Human Erythroid Differentiation Research Articles

Lysine succinylation precisely controls normal erythropoiesis.

Lysine succinylation (Ksu) has recently emerged as a protein modification that regulates diverse functions in various biological processes. However, the systemically and precise role of lysine succinylation in erythropoiesis remains to be fully elucidated. In this study, we noted a prominent increase of succinyl-CoA and lysine succinylation during human erythroid differentiation. To explore the functional significance of succinylation, we inhibited succinylation by either knock downing key succinyltransferases or overexpressing desuccinylases. Succinylation inhibition led to suppressed cell proliferation, increased apoptosis, and disrupted erythroid differentiation. In vivo overexpression of the desuccinylases SIRT5 delayed erythroid differentiation. Furthermore, integrative proteome and succinylome analysis identifies 939 succinylated proteins with 3,562 Ksu sites, distributed across various cellular compartments and involved in multiple cellular processes. Significantly, inconsistencies between protein expression levels and succinylation levels were observed, indicating that the succinylation of certain proteins may function independently of expression. Mechanistically, we implicated KAT2A-mediated succinylation of histone H3 K79, leading to chromatin remodeling and subsequently erythropoiesis regulation. Specially, we identified CYCS as a key regulator of erythropoiesis, which depends on its succinylation sites K28/K40. Taken together, our comprehensive investigation of the succinylation landscape during erythropoiesis provides valuable insights into its regulatory role and offer potential implications for erythroid-related diseases.

Complete absence of GLUT1 does not impair human terminal erythroid differentiation

AbstractThe glucose transporter 1 (GLUT1) is 1 of the most abundant proteins within the erythrocyte membrane and is required for glucose and dehydroascorbic acid (vitamin C precursor) transport. It is widely recognized as a key protein for red cell structure, function, and metabolism. Previous reports highlighted the importance of GLUT1 activity within these uniquely glycolysis-dependent cells, in particular for increasing antioxidant capacity needed to avoid irreversible damage from oxidative stress in humans. However, studies of glucose transporter roles in erythroid cells are complicated by species-specific differences between humans and mice. Here, using CRISPR–mediated gene editing of immortalized erythroblasts and adult CD34+ hematopoietic progenitor cells, we generate committed human erythroid cells completely deficient in expression of GLUT1. We show that absence of GLUT1 does not impede human erythroblast proliferation, differentiation, or enucleation. This work demonstrates, to our knowledge, for the first time, generation of enucleated human reticulocytes lacking GLUT1. The GLUT1–deficient reticulocytes possess no tangible alterations to membrane composition or deformability in reticulocytes. Metabolomic analyses of GLUT1–deficient reticulocytes reveal hallmarks of reduced glucose import, downregulated metabolic processes and upregulated AMP-activated protein kinase signaling, alongside alterations in antioxidant metabolism, resulting in increased osmotic fragility and metabolic shifts indicative of higher oxidant stress. Despite detectable metabolic changes in GLUT1–deficient reticulocytes, the absence of developmental phenotype, detectable proteomic compensation, or impaired deformability comprehensively alters our understanding of the role of GLUT1 in red blood cell structure, function, and metabolism. It also provides cell biological evidence supporting clinical consensus that reduced GLUT1 expression does not cause anemia in GLUT1–deficiency syndrome.

TXNIP-mediated inhibition of glucose metabolism is a critical step in human erythroid differentiation

TXNIP-mediated inhibition of glucose metabolism is a critical step in human erythroid differentiation

Long noncoding RNA GATA2AS influences human erythropoiesis by transcription factor and chromatin landscape modulation

AbstractLong noncoding RNAs (lncRNAs) are extensively expressed in eukaryotic cells and have been revealed to be important for regulating cell differentiation. Many lncRNAs have been found to regulate erythroid differentiation in the mouse. However, given the low sequence conservation of lncRNAs between mouse and human, our understanding of lncRNAs in human erythroid differentiation remains incomplete. lncRNAs are often transcribed opposite to protein coding genes and regulate their expression. Here, we characterized a human erythrocyte-expressed lncRNA, GATA2AS, which is transcribed opposite to erythroid transcription regulator GATA2. GATA2AS is a 2080-bp long, primarily nucleus-localized noncoding RNA that is expressed in erythroid progenitor cells and decreases during differentiation. Knockout of GATA2AS in human HUDEP2 erythroid progenitor cells using CRISPR-Cas9 genome editing to remove the transcription start site accelerated erythroid differentiation and dysregulated erythroblast gene expression. We identified GATA2AS as a novel GATA2 and HBG activator. Chromatin isolation by RNA purification showed that GATA2AS binds to thousands of genomic sites and colocalizes at a subset of sites with erythroid transcription factors including LRF and KLF1. RNA pulldown and RNA immunoprecipitation confirmed interaction between GATA2AS and LRF and KLF1. Chromatin immunoprecipitation sequencing (ChIP-seq) showed that knockout of GATA2AS reduces binding of these transcription factors genome wide. Assay for transposase-accessible chromatin sequencing (ATAC-seq) and H3K27ac ChIP-seq showed that GATA2AS is essential to maintain the chromatin regulatory landscape during erythroid differentiation. Knockdown of GATA2AS in human primary CD34+ cells mimicked results in HUDEP2 cells. Overall, our results implicate human-specific lncRNA GATA2AS as a regulator of erythroid differentiation by influencing erythroid transcription factor binding and the chromatin regulatory landscape.

