Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Dec 9.
Published in final edited form as: Blood Cells Mol Dis. 2021 Nov 17;93:102626. doi: 10.1016/j.bcmd.2021.102626

Novel histone deacetylase inhibitor CT-101 induces γ-globin gene expression in sickle erythroid progenitors with targeted epigenetic effects

Louis H Junker a, Biaoru Li b, Xingguo Zhu b, Sivanagireddy Koti a, Ryan E Cerbone a, Clifford L Hendrick a, Jose Sangerman c,d, Susan Perrine c,d, Betty S Pace b,*
PMCID: PMC9733664  NIHMSID: NIHMS1852408  PMID: 34856533

Abstract

Induction of fetal hemoglobin (HbF) expression ameliorates the clinical severity and prolong survival in persons with sickle cell disease (SCD). Hydroxyurea (HU) is the only FDA-approved HbF inducer however, additional therapeutics that produce an additive effect in SCD are needed. To this end, development of potent Class I histone deacetylase inhibitors (HDACi) for HbF induction represents a rational molecularly targeted approach. In studies here, we evaluated CT-101, a novel Class I-restricted HDACi, a Largazole derivative, for pharmacodynamics, cytotoxicity, and targeted epigenetic effects. In SCD-derived erythroid progenitors, CT-101 induced HbF expression with additive activity in combination with HU. CT-101 preferentially activated γ-globin gene transcription, increased acetylated histone H3 levels, and conferred an open chromatin conformation in the γ-globin promoter. These data indicate CT-101 represents a strong potential candidate as a molecularly targeted inducer of HbF.

Keywords: Sickle cell disease, Fetal hemoglobin, Histone deacetylase inhibitor, Epigenetics

1. Introduction

Sickle cell disease (SCD) related genetic blood disorders, affect ~100,000 people in the United States and millions of people worldwide, with a higher prevalence in low-income countries such as Sub-Saharan Africa. In fact, SCD is a designated public health burden by the World Health Organization. Epidemiologic, genetic, and therapeutic studies have clearly demonstrated that elevated fetal hemoglobin (HbF) levels reduce the clinical severity of SCD. Over the last four decades many laboratories have conducted studies to develop novel HbF inducing agents as a therapeutic strategy to treat SCD [1-22,26-36]. Recently, three drugs that enhance red blood cell viability or reduce adhesion have been US Food and Drug Administration (FDA) approved [13-15]. However, only HbF induction by hydroxyurea (HU) has been demonstrated to prolong survival and reduce organ damage [10-12]. To advance the field additional agents, which can be combined with HU are needed for those individuals non-responsive to HU alone at optimal doses. Analysis of minor genetic traits in patient populations with reduced severity, coupled with studies of the human β-globin (HBB) locus, have identified molecular targets, including the Class I histone deacetylase (HDAC) enzymes, HDAC1-3, and HDAC8, as potential therapeutic methods for inducing HbF in individuals with SCD [34-55].

A large body of work on mechanisms of γ-globin gene silencing and hemoglobin switching has demonstrated that loss of repressor complexes activates γ-globin gene transcription and HbF expression. Furthermore, broad HDAC inhibitors were among the first classes of small molecules identified that activate γ-globin gene expression in vitro and in vivo [34-55] targeting repressors such as DNA methyltransferase 1, HDAC1 and 2, BCL11A, and lysine demethylase-1, among others [37-49,55]. Pan-HDACi including valproic acid [16], sodium phenylbutyrate and arginine butyrate [17-18] have been investigated as potential treatments for SCD as HbF inducers. The latter enhanced HbF levels by 3-fold, proportions of F-cells, total hemoglobin with reduced hospitalization rates, demonstrating proof-of-concept for this class of therapeutics [18]. However, the necessity for intravenous administration limited further development.

Early in vivo studies demonstrated the ability of α-amino butyric acid to delay the γ- to β-globin switch in human infants, primates, and fetal lambs, and of hypomethylating agents such as DNA methylation trans-ferase 1 inhibitors to induce HbF in primates [3-4]. Subsequently, a transgenic mouse was established with a beta-yeast artificial chromosome (β-YAC) model. We used β-YAC mice to demonstrate the ability of α-amino butyric acid to induce HbF after pretreatment with 5-azacytizine [20]. These and other findings support epigenetic changes such as DNA hypomethylation and histone acetylation are involved in γ-globin gene reactivation. Subsequent treatment of β-YAC with the HDACi suberoylanilide hydroxaminc acid (SAHA) induced HbF [21]. Furthermore, using the BERK1 sickle mouse we demonstrated trichostatin A and SAHA induce HbF and reduce endothelial activation via inhibition of pulmonary vascular endothelial receptor VCAM-1 [22].

More recently, chemical library screenings and molecular drug modeling identified compounds including potent isoform-specific HDACi designed to induce HbF with minimal off- target side effects. In studies here, we evaluated the ability of CT-101, a novel HDACi analog of Largazole, to induce HbF alone or when combined with HU, the current standard of care. Largazole is a natural product first isolated from marine cyanobacteria by Luesch and coworkers [23]. Complete chemical synthesis of Largazole was described by Williams and coworkers with the intent of developing Largazole based analogs with HDAC class and isoform-specific inhibition [24]. Chemical synthesis of the most promising peptide isostere analog, CT-101, was accomplished in 2009 and potency as a Class I HDAC inhibitor established [25]. The prodrug CT-101 converts to the active free thiol (CT-101S) in the presence of plasma esterases and lipases. When converted to the active free sulfhydryl form, CT-101S binds to the critical zinc cofactor of specific HDAC enzymes. CT-101 may offer advantages for clinical application over pan-HDACi due to potency at nanomolar concentrations and its selectivity profile compared to first-generation HDACi.

2. Materials and methods

2.1. Synthesis of CT-101 parent and free thiol forms

Cetya's proprietary prodrug CT-101 was synthesized and the free thiol bioactive form (CT-101S) was prepared by hydrolyzing the parent CT-101 compound; purification of CT-101S was performed by preparative thin layer chromatography [25]. All compounds were stored dessicated at −20 °C under argon prior to use in studies.

2.2. Histone deacetylase inhibition assays

The free thiol form (CT-101S) of the parent compound CT-101 (Largazole peptide isostere) was provided by Dr. Robert Williams (Colorado State University, Fort Collins, CO). The other HDAC inhibitors, entinostat (#S1053), vorinostat (#S1047), panobinostat (#13280), belinostat (#S1085) and romidepsin (#R425060), were purchased from Cedarlane Laboratories, Ontario, Canada. The HDACi reference compounds Trichostatin A and TMP269 were added as internal controls to validate Class I and Class IIa inhibition profiles [29]. For in vitro HDAC inhibition assays (Reaction Biology Corp, Malvern, PA) recombinant HDAC isoforms 1–9 and HDAC -11 were combined with base reaction buffer (50 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2) and bovine serum albumin (1 mg/ml). Test articles were dissolved in dimethyl sulfoxide (DMSO) and added to the base reaction mixture to 1% of the total volume. Fluorogenic HDAC substrates were added to each sample to initiate the reaction. After incubation at 30 °C, developer was added to stop reactions and kinetic measurements were made for 1.5 h; endpoint readings were recorded once development reached a plateau. For all test articles, the IC50 values were calculated from the 10-point inhibition curves for individual HDAC isoforms and results plotted as a heat plot (Fig. 1A). Trichostatin A and TMP269 inhibitory profiles were as established.

