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Sickle cell disease (SCD) is caused by a mutation in the β-globin gene HBB1. We used a custom adenine base editor (ABE8e-NRCH)2,3 to convert the SCD allele (HBBS) into Makassar β-globin (HBBG), a non-pathogenic variant4,5. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBBS to HBBG. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBBG was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBBS base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse6 and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar β-globin represented 79% of β-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBBS-to-HBBG editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes the undesired consequences of double-strand DNA breaks. A custom adenine base editor can edit the variant of the β-globin gene that causes sickle cell disease into a non-pathogenic variant in human and mouse cells, and transplantation of the edited cells rescues sickle cell disease in mice.

The erythroblastic island (EBI), composed of a central macrophage surrounded by maturing erythroblasts, is the erythroid precursor niche. Despite numerous studies, its precise composition is still unclear. Using multispectral imaging flow cytometry, in vitro island reconstitution, and single-cell RNA sequencing of adult mouse bone marrow (BM) EBI-component cells enriched by gradient sedimentation, we present evidence that the CD11b+ cells present in the EBIs are neutrophil precursors specifically associated with BM EBI macrophages, indicating that erythro-(myelo)-blastic islands are a site for terminal granulopoiesis and erythropoiesis. We further demonstrate that the balance between these dominant and terminal differentiation programs is dynamically regulated within this BM niche by pathophysiological states that favor granulopoiesis during anemia of inflammation and favor erythropoiesis after erythropoietin stimulation. Finally, by molecular profiling, we reveal the heterogeneity of EBI macrophages by cellular indexing of transcriptome and epitope sequencing of mouse BM EBIs at baseline and after erythropoietin stimulation in vivo and provide a searchable online viewer of these data characterizing the macrophage subsets serving as hematopoietic niches. Taken together, our findings demonstrate that EBIs serve a dual role as niches for terminal erythropoiesis and granulopoiesis and the central macrophages adapt to optimize production of red blood cells or neutrophils.

In this work, we utilized a parameterization model of ektacytometry to quantify the bulk rigidity of the rigid red blood cell (RBC) population in sickle cell disease (SCD) patients. Current ektacytometry techniques implement laser diffraction viscometry to estimate the RBC deformability in a whole blood sample. However, the diffraction measurement is an average of all cells present in the measured sample. By coupling an existing parameterization model of ektacytometry to an artificially rigid RBC model, we formulated an innovative system for estimating the average rigidity of the rigid RBC population in SCD blood. We demonstrated that this method could more accurately determine the bulk stiffness of the rigid RBC populations. This information could potentially help develop the ektacytometry technique as a tool for assessing disease severity in SCD patients, offering novel insights into the disease pathology and treatment.

BACKGROUND Fetal hypoxia has been implicated in fetal growth restriction in congenital heart disease (CHD) and leads to stress erythropoiesis in utero. The objective is to assess erythropoiesis and its association with growth in newborns with CHD. METHODS Fetuses with prenatally diagnosed CHD from 2013 to 2018 were retrospectively reviewed. Pregnancies with multiple gestation, genetic abnormalities, major extra-cardiac anomalies, and placental abruption were excluded. Complete blood count tests at birth were compared to published normative values. Spearman correlation assessed associations of red blood cell (RBC) indices with birth anthropometrics and prenatal Doppler measures. RESULTS A total of 160 newborns were included. Median gestational age was 38.3 (37.3, 39.0) weeks. Infants ≥37 weeks gestation had lower hemoglobin (Hgb), hematocrit, and elevated nucleated RBC (nRBC), mean corpuscular volume, and mean corpuscular hemoglobin compared to reference. No differences in RBC indices were observed in infants

Not available.

The mature red blood cell (RBC) lacks a nucleus and organelles characteristic of most cells, but it is elegantly structured to perform the essential function of delivering oxygen and removing carbon dioxide from all other cells while enduring the shear stress imposed by navigating small vessels and sinusoids. Over the past several decades, the efforts of biochemists, cell and molecular biologists, and hematologists have provided an appreciation of the complexity of RBC membrane structure, while studies of the RBC membrane disorders have offered valuable insights into structure–function relationships. Within the last decade, advances in genetic testing and its increased availability have made it possible to substantially build upon this foundational knowledge. Although disorders of the RBC membrane due to altered structural organization or altered transport function are heterogeneous, they often present with common clinical findings of hemolytic anemia. However, they may require substantially different management depending on the underlying pathophysiology. Accurate diagnosis is essential to avoid emergence of complications or inappropriate interventions. We propose an algorithm for laboratory evaluation of patients presenting with symptoms and signs of hemolytic anemia with a focus on RBC membrane disorders. Here, we review the genotypic and phenotypic variability of the RBC membrane disorders in order to raise the index of suspicion and highlight the need for correct and timely diagnosis.

CHD8 is an ATP-dependent chromatin-remodeling factor whose monoallelic mutation defines a subtype of autism spectrum disorders (ASDs). Previous work found that CHD8 is required for the maintenance of hematopoiesis by integrating ATM-P53-mediated survival of hematopoietic stem/progenitor cells (HSPCs). Here, by using Chd8F/FMx1-Cre combined with a Trp53F/F mouse model that suppresses apoptosis of Chd8-/- HSPCs, we identify CHD8 as an essential regulator of erythroid differentiation. Chd8-/-P53-/- mice exhibited severe anemia conforming to congenital dyserythropoietic anemia (CDA) phenotypes. Loss of CHD8 leads to drastically decreased numbers of orthochromatic erythroblasts and increased binucleated and multinucleated basophilic erythroblasts with a cytokinesis failure in erythroblasts. CHD8 binds directly to the gene bodies of multiple Rho GTPase signaling genes in erythroblasts, and loss of CHD8 results in their dysregulated expression, leading to decreased RhoA and increased Rac1 and Cdc42 activities. Our study shows that autism-associated CHD8 is essential for erythroblast cytokinesis.

Etavopivat (FT-4202) is an orally administered, small-molecule allosteric activator of erythrocyte pyruvate kinase-R (PKR) in clinical development for the treatment of sickle cell disease and other hemoglobin disorders. This randomized, placebo-controlled, double-blind, first-in-human combination single-ascending dose and multiple-ascending dose phase 1 trial (NCT03815695) evaluated the safety and pharmacokinetics/pharmacodynamics of etavopivat in 90 healthy adult subjects. In 4 single-ascending dose cohorts, 8 participants were randomized 3:1 to a single oral dose of either etavopivat (n = 6) or placebo (n = 2). In four 14-day multiple-ascending dose cohorts, 12 participants were randomized 3:1 to 14 days of etavopivat (n = 9) or placebo (n = 3). In these studies, most treatment-emergent adverse events were of mild severity (grade 1) and none led to study discontinuation. Etavopivat exhibited a linear and time-independent pharmacokinetic profile (at doses ≤400 mg) and elicited the expected pharmacodynamic effects of PKR activation (decreased 2,3-diphosphoglycerate and increased adenosine triphosphate) and evidence of improved hemoglobin-oxygen affinity. In addition, pharmacodynamic responses were durable with effects continuing for 48 to 72 hours after the last dose, thereby supporting once-daily dosing. Food appeared to have no clinically meaningful effects on etavopivat exposure, thus facilitating administration with or without food. In conclusion, the evaluation of etavopivat in healthy subjects demonstrated proof of mechanism (PKR activation) without significant adverse events. This study also allowed for the selection of dose levels, projected to have an acceptable safety profile and provide therapeutic benefit, for evaluation in future trials in patients with sickle cell disease.

