LSD1 inhibition enhances robust fetal hemoglobin induction in human β0-thalassemia/HbE erythroid cells

Induction of fetal hemoglobin (HbF) ameliorates the clinical severity of β-thalassemias. Histone methyltransferase LSD1 enzyme removes methyl groups from the activating chromatin mark histone 3 lysine 4 at silenced genes, including the γ-globin genes. LSD1 inhibitor RN-1 induces HbF levels in cultured human erythroid cells. Here, the HbF-inducing activity of RN-1 was investigated in erythroid progenitor cells derived from β0-thalassemia/HbE patients. The significant and reproducible increases in γ-globin transcript and HbF expression upon RN-1 treatment was demonstrated in erythroid cells with divergent HbF baseline levels, the average of HbF induction was 17.7 + 0.8%. RN-1 at low concentration did not affect viability and proliferation of erythroid cells, but decreases in cell number was observed in cells treated with RN-1 at high concentration. Delayed terminal erythroid differentiation was revealed in β0-thalassemia/HbE erythroid cells treated with RN-1 as similar to other compounds that target LSD1 activity. Downregulation of repressors of γ-globin expression; NCOR1 and SOX6, was observed in RN-1 treatment. These findings provide a proof of concept that a LSD1 epigenetic enzymes is a potential therapeutic target for β0-thalassemia/HbE patients.

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The G9A Histone Methyltransferase BIX-01294 Reactivates ε-Globin Expression and Blocks Erythroid Differentiation in Primary Baboon Erythroid Progenitor Cell Cultures

Blood ◽

10.1182/blood.v112.11.129.129 ◽

2008 ◽

Vol 112 (11) ◽

pp. 129-129 ◽

Cited By ~ 3

Author(s):

Virryan Banzon ◽

Vinzon Ibanez ◽

Kestis Vaitkus ◽

Tatiana Kousnetzova ◽

Joseph Desimone ◽

...

Keyword(s):

Gene Silencing ◽

Cell Cultures ◽

Progenitor Cell ◽

Globin Gene ◽

Histone H3 ◽

Erythroid Differentiation ◽

Globin Genes ◽

Erythroid Cells ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cell

Abstract The development of new therapies to increase fetal hemoglobin (HbF) levels in patients with sickle cell disease and β-thalassemia depends on an increased understanding of the mechanism responsible for the developmental regulation of globin gene expression. A role for epigenetic modifications in the mechanism of of globin gene regulation is suggested by the presence of high levels of DNA methylation near the 5’ regions of developmentally silenced ε- and γ-globin genes and the ability of pharmacological inhibitors of DNA methyltransferase (DNMTase) to reactivate ε- and γ-globin expression in adults. Whether additional epigenetic modifications associated with gene silencing and DNA methylation, such as histone H3 (lys9) dimethylation, are also involved is unknown. To investigate the hypothesis that histone H3 (lys9) dimethylation may function