Fig. 1.

Fig. 1.

Open in a new tab

CT-101 shows selective histone deacetylase inhibition. A) In vitro HDAC inhibition assay was performed using the active free thiol form of CT-101 (CT-101S) and compared to currently marketed competitive compounds.Shown below is the heat plot index. B) The impact of select compounds on γ-globin RNA including to CT-101 and CT-108 (Largazole depsipeptide oxazole-thiazoline) compared to sodium 2,2-dimethylbutyrate (ST-20), valproic acid (VA) and sodium phenyl butyrate (SPB). Expression of γ-globin mRNA by RT-qPCR is represented as fold change compared to control samples.

2.3. In vitro erythroid progenitor assays

Screening assays for γ-globin induction by diverse HDAC inhibitors were first conducted in K562 cells, which were cultured in Iscove's Modified Dulbecco Medium with 10% fetal bovine serum, penicillin, and streptomycin in conditions promoting log-phase growth and 99% viability, as previously published [34]. Compounds tested included sodium 2,2-dimethylbutyrate (ST-20; 20 μM), a short chain fatty acid derivative that inhibits HDAC2, two orally active clinical pan-HDACi including valproic acid, (1 mM and 2 mM) and sodium phenylbutyrate (SPB; 0.5 mM and 1 mM), and CT108 (3 μM) and CT-101 (5 μM) for 48 h. Cells were harvested for γ-globin mRNA quantification by reverse transcription-quantitative PCR (RT-qPCR) as previously described [34].

Primary erythroid progenitors were cultured from peripheral blood mononuclear cells isolated from discarded deidentified peripheral blood of patients with SCD under Institutional Research Board exempt protocols at Boston University School of Medicine and Augusta University. Erythroid cells were generated using a modified two-phase liquid culture system, as previously described [26]. Briefly, during phase 1, peripheral blood mononuclear cells were cultured in Iscove's Modified Dulbecco medium with 15% fetal bovine serum, 15% human AB serum, 10 ng/mL Interleukin-3, 50 ng/mL Stem Cell Factor and 2 IU/mL Erythropoietin (Sigma, St. Louis, MO). Phase 2 commenced on day 7 with the same medium except Stem Cell Factor and Interleukin-3 were excluded. Test compounds including CT-101 (100 nM and 200 nM), vehicle control DMSO, hydroxyurea (HU; 75 μM), and sodium butyrate (NaB; 0.2 μM), or combined CT-101/HU (100 nM/75 μM) were added to erythroid progenitors on day 7 for 48 h or 5 days. The progenitors were harvested on day 9 or day 12 for total RNA and protein. Cell counts and viability were monitored using 0.4% trypan blue exclusion assay throughout the study period. To monitor erythroid cell maturation, Wright Giemsa staining was performed to quantify basophilic, polychromatic, and orthochromatic erythroblasts and mature red blood cells by light microscopy.

2.4. RT-qPCR analysis

TRIzol extracted total RNA from erythroid progenitors was used for RT-qPCR analysis. Expression levels of γ-globin, β-globin, and an internal control glyceraldehyde-3-phosphate (GAPDH) were quantified using standard curves generated with ΔTopo7 base plasmids carrying each gene as previously published [26]. All gene expression levels were normalized to GAPDH mRNA and control cells were normalized to one, to determine fold-change in gene expression.

2.5. Flow cytometry analysis

After treating erythroid progenitor cells for 2 or 5 days with the investigational agents, cells were fixed with 4% paraformaldehyde and stained with fluorescein isothiocyanate (FITC) conjugated anti-HbF, (ThermoScientific, Waltham, MA) and isotype control antibodies (eBioscience, San Diego, CA). Flow cytometry analysis was performed on a LSRII flow cytometer using gating parameters previously published by our group [27]. Briefly, the forward scatter and side scatter is set to gate erythroid cells, then during FLOWJO analysis, a “log F-cell” histogram (X-axis) was used to measure F-cell percentage from total erythroid cells count (Y-axis) after gating on the negative isotype control cells. The mean fluorescence intensity (MFI) was generated by FLOWJO software, to quantify the level of HbF protein per cell.

2.6. Western blot analysis

Total protein was isolated and Western blot analysis performed as previously published [26] for HbF, HbS, and β-actin protein levels with antibodies from Sigma-Aldrich (Burlington, MA). For histone acetylation levels, nuclear protein extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Kit per the manufacture's protocol (Pierce, Waltham, MA). Western blot was performed with total histone H3 (H3) and H4 and acetylated histone H3 (acH3) and acH4 antibodies (Millipore, Burlington, MA) using nuclear extracts [27]. The immuno-blots were developed using SuperSignal® West Pico Chemiluminescent Substrate (ThermoScientific, Waltham, MA) and analyzed on a Fujifilm LAS-3000 gel imager (Stamford, CT) to acquire quantitative data.

2.7. Chromatin immunoprecipitation (ChIP) assays

ChIP assays were performed as previously published [28]. Sickle erythroid progenitors were treated for 48 h with the test agents, cross-linked with 1% formaldehyde and nuclei were isolated using cell lysis buffer (50 mmol/l Tris, pH 8.0, 10 mmol/l EDTA, 0.32% SDS, and 1 × CPI). The chromatin was sonicated to an average of 400 bp fragment length using a Bioruptor (Diagenode, Denville, NJ). For each immunoprecipitation reaction chromatin equivalents of 200,000 cells were incubated with RNA PolII, acH3, total H3, and IgG (Sigma, St. Louis MO). Purified DNA was analyzed by qPCR with SYBR green (Bio-Rad) using the CFX connect real-time system, (Bio-Rad, Des Plaines, IL).

2.8. Statistical analysis

The data are reported as the mean ± standard error of the mean (SEM) of 3–5 replicates of independent experiments performed in triplicate. All data were analyzed by a two-tailed Student's t-test and p < 0.05 was considered statistically significant.

