Marrow Erythroid Cells Research Articles

Prolyl hydroxylase inhibitor desidustat attenuates autoimmune hemolytic anemia in mice

Autoimmune hemolytic anemia (AIHA) is a heterogeneous group of diseases mediated by autoantibody directed against RBCs causing hemolysis and anemia. AIHA develops rapidly or over time, depending on the triggering factor. Desidustat is a prolyl hydroxylase inhibitor clinically used for the treatment of chronic kidney disease (CKD)-induced anemia. In this study, we investigated the effect of desidustat in preclinical model of AIHA. We used rat RBC for induction of AIHA in mice. These mice were then treated with desidustat (15 mg/kg, PO, once a day) for eight weeks. Desidustat treatment increased hemoglobin, RBC and hematocrit and decreased WBC and lymphocytes. This treatment suppressed serum LDH, oxidative stress in RBCs, antibody titer and antibody deposition on RBC surface, and increased RBC lifespan. Serum and spleen iron along with spleen mass and oxidative stress were decreased by desidustat. Bone marrow iron was increased and expression of CD71 (cell surface marker for early erythroid progenitor) and TER-119 (cell surface marker for late erythroid progenitor) in bone marrow were found to be elevated by desidustat by treatment. This treatment also suppressed deposition of membrane-bound antibody in late erythroid cells. The treatment showed reduction in total splenic cells, CD71 and TER-119 positive cells in the spleen. Thus, desidustat treatment increased erythropoiesis, early maturation of bone marrow erythroid cells having longer RBC life span due to decrease in the antibody-mediated lysis of RBCs and its progenitors leading to reduced oxidative stress. Thus, desidustat can be a good therapeutic option for treatment of AIHA.

A subpopulation of human bone marrow erythroid cells displays a myeloid gene expression signature similar to that of classic monocytes.

Erythroid cells, serving as progenitors and precursors to erythrocytes responsible for oxygen transport, were shown to exhibit an immunosuppressive and immunoregulatory phenotype. Previous investigations from our research group have revealed an antimicrobial gene expression profile within murine bone marrow erythroid cells which suggested a role for erythroid cells in innate immunity. In the present study, we focused on elucidating the characteristics of human bone marrow erythroid cells through comprehensive analyses, including NanoString gene expression profiling utilizing the Immune Response V2 panel, a BioPlex examination of chemokine and TGF-beta family proteins secretion, and analysis of publicly available single-cell RNA-seq data. Our findings demonstrate that an erythroid cell subpopulation manifests a myeloid-like gene expression signature comprised of antibacterial immunity and neutrophil chemotaxis genes which suggests an involvement of human erythroid cells in the innate immunity. Furthermore, we found that human erythroid cells secreted CCL22, CCL24, CXCL5, CXCL8, and MIF chemokines. The ability of human erythroid cells to express these chemokines might facilitate the restriction of immune cells in the bone marrow under normal conditions or contribute to the ability of erythroid cells to induce local immunosuppression by recruiting immune cells in their immediate vicinity in case of extramedullary hematopoiesis.

Expression of microRNA-155 in thalassemic erythropoiesis.

Ineffective erythropoiesis (IE) is the primary cause of anemia and associated pathologies in β-thalassemia. The characterization of IE is imbalance of erythroid proliferation and differentiation, resulting in increased erythroblast proliferation that fails to differentiate and gives rise to enucleate RBCs. MicroRNAs (miRs) are known to play important roles in hematopoiesis. miR-155 is a multifunctional molecule involved in both normal and pathological hematopoiesis, and its upregulation is observed in patients with β-thalassemia/HbE. However, the expression and function of miR-155, especially in β-thalassemia, have not yet been explored. To study miR-155 expression in thalassemia, erythroblast subpopulations, CD45-CD71+Ter-119+ and CD45-CD71-Ter-119+ were collected from β IVSII-654 thalassemic bone marrow. Additionally, a two-phase culture of mouse bone marrow erythroid progenitor cells was performed. Expression of miR-155 and predicted mRNA target genes, c-myc, bach-1 and pu-1, were determined by quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and normalized to small nucleolar RNA (snoRNA) 202 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. To investigate the effect of miR-155 expression, erythroblasts were transfected with miR-inhibitor and -mimic in order to elevate and eliminate miR-155 expression, respectively. Erythroid cell differentiation was evaluated by Wright-Giemsa staining and flow cytometry. miR-155 was upregulated, both in vivo and in vitro, during erythropoiesis in β-thalassemic mice. Our study revealed that gain- and loss of function of miR-155 were involved in erythroid proliferation and differentiation, and augmented proliferation and differentiation of thalassemic mouse erythroblasts may be associated with miR-155 upregulation. miR-155 upregulation in β-thalassemic mice significantly increased the percentage of basophilic and polychromatic erythroblasts. Conversely, a significant decrease in percentage of basophilic and polychromatic erythroblasts was observed in β-thalassemic mice transfected with anti-miR-155 inhibitor. We also examined the mRNA targets (c-myc, bach-1 and pu-1) of miR-155, which indicated that c-myc is a valid target gene of miR-155 that regulates erythroid differentiation. miR-155 regulates IE in β-thalassemia via c-myc expression controlling erythroblast proliferation and differentiation.

