Co-targeting neurotransmitter signaling and CDK9 to treat diffuse midline glioma [CUT&RUN]

Aberrant neurotransmitter signaling and transcriptional dysregulation are hallmarks of glioma and represent potential therapeutic targets. In this study, we uncover mechanisms of crosstalk between monoamine neurotransmitter signaling and transcriptional regulation in diffuse midline glioma (DMG) that can be co-targeted with CDK9 inhibitors (CDK9i) and neuropsychiatric drugs as a potential treatment strategy for DMG. We identified compounds that synergize with CDK9i in DMG using targeted drug screens, then assessed DMG response to CDK9i and neurotransmitter-targeting drugs in cell culture and in orthotopic xenograft mouse models. We then used a multi-omic approach combining proteomic, transcriptomic, and chromatin profiling with phospho-proteomics, RNA-seq, CUT&RUN, and ATAC-seq in DMG cells to characterize mechanisms of synergy between CDK9i and neuropsychiatric drugs. We observe differential expression of neuronal signaling genes in both CDK9i-resistant DMG xenograft tumors and CDK9-low pediatric high grade glioma patient tumors. Additional studies reveal that drugs that modify monoamine neurotransmitter signaling, including neurotransmitter reuptake inhibitors (SSRI/SNRI) and both metabotropic neurotransmitter receptor agonists and antagonists, strongly synergize with CDK9i to reduce DMG cell viability. Co-treating DMG with CDK9i+SSRI/SNRI triggered negative feedback inhibition of autocrine neurotransmitter receptor signaling and reversed a CAMKII-dependent mechanism of CDK9i resistance. Finally, we find that CDK9i+SSRI resulted in altered histone dopaminylation at neurodevelopmental genes associated with CDK9i-resistance. These findings identify neurotransmitter signaling pathways as novel points of vulnerability in CDK9i-resistant DMG that can be targeted with FDA-approved psychiatric drugs to modulate CAMKII signaling and histone dopaminylation in pediatric brain tumors.