DMG-35. Rewriting cellular fate: reprogramming diffuse midline glioma cells into post-mitotic cells

Background Diffuse midline gliomas (DMGs) are amongst the most lethal pediatric brain tumors. Patients diagnosed with DMG face a dismal prognosis with an overall survival of 9-12 months from diagnosis. Somatic H3 K27M mutations drive global hypomethylation and maintains the stemness phenotype of these tumors. The lack of terminal differentiation provides the opportunity for pharmacological reprogramming of DMGs to non-malignant cells as shown in other cancers including breast cancer and glioblastoma. Here, we thus hypothesized that the DMG epigenome is primed for pharmacologically induced transformation into post-mitotic non-malignant cells. Methods Published protocols were assessed for their efficacy of inducing DMG cell differentiation into adipocytes and neurons. A pharmacological cocktail comprising hormones, corticosteroids, and pharmacological agonists was used to induce adipocyte formation. Furthermore, a combination of five different small molecules was used to induce neuronal differentiation. Phenotypic characteristics of reprogrammed DMG cells were assessed by monitoring proliferation, morphological manifestation, and cell-type antigen expression. Results Treatment with the adipocyte cocktail resulted in accumulation of lipid-like droplets in DMG cells and increased PPARɣ expression, a marker typical for mature adipocytes. The neuron differentiation cocktail resulted in increased expression of neuronal markers (TUJ1, MAP2 and NeuN), significant (p-value <0.001) decreased of proliferation, and morphological changes. As further proof, expression of neuronal and adipocyte markers indicating post-mitotic differentiation was validated with western blots. In vivo assays determining in situ behavior of differentiated cells are underway. Conclusion Our preliminary results suggest that H3K27M-mutant DMG cells can be differentiated into potential non-mitotic cells. Compounds used in this study are clinically relevant with most crossing the blood-brain barrier.