Proerythroblast Stage Research Articles

Identification of CNOT4 As a Positive Regulator of Erythroid Cell Growth Using a Genome Wide CRISPR Knock-out Screen

Red blood cells, also known as erythrocytes, are the principle means of oxygen delivery to the body, and inadequate or disordered erythrocyte production is a major cause of human disease. While the cell-extrinsic factors that control erythrocyte production have been well studied, our understanding of the cell-intrinsic regulation of this process is incomplete. To identify genes that regulate erythroid proliferation and/or maturation, we performed a genome-scale CRISPR knock-out screen in the immortalized human erythroid cell line, HUDEP-2. HUDEP-2 cells can be maintained at the pro-erythroblast stage and then sequentially differentiated into basophilic, polychromatic, and orthochromatic erythroblasts by changing culture conditions, which permits separate evaluation of the impact of genetic perturbations on pro-erythroblast viability and terminal erythroid differentiation capacity. To perform our screen, we used the commercially available GeCKO-v2 library, which includes 6 sgRNAs targeting virtually every protein-coding gene in the human genome. This library was packaged into lentiviral particles, each expressing Cas9, an sgRNA, and a puromycin resistance gene. HUDEP-2 cells were transduced with the pooled GeCKO-v2 library at low multiplicity of infection to ensure delivery of only one sgRNA per cell. Following puromycin selection, pro-erythroblasts were collected prior to differentiation and orthochromatic erythroblasts were sorted at day 12 of differentiation (selecting for cells that downregulated CD49d). By identifying sgRNAs that were under-represented in orthochromatic erythroblasts compared to the proerythroblasts, we identified several genes that when deleted lead to impaired erythroid differentiation. One of these genes was CNOT4, which encodes a RING E3 ligase member of the CCR4-NOT complex, a conserved transcriptional regulator. This gene has been previously reported to positively regulate JAK/STAT signaling, which is known to play a key role in erythroid growth and differentiation. Additionally, only 30 out of 1095 cell lines tested in the Depmap portal exhibit diminished growth following CNOT4 deletion, suggesting a rather specific impact on erythroid cells. To define the role of CNOT4 in erythropoiesis, we electroporated human CD34+ hematopoietic stem and progenitor cells (HSPCs) with a ribonucleoprotein (RNP) complex consisting of Cas9 protein and CNOT4-targeting sgRNA. Three unique CNOT4-targeting sgRNAs were tested. Following RNP electroporation, cells were differentiated using a four-phase erythroid differentiation culture protocol and assessed for growth, indel frequencies, and erythroid differentiation capacity. For all three sgRNAs tested, CNOT4 disruption resulted in 10-fold reduced proliferation over the course of differentiation compared to cells electroporated with non-targeting sgRNAs. Erythroid differentiation, assessed by flow cytometry using CD49d and CD233 expression, was identical in CNOT4 deleted and control cells. However, evaluation of cytospins revealed reduced cell size with diminished cytoplasm to nuclear ratio in CNOT4 deficient compared to control cells. Notably, the findings above were validated in erythroid cells differentiated from HSPCs harvested from two independent donors. Taken together, these findings suggest a novel role for CNOT4 and/or the CCR4-NOT complex in erythroid differentiation. Studies are ongoing to determine the mechanism by which CNOT4 disruption negatively impacts erythropoiesis and to examine if CNOT4 overexpression results in enhanced proliferation/differentiation of erythroid cells.

Comprehensive Characterization and Global Transcriptome Analysis of Human Fetal Liver Terminal Erythropoiesis

The fetal liver (FL) is the key erythropoietic organ during fetal development, but knowledge on human FL erythropoiesis is very limited. In this study, we sorted primary erythroblasts from FL cells and performed RNA sequencing (RNA-seq) analyses. We found that temporal gene expression patterns reflected changes in function during primary human FL terminal erythropoiesis. Notably, the expression of genes enriched in proteolysis and autophagy was up-regulated in orthochromatic erythroblasts (OrthoEs), suggesting the involvement of these pathways in enucleation. We also performed RNA-seq of in vitro cultured erythroblasts derived from FL CD34+ cells. Comparison of transcriptomes between the primary and cultured erythroblasts revealed significant differences, indicating impacts of the culture system on gene expression. Notably, the expression of lipid metabolism-related genes was increased in cultured erythroblasts. We further immortalized erythroid cell lines from FL and cord blood (CB) CD34+ cells (FL-iEry and CB-iEry, respectively). FL-iEry and CB-iEry were immortalized at the proerythroblast stage and can be induced to differentiate into OrthoEs, but their enucleation ability was very low. Comparison of the transcriptomes between OrthoEs with and without enucleation capability revealed the down-regulation of pathways involved in chromatin organization and mitophagy in OrthoEs without enucleation capacity, indicating that defects in chromatin organization and mitophagy contribute to the inability of OrthoEs to enucleate. Additionally, the expression of HBE1, HBZ, and HBG2 was up-regulated in FL-iEry compared with CB-iEry, and such up-regulation was accompanied by down-regulated expression of BCL11A and up-regulated expression of LIN28B and IGF2BP1. Our study provides new insights into human FL erythropoiesis and rich resources for future studies.

Kindlin-3 deficiency leads to impaired erythropoiesis and erythrocyte cytoskeleton.

Kindlin-3 (K3) is critical for the activation of integrin adhesion receptors in hematopoietic cells. In humans and mice, K3 deficiency is associated with impaired immunity and bone development, bleeding, and aberrant erythrocyte shape. To delineate how K3 deficiency (K3KO) contributes to anemia and misshaped erythrocytes, mice deficient in erythroid (K3KO∖EpoR-cre) or myeloid cell K3 (K3KO∖Lyz2cre), knockin mice expressing mutant K3 (Q597W598 to AA) with reduced integrin-activation function (K3KI), and control wild-type (WT) K3 mice were studied. Both K3-deficient strains and K3KI mice showed anemia at baseline, reduced response to erythropoietin stimulation, and compromised recovery after phenylhydrazine (PHZ)-induced hemolytic anemia as compared with K3WT. Erythroid K3KO and K3 (Q597W598 to AA) showed arrested erythroid differentiation at proerythroblast stage, whereas macrophage K3KO showed decreased erythroblast numbers at all developmental stages of terminal erythroid differentiation because of reduced erythroblastic island (EBI) formation attributable to decreased expression and activation of erythroblast integrin α4β1 and macrophage αVβ3. Peripheral blood smears of K3KO∖EpoR-cre mice, but not of the other mouse strains, showed numerous aberrant tear drop-shaped erythrocytes. K3 deficiency in these erythrocytes led to disorganized actin cytoskeleton, reduced deformability, and increased osmotic fragility. Mechanistically, K3 directly interacted with F-actin through an actin-binding site K3-LK48. Taken together, these findings document that erythroid and macrophage K3 are critical contributors to erythropoiesis in an integrin-dependent manner, whereas F-actin binding to K3 maintains the membrane cytoskeletal integrity and erythrocyte biconcave shape. The dual function of K3 in erythrocytes and in EBIs establish an important functional role for K3 in normal erythroid function.

