Primary Human Erythroid Progenitor Cells Research Articles

High-Throughput Screening Identifies Inhibitors for Parvovirus B19 Infection of Human Erythroid Progenitors.

Parvovirus B19 (B19V) infection can cause hematological disorders and fetal hydrops during pregnancy. Currently, no antivirals or vaccines are available for the treatment or prevention of B19V infection. To identify novel small-molecule antivirals against B19V replication, we developed a high-throughput screening (HTS) assay, which is based on an in vitro nicking assay using recombinant N-terminal amino acids 1 to 176 of the viral large nonstructural protein (NS1N) and a fluorescently labeled DNA probe (OriQ) that spans the nicking site of the viral DNA replication origin. We collectively screened 17,040 compounds and identified 2,178 (12.78%) hits that possess >10% inhibition of the NS1 nicking activity, among which 84 hits were confirmed to inhibit nicking in a dose-dependent manner. Using ex vivo-expanded primary human erythroid progenitor cells (EPCs) infected by B19V, we validated 24 compounds that demonstrated >50% in vivo inhibition of B19V infection at 10 μM, which can be categorized into 7 structure scaffolds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, the top 4 compounds were chosen to examine their inhibitions of B19V infection in EPCs at two times of the 90% maximal effective concentration (EC90). A purine derivative (P7) demonstrated an antiviral effect (EC50 = 1.46 μM) without prominent cytotoxicity (CC50 = 71.8 μM) in EPCs and exhibited 92% inhibition of B19V infection in EPCs at 3.32 μM, which can be used as the lead compound in future studies for the treatment of B19V infection-caused hematological disorders. IMPORTANCE B19V encodes a large nonstructural protein, NS1. Its N-terminal domain (NS1N) consisting of amino acids 1 to 176 binds to viral DNA and serves as an endonuclease to nick the viral DNA replication origins, which is a pivotal step in rolling-hairpin-dependent B19V DNA replication. For high-throughput screening (HTS) of anti-B19V antivirals, we miniaturized a fluorescence-based in vitro nicking assay, which employs a fluorophore-labeled probe spanning the terminal resolution site (trs) and the NS1N protein, into a 384-well-plate format. The HTS assay showed high reliability and capability in screening 17,040 compounds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, a purine derivative demonstrated an antiviral effect of 92% inhibition of B19V infection in EPCs at 3.32 μM (two times the EC90). Our study demonstrated a robust HTS assay for screening antivirals against B19V infection.

Transcriptional silencing of fetal hemoglobin expression by NonO.

Human fetal globin (γ-globin) genes are developmentally silenced after birth, and reactivation of γ-globin expression in adulthood ameliorates symptoms of hemoglobin disorders, such as sickle cell disease (SCD) and β-thalassemia. However, the mechanisms by which γ-globin expression is precisely regulated are still incompletely understood. Here, we found that NonO (non-POU domain-containing octamer-binding protein) interacted directly with SOX6, and repressed the expression of γ-globin gene in human erythroid cells. We showed that NonO bound to the octamer binding motif, ATGCAAAT, of the γ-globin proximal promoter, resulting in inhibition of γ-globin transcription. Depletion of NonO resulted in significant activation of γ-globin expression in K562, HUDEP-2, and primary human erythroid progenitor cells. To confirm the role of NonO in vivo, we further generated a conditional knockout of NonO by using IFN-inducible Mx1-Cre transgenic mice. We found that induced NonO deletion reactivated murine embryonic globin and human γ-globin gene expression in adult β-YAC mice, suggesting a conserved role for NonO during mammalian evolution. Thus, our data indicate that NonO acts as a novel transcriptional repressor of γ-globin gene expression through direct promoter binding, and is essential for γ-globin gene silencing.

Trienone analogs of curcuminoids induce fetal hemoglobin synthesis via demethylation at G\u03b3-globin gene promoter

The reactivation of γ-globin chain synthesis to combine with excess free α-globin chains and form fetal hemoglobin (HbF) is an important alternative treatment for β-thalassemia. We had reported HbF induction property of natural curcuminoids, curcumin (Cur), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), in erythroid progenitors. Herein, the HbF induction property of trienone analogs of the three curcuminoids in erythroleukemic K562 cell lines and primary human erythroid progenitor cells from β-thalassemia/HbE patients was examined. All three trienone analogs could induce HbF synthesis. The most potent HbF inducer in K562 cells was trienone analog of BDMC (T-BDMC) with 2.4 ± 0.2 fold increase. In addition, DNA methylation at CpG − 53, − 50 and + 6 of Gγ-globin gene promoter in K562 cells treated with the compounds including T-BDMC (9.3 ± 1.7%, 7.3 ± 1.7% and 5.3 ± 0.5%, respectively) was significantly lower than those obtained from the control cells (30.7 ± 3.8%, 25.0 ± 2.9% and 7.7 ± 0.9%, respectively P < 0.05). The trienone compounds also significantly induced HbF synthesis in β-thalassemia/HbE erythroid progenitor cells with significantly reduction in DNA methylation at CpG + 6 of Gγ-globin gene promoter. These results suggested that the curcuminoids and their three trienone analogs induced HbF synthesis by decreased DNA methylation at Gγ-globin promoter region, without effect on Aγ-globin promoter region.

