PF449 INACTIVATION OF THE NUCLEAR INTERACTING SET DOMAIN PROTEIN 1 IMPAIRS GATA1‐REGULATED TERMINAL ERYTHROID MATURATION AND INDUCES ERYTHROLEUKEMIA

Abstract

Background:The nuclear receptor binding SET domain protein 1 (NSD1) methyltransferase is target of recurrent genetic alterations in acute myeloid leukemia (AML) and solid cancers.Aims:To better understand its function in hematopoiesis, we genetically inactivated NSD1 in human and mouse hematopoietic cells.Methods:Expression of human NSD1 was reduced by lentiviral shRNA‐mediated knockdown. Nsd1 was inactivated in mice by crossing Nsd1 fl/fl animals with the Vav‐iCre ablator strain. Nsd1 −/− mice were characterized by measuring blood values, histology, cell cultures, flow cytometry and gene expression profiling. Molecular mechanisms were addressed by RNA‐Seq, ChIP‐Seq and proteome analysis upon induced differentiation of Nsd1 −/− cells virally expressing wildtype or a catalytic inactive Nsd1 mutant.Results:Knockdown of NSD1 mRNA significantly altered clonogenic growth of human CD34+ hematopoietic cells leading to aberrant accumulation of erythroid progenitor cells. Inactivation of Nsd1 in mice resulted in a highly penetrant lethal disease between 6–21 weeks of age. Conversely, heterozygous littermates expressed normal Nsd1 levels and remained healthy. Symptomatic mice displayed anemia, thrombocytopenia, reticulocytosis, splenomegaly and multi‐organ infiltration, with erythroblasts on peripheral blood smears. Bone marrow (BM) transplantation propagated the disease phenotype in wild type recipients, alone, or in competition. RNA sequencing of BM cells from diseased mice revealed aberrant expression of genes associated with erythroid maturation, self‐renewal and malignant transformation. In vitro terminal erythroid maturation of Nsd1 −/− erythroblasts was impaired and associated with constitutive protein expression of the erythroid transcriptional master regulator GATA1. Transactivation of selected GATA1 positively‐regulated targets was reduced, while the expression of GATA1‐repressed target genes was not affected. Similar to observations in Friend virus‐driven mouse erythroleukemia cells, overexpression of exogenous GATA1L overcame the terminal differentiation block of Nsd1 −/− erythroblasts, dependent on the integrity of the GATA1 N‐ and C‐terminal zinc‐finger domains. Notably, retroviral expression of Nsd1, but not of a catalytically‐inactive Nsd1N1918Q mutant, also rescued terminal erythroid differentiation of the cells. Early terminal maturation was associated with upregulation of erythroid regulators at mRNA and proteome level. Differentiation of Nsd1 −/− erythroblasts expressing Nsd1 was associated with increased expression of previously proposed GATA1 target genes which was accompanied by increased occupancy of GATA1 and H3K27 acetylation at promoter regions. Analysis of protein association to GATA1 in Nsd1 −/− cells expressing either catalytically active or inactive NSD1 revealed differential interactions with potent transcriptional co‐repressors.Summary/Conclusion:Collectively, our work identifies NSD1 as a novel regulator of terminal erythroid differentiation. Our study indicates that the methyltransferase activity of NSD1 promotes the co‐transcriptional repressor activity of GATA1 during erythropoiesis.