GATA-1 Overexpression Does Not Restore Deficient Erythropoiesis in Myelodysplastic Syndromes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3430-3430
Author(s):  
Alexandra Rideau ◽  
Stephane Durual ◽  
Maciej Wiznerowics ◽  
Sylvie Ruault ◽  
Vincent Piguet ◽  
...  
Keyword(s):  
Myelodysplastic Syndromes ◽  
Culture System ◽  
Cell Cycle Control ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Differentiation Markers ◽  
Cd34 Cells ◽  
Erythroleukemic Cell

Abstract Introduction: Transcription factor GATA-1 is essential for erythroid and megakaryocytic maturation. A role of GATA-1 in cell cycle control is suggested by the fact that GATA-1 mutations are associated with hematopoietic precursor proliferation and leukemogenesis and that defective GATA-1 expression is observed in in vitro cultures of erythroid myelodysplastic precursors. In order to study more in detail a potential role of GATA-1 dysregulation in myelodysplastic syndromes (MDS), we constructed lentiviral vectors with the aim to overexpress GATA-1 protein or to inhibit its production in erythroid progenitors. Methods and Results: Using RNA interference technology we tested how GATA-1 inhibition interfered with erythroid differentiation. We selected one GATA-1 specific siRNA, which abolished expression of GATA-1 protein in K562 and HEL erythroleukemic cell lines, as verified by Western blot. Interestingly, we observed in parallel to the disappearance of GATA-1 protein, decreased proliferation rates (170x for K562 and 30x for HEL after 17 days of culture) and increased apoptosis. Normal CD34+ cells cultured in our culture system and transduced with the siRNA vector were practically blocked in their erythroid differentiation: 14 % glyco+/CD36- mature erythroid cells versus 81 % in untransduced and 80 % in cultures transduced with control lentivector (obtained after 14 days of culture). Differentiation into myeloid cells was not affected. To overexpress GATA-1 we cloned the wild-type as well as a mutated, caspase-resistant, form of GATA-1 in a pWPIR-ires-GFP bicistronic lentivector. Functionality of both lentivectors was validated in HeLa cells. For the study of GATA-1 in primary human hematopoietic cells we used an in vitro culture system in which CD34+ progenitors differentiate into mature red blood cells in the presence of erythropoietin, IL-3, and SCF. Transduction of CD34+ cells with lentivectors led to increase of GATA-1 mRNA (400-fold) measured by Realtime RT-PCR and to detection of protein. No difference was observed in cell numbers, expression of erythroid differentiation markers and survival between cells transduced with control vector and with pWPIR-GATA-1-ires-GFP. CD34+ cells from 3 patients with low-risk MDS in this culture system proliferated less (15x ± 13 amplification after 14 days of culture versus 72x ± 35 for normal precursors) differentiated less, and became apoptotic earlier than normal cells. However, overexpression of GATA-1 did not restore proliferation rate, nor did it lead to increased erythroid differentiation, or increase in survival. Conclusion: GATA-1 overexpression was not able to overcome defective erythroid differentiation of myelodysplastic progenitors, nor did it increase differentiation of normal erythroid progenitors. On the other hand, GATA-1 inhibition in normal erythroid precursors led to blockage of erythroid differentiation. We therefore assume that either factors upstream of GATA-1 or additional, GATA-1 independent factors, are responsible for the myelodysplastic phenotype.

scholarly journals Fetal Hemoglobin Rescues Ineffective Erythropoiesis in Sickle Cell Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 14-15
Author(s):  
Sara El Hoss ◽  
Sylvie Cochet ◽  
Auria Godard ◽  
Hongxia Yan ◽  
Michaël Dussiot ◽  
...  
Keyword(s):  
Cell Death ◽  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Ineffective Erythropoiesis ◽  
Hypoxic Conditions ◽  
Cd34 Cells ◽  
F Cells