LSD1 Inhibitors Induce Fetal Hemoglobin in Primary Human Erythroid Cells

Significant amount of clinical literatures have supported that high level of fetal hemoglobin (HbF) improves the disease pathophysiology of β-globinopathies [sickle cell disease (SCD) and β-thalassemia]. Thus, we certainly can treat β-globinopathies by increasing the HbF level in adult erythroid cells.We originally reported that the lysine-specific histone demethylase 1 (LSD1) plays an important role in the regulation of the fetal γ-globin genes. Inhibition of LSD1 by using RNAi and monoamine oxidase inhibitor tranylcypromine (TCP) in primary human erythroid progenitor cells induces HbF to therapeutic levels. Furthermore, LSD1 inhibitor RN-1 treatment of SCD mice results in increased HbF synthesis and leads to effective improvement of many aspects of the disease pathology normally associated with SCD.Most recently, we examined thein vivo effects of some additional, publically available small molecule chemical inhibitors of LSD1 (including GSK-LSD1, LSD1-C12, LSD1-C76, OG-L002, and S2101) on HbF synthesis and erythroid physiology in SCD mice. There was a statistically significant increase in the percentage of HbF positive cells after 4 weeks of treatment with GSK-LSD1 or OG-L002 in SCD mice. Here, we report the effects of these two inhibitors in primary human erythroid cell derived from peripheral blood CD34+ cells. We isolated CD34+ cells using MACS column and cultured them using the two-phase-culture system. After seven days in expansion phase (phase 1) and three days in differentiation phase (phase 2), cells were treated with different doses of GSK-LSD1 and OG-L002 LSD1 inhibitors along with controls (DMSO, hydroxyurea and TCP) for 3 or 5 days in phase 2 culture. Flow cytometric assays showed that the percentage of HbF positive cells were significantly high when CD34+ cells treated with OG-L002 LSD1 (~50% at 0.1 µM) or GSK-LSD1 (~30% at 0.1 µM) as compared to control DMSO (~20%) after 5 days. These results suggest that GSK-LSD1 and OG-L002 could be two new promising HbF inducers based on LSD1 inhibition. These findings provide additional evidence to support that LSD1 comprises a useful molecular target for possible therapeutic intervention in treating SCD. Further study will be necessary to address the potential therapeutic effects of the compounds in SCD patients. DisclosuresNo relevant conflicts of interest to declare.

In Vivo Effects of LSD1 Inhibition By Small Chemical Inhibitors in Sickle Cell Mice

For the past decades the principal strategy for treating the β-globinopathies [sickle cell disease (SCD) and β-thalassemia] has been to devise methods for increasing the levels of fetal hemoglobin (HbF) in adult erythroid cells, as a strong clinical literature has demonstrated that high HbF levels ameliorate disease pathophysiology.We originally reported the lysine-specific histone demethylase 1 (LSD1) as a new protein involved in the regulation of the fetal γ-globin genes. Inhibition of LSD1 using either RNAi or by the momoamine oxidase inhibitor tranylcypromine in primary human erythroid progenitor cells induces HbF to therapeutic levels. Thus, inhibition of LSD1 activity presents a novel exploratory avenue as a therapeutic strategy to treat SCD. Subsequent studies using another inhibitor RN-1 showed that RN-1 treatment of SCD mice results in increased HbF synthesis and leads to effective improvement of many aspects of the disease pathology normally associated with sickle cell disease.Here we report studies designed to examine the in vivo effects of some additional, publically available small molecule chemical inhibitors of LSD1 on HbF synthesis and erythroid physiology in SCD mice. In control SCD mice treated with DMSO only, the number of HbF positive cells was about 2.5% of total in RBC, while in SCD mice exposed to GSK-LSD1, or OG-L002, there was a statistically significant increase in the percentage of HbF positive cells after 4 weeks of treatment (up to 8%, or 6% respectively). Furthermore, sickled RBCs and reticulocytes are significantly reduced while the number of mature erythrocytes increased markedly in the peripheral blood of GSK-LSD1, or OG-L002 treated SCD mice. These findings suggest that GSK-LSD1 and/or OG-L002 could be potentially new HbF inducers based on LSD1 inhibition. We believe that these experiments will be of wide general interest and will be the foundation for development of small molecule therapeutics with which to treat hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

Anemia in Patients With Resistance to Thyroid Hormone α: A Role for Thyroid Hormone Receptor α in Human Erythropoiesis

