Trienone analogs of curcuminoids induce fetal hemoglobin synthesis via demethylation at Gγ-globin gene promoter

AbstractThe reactivation of γ-globin chain synthesis to combine with excess free α-globin chains and form fetal hemoglobin (HbF) is an important alternative treatment for β-thalassemia. We had reported HbF induction property of natural curcuminoids, curcumin (Cur), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), in erythroid progenitors. Herein, the HbF induction property of trienone analogs of the three curcuminoids in erythroleukemic K562 cell lines and primary human erythroid progenitor cells from β-thalassemia/HbE patients was examined. All three trienone analogs could induce HbF synthesis. The most potent HbF inducer in K562 cells was trienone analog of BDMC (T-BDMC) with 2.4 ± 0.2 fold increase. In addition, DNA methylation at CpG − 53, − 50 and + 6 of Gγ-globin gene promoter in K562 cells treated with the compounds including T-BDMC (9.3 ± 1.7%, 7.3 ± 1.7% and 5.3 ± 0.5%, respectively) was significantly lower than those obtained from the control cells (30.7 ± 3.8%, 25.0 ± 2.9% and 7.7 ± 0.9%, respectively P < 0.05). The trienone compounds also significantly induced HbF synthesis in β-thalassemia/HbE erythroid progenitor cells with significantly reduction in DNA methylation at CpG + 6 of Gγ-globin gene promoter. These results suggested that the curcuminoids and their three trienone analogs induced HbF synthesis by decreased DNA methylation at Gγ-globin promoter region, without effect on Aγ-globin promoter region.

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Differences in BCL11A SUMOylation Are Associated with Differences in γ-Globin Expression in Primary Erythroid Cells.

Blood ◽

10.1182/blood.v114.22.458.458 ◽

2009 ◽

Vol 114 (22) ◽

pp. 458-458

Author(s):

Tatiana Kouznetsova ◽

Kestis Vaitkus ◽

Vinzon Ibanez ◽

Joseph DeSimone ◽

Donald Lavelle

Keyword(s):

Dna Methylation ◽

Western Blot ◽

Progenitor Cells ◽

Fetal Liver ◽

Globin Gene ◽

Erythroid Cells ◽

Developmentally Regulated ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Globin Promoter

Abstract Abstract 458 Increased fetal hemoglobin (HbF) levels associated with acute erythropoietic stress in man and experimental baboons have been proposed to result from increased commitment of early progenitors that preferentially express γ-globin to the terminal erythroid differentiation pathway. The increased propensity of early progenitors to preferentially express γ-globin has been hypothesized to be due to the presence of trans-acting factors favoring γ-globin expression. Because increased HbF in response to acute erythropoietic stress does not occur in transgenic human β-globin gene locus mouse models, investigation of the mechanism responsible for this phenomenon requires the use of a primate model system. We investigated the role of DNA methylation and the trans-acting factor BCL11A in the mechanism responsible for increased HbF in a primary cell culture system designed to mimic conditions associated with acute erythropoietic stress. Erythroid progenitor cells (EPC) derived from CD34+ baboon bone marrow (BM) cells cultured in Iscove's medium containing 30% fetal bovine serum supplemented with 2 U/ml Epo, 200ng/ml SCF, and 1uM dexamethasone express high levels of γ-globin (0.47+ 0.09 γ/γ+β; n=6). Bisulfite sequence analysis performed to determine whether changes in DNA methylation of 5 CpG residues within the 5' γ-globin promoter regions were associated with increased γ-globin expression showed that DNA methylation levels were similar in BM erythroid cells from normal baboons expressing very low levels of HbF (<1%), bled baboons expressing moderately elevated levels of HbF (5-10%), and cultured erythroid progenitor cells expressing highly elevated levels of HbF (30-50%). Changes in γ-globin promoter DNA methylation were thus not associated with increased γ-globin expression in EPC cultures. Further experiments were therefore performed to investigate whether differences in BCL11A expression were associated with increased γ-globin in EPC cultures. Western blot assays performed using three different anti-BCL11A monoclonal antibodies recognizing epitopes present in the N terminus, core, and C terminus detected different BCL11A isoforms in cultured EPC and normal BM erythroid cells. The size of the predominant protein band detected in cultured EPC was 125kDa, corresponding to the reported size of the in vitro transcription/translation product encoded by the BCL11A-XL transcript (Liu et al, Mol Cancer 16:18, 2006). In contrast, the size of the predominant band observed in BM erythroid cells was 220kDa. The 220kDa isoform was not observed in cultured EPC. Higher molecular weight forms of BCL11A have been observed following co-transfection of vectors encoding BCL11A and SUMO-1 (Kuwata and Nakamura, Genes Cells 13:931, 2008). Therefore we investigated whether the post-translational modification SUMOylation was responsible for the difference in the size of the 125 and 220kDa isoforms. Immunoprecipitation experiments performed using either SUMO-1 or SUMO 2/3 antibodies followed by Western blot with anti-BCL11A antibody showed that the 220 kDa isoform, but not the 125kDa isoform, was immunoprecipitated by either anti-SUMO-1 or anti-SUMO-2/3 antibody, confirming that the 220 kDA isoform, but not the 125 kDa isoform, was SUMOylated. Western blot assays performed to investigate the relative levels of these isoforms in BM erythroid cells of normal baboons, phlebotomized baboons, and early gestational age (53d) baboon fetal liver showed that expression of the 125kDa isoform was increased in bled compared to normal unbled baboons, suggesting that the deSUMOylated BCL11A isoform was increased by erythropoietic stress. The relative levels of the 125 and 220 kDa isoforms were similar in bled BM and fetal liver, indicating that SUMOylation of BCL11A was not developmentally regulated. The absolute level of BCL11A was reduced in fetal liver erythroid cells compared to BM erythroid cells consistent with observations showing that the level of BCL11A expression is developmentally regulated in man (Sankaran et al, Nature epub 2009). We conclude that BCL11A is post-translationally modified by SUMOylation in primary BM erythroid cells, but not in cultured EPC expressing high levels of HbF and suggest that modulation of the level of BCL11A SUMOylation is important in the mechanism responsible for increased HbF levels during recovery from acute erythropoietic stress. Disclosures: No relevant conflicts of interest to declare.