An Ancient Transcriptional Hub Couples Developmentally Regulated Gene Expression with Metabolism during Erythropoiesis

The α-globin genes and their regulation by the α-globin superenhancer have been extensively characterised. An adjacent gene, Nprl3, contains 4 of the 5 α-globin enhancers in mice, and 3 of 4 in humans. The neighbouring genomic positions of Nprl3 and α-globin, which share a topologically associated domain, have been maintained for >500 million years. However, the potential functional requirement of this genomic linkage has not been investigated until now. Nprl3 is conserved across Animalia and Fungi, and has been characterised to provide negative regulation of mTORC1, a critical controller of cellular metabolism. Here we report phenotypes of Nprl3-deficient erythropoiesis in mouse fetal liver and bone marrow, and in human peripheral blood. We provide molecular mechanistic insight that Nprl3 is essential for optimal erythropoiesis and for responding to fluctuating nutrient and growth factor concentrations. Finally, we show that erythroid Nprl3 function depends on transcriptional support arising from the α-globin enhancers. Analysis of Nprl3-/- fetal livers revealed severe impairment of erythroid development. On day E13.5, erythroid cells fail to develop beyond the proerythroblast stage. Loss of Nprl3 is accompanied by elevated mTORC1 signalling, confirming the canonical signalling role of Nprl3 in erythroblasts. Active mTORC1 typically inhibits autophagy; in consistence with this, we observed suppressed autophagic activity in Nprl3-/- erythroblasts. We next investigated whether the erythroid defect results from haematopoietic-intrinsic Nprl3 requirements. This was tested using competitive bone marrow - fetal liver chimera experiments, in which Nprl3-/- fetal liver cells contributed poorly to erythroid lineage reconstitution of irradiated adult bone marrow. Thus, Nprl3 regulates baseline erythropoiesis in the mouse. To discover whether NPRL3 is also required for human erythropoiesis, NPRL3-knockout was induced in primary human CD34+ progenitors using a CRISPR-Cas9 RNP-editing system. These progenitors had reduced ability to produce enucleated erythroid cells compared to their negative control counterparts. NPRL3-knockout erythroblasts also demonstrated defective mTORC1 signalling responses to iron deficiency, amino acid withdrawal and erythropoietin stimulation. These results indicate that NPRL3 has a critical role in interpreting fluctuating nutritional environments, and in tuning the metabolic response of developing erythroid cells to the extracellular milieu. Importantly, Nprl3 expression is known to be elevated in erythroid cells, increasing ~30-fold during erythroid lineage commitment due to α-globin enhancer activity. We sought to discover whether this erythroid-specific increase in Nprl3 expression is required for the functionality outlined above. Using genetic engineering to delete the Nprl3 promoter on one allele, and to delete all α-globin enhancers on the other, we eliminated all interactions between Nprl3 and α-globin regulatory elements. Embryos heterozygous for both alleles (Nprl3+/-α-globin-enhancers+/-,'Nalph') retain non-erythroid 'un-enhanced' Nprl3 expression levels, and enhancer-regulated α-globin expression. Remarkably, at E13.5, the Nalph genotype presented impaired erythropoiesis reminiscent of that observed in Nprl3-/- embryos, with development inhibited at the same stage of differentiation. Thus, we conclude that the genomic contact hub Nprl3 shares with α-globin and its enhancers bestows the erythroid-specific transcriptional upregulation required for Nprl3 to perform its important erythropoietic role. This finding suggests that the deep evolutionary coupling of these two genes has enabled the α-globin enhancers to control metabolism, as well as α-globin production, in developing erythroid cells.

Overexpression of Human TLR8 Causes Fatal Anemia in SLE-Prone Mice By Altering the Bone Marrow Erythropoietic Niche

Anemia is a common hematologic abnormality in patients with active systemic lupus erythematosus (SLE), a disease characterized by innate immune system activation by nucleic acid containing immune complexes and cell debris. Work in mouse models has shown that overactivation of several cytoplasmic innate immune sensors by either self-DNA or self-RNA specifically leads to erythroid cell death in the fetal liver while sparing other hematopoietic cell lineages.The endosomal receptors TLR7 and TLR8 both recognize ssRNA in humans. TLR7 overexpression in mice causes a lupus syndrome that includes the development of mild to moderate anemia (Hb >10) and thrombocytopenia and is associated with spleen histiocytosis, autoimmune hemolysis, erythrophagocytosis and compensatory stress erythropoiesis in the spleen. A recent study demonstrated that patients with TLR8 gain of function mutations present with immunodeficiency, inflammation and bone marrow failure. However the role of TLR8 in SLE has been difficult to study in mice because it has a 5 amino acid deletion that attenuates its RNA binding capacity.To address the role of TLR8 in SLE, we overexpressed human TLR8 in a lupus mouse model (huTLR8tg.Sle1.Yaa) using a BAC transgene. 50% of homozygous huTLR8tg.Sle1.Yaa mice developed severe anemia (Hb<9) resulting in early mortality starting at 3-4.5 months of age. This phenotype required both the Sle1 and Yaa loci that promote the formation of high titer anti-chromatin and anti-RNA antibodies and onset of nephritis at >6 months of age. There was no difference in autoantibody titers between Sle1.Yaa wt and huTLR8tg mice and early death was not due to premature onset of renal disease. All mice had normal RBC indices prior to 10 weeks of age. Anemia was associated with an increase in bone marrow (BM) trabecular bone and a decrease in erythroblastic islands (EBI) in the BM with compensatory stress erythropoiesis leading to reticulocytosis and vast splenomegaly. RBC half-life was normal even after the development of reticulocytosis, but decreased in severely anemic mice as a pre-terminal event. Using CFSE labeling of RBCs we showed that hemophagocytosis occurred in vivo as a result of expansion of phagocytic red pulp macrophages.Flow cytometry of BMs from transgenic mice showed normal erythroid progenitors but a block at the late CFU-E/early proerythroblast stage leading to overall decreased terminal erythroid differentiation. Single cell RNAseq of EBIs isolated from transgenic BMs confirmed the block in erythropoiesis. The erythroblast cluster proximal to the block had a signature of mitochondrial stress and decreased proliferation. Spleen EBIs from transgenic mice were characterized by a low frequency of early erythroblasts compared with BM EBIs. We found 6 related clusters of EBI central macrophages in the BMs of both wt and transgenic mice. In transgenic mice, most of these displayed an inflammatory and Type 1 IFN signature. One cluster (M2) expressed all the classical central macrophage phenotypic markers in wt mice but was characterized in transgenic mice by downregulation of multiple phagocytic receptors and a 5-fold decrease of VCAM1 expression. Loss of VCAM1 and downregulation of CD169 in central macrophages of transgenic BMs was confirmed by flow cytometry. By contrast spleen central macrophages from transgenic mice retained VCAM and CD169 expression.Together, these results suggest that failure of BM erythropoiesis in huTLR8 transgenic SLE.Yaa lupus-prone mice, in which the acquisition of anti-nucleic acid autoantibodies drives excess innate stimulus through TLR7 and huTLR8, is due to a block in differentiation from CFU-E to the early proerythroblast stage; this is associated with an inflammatory phenotype specifically in BM erythroblastic island central macrophages and down regulation of adhesion and phagocytic receptors. Stress erythropoiesis in the spleens is associated with vast expansion of red pulp macrophages with phagocytic properties and fatal anemia is associated with a decrease in red blood cell half-life, suggesting that excessive RBC phagocytosis, coupled with insufficient erythroblast progenitors, eventually exceeds the capability of stress erythropoiesis to replace the RBC mass. DisclosuresKalfa: Agios Pharmaceuticals, Inc.: Other: Steering Committee, Research Funding; FORMA Therapeutics, Inc: Research Funding. Paulson: Forma Therapeutics: Consultancy. Blanc: Keros Therapeutics, Inc.: Consultancy.