Single-cell multi-omics reveal stage of differentiation and trajectory-dependent immunity-related gene expression patterns in human erythroid cells.

The role of Erythroid cells in immune regulation and immunosuppression is one of the emerging topics in modern immunology that still requires further clarification as Erythroid cells from different tissues and different species express different immunoregulatory molecules. In this study, we performed a thorough investigation of human bone marrow Erythroid cells from adult healthy donors and adult acute lymphoblastic leukemia patients using the state-of-the-art single-cell targeted proteomics and transcriptomics via BD Rhapsody and cancer-related gene copy number variation analysis via NanoString Sprint Profiler. We found that human bone marrow Erythroid cells express the ARG1, LGALS1, LGALS3, LGALS9, and C10orf54 (VISTA) immunosuppressive genes, CXCL5, CXCL8, and VEGFA cytokine genes, as well as the genes involved in antimicrobial immunity and MHC Class II antigen presentation. We also found that ARG1 gene expression was restricted to the single erythroid cell cluster that we termed ARG1-positive Orthochromatic erythroblasts and that late Erythroid cells lose S100A9 and gain MZB1 gene expression in case of acute lymphoblastic leukemia. These findings show that steady-state erythropoiesis bone marrow Erythroid cells express myeloid signature genes even without any transdifferentiating stimulus like cancer.

Cimetidine Does Not Inhibit 5-Aminolevulinic Acid Synthase or Heme Oxygenase Activity: Implications for Treatment of Acute Intermittent Porphyria and Erythropoietic Protoporphyria.

Acute intermittent porphyria (AIP) is characterized by acute neurovisceral attacks that are precipitated by the induction of hepatic 5-aminolevulinic acid synthase 1 (ALAS1). In erythropoietic protoporphyria (EPP), sun exposure leads to skin photosensitivity due to the overproduction of photoreactive porphyrins in bone marrow erythroid cells, where heme synthesis is primarily driven by the ALAS2 isozyme. Cimetidine has been suggested to be effective for the treatment of both AIP and EPP based on limited case reports. It has been proposed that cimetidine acts by inhibiting ALAS activity in liver and bone marrow for AIP and EPP, respectively, while it may also inhibit the hepatic activity of the heme catabolism enzyme, heme oxygenase (HO). Here, we show that cimetidine did not significantly modulate the activity or expression of endogenous ALAS or HO in wildtype mouse livers or bone marrow. Further, cimetidine did not effectively decrease hepatic ALAS activity or expression or plasma concentrations of the putative neurotoxic porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), which were all markedly elevated during an induced acute attack in an AIP mouse model. These results show that cimetidine is not an efficacious treatment for acute attacks and suggest that its potential clinical benefit for EPP is not via ALAS inhibition.

Effect of the LSD1 inhibitor RN-1 on γ-globin and global gene expression during erythroid differentiation in baboons (Papio anubis).

Elevated levels of Fetal Hemoglobin interfere with polymerization of sickle hemoglobin thereby reducing anemia, lessening the severity of symptoms, and increasing life span of patients with sickle cell disease. An affordable, small molecule drug that stimulates HbF expression in vivo would be ideally suited to treat the large numbers of SCD patients that exist worldwide. Our previous work showed that administration of the LSD1 (KDM1A) inhibitor RN-1 to normal baboons increased Fetal Hemoglobin (HbF) and was tolerated over a prolonged treatment period. HbF elevations were associated with changes in epigenetic modifications that included increased levels of H3K4 di-and tri-methyl lysine at the γ-globin promoter. While dramatic effects of the loss of LSD1 on hematopoietic differentiation have been observed in murine LSD1 gene deletion and silencing models, the effect of pharmacological inhibition of LSD1 in vivo on hematopoietic differentiation is unknown. The goal of these experiments was to investigate the in vivo mechanism of action of the LSD1 inhibitor RN-1 by determining its effect on γ-globin expression in highly purified subpopulations of bone marrow erythroid cells enriched for varying stages of erythroid differentiation isolated directly from baboons treated with RN-1 and also by investigating the effect of RN1 on the global transcriptome in a highly purified population of proerythroblasts. Our results show that RN-1 administered to baboons targets an early event during erythroid differentiation responsible for γ-globin repression and increases the expression of a limited number of genes including genes involved in erythroid differentiation such as GATA2, GFi-1B, and LYN.

Unravelling the Mechanism behind Ineffective Erythropoiesis in Sickle Cell Disease: The Role of GATA-1

