Functional Requirements for a Samd14-Capping Protein Complex in Stress Erythropoiesis

Acute anemia induces rapid expansion of erythroid precursors and accelerated differentiation to replenish erythrocytes. Paracrine signals – involving cooperation between SCF/c-Kit signaling and other signaling inputs – are required for the increased erythroid precursor activity in anemia. Our prior work revealed that the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene increases the regenerative capacity of the erythroid system and promotes stress-dependent c-Kit signaling. However, the mechanism underlying Samd14’s role in stress erythropoiesis is unknown. We identified a protein-protein interaction between Samd14 and the α- and β heterodimers of the F-actin capping protein (CP) complex. Knockdown of the CP β subunit increased erythroid maturation in ex vivo cultures and decreased colony forming potential of stress erythroid precursors. In a genetic complementation assay for Samd14 activity, our results revealed that the Samd14-CP interaction is a determinant of erythroid precursor cell levels and function. Samd14-CP promotes SCF/c-kit signaling in CD71med spleen erythroid precursors. Given the roles of c-Kit signaling in hematopoiesis and Samd14 in c-Kit pathway activation, this mechanism may have pathological implications in acute/chronic anemia.

Mild hypobaric hypoxia influences splenic proliferation during the later phase of stress erythropoiesis

Tissue trauma and hemorrhagic shock are common battlefield injuries that can induce hypoxia, inflammation, and/or anemia. Inflammation and hypoxia can initiate adaptive mechanisms, such as stress erythropoiesis in the spleen, to produce red blood cells and restore the oxygen supply. In a military context, mild hypobaric hypoxia—part of the environmental milieu during aeromedical evacuation or en route care—may influence adaptive mechanisms, such as stress erythropoiesis, and host defense. In the present study, healthy (control), muscle trauma, and polytrauma (muscle trauma and hemorrhagic shock) mice were exposed to normobaric normoxia or hypobaric hypoxia for ∼17.5 h to test the hypothesis that hypobaric hypoxia exposure influences splenic erythropoiesis and splenic inflammation after polytrauma. This hypothesis was partially supported. The polytrauma + hypobaric hypoxia group exhibited more splenic neutrophils, fewer total spleen cells, and fewer splenic proliferating cells than the polytrauma+normobaric normoxia group; however, no splenic erythroid cell differences were detected between the two polytrauma groups. We also compared splenic erythropoiesis and myeloid cell numbers among control, muscle trauma, and polytrauma groups. More reticulocytes at 1.7 days (40 h) post-trauma (dpt) and neutrophils at 4 dpt were produced in the muscle trauma mice than corresponding control mice. In contrast to muscle trauma, polytrauma led to a reduced red blood cell count and elevated serum erythropoietin levels at 1.7 dpt. There were more erythroid subsets and apoptotic reticulocytes in the polytrauma mice than muscle trauma mice at 4 and 8 dpt. At 14 dpt, the red blood cell count of the polytrauma + normobaric normoxia mice was 12% lower than that of the control + normobaric normoxia mice; however, no difference was observed between polytrauma + hypobaric hypoxia and control + hypobaric hypoxia mice. Our findings suggest muscle trauma alone induces stress erythropoiesis; in a polytrauma model, hypobaric hypoxia exposure may result in the dysregulation of splenic cells, requiring a treatment plan to ensure adequate immune functioning.

HIF-Mediated and Non-HIF-Mediated Differential Gene Expressions in Sickle Cell Reticulocyte and Their Impact on Clinical Manifestations

