Induction Of Erythroid Differentiation Research Articles

Vitamin C derivative/AA2P promotes erythroid differentiation by upregulating CA1

Abstract Vitamin C is used to treat anaemia; however, the mechanism through which vitamin C promotes erythroid differentiation is not comprehensively understood. The in vitro erythroid differentiation induction system can reveal the differentiation mechanism and provide erythrocytes for clinical transfusion and anaemia treatment. This process can be promoted by adding small-molecule compounds. In this study, we added l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AA2P), a derivative of vitamin C, to an erythroid differentiation system induced from umbilical cord blood haematopoietic stem and progenitor cells in vitro and detected its effect on erythroid differentiation using single-cell transcription sequencing technology combined with non-targeted metabolism detection. AA2P increased the proportion of late basophilic erythroblasts, upregulating the expression of erythroid-related regulatory molecules GATA1, KLF1, ALAS2, and the globins HBG and HBB. CA1 is a target gene of AA2P, and CA1 knockdown affected the expression of globin-related genes. AA2P also increased glycolysis and decreased oxidative phosphorylation to facilitate terminal erythroid differentiation and enhanced the proliferation of early erythroid progenitors by altering the cell cycle. These results provide a reliable basis for using vitamin C to improve the efficiency of erythropoiesis in vitro and for the clinical treatment of anaemia.

BCL11A-targeted γ-globin gene induction by triterpenoid glycosides of Fagonia indica: A preclinical scientific validation of indigenous herb for the treatment of β-hemoglobinopathies

Pharmacological induction of fetal hemoglobin has proven to be a promising therapeutic intervention in β-hemoglobinopathies by reducing the globin chain imbalance and inhibiting sickle cell polymerization. Fagonia indica has shown therapeutic relevance to β-thalassemia. Therefore, we study the ethnopharmacological potential of Fagonia indica and its biomarker compounds for their HbF induction ability for the treatment of β-thalassemia. Here, we identify, compound 8 (triterpenoid glycosides) of F. indica. as a prominent HbF inducer in-vitro and in-vivo. Compound 8 showed potent erythroid differentiation, enhanced cellular proliferation, ample accumulation of total hemoglobin, and a strong notion of γ-globin gene expression in K562 cultures. Compound 8 treatment also revealed strong induction of erythroid differentiation and fetal hemoglobin mRNA and protein in adult erythroid precursor cells. This induction was associated with simultaneous downregulation of BCL11A and SOX6, and overexpression of the GATA-1 gene, suggesting a compound 8-mediated partial mechanism involved in the reactivation of fetal-like globin genes. The in vivo study with compound 8 (10 mg/kg) in β-YAC mice resulted in significant HbF synthesis demonstrated by the enhanced level of F-cells (84.14 %) and an 8.85-fold increase in the γ-globin gene. Overall, the study identifies compound 8 as a new HbF-inducing entity and provides an early “proof-of-concept” to enable the initiation of preclinical and clinical studies in the development of this HbF-inducing agent for β-thalassemia.

Abstract 4040: Asciminib activates erythroid differentiation in K562 cell line

Abstract Background: Asciminib an allosteric tyrosine kinase inhibitor (TKI) was recently approved by the FDA for managing Philadelphia positive chronic myeloid leukemia (CML) patients in chronic phase who had failed to respond to previous TKIs. In this study, we used human erythroleukemic K562 cell line to investigate the role of asciminib as an inducer of erythroid differentiation. Additionally, imatinib, which has previously been reported as a potential inducer of erythroid differentiation, was also tested on these cells, both independently as well as in combination, in order to investigate the degree of erythroid differentiation in a K562 cell line. Methodology: In this study, we investigated the effect of asciminib (5-20nM) with and without imatinib (50-200nM) in differentiating the erythroleukemic cell line K562 towards erythroid lineage. K562 cells were cultured over a 12-day period in basic (IMDM) media and an EPO-based differentiation media to select optimal medium conditions. In addition, an in-vitro drug cytotoxicity experiment (XTT) was used to determine the preliminary toxicity of asciminib with IMDM media and EPO-based differentiation media CD235a and CD71 surface marker expressions were analyzed by flow cytometry at all time-points to determine the degree of differentiation achieved by each of the conditions Moreover, to analyze and identify hemoglobin producing cells ensuing differentiation, benzidine staining was performed at indicated time points. Results: Our results suggest that asciminib, imatinib or a combination of both drugs in EPO-based differentiation media results in efficient erythroid differentiation with high levels of GPA expression (approx. 90%) and significant upregulation of globin genes (p value = 0.0001). These results were further confirmed by benzidine staining, where drug addition and dose escalation significantly increased benzidine-positive cells. Our results also demonstrated that asciminib was able to achieve maximum differentiation at a much earlier time point as compared to imatinib. Moreover, asciminib, when combined with another TKI such as imatinib, can also aid in lowering the drug concentrations being used, hence resulting in lessening the toxicity of the drug when used alone required at high doses. Conclusion: The results from this study highlighted the novel role of asciminib in erythroid differentiation using an in-vitro K562 model that can give high-levels of surface markers and globin expression. Potential clinical application of asciminib in hematological diseases that are associated specifically with a failure in the expression of globin genes should be evaluated. Keywords:K562, erythroid differentiation, asciminib, imatinib, glycophorin A, transferrin receptor, hemoglobin Citation Format: Hammad Hassan, Sarah Yousuf, Syed Muhammad Ahmed, Karim Ruknuddin, Hadiqa Raees, Sadia Habib, Fizza Iftikhar, Afsar Ali Mian, Elnasir Lalani. Asciminib activates erythroid differentiation in K562 cell line. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4040.