Background & Objectives: Ineffective erythropoiesis (IE) is an important factor in many types of anemias, including beta-thalassemia. Recently, a study by El Hoss et al provided compelling evidence for IE being an important part of the pathophysiology of sickle cell anemia (SCA) (PMID: 32855279). Furthermore, we developed a clinical index to measure IE and showed that patients with the SS genotype, but not SC, had increased IE compared to healthy donors (HD) (PMID: 34670360). However, the molecular mechanisms of IE in SCA, its relationship to disease heterogeneity and its potential as a therapeutic target, remain unknown. Importantly, GATA1, an essential erythroid transcription factor known to be downregulated in haematological conditions presenting with IE, has not been previously investigated in SCA. Thus, the aim of this project is to investigate the molecular basis of IE in SCA by focusing on GATA1 functions in erythropoiesis. Methods: HUDEP-2 cells, gene-edited sickle HUDEP-2 (SS HUDEP-2) (PMID: 32405513) cells and CD34+ cells isolated from individuals with HbSS and healthy donors, were differentiated ex vivo using a two-phase culture and (i) erythroid differentiation kinetics, (ii) markers of oxidative stress, (iii) GATA1 expression and (iv) antioxidant response factors NRF2, FOXO3 and heme-oxygenase 1 (HO-1) were assayed every other day during phase 2. IE was also studied in SS Berkeley transgenic mouse (PMID: 16166585). Both the spleen and the bone marrow were investigated in AA, AS and SS mice. Results: No significant differences were observed in the differentiation kinetics and in levels of oxidative stress in in vitro differentiated HUDEP-2 and SS HUDEP-2 cells. Moreover, GATA1 levels were similar in both cases. Importantly, ex vivo differentiation of human SS CD34+ cells showed a striking delay accompanied by high oxidative stress and a decrease in GATA1 protein levels (Figure 1) compared to normal controls. We also found a significant decrease in FOXO3, NRF2 and HO-1 levels in SS erythroblasts compared to healthy donor erythroblasts. IE was evident in SS mice compared to AA and AS controls. SS mice presented with higher levels of reactive oxygen species (ROS) in both the bone marrow and the spleen tissue. Histological studies showed that the SS Berkley mouse has a very congested bone marrow with a disorganized architecture. Moreover, we showed a significant decrease in GATA1 protein in SS spleen and bone marrow erythroid cells. Conclusion: Using ex vivo differentiation of primary SS cells and an in vivo mouse model for SCA, we show for the first time that aberrant GATA-1 levels play a key role during IE in SCA. During SS erythropoiesis, a significant reduction in the protein levels of the GATA1 erythroid master transcription factor and of FOXO3 and NF-E2, important transcriptional regulators of the oxidative stress response occur. These same changes were not seen in SS HUDEP-2 cells, suggesting that abnormalities of the bone marrow niche are central to abnormal erythropoiesis in SCA. Modification of the bone marrow niche and manipulation of GATA-1 levels may be important therapeutic tools in SCA.

Whole-Genome CRISPR-Cas9 Screening Identifies Genes Required for Human and Mouse Erythroid Development

Red blood cell (RBC) development is regulated by a few external signaling molecules which act to influence the ongoing intracellular processes responsible for producing erythrocytes. While much is known about the signaling molecules important for blood production, comparatively little is known about the genes required for successfully generating erythrocytes. Several genes have already been identified as essential for RBC development through loss-of-function studies in the mouse or in human erythroid cells in vitro. However, these studies have been limited in scope to genes that are already suspected or known to influence RBC production. Since there are ~10,000 genes expressed in erythroid cells, it is unclear how many of these genes are functionally required for erythroid development. To define the repertoire of genes required for human erythroid development, we performed a genome-scale CRISPR knock-out screen in the human erythroid progenitor cell line HUDEP-2, using the GeCKOv2 lentiviral library, which delivers Cas9 and one of 6 sgRNAs targeting virtually every gene in the genome. Following viral transduction and cell recovery for 9 days, we collected HUDEP-2 cells prior to the onset of differentiation as well as differentiated orthochromatic erythroblasts (right prior to enucleation) using flow sorting, on the basis of CD49d expression (CD49d is downregulated in the final stages of erythroid differentiation). As expected, sgRNAs targeting known erythroid essential genes (such as GATA1 and EPOR) were depleted in HUDEP-2 cells compared to the sgRNA library. Notably, we also identified genes for which sgRNAs were depleted in differentiated versus undifferentiated erythroid cells, representing genes that are likely required for terminal erythroid differentiation (such as ZFPM1, ALAS2, etc..). This supports the utility of the screen to identify novel regulators of erythropoiesis. Among highly ranked genes, NHLRC2, which was previously implicated in hemolytic anemia, was identified to be required in HUDEP-2 cells, suggesting that NHLRC2 is intrinsically required for human erythroid development. To validate the role of NHLRC2 in human erythropoiesis, we down-regulated NHLRC2 in primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) undergoing erythroid differentiation, using one of 4 independent shRNAs. NHLRC2 down-regulation resulted in impaired proliferation and differentiation of human erythroid cells in vitro. To validate the results of the genome-scale screen, we generated a secondary library dedicated to examining genes that positively or negatively regulate the final stages of erythroid differentiation, while excluding common essential genes required for the survival of more than 90% of immortalized cell lines screened by the BROAD institute. The secondary screen yielded a list of over 500 genes found to influence erythroid differentiation (FDR<0.01). One of the novel genes, VAC14, was identified as a highly ranked gene in the secondary screen for erythroid differentiation. Validation experiments demonstrated that VAC14 down-regulation using one of 3 independent shRNAs result in erythroid cell proliferation defects, both in HUDEP2 cells and in erythroid cells derived from primary human HSPCs. Since germline loss of VAC14 is embryonic lethal in the mouse, to validate the role of VAC14 in erythropoiesis in vivo, we transplanted fetal livers from Vac14 null mice (or wild-type [WT] control mice) into WT recipients and examined the VAC14 deficient hematopoietic compartment. Mice transplanted with Vac14 null HSCs exhibited a profound reduction in absolute numbers of bone marrow erythroid cells with evidence of a block in erythroid maturation. Impaired bone marrow erythropoiesis was largely (but incompletely) compensated for by splenic extramedullary erythropoiesis. Vac14 null hematopoietic cells exhibited pronounced cytoplasmic vacuolation, also affecting numerous stages of erythroid cells. Altogether, we have performed an unbiased genome-scale CRISPR knock-out screen that identified (and validated) novel genes required in erythropoiesis.

