DIPG-07. EPIGENOME SCREENING IDENTIFIES TRANSCRIPTIONAL ELONGATION AS THERAPEUTIC VULNERABILITY IN H3K27M-MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract

Mutations in the histone 3 (H3) gene (H3K27M) are the eponymous driver in diffuse intrinsic pontine gliomas (DIPGs), aggressive pediatric brain tumors for which no curative therapy currently exists. In order to identify specific epigenetic dependencies which arise as a consequence of the H3K27M mutation, we performed an shRNA screen targeting 408 genes classified as epigenetic/chromatin-associated molecules in patient-derived DIPG cell lines. This identified AFF4, a component of the super elongation complex (SEC), as both highly expressed and necessary for DIPG cells to maintain growth and self-renewal. We hypothesized that SEC overexpression occurs as a direct consequence of the H3K27M mutation and that this relative abundance overcomes repressive transcriptional regulation in order to suppresses differentiation and promote self-renewal of DIPG tumor stem cells. We found that AFF4 expression is consistently elevated in both DIPG patient samples and established cell lines relative to normal pediatric pons. We interrogated the role of AFF4 in H3K27M-mutant DIPG 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 differentiation programs under AFF4 regulatory control. Finally, we sought to determine whether CDK9, the catalytic subunit of the SEC, represents a therapeutic vulnerability in DIPG. Using ChIP-seq following CDK9 pharmacologic inhibition, we demonstrate that the loss of regulatory transcriptional input in DIPG may be reversed via CDK9 inhibition and that this acts as a potent inhibitor of DIPG cell growth both in vitro and in vivo orthotopic xenograft models. These studies represent the first pre-clinical validation of SEC inhibition as a novel therapeutic approach in DIPG.