Etavopivat is an investigational, oral, small molecule activator of erythrocyte pyruvate kinase (PKR) in development for the treatment of sickle cell disease (SCD) and other hemoglobinopathies. PKR activation is proposed to ameliorate the sickling of SCD red blood cells (RBCs) through multiple mechanisms, including reduction of 2,3-diphosphoglycerate (2,3-DPG), which consequently increases hemoglobin (Hb)-oxygen affinity; increased binding of oxygen reduces sickle hemoglobin polymerization and sickling. In addition, PKR activation increases adenosine triphosphate (ATP) produced via glycolytic flux, which helps preserve membrane integrity and RBC deformability. We evaluated the pharmacodynamic response to etavopivat in nonhuman primates (NHPs) and in healthy human subjects and evaluated the effects in RBCs from patients with SCD after ex vivo treatment with etavopivat. A single dose of etavopivat decreased 2,3-DPG in NHPs and healthy subjects. Hb-oxygen affinity was significantly increased in healthy subjects after 24 hours. After daily dosing of etavopivat over 5 consecutive days in NHPs, ATP was increased by 38% from baseline. Etavopivat increased Hb-oxygen affinity and reduced sickling in RBCs collected from patients with SCD with either homozygous hemoglobin S or hemoglobin S and C disease. Collectively, these results demonstrate the ability of etavopivat to decrease 2,3-DPG and increase ATP, resulting in increased Hb-oxygen affinity and improved sickle RBC function. Etavopivat is currently being evaluated in clinical trials for the treatment of SCD. SIGNIFICANCE STATEMENT: Etavopivat, a small molecule activator of the glycolytic enzyme erythrocyte pyruvate kinase, decreased 2,3-diphosphoglycerate in red blood cells (RBCs) from nonhuman primates and healthy subjects and significantly increased hemoglobin (Hb)-oxygen affinity in healthy subjects. Using ex vivo RBCs from donors with sickle cell disease (SCD) (homozygous hemoglobin S or hemoglobin S and C genotype), etavopivat increased Hb-oxygen affinity and reduced sickling under deoxygenation. Etavopivat shows promise as a treatment for SCD that could potentially reduce vaso-occlusion and improve anemia.

Hereditary hemolytic anemias (HHAs) comprise a heterogeneous group of anemias caused by mutations in genes coding the globins, red blood cell (RBC) membrane proteins, and RBC enzymes. Congenital dyserythropoietic anemias (CDAs) are rare disorders of erythropoiesis characterized by binucleated and multinucleated erythroblasts in bone marrow. CDAs typically present with a hemolytic phenotype, as the produced RBCs have structural defects and decreased survival and should be considered in the differential of HHAs. This article discusses the clinical presentation, laboratory findings, and management considerations for rare HHAs arising from unstable hemoglobins, RBC hydration defects, the less common RBC enzymopathies, and CDAs.

BACKGROUND Without early initiation of disease-modifying therapy, the acute and chronic complications of sickle cell anemia (SCA) begin early in childhood and progress throughout life. Hydroxyurea is a safe and effective medication that reduces or prevents most SCA-related complications. Despite recommendations to prescribe hydroxyurea for all children with SCA as young as 9 months, utilization remains low. PROCEDURE We completed a retrospective review of hydroxyurea-prescribing practices and associated clinical outcomes at our institution over a 10-year period before and after the 2014 National Heart, Lung, and Blood Institute (NHLBI) recommendations to use hydroxyurea for all children with SCA. RESULTS Hydroxyurea use more than doubled within our pediatric SCA population from 43% in 2010 to 95% in 2019. The age of hydroxyurea initiation was significantly younger during 2014-2019 compared to 2010-2013 (median 2 years vs. 6 years, p ≤ .001). With this change in clinical practice, nearly all (69/71 = 97%) children born after 2013 received disease-modifying therapy by the end of 2019, primarily hydroxyurea (93%). Concurrently, the number of SCA-related admissions significantly decreased from 67/100 patient-years in 2010 to 39/100 patient-years in 2019 (p

Hydroxyurea (hydroxycarbamide) is an effective treatment for sickle cell anaemia (SCA), but clinical responses depend primarily upon the degree of fetal haemoglobin (HbF) induction and the heterogeneity of HbF expression across erythrocytes. The number and characteristics of HbF-containing cells (F-cells) are not assessed by traditional HbF measurements. Conventional hydroxyurea dosing (e.g. fixed doses or low starting doses with stepwise escalation) produces a moderate heterocellular HbF induction, but haemolysis and clinical complications continue. Robust, pancellular HbF induction is needed to minimise or fully inhibit polymerisation of sickle haemoglobin. We treated children with hydroxyurea using an individualised, pharmacokinetics-guided regimen starting at predicted maximum tolerated dose (MTD). We observed sustained HbF induction (mean >30%) for up to 6 years, which was not dependent on genetic determinants of HbF expression. Nearly 70% of patients had ≥80% F-cells (near-pancellular), and almost half had ≥90% F-cells (pancellular). The mean HbF/F-cell content was ~12 pg. Earlier age of initiation and better medication adherence were associated with high F-cell responses. In summary, early initiation of hydroxyurea using pharmacokinetics-guided starting doses at predicted MTD can achieve sustained near-pancellular or pancellular HbF expression and should be considered an achievable goal for children with SCA treated with hydroxyurea at optimal doses. Clinical trial registration number: NCT02286154 (clinicaltrials.gov).

Autoimmune hemolytic anemia (AIHA) is a rare disease in children, presenting with variable severity. Most commonly, warm-reactive IgG antibodies bind erythrocytes at 37 °C and induce opsonization and phagocytosis mainly by the splenic macrophages, causing warm AIHA (w-AIHA). Post-infectious cold-reactive antibodies can also lead to hemolysis following the patient’s exposure to cold temperatures, causing cold agglutinin syndrome (CAS) due to IgM autoantibodies, or paroxysmal cold hemoglobinuria (PCH) due to atypical IgG autoantibodies which bind their target RBC antigen and fix complement at 4 °C. Cold-reactive antibodies mainly induce intravascular hemolysis after complement activation. Direct antiglobulin test (DAT) is the gold standard for AIHA diagnosis; however, DAT negative results are seen in up to 11% of warm AIHA, highlighting the need to pursue further evaluation in cases with a phenotype compatible with immune-mediated hemolytic anemia despite negative DAT. Prompt supportive care, initiation of treatment with steroids for w-AIHA, and transfusion if necessary for symptomatic or fast-evolving anemia is crucial for a positive outcome. w-AIHA in children is often secondary to underlying immune dysregulation syndromes and thus, screening for such disorders is recommended at presentation, before initiating treatment with immunosuppressants, to determine prognosis and optimize long-term management potentially with novel targeted medications.

Congenital dyserythropoietic anemias (CDAs) are characterized by ineffective erythropoiesis and distinctive erythroblast abnormalities; the diagnosis is often missed or delayed due to significant phenotypic heterogeneity. We established the CDA Registry of North America (CDAR) to study the natural history of CDA and create a biorepository to investigate the pathobiology of this heterogeneous disease. Seven of 47 patients enrolled so far on CDAR have CDA-I due to biallelic CDAN1 mutations. They all presented with perinatal anemia and required transfusions during infancy. Anemia spontaneously improved during infancy in three patients; two became transfusion-independent rapidly after starting interferon-α(2); and two remain transfusion-dependent at last follow-up at ages 5 and 30 y.o. One of the transfusion-dependent patients underwent splenectomy at 11 y.o due to misdiagnosis and returned to medical attention at 27 y.o with severe hemolytic anemia and pulmonary hypertension. All patients developed iron overload even without transfusions; four were treated with chelation. Genetic testing allowed for more rapid and accurate diagnosis; the median age of confirmed diagnosis in our cohort was 3 y.o compared to 17.3 y.o historically. In conclusion, CDAR provides an organized research network for multidisciplinary clinical and research collaboration to conduct natural history and biologic studies in CDA.