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A Cell-Based High-Throughput Screen For Novel Inducers Of Fetal Hemoglobin For Treatment Of Sickle Cell Disease, Cooley’s Anemia and β-Thalassemias

Blood ◽

10.1182/blood.v122.21.925.925 ◽

2013 ◽

Vol 122 (21) ◽

pp. 925-925 ◽

Cited By ~ 1

Author(s):

Kenneth R Peterson ◽

Flavia C Costa ◽

Halyna Fedosyuk ◽

Renee Neades ◽

Allen M Chazelle ◽

...

Keyword(s):

Sodium Butyrate ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Erythroid Progenitor ◽

Lead Compounds ◽

Erythroid Progenitor Cells ◽

F Cells ◽

A Cell ◽

Cooley's Anemia ◽

Globin Promoter

Abstract Decades of research has established that the most effective treatment for sickle cell disease (SCD) and Cooley’s anemia is increased fetal hemoglobin (HbF). Certain β-thalassemias may also benefit from fetal hemoglobin induction. Fetal hemoglobin normally accounts for less than 0.5% of total hemoglobin in adults; increasing levels to approximately 10% alleviates much of the pathophysiology associated with SCD. Hydroxyurea is the most widely available treatment for SCD that results in enhanced HbF production, but this drug is highly pleiotropic in its action and does not exclusively modulate γ-globin gene expression. Identification of a drug specific for inducing or reactivating γ-globin expression in pediatric and adult patients, with minimal off-target effects, continues to be an elusive goal. One hurdle has been an assay amenable to a high-throughput screen (HTS) of chemicals that displays a robust γ-globin off-on switch to identify potential lead compounds. An assay system developed in our lab to understand the mechanisms underlying the γ- to β-globin gene expression switch during development allowed us to generate a cell-based assay that was adapted for a HTS of 121,085 compounds from the libraries of the KU-HTS Laboratory (Prestwick, MicroSource, CMLD, Chembridge and ChemDiv compound libraries) and LCGC (OCL compound library). Transgenic mice were produced using a modified 213 Kb human β-globin locus yeast artificial chromosome (β-YAC). Two gene fusions were introduced into the β-YAC via homologous recombination in the host yeast, firefly luciferase was fused to the Aγ-globin promoter and Renilla luciferase was fused to the β-globin promoter. The resultant YAC was microinjected into fertilized mouse oocytes to produce transgenic mice. We used these mice to derive chemical inducer of dimerization (CID)-dependent bone marrow cells (BMCs) containing the γ-luc/β-luc β-YAC, which were employed in the HTS. We identified 232 primary screen actives that induced γ-globin 2-fold or higher. A 4-assay, 10-point dose-response secondary screen using the same CID-dependent γ-luc/β-luc β-YAC BMCs reconfirmed that 211 of these active compounds induced γ-globin ≥2-fold with minimal or no β-globin induction, minimal cytotoxicity and did not directly inhibit purified luciferase enzyme. Additional secondary assays in CID-dependent wild-type β-YAC BMCs and human primary erythroid progenitor cells confirmed the characteristics of seven of these 233 hits that were cherry-picked for further analysis. Four of the compounds were particularly promising, numbers 7, 42, 87 and 208. In CID-dependent wild-type β-YAC BMCs using the optimal dose for each compound, γ-globin mRNA induction ranged from 3- to 42-fold compared to 10-fold with sodium butyrate as measured by real-time qRT-PCR; F-cells ranged from 9.9-29.9% compared to 0.7% untreated and 15.9% treated with sodium butyrate as measured by flow cytometry. In human primary erythroid progenitor cells, the mRNA change was 1.6- to 3-fold compared to 1.75-fold with sodium butyrate and F-cells ranged from 9.1-29% compared to 5.7% untreated and 39.4% treated with sodium butyrate. Lead compounds will be tested in a pre-clinical β-YAC transgenic mouse model to determine their ability to induce HbF in vivo to aid development of these compounds for future clinical applications in hemoglobinopathies. Disclosures: No relevant conflicts of interest to declare.

