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Excessive erythrocytosis (EE) is the main sign of chronic mountain sickness (CMS), a maladaptive clinical syndrome prevalent in Andean and other high-altitude populations worldwide. The pathophysiological mechanism of EE is still controversial, as physiological variability of systemic respiratory, cardiovascular, and hormonal responses to chronic hypoxemia complicates the identification of underlying causes. Induced pluripotent stem cells derived from CMS highlanders showed increased expression of genes relevant to the regulation of erythropoiesis, angiogenesis, cardiovascular, and steroid-hormone function that appear to explain the exaggerated erythropoietic response. However, the cellular response to hypoxia in native CMS cells is yet unknown. This study had three related aims: to determine the hypoxic proliferation of native erythroid progenitor burst-forming unit-erythroid (BFU-E) cells derived from CMS and non-CMS peripheral blood mononuclear cells; to examine their sentrin-specific protease 1 (SENP1), GATA-binding factor 1 (GATA1), erythropoietin (EPO), and EPO receptor (EPOR) expression; and to investigate the functional upstream role of SENP1 in native progenitor differentiation into erythroid precursors. Native CMS BFU-E colonies showed increased proliferation under hypoxic conditions compared with non-CMS cells, together with an upregulated expression of SENP1, GATA1, EPOR; and no difference in EPO expression. Knock-down of the SENP1 gene abolished the augmented proliferative response. Thus, we demonstrate that native CMS progenitor cells produce a larger proportion of erythroid precursors under hypoxia and that SENP1 is essential for proliferation. Our findings suggest a significant intrinsic component for developing EE in CMS highlanders at the cellular and gene expression level that could be further enhanced by systemic factors such as alterations in respiratory control, or differential hormonal patterns.

Parvovirus B19 (B19V) is a human pathogenic virus associated with a wide range of clinical conditions. In pregnancy, B19V poses a potential hazard to the fetus as crossing the placental barrier and infecting erythroid progenitor cells in bone marrow and liver, it blocks fetal erythropoiesis leading to profound anemia, hydrops and/or fetal death. The virus is not regarded as a teratogen, however more scientific awareness is emerging on mechanisms and consequences of intrauterine infection and possible sequelae in the neonatal development. Reliable diagnostic procedures and fetal management strategies, including intrauterine transfusion, are established. In spite of being a recognized fetotropic agent possibly leading to fetal loss, testing for B19V is not routinely included in preconception or antenatal screenings, possibly delaying the management of B19V-complicated pregnancies. Continuous advances in B19V research will provide for better diagnostic methods and algorithms, as well as for the development of effective prophylactic interventions and novel therapeutic options.

In this issue of the JCI, Chaurasia and colleagues report an impressive ex vivo expansion of HSCs from human cord blood (CB) using cytokines and altering epigenetic modifications. The application of this protocol provides information that has potential for clinical consideration. The enhanced expansion of CB HSCs is a substantial advance over recent work from the Chaurasia and Hoffman group, in which ex vivo production of human erythroid progenitor cells from CB was promoted by chromatin modification. Moreover, this study takes advantage of information from the rapidly emerging, but not yet fully elucidated, field of epigenetics.

Studies by Qu et al1 in this issue of Blood provide new mechanistic insight into the ineffective erythropoiesis of TET2 deficiency including selective colony-forming unit–erythroid (CFU-E) hyperproliferation, heightened expression of KIT and AXL, and the rescue by KIT and AXL inhibitors of faltered late stage erythropoiesis.

Shwachman-Diamond syndrome (SDS) is one of the more common inherited bone marrow failure syndromes (IBMFS). Almost 90% of patients with SDS present mutations in the Shwachman-Bodian-Diamond syndrome gene (SBDS) which encodes for the homonymous small protein involved in ribogenesis. SDS is a multiple-organ disease mostly characterized by exocrine pancreas insufficiency, bone malformations, and more importantly bone marrow failure. Most patients with SDS present severe neutropenia, whereas thrombocytopenia and anemia are less frequent. Furthermore, 15-20% of patients develop myelodysplastic syndrome with high risk of acute myeloid leukemia (AML). STAT3 pathway is upregulated both in primary SDS leukocytes and immortalized B cells. Being STAT3 a key regulator of interleukin-6 (IL-6), we postulated that STAT3 hyper-activation could lead to a dysregulation of the IL-6 signaling cascade. Increased levels of IL-6 have been found in pediatric patients with AML and it has been associated with poorer outcomes in these patients, highlighting IL-6 as a cytokine potentially involved in the development of AML. Thus, our hypothesis is that STAT3-IL6 axis may contribute to leukemogenesis in SDS. Almost 55% of patients with SDS carry a specific nonsense mutations, namely the c.183-184TA>CT, which cause a premature termination codon (PTC). Ataluren (PTC124, PTC Therapeutics Inc, NJ) is a small PTC suppressor molecule already approved by the European Medicines Agency as a therapeutic option for Duchenne muscular dystrophy. Interestingly, we recently reported that ataluren can restore SBDS expression in bone marrow progenitors and in peripheral blood mononuclear cells isolated from patients with SDS. Moreover, we have shown that ataluren can reduce mTOR hyper-phosphorylation and excessive apoptotic rate observed in SDS leukocytes. More importantly, we reported that ataluren can improve myeloid differentiation in a small cohort of patients (Bezzerri et al, Am J Hematol 2018). In this further analysis considering an enlarged cohort of 20 SDS patients carrying nonsense mutations we found the following: Ataluren can significantly improve both myeloid colony-forming unit-granulocyte/macrophage (CFU-GM) and colony-forming unit granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM) generation from bone marrow mononuclear stem cells obtained from an enlarged cohort of 20 patients with SDS carrying nonsense mutations. Ataluren indeed almost doubled the number of CFU-GM and CFU-GEMM after 7 and 14 days of treatment.Colony-forming unit erythroid (CFU-E) generation was not affected by the treatment.Ataluren induces neutrophil maturation in SDS bone marrow mononuclear stem cells (mean increase of 61% CD16+ CD11b+ cells over untreated controls) after 24-48 hours of treatment.Consistently with STAT3 hyper-activation observed in SDS cells, here we show that patients with SDS present a significantly increased level of IL-6 in plasma (4.3-fold higher expression than the healthy control group). Also lymphoblastoid cell lines (LCL) and primary bone marrow mesenchymal stromal cells (MSC) obtained from patients with SDS show increased IL-6 release in culture supernatants compared to healthy controls (2.5-fold and 6.8-fold higher levels, respectively).Of note, ataluren can reduce IL-6 expression in SDS cells restoring normal levels both in LCL and MSC. In conclusion, these new data support the enrollment of patients for the first clinical trial for this drug in SDS. Furthermore, this study could pave the way for the use of ataluren for other nonsense mutation-mediated IBMFS where STAT3-IL6 axis and similar pro-leukemic pathways are involved. Disclosures Bezzerri: Marco Cipolli, Valentino Bezzerri, Baroukh Maurice Assael: Patents & Royalties: WO2018/050706 A1 "Method of treatment of Shwachman-Diamond syndrome". Cipolli:Marco Cipolli, Valentino Bezzerri, Baroukh Maurice Assael: Patents & Royalties: WO2018/050706 A1 "Method of treatment of Shwachman-Diamond syndrome".

Introduction: Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by macrocytic anemia, reticulocytopenia, selective deficiency of erythroid precursors, presence of congenital anomalies and an increased risk of cancer. DBA is caused by germline mutations of genes coding for ribosomal proteins (RP). Interestingly, somatic RP mutations have also been found in several malignant diseases (T-ALL, CLL, Hodgkin lymphoma, myelodysplastic syndrome (MDS) and AML). The only representative overview of malignancies in DBA was published in 2012 based on the data from the North American DBA Registry. The malignancies developed in 3% of patients (MDS and AML in 4 patients and solid tumors in 15 patients). The observed-to-expected ratio for all cancers combined was 5.4 (p Patients and methods: Czech National DBA Registry currently includes 62 patients. In cooperation with 8 national centers of pediatric and adult hematology, we collected data about patients followed with pre-malignant or malignant condition including a detailed analysis of the course of the disease, treatment and type of developed malignant disorder. In patients with an evolving MDS, a classical cytogenetic analysis, flow cytometry, mutational profile (TruSight Myeloid Sequencing Panel (Illumina) containing 54 genes) and commercial TUNEL assay for detection of apoptotic changes of erythroid cells in the bone marrow were performed. Results: Eight of 62 patients from the Registry (13%) had malignant or pre-malignant condition: four females (6.5%) had solid tumors, 3 males (4.8%) had MDS and one female (1.6%) had multiple myeloma. Age of the onset of these disorders ranged between 25-70 years. Three patients harbored RPL5 mutation, 3 patients RPL11 mutation and 2 patients RPS19 mutation. Cancer incidence was significantly higher within the RPL11 and RPL5 subgroups (p=0.0056, Fisher Exact Test). Two female patients were diagnosed with triple-negative breast carcinoma, both of them during pregnancy. The first patient died despite treatment at the age of 29 years, shortly after delivery of her second child. The second patient is currently undergoing neoadjuvant chemotherapy.One patient developed diffuse large B cell lymphoma (DLBCL), underwent chemotherapy and autologous BMT and is alive in second remission.One patient underwent hemicolectomy for colorectal adenocarcinoma at 53 years of age and is in remission 6 years after the surgery.One patient succumbed to multiple myeloma which evolved from monoclonal gammopathy of unknown significance (MGUS) after 14 years of follow-up.Three male patients developed suspected MDS at the age of 25, 28 and 29 years. Two of them had RPL5 mutation and one harbored RPS19 mutation. None of them had decrease of erythroid cells in the bone marrow, but apoptosis of erythroid progenitors was significantly increased in all cases. All 3 patients had bicytopenia in peripheral blood (anemia, leukopenia) and dysplasia in 2 or 3 hematopoietic lineages in the bone marrow, thus fulfilling criteria for MDS with multilineage dysplasia (MDS-MLD). They had no abnormalities detected by flow cytometry or cytogenetics. The patient with RPS19mut harbored ASXL1 mutation; in patient with RPL5 mut, mutational screening is ongoing. Conclusion: Our results confirmed increased incidence of cancer (13%) in patients with DBA at young age. Our cases of DLBCL and MGUS in DBA are the first published to date. In cases with suspected MDS, cytopenia and dysplastic changes in the bone marrow may either reflect specific features of ribosomopathy, or represent a severe disorder of regulatory mechanisms with propensity to clonal proliferation. International collaboration is required to refine the incidence of malignancies in DBA and to issue consensus guidelines for timely detection of solid tumors, leukemias and MDS. The best approach to DBA patients who developed MDS without harboring any mutation considered prognostically significant in MDS, is another subject of discussion. Support: NV16-32105A Disclosures No relevant conflicts of interest to declare.