in the mechanism of developmental globin gene silencing, chromatin immunopreciptation assays were performed to determine the distribution of histone H3 (lys9) dimethyl and histone H3 (lys9) acetyl throughout the β-globin gene complex in purified primary baboon bone marrow (BM) erythroid cells from phlebotomized baboons expressing low levels (5–10%) of HbF and purified erythroid cells from erythroid progenitor cell cultures expressing high levels of HbF (30–50%). In BM erythroid cells, the level of histone H3 (lys9) acetyl associated with the β-globin gene was 10–20 fold higher than with the ε- and γ-globin genes, while the level of histone H3 (lys9) dimethyl associated with the ε- and γ-globin genes was 2–4 fold higher than with the β-globin gene. In erythroid cells from day 12 erythroid progenitor cell cultures, the level of histone H3 (lys9) acetyl associated with the highly expressed γ- and β-globin genes was 10–20 fold higher than with the silent ε-globin gene, while the level of histone H3 (lys9) dimethyl associated with the ε-globin gene was 2–4 fold higher than with the γ- and β-globin genes. Therefore a reciprocal relationship was observed between levels of histone H3 (lys9) acetylation and dimethylation associated with active and inactive globin genes. Experiments were performed to further investigate the role of histone H3 (lys9) dimethyl in ε-globin gene silencing by determining the effect of the G9A histone methyltransferase inhibitor BIX-01294 on ε-globin expression. Erythroid progenitor cell cultures derived from CD34+ BM cells of three individual baboons were treated with the varying doses of the DNMTase inhibitor decitabine (0.125–1.0μM), and BIX-01294 (1.25–5μM), alone and in combination. Changes in ε- globin were assessed by real time PCR using the ΔΔCT method with α-globin as the standard. Decitabine (0.5μM) increased ε-globin 25.8±7.7 fold while BIX-01294 (2.5μM) increased ε-globin 3.09±1.16 fold. Decitabine (1μM) and BIX-01294 (2.5μM) in combination increased ε-globin 55.7±24.9 fold. BIX-01294 enhanced ε-globin expression approximately twofold at all decitabine doses ranging from 0.125–1.0μM (mean increase=103± 44.7%). BIX-01294 also blocked terminal erythroid differentiation and allowed expansion of more primitive cells as evidenced by the presence of a large population of basophilic erythroblasts at late stages of culture (day 14). These results demonstrate that BIX-01294 reactivates expression of the silenced ε-globin gene and that synergistic reactivation can be achieved using combinations of BIX-01294 and decitabine. While these results are consistent with the hypothesis that epigenetic modifications are important in the mechanism of developmental globin gene silencing, the observation that BIX-01294 blocks erythroid differentiation suggests the possible involvement of a reprogramming mechanism.