Reticulocytosis in sickle cell disease (SCD) is driven by tissue hypoxia from hemolytic anemia and vascular occlusion. Gene expression changes caused by hypoxia and other factors during reticulocytosis may impact SCD outcomes. We detected 1226 differentially expressed genes in SCD reticulocyte transcriptome compared to normal Black controls. To assess the role of hypoxia-mediating HIFs from other regulation of changes of the SCD reticulocyte transcriptome, we compared differential expression in SCD to that in Chuvash erythrocytosis (CE), a disorder characterized by constitutive upregulation of HIFs in normoxia. Of the SCD differentially expressed genes, 28% were shared between CE and SCD and thus classified as HIF-mediated. The HIF-mediated changes were generally in genes promoting erythroid maturation. We found that genes encoding the response to endoplasmic reticulum stress generally lacked HIF mediation. We then investigated the clinical correlation of erythroid gene expression for the 1226 differentially expressed genes detected in SCD reticulocytes, using clinical measures and gene expression data previously profiled in peripheral blood mononuclear cells (PBMCs) of 157 SCD patients at the University of Illinois at Chicago (UIC). Normal PBMCs contain only a small number of erythroid progenitors, but in SCD or CE PBMCs the erythroid transcriptome is enriched due to elevated circulating erythroid progenitors from heightened erythropoiesis (PMID: 32399971). We applied deconvolution analysis to assess the clinical correlation of erythroid gene expression, using a 16-gene expression signature of erythroid progenitors previously identified in SCD PBMCs. Deconvolution analysis uses the proportion of cell/tissue or specific marker genes (here the erythroid specific 16-gene signature) to dissect gene expression variation in biological samples with cell/tissue type heterogeneity. We correlated, in the 157 UIC patients, erythroid gene expression with i) degree of anemia as indicated by hemoglobin concentration, ii) vaso-occlusive severe pain episodes per year, and iii) degree of hemolysis measured by a hemolysis index. The analysis identified 231 genes associated with at least one of the complications. Increased expression of 40 erythroid specific genes, including 15 HIF-mediated genes, was associated with all three complications. These 40 genes are all upregulated in SCD reticulocytes and correlated with low hemoglobin concentration, frequent severe pain episodes, and high hemolysis index, suggesting that these manifestations may share a relationship to stress erythropoiesis-driven transcriptional activity. Expression quantitative trait loci (eQTL) contain genetic polymorphisms that associate with gene expression level, which can be viewed as a natural experiment to investigate the causal relations between gene expression change and phenotypic outcomes. To assess the causal effect of erythroid gene expression, we tested association between erythroid eQTL and the clinical manifestations in 906 SCD patients from the Walk-PHaSST and PUSH cohorts. We first mapped erythroid eQTL in the 157 UIC patients, who were previously genotyped by array, applying deconvolution algorithm on the same PBMC data for the 1226 differential genes in SCD reticulocytes, and detected 54 distinct eQTL for 30 genes at 5% false discovery rate. After adjusting for multiple comparisons, we found that the C allele of rs16911905, located in the β-globin cluster and associated with increased erythroid expression of HBD (encodes δ-globin of hemoglobin A 2), significantly correlated with lower hemoglobin concentration (β=-0.064, 95% CI -0.092 - -0.036, P=6.7×10 -6). The C allele was also associated with higher hemolytic rate (P=0.031), less frequent pain episodes (P=0.045), and increased erythroid expression of HBB here encoding sickle β-globin (P=5.1x10 -5). The association of the C allele with lower hemoglobin concentration was then validated in 242 patients from the UIC cohort (β=-0.071, 95% CI -0.13 - -0.011, P=0.023), as was the trend of association with higher hemolytic rate (P=0.0031) and less pain episodes (P=0.034). Our findings reveal HIF- and non-HIF-mediated genes in SCD stress erythropoiesis, and identify novel clinical associations for a HBD eQTL. Our study highlights the correlation of altered erythroid gene expression with SCD hemolytic and vaso-occlusive manifestations. Disclosures Saraf: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding. Gordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy.