Dynamic Changes in Lysine Succinylation As Important Regulators of Erythropoiesis

Lysine succinylation has emerged as a recently discovered protein modification that significantly impacts the chemical environment and exhibits diverse functions in various biological processes. However, the specific role of lysine succinylation in erythropoiesis has not been fully elucidated. In this study, we investigated the levels of six common acylations (acetylation, crotonylation, succinylation, propionylation, butyrylation, and malonylation) in human erythroid cells. Interestingly, we observed a prominent accumulation of lysine succinylation during human erythroid differentiation, suggesting its potential importance in this process. To explore the functional significance of succinylation, we inhibited succinylation in human erythroid progenitor cell line by disrupting the expression of the key succinyltransferases and desuccinylases. The results revealed that succinylation inhibition led to suppressed cell proliferation, increased apoptosis, and disrupted differentiation, indicating the essential role of succinylation in erythropoiesis. Furthermore, integrative proteome and succinylome analysis identifies 939 quantifiable proteins with 2,871 Ksu sites. Notably, we observed inconsistencies between alterations in protein levels and succinylation levels, suggesting that the role of succinylation in proteins' function regulation. These succinylated proteins are widely distributed in various cellular compartments and involved in multiple cell processes, indicating that succinylation is a prevalent modification in erythropoiesis. Mechanically, we identified CYCS as a key target of succinylation during erythropoiesis, emphasizing its essential role in this process. Specially, we implicated KAT2A-mediated histone succinylation in chromatin remodeling, further highlighting the regulatory significance of lysine succinylation in erythropoiesis at the epigenetic level. Collectively, our comprehensive investigation of the succinylation landscape during erythropoiesis provides valuable insights into its regulatory role and offer potential implications for erythroid-related diseases and therapeutic strategies.

Human DOCK11 Deficiency Causes Defective Erythropoiesis and Systemic Inflammation

Erythropoiesis involves significant changes in the cells, which are mediated by the plasma membrane and the actin cytoskeleton. The composition of the actin cytoskeleton and the interaction between its components are dynamically modified during erythropoiesis, however the precise role that different actin regulators play during erythroid differentiation is poorly understood. The dedicator of cytokinesis (DOCK) family member DOCK11 regulates actin cytoskeleton dynamics via its guanine nucleotide exchange factor (GEF) activity, resulting in activation of the small Rho GTPase CDC42. CDC42 has been implicated in regulating the early stages of erythroid development, as well as the terminal maturation steps involving enucleation. However, the role of human DOCK11 in hematopoietic cell function and human disease had not been defined. We analyzed a cohort of four patients from four unrelated families presenting with recurrent infections, early-onset severe immune dysregulation, systemic inflammation, as well as normocytic anemia and anisocytosis of unknown origin. Using whole-exome sequencing, we identified rare, hemizygous germline mutations in DOCK11 in these patients. Two mutations - an early stop-gain mutation and a To study the role of DOCK11 during erythropoiesis, we generated a dock11-knockout zebrafish model. We found that the dock11-knockout zebrafish embryos recapitulated the anemia and aberrant erythrocyte morphology observed in human DOCK11 deficiency. The anemia was amenable to rescue with constitutively active CDC42, suggesting that DOCK11 regulates erythrocyte numbers in a CDC42-dependent manner. As a next step we modeled human erythroid differentiation in vitro using an erythroid liquid culture system starting from purified CD34+ cells. ShRNA-mediated knockdown of DOCK11 in CD34 + cells revealed impairment of cell growth and differentiation during erythroid development. In line with moderate erythroid hypoplasia observed in the bone marrow of one of the patients, these data suggest an erythroid-intrinsic role of DOCK11 during erythropoiesis. As the patients with germline DOCK11 defects also presented with recurrent infections and systemic inflammation, we further assessed the role of DOCK11 in immune cells. In line with its role in actin dynamics, we found that human DOCK11 regulates T-cell morphology and migration, suggesting that defects in DOCK11 might impact the T cells' capability to fight infections. We further uncovered that the immune dysregulation observed in the patients involved aberrant T-cell activation and cytokine production. Mechanistically, using cells from DOCK11-deficient patients and Dock11-knockout mice, we were able to show that Dock11 regulates cytokine production at the transcriptional level by modulating the nuclear translocation of the T-cell transcription factor NFATc1, known to regulate the production of several cytokines. Collectively, we identified germline loss-of-function mutations affecting the actin regulator DOCK11 in a previously unknown disorder associating anemia and systematic inflammation. This work earmarks the DOCK11-CDC42 axis as an attractive future target for the treatment of a broader range of immune and hematological diseases

Glucose-6-phosphate dehydrogenase is dispensable for human erythroid cell differentiation in vitro

Glucose-6-phosphate dehydrogenase (G6PD) deficiency impairs cellular processes under oxidative stress. Individuals with severe G6PD deficiency still produce sufficient numbers of erythrocytes. Nevertheless, the G6PD independence of erythropoiesis remains questionable. This study elucidates the effects of G6PD deficiency on the generation of human erythrocytes. Peripheral blood-derived CD34-positive hematopoietic stem and progenitor cells (HSPCs) of human subjects with normal, moderate, and severe G6PD activities were cultured in two distinct phases: erythroid commitment and terminal differentiation. Regardless of G6PD deficiency, HSPCs were able to proliferate and differentiate into mature erythrocytes. There was no impairment in erythroid enucleation among the subjects with G6PD deficiency. To our knowledge, this study is the first report of effective erythropoiesis independent of G6PD deficiency. The evidence firmly indicates that the population with the G6PD variant could produce erythrocytes to an extent similar to that in healthy individuals.