Activin A downregulates the CD69-MT2A axis via p38MAPK to induce erythroid differentiation that sensitizes BCR-ABL-positive cells to imatinib

Induction of differentiation sensitizes chronic myeloid leukemia (CML) cells to the BCR-ABL inhibitor imatinib by mechanisms that remain unknown. We previously identified the BCR-ABL downstream effector CD69 which inhibits imatinib-induced CML cell differentiation. Herein, we found that the erythroid differentiation inducers activin A and aclacinomycin A induced expression of erythroid markers (α-globin, ζ-globin, GATA-1, and glycophorin A) and simultaneously reduced CD69 levels in K562 CML cells. Blockade of p38MAPK by SB203580 and shRNA eliminated the inhibitory effect of activin A on the promoter, mRNA, and protein levels and positive cell population of CD69. CD69 overexpression inhibited activin A-induced erythroid marker expression. Pretreatment of K562 cells with activin A to induce differentiation followed by a subtoxic concentration of imatinib caused growth inhibition and apoptosis that was reduced by CD69 overexpression. Activin A also reduced the expression of CD69's potential downstream molecule metallothionein 2A (MT2A) via p38MAPK. MT2A-knockdown reduced CD69 inhibition of activin A-induced erythroid marker expression. Furthermore, MT2A-knockdown reduced CD69 inhibition of activin A-imatinib sequential treatment-mediated growth inhibition and apoptosis in K562 and BCR-ABL-expressing CD34+ cells. These results suggest that CD69 inhibits activin A induction of erythroid differentiation-mediated CML cell sensitivity to imatinib via MT2A. Therefore, activin A induction of erythroid differentiation sensitizes BCR-ABL-positive cells to imatinib by downregulating the erythroid differentiation suppressors CD69 and MT2A.

Inflammatory Signature, Oxidative Stress, and DNA Damage Response in DBA Pathogenesis

Diamond-Blackfan anemia (DBA) is an inherited pure red cell aplasia caused mostly by pathogenic variants in genes encoding for ribosomal proteins (RPs). It was previously published that haploinsufficiency of RP leads to increased apoptosis of erythroid precursors and p53 upregulation in CD34+cells from DBA patients and in animal models. However, the exact mechanisms of p53 activation are still not completely understood. In our study, we examined the link between activation of p53 pathway, inflammation-evoked oxidative stress and DNA damage in DBA patients and cellular model.To investigate the pathogenesis of DBA, we prepared the RPL5- and RPS19-deficient murine erythroleukemia cells (MEL) by using CRISPR/Cas9 technology. The particular clones were heterozygous for different nucleotide size deletions in RPL5 or RPS19 genes resulting in decreased levels of these proteins to half when compared to control cells thus mimicking the RP haploinsuficiency in DBA patients. We examined the pathological consequences of RPL5 and RPS19 deficiency in both undifferentiated and differentiated cells; the differentiation was induced by either dimethyl sulfoxide - DMSO or hexamethylene bisacetamide - HMBA for at least 48 hours. Significantly decreased proliferation capacity and significantly increased apoptosis rate was detected for all undifferentiated RPL5- and RPS19-deficient clones in comparison with controlcells. Upon induction of erythroid differentiation, RPL5- and RPS19-deficient clones showed abnormal GATA1 expression (2-times lower than in control cells) and higher percentage of TUNEL+ apoptotic cells (RPL5-deficient cells 2.04%; RPS19-deficient cells 1.57%;controlcells 0.50%).The data from cellular models were consistent with an increased rate of apoptosis detected in the bone marrow of RPL5 and RPS19 mutant DBA patients by TUNEL assay (15-times more TUNEL+ cells in RPL5 and RPS19 mutant patients than in normal controls). These patients showed also induction of p53 in their bone marrow (p53 positive cells: 8.4% in RPL5-mutant patient and 7.3% in RPS19-mutant patient vs. 0.4% in normal controls). The activation of p53 was also confirmed in RPL5- and RPS19-deficient clones by detection of increased level of p53 phosphorylation.Because oxidative stress is known to induce p53, we examined oxidative DNA damage marker 8-oxoguanine (8-oxoG) in MEL-DBA clones. Cytospin slides of differentiated RPL5- and RPS19-deficient clones revealed elevated positivity for 8-oxoG in contrast to control cells. It is known that oxidative damage is caused by exposure of cells to inflammatory cytokines, which activate DNA damage response (DDR) through reactive oxygen species (ROS)-mediated DNA damage. We found upregulation of pro-inflammatory cytokines TNF-α and IL6 in RPL5- and RPS19-deficient cells and IL1b in RPS19-deficient cells. The increased expression of Map3k8 kinase or increased phosphorylation of p38 was detected in RPL5-deficient or RPS19-deficient cells; both of these molecules are known to play essential roles in the production of pro-inflammatory cytokines. We then analyzed the levels of pro-inflammatory cytokines in our DBA patients and observed that besides TNF-α, IL1b, and IL6 also other inflammatory cytokines (IL1a, IL4, IL10, IL12, IL17a, INF-γ, and GM-CSF) were upregulated in their serum. We also detected higher levels of ROS in differentiated MEL-DBA clones in comparison with controlcells. Increased ROS levels were also present in the serum of all DBA patients with elevated inflammatory cytokines in contrast to healthy controls. As a result of ROS production, DNA double-strand breaks can occur responding by phosphorylation of Ser-139 residue of the histone variant H2AX (γ‐H2AX). Indeed, higher γ‐H2AX positivity was seen for RPL5- and RPS19-deficient cells in comparison with control cells (mean intensity of fluorescence for RPL5-deficient cells: 2.63 ±0.34; RPS19-deficient cells: 3.29 ±0.41; and control cells: 1.02 ±0.22).Our results suggest that the defective ribosomal biogenesis is associated with the induction of inflammatory cytokine signature, oxidative stress, increased DNA damage and activation of DDR signaling. Further research is needed to elucidate the hierarchy of these processes.This study was supported by project GACR 15-13732S, AZV 16-32105A, IGA_LF_2017_015, and in part by LTAUSA17142 (BK). DisclosuresNo relevant conflicts of interest to declare.