Sickle cell disease (SCD) is an autosomal hereditary recessive disorder caused by a point mutation in the β globin gene resulting in a Glu-to-Val substitution at the 6th position of the β globin protein. The resulting abnormal hemoglobin (HbS) polymerizes under hypoxic conditions driving red blood cell (RBC) sickling (Pauling et al., 1949). While pathobiology of circulating RBCs has been extensively analyzed in SCD, erythropoiesis is surprisingly poorly documented. In β-thalassemia, ineffective erythropoiesis is characterized by high levels of apoptotic erythroblasts during the late stages of terminal differentiation, due to an accumulation of free β-globin chains (Arlet et al., 2016). Ineffective erythropoiesis is the major cause of anemia in β-thalassemia patients. In contrast, a marked decrease in life span of circulating red cells, a feature of sickle red cells, is considered to be the major determinant of chronic anemia in SCD. It is generally surmised that ineffective erythropoiesis contributes little to anemia. The bone marrow environment has been well documented to be hypoxic (0.1 to 6% O2) (Mantel et al., 2015). As hypoxia induces HbS polymerization, we hypothesized that cell death may occur in vivo because of HbS polymer formation in the late stages of differentiation characterized by high intracellular hemoglobin concentration. In the present study, using both in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. We explored the mechanistic basis of the ineffective erythropoiesis in SCD using biochemical, cellular and imaging techniques. In vitro erythroid differentiation using CD34+ cells isolated from SCD patients and from healthy donors was performed. A 2-phase erythroid differentiation protocol was used and cultures were performed at two different oxygen conditions, i.e. normoxia and partial hypoxia (5% O2). We found that hypoxia induces cell death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). This inference was supported by flow cytometry data showing higher percentages of dead cells within the non-F-cell population as compared to the F-cell population for SCD cells. Moreover, SCD dead cells showed higher levels of chaperon protein HSP70 in the cytoplasm than live cells, while no difference was detected between both subpopulations for control cells, suggesting that cell death of SCD erythroblasts was probably due to HSP70 cytoplasmic sequestration. This was supported by western-blot experiments showing less HSP70 in the nucleus of SCD erythroblasts under hypoxia, associated with decreased levels of GATA-1. At the molecular level, HSP70 was co-immunoprecipitated with HbS under hypoxia indicating that both proteins were in the same complex and suggesting interaction between HSP70 and HbS polymers in the cyotplasm. Importantly, we confirm these results in vivo by showing that in bone marrow of SCD patients (n = 5) cell loss occurs during terminal erythroid differentiation, with a significant drop in the cell count between the polychromatic and the orthochromatic stages (Figure 1). In order to specifically address the role of HbF in cell survival, we used a CRISPR-Cas9 approach to mimic the effect of hereditary persistence of fetal hemoglobin (HPFH). CD34+ cells were transfected either with a gRNA targeting the LRF binding site (-197) or a gRNA targeting an unrelated locus (AAVS1) (Weber, Frati, et al. 2020). As expected, the disruption of the LRF binding site resulted in HbF induction as shown by higher %F-cells compared to AAVS1 control. These higher levels of F-cells resulted in decreased apoptosis, under both normoxic and hypoxic conditions, clearly demonstrating the positive and selective effect of HbF on SCD cell survival (Figure 2). In summary, our study shows that HbF has a dual beneficial effect in SCD by conferring a preferential survival of F-cells in the circulation and by decreasing ineffective erythropoiesis. These findings thus bring new insights into the role of HbF in modulating clinical severity of anemia in SCD by both regulating red cell production and red cell destruction. Disclosures No relevant conflicts of interest to declare.

In vitro proliferation and differentiation of erythroid progenitors from patients with myelodysplastic syndromes: evidence for Fas-dependent apoptosis

Blood ◽  
2002 ◽  
Vol 99 (5) ◽  
pp. 1594-1601 ◽  
Author(s):  
Yann-Erick Claessens ◽  
Didier Bouscary ◽  
Jean-Michel Dupont ◽  
Françoise Picard ◽  
Josiane Melle ◽  
...  
Keyword(s):  
Myelodysplastic Syndromes ◽  
Liquid Culture ◽  
Fas Ligand ◽  
Chimeric Protein ◽  
Refractory Anemia ◽  
Erythroid Progenitors ◽  
In Vitro Proliferation ◽  
Cd34 Cells ◽  
Proliferation And Differentiation