Murine Bone Marrow Erythroid Cells Have Two Branches of Differentiation Defined by the Presence of CD45 and a Different Immune Transcriptome Than Fetal Liver Erythroid Cells.

Mouse erythropoiesis is a multifaceted process involving the intricate interplay of proliferation, differentiation, and maturation of erythroid cells, leading to significant changes in their transcriptomic and proteomic profiles. While the immunoregulatory role of murine erythroid cells has been recognized historically, modern investigative techniques have been sparingly applied to decipher their functions. To address this gap, our study sought to comprehensively characterize mouse erythroid cells through contemporary transcriptomic and proteomic approaches. By evaluating CD71 and Ter-119 as sorting markers for murine erythroid cells and employing bulk NanoString transcriptomics, we discerned distinctive gene expression profiles between bone marrow and fetal liver-derived erythroid cells. Additionally, leveraging flow cytometry, we assessed the surface expression of CD44, CD45, CD71, and Ter-119 on normal and phenylhydrazine-induced hemolytic anemia mouse bone marrow and splenic erythroid cells. Key findings emerged: firstly, the utilization of CD71 for cell sorting yielded comparatively impure erythroid cell populations compared to Ter-119; secondly, discernible differences in immunoregulatory molecule expression were evident between erythroid cells from mouse bone marrow and fetal liver; thirdly, two discrete branches of mouse erythropoiesis were identified based on CD45 expression: CD45-negative and CD45-positive, which had been altered differently in response to phenylhydrazine. Our deductions underscore (1) Ter-119's superiority over CD71 as a murine erythroid cell sorting marker, (2) the potential of erythroid cells in murine antimicrobial immunity, and (3) the importance of investigating CD45-positive and CD45-negative murine erythroid cells separately and in further detail in future studies.

The transcriptomic landscape of normal and ineffective erythropoiesis at single-cell resolution.

The anemias of myelodysplastic syndrome (MDS) and Diamond Blackfan anemia (DBA) are generally macrocytic and always reflect ineffective erythropoiesis yet result from diverse genetic mutations. To delineate shared mechanisms that lead to cell death, we studied the fate of single erythroid marrow cells from individuals with DBA or MDS-5q. We defined an unhealthy (vs healthy) differentiation trajectory using transcriptional pseudotime and cell surface proteins. The pseudotime trajectories diverge immediately after cells upregulate transferrin receptor (CD71), import iron, and initiate heme synthesis, although cell death occurs much later. Cells destined to die express high levels of heme-responsive genes, including ribosomal protein and globin genes, whereas surviving cells downregulate heme synthesis and upregulate DNA damage response, hypoxia, and HIF1 pathways. Surprisingly, 24%± 12% of cells from control subjects follow the unhealthy trajectory, implying that heme might serve as a rheostat directing cells to live or die. When heme synthesis was inhibited with succinylacetone, more DBA cells followed the healthy trajectory and survived. We also noted high numbers of messages with retained introns that increased as erythroid cells matured, confirmed the rapid cycling of colony forming unit-erythroid, and demonstrated that cell cycle timing is an invariant property of differentiation stage. Including unspliced RNA in pseudotime determinations allowed us to reliably align independent data sets and accurately query stage-specific transcriptomic changes. MDS-5q (unlike DBA) results from somatic mutation, so many normal (unmutated) erythroid cells persist. By independently tracking erythroid differentiation of cells with and without chromosome 5q deletions, we gained insight into why 5q+ cells cannot expand to prevent anemia.

Non-Permissive Parvovirus B19 Infection: A Reservoir and Questionable Safety Concern in Mesenchymal Stem Cells.

Mesenchymal stromal/stem cells (MSCs) are multipotent cells with differentiation, immunoregulatory and regenerative properties. Because of these features, they represent an attractive tool for regenerative medicine and cell-based therapy. However, MSCs may act as a reservoir of persistent viruses increasing the risk of failure of MSCs-based therapies and of viral transmission, especially in immunocompromised patients. Parvovirus B19V (B19V) is a common human pathogen that infects bone marrow erythroid progenitor cells, leading to transient or persistent anemia. Characteristics of B19V include the ability to cross the placenta, infecting the fetus, and to persist in several tissues. We thus isolated MSCs from bone marrow (BM-MSCs) and fetal membrane (FM-MSCs) to investigate their permissiveness to B19V infection. The results suggest that both BM- and FM- MSCs can be infected by B19V and, while not able to support viral replication, allow persistence over time in the infected cultures. Future studies are needed to understand the potential role of MSCs in B19V transmission and the conditions that can favor a potential reactivation of the virus.