Single-cell analysis of erythropoiesis in Rpl11 haploinsufficient mice reveals insight into the pathogenesis of Diamond–Blackfan anemia

Rpl11 haploinsufficient mice develop a macrocytic anemia similar to patients with DBA. Here, we fully characterize this model from clinical and pathophysiological perspectives. Early erythroid precursors have increased heme content and high cytoplasmic reactive oxygen species, impairing erythroid differentiation at the colony-forming unit-erythroid (CFU-E)/proerythroblast stage and subsequently. Using single-cell analyses that link a cell's surface protein expression to its total transcriptome and unbiased analyses, we found GATA1, GATA1 target gene, and mitotic spindle pathway gene transcription were the pathways that decreased the most. Expression of ribosome protein and globin genes was amplified. These changes, as well as the other transcriptional changes that were identified, closely resemble findings in mice that lack the heme export protein FLVCR and, thus, suggest that heme excess and toxicity are the primary drivers of the macrocytic anemia. Consistent with this, treating Rpl11 haploinsufficient mice with corticosteroids increased the numbers of earliest erythroblasts but failed to overcome heme toxicity and improve the anemia. Rpl11 haploinsufficient mice uniquely upregulated mitochondrial genes, p53 and CDKN1A pathway genes, and DNA damage checkpoint genes, which should contribute further to erythroid marrow failure. Together our data establish Rpl11 haploinsufficient mice as an excellent model of DBA that can be used to study DBA pathogenesis and test novel therapies.

RASA3 Regulates Stage-Specific AKT Signaling and Cell Cycle Progression in Mammalian Erythropoiesis

Inherited bone marrow failure syndromes (IBMFS) are a heterogenous group of disorders characterized by dysregulated hematopoiesis across various lineages, predisposition to malignancy, and diverse syndromic features. The scat (severe combined anemia and thrombocytopenia) mouse model has been characterized as a unique model of IBMFS. Scat carries an autosomal recessive missense mutation in the Rasa3 gene that results in RASA3 mislocalization and loss of function. RASA3 functions as a Ras-GTPase activating protein, and its loss of function in scat results in increased erythroid Ras activity, increased reactive oxygen species, and altered cell cycle progression, all culminating in delayed terminal erythroid differentiation. However, the precise mechanism of RASA3 regulation of erythroid differentiation remains undefined, and elucidation of this mechanism is crucial to identifying new therapeutic targets in inherited anemia. Considering the role of RASA3 as regulator of Ras signaling and cell cycle progression, and the importance of these processes to erythroid differentiation, we sought to first characterize the coordination of Ras signaling pathways and cell cycle progression in normal murine erythropoiesis, then observe how loss of RASA3 function alters this regulatory axis. We observed that wild type (WT) erythroblasts demonstrate population-specific influence of ERK and PI3K/AKT signaling in regulating cell cycle progression. Inhibition of both pathways with increasing doses of U0126 and LY294002, respectively, induced accumulation in G0/G1 from the proerythroblast stage until the late basophilic/polychromatic stage (U0126 vehicle vs. 1uM p= 0.0023; LY294002 vehicle vs 1uM p=0.0389). At these later stages, ERK and PI3K/AKT inhibition led to a decrease in G0/G1 percentages, suggesting a stage-specific switch in signaling mediated cell cycle regulation, with PI3K inhibition demonstrating more potent and consistent effects (U0126 vehicle vs 1uM p=0.0406, 0.0481; LY294002 vehicle vs 1uM p=0.0003, 0.0197, 0.0086, n=3). These patterns suggest that ERK and AKT may facilitate cell cycle progression past the G0/G1 checkpoint in early erythropoiesis while inducing cell cycle exit or accumulation in G0/G1 in late erythropoiesis. In scat, we previously characterized increased active Ras in erythroid cells and a delay in terminal erythroid differentiation with accumulation at the polychromatic stage. We therefore next sought to examine the potential mechanistic contribution of altered PI3K/AKT signaling and cell cycle progression to the differentiation delay seen in scat. Phospho-flow analyses demonstrate that scat bone marrow-derived basophilic and polychromatic erythroblasts have increased AKT activation compared to WT (p=0.0402, p=0.0559, respectively; n=4), with similar trends evident in scat spleen basophilic erythroblasts (p=0.064; n=4). These results are consistent with increased Ras activation in scat. Ex vivo EdU/PI analyses revealed that scat bone marrow-derived polychromatic erythroblasts demonstrate G0/G1 accumulation (p=0.0466) and decreased progression to S-phase (0.0414; n=6), also with similar trends in scat spleen basophilic erythroblasts (p=0.004; n=6). These results correlate with the observed differentiation delay in scat and indicate that RASA3 regulates stage-specific signaling and cell cycle progression during erythropoiesis. To study if cell cycle dysregulation in scat begins at an earlier stage of erythroid differentiation, we analyzed murine hematopoietic and erythroid progenitors as Ter119-, cKit+ cells expressing increasing levels of CD71 and found that both CD71lo and CD71med bone marrow-derived scat progenitors present with G0/G1 accumulation (p=0.0015, p=0.0073, respectively) and decreased progression to S-phase (p=0.0014, p=0.0241; n=7). This suggests a dynamic relationship between RASA3, Ras signaling, and cell cycle progression throughout early and late erythroid differentiation. Together, these findings support the role of RASA3 as a regulator of the signaling networks governing erythropoiesis and reveal a new targetable axis in a model of inherited bone marrow failure. Disclosures No relevant conflicts of interest to declare.

