the tumor microenvironment is unclear. In this study, we developed inducible and reversible H3.3K27M and H3.1K27M murine and patient-derived DMG cell- and mouse-models that enabled precise control of the oncohistone expression. Using these models, we demonstrate that H3K27M actively promotes tumor–neuron interactions, neuronal excitability, and glutamatergic signaling pathways that support tumor growth. Importantly, inhibition of H3K27M disrupted these tumor-associated signaling networks and partially restored neuron–immune communication within the tumor microenvironment. Together, these findings demonstrate that H3K27M extensively reshapes the tumor microenvironment in these Diffuse Midline Gliomas and provides strong rationale for directly targeting the oncohistone as a therapeutic strategy for patients with H3K27M-mutant DMG.

Lay Summary

Diffuse Midline Glioma (DMG) is a devastating childhood brain cancer. Despite decades of research, radiation remains the primary treatment and provides only temporary benefit. Most DMGs carry a mutation called H3K27M, which is an attractive target for new treatments such as directly inhibiting or removing this mutation using gene-editing. However, it remains unclear whether inhibiting H3K27M alone will be sufficient to stop the growth of established tumors. In this study, we developed human and mouse models that allow H3K27M to be turned on and off. We found that H3K27M helps tumors communicate with surrounding cells, particularly neurons. Inhibiting H3K27M disrupted tumor-promoting interactions and partially restored normal communication, supporting direct H3K27M-targeted therapies as a promising strategy for children with DMG.