Aging is an inevitable process associated with eventual deterioration of normal physiological functions. Aged hematopoiesis is associated with increased numbers of hematopoietic stem cells (HSC), but with decreased HSC functional activity (e.g. decreased engrafting capability in lethally irradiated mice and a shift in the myeloid:lymphoid bias of the engrafting HSC of the old mice, such that there are more myeloid but fewer lymphoid cells generated from HSC of the old mice). Production of HSC and progenitor (HPC) cells ex vivo is more efficient when cells are cultured in a hypoxic environment of ~ 5% oxygen tension than when cells are grown at ambient air (~21% oxygen). The bone marrow (BM) microenvironment niche that nurtures the survival and production of HSC and HPC and hematopoiesis during adult life is a hypoxic environment (~1-5% oxygen tension) compared to that of ambient air. However, almost all results of studies of young and aged mouse hematopoiesis have been based on numbers and activity of HSC and HPC that have been collected and processed in ambient air. Our recent work evaluating hematopoiesis in BM cells of young adult mice and with human cord blood cells found, through a phenomenon we designated Extra Physiological Oxygen Shock/Stress (EPHOSS), that there is a large loss of HSC with an increase of HPC within minutes of the collection of these cells in ambient air (Mantel et al., Cell, 2015). This led us to reason that perhaps what we know about aging hematopoiesis might not be entirely accurate and that a re-evaluation of aged HSC, HPC, and hematopoiesis was in order. We hypothesized that hematopoiesis in aged (~20-27 months of age) mice may not be as dysregulated as reported but that collection and processing of BM from the aged mice is more sensitive than similar cells from young (~6-16 weeks) mice to EPHOSS-induced events generated by the collection of the cells in ambient air. We evaluated BM from three different mouse strains (CB6, BALB/c, and C57Bl/6) at 20-25 months vs. 6-16 weeks of age, collected/processed in ambient air or hypoxia (3% oxygen). BM from old mice collected/processed under hypoxic conditions exhibited phenotypically increased long-term HSC and common lymphoid progenitor (CLP) numbers and decreased common myeloid progenitor (CMP) and granulocyte-macrophage progenitor (GMP) numbers when compared to old BM collected/processed under ambient air conditions. BM collected from old C57Bl/6 mice under hypoxia had increased engrafting capability more closely matching that of young BM. This was associated with a 3.14-fold increase in the number of competitive repopulating units (representative of functional HSC) in old BM collected under hypoxic conditions compared to old BM collected in ambient air as determined through limiting dilution analysis. The myeloid:lymphoid ratio of old BM collected under hypoxia matched that of young BM collected under air. This was associated with decreased cycling of CFU-GM, BFU-E, and CFU-GEMM in old BM collected/processed in hypoxia. Enhanced numbers/function of old BM HSCs collected in hypoxia is associated with changes in expression of CXCR4 (and HSC homing capability), CCR5, stress protein levels (e.g. HSP40 etc) and ROS (both total and mitochondrial). All of these noted changes demonstrated that the old BM collected/processed under hypoxic conditions more closely resemble functionally young BM. Thus, age-related differences between the HSC/HPC populations are not as drastic as previously reported and reflect the increased sensitivity of hematopoiesis from aged mice to an artificial ambient air collection procedure. Disclosures No relevant conflicts of interest to declare.

Base editing with fusions of RNA-guided DNA-binding proteins and nucleotide deaminases represents a promising approach to permanently remedy genetic blood disorders without obligatory double strand breaks, however its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. Here we purified A3A(N57Q)-BE3 protein for RNP electroporation of human peripheral blood (PB) mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs). We found that sgRNAs targeting for cytidine base editing the core GATA1 binding motif of the BCL11A +58 erythroid enhancer resulted in efficient on-target base edits (81% allele frequency) with low indels. There was similar HbF induction in erythroid progeny as compared to Cas9:sgRNA RNP nuclease mediated modification of the same target sequence (36% median HbF in base edited cells with 81% allele modifications, 39% HbF in 3xNLS-SpCas9:sgRNA#1617 nuclease edited cells with 99% indels, and 5%HbF in unedited cells). A single therapeutic base edit of the BCL11A enhancer was sufficient to ameliorate the pathobiology of both sickle cell disease (SCD) and beta-thalassemia. Base editing of CD34+ HSPCs from plerixafor mobilized PB SCD patient donors resulted in potent HbF induction (24-31% HbF level in bulk cells with 87-90% on-target allele modifications) and reduced sodium metabisulfite induced sickling (from 84% to 29% of erythrocytes). Base editing of non-mobilized PB CD34+ HSPCs from 3 beta-thalassemia patient donors potently induced gamma-globin and improved ineffective erythropoiesis in vitro, with 59-75% on-target allele modifications. Moreover highly efficient multiplex editing could be achieved in HSPCs. Simultaneous disruption of the BCL11A +58 erythroid enhancer core GATA1 motif and correction of the HBB -28A>G promoter mutation in HSPCs resulted in production of at least one therapeutic allele in 35 of 35 erythroid colonies analyzed, with each colony achieving improved alpha-to-non-alpha globin chain balance. Finally we found that base editing could be efficiently produced in engrafting HSCs. Using 3 donors, we observed 62-64% base edits at the BCL11A +58 enhancer GATA1 motif in input HSPCs with one round of electroporation and 73-85% base edits with two rounds of electroporation (separated by 24 hours), albeit with modest reduction of cell viability with tandem electroporation. We observed 70-94% human chimerism and 35-82% base edits in engrafting human cells in primary recipients and 11-61% base edits in secondary recipients as measured 16 weeks following cell infusion. Similar edit frequencies were observed across all assayed hematopoietic lineages including engrafting B-lymphocytes, erythroid precursors, and HSPCs. We observed that compared to non-engrafting progenitors, long-term engrafting HSCs favored C to T (as compared to C to G/A) editing. There was a strong correlation (Spearman r 0.86, P Disclosures No relevant conflicts of interest to declare.

Autophagy is a highly conserved pathway that degrades and recycles intracellular components. While autophagy is activated in response to cellular stress, it also contributes to hematopoietic cell differentiation. Previous studies in mice lacking core autophagy proteins have identified mitochondrial clearance as a critical function of autophagy in erythroid differentiation and maturation. Impaired autophagy results in anemia, retained mitochondria and elevated levels of reactive oxygen species in mice. However, the kinetics and regulation of mitophagy in human erythropoiesis have not been investigated. To better understand the kinetics of mitophagy in human erythropoiesis we have developed a lentiviral mitophagy reporter (MT-Keima) and used it to monitor mitochondrial clearance during erythroid differentiation of primary human CD34+ hematopoietic stem and progenitor cells (HSPCs). Measurement of mitophagy during erythroid differentiation revealed active mitochondrial clearance during early and late stages of erythropoiesis culminating with total clearance of mitochondria at the terminal stages of differentiation. Gene expression analysis during human erythroid differentiation identified the upregulation of the core autophagy program including ATG4A encoding a cysteine protease, which was restricted to the erythroid lineage. Knockdown of ATG4A in primary human HSPCs significantly reduced mitophagy in early and late erythroid cells and resulted in increased mitochondrial mass in terminally differentiated reticulocytes. Furthermore decreased expression of ATG4A, but not its paralog ATG4B, reduced the (i) expansion of erythroid progenitors, (ii) total number of erythroblasts (iii) and significantly reduced enucleation relative to luciferase controls. Finally significantly fewer erythroid colonies were found in methylcellulose culture only in HSPCs with ATG4A KD when compared to luciferase controls, while numbers of myeloid colonies were preserved, supporting a role for ATG4A selectively in the human erythroid lineage. Together these results identify ATG4A as a novel erythroid-specific regulator of mitophagy and a new potential target for the therapeutic modulation of autophagy in human erythropoiesis. Disclosures No relevant conflicts of interest to declare.

Human β-globin locus consists of at least six genes encoding components of the oxygen transport protein hemoglobin and an upstream locus control region (LCR) containing five DNase I hypersensitive sites. In addition, there are at least four conserved CTCF insulator elements surrounding the locus, which form dynamic chromatin interaction patterns across different cell types in the course of hematopoietic stem and progenitor cells (HSPCs) development. Chromatin conformation of the β-globin locus in normal adult CD34+ HSPCs reveals the formation of three topological associated domains (TADs), in which individual TAD boundaries are demarcated by CTCF sites (Figure 1). By comparing chromatin loop interactions between CD34+ HSPCs and its differentiated erythroid progenitors, we identify a chromatin loop that forms in erythroid progenitors and is not evident in the CD34+ HSPCs. Detailed examination of this loop shows that a DNase I hypersensitivity site, also called 3'HS1, overlaps with a CTCF site that forms a loop with another CTCF site adjacent to OR52A5 gene (Figure 2). To investigate the role of this specific chromatin loop in the regulation of hemoglobin gene expression, we knock out the 3'HS1 CTCF motif in adult CD34+ HSPCs under erythroid differentiation medium by CRISPR/Cas9-mediated gene editing. We find that deletion of 3'HS1 CTCF results in a 2-fold decrease of β-globin (HBB) and a 4-fold increase of the fetal hemoglobin gene encoded by γ-globin (HBG1/2) in erythroid colonies of edited CD34+ cells (Figure 3). Elevation of fetal hemoglobin upon 3'HS1 CTCF deletion was also confirmed in human umbilical cord blood-derived erythroid progenitor-2 cells (HUDEP-2), which results in a 12-fold increase of γ-globin expression (Figure 4). These results suggest that the 3'HS1 CTCF plays a crucial role in regulating the expression of fetal hemoglobin gene, however it remains unclear whether changes in chromatin structure is responsible for these changes. CTCF looping interactions have been described to form under convergent directionality. To validate the role of 3'HS1 CTCF in establishing chromatin interactions at the β-globin locus, we aim to invert this binding motif and evaluate how it disrupts chromatin organization and gene expression at this locus. Deciphering the underlying mechanisms will shed light on how three-dimensional chromatin structure is reorganized in differentiating erythroid cells as it undergoes nuclear condensation. Furthermore, elevation of fetal hemoglobin expression can potentially be a new therapeutic gene editing strategy to treat sickle cell disease and some cases of β-hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