Sickle cell anemia (SCA) is a hereditary hemoglobinopathy with a variable phenotype. There is no single biomarker that adequately predicts disease severity and can be used to monitor treatment response in patients in clinical trials and clinical care. The use of clinical outcomes, such as vaso-occlusive crises (VOC), requires long and expensive studies, sometimes with inconclusive results. To address these limitations, there are several biomarkers under study to improve the ability to predict complications and assess treatment response in both clinical and research settings. Oxygen gradient ektacytometry, also called as oxygenscan, is an assay that measures the effects of deoxygenation and reoxygenation on red blood cell (RBC) deformability and is gaining popularity in SCA research, because it captures the dynamic sickling capacity of a patient’s RBCs as they are subjected to an oxygen gradient under steady shear stress. We describe here the oxygenscan methodology and evaluate the correlation between oxygenscan parameters and more well-known biomarkers of SCA such as fetal hemoglobin (HbF), F-cells, and dense red blood cells (DRBCs). Our data indicate that the oxygenscan curve is affected by all these parameters and the result incorporates the effects of %HbF, %F-cells, RBC hydration, and RBC membrane deformability.

Inherited bone marrow failure syndromes (IBMFS) are heterogeneous disorders characterized by dysregulated hematopoiesis in various lineages, developmental anomalies, and predisposition to malignancy. The scat (severe combined anemia and thrombocytopenia) mouse model is a model of IBMFS with a phenotype of pancytopenia cycling through crises and remission. Scat carries an autosomal recessive missense mutation in Rasa3 that results in RASA3 mislocalization and loss of function. RASA3 functions as a Ras-GTPase activating protein (GAP), and its loss of function in scat results in increased erythroid RAS activity and reactive oxygen species (ROS) and altered erythroid cell cycle progression, culminating in delayed terminal erythroid differentiation. Here we sought to further resolve the erythroid cell cycle defect in scat through ex vivo flow cytometric analyses. These studies revealed a specific G0/G1 accumulation in scat bone marrow (BM) polychromatophilic erythroblasts and scat BM Ter119(−)/c-KIT(+)/CD71(lo/med) progenitors, with no changes evident in equivalent scat spleen populations. Systematic analyses of RNAseq data from megakaryocyte-erythroid progenitors (MEPs) in scat crisis vs. scat partial remission reveal altered expression of genes involved in the G1-S checkpoint. Together, these data indicate a precise, biphasic role for RASA3 in regulating the cell cycle during erythropoiesis with relevance to hematopoietic disease progression.

Fetal hypoxia has been implicated in fetal growth restriction in congenital heart disease (CHD) and leads to stress erythropoiesis in utero. The objective is to assess erythropoiesis and its association with growth in newborns with CHD.Fetuses with prenatally diagnosed CHD from 2013 to 2018 were retrospectively reviewed. Pregnancies with multiple gestation, genetic abnormalities, major extra-cardiac anomalies, and placental abruption were excluded. Complete blood count tests at birth were compared to published normative values. Spearman correlation assessed associations of red blood cell (RBC) indices with birth anthropometrics and prenatal Doppler measures.A total of 160 newborns were included. Median gestational age was 38.3 (37.3, 39.0) weeks. Infants ≥37 weeks gestation had lower hemoglobin (Hgb), hematocrit, and elevated nucleated RBC (nRBC), mean corpuscular volume, and mean corpuscular hemoglobin compared to reference. No differences in RBC indices were observed in infants34 and 34-37 weeks gestation. There was no difference in Hgb and nRBC between CHD subgroups. Neither Hgb nor nRBC were associated with birth anthropometrics or Doppler patterns.Term infants with CHD demonstrated multiple alterations in erythrocyte indices suggesting ineffective stress erythropoiesis in late gestation resulting in lower Hgb at birth. Altered erythropoiesis was not correlated to growth or Doppler patterns.Newborns with congenital heart disease (CHD) born at term gestation demonstrated altered erythropoiesis. Term newborns with CHD have decreased hemoglobin levels despite having red blood cell indices consistent with stress erythropoiesis, suggesting an incomplete compensatory response to in utero physiologic disturbances associated with CHD. The etiology is unknown; however, it may be influenced by multiple risk factors during pregnancy in the maternal-fetal dyad. Alterations in red blood cell indices were not associated with outcomes of fetal growth.

BACKGROUND: Total splenectomy (TS) and partial splenectomy (PS) are used for children with congenital hemolytic anemia (CHA), although the long-term outcomes of these procedures are poorly defined. This report describes long-term outcomes of children with CHA requiring TS or PS. PROCEDURE: We collected data from children ages 2-17 with hereditary spherocytosis (HS) or sickle cell disease (SCD) requiring TS or PS from 1996 to 2016 from 14 sites in the Splenectomy in Congenital Hemolytic Anemia (SICHA) consortium using a prospective, observational patient registry. We summarized hematologic outcomes, clinical outcomes, and adverse events to 5 years after surgery. Hematologic outcomes were compared using mixed effects modeling. RESULTS: Over the study period, 110 children with HS and 97 children with SCD underwent TS or PS. From preoperatively compared to postoperatively, children with HS increased their mean hemoglobin level by 3.4 g/dL, decreased their mean reticulocyte percentage by 6.7%, and decreased their mean bilirubin by 2.4mg/dL. Hematologic improvements and improved clinical outcomes were sustained over 5 years of follow-up. For children with SCD, there was no change in hemoglobin after PS or TS following surgery, although all clinical outcomes were improved. Over 5 years, there was one child with HS and 5 children with SCD who developed post-splenectomy sepsis. CONCLUSIONS: For children with HS, there are excellent long-term hematologic and clinical outcomes following either PS or TS. Although hemoglobin levels do not change after TS or PS in SCD, the long-term clinical outcomes for children with SCD are favorable.

Hereditary hemolytic anemias (HHA) are a heterogeneous group of anemias associated with decreased red cell survival. While there can be clinical benefit of splenectomy in many cases, splenectomy is not appropriate for all types of HHA. Additionally, there are significant risks during and following splenectomy including surgical risks, postsplenectomy sepsis, and thrombotic complications. This review discusses the diagnostic approach to HHA as well as the role of splenectomy in the management. Surgical approaches and outcomes for total and partial splenectomy are discussed.

Many hypotheses have been proposed to explain how a glutamate to valine substitution in sickle haemoglobin (HbS) can cause sickle cell disease (SCD). We propose and document a new mechanism in which elevated tyrosine phosphorylation of Band 3 initiates sequelae that cause vaso-occlusion and the symptoms of SCD. In this mechanism, denaturation of HbS and release of heme generate intracellular oxidants which cause inhibition of erythrocyte tyrosine phosphatases, thus permitting constitutive tyrosine phosphorylation of Band 3. This phosphorylation in turn induces dissociation of the spectrin-actin cytoskeleton from the membrane, leading to membrane weakening, discharge of membrane-derived microparticles (which initiate the coagulation cascade) and release of cell-free HbS (which consumes nitric oxide) and activates the endothelium to express adhesion receptors). These processes promote vaso-occlusive events which cause SCD. We further show that inhibitors of Syk tyrosine kinase block Band 3 tyrosine phosphorylation, prevent release of cell-free Hb, inhibit discharge of membrane-derived microparticles, increase sickle cell deformability, reduce sickle cell adhesion to human endothelial cells, and enhance sickle cell flow through microcapillaries. In view of reports that imatinib (a Syk inhibitor) successfully treats symptoms of sickle cell disease, we suggest that Syk tyrosine kinase inhibitors warrant repurposing as potential treatments for SCD.