Effect of Nicotinamide, 1-Methylnicotinamide, and N’-Methylnicotinamide on Erythroid Colony Formation and γ-Globin Expression in Cultured Baboon CD34+ Cells

Pharmacological treatments designed to increase Fetal Hemoglobin (HbF) levels offer great promise to alleviate the symptoms and improve the lifespan of the vast numbers of patients afflicted with sickle cell disease (SCD) and β-thalassemia. Hydroxyurea can increase HbF, but a large fraction of patients with SCD do not respond to the drug. DNMT1 and LSD1 inhibitors are the most powerful drugs to increase HbF but are limited by side effects that include neutropenia, thrombophilia and/or thrombocytopenia. The development of new, more effective, and safer pharmacological strategies to augment HbF levels in the blood thus continues to be an important goal. Previous studies have shown that γ-globin gene expression is dynamically regulated during erythroid differentiation (Papayannopoulou et al PNAS 74:2923-2927, 1977). The proportion of γ-globin gene expression is higher at earlier stages (BFUe) of erythroid differentiation than at more advanced stages (CFUe). Therefore, we suggest the hypothesis that expansion of primitive, less differentiated progenitors might favor increased γ-globin, particularly when combined other HbF-inducing drugs. To investigate this hypothesis, we have tested whether nicotinamide (NAM), the major NAM metabolite 1-methylnicotinamide (1-mNAM), and N'-methylnicotinamide (N'-mNAM), a chemical derivative of NAM, can foster expansion of erythroid colony-forming cells (BFUe and CFUe) and increase γ-globin expression of cultured baboon CD34+ cells. Previous observations have shown that NAM facilitates in vitro expansion of cord blood CD34+ cells and enhanced long term engraftment in transplanted recipients (Horwitz et al J Clin Invest 124:3121, 2014). Contrasting effects of NAM, 1-mNAM, and N'-mNAM on differentiation and proliferation of the murine erythroleukemia cell line (MEL) have been previously reported (Terada et al PNAS 76:6414, 1979; Kuykendall et al Toxicol In Vitro 21:1656, 2007). While both NAM and N'-mNAM induced MEL cell differentiation, N'-mNAM was far more potent. In contrast, 1-mNAM increased cell proliferation, reduced spontaneous differentiation, and blocked differentiation induced by NAM and N'-mNAM. The effect of all three forms of NAM was examined using bone marrow (BM) CD34+ cells from a pre-clinical non-human primate large animal model. To test the effect of NAM, 1-mNAM, and N'-mNAM on expansion of erythroid colony-forming cells, the agents (5mM) were added to liquid cultures of baboon CD34+ bone marrow cells previously expanded for 5 days in serum-free expansion media (SFEM). Colony assays were performed on d8. In two experiments total erythroid colonies (BFUe and CFUe) were 2 fold higher in cultures treated with 1-mNAM compared to untreated controls (p<0.05) while no effect was observed in cultures treated with NAM. No colonies were observed in cultures treated with N'-mNAM (Figure 1A). Observation of Wright's stained cytospin preparations showed extensive erythroid differentiation on d8 in cells treated with N'-mNAM (Figure 1B). The effect of NAM, 1-mNAM, and N'-NAM on γ-globin expression was tested in baboon CD34+ cells grown in co-culture with the AFT024 cell line. NAM, 1-mNAM, or N'-mNAM (5mM) were added to cultures on d7. Expression of γ- and β-globin mRNA was measured by RT-PCR on d17. Increased γ-globin expression (0.57±0.04 γ/γ+β)) was observed in cells treated with N'-mNAM on d7 compared to untreated controls (0.20±0.09 γ/γ+β; p<0.001). NAM and 1-mNAM had no significant effect on γ-globin gene expression (Figure 1C). These results thus show that while erythroid colonies are increased by 1-mNAM, N'-mNAM is a potent inducer of erythroid differentiation and increases γ-globin expression in primary cultures of baboon CD34+ cells. In conclusion, 1-mNAM and N'-mNAM have contrasting effects on erythroid differentiation in primary baboon CD34+ cell cultures, confirming previous experiments in the MEL cell line. Future experiments are planned to test the effect of these agents on HbF in the baboon. Disclosures Saunthararajah: EpiDestiny: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

BILIVERDIN REDUCTASE B/ FLAVIN REDUCTASE (BLVRB/FLR) EXHIBITS HEME-REGULATED, NADPH-DEPENDENT REDUCTASE ACTIVITY AND CONFERS CYTOPROTECTION IN DEVELOPING ERYTHROID CELLS