3. Results

3.1. CT-101 inhibits class I histone deacetylases

We first examined the ability of CT-101 to inhibit HDAC isoforms that utilize zinc ions as a critical cofactor, including Class I (HDAC 1, 2, 3 and 8), Class IIa (HDAC 4, 5, 7, 9), Class IIb (HDAC 6 and 10), and Class IV (HDAC 11) proteins. The active free sulfhydryl form, CT-101S, binds to the critical zinc cofactor of the specific HDAC isoforms tested. Initially, the relative HDAC inhibition by CT-101S was compared to currently approved products (Fig. 1A). In vitro inhibition was tested in singlet 10-dose IC50 mode with 3-fold serial dilution against HDACs 1-9, and 11. Addition of CT-101 resulted in inhibition of Class 1a HDAC isoforms preferentially with IC50 values at nanomolar concentrations for HDAC 1-3. Some inhibition of HDAC6 was observed with little to no inhibition of Class IIa and Class IV isoforms. Profiles for inhibition of the HDAC isoforms by commercially available compounds are shown in a heat map (Fig. 1A). In general, the other HDAC inhibitor compounds required higher concentrations, demonstrating lower potency than the CT-101. By contrast, panobinostat and belinostat, showed less selectivity and inhibited most HDAC isoforms to some degree.

3.2. CT-101 induces HbF in human K562 cells

We previously found that HbF is induced 2- to 3-fold by a large subgroup of Largazole analogs with varying degrees of cell growth inhibition (data not shown), while two candidate analogs, CT-108 and CT-101 did not inhibit cell proliferation. These analogs induced HbF expression by 4-fold and 11-fold respectively in K562 cells (Fig. 1B), without cell growth inhibition over the concentration range tested. CT-101 was active in inducing γ-globin expression over a concentration range of 10–400 nM. The level of γ-globin mRNA induction by CT-101 was maximal at 11-fold. In contrast, we observed 2.6 to 4-fold induction by other agents, including ST-20, valproic acid, and CT-108. To evaluate further potential value of CT-101, we next investigated the ability of CT-101 to induce γ-globin transcription and HbF levels in SCD erythroid progenitors under oxidative stress conditions.

3.3. CT-101 activate γ-globin gene transcription in sickle cell erythroid progenitors

A primary goal of these studies was to estimate whether CT-101 induces HbF over an effective concentration range, without causing inhibition of erythroid cell proliferation, for ultimate application in patients with SCD. Therefore, we chose to complete our studies in human primary erythroid progenitors generated from SCD patients. To address this goal, erythroid progenitors generated from peripheral blood mononuclear cells of six patients with sickle cell anemia (ages 8–35 yrs.) were treated with CT-101 100 nM and 200 nM, or DMSO as a vehicle control or HU on day 7, and harvested on day 12 for RT-qPCR analysis; untreated cells served as controls for HU-treated cells. We observed a dose-dependent 3.9-fold and 7-fold increase in γ-globin mRNA for CT-101 (100 nM and 200 nM) concentrations respectively (Fig. 2A). Of note, the level of β-globin mRNA was not changed significantly for any conditions except sodium butyrate, which decreased mRNA levels by 80%.

Fig. 2.

Fig. 2.

Open in a new tab

CT-101 selectively activates γ-globin and α-globin gene transcription in human sickle erythroid progenitors. A) Using peripheral blood nuclear cells isolated from sickle cell patients' discarded blood, we generated erythroid progenitors that were treated from day 7 to day 12 (five days) with the various test agents at the concentrations shown (Materials and Methods). Data for six subjects (n = 6) are shown as the mean + standard error of the mean (SEM) and plotted as fold change compared to CT-101 in vehicle (Veh) DMSO control. All globin mRNA levels were normalized to the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. Data are shown as the Mean ± SEM and *p < 0.05 was considered significant; **p < 0.01, ***p < 0.001. B and C) The effects of CT-101 on erythroid maturation. Erythroid progenitors treated as described in panel A were subjected to cytospin preparations and Giemsa-staineing for delination of cell morphology. Shown (panel B) is cell morphology under 100× oil immersion microscopy; quantitative data for erythroid developmental stage for 500 cells per patient (C).

The effects of CT-101 on erythroid maturation after 5 days of treatment (day 12) are shown in Fig. 2B and C. In untreated cells, we observed 9% polychromatic and 75% orthochromatic erythroblasts. Similar to HU and sodium butyrate, the number of orthochromatic erythroid cells was decreased by 42% and 56% by CT-101 (100 nM) and CT-101200 nM respectively. However, erythroid progenitors continued to grow and proliferate suggesting growth less affected. Cell treated with HU and CT-101 (75 μM/200 nM) in combination produced similar effects. There was a 10% decrease in progenitor-derived erythroid cells compared to control cultures from the same subjects, equal to the growth effects induced by HU alone (data not shown).

3.4. CT-101 induces HbF expression and increases F-cells levels in sickle erythroid progenitors

Our next set of experiments addressed the question of whether CT-101 induces HbF protein in sickle erythroid progenitors, which have intrinsic intracellular oxidative stress. Using the same culture protocol as described above, Western blot analysis demonstrated HbF levels increased compared to untreated control porgenitors from the same subject by up to 1.7-fold with CT-101 (200 nM; p < 0.01) treatment, and by 1.3-fold and 1.4-fold increase with HU or sodium butyrate, respectively (Fig. 3A and B). Of note, the level of HbS protein was not significantly changed by CT-101 or by any of the other test agents (Fig. 3C). These data suggest a preferential action of CT-101 on γ-globin gene promoter transcription, compared to β-globin.

Fig. 3.

Fig. 3.

Open in a new tab

CT-101 increased HbF levels in sickle erythroid progenitors. Cell treated under the same condition as described in Fig. 2 were analysis by Western blots for HbF, HbS, and actin levels. A) Shown is a representative gel. B) Blots were analyzed by densitometry and data generated for normalized by actin is shown in the graph. C) Blots were analyzed by densitometry and data generated for HbS normalized by actin is shown in the graph.

For a therapeutic agent to offer greatest potential for clinical application, increasing proportions of HbF-expressing cells (F-cells) and the total HbF per cell above an average baseline threshold are important parameters recognized as required for significantly reducing SCD clinical severity. Therefore, we measured proportions of erythroid progenitors expressing HbF by flow cytometry stained with an FITC-HbF antibody. We observed an increase in the percent F-cells in five of six SCD subjects' progenitors analyzed. The mean F-cells increased from 5.8% (vehicle) to 8.1% (p < 0.01) and 7.4% (p < 0.001) by CT-101 100 nM and 200 nM respectively (Fig. 4A and B). This was equivalent to a mean 30% increase in the proportion of F-cells from the same SCD subject compared to DMSO treated vehicle controls. To assess HbF protein levels per erythroid cell as an additional measure of HbF, we measured MFI by flow cytometry. The erythroid progenitors from all six subjects displayed HbF induction by CT-101 ranging from 1.8-fold to 2.4-fold above the same subjects' control progenitors (Fig. 4C and D). Furthermore, an additive effect was observed when CT-101 (100 nM and 200 nM) and HU (75 μM) were combined; average HbF increased by a mean of 2.6-fold for the group, significantly higher than HU alone.