Arginine metabolism regulates human erythroid differentiation through hypusination of eIF5A

AbstractMetabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/cationic amino acid transporter 1–dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via the activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a posttranslational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine. Notably, attenuation of hypusine synthesis in erythroid progenitors, by the inhibition of deoxyhypusine synthase, abrogates erythropoiesis but not myeloid cell differentiation. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A, and accordingly, progenitors with decreased hypusine activity exhibit diminished oxidative phosphorylation. This affected pathway is critical for eIF5A-regulated erythropoiesis, as interventions augmenting mitochondrial function partially rescue human erythropoiesis under conditions of attenuated hypusination. Levels of mitochondrial ribosomal proteins (RPs) were especially sensitive to the loss of hypusine, and we find that the ineffective erythropoiesis linked to haploinsufficiency of RPS14 in chromosome 5q deletions in myelodysplastic syndrome is associated with a diminished pool of hypusinated eIF5A. Moreover, patients with RPL11-haploinsufficient Diamond-Blackfan anemia as well as CD34+ progenitors with downregulated RPL11 exhibit a markedly decreased hypusination in erythroid progenitors, concomitant with a loss of mitochondrial metabolism. Thus, eIF5A-dependent protein synthesis regulates human erythropoiesis, and our data reveal a novel role for RPs in controlling eIF5A hypusination in HSPCs, synchronizing mitochondrial metabolism with erythroid differentiation.

HDAC6 regulates human erythroid differentiation through modulation of JAK2 signalling

Among histone deacetylases, HDAC6 is unusual in its cytoplasmic localization. Its inhibition leads to hyperacetylation of non-histone proteins, inhibiting cell cycle, proliferation and apoptosis. Ricolinostat (ACY-1215) is a selective inhibitor of the histone deacetylase HDAC6 with proven efficacy in the treatment of malignant diseases, but anaemia is one of the most frequent side effects. We investigated here the underlying mechanisms of this erythroid toxicity. We first confirmed that HDAC6 was strongly expressed at both RNA and protein levels in CD34+ -cells-derived erythroid progenitors. ACY-1215 exposure on CD34+ -cells driven in vitro towards the erythroid lineage led to a decreased cell count, an increased apoptotic rate and a delayed erythroid differentiation with accumulation of weakly hemoglobinized immature erythroblasts. This was accompanied by drastic changes in the transcriptomic profile of primary cells as shown by RNAseq. In erythroid cells, ACY-1215 and shRNA-mediated HDAC6 knockdown inhibited the EPO-dependent JAK2 phosphorylation. Using acetylome, we identified 14-3-3ζ, known to interact directly with the JAK2 negative regulator LNK, as a potential HDAC6 target in erythroid cells. We confirmed that 14-3-3ζ was hyperacetylated after ACY-1215 exposure, which decreased the 14-3-3ζ/LNK interaction while increased LNK ability to interact with JAK2. Thus, in addition to its previously described role in the enucleation of mouse fetal liver erythroblasts, we identified here a new mechanism of HDAC6-dependent control of erythropoiesis through 14-3-3ζ acetylation level, LNK availability and finally JAK2 activation in response to EPO, which is crucial downstream of EPO-R activation for human erythroid cell survival, proliferation and differentiation.

Abstract B010: Determining the role of BRD9 in human hematopoietic stem cell aging

Abstract Aging is the number one non-modifiable risk factor for cancer, including myeloid malignancies, such as Acute Myeloid leukemia (AML). Intrinsic changes within hematopoietic stem cells (HSC) with aging contribute to HSC dysfunction and may predispose to leukemia. We have previously shown that aged human HSC present a reduction of H3K27ac at active promoters and enhancers of genes associated with immune and cancer pathways. Concurrent with this epigenetic deregulation there is deregulation of immune-associated genes and downregulation of the bromodomain containing protein-9 (BRD9). This protein is a component of the non-canonical BAF (nc-BAF) chromatin remodeling complex, which binds to both active enhancers and promoters. Notably alteration of BRD9 has been shown to perturb immune and cancer associated pathways, with BRD9 regulating interferon-stimulated genes. However, little is known about the role of this protein in human hematopoiesis. Therefore, we hypothesize that chromatin remodeling due to loss of BRD9 with HSC aging leads to reduction of H3K27ac at active enhancers and promoters, contributing to HSC dysfunction. To investigate this, we treated human CD34+ hematopoietic stem and progenitor cells (HSPC) with dBRD9-A (100 nM) a chemical degrader of BRD9. Loss of BRD9 resulted in enhanced myeloid differentiation in vitro with a concurrent block in erythroid differentiation. Interestingly, amongst the myeloid populations upregulated with loss of BRD9 were monocytic myeloid derived suppressor cells (M-MDSC), immunosuppressive cells that are involved both in cancer pathogenesis and aging. Treatment with the BRD9 bromodomain specific inhibitor, I-BRD9 (1 µM), suggests that while the role of BRD9 in myeloid differentiation is largely bromodomain independent, the bromodomain may regulate erythroid differentiation. Similarly, loss of BRD9 leads to more myeloid biased colonies, suggesting that loss of BRD9 may recapitulate certain features of aged HSC. At the transcriptional level, RNA-seq revealed that loss of BRD9 resulted in few, but important differentially expressed genes, including downregulation of MPO, LIF, EPX, and GZMB (FDR < 0.05). However, gene set enrichment analysis showed that both bromodomain inhibition and loss of BRD9 resulted in downregulation of interferon and STAT3 associated genes, which are known BRD9 targets. Chromatin profiling of H3K27ac with CUT&RUN in young HSPC showed that with loss of BRD9, there is a reduction of H3K27ac at active enhancers that we had previously observed have loss of H3K27ac with human HSC aging (n=2). These active enhancers are associated with immune signaling and regulate hematopoietic transcription factors such as KLF6, RUNX1/2/3, and HIF1A. In conclusion, we have found that the chromatin remodeling protein BRD9 contributes to the reprogramming of age-associated enhancers and regulates human myeloid and erythroid differentiation in vitro, and that its loss may result in an expansion of M-MDSC, which has implications for age-related disorders such as immune dysfunction and cancer. Citation Format: Emmalee R. Adelman, Maria E. Figueroa. Determining the role of BRD9 in human hematopoietic stem cell aging. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B010.