Abnormal expression of CXCR5+CD8+ T cells and CXCL13 in severe aplastic anemia patients and their correlation with hematological parameters

Objective: To analyze the expression of C-X-C chemokine receptor 5 (CXCR5)+CD8+ T cells and plasma C-X-C motif chemokine 13 (CXCL13) in severe aplastic anemia (SAA) patients and their correlations with hematological parameters. Methods: The clinical data of 35 SAA patients in the Hematology Department of Tianjin Medical University General Hospital from January 2018 to September 2021 were retrospectively analyzed. The patients were divided into two groups according to whether they had received the medication: untreated SAA group and recovery SAA group. In untreated group, there were 18 patients who had not received any medication, with 9 males and 9 females, and aged 51 (18-76) years. In recovery SAA group, there were 17 patients who were separated from component blood transfusion after the immunosuppressive treatment with anti-thymocyte globulin (ATG) combined with cyclosporine A (CsA), with 7 males and 10 females, and aged 46 (16-70) years. Meanwhile, 20 healthy controls were also selected, including 8 males and 12 females, and aged 45(15-72) years. Peripheral blood and bone marrow samples were collected from SAA patients, while peripheral blood samples were obtained from healthy controls. Flow cytometry was used to detect the percentage of CXCR5+CD8+ T cells in peripheral blood and bone marrow samples. The concentration of plasma CXCL13 was measured by enzyme-linked immunosorbent assay (ELISA). The correlations between the percentage of CXCR5+CD8+ T cells and the concentration of CXCL13, as well as the correlations between these two parameters and the hematological parameters were analyzed by Spearman correlation analysis. Results: The proportion of CXCR5+CD8+ T cells in the bone marrow of untreated SAA group was (4.9±2.9)%, which was higher than that of recovery SAA group (2.7±1.5)%, with a statistically significant difference (t=2.34, P=0.027). The proportion of CXCR5+CD8+ T cells in peripheral blood of untreated SAA group, recovery SAA group and healthy control group was (8.4±4.2)%, (3.8±2.3)% and (2.6±2.0)% respectively. The proportion of CXCR5+CD8+ T cells in peripheral blood of untreated SAA group was higher than that of recovery SAA group and healthy control group (both P<0.05). The plasma CXCL13 concentration in untreated SAA group was (97.2±46.8) ng/L, which was significantly higher than that in recovery SAA group [(54.9±20.9) ng/L] and healthy control group [(47.6±17.3) ng/L] (both P<0.05). The proportion of CXCR5+CD8+ T cells in peripheral blood of SAA patients was positively correlated with the concentration of plasma CXCL13 (r=0.545, P<0.001). The proportion of peripheral blood CXCR5+CD8+ T cells in SAA patients was negatively correlated with white blood cell count, platelets count, percentage of neutrophils, absolute neutrophils count, percentage of reticulocytes, absolute reticulocytes count, bone marrow myeloid cells, bone marrow erythroid cells and megakaryocytes count (r=-0.556, -0.392, -0.617, -0.615, -0.395, -0.543, -0.432, -0.484 and -0.523, all P<0.05). The proportion of peripheral blood CXCR5+CD8+ T cells was positively correlated with the percentage of peripheral blood lymphocytes and bone marrow lymphoid cells (r=0.593 and 0.556, both P<0.05). Meanwhile, the concentration of plasma CXCL13 in SAA patients was negatively correlated with white blood cell count, absolute neutrophils count, percentage of reticulocytes, absolute reticulocytes count and bone marrow myeloid cells (r=-0.447, -0.446, -0.498, -0.407 and -0.456, all P<0.05), but positively correlated with bone marrow lymphoid cells (r=0.384, P<0.05). Conclusions: The proportion of CXCR5+CD8+ T cells and the concentration of plasma CXCL13 increases in SAA patients. The proportion of CXCR5+CD8+ T cells in peripheral blood is positively correlated with the concentration of CXCL13. Moreover, the proportion of CXCR5+CD8+ T cells and the concentration of CXCL13 are correlated with many hematological parameters, which may play a critical role in the immune pathogenesis of SAA.

Shape-aware fine-grained classification of erythroid cells

Fine-grained classification and counting of bone marrow erythroid cells are vital for evaluating the health status and formulating therapeutic schedules for leukemia or hematopathy. Due to the subtle visual differences between different types of erythroid cells, it is challenging to apply existing image-based deep learning models for fine-grained erythroid cell classification. Moreover, there is no large open-source datasets on erythroid cells to support the model training. In this paper, we introduce BMEC (Bone Morrow Erythroid Cells), the first large fine-grained image dataset of erythroid cells, to facilitate more deep learning research on erythroid cells. BMEC contains 5,666 images of individual erythroid cells, each of which is extracted from the bone marrow erythroid cell smears and professionally annotated to one of the four types of erythroid cells. To distinguish the erythroid cells, one key indicator is the cell shape which is closely related to the cell growth and maturation. Therefore, we design a novel shape-aware image classification network for fine-grained erythroid cell classification. The shape feature is extracted from the shape mask image and aggregated to the raw image feature with a shape attention module. With the shape-attended image feature, our network achieved superior classification performance (81.12% top-1 accuracy) on the BMEC dataset comparing to the baseline methods. Ablation studies also demonstrate the effectiveness of incorporating the shape information for the fine-grained cell classification. To further verify the generalizability of our method, we tested our network on two additional public white blood cells (WBC) datasets and the results show our shape-aware method can generally outperform recent state-of-the-art works on classifying the WBC. The code and BMEC dataset can be found on https://github.com/wangye8899/BMEC .