Erythropoiesis results from the proliferation and differentiation of pluripotent stem cells into immature erythroid progenitors (ie, erythroid burst-forming units (BFU-Es), whose growth, survival, and terminal differentiation depends on erythropoietin (Epo). Ineffective erythropoiesis is a common feature of myelodysplastic syndromes (MDS). We used a 2-step liquid-culture procedure to study erythropoiesis in MDS. CD34+ cells from the marrow of patients with MDS were cultured for 10 days in serum-containing medium with Epo, stem cell factor, insulinlike growth factor 1, and steroid hormones until they reached the proerythroblast stage. The cells were then placed in medium containing Epo and insulin for terminal erythroid differentiation. Numbers of both MDS and normal control cells increased 103fold by day 15. However, in semisolid culture, cells from patients with refractory anemia (RA) with ringed sideroblasts and RA or RA with excess of blasts produced significantly fewer BFU-Es than cells from controls. Fluorescence in situ hybridization analysis of interphase nuclei from patients with chromosomal defects indicated that abnormal clones were expanded in vitro. Epo-signaling pathways (STAT5, Akt, and ERK 1/2) were normally activated in MDS erythroid progenitors. In contrast, apoptosis was significantly increased in MDS cells once they differentiated, whereas it remained low in normal cells. Fas was overexpressed on freshly isolated MDS CD34+ cells and on MDS erythroid cells throughout the culture. Apoptosis coincided with overproduction of Fas ligand during the differentiation stage and was inhibited by Fas-Fc chimeric protein. Thus, MDS CD34+-derived erythroid progenitors proliferated normally in our 2-step liquid culture with Epo but underwent abnormal Fas-dependent apoptosis during differentiation that could be responsible for the impaired erythropoiesis.

An Unsuspected Dyserythropoiesis in Gaucher Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1344-1344
Author(s):  
Niloofar Reihani ◽  
Jean-Benoît Arlet ◽  
Thierry Billette de Villemeur ◽  
Nadia Belmatoug ◽  
Yves Colin ◽  
...  
Keyword(s):  
Gaucher Disease ◽  
Bone Lesion ◽  
Erythroid Differentiation ◽  
Abnormal Behavior ◽  
Lysosomal Storage ◽  
Erythroid Progenitors ◽  
Adhesion Properties ◽  
Clinical Complications ◽  
Cd34 Cells

Abstract Introduction: Gaucher disease (GD) is a lysosomal storage disorder, impairing glucosylceramide catabolism. GlcCer-laden macrophages transform into Gaucher cells and are responsible for the major GD symptoms (tissue infiltration, organomegaly, bone lesion). However, other signs of the disease such as anemia, spleen and bone infarcts might involve red blood cells (RBC). Despite the lysosomal characteristic of GD and the evidence of the central rdole of macrophages, our recent findings pointed out unexpected RBC abnormalities (Franco et al, Blood. 2013;121:546-555). These findings uncovered an overlooked aspect in GD showing that GD RBC exhibit abnormal rheological, morphological and membrane adhesion properties. Thus, RBC could be considered as culprit for ischemic events in GD. In this study, we analyzed the erythroid differentiation in GD to address the etiology of red cell abnormal behavior. We induced in vitro erythropoiesis from circulating GD patient progenitors. Methods: After purification of CD34+ cells from peripheral blood of Gaucher patients (n=20) or controls (CTL), erythroid progenitors were expanded in the absence of erythropoietin (EPO) and differentiated in a second step with erythropoietin. GD and CTL CD34- cells were differentiated into macrophages by addition of M-CSF in the culture medium. To determine the effect of CTL and GD macrophages on normal erythropoiesis, in vitro generated macrophages were co-cultured with CTL erythroid progenitors in the presence of EPO. Proliferation of progenitors as well as erythroid differentiation were examined by flow cytometry and cytology. Results: Our preliminary results showed: (i) a dramatic decrease in proliferation of progenitors in the first step of expansion (**p <0.005), (ii) an accelerated erythroid maturation as evaluated by the percentage of cells expressing and non-expressing Glycophorin A and C-Kit, respectively (**p <0.005), (iii) an absence of effect of Gaucher macrophages co-culture on normal erythroid terminal maturation, and (iv) a correlation between the acceleration level of maturation and the degree of anemia in GD patients as determined by hemoglobin level (**p<0.005). Conclusion: We evidenced an unexpected dyserythropoiesis characterized by accelerated erythroid differentiation in GD. The observed dyserythropoiesis is independent of macrophage defects and is intrinsic to the GD erythroid lineage. These studies by helping to understand the role of erythroid cells in the pathophysiology of GD as well as the mechanism of anemia should prove useful for the development of additive therapies to alleviate debilitating clinical complications of this morbid disease such as osteonecrosis and splenomegaly. Disclosures Le Van Kim: SHIRE: Research Funding.