Inhibition of aryl hydrocarbon receptor signaling promotes the terminal differentiation of human erythroblasts.

The aryl hydrocarbon receptor (AHR) plays an important role during mammalian embryo development. Inhibition of AHR signaling promotes the development of hematopoietic stem/progenitor cells. AHR also regulates the functional maturation of blood cells, such as T cells and megakaryocytes. However, little is known about the role of AHR modulation during the development of erythroid cells. In this study, we used the AHR antagonist StemRegenin 1 (SR1) and the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin during different stages of human erythropoiesis to elucidate the function of AHR. We found that antagonizing AHR signaling improved the production of human embryonic stem cell derived erythrocytes and enhanced erythroid terminal differentiation. RNA sequencing showed that SR1 treatment of proerythroblasts upregulated the expression of erythrocyte differentiation-related genes and downregulated actin organization-associated genes. We found that SR1 accelerated F-actin remodeling in terminally differentiated erythrocytes, favoring their maturation of the cytoskeleton and enucleation. We demonstrated that the effects of AHR inhibition on erythroid maturation were associated with F-actin remodeling. Our findings help uncover the mechanism for AHR-mediated human erythroid cell differentiation. We also provide a new approach toward the large-scale production of functionally mature human pluripotent stem cell-derived erythrocytes for use in translational applications.

Vitamin B5 and Succinyl-CoA Improve Ineffective Erythropoiesis in SF3B1 Mutated Myelodysplasia

Myelodysplastic syndrome (MDS) is a hematological clonal stem cell disease. Recurrent splicing factors mutations are reported in 50% of MDS. Interestingly, mutations in the splicing factor gene SF3B1 are over-represented in MDS with ring sideroblasts (MDS-RS), co-occurring in up to 90% of patients. In MDS-RS, anemia is the major clinical manifestation. Erythropoiesis stimulating agents (ESAs) are used to treat anemia; however, the overall response rates are 20% to 40% with a duration of response of 18-24 months. New therapeutic options are needed to improve response to ESAs treatment and delay red blood cell transfusion, which are associated with acute myeloid leukemia progression and increase in morbidity.Mutations in SF3B1 modify the recognition pattern of the 3' splice site and lead to subsequent mis-splicing of its targets. To identify critical mis-splicing events involved in the erythroid differentiation blockage, we performed splicing analysis on RNA sequencing generated from hematopoietic stem/progenitor cells undergoing differentiation. Three MDS primary samples harboring SF3B1 mutations and three age-matched healthy donors cultured under normoxia and hypoxia conditions were initially used for the analysis. High depth RNA sequencing and differential splicing analyses using rMATS identified 2,845 mis-spliced events including 200 shared between hypoxia and normoxia conditions. Here, using a cohort of 42 MDS samples, we report the mis-splicing of the coenzyme A synthase (COASY) transcript. Heme synthesis relies on succinyl-CoA synthesis, and its production itself depends on the availability of cellular CoA. We thus hypothesised that COASY mis-splicing is a key driver of ineffective erythropoiesis in MDS-RS patients.In primary hematopoietic cells, COASY is upregulated during erythroid differentiation and its silencing in CD34 + cells severely impedes the generation of mature erythroid cells CD71 - CD235a + and causes disruption in heme production. Functional characterisations of the CRISPR-CAS9 edited K562 SF3B1K700E and the SF3B1-mutated HNT-34 cell lines confirmed that COASY mis-splicing impairs COASY protein synthesis that ultimately results in 60% loss of the protein. Metabolomic analysis showed that COASY mis-splicing depletes cells in CoA and succinyl-CoA metabolites, however this phenotype can be rescued by supplementation with vitamin B5, a CoA precursor. Consequently, we showed in vitro that saturating the 40% of remaining COASY enzyme with vitamin B5 or supplementing medium with its downstream by-product, succinyl-CoA, improved erythropoietic differentiation in MDS SF3B1mut patients.In summary, our results for the first time show that SF3B1 mutations induce coenzyme A synthase (COASY) transcript mis-splicing, that consequently leads to measurable defects in metabolites essential for heme biosynthesis. Our report reveals a novel critical role of COASY in regulating normal bone marrow erythropoiesis through control of succinyl-coA during human erythroid differentiation. Remarkably, partial loss of the coenzyme A synthase in MDS-RS patients leads to disruption in the erythroid lineage as well as heme deficiency, that can be rescued by exogenous treatment with vitamin B5 or succinyl-CoA. Therefore, vitamin B5 could represent a very attractive agent to combine with existing treatments in order to increase erythroid maturation and delay red blood cell transfusion dependency in MDS-RS patients.Graphical representation: SF3B1 mutant causes mis-splicing in COASY that results in loss of protein. Deficiency in COASY triggers a downregulation of succinyl-CoA that is involved in the rate limiting step of heme synthesis. Heme deficiency subsequently impairs erythroid differentiation. Treatment of MDS SF3B1 mutant cells with vitamin B5 (precursor of CoA), or succinyl-CoA, rescues erythroid differentiation. [Display omitted] DisclosuresPlatzbecker: Geron: Honoraria; Takeda: Honoraria; Janssen: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; AbbVie: Honoraria. Wiseman: Bristol Myers Squibb: Consultancy; Novartis: Consultancy; StemLine: Consultancy; Takeda: Consultancy; Astex: Research Funding. Gribben: Abbvie: Honoraria; AZ: Honoraria, Research Funding; BMS: Honoraria; Gilead/Kite: Honoraria; Janssen: Honoraria, Research Funding; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutis: Honoraria.