Putative regulators for the continuum of erythroid differentiation revealed by single-cell transcriptome of human BM and UCB cells

Fine-resolution differentiation trajectories of adult human hematopoietic stem cells (HSCs) involved in the generation of red cells is critical for understanding dynamic developmental changes that accompany human erythropoiesis. Using single-cell RNA sequencing (scRNA-seq) of primary human terminal erythroid cells (CD34-CD235a+) isolated directly from adult bone marrow (BM) and umbilical cord blood (UCB), we documented the transcriptome of terminally differentiated human erythroblasts at unprecedented resolution. The insights enabled us to distinguish polychromatic erythroblasts (PolyEs) at the early and late stages of development as well as the different development stages of orthochromatic erythroblasts (OrthoEs). We further identified a set of putative regulators of terminal erythroid differentiation and functionally validated three of the identified genes, AKAP8L, TERF2IP, and RNF10, by monitoring cell differentiation and apoptosis. We documented that knockdown of AKAP8L suppressed the commitment of HSCs to erythroid lineage and cell proliferation and delayed differentiation of colony-forming unit-erythroid (CFU-E) to the proerythroblast stage (ProE). In contrast, the knockdown of TERF2IP and RNF10 delayed differentiation of PolyE to OrthoE stage. Taken together, the convergence and divergence of the transcriptional continuums at single-cell resolution underscore the transcriptional regulatory networks that underlie human fetal and adult terminal erythroid differentiation.

IL-6 blockade reverses bone marrow failure induced by human acute myeloid leukemia.

Most patients with acute myeloid leukemia (AML) die from complications arising from cytopenias resulting from bone marrow (BM) failure. The common presumption among physicians is that AML-induced BM failure is primarily due to overcrowding, yet BM failure is observed even with low burden of disease. Here, we use large clinical datasets to show the lack of correlation between BM blast burden and degree of cytopenias at the time of diagnosis. We develop a splenectomized xenograft model to demonstrate that transplantation of human primary AML into immunocompromised mice recapitulates the human disease course by induction of BM failure via depletion of mouse hematopoietic stem and progenitor populations. Using unbiased approaches, we show that AML-elaborated IL-6 acts to block erythroid differentiation at the proerythroblast stage and that blocking antibodies against human IL-6 can improve AML-induced anemia and prolong overall survival, suggesting a potential therapeutic approach.

Evaluating Metabolic Regulation of Human Erythropoiesis By Profiling Nutrient Transporter Expression

Recent studies have documented that cell metabolism regulates hematopoietic stem cell (HSC) renewal and lineage commitment. However, the detailed metabolic changes that occur during human erythropoiesis remain to be defined. As erythroid cell differentiation is likely to be associated with changes in metabolic requirements, we hypothesized that progenitors adapt to these metabolic modulations by altering their nutrient transporter expression profile. Using an in vitro erythroid-inducing cell culture system employing CD34+ cells from human bone marrow and peripheral blood as well as primary erythroid cells isolated from fresh bone marrow samples, we assessed the cell surface nutrient transporter profiles of progenitors at different stages of erythroid development. Quantification of cell surface nutrient transporter expression was performed using a novel scaffold of retroviral envelope receptor binding domains (RBDs) that function as specific ligands of solute carrier (SLC) nutrient transporters. This bank allowed an evaluation of diverse metabolite transporters including GLUT1/SLC2A1 glucose transporter, the PiT1/SLC20A1 and PiT2/SLC20A2 phosphate importers, the XPR1/SLC53A1 phosphate exporter, the FLVCR1 heme exporter, the RFVT1/2 (SLC52A1/SLC52A2) riboflavin importers, the CAT1/SLC7A1 arginine importer, the ASCT2/SLC1A5 glutamine transporter and the SMVT/SLC5A6 sodium-dependent multivitamin transporter. Notably, the cell surface expression profiles of these nutrient transporters, as evaluated by flow cytometry, revealed marked changes as a function of the stage of erythroid differentiation, as shown in the figure below. Specifically, while FLVCR1, RFVT1/2, and SMVT are highly expressed on erythroid progenitors, the levels of these transporters decrease starting at the proerythroblast stage. In addition, PiT1, PiT2, XPR1, and CAT1 are expressed highly during the erythroid colony forming unit (CFU-E) stage while GLUT1 gradually increases and reaches a peak at late stages of erythroid differentiation, remaining elevated on mature red cells. The noted distinct changes in transporter expression are likely a reflection of the changing demands of various nutrients during human erythropoiesis. In summary, we have established a comprehensive metabolite transporter profile at distinct stages of normal human erythropoiesis using a novel experimental strategy. These original findings form a strong foundation for future studies aiming at elucidating the metabolic requirements of normal erythropoiesis, evaluating diseases affecting red blood cell maturation due to aberrant metabolic regulation, and for identifying new therapeutic targets. Figure Disclosures Bitaudeau: Metafora-biosystems: Employment. Petit:Metafora-biosystems: Equity Ownership, Other: CEO and co-founder. Sitbon:Metafora-biosystems: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder.