Context:Patients with resistance to thyroid hormone (TH) α (RTHα) are characterized by growth retardation, macrocephaly, constipation, and abnormal thyroid function tests. In addition, almost all RTHα patients have mild anemia, the pathogenesis of which is unknown. Animal studies suggest an important role for TH and TH receptor (TR)α in erythropoiesis.Objective:To investigate whether a defect in TRα affects the maturation of red blood cells in RTHα patients.Design, Setting, and Patients:Cultures of primary human erythroid progenitor cells (HEPs), from peripheral blood of RTHα patients (n = 11) harboring different inactivating mutations in TRα (P398R, F397fs406X, C392X, R384H, A382fs388X, A263V, A263S), were compared with healthy controls (n = 11). During differentiation, erythroid cells become smaller, accumulate hemoglobin, and express different cell surface markers. We assessed cell number and cell size, and used cell staining and fluorescence-activated cell sorter analysis to monitor maturation at different time points.Results:After ∼14 days of ex vivo expansion, both control and patient-derived progenitors differentiated spontaneously. However, RTHα-derived cells differentiated more slowly. During spontaneous differentiation, RTHα-derived HEPs were larger, more positive for c-Kit (a proliferation marker), and less positive for glycophorin A (a differentiation marker). The degree of abnormal spontaneous maturation of RTHα-derived progenitors did not correlate with severity of underlying TRα defect. Both control and RTHα-derived progenitors responded similarly when differentiation was induced. T3 exposure accelerated differentiation of both control- and RTHα patient–derived HEPs.Conclusions:Inactivating mutations in human TRα affect the balance between proliferation and differentiation of progenitor cells during erythropoiesis, which may contribute to the mild anemia seen in most RTHα patients.

Parvovirus B19 NS1 protein induces cell cycle arrest at G2-phase by activating the ATR-CDC25C-CDK1 pathway.

Human parvovirus B19 (B19V) infection of primary human erythroid progenitor cells (EPCs) arrests infected cells at both late S-phase and G2-phase, which contain 4N DNA. B19V infection induces a DNA damage response (DDR) that facilitates viral DNA replication but is dispensable for cell cycle arrest at G2-phase; however, a putative C-terminal transactivation domain (TAD2) within NS1 is responsible for G2-phase arrest. To fully understand the mechanism underlying B19V NS1-induced G2-phase arrest, we established two doxycycline-inducible B19V-permissive UT7/Epo-S1 cell lines that express NS1 or NS1mTAD2, and examined the function of the TAD2 domain during G2-phase arrest. The results confirm that the NS1 TAD2 domain plays a pivotal role in NS1-induced G2-phase arrest. Mechanistically, NS1 transactivated cellular gene expression through the TAD2 domain, which was itself responsible for ATR (ataxia-telangiectasia mutated and Rad3-related) activation. Activated ATR phosphorylated CDC25C at serine 216, which in turn inactivated the cyclin B/CDK1 complex without affecting nuclear import of the complex. Importantly, we found that the ATR-CHK1-CDC25C-CDK1 pathway was activated during B19V infection of EPCs, and that ATR activation played an important role in B19V infection-induced G2-phase arrest.

Protein abundance of AKT and ERK pathway components governs cell type-specific regulation ofproliferation.

Signaling through the AKT and ERK pathways controls cell proliferation. However, the integrated regulation of this multistep process, involving signal processing, cell growth and cell cycle progression, is poorly understood. Here, we study different hematopoietic cell types, in which AKT and ERK signaling is triggered by erythropoietin (Epo). Although these cell types share the molecular network topology for pro‐proliferative Epo signaling, they exhibit distinct proliferative responses. Iterating quantitative experiments and mathematical modeling, we identify two molecular sources for cell type‐specific proliferation. First, cell type‐specific protein abundance patterns cause differential signal flow along the AKT and ERK pathways. Second, downstream regulators of both pathways have differential effects on proliferation, suggesting that protein synthesis is rate‐limiting for faster cycling cells while slower cell cycles are controlled at the G1‐S progression. The integrated mathematical model of Epo‐driven proliferation explains cell type‐specific effects of targeted AKT and ERK inhibitors and faithfully predicts, based on the protein abundance, anti‐proliferative effects of inhibitors in primary human erythroid progenitor cells. Our findings suggest that the effectiveness of targeted cancer therapy might become predictable from protein abundance.

ASH1L: A Novel Beta-Globin Gene Regulator in Humans?