Functional Requirements of a Samd14-Capping Protein Interaction in Stress Erythropoiesis

Stress erythropoiesis describes the process of accelerating red blood cell (RBC) production in anemia. Among a number of important mediators of stress erythropoiesis, paracrine signals - involving cooperation between SCF/c-Kit signaling and other signaling inputs - are required for the activation/function of stress erythroid progenitors. Whereas many critical factors required to drive erythropoiesis in normal physiological conditions have been described, whether distinct mechanisms control developmental, steady-state, and stress erythropoiesis in anemia is poorly understood. Our prior work revealed that the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene is transcriptionally upregulated in a model of acute hemolytic anemia induced by the RBC-lysing chemical phenylhydrazine. Samd14 is regulated by GATA binding transcription factors via an intronic enhancer (Samd14-Enh). In a mouse knockout of Samd14-Enh (Samd14-Enh -/-), we established that the Samd14-Enh is dispensable for steady-state erythropoiesis but is required for recovery from severe hemolytic anemia. Samd14 promotes c-Kit signaling in vivo and ex vivo, and the SAM domain of Samd14 facilitates c-Kit-mediated cellular signaling and stress progenitor activity. In addition, the Samd14 SAM domain is functionally distinct from closely related SAM domains, which demonstrates a unique role for this SAM domain in stress signaling and cell survival. In our working model, Samd14-Enh is part of an ensemble of anemia-responsive enhancers which promote stress erythroid progenitor activity. However, the mechanism underlying Samd14's role in stress erythropoiesis is unknown. To identify potential Samd14-interacting proteins that mediate its function, we performed immunoprecipitation-mass spectrometry on the Samd14 protein. We found that Samd14 interacted with α- and β heterodimers of the F-actin capping protein (CP) complex independent of the SAM domain. CP binds to actin during filament assembly/disassembly and plays a role in cell morphology, migration, and signaling. Deleting a 17 amino acid sequence near the N-terminus of Samd14 disrupted the Samd14-CP interaction. However, mutating the canonical RxR of the CP interaction (CPI) motif, which is required for CP-binding in other proteins, does not abrogate the Samd14-CP interaction. Moreover, replacing this sequence with the canonical CPI domain of CKIP-1 completely disrupts the interaction, indicating that other sequence features are required to maintain the Samd14-CP complex. Ex vivo knockdown of the β-subunit of CP (CPβ), which disrupts the integrity of the CP complex, decreased the percentage of early erythroid precursors (p<0.0001) and decreased (3-fold) progenitor activity as measured by colony formation assays (similar to knockdown of Samd14). Taken together, these data indicate that Samd14 interacts with CP via a unique CP binding (CPB) domain, and that the CP complex coordinates erythroid differentiation in stress erythroid progenitors. To test the function of the Samd14-CP complex, we designed an ex vivo genetic complementation assay to express Samd14 lacking the CPB-domain (Samd14∆CPB) in stress erythroid progenitors isolated from anemic Samd14-Enh -/- mice. Phospho-AKT (Ser473) and phospho-ERK (Thr202/Tyr204) levels in Samd14∆CPB were, respectively, 2.2 fold (p=0.007) and ~7 fold (n=3) lower than wild type Samd14 expressing cells, 5 min post SCF stimulation. Relative to Samd14, Samd14∆CPB expression reduced burst forming unit-erythroid (BFU-E) (2.0 fold) and colony forming unit-erythroid (CFU-E) (1.5 fold). These results revealed that the Samd14-CP interaction is a determinant of BFU-E and CFU-E progenitor cell levels and function. Remarkably, as the requirement of the CPB domain in BFU-E and CFU-E progenitors is distinct from the Samd14-SAM domain (which promotes BFU-E but not CFU-E), the function of Samd14 in these two cell types may differ. Ongoing studies will examine whether the function of Samd14 extends beyond SCF/c-Kit signaling and establish cell type-dependent functions of Samd14 and Samd14-interacting proteins. Given the critical importance of c-Kit signaling in hematopoiesis, the role of Samd14 in mediating pathway activation, and our discovery implicating the capping protein complex in erythropoiesis, it is worth considering the pathological implications of this mechanism in acute/chronic anemia and leukemia. Disclosures No relevant conflicts of interest to declare.