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TAF10 Interacts with GATA1 Transcription Factor and Controls Mouse Erythropoiesis

Blood ◽

10.1182/blood.v124.21.2912.2912 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2912-2912

Author(s):

Petros Papadopoulos ◽

Laura Gutierrez ◽

Jeroen Demmers ◽

Dimitris Papageorgiou ◽

Elena Karkoulia ◽

...

Keyword(s):

Transcription Factor ◽

Fetal Liver ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Fetal Liver Cells ◽

Gata1 Transcription Factor

Abstract The ordered assembly of a functional preinitiation complex (PIC), composed of general transcription factors (GTFs) is a prerequisite for the transcription of protein coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA (Spt-Ada-Gcn5-acetyltransferase), are also necessary to have tightly regulated transcription initiation. However, a new era on the role of the GTFs and specifically on the role of TFIID in tissue specific and promoter specific transcriptional regulation has emerged in the light of novel findings regarding the differentiation programs of different cell types1. TAF10 is a subunit of both the TFIID and the SAGA co-activator HAT complexes2. The role of TAF10 is indispensable for early embryonic transcription and mouse development as knockout (KO) embryos die early in gestation between E3.5 and E5.5, around the stage when the supply of maternal protein becomes insufficient3. However, when analyzing TFIID stability and transcription it was noted that not all cells and tissues were equally affected by the loss of TAF10. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. Ablation of TAF10 specifically in erythroid cells by crossing the TAF10-Lox with the EpoR-Cre mouse led to a differentiation block at around E13.5 with erythroid progenitor cells accumulating at a higher percentage (26% in the KO embryos vs 16% in the WTs at E12.5) at the double positive stage KIT+CD71+ and giving rise to fewer mature TER119+ cells in the fetal liver. At E13.5 embryos were dead with almost no erythroid cells in the fetal liver. Gene expression analysis of the fetal liver cells of the embryos revealed down-regulation of GATA1 expression and its target genes, bh1&bmaj/min globins and KLF1 transcription factor while expression of other genes known to have a role in mouse hematopoiesis remained unaffected (MYB, GATA2, PU.1). In order to get insight to the role of TAF10 during erythropoiesis we analyzed the composition of both TAF10-containing complexes (TFIID and SAGA) by mass spectrometry. We found that their stoichiometry changes slightly but not fundamentally during erythroid differentiation and development (human fetal liver erythroid progenitors, human blood erythroid progenitors and mouse erythroid progenitor cells) and no major rearrangements were generated in the composition of the TFIID as it was reported in other cell differentiation programs (e.g. skeletal differentiation, hepatogenesis). Additionally, we found GATA1 transcription factor only in the fetal liver and not in the adult erythroid cells in the mass spectrometry data of TAF10 immunoprecipitations (IPs), an interaction that we confirmed by reciprocal IP of TAF10 and GATA1 in MEL and mouse fetal liver cells. Most importantly, we checked whether TAF10 binding is enriched on the GATA1 locus in human erythroid cells during the fetal and the adult stage in erythroid proerythroblasts and we found that there is enriched binding of TAF10 in the palindromic GATA1 site in the fetal stage. Our results support a developmental role for TAF10 in GATA1 regulated genes, including GATA1 itself, during erythroid differentiation emphasizing the crosstalk between the transcriptional machinery and activators in erythropoiesis. References 1. Goodrich JA, Tjian R (2010) Unexpected roles for core promoter recognition factors in cell-type-specific transcription and gene regulation. Nature reviews Genetics 11: 549-558 2 .Timmers HT, Tora L (2005) SAGA unveiled. Trends Biochem Sci 30: 7-10 3. Mohan WS, Jr., Scheer E, Wendling O, Metzger D, Tora L (2003) TAF10 (TAF(II)30) is necessary for TFIID stability and early embryogenesis in mice. Mol Cell Biol 23: 4307-4318 Disclosures No relevant conflicts of interest to declare.