BACKGROUNDWe have previously described a unique English family with beta-thalassemia trait which was not linked to the β-globin gene locus (Thein, Wood, Wickramasinghe, & Galvin, 1993). This suggested involvement of a trans-acting factor required for full activation of the β-globin gene locus. Such a factor is likely to be a modulator of disease severity in sickle cell disease and beta-thalassemia which could provide insights for novel therapeutic targets in the beta-globinopathies. RESULTSWe applied whole genome scan (WGS) to 2 affected and 2 unaffected subjects of the English family. The familial segregation suggested a dominant transmission mode; WGS identified 15 genes as potentially causative to the phenotype, with four genes located on chromosome 1, four on chromosome 3, three on chromosome 20, and one on chromosome 6, chromosome 8, chromosome 10 and chromosome 19.Sanger sequence analysis on 23 family members spanning three generations, including the 4 individuals that were subjected to WGS, revealed that the 15 variants were not artefacts of the WGS and that all variants were present in the 2 affected but not in the 2 unaffected individuals. Furthermore we found that 4 of the 15 variants were consistently and uniquely present in all 9 affected but absent in the unaffected family members. We performed association linkage analysis using the 15 markers in the whole family, and confirmed that the phenotype was closely linked to the 4 genes that were inherited as a block spanning the centromere on chromosome 1. We concluded that the region containing these 4 genes most likely harbours the mutation causing the phenotype. Among the 4 candidate genes, 2 were not expressed in erythroid cells, but the other 2 - one encoding an integral membrane protein (LRIG2) and the other one encoding a methyl transferase (ASH1L)- were expressed in erythroid cells.Functional studies for these two genes were performed on primary human erythroid progenitor cells (hEPCs) in culture. In following the kinetics of the 2 candidates during differentiation of hEPCs, we observed that the expression of ASH1L increased at later stages of differentiation, where LRIG2 displayed a less dramatic change of expression. Moreover, ASH1L has previously been found to occupy transcribed chromatin domains and methylate histone tails in vitro (Gregory et al., 2007; Miyazaki et al., 2013; Tanaka et al., 2011). In undifferentiated mouse embryonic stem cells there is no ASH1L recruitment to the β-globin gene locus but upon erythroid differentiation the protein is recruited to the transcribed portion of the gene (Gregory et al., 2007). This suggests an involvement of ASH1L in beta-globin activation in erythroid lineages. We used shRNA lentiviruses to generate knock-down (KD) of ASH1L and obtained over 65-75% KD of the gene. In hEPCs treated with the shRNA lentivirus, we observed a slight decrease in beta-globin expression compared to the control hEPCs. The α/β-globin and α/(β+γ) globin ratios were also affected by the gene knock-down. ChIP-qPCR was performed to assess the enrichment of the ASH1L protein at β-globin promoter region. The results show that enrichment of ASH1L at the β-globin promoter correlates with the β-globin expression in cells. CONCLUSIONSThese results suggest that ASH1L is responsible for the phenotype observed in the English family and act in differentiating hEPCs as a trans-acting factor for full beta-globin gene activation. Further ChIP analysis to assess the binding of the protein to the beta-globin locus during hEPCs differentiation and under KD condition will provide us with a better understanding of the influence of the methyl transferase on β-globin activation. The replication of the patient mutation in vitro using CRISPR technology will provide the model to study fully the impact of the mutation on the phenotype described in the original paper. These findings could provide new insights for therapeutic targets for beta-globinopathies. DisclosuresNo relevant conflicts of interest to declare.

Activity of RN-1, an LSD-1 Inhibitor, on Fetal Globin Expression in Sickle Mice and Sickle Erythroid Progenitors

Sickle cell disease (SCD) is the most common monogenic disorder, afflicting millions worldwide, and causing hemolytic anemia and chronic organ damage from vaso-occlusion. Fetal hemoglobin (HbF) is an endogenous type of hemoglobin present in all humans during development, which is normally suppressed in infancy. Biochemical and clinical studies have shown that increased synthesis of HbF inhibits sickle hemoglobin (HbS) polymerization and reduces clinical severity. Concerted efforts have been made to induce the synthesis of HbF in adult erythroid cells with chemical inducers of HbF and through disruption of transcription factors in repressor complexes. As wide variability in individual responses to drug candidates have been observed in clinical trials, consistently effective HbF inducers are highly desired. We previously identified that Lysine-specific histone demethylase 1 (LSD1) is involved in the regulation of the fetal γ-globin genes, and inhibition of LSD1 using either RNAi or by the momoamine oxidase inhibitor tranylcypromine (TC) in primary human erythroid progenitor cells induces HbF to therapeutic levels. However, TC treatment has potentially problematic side effects, and at high concentrations decreases adult b-globin mRNAs and impairs erythroid maturation. We have now investigated another LSD1 inhibitor, RN-1, which is a cell-permeable TC analog that acts as a potent, irreversible inhibitor of LSD1 with a lower IC50 than TC. We investigated in vivo effects of RN-1 on γ-globin gene expression and erythroid physiology in a transgenic mouse model of SCD which expresses human α- and sickle β-globin, and has many genetic, hematologic, and pathophysiological features found in SCD patients, including irreversibly sickled RBCs, hemolytic anemia, high reticulocyte counts, hepatosplenomegaly and organ pathology. We found a robust increase in human fetal γ-globin (15-fold) and murine embryonic εY- and βH1-globin mRNAs (36 and 54-fold) and 4-fold increases in human HbF in SCD mice following repeated RN-1 treatment (at 10 μg/g body weight) within 4 weeks. Further, irreversibly sickled RBCs were significantly reduced, and RBC lifespan increased markedly in RN-1-treated SCD mice, leading to significantly decrease pathophysiologic indicators (hemolysis, splenomegaly, and organ necrosis) compared to untreated SCD mice. To begin to evaluate potential effects of RN-1 on erythroid progenitor cells from patients with SCD, peripheral blood from 5 adult SCD patients was cultured with RN-1 (0.07 to 0.25 μM) in a 2-phase progenitor assay, with mRNA analyzed on day 12 and F-reticulocytes on day 13-14 of the erythroid differentiation phase. RN-1 treated progenitors demonstrated a mean 3.4-fold higher g-globin mRNA (p=0.04) and 5% higher absolute F-reticulocytes than were observed in untreated progenitors from the same subject, with responses occurring in 5/5 subjects' assays. These preclinical studies provide additional evidence that modulating LSD-1 activity is a promising approach to inducing HbF expression as a mechanism to reduce clinical severity of SCD.Disclaimer: "Research reported in this publication was supported by the NHLBI under Award Number P50HL118006. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health"R01 DK052962 10A1R42-HL-110727 DisclosuresNo relevant conflicts of interest to declare.