Biliverdin reductase B (BLVRB), also known as flavin reductase (FLR), is especially expressed in cells having high levels of heme synthesis (e.g. developing erythroid cells, fetal and adult liver). BLVRB exhibits NADPH-dependent reductase activity toward non-alpha isomers of biliverdin, flavins and redox cycling agents, and also has pharmacological importance in the treatment of methemoglobinemia. Nonetheless, the physiological function of BLVRB is obscure. In this work, the functional role of BLVRB in developing erythroid cells in relation to its heme-associated properties is elucidated. Recombinant mouse BLVRB binds ferric heme with a submicromolar affinity in association with inhibition of the NADPH-dependent reductase activity while displaying heme peroxidase activity. The interaction of BLVRB with heme in developing erythroid cells (MEL) was demonstrated by proteomic analysis of heme peroxidase-associated proteins. Upon induction of erythroid differentiation of MEL cells, the BLVRB protein expression is markedly up-regulated. Knockdown of BLVRB in the induced MEL cells aggravates intracellular oxidation and cytotoxicity mediated by thiol depleting agent and heme synthesis inhibitor, suggesting that BLVRB may maintain the cell viability under oxidative stress or heme deficient conditions. Our study suggests that BLVRB/FLR, which is usually regarded as a biliverdin reducing enzyme in the heme degradation pathway, is an erythroid active, heme-regulated NADPH-dependent reductase. It confers cytoprotection in developing erythroid cells under oxidative stress conditions in connection with cellular heme levels. This work was supported by General Research Fund (17160916) and Health and Medical Research Fund (04151846).

Thiourea derivatives induce fetal hemoglobin production in-vitro: A new class of potential therapeutic agents for β-thalassemia

Fetal hemoglobin (HbF) induction is a cost-effective therapeutic approach for the treatment of β-hemoglobinopathies like β-thalassemia and sickle cell anemia. The present study discusses the potential of thiourea derivatives as new class of compounds that induce the fetal hemoglobin production. HbF inducing effect of thiourea derivatives was studied using experimental cell system, the human erythroleukemic K562 cell line. Erythroid induction of K562 cells was studied by the benzidine/H2O2 reaction, total hemoglobin production was estimated by plasma hemoglobin assay kit, and γ-globin gene expression by RT-qPCR, whereas fetal hemoglobin production was estimated by flow cytometry and immunofluorescence microscopy. The results indicated that newly synthesized thiourea derivative are potent inducers of erythroid differentiation of K562 cells with an increased γ-globin gene expression and fetal hemoglobin production. Moreover, these compounds showed no cytotoxic effect and inhibition on K562 cells at HbF inducing concentrations. It is important to note that hydroxyurea is a cytotoxic chemotherapeutic agent and have deleterious side effects, reflecting the need to identify new safe and effective HbF induces. These results signify thiourea derivatives as promising HbF inducers, with the potential to be studied against hematological disorders, including β-thalassemia and sickle cell anemia.

Isolation of a novel embryonic stem cell cord blood–derived population with in vitro hematopoietic capacity in the presence of Wharton's jelly–derived mesenchymal stromal cells

BackgroundRecent studies highlight the existence of a population of cord blood (CB)–derived stem cells that bare embryonic features (very small embryonic-like stem cells [VSELs]) as the most primitive CB-stem cell population. In the present study, we present for the first time a novel and high purity isolation method of VSELs with in vitro hematopoietic capacity in the presence of Wharton's jelly–derived mesenchymal stromal cells (WJ-MSCs). MethodsThe experimental procedure includes isolation upon gradually increased centrifugation spins and chemotaxis to Stromal cell-derived factor 1a (SDF-1a). Τhis cell population is characterized with flow cytometry, alkaline phosphatase (ALP) staining and qRT-PCR. The functional role of the isolated VSELs is assayed following co-culture with WJ-MSCs or bone marrow–derived mesenchymal stromal cells (BM-MSCs), whereas the stimulation of the quiescent VSEL population is verified via cell cycle analysis. The in vitro hematopoietic capacity is evaluated in methylcellulose cultures and also through induction of erythroid differentiation. ResultsThe final isolated subpopulation is characterized as a small-sized CD45/Lineage-/CXCR4+/CD133+/SSEA-4+cell population, positive in ALP staining and overexpressing the Oct3/4, Nanog and Sox-2 transcription factors. Upon the co-culture with MSCs, a stimulation of the quiescent VSEL population is observed. An impressive increase in the co-expression of the CD34+/CD45+ markers is observed following the co-culture with the WJ-MSCs, which is confirmed by the intense clonogenic ability suggesting in vitro differentiation toward all of the hematopoietic cell lineages and successful differentiation toward erythrocytes. DiscussionConclusively, we propose a novel, rapid and rather simplified isolation method of CB-VSELs, capable of in vitro hematopoiesis.

Induction of apoptosis and erythroid differentiation of human chronic myelogenous leukemia K562 cells by low concentrations of lidamycin.

Apoptosis induction and differentiation of promyelocytic leukemic cells into mature cells are major strategies for the drug-based treatment of leukemia. Lidamycin (LDM) which is a member of the enediyne antibiotic family exhibits extreme cytotoxicity. In the present study, the induction of apoptosis and differentiation in human chronic myeloid leukemia K562 cells by low concentrations of lidamycin were investigated. K562 cells were treated with lidamycin at various concentrations for 48h, and accumulated in the metaphase as determined in previous experiments. Cell viability was determined using a Cell Counting Kit-8 (CCK-8) assay and the IC50 value of lidamycin was 0.1±3.2nM. Induction of apoptosis was investigated morphologically by acridine orange/ethidium bromide (AO/EtBr) staining. Growth inhibition and apoptosis induction were observed in cells treated with low concentrations of lidamycin. In addition, western blot analysis revealed that treatment of the K562 cells with lidamycin at low concentrations upregulated the expression of caspase-8 and caspase-3. The induction of differentiation in human chronic myeloid leukemia K562 cells by lidamycin at low concentrations was also investigated. The nitroblue tetrazolium reduction ability of K562 cells was increased following treatment with lidamycin. Low concentrations of lidamycin triggered erythroid differentiation among K562 cells, indicated by morphological changes, increased hemoglobin content, and the expression of cell surface antigens such as CD71. Additionally the expression of GATA-binding factor1 (GATA-1) protein in low concentration lidamycin-treated K562 cells was increased. The results of the present study suggest that a low-concentration lidamycin exerts effects on apoptosis and erythroid differentiation induction by increasing the expression of caspases and GATA-1 protein. Lidamycin may serve a positive role in relevant targeted chemotherapy and may represent a potential candidate for chronic myelogenous leukemia differentiation-inducing treatment.

