CSIG-15. DEFINING THE ROLE OF HISTONE H3 K27 METHYLATION AND H3.3 S31 PHOSPHORYLATION IN H3.3K27M MUTANT DIFFUSE MIDLINE GLIOMAS

Abstract

Abstract Diffuse midline gliomas (DMG) are aggressive high-grade gliomas that characteristically occurs in children. Heterozygous K27M mutation occurs in histone H3.3 as early events in DMG and are sufficient to drive a global reduction of H3K27 methylation (H3K27me) on all histone H3 variants. Histone H3K27me is responsible for epigenetic gene repression, and loss of K27me is associated with epigenetic dysregulation. However, histone H3.3 has a unique Chk1 phosphorylation site, Serine 31, critical for mitotic checkpoint regulation. Masking of S31 phosphorylation results in the abrogation of cell cycle checkpoints that monitor the alignment and segregation of chromosomes. DMG cells have decreased levels of mitotic S31P and are chromosomally unstable and aneuploid. Genomic-editing of H3.3 to revert the K27M mutation restores mitotic S31P and K27me and significantly reduces the rate of chromosome instability (CIN). Expression of H3.3 K27M or a non-phosphorylatable S31A mutant in normal, diploid cells results in chromosome missegregation and cell cycle checkpoint defects; and cells fail to undergo G1 cycle arrest leading to aneuploidy. H3.3 K27M and S31A inhibit p53 accumulation. In a mouse model of DMG, H3.3K27M and H3.3S31A promoted the development of high-grade gliomas, whereas H3.3WT controls did not. H3.3S31A is WT for K27me, demonstrating that loss of S31P is oncogenic. It remains unclear if it is the H3.3 K27M mutations or the associated loss of H3K27me3 that drives the reduction of S31P. The PRC2 complex is responsible for H3K27me, catalyzed by methyltransferases subunits EZH1 and EZH2. We used genome editing to knockout EZH1 and EZH2 in DMG cells and untransformed human diploid cell lines. Deletion of EZH2 causes a loss in K27 tri-methylation, and EZH1/2 knockouts causes a global loss of K27me. Ongoing studies examine how the loss of K27me affects gene expression, proliferation, H3.3S31P, chromosome missegregation, and aneuploid.