RHEX, a novel regulator of human erythroid progenitor cell expansion and erythroblast development

Ligation of erythropoietin (EPO) receptor (EPOR) JAK2 kinase complexes propagates signals within erythroid progenitor cells (EPCs) that are essential for red blood cell production. To reveal hypothesized novel EPOR/JAK2 targets, a phosphotyrosine (PY) phosphoproteomics approach was applied. Beyond known signal transduction factors, 32 new targets of EPO-modulated tyrosine phosphorylation were defined. Molecular adaptors comprised one major set including growth factor receptor-bound protein 2 (GRB2)-associated binding proteins 1-3 (GAB1-3), insulin receptor substrate 2 (IRS2), docking protein 1 (DOK1), Src homology 2 domain containing transforming protein 1 (SHC1), and sprouty homologue 1 (SPRY1) as validating targets, and SPRY2, SH2 domain containing 2A (SH2D2A), and signal transducing adaptor molecule 2 (STAM2) as novel candidate adaptors together with an ORF factor designated as regulator of human erythroid cell expansion (RHEX). RHEX is well conserved in Homo sapiens and primates but absent from mouse, rat, and lower vertebrate genomes. Among tissues and lineages, RHEX was elevated in EPCs, occurred as a plasma membrane protein, was rapidly PY-phosphorylated >20-fold upon EPO exposure, and coimmunoprecipitated with the EPOR. In UT7epo cells, knockdown of RHEX inhibited EPO-dependent growth. This was associated with extracellular signal-regulated kinase 1,2 (ERK1,2) modulation, and RHEX coupling to GRB2. In primary human EPCs, shRNA knockdown studies confirmed RHEX regulation of erythroid progenitor expansion and further revealed roles in promoting the formation of hemoglobinizing erythroblasts. RHEX therefore comprises a new EPO/EPOR target and regulator of human erythroid cell expansion that additionally acts to support late-stage erythroblast development.

Role of Iron-Responsive Mitochondrial Metabolism and ROS in Anemia

Iron and erythropoietin (Epo) are intimately linked regulators of erythropoiesis. Moderate iron restriction suppresses erythropoiesis at the Epo-dependent, CFU-E stage, without induction of apoptosis and without suppression of other hematopoietic cell lineages. Iron modulates Epo bioactivity in patients with iron deficiency anemia (IDA) and patients with anemia of chronic disease and inflammation (ACDI). To conserve iron when supplies are low, this erythroid iron restriction response reduces iron consumption by suppressing erythropoiesis. The erythroid iron sensor is unknown. Aconitases are multifunctional iron-sulfur cluster proteins localized in the cytosol (Aco1) and mitochondria (Aco2) that convert citrate into isocitrate. We have shown that iron restriction inhibits Aco2 enzymatic activity leading to suppression of erythropoiesis in vitro, and these effects are reversed by isocitrate. Isocitrate corrects IDA in mice and ACDI in rats (Bullock GC, et al. Blood. 2010;116:97-108; Richardson CL, et al. J Clin Invest. 2013 Aug 1;123(8):3614-3623). Iron restriction also alters the cross-talk between transferrin receptor and Epo receptor signaling pathways. These results suggest that Aco2 is an iron-responsive regulator of erythropoiesis. We are investigating the downstream molecular signaling mechanisms by which iron restriction induced-inactivation of Aco2 suppresses erythropoiesis. Our novel preliminary data show that mitochondrial oxidative metabolism rates change over time during erythropoiesis and that iron restriction reduces erythroid mitochondrial metabolism 4 to 7-fold compared to iron replete controls. This iron restriction induced change in respiration is associated with a significant, 1.5 to 3-fold, increase in mitochondrial superoxide production without a corresponding increase in hydrogen peroxide. Importantly, these mitochondrial alterations are reproduced by direct inhibition of aconitase with fluoroacetate (FA) and are not due to changes in mitochondrial number. Further, isocitrate reverses the effects of iron restriction or aconitase inhibition on mitochondrial metabolism and attenuates superoxide production. Based on these data and the known role of reactive oxygen species (superoxide/hydrogen peroxide) in Epo signaling, we propose the overarching hypothesis that iron restriction inhibits mitochondrial aconitase which, in turn, alters erythroid mitochondrial metabolism and ROS signaling resulting in suppression of erythropoiesis (Figure 1). We show for the first time bioenergetics profiles from iron restricted and iron replete primary human erythroid progenitor cells undergoing erythropoiesis. We also show that moderate levels of iron restriction cause mitochondrial dysfunction and alterations in mitochondrial ROS in differentiating erythroid progenitors. The clinical relevance of this project lies in its potential for the development of new iron-free agonists and antagonists of red blood cell production. Agonists may benefit patients with anemia due to iron deficiency or chronic inflammation and antagonists may benefit patients with myeloproliferative neoplasms.Figure 1:Proposed mechanism of iron-dependent regulation of erythropoiesis [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

RHEX, a novel regulator of human erythroid progenitor cell expansion and erythroblast development.