Background Lyme disease is the most common tick-borne illness in the USA, Canada, and Europe. Clinical manifestations vary greatly, with localized skin findings functioning as early signs of the disease, followed by disseminated disease. The rarest dermatologic presentation of Lyme is a borrelial lymphocytoma, occurring distinctly in Europe and caused typically by Borrelia afzelii. Case presentation We report a case of a Caucasian 5-year-old European-American boy with slowly progressing, painless edema and erythema of his right pinna. Travel history revealed significant exposure to ticks during a recent trip to Eastern Europe. Laboratory testing for Borrelia burgdorferi demonstrated mixed positivity. He was treated with a 21-day course of amoxicillin, with complete resolution of symptoms and no sign of secondary Lyme disease. Conclusions Borrelial lymphocytoma is a rare manifestation of Lyme disease in North America, although not uncommon in Europe. Diagnosis is made by the presence of a painless erythematous swelling typically found on the ear lobe, nipples, or testes. Laboratory tests are available but with low sensitivity, therefore, a high index of suspicion is necessary for a clinical diagnosis to be made. Treatment for isolated borrelial lymphocytoma is doxycycline 4 mg/kg up to 100 mg twice daily, whereas for children less than 8 years of age amoxicillin 50 mg/kg divided three times daily, for 3–4 weeks, is preferred.

T cell-mediated responses are dependent on their secretion of key effector molecules. However, the critical molecular determinants of the secretion of these proteins are largely undefined. Here, we demonstrate that T cell activation increases trafficking via the ER-to-Golgi pathway. To study the functional role of this pathway, we generated mice with a T cell-specific deletion in SEC23B, a core subunit of coat protein complex II (COPII). We found that SEC23B critically regulated the T cell secretome following activation. SEC23B-deficient T cells exhibited a proliferative defect and reduced effector functions in vitro, as well as in experimental models of allogeneic and xenogeneic hematopoietic cell transplantation in vivo. However, T cells derived from 3 patients with congenital dyserythropoietic anemia II (CDAII), which results from Sec23b mutation, did not exhibit a similar phenotype. Mechanistic studies demonstrated that unlike murine KO T cells, T cells from patients with CDAII harbor increased levels of the closely related paralog, SEC23A. In vivo rescue of murine KO by expression of Sec23a from the Sec23b genomic locus restored T cell functions. Together, our data demonstrate a critical role for the COPII pathway, with evidence for functional overlap in vivo between SEC23 paralogs in the regulation of T cell immunity in both mice and humans.

Several members of the Ras-GTPases have been shown to play major roles as molecular switches in various signaling pathways in hematopoietic cells. The roles of Ras/MAPKs/ERK signaling pathways in EPO-induced erythroid differentiation have been explored in the past on animal models. Recently, the Ras/RalGEFs/Ral pathway has been a focus of research in oncology given its roles in tumorigenesis. However, its role in normal human erythropoiesis is poorly understood. The seven RalGEF family members are guanine nucleotide exchange factors with unique as well as overlapping roles. They bridge activated Ras to activation of RalA and RalB while they have also been shown to promote Akt activation. Recently, we identified and characterized a homozygous splicing variant (c.1580+4T>A) in RGL2 (Ral Guanine Nucleotide Dissociation Stimulator-Like 2) gene, one of the RalGEFs, in a 16 month-old female patient with a recessive form of familial syndromic pancytopenia, more severe in the erythroid lineage with transfusion-dependent anemia, hepatosplenomegaly, liver fibrosis, and cardiac dysfunction. We verified by RT-PCR that this splicing variant was associated by a decrease in the RGL2 expression levels by 70-96% in the white blood cells and reticulocytes of the patient. We hypothesized that decreased RGL2 expression leads to defective human erythropoiesis by altering terminal erythroid differentiation, erythroblast cell cycle progression and/or apoptosis. To test our hypothesis, we transduced human bone marrow derived CD34+ cells (donated by healthy volunteers under an IRB-approved research protocol in our institution) with lentivirus encoding shRNA targeting RGL2 mRNA and then cultured them in a previously validated 3-phase culture system that recapitulates erythropoiesis ex vivo. A scrambled shRNA sequence was used as control. The efficiency of the shRNAs used to knock-down (KD) RGL2 expression was initially validated by qRT-PCR after transduction of K562 cells. Our study demonstrated that knockdown of RGL2 expression resulted in decreased erythroid cell growth in ex vivo erythropoiesis accompanied by increased apoptosis. We further showed that knockdown of RGL2 expression also led to delayed terminal erythroid differentiation by flow cytometric analysis using glycophorin A (GPA), band 3, and α4-integrin as markers of maturing erythroblasts. This finding was corroborated by morphology studies using Wright staining in cytospins from RGL2-KD cultures and control. Together, our studies indicate an essential role for RGL2 and the Ras/RGL2/Ral pathway in human erythropoiesis, affecting both terminal erythroid differentiation and apoptosis. Our findings not only provide insights into regulation of normal erythropoiesis but also indicate that RGL2 may be a novel candidate gene associated with inherited bone marrow failure syndromes. Disclosures No relevant conflicts of interest to declare.

Background: Sickle cell disease (SCD) derives from a mutation in sickle hemoglobin (HbS) that leads to vaso-occlusive events that eventually cause organ failure. Based on observations from multiple labs, we hypothesize that the events linking HbS denaturation to vaso-occlusion are: HbS denaturation→oxidative stress→inhibition of erythrocyte tyrosine phosphatases→enhanced tyrosine phosphorylation of band 3→dissociation of the spectrin/actin cytoskeleton from the membrane→release of membrane-derived microparticles (MPs) and discharge of cell-free HbS→induction of microthromboses and activation of the vascular endothelium→vascular occlusion. In the studies below, we document each step of this pathway and demonstrate that inhibitors of Band 3 tyrosine phosphorylation block all downstream events. Methods: Band 3 tyrosine phosphorylation, plasma MP counts, plasma cell-free Hb concentration, sickle cell deformability, sickle cell flow through micro-capillaries, and adhesion of sickle erythrocytes to activated endothelial cells are quantitated using fresh sickle blood both before and after incubation with inhibitors of Band 3 tyrosine phosphorylation. Findings: Imatinib and other Syk kinase inhibitors block Band 3 tyrosine phosphorylation, suppress release of membrane MPs, reduce discharge of cell-free HbS, improve sickle cell deformability, enhance flow of sickle cells through microcapillaries, and reduce adherence of sickle cells to heme-activated endothelium. Interpretation: Inhibition of Band 3 Tyrosine phosphorylation restores membrane integrity and thereby suppresses the downstream sequelae that lead to vaso-occlusive events in SCD. Inhibitors of Band 3 tyrosine phosphorylation may therefore constitute a novel effective therapy for SCD. Funding Statement: NIH grants R01GM24417-40 (P.S.L.), 1RF1 NS110049-01 (J.W.), R01HL133574 (U.A.G. and J.A.L.), T32HL134622 (R.A.), and NSF #1552782 (U.A.G.). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All sickle cell blood samples were obtained following informed consent using procedures approved by the local IRBs.

Pyruvate kinase deficiency (PKD) is the most frequent red blood cell enzyme abnormality of the glycolytic pathway and the most common cause of hereditary nonspherocytic hemolytic anemia. Over 250 PKLR-gene mutations have been described, including missense/nonsense, splicing and regulatory mutations, small insertions, small and gross deletions, causing PKD and hemolytic anemia of variable severity. Alu retrotransposons are the most abundant mobile DNA sequences in the human genome, contributing to almost 11% of its mass. Alu insertions have been associated with a number of human diseases either by disrupting a coding region or a splice signal. Here, we report on two unrelated Middle Eastern patients, both born from consanguineous parents, with transfusion-dependent hemolytic anemia, where sequence analysis revealed a homozygous insertion of AluYb9 within exon 6 of the PKLR gene, causing precipitous decrease of PKLR RNA levels. This Alu element insertion consists a previously unrecognized mechanism underlying pathogenesis of PKD.

