MTAP-related increased erythroblast proliferation as a mechanism of polycythaemia vera

Polycythaemia vera (PV) is a haematological disorder caused by an overproduction of erythroid cells. To date, the molecular mechanisms involved in the disease pathogenesis are still ambiguous. This study aims to identify aberrantly expressed proteins in erythroblasts of PV patients by utilizing mass spectrometry-based proteomic analysis. Haematopoietic stem cells (HSCs) were isolated from newly-diagnosed PV patients, PV patients who have received cytoreductive therapy, and healthy subjects. In vitro erythroblast expansion confirmed that the isolated HSCs recapitulated the disease phenotype as the number of erythroblasts from newly-diagnosed PV patients was significantly higher than those from the other groups. Proteomic comparison revealed 17 proteins that were differentially expressed in the erythroblasts from the newly-diagnosed PV patients compared to those from healthy subjects, but which were restored to normal levels in the patients who had received cytoreductive therapy. One of these proteins was S-methyl-5′-thioadenosine phosphorylase (MTAP), which had reduced expression in PV patients’ erythroblasts. Furthermore, MTAP knockdown in normal erythroblasts was shown to enhance their proliferative capacity. Together, this study identifies differentially expressed proteins in erythroblasts of healthy subjects and those of PV patients, indicating that an alteration of protein expression in erythroblasts may be crucial to the pathology of PV.

LKB1 Coordinates Metabolic Pathways to Maintain Homeostasis of Haematopoietic Stem Cells and Regulate Erythropoiesis

Erythropoiesis is a highly regulated multistage process by which hematopoietic stem cells (HSCs) proliferate, differentiate and eventually form mature erythrocytes. Stem-cell maintenance and cell differentiation require homeostasis and coordination of multiple metabolisms. However, underlying mechanism of this coordination is poorly identified. Liver kinase B1 (LKB1) acts as evolutionarily conserved regulator to control cellular metabolism, cell polarity and proliferation during development and stress response. Considering that all the fundamental cellular processes regulated by LKB1 are present in erythropoiesis, we hypothesize that LKB1 may involve in orchestrating this coordination. To explore the role of LKB1 in entire erythropoiesis, we firstly crossed mice carrying loxP-flanked LKB1 alleles with EpoR-tdTomato-Cre mice, which can lead to cleavage in from HSCs to erythroblasts. LKB1fl/fl EPORcre mice developed exhaustion of HSCs, progressive severe anemia and ultimately lethality. Intriguingly, HSCs of LKB1fl/fl EPORcre mice exhibited a swiftly skewness toward the erythoid lineage when LKB1 is deleted. Nevertheless, erythroblasts of LKB1fl/fl EPORcre mice were significantly reduced. Further analysis showed that the colony forming ability of the colony-forming unit-erythroid (CFU-E) cells and subsequent terminal erythoid differentiation in LKB1fl/fl EPORcre mice were seriously impaired. In order to study erythropoiesis more specifically without the influence of stem cells, we next assessed the impact of deletion of LKB1 in the mouse erythropoietic system using GYPA-eGFP-Cre mice. LKB1fl/flGYPAcre mice showed mild anemia but had the normal lifespan.The Ter119 + cells were decreased while CFU-E cells were significantly increased. However, the colony forming ability of sorted CFU-E cells from LKB1fl/flGYPAcre mice were drastically reduced. In addition, LKB1fl/flGYPAcre mice exhibited disordered terminal erythropoiesis. Taken together, these results indicate that LKB1 may play multinomial roles in regulating stem-cell homeostasis and effective erythoid differentiation. Previous studies have shown that LKB1 controls energy metabolism via phosphorylating AMPK and regulating PGC-1 transcription to maintain homeostasis of HSCs. We found that both of p-AMPK and PGC-1 of LKB1fl/fl EPORcre mice were down-regulated in Lin - cells while Ter119 + cells have comparable p-AMPK and PGC-1. In this regard, LKB1fl/flGYPAcre mice, whether in Lin - cells or in Ter119 + cells, exhibited similar p-AMPK and PGC-1 level. Moreover, administration of ZLN005, an activator of PGC-1, partly rescued the anemia in LKB1fl/fl EPORcre mice but not in LKB1fl/flGYPAcre mice. These data imply that there is other underlying mechanism of LKB1 in erythropoiesis. To gain further mechanistic insight, we carried out proteomics analysis. Gene ontology analysis of differentially expressed proteins revealed that cholesterol metabolism related genes were significantly altered under LKB1 deficiency. We then found that LKB1 ablation led to a reduction of cholesterol level and diminishment of expression levels of primary cholesterol biosynthesis related genes. Moreover, the mature active form of SREBP2, the master transcriptional regulator of cholesterol biosynthesis, was prominently reduced. Golgi apparatus, in which SREBP2 is cleaved for activation, is intumescent or dispersed in erythroid cells of LKB1fl/fl EPORcre mice and LKB1fl/flGYPAcre mice. These results supported that loss of LKB1 impaired the cholesterol metabolism in erythropoiesis. To demonstrate Golgi apparatus-dependent cholesterol metabolism is essential for erythropoiesis, we treated LKB1fl/flGYPAcre mice with 2,3-oxidosqualene, the important intermediate in cholesterol synthesis and found that the phenotypes of LKB1fl/flGYPAcre mice were effectively restored. In parallel, 2,3-oxidosqualene treatment just slightly alleviated the anemia of LKB1fl/fl EPORcre mice. However, combination treatment with 2,3-oxidosqualene and ZLN005 was more effective in restoring phenotypes in LKB1fl/fl EPORcre mice, in contrast to ZLN005 alone. Thus, LKB1 serves as an essential metabolic regulator to coordinate energy metabolism in hematopoietic stem cells and lipid metabolism in erythoid cells, thereby maintaining homeostasis of haematopoietic stem cells and functional fitness of erythropoiesis. Disclosures No relevant conflicts of interest to declare.