Abstract 1374: The novel PARP1-selective inhibitor AZD5305 has reduced hematological toxicity when compared to PARP1/2 inhibitors in pre-clinical models

Abstract Current clinical poly (ADP-ribose) polymerase (PARP) inhibitors target both PARP1 and PARP2 and they all cause clinical cytopenias with varying severity. Understanding the mechanism underlying the hematological toxicity of these agents is key for the rational design of a best-in-class molecule with greater therapeutic potential, both as monotherapy and in combination with chemotherapy. We validated the rat as a physiologically competent translational model to investigate PARP inhibitor-driven hematological toxicity. Here we demonstrate that in comparison with a representative PARP1/2 inhibitor, olaparib, the novel highly potent PARP1-selective inhibitor and trapper, AZD5305, does not cause hematological toxicity as a monotherapy in pre-clinical rat models at predicted clinically efficacious exposures. Thus, monotherapy toxicity of PARP1/2 inhibitors likely depends on PARP2 inhibition. Next, we proceeded to investigate whether PARP1-selective inhibition would be better tolerated in chemotherapy combinations than PARP1/2 inhibition. We performed a rat in vivo study comparing daily olaparib or daily AZD5305 at matched exposures in combination with one cycle of intravenous carboplatin for 14 days. We show that olaparib and PARP1-selective AZD5305 cause comparable exacerbation of carboplatin-induced peripheral blood effects implicating PARP1 inhibition in combination-driven hematological toxicity. Importantly however, AZD5305+carboplatin showed improved hematological tolerability over olaparib+carboplatin because peripheral reticulocytes and bone marrow erythroid precursor cells recover in the presence of continuous AZD5305 but not in the presence of continuous olaparib. Importantly, this differentiation was maintained in a subsequent rat in vivo study, where daily olaparib or daily AZD5305 were combined with two three-weekly cycles of a higher dose of carboplatin to more closely mimic clinical protocol. AZD5305+carboplatin was associated with a more rapid recovery of reticulocytes, red blood cells and hemoglobin following both cycles of carboplatin. In contrast, olaparib+carboplatin was associated with a slower recovery resulting in a more sustained reduction in red cells and hemoglobin during both the first and second cycle of carboplatin. Thus, in rodents the novel potent PARP1-selective inhibitor AZD5305 has improved hematological tolerability over dual PARP1/2 inhibitors, both as a monotherapy and in carboplatin combinations. Citation Format: Sonja J. Gill, Ruth Macdonald, Carmen Pin, Rob Collins, Emilyanne Leonard, Gareth Maglennon, Andy Pike, Peter Cotton, Glen Hawthorne, Jordan Pugh, Rebecca Sargeant, Daniel Sutton, James Atkinson, Stewart Jones, Sarah Chinery, Mark Anderton. The novel PARP1-selective inhibitor AZD5305 has reduced hematological toxicity when compared to PARP1/2 inhibitors in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1374.

Malus Domestica Peel Extract Counters Carboplatin Induced Fall in Red Blood Cell Count and Bone Marrow Erythroid Cell Percentage in Mice

Introduction: The Chemotherapy-Induced Anemia (CIA) is a complex phenomenon impacted by many treatments and patient related variables. Some variations within CIA are type of chemotherapeutic agent, dose and schedule of administration of agent, type as well as stage of cancer, time of intervention, age, gender, nutritional status and renal function. The variety of patient presentation and symptomatology in CIA is very thought-provoking while setting up widespread measures to oversee ideal administration of treatments. This study will be focused on protective role of apple peel extract (APPE) on carboplatin induced fall in blood RBC count bone marrow erythroid cell percentage in mice.Aims &amp; Objective: To evaluate the protective effect of Malus domestica peel extract on carboplatin induced fall in red blood cell count and bone marrow erythroid cell percentage in mice.Place and duration of study: The study was conducted at Pharmacology Department of Postgraduate Medical institute, Lahore in June 2016. The total duration of study was 1 month.Material &amp; Methods: Forty adult male mice were randomly divided into 5 groups (n=8) and maintained on standard diet and water adlibitum. Group A was negative control. Group B was disease control wherein each animal received one dose of 125mg/kg carboplatin intraperitoneally. Group C, D &amp; E experimental groups were administered same dose carboplatin followed by the ethanolic apple peel extract in three different single oral doses of 25, 50 &amp; 75mg/kg respectively for 7 days. Blood &amp; tissue sampling was done on the 8th day. Data was analyzed using Graf Pad prism 7.Results: Acute Carboplatin anaemia resulted in 4.01±0.851012/L RBC Count &amp; 21.6±1.06% bone marrow erythroid cell percentage in Disease Control Group B. Subsequent Apple peel extract administration in a dose of 25, 50 and 75mg/kg\ countered carboplatin induced fall in Groups C, D &amp; E, simultaneously raising RBC counts &amp; bone marrow erythroid cell percentage to (6.19±0.70×1012/L &amp; 21.3±1.28%) (6.56±0.37×1012/L &amp; 25.3±1.04%) and (6.26±0.74×1012/L &amp; 23.5±1.41%.) respectively. The increase in RBC as well as erythroid cell counts was most significant with the dose of 50mg/kg and nearing that of healthy control levels (Group A)Conclusion: Apple Peel Extract effectively prevented carboplatin induced fall in RBC count and erythroid cellpercentage and produced most significant results in a dose of 50 mg/kg.