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β-Globin-Expressing Definitive Erythroid Progenitor Cells Generated from Embryonic and Induced Pluripotent Stem Cell-Derived Sacs

Blood ◽

10.1182/blood.v124.21.2674.2674 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2674-2674

Author(s):

Naoya Uchida ◽

Atsushi Fujita ◽

Thomas Winkler ◽

John F. Tisdale

Keyword(s):

Progenitor Cells ◽

Erythroid Differentiation ◽

Ips Cells ◽

Cell Fraction ◽

Erythroid Cells ◽

Erythroid Progenitor ◽

Ips Cell ◽

Erythroid Progenitor Cells ◽

Spherical Cells ◽

Induced Pluripotent

Abstract Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell (RBC) transfusion. When ES cell-derived erythroid cells are generated using embryoid bodies, these cells predominantly express embryonic type ε-globin, with lesser fetal type γ-globin and small amounts of adult type β-globin; however, no β-globin expression is detected in iPS cell-derived erythroid cells. Recently, the ES cell-derived sac (ES sac) was reported to express hemangioblast markers and could generate functional platelets (Takayama, Blood. 2008). We previously demonstrated that erythroid cells were also efficiently generated via the ES sac (2013 ASH). We extend this work to evaluate globin expression in ES sac-derived erythroid cells. We generated ES sacs from human H1 ES or iPS cells using VEGF for 15 days, as previously described. The spherical cells within ES sacs were harvested and cultured on OP9 feeder cells for 2 days, and the suspension cells were differentiated into erythroid cells using human erythroid massive amplification culture for 13 days (Blood cells Mol Dis. 2002). The globin types expressed in erythroid cells were evaluated by RT-qPCR and hemoglobin electrophoresis. When hematopoietic cell-stimulating cytokines (SCF, FLT3L, TPO, IL3, EPO, and BMP4) were added in ES sac cultures on day 9-15, we observed 1.4-fold greater amounts of GPA+ erythroid cells (p<0.05) and 1.3-fold lower ε-globin expression in ES sac-derived erythroid cells (p<0.05), suggesting that cytokine stimulation might induce more hematopoietic/stem progenitor cells (HSPC) which can be differentiated to γ- or β-globin-expressing erythroid cells. Thus, we hypothesized that the ES sac contains both primitive and definitive erythroid progenitor cells capable of ε-globin-expression or γ- or β-globin-expression upon differentiation; respectively, and that these progenitors are selectable based upon surface markers of erythroid progenitor cells or HSPCs. To investigate whether primitive erythropoiesis is switched to definitive erythropoiesis during ES sac maturation, we evaluated spherical cells within the ES sac on day 9, 12, 15, and 18 after ES sac culture. A high percentage of GPA+ erythroid cells (29.2±3.7%) were observed on as early as day9. At that time point, almost no CD34+CD45+ HSPCs were present; however, the number increased upon further ES sac maturation until day 15 (6.8±1.6%). Cells further differentiated in erythroid culture had lower ε-globin expression and higher β-globin expression (up to 13.8±1.5%) when harvested from the ES sac at later time points. These data suggest that more matured ES sacs favor less primitive erythropoiesis and more definitive erythropoiesis. On day 15, the ES sacs contained a high percentage of GPA+(CD34-) erythroid cells (68.7±4.0%) and relatively lower amounts of CD34+(GPA-) HSPCs (16.7±2.1%). Therefore, we separated GPA+ and GPA- spherical cells from ES sac by magnetic selection before further erythroid differentiation, which resulted in higher ε-globin expression (43.0±16.6% vs 4.4±1.2%, p<0.01) and lower β-globin expression (7.6±5.3x10e-7% vs 19.8±2.7%, p<0.01) from the GPA+ cell fraction. In contrast, after erythroid differentiation from CD34+ or CD34- sorted spherical cells, lower ε-globin expression (3.7±0.3% vs 17.1±0.9%, p<0.01) and higher β-globin expression (17.4±0.7 % vs 0.9±0.4 %, p<0.01) were observed from the CD34+ cell fraction. These data suggest that the ES sac contains both primitive erythroid progenitor cells in the CD34- or GPA+ cell fraction and definitive erythroid progenitor cells in the CD34+ or GPA- cell fraction. In addition, iPS sac-derived erythroid cells were generated from 2 clones of fibroblast-derived iPS cells, which demonstrated 9.0±2.6% (clone #1) and 7.3±3.7% (clone #2) of β-globin expression. These data demonstrate that similar to ES sac-derived erythroid cells, iPS cell-derived erythroid cells can produce β-globin when differentiated from iPS sacs. In conclusion, we demonstrate that human ES and iPS cells can generate both primitive and definitive erythroid progenitor cells when differentiated in ES/iPS sac. CD34 or GPA discriminates between primitive and definitive erythroid progenitor cells in ES sac. The presented differentiation and selection strategy represent an important step to develop in vitro RBC production system from pluripotent stem cells. Disclosures No relevant conflicts of interest to declare.

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Hydroxyurea Cytotoxicity and Fetal Hemoglobin Induction.

Blood ◽

10.1182/blood.v104.11.3607.3607 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3607-3607

Author(s):

Heather M. Rogers ◽

Xiaobing Yu ◽

Constance Tom Noguchi

Keyword(s):

Transcription Factor ◽

Progenitor Cells ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Erythroid Differentiation ◽