Complex Interactions in Regulation of Haematopoiesis—An Unexplored Iron Mine

Iron is one of the most abundant metals on earth and is vital for the growth and survival of life forms. It is crucial for the functioning of plants and animals as it is an integral component of the photosynthetic apparatus and innumerable proteins and enzymes. It plays a pivotal role in haematopoiesis and affects the development and differentiation of different haematopoietic lineages, apart from its obvious necessity in erythropoiesis. A large amount of iron stores in humans is diverted towards the latter process, as iron is an indispensable component of haemoglobin. This review summarises the important players of iron metabolism and homeostasis that have been discovered in recent years and highlights the overall significance of iron in haematopoiesis. Its role in maintenance of haematopoietic stem cells, influence on differentiation of varied haematopoietic lineages and consequences of iron deficiency/overloading on development and maturation of different groups of haematopoietic cells have been discussed.

Gata2a maintains cebpa and npm1a in haematopoietic stem cells to sustain lineage differentiation and genome stability

The transcription factor Gata2 is required to produce and maintain haematopoietic stem and progenitor cells (HSPCs) in development and adult haematopoiesis. Mutations in GATA2 lead to GATA2 deficiency syndrome and predispose patients to acquire leukaemia. Here we use zebrafish gata2a enhancer deletion mutants and single cell transcriptomics to understand how GATA2 mediates survival and differentiation of haematopoietic stem cells in GATA2 deficiency. gata2a mutants show marrow failure from 6 months post-fertilization (mpf), accompanied by neutropenia and erythrocytosis. Single cell transcriptional profiling of the adult kidney marrow demonstrated that HSPCs express elevated expression of erythroid- and decreased expression of myeloid genes, including cebpa. This is associated with a lineage skewing towards the erythroid fate at the expense of the myeloid fate. Thus, Gata2a is required to initiate and maintain lineage priming in HSPCs, favouring myeloid differentiation. Gata2a regulates expression of multiple targets associated with replication and DNA damage repair, including npm1a, a zebrafish NPM1 orthologue. Accordingly, mutant marrow cells show increased DNA damage associated with progressive loss of npm1a expression with age. This effect was replicated by inhibiting NPM1 activity in murine HPC7 progenitor cells. We propose that the impaired DDR leads to marrow failure in GATA2 deficiency. This leads to increased genomic instability in the surviving HSPCs, favouring acquisition of secondary leukaemogenic mutations.