Megakaryocytes (MKs) are responsible for producing anucleate platelets which are indispensable due to their roles in hemostasis, growth and development. Recent studies have also demonstrated the influence of platelets in regeneration and immune protection, thus highlighting the functionality of platelets beyond their classically defined roles. Standard models of thrombopoiesis emphasize that MK development occurs initially in the fetal liver during development, followed by long-term residency in sites of adult hematopoiesis such as the bone marrow sinusoids. Outside of the hematopoietic system, MKs have been shown to reside in the interstitium of the lung, with emerging evidence pointing to the lung microvasculature as a specialized environment that may support a significant bulk of platelet production. However, little is known about how MKs traffic to the lung and whether they are influenced by the lung microenvironment to become functionally distinct from their hematopoietic counterparts. To characterize the transcriptional signature of lung-resident MKs, we performed single cell RNA sequencing (scRNA-Seq) on primary MKs derived from syngeneic mouse lung and hematopoietic tissues. Samples were processed to obtain single cell suspensions, followed by flow cytometry to enrich for CD41 positive cells and prepared for scRNA-Seq using the 10x genomics platform. scRNA-Seq analysis revealed a developmental trajectory starting from megakaryocyte erythroid progenitors (MEPs) and ending in two distinct clusters of mature MKs which were made up of mostly lung derived MKs (Fig. 1). One of these mature clusters was made up of 91% lung MKs and possessed a unique gene enrichment profile, including marked upregulation of platelet factor 4 (PF4) and enrichment for cellular processes related to ATP production and translation. To further profile lung-resident MKs, we performed flow cytometric assessment of ploidy and maturation markers. In comparison to bone marrow derived MKs, lung derived MKs displayed a distinct ploidy profile with a significantly increased number of cells expressing CD42, a marker of MK maturation. Collectively, these findings suggest that the subset of MKs found within the lung may be in a more mature and metabolically active state to potentially facilitate increased platelet output. These studies may shed new light on the process of MK maturation, including what triggers MK endoreplication, and may advance methodologies for in-vitro platelet production. Based on the symbiotic relationship between the lung and the hematopoietic system, our ongoing studies are focused upon the interplay between the lung and resident MKs in the developmental and functional patterning of both tissue types. Disclosures No relevant conflicts of interest to declare.

none 5 no Parvovirus B19 (B19V) infection is restricted to erythroid progenitor cells (EPCs) of the human bone marrow, leading to transient arrest of erythropoiesis and severe complications mainly in subjects with underlying hematological disorders or with immune system deficits. Currently, there are no specific antiviral drugs for B19V treatment, but identification of compounds inhibiting B19V replication can be pursued by a drug repositioning strategy. In this frame, the present study investigates the activity of hydroxyurea (HU), the only disease-modifying therapy approved for sickle cell disease (SCD), towards B19V replication in the two relevant cellular systems, the UT7/EpoS1 cell line and EPCs. Results demonstrate that HU inhibits B19V replication with EC50 values of 96.2μM and 147.1μM in UT7/EpoS1 and EPCs, respectively, providing experimental evidence of the antiviral activity of HU towards B19V replication, and confirming the efficacy of a drug discovery process by drug repositioning strategy. The antiviral activity occurs in vitro at concentrations lower than those affecting cellular DNA replication and viability, and at levels measured in plasma samples of SCD patients undergoing HU therapy. HU might determine a dual beneficial effect on SCD patients, not only for the treatment of the disease but also towards a virus responsible for severe complications. Bonvicini, Francesca; Bua, Gloria; Conti, Ilaria; Manaresi, Elisabetta; Gallinella, Giorgio Bonvicini, Francesca; Bua, Gloria; Conti, Ilaria; Manaresi, Elisabetta; Gallinella, Giorgio

Axl, a member of the TAM family of receptor tyrosine kinases, mediates survival and therapy resistance of different cancer cells. The Axl ligand growth-arrest specific gene 6 (Gas6) was discovered to promote proliferation of leukemia cells in acute and chronic myeloid leukemia and Axl was identified as a potential therapeutic target in these diseases. Based on these data we investigated the role of Axl in BCR-ABL negative myeloproliferative neoplasms (MPN) and the therapeutic potential of Axl blockade in this group of diseases. We studied the effects of Axl blockade using the small molecule Axl inhibitor BGB324 and performing a lentivirus shRNA mediated knockdown of Axl in human SET-2 and murine BaF3-Jak2V617F MPN cell lines. Pharmacologic Axl blockade resulted in a significant dose dependent decrease in viability of MPN cell lines as measured by WST-1 cell viability assay. Annexin+ staining revealed an increased rate of apoptotic cells upon BGB324 treatment for SET-2 (increase by 15% at 1µM, p The finding that BGB324 inhibits growth of MPN cells was further corroborated in vivo. A xenograft tumor model with SET-2 cells was set up in vivo. SET-2 tumor bearing mice treated with BGB324 50mg/kg showed a slower tumor growth (n=8, p Furthermore, to evaluate the potential of BGB324 in primary MPN cells, peripheral blood mononuclear cells (PBMC) were isolated from MPN patients and healthy donors. Western Blot analysis showed higher levels of Axl expression by PBMC from MPN patients compared to PBMC from healthy donors. Moreover, colony-forming assays with PBMC were performed in the presence of different concentrations of BGB324. Here, a higher reduction in the number of colony forming units (BFU-E and CFU-GEMM) was observed in samples from MPN patients compared to healthy donors upon treatment with 1µM (77% vs. 5%, respectively; p In conclusion, these data indicate therapeutic potential of Axl blockade in BCR-ABL negative MPN as monotherapy and in combination with Jak2-inhibition, supporting the need for clinical investigation. Disclosures von Amsberg: Novartis: Honoraria, Speakers Bureau; Ipson: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; Astellas: Honoraria, Speakers Bureau; MSD: Honoraria, Speakers Bureau. Loges:BerGenBio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Given that both Sickle Cell Disease (SCD) and beta-thalassemia (BT) are caused by mutations in the beta-globin gene, several lentivirus-based gene addition therapies have been developed. Results from recent trials indicate that the vectors used are safe; however, their efficacy inversely correlates with the severity of patients' hemoglobinopathy. The severity of the mutations (non-beta0 vs beta0) largely influences the outcome of the gene transfer. In fact, the data indicate that a relatively low number of integrations (in the range of 1-2 copies per genome) or vector copy number (VCN) is sufficient to cure patients whose mutations are categorized as non-beta0 and express relative high levels of endogenous hemoglobins (adult hemoglobin, HbA, and/or fetal hemoglobin, HbF). In contrast, the same level of VCN alleviates the transfusion regimen of patients with beta0 mutations, but it does not cure them. In addition, the lentiviruses currently used in clinical trials were engineered by different groups and to date no one has directly compared them side by side. In light of these limitations, here we describe a study that supplies a platform for rapid screening of lentiviral vectors expressing curative hemoglobin, based on the correlation between VCN and the increase in HbA levels. We also compared newly generated lentiviral vectors to vectors currently used in clinical trials. Our ultimate goal is to generate a new vector that can increase the yield of beta globin expressed per VCN in patients' cells. Using CRISPR-Cas9 we modified the erythroid Hudep-2 cell line (Kurita et al, 2013) to generate a clonal cell line, named Hudep #M13, which, upon differentiation, produces a hemoglobin variant (HbMut) that can be discriminated from that produced by the lentiviruses (HbA). In parallel, we immortalized erythroid progenitor cells isolated from a SCD donor (SCD #13), using the HPV16-E6/E7 expression system, which was introduced into the cells by lentiviral transduction. Using Hudep #M13, we compared the correlation between gene transfer and the production of HbA for 5 novel lentiviral vectors, indicated as ALS16-20. Our new vectors include the Ankyrin insulator in the 3' LTR (Breda et al 2012), the full beta-globin gene (including the native introns), the full 3' enhancer region, a combination of different portions of the beta-globin promoter, as well as modifications and inclusion of novel genomic elements from the locus control region (LCR). Our ALS- constructs were then compared to lentiviral vectors currently utilized in clinical trials. These constructs were reproduced based on information available from the literature (Negre et al, 2015; Miccio et al, 2008; and Boulad et al, 2014) and indicated as CV-1, CV-2, and CV-3, respectively. All these vectors contain the beta-globin gene with deletions in intron 2, different portions of the beta-globin promoter and/or 3' enhancer region, and different elements and sizes of the hypersensitive sites (HS) of the LCR. In Hudep #M13, linear regression analysis of the ratio of HbA to vector copy number (VCN) for each treatment, indicates that ALS17 and ALS20 yield roughly 40, 157 and 84% more HbA per copy than CV-1, CV-2 and CV-3, respectively. Similar increment in HbA% were confirmed on primary and immortalized (SCD #13) SCD erythroblasts derived CD34+ cells isolated from patients' blood. In these specimens, ALS20 maintained a 40% HbA increase compared to CV-1, when exploring a range of VCN from 0 to 3 with a linear mixed effects model. To assess the ability of these constructs to increase hemoglobin content in vivo, we are performing murine bone marrow transplants using thalassemic hematopoietic stem cells treated with CV1 and our two most powerful vectors. Based on most recently reported data (Thompson et al, 2018), 1 copy of the vector we reproduced as CV-1, makes on average 6.8g/dL of HbA. Hence, 1 copy of our best vector has the potential to make up to 9.5g/dL HbA. This could lead to a much greater clinical impact for patient with hemoglobinopathies, especially those who require higher Hb production to become transfusion independent, like patients with the beta0 genotype. The completion of these studies will provide not only a comparative analysis of our new best vector to those already in clinical trial, but also a way to predict how much therapeutic hemoglobin per vector copy number will be produced in the clinical setting. Disclosures Casu: Aevi Genomic Medicine, Inc: Research Funding; Ionis Pharmaceuticals, Inc.: Research Funding. Kwiatkowski:bluebird bio: Consultancy, Honoraria, Research Funding; Agios Pharmaceuticals: Consultancy, Research Funding; Novartis: Research Funding; Apopharma: Research Funding; Terumo: Research Funding. Rivella:Disc Medicine: Consultancy; Protagonist: Consultancy; Ionis: Consultancy; Meira GTX: Membership on an entity's Board of Directors or advisory committees.

Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. The disease typically presents within the first year of life. Approximately 70% of DBA patients possess a mutations in one of at least 12 ribosomal proteins, with RPS19 and RPL11 being the most prevalent, accounting for over 25% and 5% of cases respectively. Current therapies for DBA have undesirable side effects, including iron overload from repeated transfusions or infections from immunosuppressive drugs and stem cell transplantation. Nemo-like Kinase (NLK) is chronically hyper-activated in RPS19- and RPL11-haploinsufficient murine and human models of DBA, as well as erythroid progenitors from DBA patients. In an RPS19-insufficient human model, genetic silencing of NLK (shRNA) increased erythroid expansion by 2.2 fold, indicating aberrant NLK activation contributes to the pathogenesis of the disease. In an independent, high-throughput kinase inhibitor screen examining progenitor expansion in RPS19-insufficient Kit+ murine cells, a number of compounds were identified that increased progenitor expansion. SB431542 and SD208 are recognized TGFβ inhibitor compounds, but were the only TGFβ inhibitors of a panel of 11 that increased progenitor expansion. Both active compounds robustly inhibited NLK activity in vitro and in vivo while the remaining 9 inhibitors had no significant impact on NLK. Both compounds increased erythroid expansion in murine (3.1 and 5.4 fold) and human (3.2 and 6.3 fold) models of DBA with no effect on wild type erythropoiesis (EC50 5 µM and 0.7 µM). No further increase in erythroid expansion was observed when NLK expression was silenced by shRNA. Virtually identical results were observed in CD34+ progenitors from 3 DBA patient bone marrow aspirates with 2.3, 1.9 and 2.1 fold increases in CD235+ erythroblast generation compared to untreated. NLK hyperactivation was limited to differentiating committed erythroid progenitors and was not detected in megakaryocytic, other myeloid progenitors or lymphoblastoid cells lines from DBA patients. During differentiation, non-erythroid lineages upregulate miR181, which results in NLK transcript degradation and loss of NLK expression. The absence of NLK in non-erythroid progenitors prevents NLK activation during ribosomal insufficiency. CRISPR/Cas9 mutation of the miR181 binding site in the NLK 3'UTR in RPS19-insufficient CD34+ HSPCs prevented NLK downregulation, increased NLK activity, and sensitized megakaryocyte and other myeloid lineages (80.5% and 76% reduction relative to controls). This is comparable to the erythroid expansion defect in RPS19-insufficiency (80.7% reduction). In differentiating erythroid progenitors, RPS19 insufficiency increased phosphorylation of the mTORC1 component Raptor (5.3 fold), reducing mTOR activity by 82%. This was restored to basal levels upon pharmacological or genetic inhibition of NLK. To compensate for a reduction in ribosomes, stimulating mTORC1 activity with leucine has been proposed to increase translational efficiency in DBA patients. Probably due to NLK phosphorylation of raptor, DBA patients did not respond as anticipated. While leucine treatment did increase mTOR activity in both control (100% to 188%) and RPS19-insufficiency (27 % to 42% of control), the combined treatment of leucine with NLK inhibition resulted in increased mTOR activity to 142% of control and significantly improved erythroid expansion. Identification of aberrantly activated enzymes, such as NLK, that are specifically expressed in erythroid progenitors, offer therapeutic promise as potential druggable targets in the clinical management of DBA that can be used in combination with existing therapies. Disclosures Glader: Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Porteus:CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Flygare:LU Holding: Patents & Royalties: Patent.

Diamond-Blackfan Anaemia (DBA) is a rare blood cell aplasia that presents clinically at approximately 2-3 months of age and its main characteristic is reduced erythroid precursors in the bone marrow, i.e. anemia. Mutations in different ribosomal protein (RP) genes have been associated with DBA, with mutations in RPS19 accounting for 20-25% of all cases. It has been proposed that RPS19 deficiency causes perturbations in ribosome biogenesis, thus activation of the p53-dependent Nucleolar Surveillance Pathway (NSP). In this context free RPs (predominantly L5 and L11) in a complex with 5S rRNA sequester the E3 ubiquitin ligase murine double minute 2 (MDM2), leading to the accumulation of p53 and subsequent activation of its transcriptional targets mediating cell cycle arrest or apoptosis. In DBA, one of the molecular mechanisms impairing the proliferation and thus reducing the number of erythroid progenitors that can progress to mature red blood cells is an elevation of p53 protein mediating activation of the NSP. In order to identify potential therapeutics that could be repurposed to prevent the activation of NSP in DBA patients, we have screened compound libraries of clinically approved therapeutics to identify pathways implicated in the p53-dependent NSP due to RPS19 deficiency. We quantitated both cell number and the level of p53 expression, identifying compounds that can result in low and high expression of p53, the latter for potential use in cancer therapy. Using an RPS19 depleted A549 cell line as a model system, the screen successfully identified different therapeutic groups. In the DBA context, we were most interested in the compounds that reduced p53 and had no negative effect on cell number. A selection of 22 molecules were re-evaluated in vitro, again using RPS19 depleted A549 cells, through the quantification of p53 protein expression and densitometry analysis. From this, 10candidates were evaluated ex vivofor their effects on proliferation using bone marrow obtained from an inducible Rps19 knockdown (DBA) mouse model. While we are currently testing a number of compounds in vivo using the Rps19 DBA mouse model (as described), one of the compounds tested thus far has demonstrated a partial rescue of the cKit+ population, no changes in erythroid precursors but interestingly a reversal of the defect in the Granulocyte-Monocyte Progenitor (GMP) population. Impairment in lineage progression in the GMP compartment has also been reported to present in bone marrow failure Shwachman-Diamond Syndrome. We are currently evaluating the mechanism by which this drug is rescuing the c-Kit+ and GMP populations in these mice. In summary, our high-throughput screening approach and follow up studies have identified a suite of novel therapies that may be beneficial for repurposing for the treatment of bone marrow failure by increasing hematopoietic progenitor cells. We plan to evaluate this, and potentially other therapies, in a clinical trial with DBA patients. Disclosures Flygare: LU Holding: Patents & Royalties: Patent.

Introduction Risk assessment is essential for guiding therapy in patients with myelodysplastic syndromes (MDS). Currently, the most widely used prognostic scoring models are the International Prognostic Scoring System (IPSS) and the revised IPSS (IPSS-R). The prognostic relevance of the percentage of erythroid precursors (EP) in bone marrow (BM) and its relationship with other biological characteristics has been poorly studied, although it has been proposed that a very low percentage of EP in BM may represent a cohort of patients with potentially adverse outcome. Our main aim was to analyze the biological and clinical features of MDS patients according to the percentage of EP in BM at diagnosis and evaluate its prognostic value on survival. Patients and Methods Data from 4,791 de novo MDS patients from the MDS Spanish Registry with available cytogenetics were collected. All patients included were diagnosed based on the 2008 WHO criteria and risk stratification was performed following the IPSS-R. Patients were distributed, according to the percentage of EP in BM, into three groups: less than 15% (EP49%). Proportions were compared by the Chi-square test. Survival curves were constructed by Kaplan-Meier method and differences between curves were evaluated by log rank tests. Multivariable analysis of survival was performed using Cox's proportional hazards regression model. P-values Results The 4,791 cases were grouped according to the percentage of erythroid cells in BM at diagnosis. In Table 1 are displayed the clinical and biological characteristics of the patients in the whole series and in the three groups based on the EP percentage in BM. The group with EP49% group presented higher rates of patients with refractory anemia with ring sideroblasts (RARS), lower number of PB and BM blasts, and more frequent high-risk cytogenetics. Conversely, the intermediate EP group with EP 15-49% showed less number of cytopenias, higher frequency of good-prognosis cytogenetics, and lower percentage of patients in higher-risk IPSS-R categories. Survival analyses confirmed that age, blast percentage in PB and BM, and IPSS-R cytogenetics are significantly associated with worse survival. A striking difference in survival within groups according to percentage of EP was also found, recognizing EP percentage as an independent risk factor for survival of MDS patients in multivariate analysis. Patients with EP50% in BM had also worse survival than the intermediate group, showing a median survival of 47 months (hazard ratio, 0.79; 95% CI, 0.67 to 0.93; P < 0.005). Conclusions Our study proves the prognostic impact of the percentage of erythroid precursors in bone marrow in MDS patients, providing evidence that the presence of EP Disclosures Díez-Campelo: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

* A.R. and M.L. have contributed equally. INTRODUCTION: Myelodysplastic syndrome (MDS) is a clonal disease that arises from the expansion of mutated hematopoietic stem cells. Oxidative stress could play an important role in the pathology of MDS since a correlation has been observed between high ROS levels and decreased survival in MDS patients. In addition, the oxidation of proteins leads to their aggregation, change and conformational changes that can cause the loss of their functions. Due to alteration of the proteome has been described in patients with MDS, in this study we considered the discovery of protein carbonylation to analyze its oxidation pattern. In addition, as the oxidative stress signaling pathways can regulate the cell cycle through p53, we analyzed p21, an important p53 target during the stress response. Finally, the possible benefit of treatment with Deferasirox as antioxidant therapy was studied. METHODS: Carbonylation pattern was analyzed in the different cell lines (n = 14, 6 MDS, 4 reactive controls and 4 MDS + DFX) by immunohistochemistry by derivatization with 2,4-dinitrophenylhydrazine (DNPH) and detection with anti-DNP antibodies. The degree of protein carbonylation was studied, also by derivatization and detection with anti-DNP, in primary cells of erythroblasts expanded for 11 days, by one-dimensional and two-dimensional electrophoresis (n = 16; 7 MDS, 6 controls, 2 reactive controls and 1 MDS + DFX). Expression levels of p21 were analyzed by qRT-PCR (n=19; 8C, 8 MDS, 3DFX). RESULTS: Immunohistochemical assays revealed a higher level of carbonylation in patients with MDS (Fig. 1a). Interestingly, Deferasirox-treated MDS patients had lower levels than non-treated patients (Fig. 1b). In addition, to evaluate the carbonylation in the erythroid precursors, the pattern of carbonylated proteins in the expanded erythroblasts was analyzed. Again, a higher level of MDS was observed compared to the control group, which was reversed after treatment with Deferasirox. Two-dimensional electrophoresis revealed that differences in carbonylation are due to a limited number of proteins or aggregates, rather than to a large spectrum of proteins. Finally, an increased p21 expression was observed in patients with myelodysplasia and, surprisingly, this effect was reversed with Deferasirox treatment. CONCLUSION: Patients with MDS showed a differential protein pattern respect to control patients, both in the myeloid and erythroid series. The increase in oxidative stress in myelodysplastic patients seems to activate signaling pathways involving p21. Treatment with Deferasirox can reverse the level of oxidation and the increase of p21 in patients with MDS. Disclosures Martinez Lopez: Celgene: Research Funding, Speakers Bureau; Jansen: Research Funding, Speakers Bureau; BMS: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau.