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Epigenetic Changes in the γ-Globin Gene Promoter in Hematopoietic Progenitor Cells at Different Stages of Differentiation.

Blood ◽

10.1182/blood.v106.11.1742.1742 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1742-1742 ◽

Cited By ~ 1

Author(s):

Mahipal Singh ◽

Kestas Vaitkus ◽

Maria Hankewych ◽

Donald Lavelle ◽

Nadim Mahmud ◽

...

Keyword(s):

Dna Methylation ◽

Promoter Region ◽

Globin Gene ◽

Gene Promoter ◽

Methylation Pattern ◽

Cell Populations ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Globin Promoter

Abstract The role of epigenetic modifications, which represent a second layer of genetic regulation, appear to play an important role during hematopoiesis. How the chromatin structure changes from a preprogrammed hematopoietic stem cell to progressively differentiated cells and how the cell’s inner and surrounding environment affects orchestrating the epigenetic modifications is not clearly understood. This investigation was initiated to determine the pattern of DNA methylation in the γ-globin gene promoter region in the cells isolated from different stages of erythroid development in baboon (P. anubis). Baboon fetal liver hematopoietic stem progenitor cells (HSPC) were purified by passage through a Miltenyi magnetic column (to deplete mature erythroblasts) followed by flow cytometric cell sorting of erythroblast depleted cells into various sub populations depending upon the expression of CD36 antigen. Three types of cell populations i.e. CD34+CD36−, CD34+CD36+ and CD34−CD36+ were collected. Clonal analysis was performed to verify the degree of differentiation of CD34+ cells based on their co-expression of CD36. CFC assays of CD34+CD36−, CD34+CD36+ and CD34−CD36+ cells revealed a cloning efficiency of 89%, 38.5% and 12% respectively, after 12 days of culture using methylcellulose media supplemented with a cocktail of growth factors and serum. Only CD34−CD36+ cells produced detectable CFU-E colonies while CD34+CD36− and CD34+CD36+ generated mostly BFU-Es. CD34+CD36− cells produced about 2 fold more BFU-Es as compared to CD34+CD36+ cell population. Moreover, the colony size also decreased as the cells progressed towards maturation. Our data suggest that CD34+CD36−cells are most primitive erythroid progenitors while CD34−CD36+ cells are the most committed mature erythroid progenitors and the CD34+CD36+ cells represent the intermediate stage. The methylation pattern of 5 CpG sites in the γ-globin promoter region in the above purified cell populations along with erythroblast fraction was assayed using bisulfite sequencing. Two independent fetuses, 56 and 58 days post conception (dpc) were analysed. Bisulfite treated genomic DNA was used to amplify the γ-globin promoter region and the PCR products were subcloned into pCR-TOPO vector. Sequences of fifteen independent clones per sample were analysed. Our results indicated almost complete methylation of the γ-globin promoter region in earlier stages of differentiation and a progressive decrease in methylation as the cells progress towards maturation. The methylation pattern observed was 98.3%, 63.3%, 28.3% and 7.4 % in 58d fetus and 92.6%, 69.2%, 26.3% and 0.0% in 56d fetus in CD34+CD36−, CD34+CD36+, CD34−CD36+ and erythroblast cell populations respectively. Only the −54 CpG site exhibited hypomethylation in the most primitive CD34+CD36− cell population. In conclusion, our results show a progressive decrease in methylation as the HSPC mature into erythroblasts and progressively accumulate more hemoglobin indicating a direct role of DNA methylation in regulation of hemoglobin production. Status of histone methylation and acetylation along with DNA methylation pattern of ε- and β-globin gene promoters in hematopoietic progenitors at various differentiated stages should enhance our understanding of γ-globin gene regulation in a non-human primate model which closely mimics human beings.

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Engineered Human Umbilical Cord Derived Erythroid Progenitor Cells (HUDEP2) with Sickle or β-Thalassemia Mutation: An in-Vitro System for Testing Pharmacological Induction of Fetal Hemoglobin

Blood ◽

10.1182/blood-2018-99-114241 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3478-3478

Author(s):

So Hyun Park ◽

Ciaran M Lee ◽

Yankai Zhang ◽

Alicia Chang ◽

Vivien A Sheehan ◽

...

Keyword(s):

