DDDR-44. REPURPOSING SSRI/SNRI 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 resistance. To identify CDK9i resistance mechanisms, we analyzed the transcriptomes of DMG xenograft tumors after three weeks of treatment with ZTR, which revealed that transcripts encoding synaptic signaling regulators such as glutamate receptors were overexpressed in surviving cells, suggesting that altered regulation of synaptic signaling 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, such as SSRIs and SNRIs (selective serotonin and serotonin/norepinephrine reuptake inhibitors), increased DMG sensitivity to an IC25 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 by BLISS analysis and show that co-treatment with CDK9i and SSRI/SNRI strongly induces the apoptotic marker cleaved caspase 3 and reduces the CDK9 target phos-Pol2Ser2. Additional phospho-proteomic analyses identified differentially phosphorylated proteins following CDK9i+SSRI treatment, including chromatin regulators, Pol2 elongation factors, and splicing regulators. Further RNAseq studies reveal that CDK9i+SSRI treatment resulted in more robust transcriptional changes than either drug alone, including the downregulation of transcripts associated with ZTR resistance such as GRIA3/4, and SYT12. These observations suggest that CDK9i +SSRI/SNRI drug combinations may overcome some of the transcriptional changes underlying CDK9i resistance. Finally, preclinical studies show that co-treatment with low doses of AZD4573 and sertraline reduces the growth of orthotopic DMG xenografts and prolongs overall survival. Taken together, these data suggest that SSRI/SNRI and CDK9i might be used in combination to synergistically reduce DMG growth and overcome CDK9i resistance.