Inhibition of S-Adenosylmethionine Synthesis Promotes Erythropoiesis Via Epigenetic Modifications

Erythroid differentiation involves global gene expression repression, chromatin condensation and enucleation, mitochondria removal and other marked cellular changes. Given the necessity for these dynamic alterations, it is hardly surprising that epigenetic modifications possess important roles for erythropoiesis. S-adenosylmethionine (SAM), a principle methyl donor for DNA and histone methylations, would be involved in this process. Yet little is known about the specific roles for SAM synthesis in erythropoiesis.SAM is synthesized from methionine and ATP via the enzymatic activity of Mat2a and we evaluated the in vivo role of SAM synthesis by treating wild type mice (C57BL/6) with a selective Mat2a inhibitor (cycloleucine). As expected, the Mat2a inhibitor administration (henceforth Mat2ai) reduced SAM in bone marrow (BM) cells (SAM; 3.17±0.43 and 0.93±0.10 area ratio for ctrl and Mat2ai, p < 0.01, n = 4 mice). Interestingly, Mat2ai increased erythropoiesis in BM (Ter119 + cell; 46.3±3.1 and 116.4±14.2×10 6 cells for ctrl and Mat2ai, p < 0.01, n = 8 mice) and in blood (hemoglobin concentrations in peripheral blood; 13.7±0.18 and 16.3±0.26 g/dl for ctrl and Mat2ai, p < 0.01, n = 8 mice). However, serum erythropoietin concentration decreased (erythropoietin; 254.2±34.1 and 42.7±5.70 pg/ml for ctrl and Mat2ai, p < 0.01, n = 10 mice). Therefore, Mat2ai promoted erythropoiesis in vivo without increasing erythropoietin. To reveal the point where the erythroid differentiation was affected, immature and mature erythroblast subsets in BM were assessed. Although immature erythroblasts were not changed by Mat2ai (24.1±2.80 and 23.8±3.86×10 6 cells for ctrl and Mat2ai, p = 0.95, n = 8 mice), mature erythroblasts in BM increased following Mat2ai (18.9±2.48 and 81.2±9.73×10 6 cells for ctrl and Mat2ai, p < 0.01, n = 8 mice). Therefore, Mat2ai promoted erythroid maturation from immature erythroblast in BM. To reveal the mechanistic insight of this promotion of erythroid maturation by Mat2ai, we performed RNA sequencing of immature erythroblast in BM. This analysis revealed that most genes were down-regulated by Mat2ai (differentially expressed genes by Mat2ai; DOWN 2578 genes, UP 72 genes). In line with this notion, transposase-accessible chromatin sequencing (ATAC-seq) of immature erythroblasts revealed that chromatin accessibility was reduced. While DNA methylation analysis (whole genome bisulfite sequence) of immature erythroblasts revealed slightly reduced global DNA methylation (approximately 2%), there were no clear correlations between changes in promotor (or gene-body) DNA methylation and transcription. This result suggests that DNA methylation changes possess limited roles for the erythroid maturation promoted by Mat2ai. On the other hand, we found that an active histone methylation mark (H3K4me3) was selectively reduced by Mat2ai and that the changes of gene expression and H3K4me3 enrichment (revealed by chromatin immunoprecipitation followed by sequencing) correlated (r = 0.66). Therefore, the loss of H3K4me3, but not the DNA methylation, might contribute to the global gene expression repression for erythroid maturation induced by Mat2ai. Finally, in vitro human erythroid differentiation analysis using CD34 + cord blood cells further revealed that therapeutic and genetic inhibition of SAM synthesis induced erythroid maturation, which was cancelled by extracellular administration of SAM. Therefore, SAM synthesis inhibition is a non-erythropoietin trigger for erythroid maturation and this process occurs in human cells.Collectively, we found that SAM synthesis inhibition promoted erythroid maturation in both mouse and human. Histone methylation alteration induced by SAM synthesis inhibition might contribute to this phenomenon. These findings may pave the way to develop a new therapeutic strategy for anemia in erythropoietin independent manner. DisclosuresHarigae: Kyowakirin: Other: Subsidies or Donations; Astellas Pharma: Other: Subsidies or Donations; Ono pharma: Honoraria, Other: Subsidies or Donations; Janssen Pharma: Honoraria; Chugai Pharma: Honoraria; Novartis Pharma: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria. Scadden: Magenta Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Editas Medicines: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Current holder of individual stocks in a privately-held company; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Dainippon Sumitomo Pharma: Other: Sponsored research; FOG Pharma:: Consultancy; Garuda Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; VCanBio: Consultancy; Inzen Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Vesicular formation regulated by ERK/MAPK pathway mediates human erythroblast enucleation