Globin Gene ◽

Fetal Hemoglobin ◽

Human Umbilical Cord ◽

Erythroid Progenitor ◽

In Vitro System ◽

Hypoxic Conditions ◽

Erythroid Progenitor Cells ◽

F Cells ◽

Hbf Induction

Abstract Introduction: Sickle cell disease (SCD) and β-thalassemia are inherited blood disorders caused by mutations in the β-globin gene (HBB). Elucidation of the multiple pathophysiologic mechanisms in SCD and β-thalassemia has resulted in an increasing efforts to identify new treatment modalities to ameliorate the consequences of the disease. However, no consistent in vitro system exists for studies of pharmacological therapies for the diseases. Human umbilical cord-derived erythroid progenitor cells (HUDEP2) are an immortalized CD34+ hematopoietic stem cell-derived erythroid precursor cell line that can differentiate into red blood cells. Here, we engineered sickle HUDEP2 and β-thalassemia HUDEP2 clonal lines through CRISPR/Cas9-mediated editing of the human HBB. We sought to establish if these engineered cell lines exhibit disease phenotypes, and if upon in vitro erythroid differentiation they produce fetal hemoglobin (HbF) in response to hydroxyurea, the only FDA-approved drug for HbF induction. Our goal is to create an in vitro system to test new HbF inducers for treating SCD or β-thalassemia. Materials and Methods: We delivered Hi-Fidelity Streptococcus pyogenes (Sp) Cas9 protein and CRISPR guide RNA as a ribonucleoprotein complex in conjunction with a single-stranded DNA donor (ssODN) template to introduce the sickle or K17X (A<T) or codon 6 [-G] β-thalassemia mutation into the HBB locus of HUDEP2 cells. Edited HUDEP2 cells were single-cell sorted into multiple 96-well plates and expanded. The genotype of the clones was determined using a probe-based droplet digital PCR assay and confirmed through Sanger sequencing. Native polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC) were used to confirm the hemoglobin phenotype. Normal parental cell line, sickle clone, and two individual β-thalassemia clones were used to test the pharmacological induction of HbF. We initiated drug treatment in the expansion phase with 30 µM hydroxyurea. Trypan Blue staining and CD71/CD233/CD235 staining determined the effect of the drugs on the viability, growth rate and erythroid development of HUDEP2 lines. After 10 days of drug treatment, differentiated HUDEP2 were analyzed for globin expression through RT-qPCR and HPLC, and HbF positive cells (F-cells) were quantified via flow cytometry. Cells were placed at 2% O2 for four hours, fixed in glutaraldehyde, stained, and viewed under magnification to assess sickling potential. Results and Discussion: We generated multiple clones with biallelic sickle or β-thalassemia mutations. Sickle HUDEP2 clones almost exclusively expressed sickle hemoglobin with low level of HbF and hemoglobin A2 (HbA2), and β-thalassemia HUDEP2 clones produced no normal adult hemoglobin, 8-10% HbF, and 26-28% HbA2. On HPLC analysis, β-Thalassemia HUDEP2 clones had an unknown tall peak (39-45%) between HbF and HbA consistent with an α-globin homotetramer (α4). When subjected to hypoxic conditions for 4 hours, sickle HUDEP2 produced sickle cells. HUDEP2 parent cells did not sickle under hypoxic conditions. Hydroxyurea induced 3.8-fold, 1.8-fold, and 1.6-fold increases in γ-globin gene (HBG) expression; 2.9-fold, 1.4-fold, and 1.4-fold increases in the percentages of F-cells; 1.4-fold, 1.2-fold, and 1.6-fold increase in the percentages of HbF in sickle, K17X(A<T) and codon 6[-G] β-thalassemia HUDEP2 clones, respectively. No change was observed in CD71/CD235 positive HUDEP2 cells in the presence hydroxyurea. This finding demonstrated that hydroxyurea treatment induces HBG expression as well as HbF and F-cells in engineered sickle and β-thalassemia HUDEP2 clones. Future work will include screening other pharmacological compounds as well as studying the mechanism of HbF induction by using HUDEP2 clones. Conclusions: Our engineered sickle and β-thalassemia HUDEP2 cell lines have properties similar to those of patient erythroid cells and respond to the known HbF inducer hydroxyurea. This in vitro model system may facilitate the drug-discovery process by enabling multimodal drug screening on a large scale with consistent and reproducible results. Acknowledgments: This work was supported by the Cancer Prevention and Research Institute of Texas grants RR140081 and RP170721 (to G.B.) and the National Heart, Lung and Blood Institute of NIH (1K08DK110448 to V.S.) Disclosures No relevant conflicts of interest to declare.

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SCF induces γ-globin gene expression by regulating downstream transcription factor COUP-TFII

Blood ◽

10.1182/blood-2008-07-170712 ◽

2009 ◽

Vol 114 (1) ◽

pp. 187-194 ◽

Cited By ~ 21

Author(s):

Wulin Aerbajinai ◽

Jianqiong Zhu ◽

Chutima Kumkhaek ◽

Kyung Chin ◽

Griffin P. Rodgers

Keyword(s):