Activated erythropoietin (EPO) receptor (EPOR) signaling causes erythrocytosis. The important role of macrophages for the erythroid expansion and differentiation process has been reported, both in baseline and stress erythropoiesis. However, the significance of EPOR signaling for regulation of macrophages contributing to erythropoiesis has not been fully understood. Here we show that EPOR signaling activation quickly expands both erythrocytes and macrophages in vivo in mouse models of primary and secondary erythrocytosis. To mimic the chimeric condition and expansion of the disease clone in the polycythemia vera patients, we combined Cre-inducible Jak2V617F/+ allele with LysM-Cre allele which expresses in mature myeloid cells and some of the HSC/Ps (LysM-Cre;Jak2V617F/+ mice). We also generated inducible EPO-mediated secondary erythrocytosis models using Alb-Cre, Rosa26-loxP-stop-loxP-rtTA, and doxycycline inducible EPAS1-double point mutant (DPM) alleles (Alb-Cre;DPM mice). Both models developed a similar degree of erythrocytosis. Macrophages were also increased in both models without increase of major inflammatory cytokines and chemokines. EPO administration also quickly induced these macrophages in wild-type mice before observable erythrocytosis. These findings suggest that EPOR signaling activation could induce not only erythroid cell expansion, but also macrophages. Surprisingly, an in vivo genetic approach indicated that most of those macrophages do not express EPOR, but erythroid cells and macrophages contacted tightly with each other. Given the importance of the central macrophages as a niche for erythropoiesis, further elucidation of the EPOR signaling mediated-regulatory mechanisms underlying macrophage induction might reveal a potential therapeutic target for erythrocytosis.

The hallmark of sickle cell disease (SCD) is polymerization of deoxygenated hemoglobin S (HbS), resulting in red blood cell (RBC) sickling, oxidative and membrane damage, hemolysis, vaso-occlusion, and end-organ damage. Exacerbating the pathogenesis of SCD, the sickle RBC (sRBC) has increased 2,3-DPG levels with decreased oxygen (O 2) affinity (increased P 50) and decreased ATP. Etavopivat, a small molecule activator of erythrocyte pyruvate kinase (PKR), increases PKR activity, resulting in decreased 2,3-DPG levels and increased ATP levels in RBCs. In a Phase 1 study in patients with SCD [NCT03815695], etavopivat significantly improved anemia and hemolysis after 2 weeks of treatment (Brown et al. Blood 2020). To evaluate the potential of etavopivat to reduce vaso-occlusive crises, exploratory studies were conducted to characterize the sRBC specific (intrinsic) and systemic effects of PKR activation. Patients with SCD received once daily etavopivat 300 or 600 mg for 2 weeks or 400 mg for up to 12 weeks. Peripheral blood was collected prior to treatment (ie, baseline), on treatment and 7-28 days post treatment. Studies to assess the sRBC intrinsic effects of PKR activation included evaluation of RBC parameters and reticulocyte counts (ADVIA ®), membrane deformability (Lorrca ®), enzyme function studies, and membrane markers by flow cytometry. Studies to assess the systemic effects of PKR activation included markers of coagulation, inflammation, and hypoxia in the 12-week cohort only. As of May 24, 2021, 15 patients who completed the 2-week dose cohorts and 7 patients treated in the 12-week dose cohort were evaluable for this analysis. The intrinsic effects of etavopivat on the sRBCs of patients receiving 2 weeks of treatment are summarized in Table 1. Etavopivat significantly increased Hb and reduced reticulocytes, including immature reticulocytes (CD71 +), suggesting that an etavopivat-mediated increase in sRBC lifespan is accompanied by a decrease in erythropoietic stress. In addition, etavopivat reduced 2,3-DPG levels thereby increasing HbS O 2 affinity (decreased P 50) resulting in a significant shift in the point of sickling (PoS) to a lower partial O 2 pressure. The deformability (EI max) of the sRBCs as measured by oxygenscan and osmoscan was significantly improved with etavopivat treatment, consistent with reduced membrane damage due to decreased HbS polymerization and improved membrane repair enabled by increased ATP production, collectively improving the health of the sRBC membrane. This improvement in membrane health was further supported by a significant reduction in the external expression of phosphatidylserine (PS) following etavopivat treatment. Finally, etavopivat significantly improved enzymatic activity not only of PKR but also superoxide dismutase and glutathione reductase, enzymes involved in reducing oxidative stress in sRBCs. This suggests that etavopivat-treated sRBCs may have an improved ability to inhibit and repair damage caused by reactive O 2 species, thereby improving overall sRBC health and function. Initial results on the effect of etavopivat on systemic biomarkers that are commonly elevated in SCD are shown in Table 2. In patients receiving etavopivat 400 mg once daily for up to 12 weeks, tumor necrosis factor-a and prothrombin 1.2, as systemic markers of inflammation and hypercoagulability, respectively, showed a significant decrease compared with baseline. Furthermore, a trend towards reduced erythropoietin levels suggests that etavopivat treatment may reduce tissue hypoxia. Patients with SCD treated with etavopivat for at least 2 weeks demonstrated a significant increase in RBC membrane deformability and improved antioxidant capacity that resulted in increased sRBC survival and decreased anemia. The reduced reticulocyte count and lowered PS surface membrane expression suggest that etavopivat-treated sRBC may have reduced adhesive properties and may thus be less likely to promote vaso-occlusion. Initial studies evaluating the downstream effects of up to 12 weeks of etavopivat treatment once daily provided evidence for a reduction in markers of inflammation and hypercoagulability, with improved O 2 delivery capacity. These initial results suggest that the multimodal effects of decreased 2,3-DPG and increased ATP by PKR activation with etavopivat may have an impact on both the anemia and vaso-occlusive events that characterize SCD. Figure 1 Figure 1. Disclosures Kalfa: Agios Pharmaceuticals, Inc.: Other: Steering Committee, Research Funding; FORMA Therapeutics, Inc: Research Funding. Telen: GlycoMimetics, Inc.: Consultancy; Novartis, Inc.: Other: Data Safety Monitoring Board; Forma Therapeutics, Inc.: Consultancy, Research Funding; CSL Behring, Inc.: Research Funding; Doris Duke Charitable Foundation: Research Funding; National Institutes of Health: Research Funding. 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. Brown: Novo Nordisk: Consultancy; Imara: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Pfizer: Research Funding; Forma Therapeutics: Research Funding. Larkin: Forma Therapeutics, Inc.: Research Funding. Ribadeneira: Forma Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Schroeder: Forma Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Wu: Forma Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Kelly: Forma Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Kuypers: Forma Therapeutics, Inc.: Research Funding.