PO-104 Role of the oncosuppressor CTCF in the erythroid cell differentiation and regulation of erythroid genes

Introduction CTCF (CCCTC-binding factor) is a highly conserved 11-zinc finger protein that was identified as a MYC regulator. CTCF has several functions including transcriptional activation or repression, insulator binding properties and global organisation of chromatin. In addition, CTCF is involved in the epigenetic regulation of many cancer related genes and is considered as an oncosuppressor. Erythropoiesis is the process of red blood cell formation originating from hematopoietic stem cells. It is highly regulated by growth factors, transcription factors and miRNAs. Previous results of our group indicate that overexpression of CTCF induces differentiation towards the erythroid lineage in leukaemia cells, accompanied with the differential expression of erythroid specific genes. In this study, we investigate the role of CTCF during erythroid differentiation and the regulation of erythroid genes by CTCF. Material and methods As experimental models we used K562, a multipotent human leukaemia cell line, and primary CD34+ hematopoietic stem cells purified from cord blood. CTCF was silenced using lentiviral particles containing shRNA specific for CTCF. Erythroid differentiation was induced by cytosine arabinoside (Ara-C) or Imatinib in K562 cells and by erythropoietin (EPO) in CD34+ cells. Erythroid differentiation was analysed by cell proliferation assays, percentage of haemoglobin producing cells (benzidine test), colony forming units and expression of erythroid markers (Western-Blot and RT-qPCR). Chromatin immunoprecipitation (ChIP) experiments were performed to study the binding of CTCF to the regulatory regions of genes involved in the erythroid differentiation. Results and discussions CTCF was silenced in K562 and CD34+ cells and erythroid differentiation was induced by Ara-C, Imatinib or EPO. Downregulation of CTCF in both K562 and CD34+ cells reduced the cell proliferation, the percentage of benzidine positive cells and the expression of erythroid marker genes. Additionally, in CD34+ cells a reduction in the number of erythroid colonies was observed. CTCF occupancy in the regulatory regions of erythroid specific genes was found. Moreover, CTCF binding to these genes changed upon induction of erythroid differentiation. These results are in agreement with our previous findings. Conclusion Our results demonstrate that CTCF downregulation inhibits erythroid differentiation through the regulation of erythroid specific genes. We suggest a pivotal role of CTCF during erythroid differentiation.

A screen for Fli-1 transcriptional modulators identifies PKC agonists that induce erythroid to megakaryocytic differentiation and suppress leukemogenesis.

The ETS-related transcription factor Fli-1 affects many developmental programs including erythroid and megakaryocytic differentiation, and is frequently de-regulated in cancer. Fli-1 was initially isolated following retrovirus insertional mutagenesis screens for leukemic initiator genes, and accordingly, inhibition of this transcription factor can suppress leukemia through induction of erythroid differentiation. To search for modulators of Fli-1, we hereby performed repurposing drug screens with compounds isolated from Chinese medicinal plants. We identified agents that can transcriptionally activate or inhibit a Fli-1 reporter. Remarkably, agents that increased Fli-1 transcriptional activity conferred a strong anti-cancer activity upon Fli-1-expressing leukemic cells in culture. As opposed to drugs that suppress Fli1 activity and lead to erythroid differentiation, growth suppression by these new Fli-1 transactivating compounds involved erythroid to megakaryocytic conversion (EMC). The identified compounds are structurally related to diterpene family of small molecules, which are known agonists of protein kinase C (PKC). In accordance, these PKC agonists (PKCAs) induced PKC phosphorylation leading to activation of the mitogen-activated protein kinase (MAPK) pathway, increased cell attachment and EMC, whereas pharmacological inhibition of PKC or MAPK diminished the effect of our PKCAs. Moreover, in a mouse model of leukemia initiated by Fli-1 activation, the PKCA compounds exhibited strong anti-cancer activity, which was accompanied by increased presence of CD41/CD61 positive megakaryocytic cells in leukemic spleens. Thus, PKC agonists offer a novel approach to combat Fli-1-induced leukemia, and possibly other cancers,by inducing EMC in part through over-activation of the PKC-MAPK-Fli-1 pathway.