Ligation of erythropoietin (EPO) receptor (EPOR) JAK2 kinase complexes propagates signals within erythroid progenitor cells (EPCs) that are essential for red blood cell production. To reveal hypothesized novel EPOR/JAK2 targets, a phosphotyrosine (PY) phosphoproteomics approach was applied. Beyond known signal transduction factors, 32 new targets of EPO-modulated tyrosine phosphorylation were defined. Molecular adaptors comprised one major set including growth factor receptor-bound protein 2 (GRB2)-associated binding proteins 1-3 (GAB1-3), insulin receptor substrate 2 (IRS2), docking protein 1 (DOK1), Src homology 2 domain containing transforming protein 1 (SHC1), and sprouty homologue 1 (SPRY1) as validating targets, and SPRY2, SH2 domain containing 2A (SH2D2A), and signal transducing adaptor molecule 2 (STAM2) as novel candidate adaptors together with an ORF factor designated as regulator of human erythroid cell expansion (RHEX). RHEX is well conserved in Homo sapiens and primates but absent from mouse, rat, and lower vertebrate genomes. Among tissues and lineages, RHEX was elevated in EPCs, occurred as a plasma membrane protein, was rapidly PY-phosphorylated >20-fold upon EPO exposure, and coimmunoprecipitated with the EPOR. In UT7epo cells, knockdown of RHEX inhibited EPO-dependent growth. This was associated with extracellular signal-regulated kinase 1,2 (ERK1,2) modulation, and RHEX coupling to GRB2. In primary human EPCs, shRNA knockdown studies confirmed RHEX regulation of erythroid progenitor expansion and further revealed roles in promoting the formation of hemoglobinizing erythroblasts. RHEX therefore comprises a new EPO/EPOR target and regulator of human erythroid cell expansion that additionally acts to support late-stage erythroblast development.

Human Parvovirus B19 DNA Replication Induces a DNA Damage Response That Is Dispensable for Cell Cycle Arrest at Phase G 2 /M

Human parvovirus B19 (B19V) infection is highly restricted to human erythroid progenitor cells, in which it induces a DNA damage response (DDR). The DDR signaling is mainly mediated by the ATR (ataxia telangiectasia-mutated and Rad3-related) pathway, which promotes replication of the viral genome; however, the exact mechanisms employed by B19V to take advantage of the DDR for virus replication remain unclear. In this study, we focused on the initiators of the DDR and the role of the DDR in cell cycle arrest during B19V infection. We examined the role of individual viral proteins, which were delivered by lentiviruses, in triggering a DDR in ex vivo-expanded primary human erythroid progenitor cells and the role of DNA replication of the B19V double-stranded DNA (dsDNA) genome in a human megakaryoblastoid cell line, UT7/Epo-S1 (S1). All the cells were cultured under hypoxic conditions. The results showed that none of the viral proteins induced phosphorylation of H2AX or replication protein A32 (RPA32), both hallmarks of a DDR. However, replication of the B19V dsDNA genome was capable of inducing the DDR. Moreover, the DDR per se did not arrest the cell cycle at the G(2)/M phase in cells with replicating B19V dsDNA genomes. Instead, the B19V nonstructural 1 (NS1) protein was the key factor in disrupting the cell cycle via a putative transactivation domain operating through a p53-independent pathway. Taken together, the results suggest that the replication of the B19V genome is largely responsible for triggering a DDR, which does not perturb cell cycle progression at G(2)/M significantly, during B19V infection.

Internal Polyadenylation of the Parvovirus B19 Precursor mRNA Is Regulated by Alternative Splicing

Alternative processing of parvovirus B19 (B19V) pre-mRNA is critical to generating appropriate levels of B19V mRNA transcripts encoding capsid proteins and small nonstructural proteins. Polyadenylation of the B19V pre-mRNA at the proximal polyadenylation site ((pA)p), which prevents generation of full-length capsid proteins encoding mRNA transcripts, has been suggested as a step that blocks B19V permissiveness. We report here that efficient splicing of the B19V pre-mRNA within the first intron (upstream of the (pA)p site) stimulated the polyadenylation; in contrast, splicing of the B19V pre-mRNA within the second intron (in which the (pA)p site resides) interfered with the polyadenylation, leading to the generation of a sufficient number of B19V mRNA transcripts polyadenylated at the distal polyadenylation site ((pA)d). We also found that splicing within the second intron and polyadenylation at the (pA)p site compete during processing of the B19V pre-mRNA. Furthermore, we discovered that the U1 RNA that binds to the 5' splice donor site of the second intron is fully responsible for inhibiting polyadenylation at the (pA)p site, whereas actual splicing, and perhaps assembly of the functional spliceosome, is not required. Finally, we demonstrated that inhibition of B19V pre-mRNA splicing within the second intron by targeting an intronic splicing enhancer using a Morpholino antisense oligonucleotide prevented B19V mRNA transcripts polyadenylated at the (pA)d site during B19V infection of human erythroid progenitors. Thus, our study reveals the mechanism by which alternative splicing coordinates alternative polyadenylation to generate full-length B19V mRNA transcripts at levels sufficient to support productive B19V infection.

Productive parvovirus B19 infection of primary human erythroid progenitor cells at hypoxia is regulated by STAT5A and MEK signaling but not HIFα.