AbstractEnucleation is a key event in mammalian erythropoiesis responsible for the generation of enucleated reticulocytes. Although progress is being made in developing mechanistic understanding of enucleation, our understanding of mechanisms for enucleation is still incomplete. The MAPK pathway plays diverse roles in biological processes, but its role in erythropoiesis has yet to be fully defined. Analysis of RNA-sequencing data revealed that the MAPK pathway is significantly upregulated during human terminal erythroid differentiation. The MAPK pathway consists of 3 major signaling cassettes: MEK/ERK, p38, and JNK. In the present study, we show that among these 3 cassettes, only ERK was significantly upregulated in late-stage human erythroblasts. The increased expression of ERK along with its increased phosphorylation suggests a potential role for ERK activation in enucleation. To explore this hypothesis, we treated sorted populations of human orthochromatic erythroblasts with the MEK/ERK inhibitor U0126 and found that U0126 inhibited enucleation. In contrast, inhibitors of either p38 or JNK had no effect on enucleation. Mechanistically, U0126 selectively inhibited formation/accumulation of cytoplasmic vesicles and endocytosis of the transferrin receptor without affecting chromatin condensation, nuclear polarization, or enucleosome formation. Treatment with vacuolin-1 that induces vacuole formation partially rescued the blockage of enucleation by U0126. Moreover, phosphoproteomic analysis revealed that inactivation of the ERK pathway led to downregulation of the endocytic recycling pathway. Collectively, our findings uncovered a novel role of ERK activation in human erythroblast enucleation by modulating vesicle formation and have implications for understanding anemia associated with defective enucleation.

Decreased PGC1\u03b2 expression results in disrupted human erythroid differentiation, impaired hemoglobinization and cell cycle exit

Production of red blood cells relies on proper mitochondrial function, both for their increased energy demands during differentiation and for proper heme and iron homeostasis. Mutations in genes regulating mitochondrial function have been reported in patients with anemia, yet their pathophysiological role often remains unclear. PGC1β is a critical coactivator of mitochondrial biogenesis, with increased expression during terminal erythroid differentiation. The role of PGC1β has however mainly been studied in skeletal muscle, adipose and hepatic tissues, and its function in erythropoiesis remains largely unknown. Here we show that perturbed PGC1β expression in human hematopoietic stem/progenitor cells from both bone marrow and cord blood results in impaired formation of early erythroid progenitors and delayed terminal erythroid differentiation in vitro, with accumulations of polychromatic erythroblasts, similar to MDS-related refractory anemia. Reduced levels of PGC1β resulted in deregulated expression of iron, heme and globin related genes in polychromatic erythroblasts, and reduced hemoglobin content in the more mature bone marrow derived reticulocytes. Furthermore, PGC1β knock-down resulted in disturbed cell cycle exit with accumulation of erythroblasts in S-phase and enhanced expression of G1-S regulating genes, with smaller reticulocytes as a result. Taken together, we demonstrate that PGC1β is directly involved in production of hemoglobin and regulation of G1-S transition and is ultimately required for proper terminal erythroid differentiation.

Characterization and Targeting TET2-Mutant HSPCs and AML

Understanding the contribution of abnormal epigenetic programs to acute myeloid leukemia (AML) is necessary for the integrated design of epigenetic-targeted therapies. The protein encoded by TET2 gene is an enzyme that catalyzes the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) as part of the process of DNA demethylation and is frequently mutated in pre-leukemic stem cells. From mice studies, TET2 knockdown/knockout models exhibit a myeloid differentiation bias and an expansion of hematopoietic stem cells (HSCs) with increased myeloproliferation. Notably, no studies to date have specifically examined the cell context effect of TET2 mutations on human hematopoiesis.To understand the mechanism of TET2 mutation-driven leukomogenesis, we have developed an in vitro/ in vivo platform using human CD34+ stem and progenitor cells that mimics TET2-mutated pre-leukemia. Using double chemically modified guide RNAs (gRNAs) coupled with Cas9 recombinant protein, we can disrupt the TET2 gene in cord blood-derived CD34+ cells (average indel frequencies=94.3%, n=6). Beyond disruption, we can modify the TET2 locus through insertion of a GFP cassette using homology driven repair to generate traceable cells. Thus, we have developed a tractable and cell-traceable model that faithfully recapitulates TET2-mutated pre-leukemia and clonal hematopoiesis of indeterminate potential (CHIP).First, we examined the effects of TET2-disruption on human erythroid differentiation in vitro by culturing bulk CD34+GFP+ cells under conditions that promote erythroid differentiation. TET2-disrupted cells produced reduced numbers of glycophorin A/ CD71 double positive erythroid cells compared to AAVS1-targeted control cells. This underscores the importance of TET2 in promoting normal erythroid differentiation, and demonstrates that our methods can give biological effects in a relevant primary human cell system.Next, we investigated the effects of TET2-disruption on hematopoietic colony formation in methylcellulose. Initial colony formation did not differ between TET2-mutant and control cells in the first plating; however, upon the second and third replating, increased numbers of colonies were observed with TET2-disruption. Analysis of indels in the colonies showed that with serial replating, there was enrichment of a 65 base pair deletion that disrupts the entire TET2 locus, suggesting that TET2 knockout cells outcompete normal unmutated hematopoietic stem/progenitor cells (HSPCs) during a month in vitro . We now know that ascorbic acid is an additional co-factor for TET enzymes to hydroxylate 5-mC to 5-hmC. Thus, we also cultured the cells overnight in high dose ascorbic acid prior to plating the cells. In both normoxic and hypoxic conditions, fewer colonies were detected in the TET2 knockout group, suggesting that high dose ascorbic acid cannot restore the TET2 enzymatic function in these cells.Lastly, we conducted in vivo engraftment studies through transplantation of CD34+ cells transfected with TET2-targeting Cas9 RNP (using 2 gRNAs without GFP), and demonstrated successful engraftment of human cells. Four months after transplant, we detected human hematopoietic engraftment with T cells, B cells, and myeloid cells, indicating that Cas9 RNP transfection did not impair engraftment ability. These cells showed high frequencies of indels within the TET2 gene. Surprisingly, we detected a significant proportion of human T cells (approximately 5%, even after transplantation of T cell-depleted CD34+ cells) that were not present with transplantation of control cells. These pilot data demonstrate that Cas9-RNP transfection did not impair engraftment ability of human HSPCs and that TET2-disrupted HSCs can give rise to hematopoietic progeny in vivo with possibly increased production of T cells. Secondary transplants are ongoing to assess for self-renewal.Here, we report the development of a CRISPR/Cas9 approach to target the TET2 gene in primary human HSPCs. We show that TET2 disruption leads to a block in erythroid differentiation, increased colony formation and replating in vitro, and increased engraftment in vivo . This model will enable further studies of effects on the epigenome through DNA methylation and hydroxymethylation studies, as well as provide an in vivo model of pre-leukemia/CHIP associated with TET2 mutations. DisclosuresNo relevant conflicts of interest to declare.