Knockdown of HCK Reduces Cell Death and Erythroid Differentiation in Human CD34+ Hematopoietic Progenitor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2860-2860
Author(s):  
Fernanda Marconi Roversi ◽  
Fernando Vieira Pericole ◽  
Adriana da Silva Santos Duarte ◽  
Karla Priscila Ferro ◽  
Flávia Adolfo Corrocher ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Specific Inhibitor ◽  
Erythroid Differentiation ◽  
Fold Change ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Myelodysplastic syndromes (MDS) are clonal disorders characterized by ineffective hematopoiesis and increased risk of transformation to acute myeloid leukemia (AML). The identification of genes and cellular pathways active in leukemia cells but not in normal hematopoietic stem/progenitor cells (HSC) may help to understand the key steps in the MDS and AML pathogenesis and lead to new approaches to further enhance the treatment of both diseases, considered incurable with non-transplantation therapy. Src kinase family (SFK) is a central mediator in multiple oncogenic signaling pathways and some SFK members (Hck, Lyn, Fgr, Fyn) had previously been described as overexpressed or activated in leukemic cells. However, to this moment, the role of hematopoietic cell kinase (HCK), the unique SFK member restricted expressed in hematopoietic cells, had not been characterized in MDS and AML pathogenesis as well as in HSC. In order to better understand the HCK importance in hematopoiesis, we used lentiviral shRNA vectors to knockdown the HCK expression in primary human CD34+ HSC. The HCK levels were reduced in approximately 70-80% (shHCK) compared to the control lentiviral shRNA (shControl-GFP). To promote erythroid differentiation, human CD34+ transduced cells were grown in methylcellulose for 7 days and in liquid media for another 6 days. During this experiment, shHCK cells showed decreased cell viability (fold change compared to shControl-GFP = 0.55, P<.0001, n=3) combined with an increase in CD71+ expression (fold change compared to shControl-GFP = 3, P<.01, n=3), indicating a delay in erythroid differentiation. As expected, shControl-GFP cells showed a decreased GATA1 expression during erythroid differentiation. Meanwhile, shHCK cells did not modulate GATA1 expression. Interestingly, without any stimulus, HCK knockdown in CD34+ cells significantly decreased apoptosis (AnnexinV+ cells) compared to shControl-GFP (fold change = 0.52, P<.01, n=4). Attempts have been made to overexpress HCK in CD34+ HSC, however more than 80% cells were apoptotic and further assays were not possible. Thus, in HSC, HCK participates of erythroid differentiation and apoptosis signaling. According to the HCK importance on HSC and that SFK inhibitors are undergoing early phase clinical testing, a specific inhibitory activity compound for HCK, named iHCK-37, had been developed by Dr Maurizzio Botta. We tested this compound on primary normal human CD34+ cells originated from healthy donors bone marrow samples and also from cord blood units. The iHCK-37 treatment did not change proliferation, survival and death of these normal CD34+ cells. Conversely, MDS and AML CD34+ cells treated with the same drug exhibited a dose-dependent growth inhibition. Likewise, following iHCK-37 treatment of MDS and AML total bone marrow mononuclear cells, the BFU-E and CFUs colony numbers were significantly decreased compared to untreated cells (vehicle). We also observed a potent in vitro antiproliferative activity of iHCK-37 against a panel of leukemia cell lines, with uM IC50 values in AML (5.0 - 5.8uM) and chronic myeloid leukemia (9.1 - 19.2uM). In addition, the combinatory in vitro treatment of iHCK-37 and 5-Azacitidine (Aza) also demonstrated additive effects relative to either drug alone. Interestingly, iHCK-37 or iHCK-37 plus Aza treatments of dysplastic and leukemia cells enhanced apoptosis and resulted in increased BAX and reduced BCL-XL protein levels. This result could be clinically relevant for MDS, as Aza is the only treatment available for higher-risk MDS, but with low response rates and frequent induced resistance and refractoriness over time. In summary, we herein have shown that HCK mRNA knockdown of normal CD34+ cells resulted in growth inhibition, decreased cell death and reduced erythroid differentiation, suggesting that HCK is essential for normal hematopoiesis. We presume that the deregulation of HCK pathway in leukemic cells might be crucial for MDS and AML pathogenesis. On the other hand, the inhibition of HCK protein activity with a specific inhibitor was able to restore the apoptotic pathways of leukemic cells, acting on cancer cells without alter any signaling of normal cells. Moreover, the specific inhibitor may have antineoplastic effect that can even be additive to current available drugs. Our study adds new insights to the role of HCK in MDS and AML as well as into potential new anticancer treatment strategies. Disclosures No relevant conflicts of interest to declare.