金沢大学医薬保健研究域薬学系, The purpose of this study was to investigate the efficacy of four different Japanese and Chinese herbal prescriptions, Ren-Shen-Yang-Rong-Tang (Ninjin'yoeito, NYT), Chai-Hu-Gui-Zhi-Gan-Jiang-Tang (Saikokeishikankyoto, SKKT), Si-Jun-Zi-Tang (Shikunshito, SKT) and Si-Wu-Tang (Shimotsuto, SMT), which are traditionally used for anemia and fatigue, against hematotoxicity in mice treated with 5-fluorouracil (5-FU). NYT 1100 mg kg-1 day-1 injected orally for 7 consecutive days before and after 5-FU injection significantly suppressed reductions in red blood cell, white blood cell and platelet counts in peripheral blood, and accelerated their recovery. Administration of SKKT also produced a slight but significant improvement in 5-FU-induced erythrocytopenia, whereas SMT and SKT could not prevent anemia. Oral injection of NYT also inhibited 5-FU-induced decreases in peripheral reticulocyte and bone marrow cell counts on day 10, and markedly hastened their recovery on day 20, in a dose-dependent manner. Erythroid progenitor colonies, such as colony forming units-erythroid and burst forming units-erythroid, formed by marrow cells from mice treated with 5-FU were significantly increased by oral administration of NYT. These findings suggest that NYT has the potential to protect against hematotoxicity, and also has hematopoietic activity, through stimulation of immature erythroid progenitor cell differentiation., 出版者許諾要件により全文公開は2010年7月より.

Erythrovirus (previously parvovirus) B19 was discovered accidentally in 1975 while screening for hepatitis B surface antigen (Chan et al, 1998). It is a small ssDNA virus that targets erythroid progenitor cells. Infection with B19 virus during pregnancy may cause serious complications in the fetus such as fetal hydrops and intrauterine fetal death. Early diagnosis and treatment is essential in the management of these complications. We conducted a survey study to assess the awareness of both front and second-line obstetric staff(GPs, midwives, senior house officers, specialist registrars and consultants) of B19 as a potential and treatable cause of hydrops fetalis. Overall there was poor awareness particularly among the front-line carers compared to the second-line carers regarding the diagnosis and management of B19 infection in pregnancy. A total of 94% of the responders agreed there was a need for educational updates on the role of B19 infection during pregnancy.

New therapeutic strategies have emerged along with our understanding that disturbances of iron homeostasis, impaired proliferation of erythroid progenitor cells, and blunted erythropoietin response occur in anemia of chronic disease. This review discusses advances in our knowledge of the causes and management of the condition.

The pathogenic parvovirus B19 (B19V) has an extreme tropism for human erythroid progenitor cells. In vitro, only a few erythroid leukemic cell lines (JK-1 and KU812Ep6) or megakaryoblastoid cell lines (UT7/Epo and UT7/Epo-S1) with erythroid characteristics support B19V replication, but these cells are only semipermissive. By using recent advances in generating large numbers of human erythroid progenitor cells (EPCs) ex vivo from hematopoietic stem cells (HSCs), we produced a pure population of CD36+ EPCs expanded and differentiated from CD34+ HSCs and assessed the CD36+ EPCs for their permissiveness to B19V infection. Over more than 3 weeks, cells grown in serum-free medium expanded more than 800,000-fold, and 87 to 96% of the CD36+ EPCs were positive for globoside, the cellular receptor for B19V. Immunofluorescence (IF) staining showed that about 77% of the CD36+ EPCs were positive for B19V infection, while about 9% of UT7/Epo-S1 cells were B19V positive. Viral DNA detected by real-time PCR increased by more than 3 logs in CD36+ EPCs; the increase was 1 log in UT7/Epo-S1 cells. Due to the extensive permissivity of CD36+ EPCs, we significantly improved the sensitivity of detection of infectious B19V by real-time reverse transcription-PCR and IF staining 100- and 1,000-fold, respectively, which is greater than the sensitivity of UT7/Epo-S1 cell-based methods. This is the first description of an ex vivo method to produce large numbers of EPCs that are highly permissive to B19V infection and replication, offering a cellular system that mimics in vivo infection with this pathogenic human virus.

Terminally differentiated cells usually do not divide and are, thus, reproductively dead. To elucidate the significance of radiation-enhanced differentiation to reproductive cell death, murine erythroid progenitor cells were gamma-irradiated in plasma clot cultures and the development of haemoglobinized clones was studied thereafter. If irradiation occurred when the cells had resumed proliferation, the total numbers of haemoglobinized clones and, in parallel, the numbers of newly haemoglobinized clones were elevated above control levels 6-24 h after 10-30 Gy and 24-48 h after 1 Gy respectively. Thereafter, clone numbers decreased below controls. This decrease was faster with the newly haemoglobinized clones, indicating that both the accumulation of haemoglobinized clones and fast exhaustion of the pool of more primitive precursors in the cultures are due to accelerated differentiation. The haemoglobinized clones appearing after irradiation were reduced in size without indication of direct cell death. We conclude that the reproductive cell death occurring in our system is due to enhancement of differentiation. Enhancement of differentiation is expressed by omission of cell cycles normally passed through by the cell progeny before terminal differentiation is reached. Dependence of differentiation enhancement on the presence of cycling cells at the time of irradiation indicates involvement of growth of essential cytoplasmic constituents during mitotic delay as observed in other cell systems.

While many cytokines are known for their inflammatory action, there is a growing interest in the tissue-protective effects of some cytokines. The prototypic tissue-protective cytokine is EPO. Initially described as neuro-protective, it is beneficial in animal models of ischemic and other types of injury. Scientists had to overcome the notion that EPO had only erythropoietic actions, was only produced by the kidney, and that its receptor was only present in erythroid progenitor cells. The use of in vitro and in vivo disease models was essential to demonstrate the protective effects of EPO. Reproducible models will be needed for the further study of the mechanism of action of EPO and for the identification of other tissue-protective cytokines. In Tissue-Protective Cytokines: Methods and Protocols, expert researchers in the field detail the key models that have been used to characterize the tissue-protective actions of cytokines. Written in the highly successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, provide step-by-step laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Thorough and intuitive, Tissue Protective Cytokines: Methods and Protocols aids scientists in continuing to study tissue-protection that will be a new field of interest of cytokine biology, both in discovering novel actions of known cytokines and in developing new drugs.

Down syndrome-associated acute megakaryoblastic leukemia (DS-AMKL) affects 1 in 500 children with DS and associates uniformly with somatic mutations in GATA1. DS-AMKL is often treated successfully; however, treatments can cause long-term side effects, such that mechanistic insights and targeted therapies are needed. Recent studies report a recurring L340F mutation in DCAF7 in DS-AMKL, at the C-terminus of the protein. A highly conserved scaffold protein, DCAF7 is a cofactor for the chromosome 21-encoded kinase, DYRK1A, and shares 100% protein sequence homology among vertebrates. In our efforts to characterize the normal function of DCAF7 in hematopoiesis and its contribution to AMKL, we have identified an unreported PDZ-binding motif (PBM) in DCAF7, which is perturbed by the L340F mutation. Overexpression of mutant (DCAF7mut), but not wild type DCAF7 (DCAF7wt), in a mouse model of DS with a Gata1 mutation leads to rapid onset of dysplastic disease characterized by peripheral blood leukopenia and thrombocytopenia, with normal red cell numbers. Bone marrow of affected mice harbors abnormal, hyperproliferative erythroid progenitors and diminished, hyperchromatic megakaryocytes, with stark depletion of granulocytic and lymphoid lineages and widespread edema. Splenic architecture is grossly abnormal, with a complete absence of white pulp, proliferation of erythroid precursors, and significant numbers of apoptotic, hyperchromatic megakaryocytes. Conversely, targeted deletion of Dcaf7 in hematopoiesis shifts the myeloid-erythroid balance toward the myeloid lineage, with increased granulopoiesis and hyperchromatic megakaryocytes in the bone marrow and lower white blood cell numbers and dramatic thrombocytopenia in the peripheral blood. Splenic architecture is largely normal, although flow cytometry reveals defective B- and T- cell development. The bone marrow knockout phenotype of Dcaf7 is strongly reminiscent of the cellular phase of primary myelofibrosis (PMF). Combined with bone marrow overexpression phenotypes, which resemble hemophagocytic syndrome, our data suggest that DCAF7 is critically important for maintaining a healthy bone marrow environment and regulating blood cell lineage choice. In order to identify functional binding partners of the DCAF7 PDZ-binding motif (PBM) that may support these functions, we performed IP mass spectrometry using DCAF7wt or DCAF7mut as bait and found two highly conserved PDZ-domain containing scaffold proteins, PAR3 and PDLIM5, that interact robustly with DCAF7wt, but not with DCAF7mut. While both DCAF7wt and DCAF7mut are able to associate with DYRK1A, the ability of DCAF7 to bring DYRK1A activity to the PAR3 and PDLIM5 signaling hubs affects the composition of DCAF7-scaffolded transcriptional complexes and downstream transcriptional outcomes. Through RNA-sequencing and Ingenuity Pathways Analysis, overexpression of both DCAF7wt and DCAF7mut in mouse bone marrow leads to the induction of interferon response genes; however, DCAF7wt also induces a pro-apoptotic gene program, whereas DCAF7mut instead promotes survival gene expression and developmental reprogramming. Consistently, expression of DCAF7wt in bone marrow progenitors causes rapid expansion and increased initial colony forming ability, but decreased replating potential compared with vector control. Conversely, expression of DCAF7mut causes increased proliferation and colony formation accompanied by enhanced replating, indicating greater self-renewal conferred by DCAF7mu. Our data indicate DCAF7 regulates the balance of proliferation, differentiation and apoptosis in hematopoietic cells and that the PBD of DCAF7 is critically important for these functions, connecting DCAF7 to signaling hubs that translate environmental cues into appropriate transcriptional outcomes. Furthermore, DCAF7 disruption causes hematopoietic disease reminiscent of human pathology, suggesting it may have broad significance in hematopoietic malignancy. Finally, our data support a model wherein DCAF7 mutation facilitates the development of malignancy by promoting the survival and proliferation of mutant clones in the bone marrow and fundamentally altering the bone marrow microenvironment. Disclosures No relevant conflicts of interest to declare.