Progenitor Cells ◽

Acute Stress ◽

Globin Gene ◽

Critical Role ◽

Fetal Hemoglobin ◽

Mapk Signaling ◽

Rapid Expansion ◽

Erythroid Progenitor ◽

Stress Erythropoiesis ◽

Erythroid Progenitor Cells

Abstract Increased fetal hemoglobin expression in adulthood is associated with acute stress erythropoiesis. However, the mechanisms underlying γ-globin induction during the rapid expansion of adult erythroid progenitor cells have not been fully elucidated. Here, we examined COUP-TFII as a potential repressor of γ-globin gene after stem cell factor (SCF) stimulation in cultured human adult erythroid progenitor cells. We found that COUP-TFII expression is suppressed by SCF through phosphorylation of serine/threonine phosphatase (PP2A) and correlated well with fetal hemoglobin induction. Furthermore, down-regulation of COUP-TFII expression with small interfering RNA (siRNA) significantly increases the γ-globin expression during the erythroid maturation. Moreover, SCF-increased expression of NF-YA associated with redox regulator Ref-1 and cellular reducing condition enhances the effect of SCF on γ-globin expression. Activation of Erk1/2 plays a critical role in SCF modulation of downstream transcriptional factor COUP-TFII, which is involved in the regulation of γ-globin gene induction. Our data show that SCF stimulates Erk1/2 MAPK signaling pathway, which regulates the downstream repressor COUP-TFII by inhibiting serine/threonine phosphatase 2A activity, and that decreased COUP-TFII expression resulted in γ-globin reactivation in adult erythropoiesis. These observations provide insight into the molecular pathways that regulate γ-globin augmentation during stress erythropoiesis.

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The -195C→G Substitution In Brazilian Hereditary Persistence of Fetal Hemoglobin Decreases NF-E1/YY1 Binding and Increases PAX1 Binding to the A Gamma Globin Promoter Region

Blood ◽

10.1182/blood.v116.21.2066.2066 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2066-2066 ◽

Cited By ~ 3

Author(s):

Fernanda Marconi Roversi ◽

Anderson Ferreira Cunha ◽

Carolina Lanaro ◽

Ana Flavia Brugnerotto ◽

Maria Emília Favero ◽

...

Keyword(s):

Dna Binding ◽

Promoter Region ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Globin Genes ◽

Dna Array ◽

Gamma Globin ◽

Nuclear Extracts ◽

Hereditary Persistence ◽

Globin Promoter

Abstract Abstract 2066 Hereditary persistence of fetal hemoglobin (HPFH) is a condition that prevents hemoglobin switching and the consequent silencing of the gamma globin genes, resulting in continued hemoglobin (Hb) F synthesis in adults. Two types of HPFH are responsible for this phenotype: deletional HPFH – deletions in the end of the beta globin locus – and non-deletional HPFH (ndHPFH) – single point mutations in the proximal promoter of both gamma globin genes. Sickle cell anemia patients or beta-thalassemia patients that present HPFH show high levels of HbF that are associated with less severe clinical course in these diseases. The development of new therapies based on the reactivation of gamma globin expression may be important for the treatment of these patients. The Brazilian ndHPFH type is characterized as a C→G substitution in the A gamma globin promoter at position –195 and the molecular mechanism responsible for the reactivation of this gene in the Brazilian ndHPFH type remains unclear. In contrast to the British ndHPFH type (-198), where the mechanism responsible for the increase of HbF levels is mediated by the raising in the affinity for the Sp1 transcription factor (TF), the Brazilian ndHPFH mutation does not affect Sp1 binding. Thus, other TF may be involved in the reactivation of the A gamma globin gene in the Brazilian ndHPFH type. The aim of this study was to investigate the mechanism involved in the reactivation or repression of the A gamma globin gene in the Brazilian ndHPFH type and identify possible TF responsible for this phenotype. In vitro primary human erythroblast cultures, derived from human CD34+ hematopoietic cells from 4 Brazilian ndHPFH type subjects and 4 control subjects, were proliferated and differentiated into late stage erythroblasts. The nuclear extracts from predominantly basophilic and polychromatic erythroblasts were used to profile TF activity using Protein-DNA Array method. The analysis of the array densitometry identified a number of TF whose DNA binding activities were either enhanced or repressed in the Brazilian ndHPFH cultures. Among the TF analyzed, the NF-E1/YY1 and the PAX-1 were selected for this study. Since this assay requires a secondary method to confirm these results, nuclear extracts were used to conduct chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). ChIP was carried out using antibodies against NF-E1/YY1 and PAX-1 to quantify the binding to these TF to the –195 A gamma globin promoter region. EMSA was performed using probes with the same sequence spotted on the array membrane to analyze the activity of NF-E1/YY1 and PAX-1. Both methods confirmed and validated the previous array results. NF-E1/YY1 is a transcription factor that represses embryonic (epsilon) and fetal (gamma) globin genes. Protein-DNA array and EMSA showed a decreased binding of NF-E1/YY1 in Brazilian ndHPFH nuclear extracts and ChIP analysis revealed diminished NF-E1/YY1 occupancy at the –195 A gamma globin promoter region of Brazilian ndHPFH. The consensus binding site for NF-E1/YY1 is a CCAN motif that is observed between the –195 and –192 position in the A gamma globin promoter region. The C→G substitution at –195 position may disrupt this DNA binding site, cause decreased NF-E1/YY1 interaction and probably allows the binding of PAX-1, a transcriptional activator with a paired box DNA-binding domain that has as a DNA binding core motif, the sequence TTCCGC. This sequence, located between the –199 and –194 position in the A gamma globin promoter, is only presente in the Brazilian type of ndHPFH. Our protein-DNA array and EMSA results showed an increased binding of PAX-1 in the Brazilian ndHPFH nuclear extracts and quantitative ChIP analysis with anti-PAX-1 antibody showed that PAX-1 binds to the –195 A gamma globin promoter region only in the presence of this C→G substitution. These results suggest that the –195 site (C→G) in the A gamma globin promoter region may decrease NF-E1/YY1 binding and increase PAX-1 binding in this DNA region, probably resulting in the reactivation of the A gamma globin gene. The increase in the HbF levels in the Brazilian ndHPFH occurs differently from the British ndHPFH type and represents a novel mechanism of A gamma globin reactivation. Such findings may lead to the development of future therapeutic strategies for HbF induction in the treatment of other hemoglobinopathies. Support by FAPESP and CNPq. Disclosures: No relevant conflicts of interest to declare.

