Abstract
Mutations in the histone 3 (H3) gene (H3K27M) are the eponymous driver in diffuse midline gliomas (DMGs). Emerging understanding of H3K27M biology suggests that secondary cooperating factors are required to promote oncogenesis. We have 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 drive a gene expression program which suppresses scheduled transcription of pro-differentiation pathways and promotes self-renewal of tumor stem cells. We initially performed an shRNA screen targeting 408 genes classified as epigenetic/chromatin-associated molecules in a patient-derived DMG cell line. We validated initial hits in secondary screens using multiple shRNA constructs. Live cell imaging systems were utilized to assay growth and neurosphere formation. Finally, we employed RNA-seq-based gene expression analysis to delineate downstream transcriptional changes under AFF4 regulatory control. We found that AFF4 expression is consistently elevated in DMG lines relative to normal pons, and AFF4 inhibition results in decreased rates of growth and stem cell maintenance uniquely within the context of the H3K27M mutation. Further mechanistic studies will lay the groundwork for pre-clinical validation of SEC inhibition as a novel therapeutic approach in DMG.