The Glucocorticoid Receptor-Dependent Stress Response in Human Erythropoiesis Is BCL11A-Dependent

Extrapolations from data in mouse models and from human erythroid cells expanded ex vivo with the glucocorticoid receptor (GR) agonist Dexamethasone (Dex), suggest that GR plays an important role in the regulation of stress erythropoiesis 1. However, the mechanistic details of stress erythropoiesis are still poorly understood. By exploring the effects of Dex on erythroid expansion of CD34+ cells from a large number of healthy adult donors (n=25), we documented that Dex expands a population of immature erythroid cells which express high levels of BCL11A. In addition, in these cells BCL11A is mostly in the nuclei, compared to cells grown without Dex. These results suggest that GR regulates the transcriptional activity of BCL11A. To validate the role of BCL11A in the Dex response, we made observations in cells from patients with BCL11A deficiencies 2. We found that BCL11A-deficient CD34+ cells generate lower numbers of maturing erythroid cells compared to controls with Dex addition, suggesting that they respond poorly to Dex (Fig 1). Of note, RNAseq analyses indicate that erythroid cells expanded from the patients express levels of BCL11A lower than that of their parent cells while the levels of expression of other genes known to mediate the response to Dex of erythroid cells (ZFP36L2, CDKNIC and PPARA 3-5) expressed by these cells is normal. Further we extended our observations to Cushing's patients with highier cortisol levels, before(V1) and after their treatment(V2) using concurrent age and weight matched healthy controls (MC). There is no significant difference in frequency of CD34+ cells among V1, V2 and MC (range 0.3-2% in all cases). CD34 pos cells from all groups express similar levels of cKIT, IL-3Rβ, CXCR4 and CALR. However, a greater proportion of CD34+ cells from V1 express CD36 and CD110 (the thrombospondin and thrombopoietin receptor) and CD133 (the hematopoietic stem cell marker prominin) than those from V2 and MC (60-80% vs 10-20%), suggesting that the circulating progenitor cells from active Cushing's patients are a unique population likely generated in response to their high cortisol levels given that the phenotype of CD34+ cells from the blood of V2 patients is similar to that found in MC. The phenotype of CD34+ cells from Cushing's patients in vivo is similar to that of CD34+ cells generated in vitro after 2-4 days of culture with Dex 3,6,7. V1 progenitor cells generate similarly large number of immature erythroid cells in culture with and without Dex. By contrast with normal cells, these active cells also express lower levels of the cytoplasm-restricted form of GRα (GRS203) and greater levels of GILZ and BCL11A than normal or V2 cells. In conclusion, the data presented here indicate that GR activation switches the erythroid differentiation program from the steady-state to the stress mode and that activation of BCL11A is part of the response of erythroid cells to Dex. References 1) Varricchio et al Am J Blood Res. 2014;4:53; 2) Funnell et al Blood 2015; 126:89; 3) Ashley et al JCI 2020;130:2097; 4) Zhang L et al Nature 2013;499:92; 5) Lee et al Nature 2015;522:474; 6) Heideveld et al Haematologica 2015; 100:1396; 7) Xiang et al Blood 2015;125:1803. Figure 1 Figure 1. Disclosures Migliaccio: Dompe farmaceutici Spa R&D: Other: received funding for reserach .

Monocytes from Patients with Polycythemia Vera Express Molecules Related to Stress Erythropoiesis and Have Increased Erythrocyte Phagocytosis