Etavopivat is a small molecule activator of erythrocyte pyruvate kinase (PKR), that increases PKR activity, resulting in decreased 2,3-DPG and increased ATP in red blood cells (RBCs) of healthy volunteers (HV) and patients (pts) with sickle cell disease (SCD) (Kalfa 2019, Brown 2020). Based on initial safety, pharmacokinetic (PK) and pharmacodynamic (PD) data for HVs and pts with SCD, we performed multiple-dose studies in pts with SCD (NCT03815695): 2-wk multiple ascending dose (MD) cohorts to identify the once daily etavopivat dose that provides maximum PD activity with an acceptable safety profile and a 12-wk open label (OL) study to further characterize the safety and clinical activity at the maximum PD dose. These data are presented here. In the completed MD cohorts, 20 pts with SCD were randomized 8:2 to receive etavopivat (300 mg, then 600 mg) or placebo (PBO) once daily for 2 wks. In the ongoing OL cohort, up to 20 pts will receive etavopivat 400 mg once daily for 12 wks. Safety assessments included adverse events (AEs), vital signs, electrocardiograms, and peripheral blood laboratory parameters. PK/PD blood sampling was performed for up to 72 h after last dose and at end of study visit. RBC function studies were performed to assess membrane deformability (Lorrca ®). Enrollment in the MD cohorts (n=17 HbSS, n=2 HbSβ+thalassemia, n=1 HbSC) is complete and data unblinded (n=8: 300 mg etavopivat; n=8: 600 mg etavopivat; n=4: PBO). As of July 13, 2021, 11 pts (n=10 HbSS, 1 HbSC) have been treated in the OL cohort: median treatment duration was 12 (range 1-12) wks, 6 pts completed 12 wks of treatment. In MD pts, etavopivat demonstrated dose-proportional PK with overlapping PD responses (decreased 2,3-DPG and increased ATP), confirming prior results in HVs that etavopivat 400 mg once daily provides near maximal PD activity. Etavopivat was well tolerated in both MD and OL cohorts. AEs were reported in 1 of 4 (25%) MD PBO pts, most were grade (gr) ≤3, with 1 gr 4 blood creatine phosphokinase (CPK) increase. 13 of 16 (81%) etavopivat-treated MD pts reported AEs, most were gr 1/2 and commonly (>2 pts) included sickle cell pain (n=6 [38%]), headache (n=5 [31%]), and nausea (n=3 [19%]). One pt had a serious AE (SAE) of gr 3 vaso-occlusive crisis (VOC) after completion of etavopivat (considered unrelated). In the OL cohort, AEs were reported in 7 of 11 (64%) pts who received at least 1 wk of etavopivat. AEs reported in >1 pt were headache and VOC (n=2 [18%] each). Most AEs were gr 1/2; one pt had SAEs of gr 3 acute chest syndrome and VOC (unrelated), one pt had an SAE of gr 3 deep vein thrombosis (possibly related), and one pt had an AE of gr 4 transient blood CPK increase (unrelated). Hematologic and hemolytic parameters were significantly improved at end of treatment in both MD and OL cohorts (Table 1); 11 of 15 (73%) evaluable MD pts achieved a Hb increase ≥1g/dL over baseline (mean 1.1 g/dL, P1g/dL Hb increase over baseline (mean 1.39 g/dL). Reductions in ARC, indirect bilirubin and LDH were also observed. Of 9 pts on etavopivat for at least 4 wks, 8 (89%) reported an increase in Hb >1g/dL, and the highest mean Hb increase was 1.81 g/dL during active treatment. Etavopivat-treated RBCs from MD pts (n=14) demonstrated improved functional health, including point of sickling and deformability. The improved deformability persisted up to 1 wk after etavopivat treatment in 36% of pts. Similar results were observed in the initial OL pts. Data on additional treated pts will be presented. Etavopivat 400 mg once daily for up to 12 wks was well-tolerated, with a safety profile consistent with underlying SCD. Increases in Hb >1 g/dL were observed in 89% of pts and maintained throughout 12 wks of treatment in the majority (83%) of pts. Increased Hb and a significant reduction in ARC indicated that etavopivat enhanced survival of sickle RBCs and significantly improved the severe anemia associated with SCD. These longer-living sickle RBCs have improved membrane health that may further reduce the risk of VOCs and end-organ damage. These results support further evaluation of etavopivat in the ongoing Phase 2/3 Hibiscus Study in pts with SCD (NCT04624659). Figure 1 Figure 1. Disclosures Brown: Imara: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Novo Nordisk: Consultancy; Forma Therapeutics: Research Funding; Pfizer: Research Funding; Global Blood Therapeutics: Consultancy, Research Funding. 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. Idowu: Pfizer: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Forma Therapeutics, Inc.: Research Funding; Ironwood: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kalfa: Agios Pharmaceuticals, Inc.: Other: Steering Committee, Research Funding; FORMA Therapeutics, Inc: Research Funding. Geib: Forma Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Forsyth: Forma Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Schroeder: Forma Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Wu: Forma Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Kelly: Forma Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Telen: GlycoMimetics, Inc.: Consultancy; Novartis, Inc.: Other: Data Safety Monitoring Board; Forma Therapeutics, Inc.: Consultancy, Research Funding; CSL Behring, Inc.: Research Funding; Doris Duke Charitable Foundation: Research Funding; National Institutes of Health: Research Funding.

Background Sickle cell disease (SCD) and thalassemia are inherited hemoglobinopathies characterized by lifelong anemia. In SCD, a single mutation in the β-globin gene results in sickle hemoglobin (HbS) that polymerizes upon deoxygenation, causing red blood cells (RBCs) to sickle leading to a variety of complications. In thalassemia, mutation(s) in α- or β-globin genes result in reduced or absent adult Hb causing ineffective erythropoiesis and downstream complications. The resultant anemias are exacerbated by impaired RBC function due to decreased ATP content. Supportive care and agents like hydroxyurea are used most in SCD, with a subset of patients (pts) on regular transfusions. Regular or episodic transfusions, with their own set of complications, are the mainstay of treatment for thalassemias. Etavopivat, a potent, selective, oral, small molecule activator of erythrocyte pyruvate kinase (PKR) increases ATP and decreases 2,3 diphosphoglycerate (2,3-DPG). In a Phase 1 study, etavopivat 300‒600 mg once daily in pts with SCD not regularly transfused was well-tolerated, improved hematological markers, decreased hemolysis, and improved markers of RBC functional health (Brown et al. EHA 2021 # EP1202). Etavopivat 200 and 400 mg once daily (dose levels predicted to provide the desired pharmacodynamic response profiles) is being evaluated in a Phase 2/3 study of pts with SCD who are not on chronic transfusions (The Hibiscus Study, NCT04624659). Herein we describe the design of a Phase 2, open-label, multicenter study (NCT04987489) evaluating the efficacy and safety of etavopivat in pts with: SCD on chronic transfusions (Cohort A), transfusion-dependent thalassemia (Cohort B), and non-transfusion-dependent thalassemia (Cohort C). Study Design and Methods Up to 20 pts aged 12-65 years will be enrolled in each of the three cohorts described above. Pts will receive etavopivat 400 mg once daily for 48-wks (Figure). In cohorts A and B, pts must have received ≥6 RBC units in the 24 wks before the first dose of etavopivat without a >35-day transfusion-free period during that period and be on iron chelation therapy for >3 months before enrollment. Additionally, pts in Cohort A should have received ≥24 monthly transfusions for the prevention or treatment of primary stroke. Pts in Cohort C should have a Hb ≤10 g/dL. Key exclusion criteria include significant infection, hepatic/renal dysfunction, history of malignancy/cardiac/pulmonary disease, a drug malabsorption disorder, prior/concomitant therapies ≤3 months before the first dose (eg, chronic systemic glucocorticoids, new chelation therapy). Baseline assessments will include medical, disease, transfusion, and medication histories. Transfusions received during the study (every ~3-5 wks) will be recorded and include Hb values before and ≥15 minutes after transfusion, number of RBC units, and volume of packed RBCs. If a pt has an increase in pre-transfusion Hb of ≥1.0 g/dL compared with their baseline pre-transfusion Hb, the Investigator may delay transfusion by 1 wk or reduce the number of RBC units transfused. In pts with SCD, RBC exchange may also be performed. The primary endpoint for Cohorts A/B is erythroid response defined as the proportion of pts with ≥20% reduction in transfusions over a continuous 12-wk treatment period versus baseline, and for Cohort C is Hb response at Wk 12 defined as an increase in Hb of ≥1.0 g/dL from baseline. For Cohorts A/B, secondary and exploratory endpoints include the proportion of pts with a reduction in transfusions over 12 wks of ≥33% and ≥50%, respectively, and a reduction in transfusions over 12, 24, and 48 wks; and for Cohort C, Hb response at Wks 24 and 48, and changes from baseline in Hb over 12, 24, and 48 wks. The following additional endpoints will be assessed for all cohorts: changes from baseline in quality of life as assessed via the Short Form Health Survey and Patient-Reported Outcome Measurement Information System Fatigue Scale; changes from baseline in the levels of serum ferritin at 12, 24, and 48 wks; liver iron at 48 wks; 2,3-DPG and ATP; pharmacokinetics; and safety. All primary endpoints will be analyzed using a 1-sided test at α=0.025. Summary Etavopivat is a novel, investigational, PKR activator designed to improve the functional health of RBCs. This Phase 2 study will assess the safety of etavopivat and its impact on Hb levels and transfusion burden in pts with SCD or thalassemia. Figure 1 Figure 1. Disclosures Lal: Chiesi: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; bluebird bio, Inc.: Research Funding; Protagonist Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharmaceuticals: Consultancy; Insight Magnetics: Research Funding; Novartis: Research Funding; Terumo Corporations: Research Funding; La Jolla Pharmaceutical Company: Research Funding. Brown: Novo Nordisk: Consultancy; Novartis: Consultancy, Research Funding; Imara: Consultancy, Research Funding; Forma Therapeutics: Research Funding; Pfizer: Research Funding; Global Blood Therapeutics: Consultancy, Research Funding. Coates: Apo Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Chiesi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Forma Pharma: Consultancy; Sangamo: Consultancy; UpToDate: Patents & Royalties; Vifor Pharma: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kalfa: Agios Pharmaceuticals, Inc.: Other: Steering Committee, Research Funding; FORMA Therapeutics, Inc: Research Funding. Kwiatkowski: Bioverativ: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; CRISPR: Research Funding; Imara: Consultancy, Research Funding; Silence Therapeutics: Consultancy; Sangamo: Research Funding; bluebird bio: Consultancy, Research Funding; Chiesi: Research Funding; Vertex: Research Funding. Brevard: Forma Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Potter: Forma Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company; Bristol Myers Squibb: Ended employment in the past 24 months. Wood: Forma Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Sheth: Bluebird bio: Consultancy; CRISPR: Consultancy; Bristol Myers Squibb: Consultancy, Research Funding; Imara: Research Funding; Chiesi: Consultancy; Agios: Consultancy; Dispersol: Research Funding.