Cell Toxicity ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

High Concentrations ◽

Globin Gene Expression

Abstract An important treatment strategy for sickle cell anemia is to increase fetal hemoglobin (HbF) in circulating erythrocytes. We use hydroxyurea (HU) as a model compound to induce HbF in adult human erythroid progenitor cells to examine the relationship between cell toxicity and globin gene expression. HU inhibits ribonucleotide reductase and its use is limited by hematopoietic toxicity at high dose. Cultures of primary human hematopoietic progenitor cells were stimulated with erythropoietin (EPO) and the effect of increasing doses of HU (from 1 to 200 mM) was determined on cell proliferation and differentiation, globin production, and erythroid transcription factors expression. At the lowest concentration (1 mM) we observed a minimal increase in cell proliferation with little change in % benzidine positive cells after 12 days of culture with EPO. As HU concentration increased, proliferation and % benzidine positive cells decreased, with concentrations of 100 and 200 mM being highly toxic, reducing cell number by 10 fold or more. Analysis of globin gene expression indicates that low concentrations of HU increase both g-globin and b-globin, resulting in only a modest increase in the g/(g+b) ratio compared with control. The g/(g+b) ratio increases with increasing HU concentration reaching a value of 0.25 or greater for concentrations of 50 mM or more, and approaching 1.0 at 200 mM, a consequence of the suppression of b-globin expression. This concentration of HU also inhibited g-globin expression, so that although the g/(g+b) ratio is quite high, it is at a cost in overall globin production and cell toxicity. Hemoglobin expression is determined primarily at the transcription level. We examined expression of GATA-1, GATA-2, SCL/Tal-1 and EKLF as regulatory proteins critical to erythropoiesis. We found that HU affects expression of select transcription factors associated with erythroid differentiation. EPO induction of GATA-1, a zinc-finger transcription factor required for survival and differentiation of erythroid progenitor cells, is delayed with HU, and the peak level of GATA-1 decreases at mid- and high concentrations, falling by 10 fold or more at 100 mM or greater. At the lowest concentration (1 mM) GATA-1 increases higher than the control. HU also delays EPO induction of SCL/Tal-1, a basic-helix-loop-helix transcription factor that positively regulates erythroid differentiation and is required for the production of mature erythrocytes, and EKLF, a zinc-finger transcription factor necessary for induction of b-globin in adult erythroid cells that acts by direct binding to the b-globin promoter. At the lowest concentration (1 mM), the delay in EPO induction of SCL/Tal-1 and EKLF is followed by a marked increase leading to peak levels greater than the control. At mid- and high concentrations, overall levels of SCL/Tal-1 and EKLF are reduced. GATA-2, a member of the GATA-family that plays a critical role in proliferation and survival of early erythroid progenitor cells, is down-regulated with EPO stimulation and is not markedly affected by HU. Therefore, HU concentration is crucial in optimizing the production of HbF. At low levels, HU increases both b- and g-globin resulting in small increases in g/(g+b) ratio, while at high concentrations the maximal increases in g/(g+b) ratio are concomitant with cytotoxicity. These data explain in part the importance of the maximum tolerated dose to achieve maximum increase in %HbF in hydroxyurea therapy.μμμγβγγβγγβμμβγγγβμμμβγγγβγγβββ

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Activity of RN-1, an LSD-1 Inhibitor, on Fetal Globin Expression in Sickle Mice and Sickle Erythroid Progenitors

Blood ◽

10.1182/blood.v126.23.961.961 ◽

2015 ◽

Vol 126 (23) ◽

pp. 961-961 ◽

Cited By ~ 1

Author(s):

Shuaiying Cui ◽

Jose Sangerman ◽

Seyed Mehdi Nouraie ◽

Yan Dai ◽

Oluwakemi Owoyemi ◽

...

Keyword(s):