scholarly journals GPS2 promotes erythroid differentiation by control of the stability of EKLF protein

Blood ◽  
2020 ◽  
Vol 135 (25) ◽  
pp. 2302-2315
Author(s):  
Wen-Bing Ma ◽  
Xiao-Han Wang ◽  
Chang-Yan Li ◽  
Huan-Huan Tian ◽  
Jie Zhang ◽  
...  
Keyword(s):  
Thyroid Hormone Receptor ◽  
Fetal Liver ◽  
Severe Combined Immunodeficiency ◽  
Interleukin 2 ◽  
Flow Cytometric Analysis ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
The Stability

Abstract Erythropoiesis is a complex multistage process that involves differentiation of early erythroid progenitors to enucleated mature red blood cells, in which lineage-specific transcription factors play essential roles. Erythroid Krüppel-like factor (EKLF/KLF1) is a pleiotropic erythroid transcription factor that is required for the proper maturation of the erythroid cells, whose expression and activation are tightly controlled in a temporal and differentiation stage-specific manner. Here, we uncover a novel role of G-protein pathway suppressor 2 (GPS2), a subunit of the nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid hormone receptor corepressor complex, in erythrocyte differentiation. Our study demonstrates that knockdown of GPS2 significantly suppresses erythroid differentiation of human CD34+ cells cultured in vitro and xenotransplanted in nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor γ-chain null mice. Moreover, global deletion of GPS2 in mice causes impaired erythropoiesis in the fetal liver and leads to severe anemia. Flow cytometric analysis and Wright-Giemsa staining show a defective differentiation at late stages of erythropoiesis in Gps2−/− embryos. Mechanistically, GPS2 interacts with EKLF and prevents proteasome-mediated degradation of EKLF, thereby increasing EKLF stability and transcriptional activity. Moreover, we identify the amino acids 191-230 region in EKLF protein, responsible for GPS2 binding, that is highly conserved in mammals and essential for EKLF protein stability. Collectively, our study uncovers a previously unknown role of GPS2 as a posttranslational regulator that enhances the stability of EKLF protein and thereby promotes erythroid differentiation.

Pomalidomide Augments Fetal Hemoglobin Production In Primary Erythroid Cells By a Novel Mechanism Modulating BCL11A But Not KLF-1

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 314-314
Author(s):  
Abena O. Appiah-Kubi ◽  
Lionel Blanc ◽  
Sharon A. Singh ◽  
Sebastien Didier ◽  
Sehba Dsilva ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Culture System ◽  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Cell Production ◽  
Expression Levels ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
F Cells

Abstract Fetal hemoglobin (HbF) is a known modifier of sickle cell disease (SCD) severity. KLF-1 is a regulator of the globin switch. It does so by increasing beta-globin production and up-regulating BCL11A, a repressor of HbF synthesis. Pomalidomide, a second generation immunomodulatory drug (IMiD), regulates HbF and F-cell production during erythropoiesis in human CD34+ cells. The mechanism by which pomalidomide enhances F-cell production is not well understood. In this study, CD34+ cells were obtained after purification of peripheral blood and positive selection and cultured using a three-phase in vitro liquid culture system which recapitulates erythropoiesis, including terminal differentiation and enucleation, in the presence of no drug, pomalidomide, hydroxyurea, or dimethyl sulfoxide (DMSO; vehicle control). Erythroid differentiation was assessed morphologically and by flow cytometry using the transferrin receptor and glycophorin A as markers of erythroid maturation. Flow cytometry was used to quantify F-cells. RT-qPCR was used to quantify mRNA expression of BCL11A, KLF-1, and gamma-globin. Western blot was used to measure the total expression levels of BCL11A. In this culture system pomalidomide increased F-cells more than hydroxyurea in both SCD and normal control erythroid cultures. There was a significant decrease in BCL11A expression levels, a repressor of HbF synthesis, with pomalidomide but not with hydroxyurea. This decrease was seen in both SCD and normal samples. KLF-1 was not affected by pomalidomide. These findings suggest a very different mechanism of action for pomalidomide versus hydroxyurea in increasing F-cell production. Pomalidomide appears to target the erythroid specific BCL11A but not the more pleiotropic transcription regulator KLF-1. Since the F-cell production was augmented in the presence of pomalidomide in controls as well as SCD erythroid cultures this study suggests a role for pomalidomide in the pharmacologic augmentation of fetal hemoglobin levels, perhaps in addition to hydroxyurea, not only in SCD but in any beta-hemoglobinopathy. Disclosures: Chan: BioTheryX Inc: Employment.

