Abstract
BACKGROUND: With no successful treatment options available, diffuse midline glioma (DMG) are fatal brain tumors with a median survival of ∼12 months. DMG are characterized by transcriptional dysregulation induced by the H3K27M driver mutations. Since these histone mutations cannot yet be directly targeted pharmacologically, our goal is to identify druggable targets of aberrant transcription in DMG. Previous studies identified RNA polymerase 2 (Pol2)-targeting cyclin-dependent kinase 9 inhibitors (CDK9i) and histone deacetylase inhibitors (HDACi) as potential DMG therapeutics, and CDK9i and HDACi are under investigation in ongoing glioma clinical trials. This study aims to develop CDK9i+HDACi combination therapies for DMG. METHODS: We determined the consequences of CDK9i+HDACi combination treatment in DMG using in vitro dose curve analyses, orthotopic xenograft models, and molecular profiling through immunofluorescence imaging, Western blotting, flow cytometry and RNA-seq analysis. RESULTS: We find that CDK9i+HDACi combination therapy synergistically reduces DMG growth in vitro and alters DMG protein post-translational modifications associated with gene regulation by reducing phos-Pol2-Ser2 and increasing histone acetylation. Further RNA-seq analysis identified unique transcriptional changes induced by CDK9i+HDACi, including the downregulation of genes associated with mitochondrial function, cell division, and DNA repair. In line with these gene expression changes, additional studies suggest that CDK9i+HDACi treatment induces DNA damage and cell cycle defects. Finally, preliminary pre-clinical studies using an orthotopic DMG xenograft model reveal that CDK9i and HDACi treatment reduces DMG tumor growth as measured by bioluminescent imaging. CONCLUSIONS: Together, these studies suggest that CDK9i and HDACi co-treatment alters DMG protein modifications and gene expression leading to cell cycle defects, DNA damage, and reduced tumor growth. In view of the proposed synergistic role of CDK9i and HDACi in DMG, additional ongoing studies are examining if this combination may represent a novel approach for the treatment of DMG.