Bone marrow sinusoidal endothelium controls terminal erythroid differentiation and reticulocyte maturation

Within the bone marrow microenvironment, endothelial cells (EC) exert important functions. Arterial EC support hematopoiesis while H-type capillaries induce bone formation. Here, we show that BM sinusoidal EC (BM-SEC) actively control erythropoiesis. Mice with stabilized β-catenin in BM-SEC (Ctnnb1OE-SEC) generated by using a BM-SEC-restricted Cre mouse line (Stab2-iCreF3) develop fatal anemia. While activation of Wnt-signaling in BM-SEC causes an increase in erythroblast subsets (PII–PIV), mature erythroid cells (PV) are reduced indicating impairment of terminal erythroid differentiation/reticulocyte maturation. Transplantation of Ctnnb1OE-SEC hematopoietic stem cells into wildtype recipients confirms lethal anemia to be caused by cell-extrinsic, endothelial-mediated effects. Ctnnb1OE-SEC BM-SEC reveal aberrant sinusoidal differentiation with altered EC gene expression and perisinusoidal ECM deposition and angiocrine dysregulation with de novo endothelial expression of FGF23 and DKK2, elevated in anemia and involved in vascular stabilization, respectively. Our study demonstrates that BM-SEC play an important role in the bone marrow microenvironment in health and disease.

Plasmodium vivax infection compromises reticulocyte stability

The structural integrity of the host red blood cell (RBC) is crucial for propagation of Plasmodium spp. during the disease-causing blood stage of malaria infection. To assess the stability of Plasmodium vivax-infected reticulocytes, we developed a flow cytometry-based assay to measure osmotic stability within characteristically heterogeneous reticulocyte and P. vivax-infected samples. We find that erythroid osmotic stability decreases during erythropoiesis and reticulocyte maturation. Of enucleated RBCs, young reticulocytes which are preferentially infected by P. vivax, are the most osmotically stable. P. vivax infection however decreases reticulocyte stability to levels close to those of RBC disorders that cause hemolytic anemia, and to a significantly greater degree than P. falciparum destabilizes normocytes. Finally, we find that P. vivax new permeability pathways contribute to the decreased osmotic stability of infected-reticulocytes. These results reveal a vulnerability of P. vivax-infected reticulocytes that could be manipulated to allow in vitro culture and develop novel therapeutics.

A Proposed Concept for Defective Mitophagy Leading to Late Stage Ineffective Erythropoiesis in Pyruvate Kinase Deficiency

Pyruvate kinase deficiency (PKD) is a rare congenital hemolytic anemia caused by mutations in the PKLR gene. Here, we review pathophysiological aspects of PKD, focusing on the interplay between pyruvate kinase (PK)-activity and reticulocyte maturation in the light of ferroptosis, an iron-dependent process of regulated cell death, and in particular its key player glutathione peroxidase 4 (GPX4). GPX4 plays an important role in mitophagy, the key step of peripheral reticulocyte maturation and GPX4 deficiency in reticulocytes results in a failure to fully mature. Mitophagy depends on lipid oxidation, which is under physiological conditions controlled by GPX4. Lack of GPX4 leads to uncontrolled auto-oxidation, which will disrupt autophagosome maturation and thereby perturb mitophagy. Based on our review, we propose a model for disturbed red cell maturation in PKD. A relative GPX4 deficiency occurs due to glutathione (GSH) depletion, as cytosolic L-glutamine is preferentially used in the form of α-ketoglutarate as fuel for the tricarboxylic acid (TCA) cycle at the expense of GSH production. The relative GPX4 deficiency will perturb mitophagy and, subsequently, results in failure of reticulocyte maturation, which can be defined as late stage ineffective erythropoiesis. Our hypothesis provides a starting point for future research into new therapeutic possibilities, which have the ability to correct the oxidative imbalance due to lack of GPX4.

