THE ONCOHISTONE H3.3K27M DRIVES DIFFUSE INTRINSIC PONTINE GLIOMA INDEPENDENT OF FUNCTIONAL EZH2

Diffuse Intrinsic Pontine Glioma (DIPG) is an incurable childhood brain tumor. Recent studies demonstrate that the replication independent oncohistone H3.3K27M, mutated in 65% of DIPG cases, inhibits Enhancer of zeste homolog 2 (EZH2), the enzymatic component of Polycomb Repressor Complex 2 (PRC2), leading to a global reduction in H3K27me3 levels and gene de-repression. Conflicting data exists supporting both an oncogenic and a tumor suppressor role for EZH2 in cancer. The present work addressed the functional significance of EZH2 and its cross-talk with the mutant histone H3.3K27M, in the context of DIPG pathogenesis. Brainstem tumors were established by intracranial injections of NTv-a; EZH2f/fneonatal pups using Replication Competent Avian Sarcoma leucosis virus long terminal repeat with splice acceptor (RCAS) viruses, expressing PDGF-B p53 shRNA, and RCAS-Cre/Y. Immunohistochemical staining (IHC) for Ki-67, H3K27me3, GFAP, Olig-2 and Nestin were performed on the Discovery ULTRA (Ventana). Cell proliferation assay (BrdU) was performed in neurosphere cultures established from brainstems of NTv-a; EZH2f/fneonates, using the EZH2 inhibitor, EPZ011989 (1mM, Epizyme) for 5 days. Ezh2 deletion in NTv-a; Ezh2f/fmice exacerbated DIPG pathogenesis, indicated by a 2.5-fold higher Ki-67 immunostaining (pEzh2deletion in neurospheres from Ntv-a; Ezh2f/fneonates enhanced cell proliferation as did the addition of EPZ011989. Superimposition of the H3.3K27M mutant histone in this background, significantly shortened tumor latency, with a median survival of 54 days compared to 70 days in H3.3 wild type histone control group (p<0.05) and 50% incidence of Grade IV tumors. These results suggest that H3K27M may be oncogenic even in the absence of EZH2. FUTURE Current studies aim to analyze the genetic and epigenetic landscape of these tumors using RNA-Seq and functional assays to determine whether EZH2 inhibitors will be a viable option for children with DIPG in the clinic.