Generation and Functional Analysis of Congenital Dyserythropoietic Anemia (CDA) Patient-Specific Induced Pluripotent Stem Cells

The congenital dyserythropoietic anemias (CDAs) are inherited red blood cell disorders representing ineffective erythropoiesis and dyserythropoietic changes in the bone marrow. We recently diagnosed a female patient with undiagnosed congenital anemia as type IV CDA caused by a heterozygous missense mutation of the erythroid-specific transcription factor, KLF1; c.973G>A, p. E325K. Although the mutation has been reported in a male patient characterized as hydrops fetalis, severe neonatal jaundice and transfusion-dependent anemia (Arnaud L et al., Am J Hum Genet, 2010), the proband showed relatively mild phenotype showing moderate dyserythropoietic anemia. In order to investigate the pathological significance of mutant KLF1 during erythroid cell development and differentiation, we generated induced pluripotent stem cells (iPSCs) from peripheral blood of the CDA patient (CDA-iPSCs), and utilized these cells to establish in vitro CDA model for better understanding of its molecular basis. CDA-iPSCs were generated from T lymphocytes in peripheral blood mononuclear cells. Hematopoietic precursors were induced from CDA-iPSCs by embryoid bodies formation. CD34(+) precursor cells were isolated and further cultured in liquid culture with cytokine cocktail (erythropoietin (EPO), interleukin (IL)-3, and stem cell factor (SCF)) for additional 1-3 weeks. Flow cytometric analysis showed that CDA-iPSC-derived cells contained significantly lower percentage of CD235a(+)/CD71(+) erythroid lineage cells than the cells derived from control iPSCs, and lack expression of the adhesion molecule CD44, which is known to be down regulated in peripheral blood erythroid cells of CDA patients (Arnaud L et al., Am J Hum Genet, 2010). In addition, colony-forming unit (CFU) assay indicated that CD34(+) fraction derived from CDA-iPSCs contained a lower number of erythroid colony-forming cells and the most of the cells in these colonies are morphologically abnormal, in comparison with control iPSCs. We next evaluated mRNA expression levels of fetal (HBG1 and HBG2), embryonic (HBE), and adult (HBB) globins, resulting that HBG1 and HBG2 were significantly increased in CDA-iPSCs-derived erythroid lineage cells, whereas HBE showed no significant change and HBB was decreased in CDA-iPSCs-derived erythroid lineage cells. However, BCL11A, one of the target genes of KLF1 and also known as a suppressor of HBG1 and HBG2, was not decreased in the presence KLF1 gene mutation, indicating that elevated HBG1 and HBG2 in CDA-iPSCs-derived erythroid cells was mediated by other mechanism like Leukemia/lymphoma Related Factor (LRF; Masuda T et al., Science, 2016). Here we suggest that our model provides insights on understanding the mechanisms of type IV CDA and the effect of KLF1 gene mutation on clinical phenotype and it would be a useful tool for drug screening and identification of novel biomarker for the rare congenital anemia.Figure 1 Induction of erythroid differentiation from human iPSCsFigure 2 Flow cytometric analysis of erythroid cells induced from CDA-iPSCsFigure 3 RT-PCR analysis of erythroid cells induced from CDA-iPSCs DisclosuresKohara:SBI Pharmaceuticals: Research Funding. Miyamoto:SBI Pharmaceuticals: Research Funding. Tani:Oncolys BioPharma: Equity Ownership; SymBio Pharmaceuticals: Consultancy, Equity Ownership; SBI Pharmaceuticals: Research Funding; Shinnihonseiyaku: Research Funding.

MicroRNA 200a inhibits erythroid differentiation by targeting PDCD4 and THRB.

Previous studies on erythropoiesis revealed that microRNAs (miRNAs) play a critical role in erythroid differentiation. Given the abundance of identified miRNAs and the limited understanding of erythroid miRNAs, additional examination is required. Here, two sets of erythroid differentiation miRNome data were analysed to screen for novel erythroid-inhibiting miRNAs. MIR200A was selected based on its pattern of downregulated expression in the miRNome datasets during induction of erythroid differentiation. Overexpression of MIR200A in K562 and TF-1 cells confirmed its inhibitory role in erythroid differentiation. Further invivo study indicated that overexpression of mir200a inhibited primitive erythropoiesis of zebrafish. Transcriptome analyses after MIR200A overexpression in TF-1 cells indicated a significant role in regulating erythroid function and revealed potential regulation networks. Additionally, bioinformatics and experimental analyses confirmed that PDCD4 (programmed cell death 4) and THRB (thyroid hormone receptor, beta) are both targets of MIR200A-3p. Gain- and loss-of-function studies of PDCD4 and THRB revealed that the two targets were capable of promoting erythroid gene expression. Overall, our results revealed that microRNA 200a inhibits erythroid differentiation by targeting PDCD4 and THRB.

Induction of an Erythroid but Not Megakaryocytic Expression Signature in Myeloid Cells By in Vitro 5-Aza-2'-Deoxycytidine Treatment

