Acute erythroleukemia (AML-M6 or AEL) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing three genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (e.g. DNMT3A, TET2 or IDH2), and undefined cases with low mutational burden. We established an erythroid vs. myeloid transcriptomics-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, more than 25% of AEL patients, including in the genetically-undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1 binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicates that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.

The nuclear receptor binding SET domain protein 1 (NSD1) is recurrently mutated in human cancers including acute leukemia. We show that NSD1 knockdown alters erythroid clonogenic growth of human CD34 + hematopoietic cells. Ablation of Nsd1 in the hematopoietic system of mice induces a transplantable erythroleukemia. In vitro differentiation of Nsd1 −/− erythroblasts is majorly impaired despite abundant expression of GATA1, the transcriptional master regulator of erythropoiesis, and associated with an impaired activation of GATA1induced targets. Retroviral expression of wildtype NSD1, but not a catalytically-inactive NSD1 N1918Q SET-domain mutant induces terminal maturation of Nsd1 −/− erythroblasts. Despite similar GATA1 protein levels, exogenous NSD1 but not NSD N1918Q significantly increases the occupancy of GATA1 at target genes and their expression. Notably, exogenous NSD1 reduces the association of GATA1 with the co-repressor SKI, and knockdown of SKI induces differentiation of Nsd1 −/− erythroblasts. Collectively, we identify the NSD1 methyltransferase as a regulator of GATA1-controlled erythroid differentiation and leukemogenesis.

Recurrent epigenomic alterations associated with multiple human pathologies have increased the interest in the nuclear receptor binding SET domain protein 1 (NSD1) lysine methyltransferase. Here, we review the current knowledge about the biochemistry, cellular function and role of NSD1 in human diseases. Several studies have shown that NSD1 controls gene expression by methylation of lysine 36 of histone 3 (H3K36me1/2) in a complex crosstalk with de novo DNA methylation. Inactivation in flies and mice revealed that NSD1 is essential for normal development and that it regulates multiple cell type-specific functions by interfering with transcriptional master regulators. In humans, putative loss of function NSD1 mutations characterize developmental syndromes, such as SOTOS, as well as cancer from different organs. In pediatric hematological malignancies, a recurrent chromosomal translocation forms a NUP98-NSD1 fusion with SET-dependent leukemogenic activity, which seems targetable by small molecule inhibitors. To treat or prevent diseases driven by aberrant NSD1 activity, future research will need to pinpoint the mechanistic correlation between the NSD1 gene dosage and/or mutational status with development, homeostasis, and malignant transformation.

We have uncovered a novel role for the nuclear receptor-binding SET domain protein 1 (NSD1) in human and murine erythroid differentiation. Mechanistically, we found that the histone methyltransferase activity of NSD1 is essential for chromatin binding, protein interactions and target gene activation of the erythroid transcriptional master regulator GATA1.

The prognosis of stage III/IV Merkel cell carcinoma (MCC) is very poor. The Phosphatidylinositol 3-kinase p110δ specific inhibitor idelalisib has recently been reported to induce complete clinical remission in a stage IV MCC patient. Here we assessed the expression of p110δ in primary MCC and MCC cell lines including its functionality.Immunofluorescence microscopy revealed a specific cytoplasmic p110δ expression in 71.4% of the tested MCCs and in all tested MCC cell lines. Compared to the B cell leukemia cell line REH all MCC cell lines, except MKL-1, revealed a lower response towards the treatment with idelalisib. MKL-1 showed a 10-fold higher IC50 compared to REH which was accompanied by a significant decrease of Akt phosphorylation. However, treating the MCC cells with the specific PI3K p110α subunit inhibitor BYL719 led to a more effective decrease of the cell viability compared to idelalisib: WaGa cells 30-fold, PeTa cells 15-fold and all other MCC cell lines 3-fold.Although PI3K p110δ is expressed in the majority of MCCs and cell lines its inhibition by idelalisib alone does not suffice to effectively affect MCC cells viability.

Malignancies of the erythroid lineage are rare but aggressive diseases. Notably, the first insights into their biology emerged over half a century ago from avian and murine tumor viruses-induced erythroleukemia models providing the rationale for several transgenic mouse models that unraveled the transforming potential of signaling effectors and transcription factors in the erythroid lineage. More recently, genetic roadmaps have fueled efforts to establish models that are based on the epigenomic lesions observed in patients with erythroid malignancies. These models, together with often unexpected erythroid phenotypes in genetically modified mice, provided further insights into the molecular mechanisms of disease initiation and maintenance. Here, we review how the increasing knowledge of human erythroleukemia genetics combined with those from various mouse models indicate that the pathogenesis of the disease is based on the interplay between signaling mutations, impaired TP53 function, and altered chromatin organization. These alterations lead to aberrant activity of erythroid transcriptional master regulators like GATA1, indicating that erythroleukemia will most likely require combinatorial targeting for efficient therapeutic interventions.

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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 shR-NA-mediated knockdown. Nsd1 was inactivated in mice by crossing Nsd1fl/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 Nsd1 N1918Q 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 t...