Abstract LB-186: Somatic Y641 mutations in EZH2 alter the balance of di- and tri-methylation of H3K27 in diffuse large B-cell lymphomas

Abstract

Abstract EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) responsible for methylating histone H3 on lysine 27 (H3K27) and repressing transcription of target genes. PRC2 activity is essential for maintaining the self-renewal capacity of embryonic and adult stem cells and the dynamic regulation of this activity is critical for proper development and differentiation. Dysregulation of H3K27 methylation is implicated in tumorigenesis and occurs through multiple mechanisms. Elevated levels of EZH2 are known to correlate with poor prognosis in a number of solid tumors including prostate and breast. Inactivating mutations in UTX, an H3K27 demethylase which acts in opposition to EZH2, have been described in several tumor types including multiple myeloma, esophageal squamous cell carcinoma, and renal cell carcinoma. More recently, somatic mutations in EZH2 were identified in myelodysplastic syndrome (MDS), follicular lymphoma (FL), and GCB diffuse large B cell lymphoma (DLBCL). While the mutations in MDS are often homozygous and encompass missense, nonsense, and frame shift mutations at multiple regions of the protein, the mutations in DLBCL and FL are heterozygous and occur at a single residue (Y641) suggesting that the effect of these mutations on PRC2 activity could be quite different between MDS and lymphoma. Utilizing a biochemical approach with recombinant PRC2 containing either wild-type or mutant EZH2, we demonstrate that Y641 mutants exhibit an altered substrate preference. In contrast to wild-type EZH2 which prefers an unmodified or mono-methylated K27 residue, Y641 mutants act primarily on a di-methylated H3K27 with little activity for unmodified or mono-methylated K27. Consistent with these biochemical data, we find that when compared to wild-type lymphoma cell lines those harboring Y641 mutations have elevated levels of H3K27me3 and reduced H3K27me2. To further characterize the substrate specificity of PRC2 complexes containing either WT or mutant EZH2, we utilized an epigenetic peptide library which contained unmodified and modified peptides from histone H2A, H2B, H3 and H4. In addition, we have conducted cell-based studies to understand the effect of these mutations on H3 methylation and the regulation of EZH2 target genes. These data and the implications of these findings for the treatment of FL and DLBCL will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-186. doi:10.1158/1538-7445.AM2011-LB-186