DMG-57. Targeting neurotransmitter signaling to overcome resistance to CDK9 inhibitors in diffuse midline glioma

Abstract Diffuse midline glioma (DMG) is a subset of aggressive brain tumors with few effective therapies. Cyclin-dependent kinase 9 (CDK9i) inhibitors such as zotiraciclib (ZTR) are in clinical trials for glioma, but CDK9i treatment alone may be ineffective due to the development of drug resistance. We analyzed the transcriptomes of DMG xenograft tumors after three weeks of treatment with ZTR revealing that genes encoding synaptic proteins like serotonin and glutamate receptors were up-regulated in the surviving cells, suggesting that altered synaptic signaling pathways may mediate CDK9i resistance. We also performed drug synergy screens using a library of FDA-approved and bioactive compounds and found that drugs that modulate neurotransmitter signaling like SSRIs and SNRIs increased DMG sensitivity to a low dose of ZTR. Follow-up studies confirm that combinations of structurally distinct CDK9i (ZTR, AZD4573) and SSRI/SNRI (sertraline, duloxetine) synergistically reduce DMG growth in neurospheres. CDK9i+SSRI combination treatment was also sufficient to reduce tumor growth and improve survival in a DMG xenograft model. Through phosphoproteomics, we find that an acute treatment with CDK9i+SSRI resulted in unique changes in protein phosphorylation compared to either CDK9i or SSRI treatment alone. Differentially phosphorylated proteins induced by CDK9i+SSRI treatment included chromatin remodelers, Pol2 elongation factors, and splicing regulators, indicating that our combination therapy may alter DMG gene regulation. Indeed, further RNAseq and chromatin profiling studies reveal that CDK9i+SSRI treatment resulted in altered histone dopaminylation and Pol2 phosphorylation across the genome and led to more robust transcriptional changes than either CDK9i or SSRI treatment alone. Comparing the transcriptomes of the CDK9i+SSRI-treated DMG cells to our ZTR-resistance gene signature revealed that many of the transcripts induced in the ZTR-resistant DMG tumors were downregulated by CDK9i+SSRI combination therapy. These findings suggest that CDK9i+SSRI/SNRI drug combinations alter DMG signaling to chromatin to overcome transcriptional changes underlying CDK9i resistance and reveal a connection between dysfunctional neurotransmitter signaling, histone dopaminylation and aberrant gene expression in DMG.