Fig. 4.

Fig. 4.

Open in a new tab

CT-101 increases F-Cells and fetal hemoglobin levels in sickle erythroid progenitors. Erythroid progenitors generated as described in Fig. 2 above were stained with anti-FITC HbF antibody and analyzed by flow cytomtery (Materials and Methods). A) The percentage of erythroid progenitors positive for HbF protein (%F-cells) was quantified and plotted in the graph. Data are shown as the Mean ± SEM and *p < 0.05 was considered significant. B) The level of HbF protein in erythroid cells was measured by mean fluorenee intensity (MFI) using flow cytometry analysis. Shown is the quantitative data generated by FlowJo analysis.

3.5. CT-101 increase levels of acetylated histone H3 in the γ-globin gene promoter

To gain insights into the molecular mechanisms of γ-globin gene activation by CT-101, we quantified changes in nuclear acetylated histone H3 (acH3) and acH4 protein levels produced by CT-101 using Western blot analysis. As shown in Fig. 5A, the level of acH3 (n = 6) was increased in a dose-dependent manner 1.8-fold and 2.0-fold by CT-101 100 nM and 200 nM respectively, compared to a 3-fold increase by the pan-HDACi sodium butyrate. Interestingly, combination treatment with CT-101 and HU increased acH3 2.7-fold. Similar findings were observed for acH4 at both CT-101 concentrations and the additive effect for combined treatment with HU (Fig. 5B).

Fig. 5.

Fig. 5.

Open in a new tab

CT-101 increases nuclear acetylated histone levels in the γ-globin promoter and locus control region-DNAse I hypersensitivity site 2 enhancer (LCR-HS2). Sickle erythroid cells treated under the same conditions as described in Fig. 2 were used for Western blot and ChIP assay. A) A representative Western blot gel is shown above the quantitative data generated by densitometry analysis for total histone H3 (H3) and acetylated H3 (acH3). Data are shown as the Mean ± SEM and *p < 0.05 was considered significant. B) Shown is a representative Western blot for total histone H4 (H4) and acetylated H4 (acH4) and the quantitative data generated by densitometry analysis is summarized in the graph. C–F) Chip assay was performed using SCD erythroid progenitors and RNA Polll and acH3 antibody for immunoprecipitation reactions (Material and Methods). Shown is the chromatin enrichment for the proximal γ-globin promoter (C), βS-globin promoter (D), LCR-HS2 (E) and negative control region located at −1225 Gγ cyclic AMP response element (G-CRE) (F).

Finally, we performed ChIP assay (Material and Methods) to determine the ability of CT-101 to increase acH3 levels in the HBB locus, which facilitates open chromatin conformations essential for gene transcription [8,30]. Immunoprecipitations were performed with RNA Polll, H3 and acH3 antibodies and IgG to control for non-specific in vivo binding. Chromatin enrichment was quantified in the proximal γ-globin and β-globin promoters, the locus control region-hypersensitive site 2 (LCR-HS2) and the negative control region around the −1225 Gγ-globin cyclic AMP response element (G-CRE) [31]. In parallel to the general increase in nuclear acH3 and acH4 levels (Fig. 5A and B), CT-101 100 mM and 200 nM mediated chromatin enrichment in the γ-globin promoter for acH3 (3.8-fold) (Fig. 5C). In contrast, the level of total H3 was not significantly changed. There was also increased RNA Polll chromatin enrichment in the γ-globin promoter region for all treatment conditions, consistent with increased gene transcription rates.

ChIP assays in the βs-globin proximal promoter showed RNA Polll and acH3 chromatin enrichment similar to the level observed for vehicle treated cells, and these levels were not changed by any of the test agents including CT-101 (Fig. 5D). We also studied change in chromatin enrichment in the LCR-HS2, since it is required for sequential enhancement of globin gene transcription during erythropoiesis and development [8]. Interestingly, we observed increased RNA Polll and acH3 chromatin enrichment in the LCR-HS2 with both concentrations of CT-101, HU/CT-101 and sodium butyrate (Fig. 5E). By contrast, low-level chromatin enrichment for RNA PollI and acH3 in the G-CRE was observed, which supports specificity of the test agent treatments (Fig. 5F).

4. Discussion

For decades, discovery of small molecules to reactivate γ-globin gene expression has been a highly sought-after strategy for treatments for SCD. Formation of hemoglobin hybrid molecules (α2γβS) which significantly decrease HbS polymerization through HbF induction ameliorates clinical disease severity, with certain threshold targets correlating with a benign clinical course [32]. Many different classes of agents including inhibitors of DNA methyltransferase (5-azacytidine, decitabine), methyl-CpG-binding domain protein 2 (MBD-2), HDACi and drug combinations were demonstrated four decades ago to induce HbF expression. Short chain fatty acid derivative pan- and selective- HDACi (sodium phenylbutyrate, arginine butyrate, valproate, 2,2 dimethylbutyrate) and panobinostat, among others, have shown clinical activity as HbF inducers [17-18,33]. Clinical trials in β-thalassemia and SCD patients with pulse butyrate showed robust HbF induction [18], although requirement for intravenous administration limited further development. Two oral HDACi were tested in early phase clinical trials in adults with SCD. The first was Zolinza (vorinostat), a pan-HDACi approved for treatment of T-cell lymphoma, induced HbF levels in three patients with Hodgkin lymphoma [35-36]. Subsequently, a Phase I/II study of vorinostat [37] enrolled 5 subjects with SCD who tolerated treatment without adverse effects over 12 weeks. Three of five subjects showed an increase in HbF, leading the investigators to speculate that higher weekly doses or development of HDAC1 or HDAC2 selective agents might facilitate higher HbF induction. Likewise, three patients with Hodgkin's lymphoma treated with Farydak (panobinostat) had a 2-fold increase in HbF over 3 months of therapy [38]. Subsequently, a Phase I trial of panobinostat was conducted in SCD patients at Augusta University refractory or unresponsive to HU therapy (NCT01245179; ClinicalTrials.gov). This trial administered low doses of panobinostat (10 mg per day), 3 days per week, before closing due to funding limitations. Nine subjects tolerated panobinostat without dose limiting or serious adverse effects, such as the thrombocytopenia and cardiac arrhythmias observed in cancer patients at higher doses. These studies illustrate the potential for HDAC inhibitors to provide a clinically rational therapeutic approach for SCD.