Gene Therapy for Beta Thalassemia: Preliminary Results from the PHASE I/II Tiget-Bthal Trial of Autologous Hematopoietic Stem Cells Genetically Modified with GLOBE Lentiviral Vector

Gene Therapy for Beta Thalassemia: Preliminary Results from the PHASE I/II Tiget-Bthal Trial of Autologous Hematopoietic Stem Cells Genetically Modified with GLOBE Lentiviral Vector

EpoR-tdTomato-Cre mice enable identification of EpoR expression in subsets of tissue macrophages and hematopoietic cells

AbstractThe erythropoietin receptor (EpoR) has traditionally been thought of as an erythroid-specific gene. Notably, accumulating evidence suggests that EpoR is expressed well beyond erythroid cells. However, the expression of EpoR in non-erythroid cells has been controversial. In this study, we generated EpoR-tdTomato-Cre mice and used them to examine the expression of EpoR in tissue macrophages and hematopoietic cells. We show that in marked contrast to the previously available EpoR-eGFPcre mice, in which a very weak eGFP signal was detected in erythroid cells, tdTomato was readily detectable in both fetal liver (FL) and bone marrow (BM) erythroid cells at all developmental stages and exhibited dynamic changes during erythropoiesis. Consistent with our recent finding that erythroblastic island (EBI) macrophages are characterized by the expression of EpoR, tdTomato was readily detected in both FL and BM EBI macrophages. Moreover, tdTomato was also detected in subsets of hematopoietic stem cells, progenitors, megakaryocytes, and B cells in BM as well as in spleen red pulp macrophages and liver Kupffer cells. The expression of EpoR was further shown by the EpoR-tdTomato-Cre–mediated excision of the floxed STOP sequence. Importantly, EPO injection selectively promoted proliferation of the EpoR-expressing cells and induced erythroid lineage bias during hematopoiesis. Our findings imply broad roles for EPO/EpoR in hematopoiesis that warrant further investigation. The EpoR-tdTomato-Cre mouse line provides a powerful tool to facilitate future studies on EpoR expression and regulation in various non-hematopoietic cells and to conditionally manipulate gene expression in EpoR-expressing cells for functional studies.

In Vivo Characterization of Ftx-6058, a Novel Small Molecular Fetal Hemoglobin Inducer for Sickle Cell Disease

Sickle cell disease (SCD) results from genetic mutation in the β-globin gene encoding a subunit of the adult form of hemoglobin (HbA), leading to red blood cell (RBC) deformation and disease pathology. It has been demonstrated that reactivation of the fetal ortholog of the hemoglobin beta subunit, HBγ (also referred to as HBG proteins), can prevent or reduce disease-related pathophysiology. In SCD, the presence of HBG protein in hemoglobin tetramers prevents sickle hemoglobin polymerization under deoxygenated conditions and therefore may be of therapeutic benefit in SCD. FTX 6058, a novel orally bioavailable small molecule, is in development for the treatment of sickle cell disease (SCD) by Fulcrum Therapeutics. FTX-6058 was demonstrated to inhibit the novel biological target and elevate the expression of HBγ, resulting in induction of fetal hemoglobin (HbF) tetramer in differentiated human primary CD34+ cells. The in vivo target engagement (TE) and pharmacologic effects of FTX-6058 were characterized in wild-type CD-1 mice and humanized Townes SCD mice, with TE also confirmed in non-human primates. In CD-1 mice, once-daily (QD) FTX-6058 oral administration induced TE in a time- and dose-dependent manner and most markedly in erythroid lineage (Ter119+) cells derived from bone marrow, the putative therapeutic compartment, and increased transcript levels of Hbb-bh1, a murine embryonic hemoglobin surrogate for human HBG gene. Steady state TE in circulating monocytes, following repeated QD FTX-6058 administration, correlated well with that in bone marrow-derived erythroid cells, suggesting peripheral monocytes as a suitable surrogate for assessing erythroid TE activity in Fulcrum's Phase 1 study. In non-human primate cynomolgus monkeys, QD oral dosing of FTX-6058 as early as for 7 days induced robust and comparable TE in bone marrow derived CD34+ erythroid progenitors and circulating monocytes, further supporting the use of monocytes to assess TE in bone marrow. Mouse data also provided evidence of the reversibility of TE effects once dosing is stopped. In repeat-dose studies in the humanized Townes SCD mouse model, FTX-6058 was superior to standard of care hydroxyurea as measured by human HBG1 transgene induction and increased %F-cells and HBG1 protein levels. Furthermore, the induction of %F cells was sustainable for several days after dosing cessation. These in vivo studies have demonstrated that FTX-6058 engages its novel biological target in multiple preclinical models and induces HbF expression at plasma concentrations likely to b e readily achievable in clinic, and peripheral monocytes is a suitable surrogate for assessing TE in bone marrow erythroid cells, which could be beneficial to patients with SCD. Disclosures Xie: Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Roth:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Efremov:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Silver:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Ronco:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Thompson:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Stickland:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Moxham:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Wallace:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company.