Progenitor Cells ◽

Hemolytic Anemia ◽

Conflicts Of Interest ◽

Globin Gene ◽

Clinical Severity ◽

Globin Genes ◽

Erythroid Progenitors ◽

Irreversible Inhibitor ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells

Abstract Sickle cell disease (SCD) is the most common monogenic disorder, afflicting millions worldwide, and causing hemolytic anemia and chronic organ damage from vaso-occlusion. Fetal hemoglobin (HbF) is an endogenous type of hemoglobin present in all humans during development, which is normally suppressed in infancy. Biochemical and clinical studies have shown that increased synthesis of HbF inhibits sickle hemoglobin (HbS) polymerization and reduces clinical severity. Concerted efforts have been made to induce the synthesis of HbF in adult erythroid cells with chemical inducers of HbF and through disruption of transcription factors in repressor complexes. As wide variability in individual responses to drug candidates have been observed in clinical trials, consistently effective HbF inducers are highly desired. We previously identified that Lysine-specific histone demethylase 1 (LSD1) is involved in the regulation of the fetal γ-globin genes, and inhibition of LSD1 using either RNAi or by the momoamine oxidase inhibitor tranylcypromine (TC) in primary human erythroid progenitor cells induces HbF to therapeutic levels. However, TC treatment has potentially problematic side effects, and at high concentrations decreases adult b-globin mRNAs and impairs erythroid maturation. We have now investigated another LSD1 inhibitor, RN-1, which is a cell-permeable TC analog that acts as a potent, irreversible inhibitor of LSD1 with a lower IC50 than TC. We investigated in vivo effects of RN-1 on γ-globin gene expression and erythroid physiology in a transgenic mouse model of SCD which expresses human α- and sickle β-globin, and has many genetic, hematologic, and pathophysiological features found in SCD patients, including irreversibly sickled RBCs, hemolytic anemia, high reticulocyte counts, hepatosplenomegaly and organ pathology. We found a robust increase in human fetal γ-globin (15-fold) and murine embryonic εY- and βH1-globin mRNAs (36 and 54-fold) and 4-fold increases in human HbF in SCD mice following repeated RN-1 treatment (at 10 μg/g body weight) within 4 weeks. Further, irreversibly sickled RBCs were significantly reduced, and RBC lifespan increased markedly in RN-1-treated SCD mice, leading to significantly decrease pathophysiologic indicators (hemolysis, splenomegaly, and organ necrosis) compared to untreated SCD mice. To begin to evaluate potential effects of RN-1 on erythroid progenitor cells from patients with SCD, peripheral blood from 5 adult SCD patients was cultured with RN-1 (0.07 to 0.25 μM) in a 2-phase progenitor assay, with mRNA analyzed on day 12 and F-reticulocytes on day 13-14 of the erythroid differentiation phase. RN-1 treated progenitors demonstrated a mean 3.4-fold higher g-globin mRNA (p=0.04) and 5% higher absolute F-reticulocytes than were observed in untreated progenitors from the same subject, with responses occurring in 5/5 subjects' assays. These preclinical studies provide additional evidence that modulating LSD-1 activity is a promising approach to inducing HbF expression as a mechanism to reduce clinical severity of SCD. Disclaimer: "Research reported in this publication was supported by the NHLBI under Award Number P50HL118006. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health" R01 DK052962 10A1 R42-HL-110727 Disclosures No relevant conflicts of interest to declare.

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

Scientific Reports ◽

10.1038/s41598-021-87738-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Khanita Nuamsee ◽

Thipphawan Chuprajob ◽

Wachirachai Pabuprapap ◽

Pornrutsami Jintaridth ◽

Thongperm Munkongdee ◽

...

Keyword(s):

Dna Methylation ◽

Promoter Region ◽

Globin Gene ◽

Gene Promoter ◽

Fetal Hemoglobin ◽

K562 Cells ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Hbf Induction ◽

Globin 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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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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Soluble Guanylate Cyclase Modulators Effect Fetal Hemoglobin Levels through a Non-cGMP-Protein Kinase G Signaling Pathway

Blood ◽

10.1182/blood-2018-99-120306 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1060-1060

Author(s):

Bin Sun ◽

Valerie Miller ◽

Karin P Potoka ◽

Yukio Nakamura ◽

Mark T Gladwin ◽

...

Keyword(s):

Signaling Pathway ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Active Site ◽

Soluble Guanylate Cyclase ◽

Fetal Hemoglobin ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Hbf Induction