Eltrombopag Improves Erythroid Differentiation in a Human iPSC Model of Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a congenital bone marrow (BM) failure syndrome primarily characterized by defective erythropoiesis. In most patients, pathogenic heterozygous mutations have been identified in genes encoding ribosomal proteins (RP). The resulting RP haploinsufficiency was recently shown to delay globin protein translation in erythroid cells, whereas synthesis of heme, the nonprotein iron-containing component of hemoglobin, proceeds normally (Yang et al., Sci Transl Med 2016). Because heme is first synthesized at or just before the proerythroblast stage, free heme is in excess of globin in these cells. High levels of free heme induce proerythroblast cell death, and erythroid differentiation thus halts at the earlier BFU-E/CFU-E progenitor stage. Consistent with these observations, inhibition of heme synthesis with succinylacetone was previously shown to improve erythroid differentiation of DBA marrow cells in vitro. In this study, we investigated whether eltrombopag (Epag), an FDA-approved mimetic of thrombopoietin that promotes trilineage hematopoiesis in subjects with acquired BM failure (Olnes et al., NEJM 2012; Townsley et al., NEJM 2017), could rescue erythropoiesis in DBA. We hypothesized that Epag might inhibit heme synthesis by restricting iron availability due to its robust intracellular iron chelating properties, leading to decrements in iron-induced reactive oxygen species (ROS) and increased proerythroblast survival and maturation. To test this possibility,we first established an induced pluripotent stem cell (iPSC) model of DBA by reprogramming mononuclear cells (MNCs) from a patient with inactivating mutations in RPS19, the most commonly mutated gene in DBA. We also generated a control isogenic iPSC line by CRISPR/Cas9-mediated correction of RPS19 point mutations in the established DBA iPSC line.RPS19 haploinsufficiency was confirmed by Western blot and the expected reduction in 40S/60S ribosomal subunit ratio was detected by polysome profiling of DBA iPSCs. This phenotype normalized in the isogenic iPSCs. Both DBA and isogenic iPSC lines, and iPSCs derived from a healthy donor, were then subjected to hematopoietic differentiation for 21 days using the STEMdiffTMmonolayer-based approach (Ruiz et al., BioRxiv 2019). Hematopoietic cells were harvested between day 19 and 21 of culture when maximum erythroid production is observed in this system. Normal and isogenic iPSCs efficiently gave rise to erythroid cells at various stages of maturation, including CD71+CD45+EPOR+primitive erythroid progenitors (P1), CD71+CD45loEPOR-proerythroblasts (P2), and more mature CD71+CD45-EPOR-erythroblasts (P3) (Figure). In contrast, the majority of erythroid cells detected after differentiation of DBA iPSCs were comprised within P1 with limited maturation to P2 and P3, consistent with a block in differentiation at the early erythroid progenitor stage (Figure). Furthermore, in colony forming unit (CFU) assays, DBA iPSCs generated numbers of myeloid colonies (CFU-G, CFU-M and CFU-GM) comparable to normal and isogenic iPSCs, but erythroid colonies (BFU-E and CFU-E) were undetectable, in keeping with DBA progenitor's inability to differentiate in vitro. Next, DBA iPSCs were differentiated in the presence of Epag 3 µg/mL from day 10 to 21 of culture. Addition of Epag improved late erythroid maturation, as indicated by reduced percentages of early progenitors (P1) and a concomitant increase in more mature P2 and P3 erythroblastic populations (Figure). Investigations are ongoing to confirm Epag-mediated iron restriction and decreased heme synthesis as the primary molecular mechanism underpinning the improved erythroid maturation observed in this study. Overall, our data indicate that directed differentiation of DBA iPSCs recapitulates early erythroid maturation defects in vitro, and erythropoiesis can be rescued in part by addition of Epag during culture. These results suggest that Epag may improve red blood cell production in patients with DBA. Figure Disclosures No relevant conflicts of interest to declare.

The Condensin II Subunit, NcapH2, Is Required for Proper Embryonic Erythroid Terminal Maturation and Embryonic Viability in a Primary Mouse Model

The process of nuclear condensation is an integral part of erythroid differentiation. We have previously shown that loss of the histone methyltransferase, Setd8, results in severe embryonic (primitive) erythroid anemia due to loss of chromatin condensation and proper erythroid terminal maturation (Malik 2017). Setd8 mediated H4K20 mono-methylation is an important mediator of higher order chromatin condensation and can recruit members of the Condensin II complex (Lau 2015). ondensin I and II complexes are five protein, large complexes responsible for DNA organization and packaging during mitosis. In eukaryotes, the complexes are composed of conserved SMC components, with each Condensin having a unique kleisin bridge protein and two HEAT subunits. Subunits of Condensin II are highly expressed in erythroid cells, suggesting an erythroid specific function. Overexpression of the Condensin II HEAT subunit, Cap-G2, accelerates erythroid differentiation in MEL cells (Xu 2006). To interrogate the role of Condensin II in a primary model, we mated endogenous EpoR driven cre expressing mice with mice containing a floxed allele around Condensin II kleisin subunit, NcapH2. Embryos homozygous for EpoR specific excision of NcapH2 suffered from severe primitive erythroid anemia with visible anemia beginning at embryonic day (E11.5) and lethality occurring at approximately E13.5. Peripheral blood analysis at E11.5 was significant for erythroblasts with enlarged cell size and nuclear size, as well erythroblasts with dysplastic appearing nuclei. Knockout embryos at E12.5 exhibited peripheral blood anemia, with an increase in the number of dysplastic appearing cells, and decreased fetal liver coloration suggesting a failure of both primitive and fetal definitive erythropoiesis. Failure of the embryonic erythroid lineage to expand was confirmed in a primary cell culture system in which loss of proliferation was limited to mid to late stage precursors, suggesting that Condensin II function is critical beyond the proerythroblast stage of maturation when nuclear condensation is occurring and mass spectrometry demonstrates that H4K20 mono-methylation accumulates. Characterization of primitive erythroblasts from knockout embryos showed increased cell size, increased scatter in the size of the nucleus, and a high frequency of cells with punctate nuclear staining. In addition, the subset of erythroid cells with punctate nuclei did not appear to have a nuclear envelope, suggesting a pre-mitotic error, likely during G2 of the cell cycle. Indeed, knockout cells showed a buildup of cells in G0-G1 of the cell cycle, suggesting arrest following a defective cell division. Together, our data suggests that Condensin II is required during erythroid maturation for proper cell cycle progression and chromatin organization, and that chromatin modifications, such H4K20me1, may be a required mechanism to control the higher-level organization of chromatin. This control is required in red cell precursors, as they undergo rapid expansion, maturation, and nuclear condensation simultaneously. Understanding these mechanisms will give valuable insight into the unique sensitivity of red cells to chromatin perturbations observed in bone marrow failure and myelodysplastic syndromes. Disclosures No relevant conflicts of interest to declare.

The dynamic emergence of GATA1 complexes identified in in vitro embryonic stem cell differentiation and in vivo mouse fetal liver.