Loss of FoxM1 Promotes Erythroid Differentiation through Increased Proliferation of Erythroid Progenitors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 933-933
Author(s):  
Minyoung Youn ◽  
Elena Bibikova ◽  
Corinne LaVasseur ◽  
Bertil Glader ◽  
Kathleen Sakamoto ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Fold Increase ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Human Cord Blood ◽  
Cd34 Cells ◽  
Multipotent Progenitor Cells ◽  
Foxm1 Expression ◽  
Cord Blood Cd34

Abstract FoxM1 belongs to the fork head/winged-helix family of transcription factors and regulates a network of proliferation-associated genes including the G2/M transition, chromosome segregation, and spindle assembly. FoxM1 expression is commonly upregulated in a number of human cancers such as liver, ovarian, breast, prostate, colon, and brain tumors. Its abnormal upregulation has been shown to be a key driver of cancer progression and an initiating factor of oncogenesis. In normal cells, FoxM1 is highly expressed in multipotent progenitor cells and inhibits differentiation of the progenitors, suggesting that FoxM1 plays in a role in the maintenance of multipotent progenitor cells. However, the exact molecular mechanism by which FoxM1 regulates stem/progenitor cells is still uncharacterized. In this study, we have examined the role of FoxM1 in normal hematopoiesis using human cord blood CD34+ cells. To investigate the role of FoxM1 in normal hematopoiesis, we infected human cord blood CD34+ cells with FoxM1 shRNA lentivirus and observed blood cell differentiation using FACS analysis with a range of cell surface markers. We found that knockdown of FoxM1 resulted in an increase of the erythroid population (CD71+/GlyA+), a decrease of the myeloid population (CD11b+), and an unchanged megakaryocyte population (CD41a+) in two phase liquid culture system. Overall, we found a 2-fold increase in the erythroid population compared to the myeloid population. Importantly, methylcellulose colony assays also demonstrated increased numbers of CFU-E colonies (2-2.5 fold increase compared to control) and decreased numbers of CFU-GM colonies in FoxM1 knockdown cells. Taken together, these findings imply a role for FoxM1 in normal erythropoiesis. To better define the function of FoxM1 in hematopoietic cells, we sorted distinct populations of cells based on their cell surface marker expression and quantitated FoxM1 expression level by RT-qPCR. FoxM1 had a 3-fold increased expression in CD71+ (erythroid) cells compared to CD11b+ (myeloid) cells. Additionally, we found FoxM1 expression was particularly elevated in the BFU-E and CFU-E stages of erythropoiesis, suggesting a functional role for FoxM1 in erythroid progenitor proliferation. Finally, to study the potential molecular mechanism of FoxM1 in normal hematopoiesis, we analyzed cell cycle progress in FoxM1 knockdown cells with DAPI staining. We found increased S and G2/M phases in FoxM1 knockdown cells, which was significant only in the CD71+ (erythroid) population and not in the unsorted cell populations. We also detected an increase of BrdU+ cells in FoxM1 knockdown CD71+ population by BrdU incorporation assay, indicating faster proliferation of erythroid progenitors with FoxM1 knockdown. These findings suggest a novel function of FoxM1 in normal human hematopoiesis, in which FoxM1 deficiency leads to increased proliferation of erythroid progenitors resulting in increased erythroid differentiation. Our data indicate that FoxM1 inhibitors, such as Thiostrepton or FDI-6, may be beneficial in treating patients with anemia. Disclosures No relevant conflicts of interest to declare.

Comprehensive Analysis of Aberrant RNA Splicing in Myelodysplastic Syndromes

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 826-826 ◽  
Author(s):  
Yusuke Shiozawa ◽  
Sato Sato-Otsubo ◽  
Anna Gallì ◽  
Kenichi Yoshida ◽  
Tetsuichi Yoshizato ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Rna Sequencing ◽  
Myelodysplastic Syndromes ◽  
Rna Splicing ◽  
Comprehensive Analysis ◽  
Erythroid Cells ◽  
Splice Sites ◽  
Cd34 Cells