Human parvovirus B19 (B19V) causes a variety of human diseases. Disease outcomes of bone marrow failure in patients with high turnover of red blood cells and immunocompromised conditions, and fetal hydrops in pregnant women are resulted from the targeting and destruction of specifically erythroid progenitors of the human bone marrow by B19V. Although the ex vivo expanded erythroid progenitor cells recently used for studies of B19V infection are highly permissive, they produce progeny viruses inefficiently. In the current study, we aimed to identify the mechanism that underlies productive B19V infection of erythroid progenitor cells cultured in a physiologically relevant environment. Here, we demonstrate an effective reverse genetic system of B19V, and that B19V infection of ex vivo expanded erythroid progenitor cells at 1% O2 (hypoxia) produces progeny viruses continuously and efficiently at a level of approximately 10 times higher than that seen in the context of normoxia. With regard to mechanism, we show that hypoxia promotes replication of the B19V genome within the nucleus, and that this is independent of the canonical PHD/HIFα pathway, but dependent on STAT5A and MEK/ERK signaling. We further show that simultaneous upregulation of STAT5A signaling and down-regulation of MEK/ERK signaling boosts the level of B19V infection in erythroid progenitor cells under normoxia to that in cells under hypoxia. We conclude that B19V infection of ex vivo expanded erythroid progenitor cells at hypoxia closely mimics native infection of erythroid progenitors in human bone marrow, maintains erythroid progenitors at a stage conducive to efficient production of progeny viruses, and is regulated by the STAT5A and MEK/ERK pathways.

MicroRNA-15a and -16-1 act via MYB to elevate fetal hemoglobin expression in human trisomy 13

Many human aneuploidy syndromes have unique phenotypic consequences, but in most instances it is unclear whether these phenotypes are attributable to alterations in the dosage of specific genes. In human trisomy 13, there is delayed switching and persistence of fetal hemoglobin (HbF) and elevation of embryonic hemoglobin in newborns. Using partial trisomy cases, we mapped this trait to chromosomal band 13q14; by examining the genes in this region, two microRNAs, miR-15a and -16-1, appear as top candidates for the elevated HbF levels. Indeed, increased expression of these microRNAs in primary human erythroid progenitor cells results in elevated fetal and embryonic hemoglobin gene expression. Moreover, we show that a direct target of these microRNAs, MYB, plays an important role in silencing the fetal and embryonic hemoglobin genes. Thus we demonstrate how the developmental regulation of a clinically important human trait can be better understood through the genetic and functional study of aneuploidy syndromes and suggest that miR-15a, -16-1, and MYB may be important therapeutic targets to increase HbF levels in patients with sickle cell disease and β-thalassemia.

Pharmacological Activation of Guanine Nucleotide Exchange Factors for the Small GTPase Rap1 Recruits High-Affinity β1Integrins as Coreceptors for Parvovirus B19: ImprovedEx VivoGene Transfer to Human Erythroid Progenitor Cells

Parvovirus B19 has potential as a gene therapy vector because of its restricted tropism for human erythroid progenitor cells in the bone marrow. B19 binds to the cell surface through P antigen and we identified activated beta(1) integrins as coreceptors for internalization. Because differentiation with phorbol ester induces beta(1) integrin coreceptor activity, but cell differentiation is not desirable in gene transfer to human progenitor cells and one of the downstream effectors of phorbol esters is the small GTPase Rap1, the role of Rap1 in the recruitment of beta(1) integrins on hematopoietic cells was examined. Expression of a constitutively active Rap1 (63E) was sufficient to recruit beta(1) integrin coreceptors in erythroleukemic K562 cells by inducing high-affinity integrin conformation. A crucial role of actin polymerization in Rap1-mediated beta(1) integrin recruitment was documented by complete inhibition of the 63E Rap1 effect with low-dose cytochalasin D and by the ability of a constitutively active mutant of the actin cytoskeleton regulator Rac1 to sensitize K562 cells to the pharmacological activation of endogenous Rap1, using the Rap1 exchange factor-specific 8-pCPT-2'-O-Me-cAMP [8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate]. Interestingly, in primary human erythroid progenitor cells, 8-pCPT-2'-O-Me-cAMP was sufficient to significantly increase B19-mediated gene transfer, suggesting that these cells possess the cytoskeleton organization capacity required for efficient recruitment of beta(1) integrins by brief pharmacological stimulation of Rap1 GTP loading. Because 8-pCPT-2'-O-Me-cAMP has been implicated in enhanced homing of progenitor cells, these results identify a novel tool with which to optimize ex vivo B19-mediated gene transfer and potentially improve homing of transduced cells by Rap1-beta(1) integrin activation with 8-pCPT-2'-O-Me-cAMP.

High Throughput Screen To Identify Small Molecules That Differentially Regulate Expression of the Globin Genes.