GATA1 is an essential transcriptional regulator of myeloid hematopoietic differentiation towards red blood cells. During erythroid differentiation, GATA1 forms different complexes with other transcription factors such as LDB1, TAL1, E2A and LMO2 (“the LDB1 complex”) or with FOG1. The functions of GATA1 complexes have been studied extensively in definitive erythroid differentiation; however, the temporal and spatial formation of these complexes during erythroid development is unknown. We applied proximity ligation assay (PLA) to detect, localize and quantify individual interactions during embryonic stem cell differentiation and in mouse fetal liver (FL) tissue. We show that GATA1/LDB1 interactions appear before the proerythroblast stage and increase in a subset of the CD71+/TER119− cells to activate the terminal erythroid differentiation program in 12.5 day FL. Using Ldb1 and Gata1 knockdown FL cells, we studied the functional contribution of the GATA1/LDB1 complex during differentiation. This shows that the active LDB1 complex appears quite late at the proerythroblast stage of differentiation and confirms the power of PLA in studying the dynamic interaction of proteins in cell differentiation at the single cell level. We provide dynamic insight into the temporal and spatial formation of the GATA1 and LDB1 transcription factor complexes during hematopoietic development and differentiation.

A Novel Role for PIEZO1 in Calcium Homeostasis during Erythropoiesis

In erythrocytes, the mechanosensitive ion transporter PIEZO1 is crucial to maintain cellular volume homeostasis during circulation. Gain of function mutations in the PIEZO1 gene lead to prolonged Ca2+ influx resulting in hereditary xerocytosis (HX). This rare disorder of erythrocyte hydration is characterized by dehydration caused by increased Gardos channel activation. HX patients display a mild to moderate degree of hemolysis, which is generally compensated by an increased reticulocyte count. In addition to its function in erythrocytes we found that PIEZO1 RNA is also expressed at the proerythroblast stage suggesting a role during erythropoiesis. We therefore hypothesize that disturbed Ca2+ homeostasis due to inappropriate PIEZO1 channel function leads to defects in erythroid differentiation.Primary erythroblasts were cultured from peripheral blood mononuclear cells from an HX patient, heterozygous for the previously reported c.6262C>G p.Arg2088Gly mutation in PIEZO1 (Glogowska, 2017 Blood 130(16): 1845-1856). Primary erythroblasts displayed a twofold decreased proliferation compared to healthy controls. Incubating erythroblasts from a healthy control with the PIEZO1 agonist Yoda1 led to a dose dependent decrease in erythroblast proliferation, thereby recapitulating the patient's phenotype. In addition, Yoda1 treatment was accompanied with increased cell death and reduced enucleation. In HX patient's erythroblasts basal Ca2+ levels were higher compared to control cells. In agreement with this, Yoda1-induced PIEZO1 activation led to a dose dependent Ca2+ influx. Yoda1 caused Erk1/2 and SAPK/JNK phosphorylation as well as NFATc2 dephosphorylation, suggesting that PIEZO1 triggers calcium-dependent signal transduction, possibly via calcineurin activation. Indeed, Yoda1-induced NFATc2 dephosphorylation was blocked and SAPK/JNK phosphorylation was inhibited upon pre-incubation of erythroblasts with the calcineurin inhibitor FK506.In conclusion, we found that PIEZO1 has a role in regulating Ca2+ homeostasis during erythropoiesis possibly through activation of calcineurin. Inappropriate PIEZO1 channel activation in erythroid cells results in cell death and proliferation/differentiation defects. This novel feature adds to the complexity of the phenotypic expression of HX. Disclosuresvan Wijk:RR Mechatronics: Research Funding; Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Human Acute Myeloid Leukemia Inhibits Normal Erythroid Differentiation through the Paracrine Effects of IL-6

Acute Myeloid Leukemia (AML) is an aggressive cancer resulting in severe cytopenias related to bone marrow (BM) failure. The common assumption for AML-induced BM failure is overcrowding due to clonal expansion of immature myeloid blasts, leading to failure of normal hematopoiesis. However, in a cohort of 293 AML patients, we found that disease burden (% of blasts determined on diagnostic BM aspirate) did not predict severity of cytopenia (hemoglobin rs=-0.053; p=0.49; WBC rs=-0.030, p=0.70; platelet rs=0.091, p=0.026), strongly arguing against simple crowding as the main mechanism underlying AML-induced BM failure. Thus, the goal of our study is to identify novel mechanism(s) associated with AML-induced BM failure, potentially enabling development of new therapies to improve AML management and reverse morbidity.Conventional xenograft models of human AML do not typically exhibit cytopenias, making them unsuitable to study AML-induced BM failure. We speculated that in the mouse, increased splenic extramedullary hematopoiesis compensated for failed intramedullary hematopoiesis due to AML. To test this hypothesis, we performed surgical splenectomy on NSG mice prior to their transplantation with human AML. Strikingly, splenectomized NSG mice engrafted with primary human AML at a 30-70% disease burden (n=8 for primary AML samples, n=5-10 for each group of splenectomized NSG mice) developed leukopenia and severe anemia compared to sham-operated AML-engrafted controls. AML-engrafted splenectomized NSG mice showed early mortality compared to AML-engrafted NSG mice with intact spleens (10.1 weeks vs. 34.2 weeks p<0.0001). Thus, our splenectomized murine xenograft model uniquely recapitulates human disease and allows for novel characterization of AML-induced BM failure.Utilizing our model, splenectomized NSG mice engrafted with human AML demonstrated depletion of normal hematopoietic progenitors (HSPCs) including HSCs (11.1 fold p<0.0001), MPPs (8.90 fold, p<0.001)), CMPs (11.4 fold p<0.0001), MEPs (1.76 fold p=0.0005) and GMPs (5.07 fold p<0.0001). Focusing on anemia, AML also depleted erythroid progenitors including proerythroblasts (1.39 fold p=0.04), normoblasts (4.96 fold p<0.0001), late normoblasts (10.0 fold p<0.0001), and reticulocytes (4.34 fold p=0.007). The relative preservation of proerythroblasts and depletion of normoblasts indicated that AML blasts impart a specific in vivo differentiation blockade in erythroid differentiation.To explore mechanisms by which AML blasts inhibit normal HSPCs, we generated conditioned media (CM) from patient-derived AML blasts, and found that AML-CM suppressed BFU-E colony formation from normal HSPCs (3.1-5.1 fold). Furthermore, in a direct co-culture system with AML blasts and CD34+ HSPCs, AML blasts inhibited erythroid differentiation from the CFU-E to normoblast stage by 81%. Removing CD34+ cells from the AML co-culture allowed cells to resume differentiation to the proerythroblast stage. These experiments demonstrate that AML imparts a differentiation blockade along the MEP-proerythroblast axis in a cell non-autonomous, reversible fashion.Using cytokine array analysis, we identified elevated IL-6 levels in AML sample-derived CM (n=10, 2841±766.4 pg/ml, 7.80 fold increase, p=0.03) compared to CD34+-derived CM (n=5, 364±36.0 pg/ml). Increased IL-6 was also found in BM aspirates from human AML engrafted splenectomized NSG mice (715±125pg/ml, p=0.001) compared to mice engrafted with normal CD34+ HSPCs (undetectable). Thus, we hypothesized that IL-6 produced by AML blasts acts as a paracrine factor to suppress erythropoiesis. Consistent with this hypothesis, an IL-6 neutralizing antibody reversed the inhibition of BFU-E formation imparted by AML-CM. Furthermore, the addition of recombinant IL-6 to liquid cultures of erythroid differentiation resulted in a 23% reduction in proerythroblasts.Together, our data suggests that (1) overcrowding is not the primary mechanism resulting in BM failure in AML; (2) AML blasts play a previously unrecognized role in imparting a differentiation blockade along the MEP-proerythroblast axis, resulting in progressive anemia; and (3) this differentiation blockade is at least partially attributable to IL-6 secreted from AML blasts as a paracrine factor. DisclosuresNo relevant conflicts of interest to declare.