Introduction: Aberrant DNA methylation is frequently found in hematologic malignancies where it is associated with altered gene expression. DNA hypomethylating agents (DNMTi), e.g. 5-aza-2'-deoxycytidine (DAC), are used for both global and gene-specific in vivo demethylation and offer a therapeutic option in myelodysplastic syndromes (MDS) and AML. DNMTi have already been utilized to upregulate suppressed fetal hemoglobin (HbF) in adult patients (pts) suffering from hemoglobinopathies. Here we systematically investigated the potential of DAC for in vitro induction of erythroid differentiation as well as HbF expression in the bipotent myeloid leukemia cell line K562 and in vivo in a clinical treatment situation in MDS pts.Methods and Results: We treated K562 cells with non-toxic concentrations of DAC (100 nM, three 24 hour pulses), hemin (50 nM) and phorbol myristate acetate (PMA, 5 nM). DAC treatment led to morphological changes indicating erythroid but not megakaryocytic differentiation. This was confirmed by benzidine staining where DAC (13% positive cells) and hemin (58%) but not PMA treated cells (0%) became positive for hemoglobin synthesis. Lack of CD41 detection by FACS analysis for DAC and hemin indicated absence of megakaryocytic differentiation. Transcriptome profiling by mRNA expression arrays (Affymetrix GeneChip® HG U133 Plus 2.0) revealed highest similarity between hemin and DAC treatment by unsupervised hierarchical clustering, followed by vehicle control and untreated cells. The transcriptome of PMA treated cells clustered most distantly to all other treatments. Both, DAC and hemin induced moderately balanced up- and downregulation of transcripts to an almost identical extent. 1414 transcripts were >2 fold upregulated and 1505 were >2 fold downregulated upon DAC treatment, whereas 1548 were up- and 2404 were downregulated in hemin treated cells, respectively. The extent of transcriptome dynamics was considerably stronger upon PMA treatment, where 4196 and 3780 transcripts were up- and downregulated, respectively. When intersecting transcriptome changes between the 3 drug treatments (Fig. 1), 368 out of 1548 (23.7%) upregulated transcripts in hemin treated cells were concordantly upregulated upon DAC treatment. The overlap of upregulated transcript was lower compared to PMA treated cells (14.9%). GO analyses of upregulated transcripts identified terms related to erythropoesis and iron metabolism among the top regulated groups of transcripts in DAC treated cells whereas terms related to megakaryocytic differentiation did not show significance. Particularly strong differences of transcripts were observed for a1-, a2-, Ag-, e- and z-globin expression upon DAC and hemin treatment, whereas b- and d-globin were expressed at low levels. These changes were not observed for PMA treated cells. Induction of a- and Ag-globin on mRNA level resulted in enrichment of a- and Ag-globin protein to 15.8% of total cellular protein amount, and consequently in HbF formation in K562 cells as assessed by reversed phase and anion exchange chromatography. HbF levels in peripheral blood were measured from 16 MDS pts, median age 74 years (range 66-78) also treated with a 3-day DAC schedule. Median HbF fraction at baseline was 0.4% (0.1-3.9%) of total hemoglobin with 6 pts (37.5%) exhibiting increased HbF levels (>1%) already before treatment. In 13/16 (81%) pts, increase of HbF with a median increment of 1.2% (range 0.3-3.7%) was observed. In 3 pts, HbF decreased over the treatment course. Median number of courses until maximum increment was 3 (2-6). HbF levels in 2 pts with AML and 1 with pancreatic cancer treated with nucleoside analogues without demethylating activity (cytosine arabinoside and gemcitabine, respectively) according to standard chemotherapy protocols served as control group and did not show comparable increments.Conclusions: We describe an erythroid differentiation program, from transcriptome level to HbF protein formation, induced by the hypomethylating agent DAC in the bipotent cell line K562. This DAC-mediated differentiation process is specific for erythropoesis but not megakaryopoesis. This is substantiated by in vivo upregulation of HbF upon DAC adminstration in MDS pts. Therefore, we propose to utilize HbF expression as potential biomarker during DAC treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Thalassemia Modular Stratification System for Personalized Therapy of Beta-Thalassemia (THALAMOSS)

Thalassemia Modular Stratification System for Personalized Therapy of Beta-Thalassemia (THALAMOSS)

Thioredoxin-interacting protein regulates the differentiation of murine erythroid precursors

Thioredoxin-interacting protein (TXNIP) is involved in various cellular processes including redox control, metabolism, differentiation, growth, and apoptosis. With respect to hematopoiesis, TXNIP has been shown to play roles in natural killer cells, dendritic cells, and hematopoietic stem cells. Our study investigates the role of TXNIP in erythropoiesis. We observed a rapid and significant increase of TXNIP transcript and protein levels in mouse erythroleukemia cells treated with dimethyl sulfoxide or hexamethylene bisacetamide, inducers of erythroid differentiation. The upregulation of TXNIP was not abrogated by addition of the antioxidant N-acetylcysteine. The increase of TXNIP expression was confirmed in another model of erythroid differentiation, G1E-ER cells, which undergo differentiation upon activation of the GATA1 transcription factor. In addition, we showed that TXNIP levels are induced following inhibition of p38 or c-Jun N-terminal kinase (JNK)mitogen-activated protein kinases. We also observed an increase in iron uptake and a decrease in transferrin receptor protein upon TXNIP overexpression, suggesting a role in iron homeostasis. Invivo, flow cytometry analysis of cells from Txnip(-/-) mice revealed a new phenotype of impaired terminal erythropoiesis in the spleen, characterized by a partial block between basophilic and late basophilic/polychromatic erythroblasts. Based on our data, TXNIP emerges as a novel regulator of terminal erythroiddifferentiation.

Abnormal Mirna Expression Profile and Cytokine Production in Myelodysplastic Vascular Niche

