EZH2 (Enhancer of Zeste Homolog 2) is the catalytic component of the Polycomb Repressive Complex group 2 (PRC2), which establishes the repressive epigenetic mark histone 3 lysine 27 trimethylation (H3K27me3), resulting in silencing of gene expression. EZH2 was originally identified as a tumor suppressor since loss-of-function events were observed in myelodysplastic syndrome, acute myeloid, and T-cell leukemias. However, next-generation sequencing identified recurrent activating point mutations at tyrosine residue 641 (Y641) in 25% of germinal center diffuse large B-cell lymphoma and 15% of follicular lymphomas, suggesting that EZH2 also functions as an oncogene in a cell type-dependent manner. Using a faithful genetically engineered mouse model, we previously demonstrated that the Ezh2Y641F mutation drives formation of B-cell lymphoma as a single event and in cooperation with Bcl2 amplification or p53 loss. Unexpectedly, at the chromatin level, expression of Ezh2Y641F did not monotonically increase abundance of global H3K27me3 but rather resulted in redistribution of this mark across the genome, exhibiting loss of H3K27me3 at many loci, with direct effects on transcription. Others have shown that EZH2 mediates germinal center proliferation via repression of p21 and cooperation with Bcl6; however, given the global effect of Ezh2Y641F on chromatin, the oncogenic mechanisms of these mutations remain underexplored. These include the timing of the mutation during hematopoietic development, its role during differentiation, and the oncogenic activity of downstream targets. In order to investigate the effect of Ezh2Y641 mutations on hematopoietic development and differentiation, we used Cre alleles to drive expression of the mutant protein at different stages of hematopoietic development. Our data demonstrate that expression of Ezh2Y641 in early B cells is sufficient to drive oncogenic transformation. However, expression of Ezh2Y641 in hematopoietic stem cell (HSC) is not sufficient to drive oncogenic transformation despite the presence of mature healthy B cells. Additionally, expression of Ezh2Y641F in HSCs results in loss of self-renewal, differentiation bias towards the lymphoid lineages, and a partial block at the pre-pro B-cell stage. With regards to direct downstream targets, we identified upregulation of several HoxC cluster genes in Ezh2Y641F-mutant B cells prior to transformation. Upregulation of these genes is mediated via loss of H3K27me3 at the HoxC cluster and focal gains of H3K27 acetylation (H3K27ac). Overall, our findings underline the complicated biology and oncogenic activity of EZH2Y641 mutations in lymphoma, shed light on the effect of these mutations during hematopoietic development, and underscore some of the consequences of the paradoxical loss of H3K27me3 at certain loci.

Citation Format: Samantha J. Nixon, Sarah Zimmerman, Jeremy M. Simon, George P. Souroullas. Understanding the properties and oncogenic mechanisms of EZH2 Y641 mutations in lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-08.