Developmental Stage and Cellular Context Determine Oncogenic and Molecular Outcomes ofEzh2Y641FMutation in Hematopoiesis

Abstract Mutations in the histone methyltransferase EZH2, particularly the Y641 hotspot mutation, have been implicated in hematologic malignancies, yet the effect of timing and cellular context on their oncogenic potential has remained unknown. In this study, we utilized a conditional allele with tissue-specific Cre drivers to investigate the effects of Ezh2Y641F mutations at various stages of development, with a focus on the hematopoietic system. We found that ubiquitous heterozygous Ezh2Y641F expression at birth, or conditional expression in hematopoietic or mesenchymal stem cells, led to decreased survival due to hematopoietic defects and bone marrow failure, with no evidence of malignancy. In contrast, Ezh2Y641F expression in committed B cells drives lymphoma formation, highlighting the lineage-specific oncogenic activity of the mutation. Transcriptomic analysis of B cell progenitors revealed key pathway alterations between Cre models such as altered IL2-Stat5 signaling pathway, differential expression of E2F targets, and altered GTPase pathway expression driven by upregulation of Guanylate Binding Proteins (GBPs) in Mx1-Cre Ezh2Y641F pro-B cells. We further found that the GBP locus is regulated by Ezh2-mediated H3K27me3, it is associated with poorer survival in Acute Myeloid Leukemia patients and has variable effects on apoptosis in human lymphoma and leukemia cell lines. These findings suggest that the Ezh2Y641F mutation may alter immune regulatory pathways, cell differentiation and apoptosis, with potential implications for disease progression. Our results highlight the critical role of mutation timing and cellular context in EZH2-driven hematopoietic disease, resulting in distinct downstream changes that shape the oncogenic impact of EZH2. Competing Interest Statement The authors have declared no competing interest. Footnotes Small changes to the text to improve clarity and discussion. Addition of Supp. Table 2 that lists all primer sequences used in experiments. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE270480