Abstract Introduction Splicing factor (SF) mutations represent a novel class of driver mutations highly prevalent in myelodysplastic syndromes (MDS), where four genes, including SF3B1, SRSF2, U2AF1, and ZRSR2, are most frequently affected. SF3B1 and SRSF2 mutations show prominent specificity to RARS/RCMD-RS and CMML subtypes, respectively. Although abnormal RNA splicing is thought to play a central role in the pathogenic mechanism of mutated SFs, little is known about exact gene targets, whose abnormal splicing is implicated in the pathogenesis of MDS or about the molecular mechanism that explains the unique subtype specificity of SF mutations, especially to those subtypes characterized by increased ring sideroblasts. Methods To address these issues, comprehensive analysis of abnormal RNA splicing was performed for a total of 336 MDS patients with different SF mutations. High-quality RNA was extracted from bone marrow mononuclear cells (BM/MNCs) and/or CD34+ cells and subjected to high-throughput sequencing, followed by exhaustive detection of splicing junctions for all relevant reads. Aberrant splicing events associated with different SF mutations were explored by comparing observed splicing junctions between samples with and without mutations. To specifically determine the role of SF3B1 mutations in ring sideroblast formation, CD34+ bone marrow cells from 13 patients with or without SF3B1 mutations were differentiated in vitro into erythroid cells. RNA sequencing was performed on cells recovered on day 7 and day 14 and differentially spliced genes in erythroid cells between SF3B1-mutated and unmutated samples were investigated. Results SF3B1, SRSF2, U2AF1, and ZRSR2 were mutated in 28%, 18%, 5%, and 7% of the patients, respectively. First, we compared SF3B1-mutated samples with those without known SF mutations. RNA sequencing of CD34+ cells revealed 230 splicing events significantly enriched in SF3B1-mutated cases, of which 90% (n = 206) were caused by misrecognition of 3' splice sites. A similar result was obtained in the experiment for BM/MNCs, where 177 (83%) out of 206 splicing events significantly enriched in SF3B1-mutated samples were caused by misrecognition of 3' splice sites. These observations were in accordance with the known function of SF3B1 in branch point recognition in the U2 snRNP complex. In both BM/MNCs and CD34+ cells, approximately 70% of the unusual 3' splice sites were located from 5 to 25 bases downstream from the authentic junctions. The bases immediately upstream to these 3' splice sites were more often pyrimidines, which was not accordance with the general rule: the bases next to 3' splice sites are purines, especially guanines. About 50% of these altered 3' splice sites resulted in frameshift, indicating that SF3B1 mutations cause deleterious effects in many genes simultaneously. Next, to explore the genes whose abnormal splicing is responsible for increased ring sideroblast formation, RNA sequencing was carried out for erythroid progenitor cells differentiated in vitro from CD34+ cells from MDS patients with or without SF3B1 mutations. We found that a total of 146 altered 3' splice sites were significantly associated with SF3B1 mutations, of which 87 were overlapped to the aberrant splice sites shown to be enriched in SF3B1 mutated primary MDS specimens. These splice sites were found in genes involved in heme biosynthesis, cell cycle progression, and DNA repair and their consequence was mostly deleterious due to aberrant frameshifts. Abnormal splicing events associated with other SF mutations were also identified. Among these, the most common abnormalities associated with mutated SRSF2 and U2AF1 were alternative exon usage. Misrecognition of 3' splice sites was also common in U2AF1-mutated cases. ZRSR2 mutations were associated with retentions of U12 introns, which is consistent with the known role of ZRSR2 as an essential component of the minor (U12-type) spliceosome. Conclusion SF mutations were associated with characteristic abnormal splicing changes in primary MDS samples as well as in vitro cultured cells. Our results provide insights into the pathogenic role of SF mutations in MDS. Disclosures No relevant conflicts of interest to declare.

scholarly journals The role of Ikaros in human erythroid differentiation

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1138-1146 ◽  
Author(s):  
Marilyne Dijon ◽  
Florence Bardin ◽  
Anne Murati ◽  
Michèle Batoz ◽  
Christian Chabannon ◽  
...  
Keyword(s):  
Culture System ◽  
Myeloid Cells ◽  
Erythroid Differentiation ◽  
Relative Increase ◽  
Dominant Negative ◽  
Erythroid Cells ◽  
Cd34 Cells ◽  
First Time ◽  
Erythroid Development