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Identification of New and Diverse Inducers of Fetal Hemoglobin with High Throughput Screening (HTS)

Blood ◽

10.1182/blood.v116.21.4277.4277 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4277-4277 ◽

Cited By ~ 2

Author(s):

Jose I Sangerman ◽

Michael S Boosalis ◽

Ling Shen ◽

Sarah Haigh ◽

Ada Kane ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Globin Gene ◽

Clinical Stage ◽

Gene Promoter ◽

Fetal Hemoglobin ◽

Luciferase Reporter ◽

Significant Activity ◽

Globin Mrna ◽

Globin Promoter

Abstract Abstract 4277 Pharmacologic augmentation of fetal hemoglobin (HbF, γ-globin) production, to replace diminished β-globin chains in the β-thalassemias and to inhibit HbS polymerization in sickle cell disease, is a definitive therapeutic modality. Despite long-term efforts, regulatory approval has been obtained for only one chemotherapeutic agent. Pharmacologic reactivation of high-level HbF expression with non-cytotoxic, tolerable therapeutics is still an unmet medical need for this global health burden. To investigate potential therapeutic libraries for unrecognized HbF inducers, we developed a high-throughput screening (HTS) program to interrogate diverse chemical libraries, including a library of FDA-approved and clinical stage drugs. This program has identified unexpected new and highly potent HbF-inducing drugs, some of which are already in clinical use for other medical indications and have established safety profiles. A human cell-based assay which was previously used in low throughput assays, utilizing a 1.4-kilobase (kb) KpnI-BglII fragment of the HS2 of the locus control region (LCR) linked to the γ-globin gene promoter and the enhanced green fluorescent protein (EGFP) reporter gene, was adapted for high throughput screening and employed as the primary screen. Cytotoxic activity was assayed in a simultaneous counter screen. A number of hits were identified as being more potent than positive controls (such as butyrate). Several hits were immediately eliminated from further development as potential hemoglobinopathy therapeutics because of cytotoxicity (e.g., Idarubicin) or undesirable off-target effects, but nonetheless validated the HTS itself and were validated in secondary confirmatory assays as highly-potent HbF-inducers. The HTS assay identified 8 FDA-approved drugs as potent inducers of γ-globin gene expression, with activity at 1–2 logs lower concentrations (1000-fold higher potency) than prior generation therapeutic candidates. The γ-globin-specificity of hits was determined in a secondary assay employing a stably-transfected dual-luciferase reporter construct containing the LCR and the β-globin promoter linked to renilla luciferase and the Aγ-globin promoter linked to firefly luciferase (μLCRβprRlucAγprFluc cassette). Clinical-stage or clinically-approved agents, including Ambroxol at 1 μM, Desloratadine at 1 μM, Resveratrol at 10 μM, Benserazide at 5 μM, the HDAC inhibitor MS-275 at 5 μM, and an established bioactive, NSC-95397, at 1 μM were all significantly more active in this assay than Butyrate at 2000 μM, with MS-275 and Resveratrol being the most active. These drugs were then assayed for their ability to induce γ-globin mRNA expression in cultured primary human erythroid progenitors, at concentrations which are pharmacologically achievable in humans. Drugs significantly more active in γ -globin mRNA induction than the positive control (2-fold induction) in this system included Ambroxol (3-fold), Desloratadine (up to 6-fold), Resveratrol (up to 3-fold), Benserazide (up to 5-fold), and MS-275 (up to 3.7-fold). Two agents were subsequently studied in anemic baboons, and demonstrated in vivo induction of γ-globin mRNA, HbF, and F-reticulocytes. Unexpectedly, rises in total hemoglobin (>1 gm/dL) also occurred with 2 agents. Thus, a panel of structurally- and functionally-unrelated compounds demonstrate greater HbF-inducing activity, with up to 1000-fold higher potency, than current HbF-inducers which have significant activity in clinical trials. Some of the drugs identified by HTS have entirely benign safety profiles. These candidates could be clinically evaluated rapidly and at significantly less cost than new chemical entities, which require extensive toxicology, manufacturing, and clinical evaluation. These findings demonstrate the utility of a high-throughput screening program based on γ-globin gene promoter induction. Disclosures: No relevant conflicts of interest to declare.

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The Effects of Environmental Cues on Hematopoietic Stem Cell Colony Fetal Hemoglobin Production.