Multiple transcriptional and epigenetic regulators of γ-globin expression mediate gene silencing, including MBD-2, BCL11A, KLF1, LRF, NuRD complex, LSD-1 and others [34,39-46,57]. The zinc finger transcription factors BCL11A and LRF independently mediate γ-globin gene silencing during development [43,47]. A genome-wide association study discovered BCL11A as a repressor of γ-globin which accounts for ~30% of HbF levels [5] and mediates interaction with HDAC1 and HDAC2 as shown by co-immunoprecipitation experiments [44]. Disruption of the NuRD complex containing HDAC1 and HDAC2, among other proteins, abrogates γ-globin gene silencing in multiple erythroid model systems [39,48-49]. Chui et al. first showed that sodium butyrate induces γ-globin expression via BCL11A silencing. Previous strategies to develop isoform-specific HDACi involved a chemical genetic strategy combining focused libraries of biased chemical probes and reverse genetics using RNA interference [36]. Data support silencing Class I HDAC specifically, HDAC1, HDAC2, and HDAC3 as molecular targets mediated γ-globin gene silencing through disruption of the NuRD complex and chromatin accessibility.

The inhibitory action of CT-101 primarily targets Class I HDACs found in the NuRD repressor complex involved in γ-globin gene regulation during development. Here, we demonstrate the ability of CT-101 to induce HbF in primary sickle cell erythroid progenitors. Small molecule therapeutics offer greater logistical feasibility as global therapies compared to other options such as bone marrow transplant and gene therapy, which require stem cell harvest followed by chemoablation of the bone marrow conducted in sophisticated advanced academic institutions [50-51].

This study evaluated a novel synthetic Largazole analog, CT-101 as a potent and selective Class I HDACi with IC50 levels as low as 4 nM. Inhibition by currently approved products is variable, and pan-type inhibitors exhibit less selectivity across the 11 HDAC isoforms. Some approved products inhibit all HDAC isoforms to some degree as measured by in vitro inhibition assays. CT-101 shows both selective inhibition mainly for Class I HDAC and high potency, both important for development of targeted drug therapies. Results from in vitro inhibition assays for CT-101 shows a comparable level of Class I HDAC inhibition observed with panobinostat and up to 100-fold lower potency against Class I HDAC isoforms. The HDAC inhibitory profile of CT-101 supports Class I HDACs and HDAC 6 targeting, with minimal effects on HDAC IIa and IV isoforms.

The Class I HDAC isoforms localize in the nucleus and are most important for the epigenetic regulation of gene expression mediated by HDAC enzymes [52]. Inhibition of Class I HDAC isoforms is a preferred drug profile to limit off-target effects produced by first generation approved agents. The undesirable side effects of thrombocytopenia, neutropenia, and fatigue have limited dosing of first-generation FDA-approved HDACi, such as Beleodaq, Farydak, Istodax, and Zolinza [53]. The potency and selectivity of CT-101 may offer a more effective HDACi with fewer off-target effects for treatment of non-malignant diseases such as the β-hemoglobinopathies.

Our findings herein demonstrate the ability of CT-101 to activate γ-globin transcription selectively and increase F-cells and HbF levels by flow cytometry and Western blot analysis. Furthermore, combined treatment of CT-101 and HU produced an additive effect on HbF levels. Interestingly, CT-101 did not alter β-globin transcription, which is similar to findings previously published in normal erythroid progenitor treated with SAHA (pan-HDACi) and the HDAC1 and HDAC3 inhibitor, NK75 [36]. CT-101 produce limited cell toxicity after 5 days of treatment shown by slightly reduced sickle erythroid maturation; however, cell numbers continued to increase. Studies by Esrick et al [54] demonstrated HDAC1 and HDAC2 inhibition had no effect on cell proliferation or cell cycle phase. Furthermore, combining HDAC2 knock-down with HU treatment produced an additive effect similar to our findings.

The identification of druggable targets to reactivate γ-globin gene transcription during adult development has enabled rational approaches with more than 70 small molecules reported to induce HbF expression through different mechanisms. Epigenetic mechanisms including histone hypo-acetylation and DNA hypermethylation are required to achieve developmentally regulated hemoglobin switching [55]. Therefore, inhibition of HDAC enzyme activity should result in histone hyper-acetylation, increased chromatin accessibility, and γ-globin gene transcription. In our studies, CT-101 increased histone H3 and H4 acetylation in sickle erythroid progenitors associated with hyperacetylation of both proteins. Finally, ChIP assay in the proximal γ-globin and β-globin promoters and the enhancer element LCR-HS2 demonstrated that CT-101 mediated a significant increase in acH3 in the γ-globin promoter and LCR-HS2, without altering acetylation in the β-globin promoter. This is consistent with the lack of increase in β-globin mRNA by RT-qPCR in CT-101 treated sickle erythroid progenitors. These data are consistent with previous findings using chromosome conformation capture to monitor the chromatin structure of the HBB locus during switching [56].

Two prior HDAC inhibitors, SAHA and NK57, mediate conformational change characterized by increased contact between the LCR and the γ-globin and ε-globin promoters. As the main activity of CT-101 is inhibition of HDAC1 and HDAC3, we expect similar effects as NK57 on HBB chromatin structure. These data support rational design of inhibitors of Class I HDAC isoforms for HbF induction and provide a new molecularly targeted, potential therapy for inducing HbF. The absence of additive toxicity with HU is encouraging for combination applications, particularly for those patients who do not tolerate optimal doses of HU, as HbF induction has been recognized as a preferred method of ameliorating SCD for more than 50 years [55] due to its complimentary mechanism of action that can be used with recently approved therapeutics [58].

Acknowledgements

We gratefully acknowledge the significant contributions of Dr. Robert M. Williams on design and synthesis of the CT-compounds prior to his passing. We would like to thank Ms. Krystle Stone, research assistant in the Pediatric Sickle Cell Program at Augusta University for faithfully collecting discarded blood samples from sickle cell patients on chronic transfusions, used to isolate peripheral blood mononuclear cells for primary cultures.

Funding

This work was funded by National Heart, Lung, and Blood Institute grants R41 HL-136068 (RMW and SP), R01 DK-52962 (SP) and R42 HL136068 (BSP, RMW, LJ).

Footnotes

CRediT authorship contribution statement

LJ contributed to experimental design, data analysis and contributed to writing and editing the paper. BL contributed to experimental design, performed experiments, collected and analyzed data and reviewed and edited the manuscript. XZ performed histone acetylation protein work, ChIP assay design, data collection and analysis, and reviewed and edited the manuscript. SK and REC performed the synthesis of the test compound CT-101. CH contributed to experimental design and drafting of manuscript. RMW supervised the synthetic chemistry used to produce CT-101, prior to his passing. SP directed experiments on activity of test agents and contributed to writing and editing of the paper. JS performed experiments on activity of the test compounds. BP supervised the design and execution of the experiments and contributed to writing and editing the paper.

Declaration of competing interest

LJ, CH, SK, and REC are employed by Cetya Therapeutics and CH serves on the Board of Directors of Cetya. None of the other authors have declared any relevant conflicts of interest.