Severe congenital neutropenia (SCN) is frequently associated with mutations in the ELANE gene encoding neutrophil elastase (NE), an azurophilic granule protein which represents a major fraction of all protein synthesized by neutrophilic promyelocytes and myelocytes. The reduction of mutant ELANE translation inversely correlates with neutrophil differentiation. Chronic administration of granulocyte colony-stimulating-factor (G-CSF) has been successfully used to treat some of these patients, especially those ones with mutations in C-terminus translating exons-4 and -5. Patients with proximal mutations of ELANE tend to be resistant to G-CSF therapy. The SCN pathobiology is poorly understood due to the lack of efficient disease animal models, and difficulty in obtaining relevant primary cell populations from patients. We have demonstrated that ELANE exon-3 mutations, sensitive to higher doses of G-CSF administration, cause maturation arrest and apoptosis of promyelocyte and myelocyte stages due to mutant NE inducing unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. We have also shown that SCN patients with mutations in ELANE at its translation initiation codon (ELANE-ATG mut) result in synthesis of alternate truncated neutrophil elastase peptides from downstream in-frame ATG due to the presence of internal ribosomal entry sites. However, the mechanisms of SCN pathogenesis associated with mutations at the translation initiation codon of ELANE gene associated with G-CSF resistance remain unclear. We employed a disease-in-a-dish approach by directed hematopoietic and granulopoietic differentiation of gene edited (CRISPR/Cas9) isogenic patient-derived iPSC lines. We used four iPSC lines (healthy donor 28L and its start codon knock-in isogenic 28L-GTG; and patient-derived ATG mut SCN110 and its isogenic corrected line SCN110C) to evaluate if ELANE start codon mutations are both necessary and sufficient to cause disease pathogenesis, and to unravel the underlying molecular mechanisms. We found that myeloid precursors derived from 28-GTG and SCN110 iPSC lines generate low molecular weight truncated NE that is detected by a C-terminal region (C225-H238) specific anti-NE antibody (Ab). The survival, expansion and differentiation of ELANE start codon mutant hematopoietic progenitors were significantly reduced in cytokine supplemented in vitro myelopoiesis cultures, and the correction of the mutation (SCN110C) restored the granulocytic precursor expansion and differentiation. The reduced expansion in 28L-GTG and SCN110 lines was associated with both increased apoptosis and expression of proapoptotic BH3-only proteins in comparison to their isogenic 28L and SCN110C lines. However, this apoptosis was not associated with UPR/ER-stress in ELANE-ATG mut myeloid precursors. Granulopoietic differentiation of the 28L-GTG and SCN110 iPSC derived hematopoietic progenitors was significantly reduced, while it was rescued in corrected isogenic lines. Correlating with their clinical behavior, high-dose G-CSF in vitro did not restore the granulopoietic differentiation of ELANE ATG mutant hematopoietic progenitors. Mechanistically, 28L-GTG and SCN110 myeloid precursors contained high-molecular weight, sodium dodecyl sulfate (SDS) resistant NE bands that were detected by anti-NE Ab specific for the C-terminal (C225-238), but not the N-terminal region (C19-38), suggesting that truncated mutant NE generates insoluble NE aggregates. The association of truncated NE with classical autophagy marker LC3B strongly suggested the activation of an aggrephagy process in these cells. We found that SERF1 (small EDRK-rich factor), an RNA-chaperoning protein, known to localize in misfolded protein aggregates of neurodegenerative diseases, was highly upregulated in GTG knock-in and SCN patient myeloid precursors. SERF1 was distributed in both nucleus and cytoplasm, co-localized and physically interacted with mutant truncated NE in the cytoplasm of ELANE-ATG mut cells as shown by proximity ligation assays. Silencing of SERF1 made the survival and differentiation of 28L-GTG and SCN110 derived myeloid precursors sensitive to G-CSF. ELANE translation initiation mutant was associated with truncated NE aggregates and induced apoptosis mediated by SERF1. This data opens a path for therapeutic intervention of G-CSF resistant ELANEmut SCN. Disclosures Kalfa: Agios Pharmaceuticals, Inc.: Other: Steering Committee, Research Funding; FORMA Therapeutics, Inc: Research Funding. Lutzko: Aruvant Sciences: Patents & Royalties: preclinical vector development. Cancelas: Hemerus LLC: Research Funding; Vascular Solutions Inc.: Research Funding; Westat Inc: Research Funding; Fresenius-Kabi LLC: Research Funding; Cytosorbents Inc: Research Funding; Hemanext: Membership on an entity's Board of Directors or advisory committees, Research Funding; TerumoBCT: Research Funding; Cerus Co: Research Funding; University of South Florida/MEQU Inc: Research Funding.