Fetal Hemoglobin Induction with Forced Chromatin Looping in Gene-Modified Erythroid Cells Differentiated from Rhesus Hematopoietic Progenitor Cells

Substantial advances in genome engineering offer new therapies for hemoglobin (Hb) disorders including sickle cell disease. Fetal hemoglobin reactivation remains a viable strategy, as recently demonstrated in human erythroid cells with an artificial looping molecule ZF-Ldb1, tethering Ldb1 to the γ-globin promoter by specific zinc finger proteins (Deng et al., 2014). Large animal preclinical testing can improve the therapeutic potential of such approaches by optimizing safety and efficacy issues before launching clinical trials in humans. A robust in vitro erythroid evaluation system, however, does not exist to determine if fetal hemoglobin reactivation can be modeled in the rhesus macaque. Therefore, in the current work, we aimed to develop an efficient in vitro erythroid differentiation model from rhesus hematopoietic progenitor cells with Hb production at detectable protein levels, and to evaluate rhesus fetal hemoglobin reactivation with ZF-Ldb1.To adapt a human erythroid differentiation method to rhesus cells, we used C3H or OP9 feeder cells to culture rhesus peripheral blood mononuclear cells (PBMCs) with HEMA differentiation media containing 10ng/ml stem cell factor (SCF), 1ng/ml interleukin-3 (IL3), 2U/ml erythropoietin (Epo), and 20% fetal bovine serum (FBS) (Migliaccio et al., 2010). Two weeks after erythroid differentiation, robust Hb production evaluated by HB electrophoresis was obtained on both C3H and OP9 feeder cells but not in feeder-free conditions. Increased concentration of SCF (20x), IL3 (10x), and Epo (1.5x) allowed for Hb production even in feeder-free conditions; however, fetal Hb was predominant (due to 20x SCF). Both conditions provided similar levels of enucleation (39±6.3% vs 29±5.6%, ns), indicating efficient differentiation. We then compared HEMA media with serum-free StemSpan-based differentiation media to evaluate transduction with a GFP-encoding lentiviral vector at MOI 2.5 two days after initiating differentiation. Transduction was almost completely inhibited (0.4±0.5%) in HEMA media, while StemSpan-based transduction media followed by HEMA differentiation media resulted in significant increase of transduction efficiency (41±8.5%, p<0.01). These data demonstrate that feeder cells are required for rhesus erythroid differentiation in HEMA media, while FBS strongly interferes lentiviral transduction.Next, we sought to establish a reverse phase HPLC method to quantitatively analyze rhesus globins among differentiated erythroid cells. Peaks on the HPLC chromatogram were eluted and run in liquid chromatography-mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) for protein identification. We identified β-globin and γ-globin (HBG1 and HBG2) peaks using both rhesus peripheral blood (99±26% and 0.65±0.28%) and in vitro differentiated rhesus erythroid cells (63±13% and 37±16%), which is essential for Hb switching analysis. In addition, human β-globin and T87Q β-globin (with anti-sickling mutation) proteins were also separated from rhesus globins in the HPLC chromatogram and were detectable for quantitation.Finally, we evaluated whether fetal Hb expression can be induced by the ZF-Ldb1 molecule in rhesus erythroid cells. We transduced differentiating rhesus CD34+ cells with ZF-Ldb1-encoding lentiviral vectors that are driven by an erythroid specific ankyrin-1 promoter, and contain GFP along with a β-globin locus control region (LCR) enhancer or YFP without the LCR. After erythroid differentiation, we obtained both LCR/ZF-Ldb1/GFP+ (9.4%) and ZF-Ldb1/YFP+ (1.3%) rhesus erythroid cells, and higher γ-globin RNA expression was detected for GFP+ and YFP+ fractions (68.1±1.5% and 59.8±2.1%, respectively), as compared to GFP- and YFP- controls (28.1±3.1% and 31.8±1.9%, p<0.05). These data demonstrate successful γ-globin induction with ZF-Ldb1 vectors in the rhesus macaque.In conclusion, we developed an efficient rhesus erythroid cell differentiation protocol from hematopoietic progenitor cells with robust Hb production detectable at protein levels, analyzed in a newly established HPLC method. The ZF-Ldb1 vector transduction allows for induction of γ-globin expression in rhesus erythroid cells. Further studies are warranted to optimize gene modification and transplantation of rhesus hematopoietic progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