The bone marrow (BM) vascular niche has an important role in supporting adult hematopoietic stem cells (HSC) proliferation and differentiation. Using expanded circulating endothelial colony forming cells (ECFCs) of patients with low risk MDS patients and healthy donors as a surrogate of the vascular niche, we previously demonstrated in MDS patients an impaired talking between endothelial and hematopoietic cells. Molecular studies have revealed that MDS ECFCs show the promoter hypermethylation of p15INK4b, DAPK1, CDH1 and SOCS1 genes and have an altered gene expression profile, including adhesion, angiogenesis, cell activation, cell survival and apoptosis pathways. We also demonstrated that the Wingless and int (Wnt) pathway, one of the main regulators of cell-to-cell interactions in hematopoietic tissues (in particular Wnt3 and Wnt5) was down regulated at multiple levels in MDS ECFCs in comparison to the normal counterpart.As normally ECFCs regulate hematopoiesis by autocrine/paracrine secretion of various soluble mediators such as growth factors, cytokines and chemokines, we also investigated their production by MDS ECFCs. Moreover, since the release of angiogenic factors from BM microvasculature is regulated by specific microRNA (miRNA), we investigated the expression profile of a set of 84 miRNAs including those with predictable effects on the Wnt pathway using the miScript miRNA PCR array (Qiagen, Milan, Italy). We cultured normal CD34+ over a layer of MDS and normal ECFCs for 8 days in different cytokine-containing media able to induce selective granulocytic-macrophage, erythroid or megakaryocyte differentiation. At baseline and after 5 and 8 days of co-culture, we collected supernatants that were analyzed by Bio-Plex Pro™ human cytokine 27-plex immunoassay using Bio-Plex 200 system (Bio-Rad, Milan, Italy).We observed that in co-cultures with normal hematopoietic cells and MDS ECFCs, the level of several soluble mediators was significantly different in normal or MDS ECFC co-coltures. In particular, MDS co-cultures produced significant higher amounts of IL-1b, IL5, IP10, IL13 and VEGF (p≤0.05). Conversely, IL6, IL10, IL8 and RANTES were produced in lower quantities in MDS co-cultures (p≤0.05). These soluble mediators have a pleiotropic role and affect, directly or indirectly, different cellular pathways such as cell adhesion, hematopoietic differentiation, cell survival and angiogenesis. We also observed that their alterations were present in all the three lineages of differentiation but were more marked in megakaryocytic and myeloid than in erythroid differentiation. Moreover, the miRNA array revealed that MDS ECFCs contained significant lower amounts of miR-142-3p, miR-424-5p and miR-196b-5p (p=0.003, p=0.001 and p=0.033, respectively). These miRNAs are all variously involved in hematopoiesis or angiogenesis; in particular, miR-142-3p has been demonstrated to modulate the Wnt pathway and to down-regulate the expression of BMP4, a powerful inducer of erythroid differentiation which was highly expressed by MDS ECFCs.Considering that the addition of Wnt3 and Wnt5 to cultures restored only partially the normal differentiation of hematopietic cells, the abnormal secretion of soluble mediators as well as the altered expression of miRNAs might contribute to the dysfunction of vascular niches leading to myelodysplasia.This study was supported by AIRC grant to LML. DisclosuresNo relevant conflicts of interest to declare.

Erythroid induction of K562 cells treated with mithramycin is associated with inhibition of raptor gene transcription and mammalian target of rapamycin complex 1 (mTORC1) functions

Rapamycin, an inhibitor of mTOR activity, is a potent inducer of erythroid differentiation and fetal hemoglobin production in β-thalassemic patients. Mithramycin (MTH) was studied to see if this inducer of K562 differentiation also operates through inhibition of mTOR. We can conclude from the study that the mTOR pathway is among the major transcript classes affected by mithramycin-treatment in K562 cells and a sharp decrease of raptor protein production and p70S6 kinase is detectable in mithramycin treated K562 cells. The promoter sequence of the raptor gene contains several Sp1 binding sites which may explain its mechanism of action. We hypothesize that the G+C-selective DNA-binding drug mithramycin is able to interact with these sequences and to inhibit the binding of Sp1 to the raptor promoter due to the following results: (a) MTH strongly inhibits the interactions between Sp1 and Sp1-binding sites of the raptor promoter (studied by electrophoretic mobility shift assays, EMSA); (b) MTH strongly reduces the recruitment of Sp1 transcription factor to the raptor promoter in intact K562 cells (studied by chromatin immunoprecipitation experiments, ChIP); (c) Sp1 decoy oligonucleotides are able to specifically inhibit raptor mRNA accumulation in K562 cells. In conclusion, raptor gene expression is involved in mithramycin-mediated induction of erythroid differentiation of K562 cells and one of its mechanism of action is the inhibition of Sp1 binding to the raptor promoter.

The over-expression of aquaporin-1 alters erythroid gene expression in human erythroleukemia K562 cells.

Aquaporin genes are differentially expressed in primitive versus definitive erythropoiesis. Our previous research results showed that over-expression of aquaporin-1 (AQP1) gene greatly promotes the erythroid differentiation of erythroleukemia K562 cells, using benzidine staining and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) analysis for representative erythroid-related genes, including γ-globin. But the molecular mechanisms underlying erythroid-specific gene regulation remain unknown. In this study, we demonstrated that AQP1 induced hemoglobins expression and altered erythroid gene expression by microarray analysis in K562 cells. The retroviral expression vector of AQP1 (pBABE-puro-AQP1) was constructed and infected K562 cells to establish a stable AQP1 over-expression cell line (K562-AQP1). AQP1 over-expression effectively inhibited cell proliferation and induced cell growth arrest in G1 phase of K562 cells. Then microarray profile was applied to analyze the differentially expressed genes which involved the mechanism of AQP1 in erythroid differentiation induction. The DAVID functional annotation clustering tool was used to identify biological functions enriched with the differentially expressed genes (n = 466 genes) and to group genes into clusters based on their functional similarity. Significant enrichment of genes involved in "oxygen transporter activity" (p = 3.8E-7) including hemoglobins (HBD, HBG, HBB, HBE1, and HBQ1), HEMGN, and EBP42 were validated by qRT-PCR. Moreover, silencing of HEMGN by RNA interference in K562-AQP1 cells resulted in down-regulation of these genes. These data provide a better understanding of the role of AQP1 in erythroid differentiation, by promoting HEMGN induction and other potential signaling pathways associated with hemoglobin induction.