Stress erythropoiesis (SE) is characterized by an increase in erythropoietic activity in the bone marrow and in extramedullary sites. The central macrophage present in the erythroblastic island (EBI) plays a key role in regulating SE through the expression of molecules mediating cell adhesion, iron metabolism, and those capable of identifying and engulfing damaged and senescent erythrocytes. Those receptors are also expressed in monocytes (MC), suggesting that their monocytic expression could be involved in erythropoiesis and erythrophagocytosis. CD14 +CD16 + intermediate MC (I-MC) express markers that are typically found in EBI macrophages and, together with CD14 +CD16 - classic MC (C-MC), are able to remove circulating iron. Polycythemia vera (PV) is characterized by autonomous overproduction of red blood cells (RBCs) with extramedullary hematopoiesis most often caused by an acquired JAK2 V617F mutation, resulting in a state of chronic SE. The depletion of macrophages from EBIs in animal model of PV reverses splenomegaly and erythrocytosis indicating that they are essential for the development of chronic SE. It is unknown if MC play the same role in humans or if they have different expressions of those key molecules, which could contribute to the severity of the disease. We aimed to investigate the role of MCs in SE present in PV by characterizing the expression of molecules relevant to RBC adhesion, anti-inflammation, erythrophagocytosis, and iron metabolism in MCs from PV patients and from healthy controls (HC). Peripheral blood MCs were isolated from HC (n=21) and PV patients (n=17) and phenotyped by flow cytometry (FC) for sialoadhesin (CD169), VCAM1 (CD106), the receptor for the hemoglobin-haptoglobin complex (CD163), mannose receptor (CD206), SIRPα (CD172), ferroportin (Fpn), and separated into subtypes according to expression of CD14 and CD16. We also evaluated MC erythrophagocytosis by determining positivity for intracellular glycophorin (CD235a) (nHC=13 and nPV=17). In PV, we observed significantly higher expression of CD169 and CD106, and lower expression of CD172 in C-MC (1,167±216.6 vs 1,834±241.9, p=0.009; 1,427±217.1 vs 2,849±182.3, p=0.0004; 104,707±9,546 vs 77,070±8,756, p=0.0428, respectively); higher CD169 and CD106 in I-MC (2,221±322.4 vs 3,150±321.8, p=0.0371; 2,186±201.7 vs 2,721±153.5, p=0.0238, respectively); and higher CD206, CD163, CD172, and CD106 in CD14 lowCD16 + non-classical MCs (NC-MC) (181.2±8.5 vs 268.6±13.4, p<0.0001; 312.3±15.1 vs 368.8±12.1, p=0.0174; 13,923±2256 vs 22,792±3211, p=0.0161; 1,234±96 vs 1,498±58.9 p=0.004, respectively). Fpn expression was not significantly different. A lower expression of CD172 in the C-MC suggests less inhibitory signaling for erythrophagocytosis in those cells. Although C-MC and I-MC have been previously linked to erythropoiesis, we saw a larger number of the investigated molecules being more expressed in NC-MC, supporting their possible involvement in regulating erythropoiesis. Our results suggest that MCs in PV could be more likely to attach erythroid cells and could therefore contribute to form EBIs if differentiated to macrophages. Higher molecule expression was associated to a higher percentage of MCs containing intracellular CD235a (0.35±0.07 vs 4.48±1.08, p<0.0001). This is evidence of circulating PV MCs performing erythrophagocytosis and supports a role for them in RBC clearance. Our findings reveal an increase in the expression of markers relevant to the adhesion of erythrocytes to MCs in all MC subsets from PV patients along with more frequent erythrocyte phagocytosis in circulating cells. Further studies should yield better understanding of the role of MCs in SE and in the formation of EBIs, providing future targets for the treatment of chronic SE in PV patients. Disclosures Fertrin: Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Costa: Novartis: Consultancy.