Long non-coding RNA-dependent mechanism to regulate heme biosynthesis and erythrocyte development

In addition to serving as a prosthetic group for enzymes and a hemoglobin structural component, heme is a crucial homeostatic regulator of erythroid cell development and function. While lncRNAs modulate diverse physiological and pathological cellular processes, their involvement in heme-dependent mechanisms is largely unexplored. In this study, we elucidated a lncRNA (UCA1)-mediated mechanism that regulates heme metabolism in human erythroid cells. We discovered that UCA1 expression is dynamically regulated during human erythroid maturation, with a maximal expression in proerythroblasts. UCA1 depletion predominantly impairs heme biosynthesis and arrests erythroid differentiation at the proerythroblast stage. Mechanistic analysis revealed that UCA1 physically interacts with the RNA-binding protein PTBP1, and UCA1 functions as an RNA scaffold to recruit PTBP1 to ALAS2 mRNA, which stabilizes ALAS2 mRNA. These results define a lncRNA-mediated posttranscriptional mechanism that provides a new dimension into how the fundamental heme biosynthetic process is regulated as a determinant of erythrocyte development.

Puromycin Selection Confounds the RNA-Seq Profiles of Primary Human Erythroblasts

Lentiviral transduction followed by puromycin selection is a well-recognized procedure for gene transfer and expression experiments using a variety of cell types including human hematopoietic stem and progenitor cells. Despite its widespread application, research regarding the potential effects of bacterial puromycin N-acetyltransferase (pac) gene expression in mammalian cell cultures is incomplete. Here the potential for puromycin selection to affect transcriptome profiles was examined using a well-studied model for human erythropoiesis. Experiments were performed using primary CD34(+) cells from six adult healthy human donors transduced with two commercially available pac-encoding lentiviral vectors and compared to non-transduced control cells. RNA-Seq gene expression profiles were generated at the proerythroblast stage of differentiation, then differential gene expression was analyzed with DEseq2 in R-Studio software. Inter-donor variation in the gene expression profiles and variations between puromycin selected populations after transduction of the separate lentiviral vectors was manifested by significant differences in the RNA detection levels of less than 0.1%. However, puromycin selection after pac gene transduction caused significant changes in over 5% of the mRNA when compared to non-transduced controls. The results suggest that consideration should be given for the potential of puromycin selection to confound the interpretation of RNA-Seq transcriptome profiles.

Role of Activated Pleckstrin-2 and Down-Stream Effects on Ineffective Erythropoiesis in β-Thalassemic Mice

Erythropoiesis involves stem cell differentiation to mature red blood cells (RBCs). Erythropoietin (Epo) is essential for erythroipoiesis, and Epo binding to Epo receptor triggers a complicated and incompletely understood set of potentially related molecular signals influencing cell survival, differentiation, and enucleation. Although Epo is associated with increased survival of erythroid precursors, it induces reactive oxygen species (ROS), and high Epo concentration has an anti-enucleation effect in vitro.i Furthermore, diseases of ineffective erythropoiesis, e.g. β-thalassemia, are associated with increased Epo and ROS concentrations implicated in the expansion of and damage to erythroid precursors, respectively. Treating erythroblasts with low dose ROS scavenger promotes enucleation, but high dose ROS scavenger leads to cell death,i suggesting that an optimal ROS concentration is integral to effective erythropoiesis. We and others have shown that ROS is increased in β-thalassemic erythroid precursors, but despite increased ROS, erythroid precursor apoptosis is not increased. We hypothesize that compensatory mechanisms prevent the ill-effects of increased ROS on erythroid precursors. Our prior experiments demonstrate disordered erythropoiesis in β-thalassemic (th1/th1) mice, restored in transferrin-treated th1/th1 mice,ii despite which, ROS remained increased in erythroid precursor from transferrin-treated th1/th1 mice. To identify mechanisms responsible for transferrin's effect, we performed RNA seq analysis of erythroblasts from wild type (WT), th1/th1, and transferrin-treated th1/th1 mice. We identified increased pleckstrin-2 (plek2) in th1/th1 relative to WT mice, normalized in transferrin-treated th1/th1 mice. We hypothesize that plek2 activation counteracts the ill effects of ROS and promotes enucleation in β-thalassemia. Using confocal microscopy, we demonstrate that 1) plek2 co-localizes with actin on the cell membrane after the pro-erythroblast stage but is in the nucleus throughout terminal erythropoiesis in WT mice; 2) membrane-associated plek2 is present earlier, in pro-erythroblasts, and remains membrane-associated until orthochromatophilic stage in th1/th1 mice; and 3) plek2 localization is normalized in transferrin-treated th1/th1 mice. Because plek2 activation leads to its association with the cell membrane and plek2 activation is increased in th1/th1 erythroblasts, we set out to explore the role of plek2 activation on ineffective erythropoiesis in transferrin-treated th1/th1 mice. Prior publications propose that plek2 interacts with and results in the phosphorylation of cofilin, preventing cofilin's translocation to the mitochondria as part of the apoptosis pathway in response to increased ROS.iii We demonstrate decreased in mitochondria cofilin localization and increased cellular p-cofilin in th1/th1 erythroblasts, normalized after transferrin treatment. These data suggest that despite an increase in ROS, plek2 and its induction of p-cofilin inhibit apoptosis in β-thalassemic erythroid precursors. Furthermore, in light of a prior report of an anti-enucleation effect of plek2 in vitroiii and the known regulation of enucleation by RacGTPasesiv, we hypothesize that plek2 activation triggers RacGTPase and prevents enucleation in th1/th1 mice. We demonstrate that in addition to changes in erythroblast RacGTPase concentration, membrane co-localization between plek2 and RacGTPase is enhanced and occurs earlier in th1/th1 erythroid differentiation relative to WT, normalized after transferrin treatment. Lastly, cleavage of Rho-associated kinase, Rock1, associated with enucleation,v is also decreased in th1/th1 erythroblasts, enhanced after transferrin treatment. Taken together, we speculate that plek2 haplo-insufficiency benefits β-thalassemic mice by enabling apoptosis of ineffective erythroblasts, or result in worsening,possibly lethal, phenotype in light of the direct or indirect (through effects on RacGTPase or Rock1) role of plek2 in enucleation. Presently, we are testing this hypothesis by generating plek2+/- and plek-/- β-thalassemic mice. In conclusion, we demonstrate the important compensatory role of plek2 in β-thalassemic erythropoiesis. i Zhao Exp Hematol 2016ii Liu Blood 2013 iii Zhao haematol 2014iv Ji Nat Cell Bio 2008v Gabet Cell Death Diff 2011 DisclosuresNo relevant conflicts of interest to declare.

