PDTM-41. SUPER ELONGATION COMPLEX-MEDIATED TRANSCRIPTIONAL DEPENDENCY IN H3K27M-MUTANT DIFFUSE MIDLINE GLIOMAS

Abstract

Mutations in the histone 3 (H3) gene (H3K27M) are the eponymous driver in diffuse midline gliomas (DMGs), aggressive pediatric brain tumors for which no curative therapy currently exists. Emerging understanding of H3K27M biology suggests that secondary factors are required to promote oncogenesis. In order to identify specific epigenetic co-operators, we performed an shRNA screen targeting 408 genes classified as epigenetic/chromatin-associated molecules in patient-derived DMG cell lines. This identified AFF4, a component of the super elongation complex (SEC), as critical for DMG viability. We hypothesized that AFF4 promotes DMG tumorigenesis by co-operating with the H3K27M mutation to suppress scheduled transcription of pro-differentiation pathways and promote self-renewal of tumor stem cells. We found that AFF4 expression is consistently elevated in both DMG patient samples and established cell lines relative to the normal pediatric pons. We interrogated the role of AFF4 in H3K27M-mutant DMG using an shRNA lentiviral approach. Using live cell imaging, we demonstrate a significant decrease in in vitro clonogenicity and stem cell maintenance following AFF4 depletion. We employed RNA-seq-based gene set enrichment analysis to delineate downstream transcriptional changes under AFF4 regulatory control. Finally, we sought to determine whether CDK9, the catalytic subunit of the SEC, represents a therapeutic vulnerability in DMG. Using a combination of CDK9 overexpression and pharmacologic inhibition, we demonstrate that the disordered regulatory input of the SEC in DMG is dependent on the kinase activity of CDK9 and that this may be exploited through small molecule inhibition in both in vitro and in vivo patient-derived xenograft models. These studies represent the first pre-clinical validation of SEC inhibition as a novel therapeutic approach in pediatric DMG.