Blood ◽

10.1182/blood.v116.21.3860.3860 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3860-3860

Author(s):

Jennifer L Nichols ◽

Donald Lavelle ◽

Joseph DeSimone

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Globin Gene ◽

Gene Promoter ◽

Feeder Layer ◽

Liquid Media ◽

Feeder Layers ◽

Globin Promoter ◽

Cells Cultured ◽

In Cells

Abstract Abstract 3860 Objective. Increased fetal hemoglobin (HbF) levels are associated with increased life span and reduced pain crises in patients with Sickle Cell disease. An increased understanding of the mechanisms controlling HbF expression would be important to develop new therapies to increase HbF. The objective of these experiments was to test the hypothesis that interactions between bone marrow (BM) erythroid progenitor cells and the stromal microenvironment influence HbF expression. Materials and Methods. Baboon CD34+ BM cells were harvested and purified by immunomagnetic column chromatography using the 12.8 anti-CD34 mouse monoclonal antibody and immunomagnetic microbeads conjugated to rat anti-mouse IgM (Miltenyi). CD34+ BM Cells were grown in liquid cultures in Iscove's media containing 30% fetal bovine serum, 200ng/ml stem cell factor, 2u/ml erythropoietin, and 1uM dexamethasone in the presence of an AFT024 mouse fetal liver cell line and an OP9 mouse bone marrow cell line as feeder layers, in methylcellulose media, and in liquid media. Globin chain synthesis in cultures was measured on d11 and d14 by biosynthetic radiolabeling. Cells were incubated overnight in leucine-free α-MEM media containing 50uCi/ml [3H leucine]. Globin chains were separated by High Performance Liquid Chromatography (HPLC), and the radioactivity in each fraction was determined by liquid scintillation counting. To correlate changes in γ-globin expression with DNA methylation of the γ-globin gene promoter, the methylation state of 5 CpG sites within the γ-globin promoter region was determined by bisulfite sequencing. Erythroid cells were purified from cultures on d14 by immunomagnetic column chromatography using mouse anti-baboon RBC monoclonal antibody. DNA was isolated from purified erythroid cells using Qiagen kits. Bisulfite modification was performed using Epitect bisulfite kits. Two rounds of PCR amplification were performed using nested primers flanking 5 CpG residues within the baboon γ-globin gene promoter. Minilysate DNA was prepared from at least 10 independent clones for each sample. Sequence analysis of purified DNA samples was performed at the University of Illinois DNA Sequence facility. Results. Elevated levels of γ-globin chain synthesis were observed in cells cultured in methylcellulose and liquid media in the absence of a feeder layer when compared to cells grown in the presence of feeder layers. On d11, the γ/γ+β chain synthetic ratio was 0.623 in cells cultured in methylcellulose and 0.324 in cells cultured in liquid media, compared to 0.092 in cells cultured in the presence of AFT024 feeder layers and 0.178 in cells cultured in the presence of OP9 feeder layers. On d14, the γ/γ+β chain synthetic ratio was 0.663 in cells cultured in methylcellulose and 0.349 in cells cultured in liquid media, compared to 0.135 in cells cultured in the presence of AFT024 feeder layers and 0.252 in cells cultured in the presence of OP9 feeder layers. The level of DNA methylation (%dmC) of 5 sites in the γ-globin gene promoter negatively correlated with levels of HbF production. On d14, the level of DNA methylation was 79% in cells cultured in methylcellulose, 72% in cells cultured in liquid media, and 97% in cells grown in the presence of AFT024 feeder layers. Conclusions. Cells grown in the presence of AFT024 feeder layers expressed physiologic levels of γ-globin and cells grown in the presence of OP9 feeder layers expressed midrange levels of γ-globin. Significantly increased γ-globin expression was observed in cells cultured in the absence of a feeder layer. The γ-globin promoter in cells grown in the presence of the AFT024 feeder layer exhibited a significantly higher degree of methylation than in cells grown in methylcellulose and liquid media. These results show that interactions between erythroid progenitor cells and the stromal microenvironment can influence both the level of γ-globin expression and the DNA methylation of the γ-globin gene promoter. These results have clinical relevance; if the bone marrow microenvironment could be effectively altered in vivo, methylation of the γ-globin promoter could be decreased and HbF production increased. Further experiments must be performed to determine what is mediating the methylation of the γ-globin promoter when cells are grown on these feeder layers. Disclosures: No relevant conflicts of interest to declare.

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Activation of the beta-globin promoter by the locus control region correlates with binding of a novel factor to the CAAT box in murine erythroleukemia cells but not in K562 cells

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6969-6983.1993 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6969-6983

Author(s):

N L Delvoye ◽

N M Destroismaisons ◽

L A Wall

Keyword(s):