Mapping Human Pluripotent Stem Cell-derived Erythroid Differentiation by Single-cell Transcriptome Analysis

There is an imbalance between the supply and demand of functional red blood cells (RBCs) in clinical applications. This imbalance can be addressed by regenerating RBCs using several in vitro methods. Induced pluripotent stem cells (iPSCs) can handle the low supply of cord blood and the ethical issues in embryonic stem cell research, and provide a promising strategy to eliminate immune rejection. However, no complete single-cell level differentiation pathway exists for the iPSC-derived erythroid differentiation system. In this study, we used iPSC line BC1 to establish a RBC regeneration system. The 10X Genomics single-cell transcriptome platform was used to map the cell lineage and differentiation trajectory on day 14 of the regeneration system. We observed that iPSC differentiation was not synchronized during embryoid body (EB) culture. The cells (on day 14) mainly consisted of mesodermal and various blood cells, similar to the yolk sac hematopoiesis. We identified six cell classifications and characterized the regulatory transcription factor (TF) networks and cell–cell contacts underlying the system. iPSCs undergo two transformations during the differentiation trajectory, accompanied by the dynamic expression of cell adhesion molecules and estrogen-responsive genes. We identified erythroid cells at different stages, such as burst-forming unit erythroid (BFU-E) and orthochromatic erythroblast (ortho-E) cells, and found that the regulation of TFs (e.g., TFDP1 and FOXO3) is erythroid-stage specific. Immune erythroid cells were identified in our system. This study provides systematic theoretical guidance for optimizing the iPSC-derived erythroid differentiation system, and this system is a useful model for simulating in vivo hematopoietic development and differentiation

Impairment of human terminal erythroid differentiation by histone deacetylase 5 deficiency

AbstractHistone deacetylases (HDACs) are a group of enzymes that catalyze the removal of acetyl groups from histone and nonhistone proteins. HDACs have been shown to have diverse functions in a wide range of biological processes. However, their roles in mammalian erythropoiesis remain to be fully defined. This study showed that, of the 11 classic HDAC family members, 6 (HDAC1, -2, -3, and HDAC5, -6, -7) are expressed in human erythroid cells, with HDAC5 most significantly upregulated during terminal erythroid differentiation. Knockdown of HDAC5 by either short hairpin RNA or small interfering RNA in human CD34+ cells followed by erythroid cell culture led to increased apoptosis, decreased chromatin condensation, and impaired enucleation of erythroblasts. Biochemical analyses revealed that HDAC5 deficiency resulted in activation of p53 in association with increased acetylation of p53. Furthermore, although acetylation of histone 4 (H4) is decreased during normal terminal erythroid differentiation, HDAC5 deficiency led to increased acetylation of H4 (K12) in late-stage erythroblasts. This increased acetylation was accompanied by decreased chromatin condensation, implying a role for H4 (K12) deacetylation in chromatin condensation. ATAC-seq and RNA sequencing analyses revealed that HDAC5 knockdown leads to increased chromatin accessibility genome-wide and global changes in gene expression. Moreover, pharmacological inhibition of HDAC5 by the inhibitor LMK235 also led to increased H4 acetylation, impaired chromatin condensation, and enucleation. Taken together, our findings have uncovered previously unrecognized roles and molecular mechanisms of action for HDAC5 in human erythropoiesis. These results may provide insights into understanding the anemia associated with HDAC inhibitor treatment.

An IDH1-vitamin C crosstalk drives human erythroid development by inhibiting pro-oxidant mitochondrial metabolism

The metabolic changes controlling the stepwise differentiation of hematopoietic stem and progenitor cells (HSPCs) to mature erythrocytes are poorly understood. Here, we show that HSPC development to an erythroid-committed proerythroblast results in augmented glutaminolysis, generating alpha-ketoglutarate (αKG) and driving mitochondrial oxidative phosphorylation (OXPHOS). However, sequential late-stage erythropoiesis is dependent on decreasing αKG-driven OXPHOS, and we find that isocitrate dehydrogenase 1 (IDH1) plays a central role in this process. IDH1 downregulation augments mitochondrial oxidation of αKG and inhibits reticulocyte generation. Furthermore, IDH1 knockdown results in the generation of multinucleated erythroblasts, a morphological abnormality characteristic of myelodysplastic syndrome and congenital dyserythropoietic anemia. We identify vitamin C homeostasis as a critical regulator of ineffective erythropoiesis; oxidized ascorbate increases mitochondrial superoxide and significantly exacerbates the abnormal erythroblast phenotype of IDH1-downregulated progenitors, whereas vitamin C, scavenging reactive oxygen species (ROS) and reprogramming mitochondrial metabolism, rescues erythropoiesis. Thus, an IDH1-vitamin C crosstalk controls terminal steps of human erythroid differentiation.