Several lines of evidence indicate that the pharmacological activation of fetal hemoglobin is an effective therapy for sickle cell anemia and beta thalassemia, but novel treatments for these diseases are needed. We developed and validated a high throughput assay to detect differential regulation of the globin genes and utilized this assay in a small molecule screen to identify novel compounds that increase the relative expression of gamma globin. In our assay, transcripts for the alpha, beta, delta, epsilon, gamma, theta, and zeta globin genes are amplified by multiplexed ligation-mediated PCR. Labeled amplicons are captured on different fluorescent microspheres using molecular barcodes, and the relative abundance of labeled amplicons is detected by high speed flow cytometry. To recapitulate the activity of compounds in the bone marrow of patients as accurately as possible, the screen was performed using primary human erythroid progenitor cells cultured in vitro. The assay was adapted to 384-well format with robotic liquid handling. In validation studies, the assay detected the expected increases in globin gene expression during erythroid differentiation, increased gamma globin expression in umbilical cord blood progenitor cells, and increased gamma globin expression in cells treated with known inducers of fetal hemoglobin including hydroxyurea and sodium butyrate. We screened a library of 1040 known bioactive compounds, 75% of which are FDA approved drugs, and a library of 600 compounds produced by diversity oriented synthesis that have been shown to inhibit histone deacetylase (HDAC) activity. In the screen, we rediscovered previously identified globin gene regulators, further validating our globin assay. For example, corticosteroids, known activators of fetal hemoglobin, increased the relative expression of gamma globin. Thyroid hormone specifically increased expression of delta globin, consistent with clinical observations that hemoglobin A2 levels are increased in hyperthyroidism and decreased in hypothyroidism. We identified ten novel compounds from the diversity oriented synthesis library that powerfully induce expression of the gamma globin gene relative to beta globin. Moreover, HDAC inhibition reversed the ontogeny of globin gene expression, coordinately increasing expression of fetal and embryonic relative to the adult globin genes. Relative to beta globin gene expression, gamma and epsilon globin were induced while delta globin was unaffected by HDAC inhibitors; relative to alpha globin expression, zeta globin was increased and theta globin was unaffected. The identification of compounds that differentially regulate globin gene expression may provide lead compounds for the development of novel therapies for sickle cell disease and beta thalassemia and may help elucidate the molecular events underlying switching of the globin genes during normal development.

Characterization of a Newly Discovered Human Alpha-Globin Gene.

It is well known that the globin genes are organized within the human genome as two gene clusters on chromosomes 16 and 11 (α cluster: ζ2-ζ1-α2-α1-# and β cluster: ε-Gγ-Aγ-δ-β). Switching in the expression patterns of those genes during human ontogeny generally follows a pattern determined by their genomic arrangement, but the genetic mechanism is not defined. In an effort to better understand the γ-to-β globin switching phenomenon, gene expression patterns from reticulocyte RNA were studied. cDNA were generated from the reticulocytes of 28 separate donors (14 cord blood; 14 adult blood) and analyzed using Affymetrix HG-U133 arrays. Quantitative PCR was used to confirm the array expression patterns. Among those cDNA identified as having expression patterns that were relatively decreased among adult reticulocytes, we were surprised by expression associated with a cluster of expressed sequence tags (EST) located within the α-globin locus in the region between the ζ1 and α2 genes. In the context of the 44,229 arrayed probes, high-level expression from that EST cluster was detected (ranked < 25th). Informatic analysis revealed alignment with the second and third exons previously described for the Ψα2 globin gene, but no significant similarity with the first exon of Ψα2. Based upon these comparisons, we used adult human reticulocyte RNA to amplify a cDNA clone for further study (GenBank: AY698022; named μ-globin). Sequencing of that clone revealed an insert that encodes a predicted 423 nt. open reading frame that is one amino acid residue shorter than the α2-globin gene. While the mature μ-globin mRNA results from splicing of two introns, it lacks a canonical Kozak sequence. BLASTP and CLUSTALW analyses revealed the highest level of homology with the avian αD-globin protein with a sequence identity of 55% (78/141 amino acids). In addition, the predicted heme- and globin-binding amino acids of μ-globin and avian αD-globin are largely conserved. Experimentally, we analyzed the expression level of the μ-globin transcripts using quantitative real-time PCR. Non-erythroid tissues including whole brain, white blood cells, and the Jurkat cell line demonstrated only background levels. The expression level of μ-globin (copies per ng cDNA) was 3.04x10(4) ± 1.68x10(3) in fetal liver, 1.71x10(5) ± 9.51x10(4) in cord reticulocytes, 1.15x10(4) ± 1.19x10(3) in bone marrow, and 2.17x10(4) ± 6.84x10(3) in adult reticulocytes. Based upon the consistent decrease in μ-globin expression between fetal and adult erythroid tissues, we measured the expression of γ-globin in those tissues for comparison. The expression level of γ-globin (copies per ng cDNA) was 2.49x10(6) ± 1.48x10(5) in fetal liver, 1.49x10(8) ± 7.88x10(7) in cord reticulocytes, 3.84x10(3) ± 3.83x10(2) in bone marrow, and 5.70x10(5) ± 5.80x10(5) in adult reticulocytes. To determine if μ-globin expression is regulated during erythropoiesis, we assayed primary human erythroid progenitor cells cultured in erythropoietin. The expression pattern of μ-globin was similar to the other globin genes increasing from background levels on day 0 to maximum levels in committed erythroblasts (3.56x10(5) ± 1.99x10(4) copies per ng cDNA). These results suggest that the human genome encodes a previously unrecognized α-globin that most closely resembles avian αD-globin. Expression of that gene is decreased during the fetal-to-adult transition of human ontogeny and regulated during erythropoiesis.