Regulation of Fetal Hemoglobin Expression By the VHL-HIF1α Oxygen Sensing System

Defining the mechanisms that control the perinatal switch from γ-globin (HBG1 and HBG2) to β-globin (HBB) gene expression in human red blood cells (RBCs) has informed novel approaches to reactivate fetal hemoglobin (HbF, α2γ2) therapeutically for sickle cell disease and β-thalassemia. However, one longstanding unsolved problem is to explain how HbF becomes elevated in conditions such as blood loss, hypoxia and hemolysis. These conditions are associated with accelerated RBC production, also referred to as stress erythropoiesis, driven by activation of hypoxia-inducible factor (HIF) via a canonical O 2 sensing pathway. At high O 2 levels ("normoxia"), O 2-dependent prolyl hydroxylase domain (PHD) enzymes hydroxylate HIFα, thereby targeting it for ubiquitination by the von Hippel-Lindau (VHL) E3 ubiquitin ligase complex, followed by proteasomal degradation. At low O 2 tension (hypoxia), PHD activity is reduced, causing HIF1α to accumulate, dimerize with constitutively expressed HIF1β, and bind hypoxia response elements (HREs) to activate a broad array of genes that facilitate hypoxic adaptation. We identified VHL and HIF1α, as negative and positive regulators of HbF expression, respectively. Disruption of the VHL gene in CD34 + hematopoietic stem and progenitor cells (HSPCs) by transfection with ribonucleoprotein (RNP) consisting of Cas9 and VHL-targeting guide RNA increased HbF expression from 7.5% ±1.2% in control cells to 30.9% ± 4.8% (mean ± SD, P<0.0001) in RBC progeny generated by in vitro differentiation. Similarly, γ-globin mRNA was induced by 5 -fold after disruption of VHL in HUDEP-2 cells, an immortalized erythroid line that expresses mainly adult hemoglobin (HbA, α2/β2). Mass spectrometry and transcriptome analysis of VHL-disrupted CD34 + HSPCs revealed increased HIF1α protein expression with no change in the corresponding mRNA. Inhibition of HIF1A mRNA by RNA interference suppressed γ-globin induction in VHL-/- HUDEP-2 clones and in RBCs generated from VHL RNP-treated CD34 + cells, indicating VHL knockout induced HbF through HIF1α protein accumulation. CUT&RUN analysis of VHL-depleted erythroblasts revealed HIF1α/HIF1β heterodimer occupancy at BGLT3, a long-noncoding RNA locus approximately 2.7kb 3' of HBG1. The HIF1α-bound BGLT3 locus contains two canonical HREs (ACGTG) separated by 13 bp. Disruption of each HRE motif in VHL-/- HUDEP-2 cells by base editing caused additive reductions in BGLT3 HIF1α occupancy and γ-globin expression. Mechanistically, VHL depletion caused the accumulation of HIFα/β heterodimers at BGLT3, recruitment of the transcriptional activators GATA1 and P300, and targeted chromatin accessibility to establish the active enhancer mark H3K27ac. These changes were accompanied by altered chromosome conformation to favor long-range interactions between the γ-globin loci (HBG1 and HBG2) and the locus control region, a powerful upstream enhancer. Treatment of healthy donor CD34 + HSPCs-derived erythroblasts with the clinically approved PHD inhibitor FG-4592 (Roxadustat) caused HbF to increase from 4.76%±1.22% at baseline to 12.86 ± 2.40% (mean ± SD in 3 biological replicates, P<0.01). Treatment of the same erythroblasts with FG-4592 and hydroxyurea, a widely used SCD drug that acts partly by inducing HbF, caused HbF to increase from 4.76% ± 1.22% at baseline to 24.1% ± 5.3% (mean ± SD in 3 biological replicates, P<0.01), indicating an additive effect. Our findings link developmental globin gene regulation with O 2 sensing, provide a mechanism for HbF induction during stress erythropoiesis, and identify a novel therapeutic approach for β-hemoglobinopathies. Disclosures Blobel: Pfizer: Consultancy; Fulcrum Therapeutics, Inc.: Consultancy. Weiss: Beam Therapeutics: Current holder of stock options in a privately-held company; Forma Therapeutics: Consultancy; Cellarity Inc.: Consultancy; Novartis: Consultancy.

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. Disclosures Kalfa: Agios Pharmaceuticals, Inc.: Other: Steering Committee, Research Funding; FORMA Therapeutics, Inc: Research Funding. Paulson: Forma Therapeutics: Consultancy. Blanc: Keros Therapeutics, Inc.: Consultancy.

Nitric Oxide Dependent Metabolism Regulates the Proliferation and Differentiation of Stress Erythroid Progenitors