Although the clinical course of patients with myelofibrosis (MF) is punctuated by the development of extramedullary hematopoiesis leading to symptomatic splenomegaly, the spleen is not a hematopoietic organ in normal adults. Spleens from patients with MF are characterized by increased numbers of hematopoietic cells and increased micro vessel density within the red pulp. Previously, we reported that the siderophore, lipocalin-2 (LCN2), was elaborated by MF myeloid cells which leads to increased MF marrow fibrosis, osteoblastogenesis,and depletion of the pool of normal hematopoietic cells but promotion of the proliferation of MF cells (Lu M, Blood, 2015). Using nanostring nCounter technology, we have now demonstrated that LCN2 transcript levels were significantly higher in MF spleen (4 fold) than in normal spleen samples (p=0.008) and that LCN2 transcript levels were only modestly increased in MF marrow (p=0.48). The data identified the spleen as a major source of LCN2 in MF patients. These findings led us to further examine the effects of LCN2 on MF spleen CD34+ cells and micro environmental cells. Normal spleen adherent cells (AC) were treated with LCN2 and conditioned media (CM) were collected. CD34+ cells isolated from normal BM or MF spleen were cultured with this CM for an additional 5 days. LCN2 treated AC CM increased MF hematopoietic cell numbers by 2 fold and increased MF CFU-GM and BFU-E numbers by 40% (p We, then, cultured MF splenic and normal BM CD34+ cells in media alone and in a co-culture system with MF splenic endothelial cells in the absence of exogenous cytokines. The total MF and normal BM cell number was dramatically increased when they were co-cultured with EC as compared to the CD34+ cells cultured alone. The absolute MF CD34+ cell number was increased more than 50 fold (p Disclosures No relevant conflicts of interest to declare.

High Mobility Group AT Hook 2 (HMGA2) is a non-histone chromatin protein that regulates gene transcription and controls cell proliferation, survival and self-renewal of stem cells. HMGA2 is expressed at a low level in normal adult hematopoietic progenitors but is highly expressed in hematopoietic progenitors of patients with Myelofibrosis (MF). However, the contribution of HMGA2 to the pathogenesis of MF remains unknown. MF is the deadliest form of myeloprolifearative neoplasm (MPN) characterized by deposition of fibrous tissues in the bone marrow, increased megakaryopoiesis, ineffective erythropoiesis and extramedullary hematopoiesis. Median survival of patients with MF is less than 6 years. The JAK2V617F mutation has been found in 50-60% patients with MF. However, it is not clear whether JAK2V617F mutation alone is sufficient to cause MF. Interestingly, up-regulation of HMGA2 expression has been found in association with the JAK2V617F mutation in a significant percentage of patients with MF. To understand the role of JAK2V617F mutation in the pathogenesis of MPN, we previously generated a conditional Jak2V617F knock-in mouse. We observed that expression of heterozygous Jak2V617F in mouse hematopoietic compartments is sufficient to induce a polycythemia vera (PV)-like MPN. Recently, we have shown that deletion of EZH2 promotes the development of MF in Jak2V617F knock-in mice and EZH2 deletion increases the expression of HMGA2 in hematopoietic progenitors of EZH2-deleted Jak2V617F mice. To directly assess the effects of concomitant expression of HMGA2 and heterozygous Jak2V617F in mice hematopoietic compartments, we expressed control vector or HMGA2 in wild type and heterozygous Jak2V617F knock-in mice BM by lentiviral transduction and performed bone marrow transplantation into lethally irradiated C57BL/6 recipient mice. Whereas recipients of vector-transduced Jak2V617F knock-in BM cells exhibited a PV-like MPN characterized by increased red blood cells (RBC), hemoglobin, hematocrit and platelets in their peripheral blood, recipients of HMGA2-transduced Jak2V617F knock-in BM showed reduced hemoglobin and hematocrit parameters compared with recipients of vector-expressing Jak2V617F BM cells. Interestingly, peripheral blood neutrophil and platelet counts were further increased in transplanted animals receiving HMGA2-transduced Jak2V617F BM cells. Expression of HMGA2 also resulted in significantly larger spleen size in the transplanted animals receiving HMGA2-expressing Jak2V617F BM cells. Flow cytometric analysis showed significant increase in megakaryocytic precursors (CD41+) but decrease in erythroid precursors (CD71+/Ter119+) in the BM and spleens of transplanted animals receiving HMGA2-expressing Jak2V617F BM compared with control vector-expressing Jak2V617F BM. Furthermore, the frequency of hematopoietic stem/progenitor cells (LSK; Lin-Sca-1+c-kit+) was significantly increased in recipients of HMGA2-transduced Jak2V617F knock-in BM compared with control vector-transduced Jak2V617F knock-in BM or HMGA2-transduced wild type BM. Histopathologic analysis revealed extensive fibrosis in the BM and spleens from recipients of HMGA2-expressing Jak2V617F mice at 32 weeks after transplantation while BM and spleens from recipients of vector-transduced Jak2V617F knock-in BM or HMGA2-transduced wild type BM showed very little or no fibrosis at this age. Together, these data suggest that expression of HMGA2 promotes megakaryopoiesis and accelerates the development of MF in mice expressing Jak2V617F. To gain insights into the mechanisms by which expression of HMGA2 accelerates the development of MF in Jak2V617F mice, we performed RNA-sequencing analysis on purified LSK (Lin-Sca-1+c-kit+) cells. Gene set enrichment and pathway analyses revealed that the genes related to chemokine, TGF-β, MAP Kinase, PI3 kinase-Akt, mTOR and WNT signaling pathways were up-regulated in HMGA2-expressing Jak2V617F mice LSK compared with vector-expressing Jak2V617F LSK cells. We also found that HMGA2 directly binds to the promoter regions of some of these target genes and regulate their expression. Further studies will validate the targets of HMGA2 and determine their contribution in MF mediated by Jak2V617F. In conclusion, our studies show that expression of HMGA2 cooperates with Jak2V617F in the development of MF. Disclosures No relevant conflicts of interest to declare.

Primary Familial and Congenital Polycythemia (PFCP) is a non-malignant pathology of the erythroid lineage, characterized by an isolated increase of the red cell mass without evolution into myelofibrosis or acutisation. Around twenty constitutive non-sense and missense mutations located in the exon 8 of the erythropoietin receptor (EPOR) gene have been described so far. They all lead to the truncation of the C-terminal part of the protein and the loss of several cytoplasmic tyrosines. The erythropoietin (EPO) hypersensitivity of the PFCP erythroid progenitors is usually explained by the disappearance of these negative signaling regulation and internalization domains (Figure 1a). Nonetheless, relatively few functional studies have been carried out. We therefore investigated the mechanism of EPOR mutations in PFCP. We identified and extensively studied a new constitutive heterozygous frameshift EPOR mutation, p.Gln434Profs*11, which generates a new 11 amino acid (AA) C-terminal tail and a STOP codon at position 444, leading to the truncation of 63 AA of the wild-type receptor (Figure 1c). The primary progenitor cells displayed a major hypersensitivity to EPO, similar to Polycythemia Vera (PV) patients, as well as a spontaneous and persistent JAK2 and STAT5 phosphorylation, compared to the control cells. To study the mechanism of this new EPOR mutant, Ba/F3 cells were transduced with different retroviruses encoding either the HA-tagged wild-type EPOR (EPOR WT)or a truncated receptor at position 444, p.Gln444* (EPOR STOP) or the frameshift EPOR p.Gln434Profs*11mutation (EPOR FS), identical to the patient's mutation (Figure 2). As observed in primary cells, EPOR FS conferred a spontaneous STAT5 phosphorylation and a 4- to 5-fold EPO hypersensitivity to Ba/F3 cells (IC50 of 0.003 U/mL vs 0.01 U/mL) compared to both EPOR WT and EPOR STOP. As expected, the loss of negative regulatory domains in the C-terminal part of the receptor induced a persistent STAT5 activation in EPOR FS and EPOR STOP Ba/F3 cells. Moreover, EPOR FS was more stable (half-life of 120 minutes vs 60 minutes) and displayed a higher level of localization at the cell surface (more than 2-fold), compared to EPOR WT and EPOR STOP. However, no modification of the EPOR FS internalization pattern was observed during 125I-EPO labeling experiments and cytometry analysis. Furthermore, a dileucine motif, known to be a potential clathrin-dependent endocytosis site, is lost in the new C-terminal tail of EPOR FS mutant, yet its abrogation in EPOR WT and EPOR STOP did not modify the phenotype of Ba/F3 cells. Therefore, unlike previous reports, the major EPO hypersensitivity induced by EPOR p.Gln434Profs*11 cannot be explained by the receptor truncation, but rather by the appearance of a new C-terminal tail that confers spontaneous signaling. We wondered if this model could be extended to other EPOR mutations already described in PFCP (Figures 1a-b and 2). We therefore measured the impact on Ba/F3 cells proliferation of the frameshift EPOR p.Pro438Metfs*6 and its non-sense mutant counterpart, p.Pro443*, which retains the tyrosine at position 426, a binding site for the negative signaling regulators SOCS3 and CIS. EPO-hypersensitivity (4- to 5-fold) was only induced by EPOR p.Pro438Metfs*6, suggesting a common mechanism for the frameshift EPOR mutations. Interestingly, two proximal non-sense mutations, EPOR p.Glu399* and p.Glu425*, lacking 7 of the 8 cytoplasmic tyrosines that compose EPOR negative regulatory and internalization domains, were also able to confer a high EPO hypersensitivity to Ba/F3 cells. To our knowledge this is the first extensive functional study of EPOR mutations in PFCP. We highlighted that this pathology is much more complex than expected, since different mechanisms are involved in the EPO hypersensitivity phenotype, according to the type of EPOR mutation. Indeed, extensive truncations are sufficient by themselves to confer the EPO hypersensitivity phenotype due to the loss of all negative regulatory and internalization domains, whereas more distal truncations induced by frameshift mutants confer EPO hypersensitivity because of the appearance of a new C-terminal tail. The latter, by increasing EPOR stability at the cell surface, may cause pre-activation of both receptor and JAK2, constitutive signaling and hypersensitivity to EPO close to that of JAK2V617F-positive PVs. Disclosures No relevant conflicts of interest to declare.

The human pathogenic parvovirus B19 cannot be grown in standard tissue culture but propagates in human bone marrow, where it is cytotoxic to erythroid progenitor cells. We now show that parvovirus B19 can replicate in cynomolgus bone marrow. Cynomolgus monkeys may be a suitable animal model for pathogenesis studies of parvovirus B19.

Erythropoietin (Epo) is a polypeptide hormone produced in kidney. It stimulates the proliferation of erythroid progenitor cells in response to hypoxia. In this issue of Blood , the report of Singbrant et al clearly shows the indirect effect of Epo on bone remodeling and its importance for the Epo

The glycoprotein EPO is the hormon responsibile for regulation of erythroid progenitor cells in the born marrow; rhEPO widely used as important therapeutic preparate for correcting certain types of anemia. It was established three recombinant CHO cell line producing human erythropoietin (EPO): CHO-PE, CHO-972, CHO-23. The recombinant cells were different by gene constructions. In present work we have made a comparative study of morphology, cultural and productive properties of that three cell lines.