Abstract Sickle cell anemia results from a point mutation in both alleles of the β-globin gene. This homozygous mutation ultimately leads to a structural alteration of the hemoglobin protein that promotes polymerization of the mutant sickle hemoglobin tetramer (HbS) upon deoxygenation. HbS polymerization results in rigid, sickle-shaped RBCs with increased cell-to-cell adhesion properties. These sticky and rigid RBCs are prone to become trapped in small capillary networks leading to ischemia-reperfusion injury, endothelial damage and the hallmark pain crisis of sickle cell anemia. Neonates are protected from deoxy-HbS polymerization by high levels of fetal hemoglobin (HbF). HbF is composed of two α-globin and two γ-globin subunits(α2γ2), The γ-globin molecule cannot interact with deoxy-βS-globin polymers, which makes HbF an effective inhibitor of deoxy-HbS polymerization. The level of HbF required to reduce the symptoms of sickle cell anemia is 20-25%, but levels as low as 9% can prolong red cell survival. Treatment with hydroxyurea (HU) induces HbF and reduces the hematologic and clinical consequences of sickle cell anemia. Basal and inducible HbF levels are important in predicting the severity of sickle cell anemia and are highly phenotypically variable among patients, leading to varied responses to treatment. Patients are also variably susceptible to HU-induced cytopenias, which limits the use of HU in certain patients. HU is currently the only FDA-approved HbF-inducer for the treatment of sickle cell anemia and there is clearly a need for alternative HbF-inducers. Understanding the signaling pathways that regulate HbF induction will lead to novel therapeutic targets for sickle cell anemia. The soluble guanylate cyclase/cyclic guanosine monophosphate-dependent protein kinase (sGC/PKG) signaling pathway potentially links HU to the induction of HbF expression. In this study we investigated the direct role of sGC in HbF induction using novel pharmacologic modulators of sGC. Nitric oxide (NO) activates sGC by binding to the ferrous iron (Fe2+) in the active site heme moiety. Once activated, sGC converts GTP to cGMP, which in-turn activates PKG. Reactive oxygen species (ROS) oxidize the active site heme of sGC leading to NO-insensitivity. We tested the ability of a novel sGC activator, BAY 58-2667, to induce γ-globinin primary and immortalized (HUDEP-2) human erythroid progenitor cells. BAY 54-6544 binds to heme-free inactivated sGC to restore its guanylyl cyclase activity independent of NO. We also tested the ability of the sGC stimulator, BAY 41-2272, to induce γ-globinin primary and HUDEP-2 human erythroid progenitor cells. BAY 41-2272, binds to the ferrous iron at the active site of non-oxidized sGC to stimulate guanylyl cyclase activity in a synergistic manner with NO. We compared g-globin mRNA and protein expressionin the primary and immortalized human erythroid progenitors after treatment with different concentrations and combinations of BAY 54-6544, BAY 41-2667 and HU. We also evaluated g-globin induction in cellstreated with the pan-phosphodiesterase inhibitor IBMX and a synthetic cGMP analog. Although we see robust induction of cGMP and activation of PKG with all treatments, we only see significant induction of g-globin expression in the HU treated cells. This data suggests that the induction of HbF occurs through a non-sGC/PKG-dependent signaling pathway. These data demonstrate a very limited induction of γ-globin by BAY 54-6544 and BAY 41-2667 that appears to be disproportionate to, and independent of, cGMP/PKG signaling. These data also demonstrate, for the first time, that HU treatment of the immortalized HUDEP-2 cell line induces γ-globin expression more consistently than in primary erythroid progenitors. Disclosures No relevant conflicts of interest to declare.

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Metformin induces FOXO3-dependent fetal hemoglobin production in human primary erythroid cells

Blood ◽

10.1182/blood-2017-11-814335 ◽

2018 ◽

Vol 132 (3) ◽

pp. 321-333 ◽

Cited By ~ 16

Author(s):

Yankai Zhang ◽

Alireza Paikari ◽

Pavel Sumazin ◽

Carly C. Ginter Summarell ◽

Jacy R. Crosby ◽

...

Keyword(s):

Progenitor Cells ◽

Fetal Hemoglobin ◽

Dependent Manner ◽

Metformin Treatment ◽

Erythroid Cells ◽

Erythroid Progenitor ◽

Functional Studies ◽

Erythroid Progenitor Cells ◽

Key Points ◽

Hemoglobin Production

Key Points Functional studies in human primary erythroid progenitor cells support a role for FOXO3 in γ-globin regulation. Metformin treatment of human primary erythroid progenitor cells increases fetal hemoglobin in a partially FOXO3-dependent manner.