Infection and tissue damage induce inflammation, which increases myelopoiesis at the expense of steady state erythropoiesis. Stress erythropoiesis is induced to compensate for the loss of erythroid output until the inflammation is resolved and bone marrow erythropoiesis can resume. Steady state erythropoiesis constantly produces erythrocytes, while stress erythropoiesis generates a bolus of new erythrocytes through the rapid expansion of immature progenitor cells which is followed by the synchronous differentiation of progenitors. We hypothesized that the proliferation of early progenitor cells and their transition to differentiation is regulated by changes in metabolism. Metabolomics and isotope tracing analysis was performed to assess the intracellular metabolic profiles in proliferating progenitors isolated from in vitro stress erythropoiesis cultures. We observed an active engagement of glucose metabolism in glycolysis and anabolic biosynthesis, while the levels of TCA intermediates suggested that TCA cycle and mitochondrial respiration were blocked. Concomitantly, inducible nitric oxide synthase (iNOS) was induced in progenitor cells to increase the production of nitric oxide (NO), which was demonstrated to be crucial for proliferating progenitor metabolism. Inhibition or genetic mutation of iNOS decreased NO levels resulting in the suppression of progenitor proliferation in vitro and in vivo. As evaluated by RNA-seq, inhibition of iNOS suppressed cell proliferation-related pathways including cell cycle and nucleotide metabolism, while upregulating erythroid differentiation genes. These data suggest that iNOS-derived NO production establishes a metabolism that promotes the proliferation of progenitor cells while inhibiting their differentiation. Notably, proliferating progenitor cells displayed low levels of the metabolite itaconate and decreased expression of Immunoresponsive gene 1 (Irg1), the enzyme that catalyzes itaconate synthesis from cis-aconitate. Further analysis showed that the addition of 4-Octyl itaconate (OI), a cell-permeable itaconate derivative, inhibited iNOS-derived NO production by activating nuclear factor erythroid 2-related factor 2 (Nrf2), which in turn impaired progenitor expansion. These results indicate that itaconate production is inhibited to enable the accumulation of NO and the NO dependent metabolism required for progenitor cell proliferation during the initial expansion stage of stress erythropoiesis. In contrast, the transition to differentiation is marked by elevated itaconate synthesis, Nrf2 activation, and attenuated iNOS expression. We hypothesized that the inhibition of NO production alters metabolism and in concert with new cell signaling removes the NO-dependent inhibition of erythroid program, which allows the differentiation of progenitor cells. We tested this mechanism by examining the effects of iNOS inhibitors and mutants in iNOS, Irg1 and Nrf2 on progenitor cells isolated from differentiation cultures. iNOS deficiency led to the activation of erythroid transcriptional program, and increased numbers of mature progenitors as well as stress BFU-Es. In contrast, Irg1 and Nrf2 mutants showed impaired transition to erythroid differentiation, while they had elevated iNOS expression and NO production. Further analysis showed that treatment with either OI or iNOS inhibitor inhibited NO production in Irg1 and Nrf2 deficient progenitors, and consequently rescued the defects in erythroid differentiation. These data support a model in which inflammation inhibits steady state erythropoiesis, while at the same time promoting stress erythropoiesis to maintain homeostasis. Our work reveals a dynamic and tight coordination between pro-inflammatory signals and progenitor cell metabolism in regulating the proliferation and differentiation of stress erythroid progenitors, and highlights the therapeutic potential of targeting metabolic and inflammatory signaling pathways in inflammatory anemia. Disclosures Paulson: Forma Therapeutics: Consultancy.

Canonical Wnt; a safeguard and threat for Erythropoiesis

Myeloid dysplastic syndrome (MDS) reflects a preleukemic BM disorder with limited treatment options and poor disease survival1. As only a minority of MDS patients is eligible to curative hematopoietic stem cell (HSC) transplantation, there is an urgent need to develop alternative treatment options. Chronic activation of Wnt/β-catenin has been implicated to underlie MDS formation and recently assigned to drive MDS transformation to acute myeloid leukemia (AML). Wnt/β-catenin signaling therefore may harbor a pharmaceutical target to treat MDS and/or prevent leukemia formation. However, targeting the Wnt/β-catenin pathway will also affect healthy hematopoiesis in MDS patients.The control of Wnt/β-catenin on healthy hematopoiesis is poorly understood. Whereas Wnt/β-catenin is dispensable for steady-state erythropoiesis, its activity is essential for stress erythropoiesis in response to BM injury and anemia. Manipulation of Wnt/β-catenin signaling in MDS may therefore deregulate stress erythropoiesis and even increase anemia severity. Here we provide a comprehensive overview of the most recent and established insights in the field to acquire more insight into the control of Wnt/β-catenin signaling on healthy and inefficient erythropoiesis as seen in MDS.