References

  • [1].Steinberg MH, Rodgers GP, Pharmacologic modulation of fetal hemoglobin, medicine, (Baltimore) 80 (5) (2001. Sep) 328–344. [DOI] [PubMed] [Google Scholar]
  • [2].Wilber A, Nienhuis AW, Persons DA, Transcriptional regulation of fetal to adult hemoglobin switching new therapeutic opportunities, Blood 117 (15) (2011. Apr 14) 3945–3953, 10.1182/blood-2010-11-316893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [3].Ginder GD, Epigenetic regulation of fetal globin gene expression in adult erythroid cells, Translat Res 165 (1) (2015) 115, 10.1016/j.trsl.2014.05.002. Epub2014 May 11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].Perrine SP, Pace BS, Faller DV, Targeted fetal hemoglobin induction for treatment of beta hemoglobinopathies, Hematol. Oncol. Clin. North Am 28 (2) (2014. Apr) 233–248, 10.1016/j.hoc.2013.11.009. [DOI] [PubMed] [Google Scholar]
  • [5].Uda M, Galanello R, Sanna S, Lettre G, Sankaran VG, Chen W, Usala G, Busonero F, Maschio A, Albai G, Piras MG, Sestu N, Lai S, Dei M, Mulas A, Crisponi L, Naitza S, Asunis I, Deiana M, Nagaraja R, Perseu L, Satta S, Cipollina MD, Sollaino C, Moi P, Hirschhorn JN, Orkin SH, Abecasis GR, Schlessinger D, Cao A, Genome-wide association study shows BCL11A associated with persistent fetal hemoglobin and amelioration of the phenotype of beta-thalassemia, Proc. Natl. Acad. Sci. U. S. A 105 (5) (2008. Feb 5) 1620–1625, 10.1073/pnas.0709089105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Sedgewick AE, Timofeev N, Sebastiani P, So JCC, Ma ESK, Chan LC, Fucharoen G, Fucharoen S, Barbosa CG, Vardarajan BN, Farrer LA, Baldwin CT, Steinberg MH, Chui DHK, BCL11A is a major HbF quantitative trait locus in three different populations with beta-hemoglobinopathies, Blood Cells Mol. Dis 41 (3) (2008. Nov-Dec) 255–258, 10.1016/j.bcmd.2008.06.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Murray N, Serjeant BE, Serjean GR, Sickle cell-hereditary persistence of fetal haemoglobin and its differentiation from other sickle cell syndromes, Br. J. Haematol 69 (1) (1988. May) 89–92, 10.1111/j.1365-2141.1988.tb07607.x. [DOI] [PubMed] [Google Scholar]
  • [8].Perrine RP, Pembrey ME, John P, Perrine S, Shoup F, Natural history of sickle cell anemia in Saudi Arabs. A study of 270 subjects, Ann. Intern. Med 88 (1978) 1–6. [DOI] [PubMed] [Google Scholar]
  • [9].Platt OS, Brambilla DJ, Rosse WF, Milner PF, Castro O, Steinberg MH, Klug PP, Mortality in sickle cell disease. Life expectancy and risk factors for early death, N. Engl. J. Med 330 (1994) 1639–1644, 10.1056/NEJM199406093302303. [DOI] [PubMed] [Google Scholar]
  • [10].Charache S, Terrin ML, Moore RD, Dover GJ, Barton FB, Eckert SV, McMahon RP, Bonds DR, Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia, investigators of the multicenter study of hydroxyurea in sickle cell anemia, N. Engl. J. Med 332 (20) (1995) 1317–1322, 10.1056/NEJM199505183322001. [DOI] [PubMed] [Google Scholar]
  • [11].Steinberg MH, McCarthy WF, Castro O, Balias SK, Armstrong FD, Smith W, Ataga K, Swerdlow P, Kutlar A, DeCastro L, Waclawiw MA, The risks and benefits of long-term use of hydroxyurea in sickle cell anemia: a 17.5-year follow-up, Am. J. Hematol 85 (2010) 403–408, 10.1002/ajh.21699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Dhar A, Leung TM, Appiah-Kubi A, Gruber D, Aygun B, Sergano O, Mitchell E, Longitudinal analysis of cardiac abnormalities in pediatric patients with sickle cell anemia and effect of hydroxyurea therapy, Blood Adv. 5 (21) (2021) 4406–4412, 10.1182/bloodadvances.2021005076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Niihara Y, Miller ST, Kanter J, Lanzkron S, Smith WR, Hsu LL, Gordeuk VR, Viswanathan K, Sarnaik S, Osunkwo I, Guillaume E, Sadanandan S, Sieger L, Lasky JL, Panosyan EH, Blake OA, New TN, Bellevue R, Tran LT, Razon RL, Stark CW, Neumayr LD, Vichinsky EP, Investigators of the Phase 3 Trial of l-Glutamine in Sickle Cell Disease, A phase 3 Trial of l-glutamine in sickle cell disease, N. Engl. J. Med 379 (3) (2018. Jul 19) 226–235, 10.1056/NEJMoa1715971. [DOI] [PubMed] [Google Scholar]
  • [14].Ataga KI, Kutlar A, Kanter J, Liles D, Cancado R, Friedrisch J, Guthrie TH, Knight-Madden J, Alvarez OA, Gordeuk VR, Gualandro S, Colella MP, Smith WR, Rollins SA, Stocker JW, Rother RP, Crizanlizumab for the prevention of pain crises in sickle cell disease, N. Engl. J. Med 376 (5) (2017. Feb 2) 429–439, 10.1056/NEJMoa1611770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Vichinsky E, Hoppe CC, Ataga KI, Ware RE, Nduba V, El-Beshlawy A, Hassab H, Achebe MM, Alkindi S, Brown RC, Diuguid DL, Telfer P, Tsitsikas DA, Elghandour A, Gordeuk VR, Kanter J, Abboud MR, Lehrer-Graiwer J, Tonda M, Intondi A, Tong B, Howard J, H.O.P.E.Trial Investigators, A phase 3 randomized trial of voxelotor in sickle cell disease, N. Engl. J. Med 381 (6) (2019. Aug 8) 509–519, 10.1056/NEJMoa1903212, 381(6). [DOI] [PubMed] [Google Scholar]
  • [16].Selby R, Nisbet-Brown E, Basran RK, Chang L, Olivieri NF, Valproic acid and augmentation of fetal hemoglobin in individuals with and without sickle cell disease, Blood 90 (2) (1997. Jul 15) 891–893, 10.1182/blood.V90.2.891. [DOI] [PubMed] [Google Scholar]
  • [17].Perrine SP, Ginder GD, Faller DV, Dover GJ, Ikuta T, Witkowska HE, Cai SP, Vichinsky EP, Olivieri NF, A short-term trial of butyrate to stimulate fetal-globin-gene expression in the beta-globin disorders, N. Engl. J. Med 328 (2) (1993) 81–86, 10.1056/NEJM199301143280202. [DOI] [PubMed] [Google Scholar]