Rap-536 Induces Reticulocyte Maturation in Wild-Type Mice, Increases Survival of Beta-Thalassemic Reticulocytes, and Increases Red Blood Cells in a Mouse Model of Alpha-Thalassemia

Luspatercept is a recombinant fusion protein that binds and sequesters several endogenous transforming growth factor-beta superfamily ligands, including growth differentiation factor 11, thereby diminishing Smad2/3 signaling in target cells involved in erythropoiesis. Luspatercept, and its murine analog RAP-536, have been shown to act as erythroid maturation agents via their effects on late-stage erythropoiesis by inducing erythroblast maturation, leading to increases in red blood cells (RBCs) and hemoglobin (Hb). This study demonstrated that thalassemic (th3/+) reticulocytes are unstable, and that RAP-536, in addition to its function as an erythroid maturation agent, modulates the maturation of wild-type (WT) and th3/+ reticulocytes. Furthermore, RAP-536 treatment increased RBCs and decreased bilirubin in a mouse model of alpha-thalassemia (129S-Hba-a1tm1Led/J). To examine whether acute RAP-536 treatment acts on reticulocytes and alters reticulocyte levels in blood, the blood of WT mice was analyzed 3, 12, and 24 hours, and 2, 3, 4, and 7 days after a single dose of RAP-536 (10 or 30 mg/kg) or vehicle. RAP-536 treatment increased RBCs, Hb, and hematocrit significantly at all time points, compared with vehicle. However, in mice treated with RAP-536, reticulocytes in blood decreased significantly on Days 2, 3, and 4 and returned to normal baseline levels on Day 7. Analysis of reticulocyte subpopulations in blood 3 days after RAP-536 treatment showed that the relative percentages of immature reticulocytes (CD71+ or high RNA content) within the blood reticulocyte population decreased, suggesting that reticulocytes released from the bone marrow (BM) were more mature and/or reticulocytes matured faster in blood. A quantitative pharmacology (QP) model was developed to explore which RAP-536-induced modulations of erythropoiesis in WT mice can simulate the experimental observations. The model represents erythroblast, reticulocyte, and RBC (erythrocyte) maturation stages in BM, peripheral blood, and spleen, in the presence or absence of a RAP-536 effect. The QP model consists of a system of ordinary differential equations, with homeostatic parameter values assigned from literature or experimental measures, and RAP-536-perturbed parameter values regressed by fitting the model to erythropoiesis data of RAP-536-treated WT mice. Comparison of model parameters for homeostatic versus RAP-536-perturbed states indicated that RAP-536 leads to an increase in the erythroblast-to-reticulocyte and reticulocyte-to-RBC conversion rates, the transfer of BM reticulocytes to blood, and a delayed increase in erythroblast production. To directly test whether RAP-536 treatment affects reticulocyte development in blood, comparative blood transfusion experiments were performed. Biotinylated GFP+ blood from WT mice (C57BL/6-Tg(UBC-GFP)30Scha/J) and biotinylated GFP− blood from th3/+ beta-thalassemic mice (B6.129P2-Hbb-b1tm1Unc Hbb-b2tm1Unc/J) were co-transfused into GFP− WT recipient mice (C57BL/6J), which were subsequently treated with RAP-536 or vehicle. In the donor reticulocyte population, th3/+ reticulocyte percentage decreased continuously up to 3 days after transfusion, suggesting that many of the th3/+ reticulocytes were eliminated before they could form RBCs. However, compared with vehicle, RAP-536 treatment led to increased persistence of the relative percentages of th3/+ reticulocytes (Figure A). Consequently, 7 days after transfusion, when most reticulocytes have matured to RBCs, the percentage of th3/+ RBC among donor RBCs was higher with RAP-536 (Figure B). Finally, treatment of an alpha-thalassemia mouse model (129S-Hba-a1tm1Led/J) with RAP-536 10 mg/kg for 8 weeks increased RBCs and hematocrit and reduced serum bilirubin, compared with vehicle. These results suggest that RAP-536 is, as previously shown, an erythroid maturation agent, which also modulates reticulocyte maturation in blood. In WT mice, RAP-536 modulated blood reticulocyte dynamics consistent with faster maturation. RAP-536 also prolonged the persistence of th3/+ reticulocytes and maintained a higher frequency of th3/+ RBCs. These data, together with the finding that RAP-536 reduces hemolysis in an experimental alpha-thalassemia disease model, suggest that luspatercept has the potential to improve anemias associated with hemolysis and/or reticulocytosis. Disclosures Acar: Bristol Myers Squibb: Ended employment in the past 24 months. Jupelli:Bristol Myers Squibb: Current Employment. Abbiati:Bristol Myers Squibb: Current Employment. Ramanathan:Acceleron Pharma: Current Employment, Current equity holder in publicly-traded company. Santini:Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Ratushny:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Dunshee:Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Genentech Inc.: Current Employment, Current equity holder in publicly-traded company. Lopes de Menezes:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. MacBeth:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Suragani:Acceleron Pharma: Current Employment, Current equity holder in publicly-traded company. Loos:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Schwickart:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