Role of Pleckstrin 2 in Improved Erythropoiesis of Transferrin-Treated Beta-Thalassemic Mice

Erythropoiesis is a process during which multipotent hematopoietic stem cells proliferate, differentiate and ultimately produce enucleated reticulocytes. Terminal erythroid differentiation begins at the morphologically recognizable pro-erythroblast (pro-E) stage and is completed when orthochromatic erythroblasts (ortho-E) expel their nuclei to produce reticulocytes. Progressive differentiation between these stages occurs in homologous cell division progressively doubling proportions of pro-E, basophilic (baso-E), polychromatophilic (poly-E), and ortho-E, and multiple signaling pathways are involved in the generation of enucleated erythroid cells, including multiple steps requiring actin cytoskeleton reorganization. We have previously shown that β-thalassemic mice (th1/th1) demonstrate a disordered progression from pro-E to baso-E and that exogenous transferrin therapy restores normal proportion of early stage erythroid precursors in th1/th1 mice (Liu Blood 2013). To identify genes that play novel function in different stages of terminal erythropoiesis, we performed RNA seq analysis of sorted bone marrow pro-E from WT, th1/th1, and transferrin-treated th1/th1 mice. We identify pleckstrin-2 (plek2) as a gene of interest with a 15-fold increase in plek2 mRNA expression in th1/th1 relative to WT mice, normalized in transferrin-treated th1/th1 mice. Plek2 is an actin binding protein, like pleckstrin-1, contains a central DEP domain known to bind RacGTPase, is Epo dependent, and is expressed in all stages of terminal erythropoiesis. We evaluate plek2 mRNA and protein expression in sorted bone marrow erythroid precursors from WT, th1/th1, and transferrin-treated th1/th1 mice. Our data demonstrates a statistically significant increase in plek2 mRNA in th1/th1 relative to WT mice, with the highest expression of plek2 in poly-E, normalized in transferrin-treated th1/th1 mice (Figure 1A). A similar pattern of increased protein concentration in th1/th1 relative to WT mice and normalization in transferrin-treated th1/th1 mice is evident in sorted bone marrow samples (Figure 1B). Prior in vitro studies demonstrate that membrane localization of plek2 is required for erythroid differentiation. Thus, we performed sub-cellular fractionation in bone marrow erythroid precursors and determined for the first time that in sorted erythroblasts from WT bone marrow, plek2 is found exclusively in the cytoplasm in pro-E and in both cytoplasm and membrane from baso-E to ortho-E (Figure 2), co-localized with actin filaments in the membrane (data not shown). In contrast, sorted erythroblasts from th1/th1 bone marrow reveal membrane-associated plek2 starting from pro-E, demonstrating earlier co-localization with actin filaments (data not shown) and suggesting an earlier activation of plek2 and consequent actin cytoskeleton reorganization during erythroid differentiation in th1/th1 mice, normalized in transferrin-treated th1/th1 mice (Figure 2). Erythropoiesis involves a complicated and incompletely understood set of potentially related molecular signals influencing cell survival, differentiation, enucleation, and release into the circulation. For example, although Epo increases survival, Epo signaling also activates RacGTPases, inhibiting enucleation. Recent in vitro data demonstrates that knockdown of plek2 affected enucleation with significantly lower reticulocyte count. Although the involvement of RacGTPase in plek2-mediated erythroid differentiation has not been explored, we hypothesize that plek2 activation triggers RacGTPase and prevents enucleation in th1/th1 mice. Our data demonstrates that RacGTPase concentration is increased in sorted bone marrow erythroid precursors from th1/th1 relative to WT mice and normalized in transferrin-treated th1/th1 mice (Figure 1B). These results suggest that plek2 plays an important role in erythropoiesis likely as a key factor in the improved enucleation of transferrin-treated th1/th1 mice. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Evidence that the expression of transferrin receptor 1 on erythroid marrow cells mediates hepcidin suppression in the liver

Hepcidin is the key regulator of iron absorption and recycling, and its expression is suppressed by red blood cell production. When erythropoiesis is expanded, hepcidin expression decreases. To gain insight into the stage of erythroid differentiation at which the regulation might originate, we measured serum hepcidin levels in archived pure red cell aplasia samples from patients whose block in erythroid differentiation was well defined by hematopoietic colony assays and marrow morphologic review. Hepcidin values are high or high normal in pure red cell aplasia patients in whom erythropoiesis is inhibited prior to the proerythroblast stage, but are suppressed in patients with excess proerythroblasts and few later erythroid cells. These data suggest that the suppressive effect of erythropoietic activity on hepcidin expression can arise from proerythroblasts, the stage at which transferrin receptor 1 expression peaks, prompting the hypothesis that transferrin receptor 1 expression on erythroid precursors is a proximal mediator of the erythroid regulator of hepcidin expression. Our characterization of erythropoiesis, iron status, and hepcidin expression in mice with global or hematopoietic cell-specific haploinsufficiency of transferrin receptor 1 provides initial supporting data for this model. The regulation appears independent of erythroferrone and growth differentiation factor 15, supporting the concept that several mechanisms signal iron need in response to an expanded erythron.