  • [18].Atweh GF, Sutton M, Nassif I, Boosalis V, Dover GJ, Wallenstein S, Wright E, McMahon L, Stamatoyannopoulos G, Faller DV, Perrine SP, Sustained induction of fetal hemoglobin by pulse butyrate therapy in sickle cell disease, Blood 93 (6) (1999) 1790–1797, 10.1182/bloodV93.6.1790.406k27_1790_1797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Perrine SP, Swerdlow P, Faller DV, Qin G, Rudolph AM, Reczek J, Kan YW, Butyric acid modulates developmental globin gene switching in man and sheep, Adv. Exp. Med. Biol 271 (1989) 177–183, 10.1007/978-1-4613-0623-8_18. [DOI] [PubMed] [Google Scholar]
  • [20].Pace B, Li Q, Peterson K, Stamatoyannopoulos G, Alpha-amino butyric acid cannot reactivate the silenced gamma gene of the beta locus YAC transgenic mouse, Blood 84 (12) (1994. Dec 15) 4344–4353, 10.1182/blood.V84.12.4344.bloodjournal84124344. [DOI] [PubMed] [Google Scholar]
  • [21].Johnson J, Qian XH, Hunter R, McElveen R, Pace BS, Scriptaid, a novel histone deacetylase inhibitor induces gamma-globin gene expression in erythroid progenitors, Cell Mol. Biol 51 (2005) 229–238. [PubMed] [Google Scholar]
  • [22].Hebbel RP, Vercellotti GM, Pace BS, Solovev AN, Kollander R, Abanonu CF, Nguyen J, Vineyard JV, Belcher JD, Abdulla F, Osifuye S, Eaton JW, Kelm RJ Jr., Slungaard A, The HDAC inhibitors trichostatin a and suberoylanilide hydroxamic acid exhibit multiple modalities of benefit for the vascular pathobiology of sickle transgenic mice, Blood 115 (12) (2010. Mar 25) 2483–2490, 10.1182/blood-2009-02-204990. Epub 2010 Jan 6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [23].Taori K, Paul VJ, Luesch H, Structure and activity of Largazole, a potent antiproliferative agent from the Floridian Marine Cyanobacterium Symploca sp. J Am Chem Soc. 130 (2008) 1806–1807, 10.1021/ja7110064. [DOI] [PubMed] [Google Scholar]
  • [24].Bowers A, West N, Taunton J, Schreiber SL, Bradner JE, Williams RW, Total synthesis and biological mode of action of Largazole: a potent class I histone deacetylase inhibitor, J Am Chem Soc. 130 (2008) 11219–11222, 10.1021/ja8033763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Bowers AA, Greshook TJ, West N, Estiu G, Schreiber SL, Wiest O, Williams RM, Bradne JE, Bowers AA, Greshook TJ, West N, Estiu G, Schreiber SL, Wiest O, Williams RM, Bradner JE, J. Am. Chem. Soc 131 (8) (2009. Mar 4)) 2900–2905, 10.1021/ja807772w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [26].Li B, Zhu X, Hossain M, Guy C, Xu X, Bungert J, Pace BS, Fetal hemoglobin induction in sickle erythroid progenitors using a synthetic zinc finger DNA-binding domain, Haematologica 103 (9) (2018. Sep) e384–e387, 10.3324/haematol.2017.185967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Zhu X, Li B, Pace BS, NRF2 mediates γ-globin gene regulation and fetal hemoglobin induction in human erythroid progenitors, Haematologica 102 (8) (2017) e285–e288, 10.3324/haematol.2016.160788, pii: haematol.2016.160788, 2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Zhu X, Pace BS, Loss of NRF2 function exacerbates the pathophysiology of sickle cell disease in a transgenic mouse model, Blood 130 (Suppl_1) (2017. Dec 7) 960, 10.1182/blood.V130.Suppl_1.960.960. pii: blood-2017-10-810531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [29].Howitz KT, Screening and profiling assays for HDACs and sirtuins, Drug Discov. Today Technol 18 (2015) 38–48, 10.1016/j.ddtec.2015.10.008. [DOI] [PubMed] [Google Scholar]
  • [30].Tolhuis B, Palstra RJ, Splinter E, Grosveld F, de Laat W, Mol. Cell 10 (6) (2002. Dec) 1453–1465, 10.1016/s1097-2765(02)00781-5. [DOI] [PubMed] [Google Scholar]
  • [31].Sangerman J, Lee MS, Yao X, Oteng E, Hsiao CH, Li W, Zein S, Ofori-Acquah SF, Pace BS, Mechanism for fetal hemoglobin induction by histone deacetylase inhibitors involves gamma-globin activation by CREB1 and ATF-2, Blood 108 (10) (2006. Nov 15) 3590, 10.1182/blood-2006-01-023713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Nobuchi CT, Schechter AN, Sickle hemoglobin polymerization in solution and in cells, Annu. Rev. Biophys. Chem 14 (1985) 239–263, 10.1146/annurev.bb.14.060185.001323. [DOI] [PubMed] [Google Scholar]
  • [33].Saunthararajah Y, Hillery CA, Lavelle D, Molokie R, Dorn L, Bressler L, Gavazova S, Chen YH, Hoffman R, DeSimone J, Effects of 5-aza-2'-deoxycytidine on fetal hemoglobin levels, red cell adhesion, and hematopoietic differentiation in patients with sickle cell disease, Blood 102 (12) (2003. Dec 1) 3865–3870, 10.1182/blood-2003-05-1738. [DOI] [PubMed] [Google Scholar]
  • [34].Dai Y, Sangerman J, Luo HY, Fucharoen S, Chui DH, Faller DV, Perrine SP, Therapeutic fetal-globin inducers reduce transcriptional repression in hemoglobinopathy erythroid progenitors through distinct mechanisms, Blood Cells Mol. Dis 56 (2016) 62–69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [35].Jing B, Jin J, Xiang R, Liu M, Yang L, Tong Y, Xiao X, Lei H, Liu W, Xu H, Deng J, Zhou, Wu Y, Vorinostat and quinacrine have synergistic effects in T-cell acute lymphoblastic leukemia through reactive oxygen species increase and mitophagy inhibition, Cell Death Dis. 9 (6) (2018. May 22) 589, 10.1038/S41419-018-0679-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [36].Bradner JE, Mak R, Tanguturi SK, Mazitschek R, Haggarty SJ, Ross K, Chang CY, Bosco J, West N, Morse E, Lin K, Shen JP, Kwiatkowski NP, Gheldof N, Dekker J, DeAngelo DJ, Carr SA, Schreiber SL, Golub TR, Ebert BL, Chemical genetic strategy identifies histone deacetylase 1 (HDAC1) and HDAC2 as therapeutic targets in sickle cell disease, Proc. Natl. Acad. Sci. U. S. A 107 (28) (2010. Jul 13) 12617–12622, 10.1073/pnas.1006774107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [37].Okam MM, Esrick EB, Mandell E, Campigotto F, Neuberg DS, Ebert BL, Phase 1/2 trial of vorinostat in patients with sickle cell disease who have not benefitted from hydroxyurea, Blood 125 (23) (2015. Jun 4) 3668–3669, 10.1182/blood-2015-03-635391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [38].Oki Y, Copeland A, Younes A, Clinical development of panobinostat in classical Hodgkin’s lymphoma, Expert. Rev. Hematol 4 (3) (2011) 245–252, 10.1586/ehm.11.24. [DOI] [PubMed] [Google Scholar]