VPS4A mutations Cause a Syndrome with Dyserythropoiesis, Hemolytic Anemia, and Neurodevelopmental Delay

The Congenital Dyserythropoietic Anemia Registry (CDAR, ClinicalTrials.gov Identifier: NCT02964494) was created to investigate the natural history, biology, and molecular pathogenetic mechanisms of CDA. To date, there are 6 genes known to cause CDA (CDAN1, C15orf41, SEC23B, KIF23, KLF1, GATA1). However, 57% of patients registered in CDAR so far (17 out of 33 patients) have an unidentified genetic cause. We have utilized whole exome sequencing (WES) in family-trio design to search for novel candidate gene mutations that may be responsible for the disease. Three unrelated patients with dyserythropoiesis, hemolytic anemia, and neurodevelopmental delay were found to have missense mutations in the gene VPS4A which encodes an ATPase that participates with the ESCRT III machinery in endosomal vesicle trafficking, centrosome localization, and the abscission step of cytokinesis. It has been shown to play an essential role in division of HeLa cells in vitro where it concentrates at the spindle poles during mitosis and at the midbody during cytokinesis. The aim of this work is to validate the pathogenetic role of these VPS4A variants in CDA and further investigate the role of VPS4A in erythropoiesis. Patients 1 and 3 had de novo mutations (R284W and G203A) and transfusion-dependent anemia with presence of binucleated erythroblasts in the bone marrow resembling CDA type I. Of note, the patients' erythroblasts exhibited cytoplasmic bridges (Figure 1A) rather than the nuclear chromatin bridges observed in CDA-I. Patient 2, offspring of consanguineous parents, presented with hemolytic anemia and was found to have a homozygous mutation (A28V) in a highly conserved alanine residue in the microtubule-interacting domain (MIT) of VPS4A. She had rare evidence of dyserythropoiesis with fewer than 3% binucleated erythroblasts in bone marrow studies. All three patients had significant neurodevelopmental delay with axial hypotonia and appendicular hypertonia. Flow cytometry analysis of peripheral blood from each of these patients revealed a unique cell population which is negative for RNA (by thiazole orange) but still CD71 positive suggesting that loss of VPS4A function also impacts reticulocyte maturation, likely because of defective endosomal vesicle trafficking. Using CD34+ cells in ex vivo erythropoiesis cultures, we first confirmed that VPS4A is expressed in human erythroblasts and localizes at the spindle poles and midbody during mitosis and cytokinesis in these cells. RNA isolated from reticulocytes from patients 1 and 2 was assessed for expression of VPS4A and the paralogous VPS4B. Samples from patient 1 had reduced expression of VPS4A (<50%) and only slight increase (2-3%) in VPS4B while samples from patient 2 had normal expression of VPS4A, but 10-20x increase in VPS4B highlighting the variable effects of these mutations on protein expression, function, and disease phenotype. Using iPSCs derived from patient 1 peripheral blood mononuclear cells (PBMCs), we have modeled the disease with in vitro erythropoiesis cultures. We observed decreased VPS4A expression and mislocalization in dividing erythroblasts produced from the patient-derived iPSCs. Moreover, the erythroblasts cultured from patient 1 demonstrated an increased rate of being binucleated and frequently maintained cytoplasmic bridges as seen in the patient's bone marrow (Figure 1B). In summary, VPS4A plays a critical role in human erythroblast mitosis, cytokinesis, and reticulocyte maturation and mutations in VPS4A cause congenital dyserythropoietic and hemolytic anemia. The CDA registry is critical to allow systematic gene and pathway evaluation, enable novel treatment considerations, and reveal molecular mechanisms essential for erythropoiesis. Disclosures Kalfa: Agios: Other: local PI of clinical research trial; FORMA: Other: sponsored research agreement.

Formin Links Erythroid Cytoskeleton to Organelle Clearance through Escrt-III Complex during Reticulocyte Maturation

The final stages of mammalian terminal erythropoiesis involve cell cycle exit of orthochromatic erythroblast, enucleation of the condensed nucleus, and organelle clearance of the nascent reticulocytes. Although many critical factors in these processes have been discovered over the past years, several key questions remain unanswered. For example, what are the factors that regulate the exit of the last mitosis of erythroblast for enucleation? How does the nascent reticulocyte separate from the extruded nucleus? What are the signals involved in regulating the clearance of organelles in reticulocyte? Answers to these questions with mechanistic insights are important not only for our understanding of the basic biology of terminal erythropoiesis and pathophysiology of many red cell-related diseases, but also to provide clues for efficient strategies for in vitro or ex vivo generation of red blood cells in transfusion medicine. Our work on formin family proteins, enzymes involved in linear actin filament polymerization, in erythropoiesis may shed light on the clues to these questions. We show in our published work that mDia2, one of the diaphanous-related formins, plays critical roles in enucleation and cytokinesis of erythroblasts. However, the mechanism of how mDia2 regulates these processes is unclear. In this study, we used mDia2 hematopoietic-specific knockout mouse model and revealed that mDia2 controls the motility of the nascent reticulocyte that is required for the detachment of the pyknotic nucleus. Reticulocytes in mDia2 deficient mice are rigid with extended spectrin chains, possibly due to disrupted actin protofilaments. Indeed, a stochastic optical reconstruction (STORM) high resolution microscopy analysis revealed that actin protofilaments were completely disrupted with loss of mDia2 in the reticulocytes. Using immuno-gold stain and electron microscopy, we further found that mDia2 localized at the junctional complex, confirming its critical role in the polymerization of actin protofilaments and maintenance of erythroid cytoskeleton. In addition to the cytoskeleton defects, reticulocytes from mDia2 deficient mice also showed enlarged volume with many organelles failed to be eliminated. Flow cytometry analyses showed that several membrane proteins destined to be downregulated, as well as mitochondria and lysosome markers, remained high in mDia2 deficient reticulocytes. We also performed a tandem mass tagging (TMT) mass spectrometry, which revealed numerous chromatin-associated proteins that failed to be downregulated. Together, these results demonstrated an important role of mDia2 in the reticulocyte maturation. The erythroid phenotypes in mDia2 deficient mice, including failure of cytokinesis and organelle clearance, prompted us to investigate whether there are any defects in ESCRT complexes that are cellular components essential for these processes. Indeed, several ESCRT III complex and associated proteins, including Chmp5, Vta1, and Usp8, were significantly downregulated. mDia2 is known to function though actin polymerization to influence the transcriptional activity of SRF. We found that Chmp5 was a novel target of SRF through ChIP assay. Transplantation of mDia2 knockout c-kit positive progenitors transduced with Chmp5 into the lethally irradiated WT recipient mice dramatically reduced the percentages of bi-nuclear erythroblasts and reverted anemia. Consistent with phenotypes of Chmp5 knockout cells where increased late endosome and lysosome are commonly found, markers for late stage endosome and lysosome were significantly increased in mDia2 deficient erythroblasts and reticulocytes. These data support that mDia2 regulates endosome/MVB and lysosome discharge through Chmp5 during reticulocyte maturation. More importantly, overexpression of Chmp5 largely rescued the defects in lysosome and mitochondria clearance in mDia2 deficient reticulocytes. Our study reveals mDia2 formin as a master regulator of the late stage terminal erythropoiesis in the maintenance of erythroid cytoskeleton and organelle clearance. The novel mDia2-SRF-ESCRT III complex pathway provides the first signaling axis that connects erythroid cytoskeleton to reticulocyte maturation, which may open a new field in signaling networks that modulate enucleation to reticulocyte formation. Disclosures Ji: Longbiopharma: Consultancy.