In alloimmune anemia of the newborn, the level of hemolysis caused by the presence of antibodies to antigens of the Kell blood-group system is less than that caused by antibodies to the D antigen of the Rh blood-group system, and the numbers of reticulocytes and normoblasts in the baby's circulation are inappropriately low for the degree of anemia. These findings suggest that sensitization to Kell antigens results in suppression of fetal erythropoiesis as well as hemolysis.We compared the growth in vitro of Kell-positive and Kell-negative hematopoietic progenitor cells from cord blood in the presence of human monoclonal anti-Kell antibodies and anti-D antibodies and serum from women with anti-Kell antibodies.The growth of Kell-positive erythroid progenitor cells (erythroid burst-forming units and colony-forming units) from cord blood was markedly inhibited by monoclonal IgG and IgM anti-Kell antibodies in a dose-dependent fashion (range of concentrations, 0.2 to 20 percent), but monoclonal anti-D antibodies had no effect. The growth of these types of cells from Kell-negative cord blood was not affected by either type of antibody. Neither monoclonal anti-Kell antibodies nor monoclonal anti-D antibodies inhibited the growth of granulocyte or megakaryocyte progenitor cells from cord blood. Serum from 22 women with anti-Kell antibodies inhibited the growth of Kell-positive erythroid burst-forming units and colony-forming units but not of Kell-negative erythroid burst-forming units and colony-forming units (P0.001 for the difference between groups). The maternal anti-Kell antibodies had no inhibitory effects on granulocyte-macrophage or mega-karyocyte progenitor cells from cord blood.Anti-Kell antibodies specifically inhibit the growth of Kell-positive erythroid burst-forming units and colony-forming units, a finding that supports the hypothesis that these antibodies cause fetal anemia by suppressing erythropoiesis at the progenitor-cell level.

We read with interest the article by Heinrich et al[1][1] on apparent erythroid-specific expression of green fluorescent protein (GFP) and Cre-recombinase as a result of a “knock-in” mutation into erythropoietin receptor ( EpoR ) gene. Currently, evaluation of erythroid progenitors relies on the

Abstract 2379 Introduction: The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression. This is achieved by ubiquitinylation of various cell cycle regulators to tag them for proteasomal degradation. APC/C in conjunction with its adaptor protein Cdh1 (APC/CCdh1), both stabilizes G1-phase and is involved in the induction of cell cycle arrest and differentiation. Here, we have analyzed the influence of APC/CCdh1 on self-renewal capacity and differentiation potential and kinetics of human hematopoietic stem cells (HSCs). Methods: In order to study the expression levels of Cdh1 among different hematopoietic lineages, we stained mononuclear cells from bone marrow of healthy donors with antibodies against the cell surface markers CD11b, Glycophorin A, CD41a, CD34, CD3 and CD19, and isolated these subsets via cell sorting. We extracted protein from these subsets and performed Western Blot analysis. We established a strong lentiviral Cdh1 knock down (kd) in CD34+ cells and performed colony forming cell (CFC) assays: 1×104 Cdh1-kd-CD34+ cells and CD34+ cells transduced with a control vector (ctrl-CD34+) were plated in MethoCult H4534 and MethoCult H4534 + EPO (1 IU/ml). At 14 days, CFU-GEMM, CFU-GM, CFU-G, CFU-M and BFU-E were scored. CD11b- and Glycophorin A-FACS, FACS analysis of propidium iodide staining and Pappenheim's staining were carried out on individually picked colonies in order to assess differentiation kinetics and cell cycle distribution. Self-renewal capacity of ctrl- and Cdh1-kd-CD34+ cells was examined by performing replating assays (secondary CFCs) with the obtained CFU-GEMM colonies as previously described (Katayama et al., BMT, 1999). Secondary colonies were analyzed after another 14 days of incubation. Results: We observed Cdh1 protein levels to significantly vary among hematopoietic cell subsets: The highest Cdh1 levels were detected in CD34+ cells, lower levels in cells of the lymphoid lineage (CD3+; CD19+) and only marginal expression levels in cells arising from myeloid progenitors (CD11b; Glycophorin A; CD41a). By correlating Cdh1 levels of the individual cell subsets with their cell cycle profiles, we were able to exclude the possibility that this was merely due to differences in cell cycle distribution. Analysis of the CFC assays performed with the lentiviraly infected CD34+ cells showed a considerable decrease of about 40% in the number of BFU-E and 35% in the number of CFU-G- and CFU-M-numbers, when Cdh1-kd-CD34+ cells were plated compared to ctrl-CD34+ cells. Furthermore, we observed an increase of CFU-GEMMs with Cdh1 depletion. The expression levels of the cell surface markers CD11b and Glycophorin A were 10–20% lower among the colonies arisen from Cdh1-kd-CD34+ cells vs. ctrl-CD34+ cells. Upon manual counting of Pappenheim stained preparations, we found the early stages of both erythroid and myeloid differentiation to be more prevalent in the Cdh1-kd colonies. When studying the replating capacity, we observed that Cdh1-depleted cells gave rise to almost twice as many secondary colonies as compared to ctrl-cells. There was no difference with regard to the relative proportions of the colony types. Interestingly, tracking of GFP, which had been used as a reporter-gene for the lentiviral transduction of the ctrl- and Cdh1-kd-cells, showed that it was enhanced in Cdh1-kd-secondary colonies compared to ctrl-secondary colonies. Conclusion: By analyzing human bone marrow subsets we observed that Cdh1 levels diminish from HSCs to mature lymphoid and, to an even greater extent, mature myeloid cells, suggesting that Cdh1 is important to induce differentiation but dispensable for maintaining the differentiated state. Our in vitro results are consistent with an important role of APC/CCdh1 in both myeloid and erythroid differentiation of HSCs. The data suggests that depletion of Cdh1 in HSC interferes with normal differentiation both by decreasing the number of mature lineage progenitors and by delaying individual cell maturation. HSCs deficient in Cdh1 seem to increasingly undergo self-renewal. The stronger the Cdh1-kd the more likely the generation of secondary colonies appeared to be. In vivo models may be particularly helpful to further elucidate these phenomena. Disclosures: No relevant conflicts of interest to declare.

Abstract 5087 Purpose The primary goal of this study was to determine if exposure to recombinant human erythropoietin (rHuEPO) increases survival of RBCs in rats. A secondary objective was to test if rHuEPO prolongs survival of RBC by interaction with erythroid progenitor cells or by affecting cells of the reticuloendothelial system. Methods Normal male Wistar rats (337 ± 8 g) were divided into four groups (n=2). Animals in groups 0 and 1 were not treated whereas animals groups 2 and 3 received multiple doses of rHuEPO (Epogen, Amgen, CA) 450 IU/kg three times a week for two weeks via intravenous injections in the tail vein. Subsequently, blood samples (∼ 1-2 mL) were collected from all animals and labeled with water-soluble biotin as a tag for RBC. The biotinylated RBCs were injected back to animals according to the following pattern. Group 1 received blood from Group 2 and Group 2 from Group 1. Animals from Groups 0 and 3 were injected back with their own blood. Two days were allowed for a system equilibration. Next, blood samples (∼ 100 mL) were drawn weekly from each animal until the signal reached the limit of quantification. The biotinylated cells were detected by streptavidin conjugated to phycoerythin and analyzed by flow cytometry (FACSCalibur, Becton-Dickinson). The RBC absolute count was determined by a hematology analyzer (BC-2800 Vet, Mindray, China). The survival data were represented as the fraction of surviving cells. For quantification of RBC survival, the times necessary to reduce the size of the labeled sample by 25, 50, and 75% (T25, T50, and T75) were used. Results The mean survival curves for each group were compared to the control rats (Group 0). The survival curve for animals in Group 3 was distinctly higher that the survival curve for the control which was reflected by the T50 values of 31.5±1.6 and 13.6±0.1 days, respectively. This implies that the survival of RBCs is prolonged by the treatment with rHuEPO. If the effect of erythropoietin on RBC survival is mediated by its interaction with the erythroid progenitor cells, then an increase in the RBC survival in Group 1 is expected. This was somewhat confirmed by the survival curve for Group 1 that was above the control survival curve, however not to the extent of Group 3. Contrary, the survival curve for Group 2 overlapped with the control, supporting the hypothesis that the effect of rHuEPO on RBC survival is not mediated by its interaction with the reticuloendothelial system. The T50 values for Group 1 and 2 were 24.3±7.4 and 15.6±3.0 days, respectively. Conclusions The survival patterns of RBCs following two week long exposure to rHuEPO imply that their lifespan is prolonged when compared to control rats. Lack of an increase of RBC survival for Group 1 and a moderate increase of RBC survival for Group 2 support the endowment hypothesis that rHuEPO prolongs survival of RBCs possible by enhancing viability of the erythroid precursors. Disclosures No relevant conflicts of interest to declare.

Congenital hypoplastic (Diamond-Blackfan) anemia is a rare macrocytic anemia, generally presenting during infancy or childhood. The condition usually occurs sporadically or in a pattern consistent with autosomal recessive inheritance, although autosomal dominant transmission has been proposed in some kindreds. We report the largest known kindred of congenital hypoplastic anemia, with at least 7 affected individuals over 3 generations, and propose that studies of this kindred may be useful for identifying the mechanism by which their genetic abnormality results in congenital hypoplastic anemia. Erythropoietic investigations on relatives show no inhibitors of erythropoiesis in serum, T-lymphocytes, or macrophages. Their erythroid progenitor cells (CFU-E and BFU-E) were generally quantitatively normal, and were capable of rapid proliferation, as judged by cell-cycle shortening. However, their erythroid progenitors displayed a relative insensitivity to recombinant erythropoietin, and produced relatively few normoblasts per erythroid progenitor cell. We propose that these and subsequent studies may be helpful in selecting candidate genes responsible for the molecular defect in this kindred.

Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders, which can be difficult to diagnose based only on morphologic bone marrow examination. Karyotyping can be useful in diagnosing borderline cases. However, only 40% of patients with MDS have an abnormal karyotype. Flow cytometry(FCM) approaches have been described but not clearly defined in MDS due to use of different criteria. We devised a 10-parameter FCM panel, mainly including myeloid and erythroid maturation markers, to differentiate MDS marrows from normal marrows. Design: Bone marrow from 91 patients with cytopenia(s) or anemia were included in the study(10/2005–7/2006). Cases were divided into 3 groups: 1. normal, not morphologically suspicious for MDS, 46 cases, 2. equivocal, morphologically suggestive but not diagnostic of MDS, 20 cases, 3. morphologically diagnostic of MDS, 25 cases. 10 FCM paremeters were performed and scored: 1. hypogranularity, 2. aberrant expression of CD56, 3. lack of CD10 expression, 4. decreased CD64 expression, 5. & 6. lack of CD13 or CD33 expression, 7. & 8. abnormal CD13/CD16 or CD16/CD11b pattern, 9. increase of CD 34 expression gating on all cells excluding erythrocytes, 10. decreased expression of CD71/Glycophorin gating on erythroid precursors. Karyotypings were analyzed. Results: 1. and 2. (see Table 1 and 2). 3. Karyotyping were anlayzed in 86 cases. Cytogenetic abnormalities were found in 2.2% (1/44) of normal group, 5.3% (1/19) of equivocal group and 34.8% (8/43) of MDS group. In MDS group 7 of 8 patients (87.5%) who had both morphologic and cytogenetic diagnosis of MDS were scored >=3 of 8 FCM parameters. Conclusions: Study showed the 8-parameter FCM panel was more predictive of MDS than 10-parameters one. Both CD13/CD16 & CD16/CD11b patterns were considered to be non-specific. In 8-parameter panel, zero score tended to rule out MDS, while score >=3 suggested MDS. The most specific FCM parameters were hypogranularity, CD34, aberrant expression of CD56 or lack of CD10 expression by mature granulocytes. CD71/Glycophorin A might be useful in identifying dysplasia in erythroid lineage. | Parameters/Group | Normal Group(A)(n=46) | Equivocal Group(B)(n=20) | MDS Group(C)(n=25) | CHITEST(P value)(GRoup A vs C) | |:-------------------------------------------------------------------------------------------------------------- | --------------------- | ------------------------ | ------------------ | ------------------------------ | | * These two parameters were deleted in the 8-parameter panel due to their high frequency seen in normal group. | | Hypogranularity | 4 (8.7%) | 6 (30%) | 18 (72%) | 0.0001 | | CD56 | 0 (0.0%) | 1 (5%) | 6 (24%) | 0.0005 | | CD10 | 7 (15.2%) | 7 (35%) | 15 (60%) | 0.0001 | | CD64 | 6 (13.1%) | 2 (10%) | 6 (24%) | 0.2396 | | CD13 | 0 (0.0%) | 0 (0.0%) | 2 (8.0%) | 0.0516 | | CD33 | 0 (0.0%) | 2 (10 %) | 0 (0%) | | | CD13/CD16* | 23 (50.0%) | 7 (35%) | 21 (84%) | 0.0047 | | CD34 | 8 (17.4%) | 7 (35%) | 13 (52%) | 0.0026 | | CD71 | 2 (4.3%) | 5 (25%) | 12 (48%) | 0.0001 | | CD16/CD11b* | 27 (58.7%) | 8 (40%) | 22 (88%) | 0.0107 | Table 1. Comparison of FCM Parameters in Patients with/without MDS | Score/Group | Normal Group (A)* (n=46) | Equivocal Group (B)* (n=20) | MDS Group (C)* (n=25) | |:-------------------------------------------------------------------------------- | ------------------------ | --------------------------- | --------------------- | | * Fish Exact T Test: Group A vs C: p3 | 0 (0.0%) | 3 (15%) | 17 (68%) | | *Mean score +/− SD | 0.65+/−1.06 | 1.55+/−1.54 | 2.8+/−1.65 | Table 2. Scoring with 8 FCM Parameters in Patients with/without MDS

Since exogenous hemin has been shown to exert a variety of stimulatory effects on erythroid cells, including the augmentation of hemoglobin synthesis, we determined its effect on early stages of erythroid development by employing clonal cells assays. The addition of hemin at a concentration of 2 X 10(-4) M to cultures of normal murine marrow substantially increased the observed number of primitive BFU-E, which was in contrast to its lack of an effect on more mature erythroid colony-forming cells. This cell-specific enhancement of primitive BFU-E resulted in marrow frequencies equivalent to or exceeding those reported in the presence of “burst-promoting activity.” In the presence of hemin, the number of BFU-E was also observed to be linearly related to the number of cells plated at very low plating densities, and the cell titration curve was observed to extrapolate to the origin. The evidence suggests that hemin may be a primary growth regulator of early developmental stages of erythroid progenitor cells.

In vitro colony formation of bone-marrow erythroid progenitor cells in patients with paroxysmal nocturnal haemoglobinuria (PNH) was examined. The numbers of early and late erythroid progenitors (BFU-E and CFU-E) showed wide variations; two cases out of eight cases of PNH showed decreased erythroid colony formation, but other cases showed normal or rather increased colony formation of BFU-E and CFU-E. The number of erythroid progenitors in patients with PNH may be related to the marrow cellularity.

The synthesis of γ- and β-globin chains was investigated in erythroid cultures from first and second trimester fetuses as well as neonates in order to examine whether the differences in production of HbA and F in the fetal and newborn ontogenetic stages reflect in vivo predetermined differences in commitment among erythroid progenitors. Cultures of erythroid progenitors from first and second trimester fetuses (12 experiments) produced amounts of HbF that were similar to those synthesized in vivo by fetal liver erythro-blasts and reticulocytes (range of HbF production: in vivo, 84%-93%; in culture, 80%-94%). There was no difference in HbF synthesis in cultures of fetal liver or peripheral blood-origin progenitors from the same fetus. In cultures of circulating progenitors from full-term newborns (nine experiments), amounts of fetal hemoglobin that are expected for the newborn ontogenetic stage (range of HbF in culture: 55%-80% of total Hb) and that are close to the levels of HbF production in cord-blood reticulocytes of the same fetuses, were observed. Varying the concentration of erythropoietin by 40-fold produced only minor changes in the proportion of γ and β chain synthesis in culture. The findings show that the fetal or newborn erythroid stem cells are programmed in vivo for expressing the characteristic, for each ontogenetic stage, pattern of non-α-globin synthesis. This is consistent with the proposition that the regulation of γ- and β-chain synthesis during development takes place at the level of erythroid progenitor cells rather than in the terminally mature erythron.

The addition of normal mouse serum to otherwise normally cultured Friend erythroid progenitor cells has a significant inhibitory effect on the ability of the cells to differentiate during dimethylsulfoxide stimulation. In contrast, heterologous sera from five different species showed no inhibition of erythroid differentiation when compared to the non-mouse serum-treated control cells. Experiments designed to determine the nature of the serum inhibitory factor led to the following conclusions: (a) delipidating the serum prior to its use does not remove the inhibitory factor; (b) the differentiation-inhibiting factor (DIF) is nondialyzable and (c) the factor is stable to both heat (56°C for 60 min) and repeated freezing. While the nature of the serum inhibitory factor remains to be determined, the evidence obtained so far indicates that the inhibitory response is not due to a cytotoxic effect on the cells, but rather an interaction with the cells in such a manner so as to modulate the expression of the erythroid cell program.

Ten patients with preleukemia were studied by the erythroid cell clonal culture technique. In nine of these patients, erythroid colonies derived from peripheral blood BFU-E were not observed, while the other patient had markedly decreased peripheral blood BFU-E-derived erythroid colonies in vitro. In three patients, marrow cells were also cultured and no BFU-E-derived erythroid colonies were detected. These studies indicate that immature erythroid progenitor cells, BFU-E, in patients with preleukemia are either markedly decreased in number or grossly defective in their proliferative or differentiative capacities.

Erythroid progenitor cells in +/+ and Sl/Sld fetal livers manifested as burst-forming units-erythroid (BFU-E) and colony-forming units- erythroid (CFU-E) were assayed in vitro during early development. The proportion of BFU-E was higher as mutant than in normal fetal livers. On the other hand, the proportion of CFU-E was less in the mutant than in the normal. These results suggest that the defect in Sl/Sld fetal hepatic erythropoiesis is expressed at the steps of differentiation that effect the transition from BFU-E to CFU-E.

Based on studies using anti-Ia-like heteroantisera, it has been concluded that human erythroid progenitor cells (CFU-E and BFU-E) express Ia-like antigens. We attempted to confirm these findings using a well-characterized anti-Ia-like heteroantiserum and hybridoma-derived monoclonal antibodies against Ia-like antigens. Specificity of the monoclonal antibodies was demonstrated by the pattern of reactivity against a panel of Ia-positive and Ia-negative cells and by immunochemical techniques. Normal human bone marrow or peripheral blood buffy coat cells were incubated with anti-Ia-like heteroantiserum or monoclonal antibody, with and without complement, washed, and plated with the appropriate humoral stimulator in semisolid media for determination of CFU-C, BFU-E, and CFU-E. Anti-Ia-like heteroantiserum produced complement-dependent inhibition of CFU-C-, BFU-E, and CFU-E-derived colony formation. Monoclonal anti-Ia-like antibody caused complement-dependent inhibition of CFU-C that persisted to a titer of 1:1000, but did not inhibit colony formation by BFU-E and CFU-E. Our results suggest either that erythroid progenitor cells do not express Ia-like antigens or that these antigens, if present, are expressed at very low density or in altered configuration, thus rendering them apparently unreactive with our monoclonal antibodies in a cytotoxicity assay.

The plasma clot culture system was utilized to support the growth of erythroid-committed stem cells from the bone marrow of five patients with homozygous β+-thalassemia and the peripheral blood of one patient doubly heterozygous for β°-thalassemia and Hb Lepore. Addition of erythropoietin to the cultures resulted in abundant colony growth (76–185 CFU-E and 3.5–11 BFU-E/6 × 104 bone marrow cells). Eight BFU-E/105 cells were assayed from the peripheral blood of the β°-thalassemia/Hb Lepore patient. Globin chain synthetic ratios of CFU-E- and BFU-E-derived colonies were determined by CMC column chromatography after labeling of the cultures with 3H-leucine for 24 hr prior to peak growth. Absent or decreased β-globin chain synthesis was observed in all cases; β/α synthetic ratios obtained in culture were quantitatively similar to those obtained in the starting bone marrow or peripheral blood cells. Study of globin chain synthesis in colonies derived from peripheral blood BFU-E of the patient with β°-thalassemia/Hb Lepore demonstrated synthesis of a labeled peak consistent with Lepore 60 chains; a similar labeled peak was barely discernable in the study of the starting peripheral blood. We conclude that thalassemic erythroid progenitor cells express in vitro the same program of abnormal globin chain synthesis as in vivo and that the plasma clot culture technique provides a valuable system for the study of the molecular defect in nucleated thalassemic erythroid cells.