Abstract Ikaros—a factor that positively or negatively controls gene transcription—is active in murine adult erythroid cells, and involved in fetal to adult globin switching. Mice with Ikaros mutations have defects in erythropoiesis and anemia. In this paper, we have studied the role of Ikaros in human erythroid development for the first time. Using a gene-transfer strategy, we expressed Ikaros 6 (Ik6)—a known dominant-negative protein that interferes with normal Ikaros activity—in cord blood or apheresis CD34+ cells that were induced to differentiate along the erythroid pathway. Lentivirally induced Ik6-forced expression resulted in increased cell death, decreased cell proliferation, and decreased expression of erythroid-specific genes, including GATA1 and fetal and adult globins. In contrast, we observed the maintenance of a residual myeloid population that can be detected in this culture system, with a relative increase of myeloid gene expression, including PU1. In secondary cultures, expression of Ik6 favored reversion of sorted and phenotypically defined erythroid cells into myeloid cells, and prevented reversion of myeloid cells into erythroid cells. We conclude that Ikaros is involved in human adult or fetal erythroid differentiation as well as in the commitment between erythroid and myeloid cells.

scholarly journals Heterozygous Mutations in DDX41 Cause Erythroid Progenitor Cell Defects

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 148-148
Author(s):  
Timothy M Chlon ◽  
Emily Stepanchick ◽  
Analise Sulentic ◽  
Kathleen Hueneman ◽  
Daniel Starczynowski
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Progenitor Cells ◽  
Liquid Culture ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Colony Assays

Abstract Germline mutations in the RNA Helicase gene DDX41 cause inherited susceptibility to Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). These mutations are always heterozygous and are typically frameshifts, causing loss of protein expression. We recently reported that at least one functional copy of DDX41 is essential for hematopoiesis, and that DDX41 is required for ribosome biogenesis. While biallelic DDX41 mutations cause dramatic defects in hematopoiesis, the role of heterozygous mutations in Myelodysplastic Syndrome pathogenesis is not yet understood. Recent clinical studies have pointed out that some patients bearing germline DDX41 mutations have idiopathic cytopenias of unknown significance (ICUS) prior to MDS onset, suggesting that underlying hematopoietic defects precede and potentially contribute to the onset of MDS/AML (Choi et al., Haemotologica 2021). It has also been noted that the majority of DDX41-mutant MDS patients have refractory anemia, indicating that the erythroid lineage is particularly effected in these patients (Sebert et al., Blood 2019). Since ribosome defects are a common cause of inherited anemias and also contribute to MDS pathogenesis, we characterized the effect of heterozygous DDX41 mutations on erythropoiesis in murine and human models. Mice that have been transplanted with Ddx41 +/- bone marrow develop anemia at 12-15 months post-transplant, indicating that detection of erythroid defects in vivo is aging-dependent. We characterized the effect of heterozygosity of Ddx41 on erythroid progenitor function in vitro and found that Ddx41 +/- bone marrow from young mice yields fewer BFU-E in colony assays but comparable numbers of myeloid colonies. Liquid culture erythroid differentiation of Ddx41 +/- bone marrow produces fewer CD71+ Ter119+ progenitors than controls. To characterize the effect of heterozygous DDX41 mutations on human erythropoiesis, we generated induced pluripotent stem cells bearing heterozygous frameshift mutations in DDX41 using CRISPR. We found that these DDX41 +/- iPSC lines produced CD43+/CD34+ hematopoietic progenitor cells (HPC) with equal efficiency as unmodified control iPSC. However, once these HPC were induced to differentiate down the erythroid lineage in liquid culture, they made fewer CD71+ GLYA+ erythroid progenitors and fewer hemoglobinized cells. The DDX41 +/- HPC also produced fewer BFU-E in colony assays. Mechanistically, we found that the in vitro-derived erythroid progenitors from both mice and human iPSC had decreased protein translation, suggesting that ribosome defects underlie the observed erythroid differentiation defects. In diseases such as Diamond Blackfan Anemia and Dyskeratosis Congenita, ribosome defects lead to p53 activation which reduces cell cycle progression in erythroid progenitors. To test the role of p53 in the erythroid defects caused by Ddx41 heterozygosity, we crossed Ddx41 +/- mice with p53-knockout mice and found that loss of p53 fully rescued the BFU-E colony formation of Ddx41 +/- bone marrow HPC. We confirmed this finding using CRISPR-mediated knockout of p53 in Ddx41 +/- BM HPC. Collectively, these results suggest that a mild ribosome defect in DDX41 +/- HPC causes a deficit in erythropoiesis that results in anemia with aging. It is likely that this anemia causes stress in the bone marrow and a selective environment in which malignant hematopoietic stem and progenitor cells arise, leading to MDS and AML. Disclosures Starczynowski: kurome Inc: Consultancy.

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