Control Region ◽

Globin Gene ◽

Gene Promoter ◽

K562 Cells ◽

Locus Control Region ◽

Model Systems ◽

Beta Globin ◽

Wild Type ◽

Globin Promoter ◽

Murine Erythroleukemia

Four distinct factors in extracts from murine erythroleukemia (MEL) cells interacted with the human beta-globin gene promoter CAAT box: CP1, GATA-1, and two novel factors, denoted a and b, one of which is highly inducible in the MEL system. GATA-1 binding to the CAAT element was very unstable (half-life < 1 min), whereas bindings of a, b, and CP1 were comparatively stable, with half-lives of 18, 19, and 3.5 min, respectively. Stable transfections of MEL cells showed that in the presence of the beta-globin locus control region (LCR), the wild-type CAAT box, a mutant which bound to GATA-1 with increased stability over the normal sequences, and a mutant which bound a, b, and CP1 specifically could all stimulate transcription greater than ninefold over that induced by a null CAAT mutation in both uninduced and terminally differentiated MEL cells. A mutant which bound the a and b factors specifically gave only a twofold stimulation of promoter activity, and this lower activity correlated with a decrease in the stability of binding of the b protein. On the other hand, CP1 binding alone did not stimulate transcription. Taken together, these results suggest that in the context of the wild-type beta-globin CAAT element the b factor stimulates transcription directed by the LCR in MEL cells, although the LCR can also function through more stable GATA-1-binding sequences. However, in K562 cells, the wild-type beta-globin CAAT box alone was unable to stimulate gene expression directed by the LCR and high levels of transcription were obtained only upon inclusion of more upstream beta-globin promoter sequences. In contrast, a construct containing only the A gamma-globin CAAT box region did give high expression levels in K562 cells. Thus, there is a fundamental difference in the way the LCR functions in these two model systems in terms of its requirements at the promoter level.

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Neither DNA Hypomethylation or Changes in the Kinetics of Erythroid Differentiation Account for 5-Azacytidine’s Ability To Induce Human Fetal Hemoglobin.

Blood ◽

10.1182/blood.v110.11.572.572 ◽

2007 ◽

Vol 110 (11) ◽

pp. 572-572

Author(s):

Rodwell Mabaera ◽

Michael R. Greene ◽

Christine A. Richardson ◽

Sarah J. Conine ◽

Christopher H. Lowrey

Keyword(s):

Dna Methylation ◽

Promoter Methylation ◽

Fetal Hemoglobin ◽

Global Dna Methylation ◽

Unexpected Finding ◽

Erythroid Cells ◽

Dna Hypomethylation ◽

Globin Mrna ◽

Hbf Induction ◽

Globin Promoter

Abstract 5-Azacytidine (5-Aza) is among the most potent inducers of fetal hemoglobin (HbF) in patients with β-thalassemia and sickle cell disease. Two models have been proposed to explain this activity. The first model is based on the drug’s ability to inhibit DNA methyltransferase enzymes causing global DNA hypomethylation, including the promoters of the fetal globin genes, resulting in their expression during adult erythropoiesis. The second model is based on the drug’s other well-know property - cytotoxicity. In this model, a cohort of differentiating erythroblasts either dies or undergoes growth arrest during drug exposure. The marrow then responds with a rapid proliferation of erythroblasts which express high levels of fetal globin mRNA and HbF. To determine which model best explains HbF induction by 5-Aza, we used an in vitro differentiation system in which CD34+ cells from normal donors are cultured with SCF, IL-3 and Flt-3 ligand for 7 days and then switched to Epo for an additional 14 days. This results in >90% erythroid cells, a 2,000-fold expansion in cell number and Hb expression similar to that seen in normal red cells (∼99% HbA, ∼1% HbF). To model common 5-Aza dosing schedules, we treated cultures daily with doses ranging from 0 to 1000 nM. Near-maximal induction of γ-globin mRNA (∼2-fold increase in γ/γ+β) and HbF (from 1% to 40%) occurred at the 300 nM dose level. While this dose was associated with hypomethylation of the 7 CpGs between −256 and +50 bp of the γ promoter (59% +/− 14% methylated CpGs vs. 92% +/− 8.1% in untreated controls, p < 0.001), no changes were found in cell expansion rate, differentiation kinetics (as judged by glycophorin A expression) or cell cycle distribution. These results argue against the cytotoxicity model. We also observed that global DNA methylation was not significantly changed by the 300 nM dose of 5-Aza. Because 5-Aza is incorporated into both RNA and DNA, it affects many cellular processes beyond DNA methylation. To determine if DNA hypomethylation is sufficient for γ-globin mRNA and HbF induction, we treated differentiating cells with DNMT1 siRNA. This resulted in transiently decreased DNMT1 mRNA and protein and lowered global DNA methylation. γ-globin promoter methylation decreased to levels equivalent to those seen with 300 nM 5-Aza (61% +/− 7% of CpGs methylated vs. 88% +/− 8% in controls, p < 0.001) but did not induce γ-globin mRNA or HbF. To further evaluate this unexpected finding we used a lentiviral vector expressing DNMT1 shRNA. This suppressed DNMT1 mRNA and protein throughout differentiation and decreased total γ-globin promoter methylation from 98% +/− 4% in control cells, to 20% +/− 11% (p<0.001) in treated cells. This level of γ promoter methylation is similar to that seen in fetal erythroid cells (13% +/− 7%). Global CpG methylation was also decreased vs. controls (30% +/− 10% vs. 83% +/− 11%, p<0.001). Again, despite significant γ promoter and global hypomethylation, no induction of γ-globin mRNA or HbF was observed. These results suggest that neither model explains 5-Aza’s ability to induce fetal Hb and that alternative mechanisms await discovery. If verified, these findings have important implications for the future development of clinically useful HbF inducing agents.

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