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Identification Of BCL11A Structure-Function Domains For Fetal Hemoglobin Silencing

Blood ◽

10.1182/blood.v122.21.435.435 ◽

2013 ◽

Vol 122 (21) ◽

pp. 435-435 ◽

Cited By ~ 2

Author(s):

Jian Xu ◽

Daniel E. Bauer ◽

Cong Peng ◽

Elenoe C. Smith ◽

Stuart H. Orkin

Keyword(s):

Structure Function ◽

Zinc Finger ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Gene Repression ◽

Functional Domains ◽

Globin Genes ◽

Erythroid Cells ◽

Zinc Finger Motifs ◽

Hbf Induction

Abstract Reactivation of fetal hemoglobin (HbF, α2γ2) expression in adults ameliorates the clinical symptoms in patients with the major β-hemoglobin disorders, sickle cell disease (SCD) and β-thalassemias. The zinc-finger protein BCL11A is a major modulator of hemoglobin switching and HbF silencing. BCL11A was initially identified by genome-wide association studies (GWAS) as a new HbF-associated gene. Down-regulation of BCL11A in primary human erythroid cells induces HbF expression. Knockout of BCL11A in mice impairs HbF silencing in adult erythroid cells. Most importantly, inactivation of BCL11A alone in humanized SCD mice corrects the hematologic and pathologic defects through high-level HbF induction. These studies established BCL11A as a genetically and functionally validated transcriptional regulator of HbF switching and silencing. In human and mouse erythroid cells, BCL11A is expressed as several isoforms, yet their individual roles in globin gene expression remain unexplored. Furthermore, the functional domains within the BCL11A protein responsible for its activity in HbF repression are largely unknown. To further understand the mechanistic roles of BCL11A in globin expression, we established a functional assay based on a BCL11A-null erythroid cell line generated by transcription activator-like effector nucleases (TALENs)-mediated deletion of an obligate erythroid-specific enhancer of BCL11A in murine erythroleukemia (MEL) cells. In the BCL11A-null cells, the expression of β-like embryonic globin genes is markedly induced (>200-fold), consistent with the role of BCL11A in repression of murine embryonic globin genes. To examine the activity of known BCL11A isoforms in HbF silencing, we expressed various BCL11A isoforms in these engineered BCL11A-null cells. Ectopic expression of full-length BCL11A-XL isoform, but not the alternatively spliced, C-terminally truncated L isoform, restored the full repression of β-like embryonic globins in BCL11A-null cells. Since XL and L differ only by 91 amino acids containing three tandem C2H2-type zinc finger motifs, these results indicate that the C-terminal zinc finger motifs are indispensable for BCL11A-mediated transcriptional repression. To systemically define BCL11A functional domains for globin gene repression, we next generated a panel of BCL11A mutant cDNAs, including deletion of the N-terminal NuRD-interacting motif and one or more C2H2-type zinc finger domains. Analysis of various BCL11A mutants in the functional rescue assay identified several functional domains, including the N-terminal NuRD-interacting motif and five out of the six C2H2 zinc fingers, that are required for BCL11A-mediated repression. These findings provide the foundation for further molecular analysis of BCL11A functional domains in globin gene repression. BCL11A is known to interact with several transcriptional co-repressor complexes including Mi-2β/NuRD/HDAC1/HDAC2, LSD1/CoREST and SWI/SNF complexes, occupy discrete regions within the human β-globin cluster, and promote long-range chromosomal interactions. Our results suggest that BCL11A functional domains may be involved in protein-protein interactions, protein homo-/heterodimerization, and/or chromatin/DNA association that are required for its activity in HbF silencing. In summary, we demonstrate that several functional domains on BCL11A protein are indispensable for its transcriptional activity in HbF silencing. Further focused studies of BCL11A structure-function domains in HbF silencing not only will advance our understanding of the molecular mechanisms by which BCL11A controls the clinically important fetal-to-adult globin switch, but may identify novel cellular targets for therapeutic HbF induction in β-hemoglobinopathies. Disclosures: No relevant conflicts of interest to declare.

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