Elevated P21 (CDKN1a) Mediates Apoptosis of Beta-Thalassemic Erythroid Cells in Mice but Its Ablation Doesn't Improve Erythroid Maturation

Beta-thalassemias are caused by mutations in the β-globin gene leading to anemia. In β-thalassemia, excessive accumulation of unpaired α globin chains in erythroblasts, triggers redox-mediated reactions, which is associated with increased production of immature erythroid precursors that fail to mature. This impaired maturation is in part due to increased apoptosis of late maturing erythroblasts in β-thalassemic patients that aggravates anemia despite enhanced erythropoiesis leading to what is called ineffective erythropoiesis and ultimately resulting in extramedullary expansion of hematopoiesis. The mechanism of apoptosis in beta-thalassemia remains poorly understood. To investigate this, we examined the status of mediators of stress response during erythroid cell maturation ofHbbth3/+ (th3/+) mice, a model that mimics the beta-thalassemia intermedia phenotype in humans. We found that both Foxo3 and p53 were prematurely activated in th3/+ beta-thalassemic erythroblasts as compared to wild type controls. We crossed Hbbth3/+ (th3/+) and Foxo3-/- mice and found that red blood cell (RBC) count and hemoglobin content were improved (by 1g/L, n=10), and erythroblast apoptosis was decreased to similar levels as in the WT during erythroblast maturation of double mutant mice. However, loss of Foxo3 did not ameliorate the splenomegaly of th3/+mice. We also found that p53 direct target, p21 the cyclin-dependent kinase inhibitor was greatly upregulated in th3/+erythroblasts as well as in beta-thalassemic patients' erythroblasts. To address the contribution of p21, we crossed p21-/- and Th3/+. It showed a significant decrease of apoptosis in CD45- TER119+ erythroblasts both in the bone marrow and spleen of double mutant mice (30% and 23% reduction respectively, n=6 mice each genotype). Although, as in beta-thalassemic patients, serum erythropoietin (Epo) was elevated in the peripheral blood of th3/+mice, the double mutant mice had significantly lower level of Epo than th3/+ (45% reduction, n=3 mice per genotype).In p21-/-th3/+, CD45- TER119+ cells also showed lesser ROS accumulation(12% less, n=3 per genotypes). However, to our surprise, the deletion of p21 on beta-thalassemic background did not have any effect on splenomegaly (n=6 mice each genotype), complete blood count, hemoglobin, RBC production or bone marrow erythroid cell maturation (n=12 mice each genotype). To further examine the underlying mechanism, we analyzed cell cycle in double mutant p21-/-th3/+ erythroblast at distinct stages of maturation identified by CD45, TER119, CD44 and cell size (n=3 mice per genotype) using ki67 staining at distinct stages of maturation. We found p21-/-th3/+erythroblasts proliferate much less than their th3/+ counterparts (basophilic erythroblasts G2 14% less, polychromatic erythroblasts 20% less, p&amp;lt;0.05 n=3 mice per genotype). This may partially explain lack of improvement of RBC production and anemia despite enhanced erythroblast survival. ROS levels were also reduced in double mutant p21-/-th3/+ erythroblasts as compared to controls. Next we investigated the status of p53 and Foxo3 in double mutant p21-/-th3/+ erythroblasts as compared to controls. We confirmed as we had observed earlier that nuclear p53 and Foxo3 expression were greater in th3/+ primitive erythroid (TER119-/low, c-KIT+, CD71Hi) cells than in wild type (n=3 mice per genotype) controls. Strikingly, the double mutant p21-/-th3/+ erythroblasts exhibited the greatest nuclear Foxo3 in all four groups, while nuclear p53 was dramatically reduced by over 80% (n=2 mice each genotype. Each mouse taking &amp;gt;=30 cells to calculate nuclear MFI) even as compared to wild type. These combined studies suggest that ameliorating apoptosis may not improve anemia in beta-thalassemia. Disclosures Liang: Hemogenyx Pharmaceuticals LLC: Current Employment.

Cytogenetic Effects of Carbendazim on Mouse Bone Marrow Cells

A study of the genotoxic activity of fungicide carbendazim was carried out. It was demonstrated that the tested technical grade active ingredient of carbendazim in the dose range of 125–2000 mg/kg body weight induced a statistically significant dose-dependent increase in the frequency of micronucleated polychromatic (PCE) and normochromatic erythrocytes (NCE) in the bone marrow of CD-1 mice. After administration of carbendazim at doses of 250–2000 mg/kg body weight, the accumulation of cells containing two or more micronuclei was observed. According to the results of comet assay, there was no increase in the level of breaks and alkali-labile sites in the DNA of the mouse bone marrow and liver cells after administration of technical grade carbendazim at doses up to 2000 mg/kg body weight. It is therefore suggested that the observed accumulation of cells with micronuclei is due to the aneugenic effect of carbendazim. In addition, a dose-dependent increase in the proportion of macrocytes/gigantocytes and dumbbell-shaped cells with constrictions among PCEs and NCEs was observed, which might be indicative of the inhibitory effect of the test pesticide on cytokinesis. The accumulation of polychromatophiles with pyknotic nuclei after exposure to carbendazim is probably caused by the distortion of nuclear extrusion. A possible general mechanism underlying the impairment of karyokinesis, cytokinesis, and nuclear extrusion in mammalian bone marrow erythroid cells is discussed.