Sickle cell anemia (SCA), caused by a mutant β-globin gene, results in polymerization of the abnormal hemoglobin S and sickle shaped RBCs that cause vascular occlusions, chronic hemolytic anemia and cumulative organ damage. We have reported sickle nephropathy from elevated levels of angiotensin-II (AT), the bioactive peptide of the renin-angiotensin system (RAS), by signaling through the AT receptor-1 (AT1R), in both mice and humans with SCA, and that occurs in the absence of hypertension or hyper-reninemia [Roy et al, AJH, 2018]. Herein, we investigated the mechanisms underlying the hyperangiotensinemia in SCA. We found that AT levels are elevated due to higher oxidized state of its precursor, angiotensinogen (ATGN), which results from the elevated reactive oxygen species (ROS) in SCA. Oxidized ATGN is more rapidly converted to AT. Hence, the high ROS in SCA increases AT production. Blockade of AT-AT1R signaling in SCA mice, either globally (with captopril that reduces AT production, or losartan that blocks AT1R, or by constitutive genetic knockout of AT1R in SCA mice [SCA AT1R-/-]), or in erythroid cells by an erythroid-specific AT1R knockout (AT1Rf/f EpoR Cre+ termed eCre+) in SCA mice (SCA eCre+) resulted in significant reduction in RBC ROS (Figure 1a). These data show that AT-AT1R signaling in turn generates ROS in sickle erythroid cells, thus creating a positive feedback loop of ↑ROS -->↑RAS -->↑ROS, which causes, and perpetuates the hyperangiotensinemia seen in SCA. The AT-mediated ROS-RAS loop is driven largely by sickle erythropoiesis: SCA eCre+ mice have reduced RBC ROS, hence have reduced AT, and do not develop nephropathy. Surprisingly, while global AT1R deficiency in WT mice (WT AT1R-/-) had no RBC phenotype, SCA AT1R-/- mice developed profound anemia, suggesting AT signaling may be important for the stress erythropoiesis (Stress-E) state, present in SCA. AT is known for its role as a renal erythropoietin (Epo) secretagogue, but its role in Stress-E is unknown. Induction of Stress-E in WT AT1R-/- mice (with phenylhydrazine or daily phlebotomies) resulted in higher anemia in WT AT1R-/- mice than in WT AT1R+/+ mice, suggesting AT1R signaling is important for Stress-E, not baseline erythropoiesis (Base-E). However, WT mice with erythroid-specific deficiency of AT signaling (WT eCre+), when stressed, were able to maintain hemoglobin comparable to their controls (WT eCre-), with an exponential increase in Epo level. Epo levels were high in SCA mice to begin with but were insufficient to compensate with loss of AT signaling in SCA AT1R-/- mice, resulting in development of anemia. RNAseq analysis of sorted Stress-E nucleated erythroid precursors and enucleated erythrocytes in WT AT1R-/- mice showed a remarkable downregulation of the Hedgehog, BMP4, KIT and WNT Stress-E signaling pathways, when compared to WT (AT1R+/+) mice, further confirming that AT signaling is critical, and conceivably upstream to these established Stress-E pathways. However, AT-AT1R signaling in Stress-E states is a double-edged sword: on one hand it was essential to sustain erythropoiesis, while on the other hand it resulted in significant ROS production. This was seen in SCA, and in WT mice where Stress-E was associated with higher RBC ROS (Figure 1a). AT is known to activate NADPH oxidase (NOX), to generate ROS in other cell types. But pharmacological inhibition of NOX signaling, or genetic deficiency of Gp22phox (a common subunit of NOX isoforms) in SCA mice did not lower RBC ROS. However, AT-AT1R signaling in Stress-E increased the mitochondrial mass in erythroid precursors, and also resulted in retention of dysfunctional mitochondria with lower membrane potential in the enucleated erythrocytes, a source of high ROS (Figure 1b-c). Hence, AT signaling in Stress-E inhibited mitophagy, which is required for clearance of mitochondria after enucleation. The transcriptome profile corroborated these findings: With Stress-E, WT AT1R-/- mice had upregulation of genes involved in mitophagy, and genes involved in maintaining mitochondrial integrity/quality and cellular redox homeostasis under oxidative stress. Taken together, our results show that AT signaling plays a critical role in increasing erythroid cell mass during Stress-E, but also downregulates mitophagy, antioxidant genes, and results in increased retention of dysfunctional mitochondria, which are the source of high RBC ROS that mediates end-organ injury. Disclosures Kalfa: Forma Therapeutics, Inc: Research Funding; Agios Pharmaceuticals, Inc: Consultancy, Research Funding. Malik:Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents & Royalties.

Introduction: Hydroxyurea has well-described clinical and laboratory benefits in sickle cell anemia (SCA). Traditionally, hemoglobin concentration (Hb) and fetal hemoglobin (HbF) are used to assess treatment response, but these aggregate parameters do not fully capture the range of erythrocyte abnormalities in SCA. Dense red blood cells (DRBCs) are defined as RBCs with density >1.11 g/mL as measured by density-gradient fractionation methods, which are tedious and require technical expertise. This subpopulation of dehydrated RBCs has been associated with increased SCA severity but has not been extensively studied in children. Here we describe the utility of rapid, automated DRBC quantitation and show that the percent of DRBCs (%DRBCs) is a robust biomarker of hydroxyurea response in children with SCA. Methods: The Therapeutic Response Evaluation and Adherence Trial (TREAT, NCT02286154) was a single-center prospective study that demonstrated individualized, pharmacokinetic (PK)-guided hydroxyurea dosing in young patients (mean age 12.1 months at initiation) results in robust and sustained HbF levels >30-40% for most adherent patients. The longitudinal follow-up phase of TREAT aims to comprehensively evaluate hematologic parameters and organ function to demonstrate the long-term benefits of this treatment strategy. During clinic visits (every 3-6 months), TREAT participants had complete blood counts (CBCs) with the ADVIA® 2120i system. This automated analyzer quantifies hyperchromic RBCs within seconds by directly measuring cell hemoglobin concentration, where DRBCs are known to correspond to RBCs with a measured MCHC >41 g/dL. Data were analyzed at baseline and after at least 6 months of hydroxyurea therapy (M≥6). α- and β-globin genotypes were determined for all patients. Results: Thirty-three TREAT participants had ADVIA data available for analysis (Table). All had homozygous SCA except one with sickle-β0-thalasemia; 17 (51.5%) were male. The median age at hydroxyurea initiation was 10.3 months (mean 26.1 months; range 6 months - 17 years). The median duration of hydroxyurea therapy was 27.7 months (mean 31.5). At baseline, median %DRBCs was 2.1%. Baseline %DRBCs were directly correlated with age at initiation of hydroxyurea (p=0.022) and inversely correlated with baseline HbF (p=0.001). After initiation of hydroxyurea (M≥6), there was a 47.6% reduction in %DRBCs (p=0.001). Median %DRBCs at M≥6 was directly correlated with absolute reticulocyte (ARC) (p=0.002), absolute neutrophil (ANC) (p=0.003) and platelet counts (p Conclusions: DRBCs can be rapidly quantified without the tedium of classical density-gradient fractionation methods using the automated ADVIA 2120i hematology analyzer, which directly measures hyperchromic RBCs. These data from TREAT demonstrate the feasibility of serial %DRBC measurements in clinical trials and clinical practice. TREAT participants had low baseline DRBCs compared to untreated adult SCA patients (reported mean ≈12%), reflecting the young age of these patients. Nevertheless, there was a significant decline in %DRBCs with treatment that correlated strongly with the known laboratory benefits of hydroxyurea. In conclusion, automated DRBC measurements are a robust biomarker that can be incorporated in a panel of laboratory measurements of response to hydroxyurea (and severity of SCA). A larger, multicenter study of PK-guided hydroxyurea therapy (HOPS, NCT03789591) is currently underway in 11 US sickle cell centers and will provide generalizable data on DRBC responses with optimal hydroxyurea therapy. Disclosures Malik: Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents & Royalties. Kalfa:Agios Pharmaceuticals, Inc: Consultancy, Research Funding; Forma Therapeutics, Inc: Research Funding.