DMG-10. Synergistic CDK9 and BET inhibition overcomes therapeutic resistance in H3K27M pediatric diffuse midline gliomas by reprogramming DNA damage response

Abstract Among pediatric high-grade gliomas (pHGGs), diffuse midline glioma (DMG) harboring H3K27M mutation stands out as an aggressive brain tumor with dismal overall survival. The mutation in the histone H3 genes results in epigenetic changes affecting H3K27 acetylation and trimethylation, hence, affecting gene expression. Although significant progress has been made in understanding its biology, there are currently no effective treatment strategies that specifically target the unique pathology of it. Therefore, the aim of this study was to develop a specific combination therapy for H3K27M DMG. Epigenetic drug screens of nine H3K27M, two H3G34R/V, three wildtype pHGGs and three human non-malignant control cell lines revealed hits targeting cyclin-dependent kinase 9 (CDK9), bromodomain and extra-terminal domain (BET) and aurora kinase with H3K27M-selective potency. These were used in combination and synergy screens, and the most potent combination partners were identified to be CDK9 inhibitor (CDK9i) and BET inhibitor (BETi) affecting transcription elongation as a specific vulnerability in H3K27M DMGs. High content microscopy imaging of 3D tumoroids revealed that the CDK9i-BETi combination was specifically and significantly more cytotoxic for H3K27M DMGs compared to H3G34V/H3 WT pHGG and healthy control cell models. Moreover, the identified combination therapy was found to be either additive or synergistic with ionizing radiation and can therefore be effectively integrated in standard-of-care therapy. Bulk RNA sequencing and proteomics demonstrated that CDK9i and BETi downregulated DNA damage response (DDR) pathways. Combination therapy caused DNA damage accumulation and induced apoptosis in H3K27M DMG models in vitro. It also facilitated tumor shrinkage in zebrafish early larvae bearing H3K27M DMG cells but showed limited effect in those injected with H3G34V pHGG cells. Overall, combination of CDK9i and BETi demonstrates selectivity for H3K27M DMG in vitro and in vivo by synergistically downregulating DDR pathways, inducing DNA damage and apoptosis, hence holds potential for future clinical trials.