  • [39].Amaya M, Desai M, Gnanapragasam MN, Wang SZ, Zhu SZ, Williams DC Jr., Ginder GD, Mi2beta-mediated silencing of the fetal gamma-globin gene in adult erythroid cells, Blood 121 (17) (2013. Apr 25) 3493–3501, 10.1182/blood-2012-11-466227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].Borg J, Patrinos GP, Felice AE, Philipsen S, Erythroid phenotypes associated with KLF1 mutations, Haematologica 96 (5) (2011. May) 635–638, 10.3324/haematol.2011.043265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [41].Canver MC, Smith EC, Sher F, Pinello L, Sanjana NE, Shalem O, Chen DD, Schupp PG, Vinjamur DS, Garcia SP, Luc S, Kurita R, Nakamura Y, Fujiwara Y, Maeda T, Yuan G-C, Zhang F, Orkin ST, Bauer DE, BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis, Nature 527 (7577) (2015. Nov 12) 192–197, 10.1038/nature15521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [42].Chen Z, Luo H, Steinberg MH, Chui DHK, BCL11A represses HBG transcription in K562 cells, Blood Cells Mols Dis. 41 (2009) 144–149. [DOI] [PubMed] [Google Scholar]
  • [43].Masuda T, Wang X, Maeda M, Canver MC, Sher F, Funnell APW, Fisher C, Suciu M, Martyn GE, Norton LJ, Kurita R, Nakamura Y, Xu J, Higgs DR, Crossley M, Bauer DE, Orkin SH, Kharchenko PV, Maeda T, Zhu C, Transcription factors LRF and BCL11A independently repress expression of fetal hemoglobin, Science 351 (6270) (2016. Jan 15) 285–289, 10.1126/science.aad3312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [44].Sankaran VG, Menne TF, Xu J, Akie TE, Lettre G, Handel BV, Mikkola HKA, Hirschhorn JN, Cantor AB, Orkin SH, Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A, Science 322 (5909) (2008. Dec 19) 1839–1842, doi: 10.1126/science.1165409. [DOI] [PubMed] [Google Scholar]
  • [45].Shi L, Cui S, Engel JD, Tanabe O, Lysine specific demethylase 1 is a therapeutic target for fetal hemoglobin induction, Nat. Med 19 (3) (2013. Feb 17) 291–294, 10.1038/nm.3101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [46].Zhou D, Liu K, Sun CW, Pawlik KM, Townes TM, KLF1 regulates BCL11A expression and gamma- to beta-globin gene switching, Nat. Genet 42 (9) (2010. Aug 1) 742–744, 10.1038/ng.637. [DOI] [PubMed] [Google Scholar]
  • [47].Kurita R, Suda N, Sudo K, Miharada K, Hiroyama T, Tani K, Nakamura Y, Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells, PLoS One 8 (3) (2013. Mar 22), e59890, 10.1371/journal.pone.0059890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [48].Gnanapragasam MN, Scarsdale JN, Amaya ML, Webb HD, Desai MA, Walavalkar NM, Wang SZ, Zhu SZ, Ginder GD, Williams DC Jr., p66Alpha-MBD2 coiled-coil interaction and recruitment of Mi-2 are critical for globin gene silencing by the MBD2-NuRD complex, Proc. Natl. Acad. Sci. U. S. A 108 (18) (2011. May 3) 7487–7492, 10.1073/pnas.1015341108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [49].Rupon JW, Wang SZ, Gaensler K, Lloyd J, Ginder GD, Methyl binding domain protein 2 mediates gamma-globin gene silencing in adult human betaYAC transgenic mice, Proc. Natl. Acad. Sci. U. S. A 103 (17) (2006) 6617–6622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [50].Suragani RNVS, Cawley SM, Li R, Wallner S, Alexander MJ, Mulivor AW, Gardenghi S, Rivella S, Grinberh AV, Pearsall RS, Kumar R, Modified activin receptor IIB ligand trap mitigates ineffective erythropoiesis and disease complications in murine-thalassemia, Blood 123 (25) (2014. Jun 19) 3864–3872, 10.1182/blood-2013-06-511238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [51].Paulson RF, Targeting a new regulator of erythropoiesis to alleviate anemia, Nat. Med 20 (2014. Apr 7) 334–335, 10.1038/nm.3524. [DOI] [PubMed] [Google Scholar]
  • [52].Milazzo G, Mercatelli D, Di Muzio G, Triboli L, De Rosa P, Perini G, Giorgi FM, Histone deacetylases (HDACs): evolution, specificity, role in transcriptional complexes, and pharmacological actionability, Genes 11 (2020. May 15) 556, 10.3390/genes11050556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [53].Eckschlager T, Pich J, Stiborova M, Hrabeta J, Histone deacetylase inhibitors as anticancer drugs, Int. J. Mol. Sci 18 (2017. Jul 1) 1414, 10.3390/ijms18071414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [54].Esrick EB, McConkey M, Lin K, Frisbee A, Ebert BL, Inactivation of HDAC1 or HDAC2 induces gamma globin expression without altering cell cycle or proliferation, Am. J. Hematol 90 (7) (2015. Jul) 624–628, 10.1002/ajh.24019. Epub 2015 May 28 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [55].Molokie R, DeSimone J, Lavelle D, Epigenetic regulation of hemoglobin switching in non-human primates, Semin. Hematol 58 (1) (2021. Jan) 10–14, 10.1053/j.seminhematol.2020.12.001. Epub 2020 Dec 28 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [56].Dekker J, Rippe K, Dekker M, Kleckner N, Capturing chromosome conformation, Science 295 (5558) (2002. Feb 15) 1306–1311, 10.1126/science.1067799. [DOI] [PubMed] [Google Scholar]
  • [57].Stamatoyannopoulos G, Grosveld F, Hemoglobin switching, in: The Molecular Basis of Blood Disease, 3rd ed., Saunders, Philadelphia, 2001. [Google Scholar]
  • [58].Henry ER, Metaferia E, Li Q, Harper J, Best RB, Glass KE, Cellmer T, Dunkelberger EB, Conrey A, Thein SL, Bunn HF, Eaton WA, Treatment of sickle cell disease by increasing oxygen affinity of hemoglobin, Blood (2021) 1171–1181, 10.1182/blood.2021012070. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES