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

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.

Benserazide racemate and enantiomers induce fetal globin gene expression in vivo: Studies to guide clinical development for beta thalassemia and sickle cell disease.

Increased expression of developmentally silenced fetal globin (HBG) reduces the clinical severity of ß-hemoglobinopathies. Benserazide has a relatively benign safety profile having been approved for 50 years in Europe and Canada for Parkinson's disease treatment. Benserazide was shown to activate HBG gene transcription in a high throughput screen, and subsequent studies confirmed fetal hemoglobin (HbF) induction in erythroid progenitors from hemoglobinopathy patients, transgenic mice containing the entire human ß-globin gene (ß-YAC) and anemic baboons. The goal of this study is to evaluate efficacies and plasma exposure profiles of benserazide racemate and its enantiomers to select the chemical form for clinical development. Intermittent treatment with all forms of benserazide in ß-YAC mice significantly increased proportions of red blood cells expressing HbF and HbF protein per cell with similar pharmacokinetic profiles and with no cytopenia. These data contribute to the regulatory justification for development of the benserazide racemate. Additionally, dose ranges and frequencies required for HbF induction using racemic benserazide were explored. Orally administered escalating doses of benserazide in an anemic baboon induced γ-globin mRNA up to 13-fold and establish an intermittent dose regimen for clinical studies as a therapeutic candidate for potential treatment of ß-hemoglobinopathies.

Therapeutic fetal-globin inducers reduce transcriptional repression in hemoglobinopathy erythroid progenitors through distinct mechanisms.

Pharmacologic augmentation of γ-globin expression sufficient to reduce anemia and clinical severity in patients with diverse hemoglobinopathies has been challenging. In studies here, representative molecules from four chemical classes, representing several distinct primary mechanisms of action, were investigated for effects on γ-globin transcriptional repressors, including components of the NuRD complex (LSD1 and HDACs 2-3), and the downstream repressor BCL11A, in erythroid progenitors from hemoglobinopathy patients. Two HDAC inhibitors (MS-275 and SB939), a short-chain fatty acid derivative (sodium dimethylbutyrate [SDMB]), and an agent identified in high-throughput screening, Benserazide, were studied. These therapeutics induced γ-globin mRNA in progenitors above same subject controls up to 20-fold, and increased F-reticulocytes up to 20%. Cellular protein levels of BCL11A, LSD-1, and KLF1 were suppressed by the compounds. Chromatin immunoprecipitation assays demonstrated a 3.6-fold reduction in LSD1 and HDAC3 occupancy in the γ-globin gene promoter with Benserazide exposure, 3-fold reduction in LSD-1 and HDAC2 occupancy in the γ-globin gene promoter with SDMB exposure, while markers of gene activation (histone H3K9 acetylation and H3K4 demethylation), were enriched 5.7-fold. These findings identify clinical-stage oral therapeutics which inhibit or displace major co-repressors of γ-globin gene transcription and may suggest a rationale for combination therapy to produce enhanced efficacy.

Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS) Are Active In Vivo in Anemic Baboons and Transgenic Mice.

High-level fetal (γ) globin expression ameliorates clinical severity of the beta (ß) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.

Butyrate histone deacetylase inhibitors.

In addition to being a part of the metabolic fatty acid fuel cycle, butyrate is also capable of inducing growth arrest in a variety of normal cell types and senescence-like phenotypes in gynecological cancer cells, inhibiting DNA synthesis and cell growth in colonic tumor cell lines, suppressing hTERT mRNA expression and telomerase activity in human prostate cancer cells, and inducing stem cell differentiation and apoptosis by DNA fragmentation. It regulates gene expression by inhibiting histone deacetylases (HDACs), enhances memory recovery and formation in mice, stimulates neurogenesis in the ischemic brain, promotes osteoblast formation, selectively blocks cell replication in transformed cells (compared to healthy cells), and can prevent and treat diet-induced obesity and insulin resistance in mouse models of obesity, as well as stimulate fetal hemoglobin expression in individuals with hematologic diseases such as the thalassemias and sickle-cell disease, in addition to a multitude of other biochemical effects in vivo. However, efforts to exploit the potential of butyrate in the clinical treatment of cancer and other medical disorders are thwarted by its poor pharmacological properties (short half-life and first-pass hepatic clearance) and the multigram doses needed to achieve therapeutic concentrations in vivo. Herein, we review some of the methods used to overcome these difficulties with an emphasis on HDAC inhibition.

Role of STAT3 and GATA-1 interactions in gamma-globin gene expression.

OBJECTIVE: We previously demonstrated a silencing role for signal transducers and activators of transcription 3 (STAT3) in gamma-globin gene regulation in primary erythroid cells. Recently, GATA-1, a key transcription factor involved in hematopoietic cell development, was shown to directly inhibit STAT3 activity in vivo. Therefore, we completed studies to determine if interactions between these two factors influence gamma-globin gene expression. MATERIALS AND METHODS: Chromatin immunoprecipitation assay was used to ascertain in vivo protein binding at the gamma-globin 5' untranslated region (5'UTR); protein-protein interactions were examined by coimmunoprecipitation analysis. In vitro protein-DNA binding were completed using surface plasmon resonance and electrophoretic mobility shift assay. Activity of a luciferase gamma-globin promoter reporter and levels of gamma-globin messenger RNA and fetal hemoglobin in stable K562 cell lines overexpressing STAT3 and GATA-1, were used to determine the influence of the STAT3/GATA-1 interaction on gamma-globin gene expression. RESULTS: We observed interaction between STAT3 and GATA-1 in K562 and mouse erythroleukemia cells in vivo at the gamma-globin 5'UTR by chromatin immunoprecipitation assay. Electrophoretic mobility shift assay performed with a 41-base pair gamma-globin DNA probe (gamma41) demonstrated the presence of STAT3 and GATA-1 proteins in complexes assembled at the gamma-globin 5'UTR. A consensus STAT3 DNA probe inhibited GATA-1-binding in a concentration-dependent manner, and the converse was also true. Enforced STAT3 expression augmented its binding at the gamma-globin 5'UTR in vivo and silenced gamma-promoter-driven luciferase activity. Stable enforced STAT3 expression in K562 cells reduced endogenous gamma-globin messenger RNA level. This effect was reversed by GATA-1. CONCLUSION: These data provide evidence that GATA-1 can reverse STAT3-mediated gamma-globin gene silencing in erythroid cells.

Failure of terminal erythroid differentiation in EKLF-deficient mice is associated with cell cycle perturbation and reduced expression of E2F2.

Erythroid Krüppel-like factor (EKLF) is a Krüppel-like transcription factor identified as a transcriptional activator and chromatin modifier in erythroid cells. EKLF-deficient (Eklf(-/-)) mice die at day 14.5 of gestation from severe anemia. In this study, we demonstrate that early progenitor cells fail to undergo terminal erythroid differentiation in Eklf(-/-) embryos. To discover potential EKLF target genes responsible for the failure of erythropoiesis, transcriptional profiling was performed with RNA from wild-type and Eklf(-/-) early erythroid progenitor cells. These analyses identified significant perturbation of a network of genes involved in cell cycle regulation, with the critical regulator of the cell cycle, E2f2, at a hub. E2f2 mRNA and protein levels were markedly decreased in Eklf(-/-) early erythroid progenitor cells, which showed a delay in the G(1)-to-S-phase transition. Chromatin immunoprecipitation analysis demonstrated EKLF occupancy at the proximal E2f2 promoter in vivo. Consistent with the role of EKLF as a chromatin modifier, EKLF binding sites in the E2f2 promoter were located in a region of EKLF-dependent DNase I sensitivity in early erythroid progenitor cells. We propose a model in which EKLF-dependent activation and modification of the E2f2 locus is required for cell cycle progression preceding terminal erythroid differentiation.

Ankyrin-linked hereditary spherocytosis in an African-American kindred.

Mutations of ankyrin-1 are the most frequent cause of the inherited hemolytic anemia, hereditary spherocytosis (HS), in people of European ancestry. Ankyrin-1, which provides the primary linkage between the erythrocyte membrane skeleton and the plasma membrane, has numerous isoforms generated by alternative splicing, alternate polyadenylation, use of tissue-specific promoters, and alternate NH(2) or COOH-termini. Mutation detection in erythrocyte membrane protein genes, including ankyrin, has been a challenge, primarily due to the large size of these genes, and the apparent frequent occurrence of HS-associated null alleles. Using denaturing high-performance liquid chromatography (DHPLC), we screened the ankyrin gene of the proband of a large, three generation African-American kindred with ankyrin-deficient HS. DHPLC yielded an abnormal chromatogram for exon 1. Examination of the corresponding exon 1 sequence in genomic DNA from the proband revealed heterozygosity for a mutation of the initiator methionine (ATG to ATA Met 1 Ile). Coupled in vitrotranscription/translation studies with rabbit reticulocyte lysates demonstrated that the wild-type ankyrin erythroid cDNA initiates only from the known initiator methionine, indicating that the use of alternate initiator methionine is not a mechanism of isoform diversity in erythroid cells. The mutant ankyrin allele, unlike some initiator methionine mutations that utilize downstream codons for translation initiation, was associated with a null allele. This is the first report describing ankyrin-linked HS in an African-American kindred.

Mechanism for fetal hemoglobin induction by histone deacetylase inhibitors involves gamma-globin activation by CREB1 and ATF-2.

The histone deacetylase inhibitors (HDA-CIs) butyrate and trichostatin A activate gamma-globin expression via a p38 mitogen-activating protein kinase (MAPK)-dependent mechanism. We hypothesized that down-stream effectors of p38 MAPK, namely activating transcription factor-2 (ATF-2) and cyclic AMP response element (CRE) binding protein (CREB), are intimately involved in fetal hemoglobin induction by these agents. In this study, we observed increased ATF-2 and CREB1 phosphorylation mediated by the HDACIs in K562 cells, in conjunction with histone H4 hyperacetylation. Moreover, enhanced DNA-protein interactions occurred in the CRE in the (G)gamma-globin promoter (G-CRE) in vitro after drug treatments; subsequent chromatin immunoprecipitation assay confirmed ATF-2 and CREB1 binding to the G-CRE in vivo. Enforced expression of ATF-2 and CREB produced (G)gamma-promoter trans-activation which was abolished by a 2-base pair mutation in the putative G-CRE. The data presented herein demonstrate that gamma-gene induction by butyrate and trichostatin A involves ATF-2 and CREB1 activation via p38 MAPK signaling.

Alterations in expression and chromatin configuration of the alpha hemoglobin-stabilizing protein gene in erythroid Kruppel-like factor-deficient mice.

Erythroid Krüppel-like factor (EKLF) is an erythroid zinc finger protein identified by its interaction with a CACCC sequence in the beta-globin promoter, where it establishes local chromatin structure permitting beta-globin gene transcription. We sought to identify other EKLF target genes and determine the chromatin status of these genes in the presence and absence of EKLF. We identified alpha hemoglobin-stabilizing protein (AHSP) by subtractive hybridization and demonstrated a 95 to 99.9% reduction in AHSP mRNA and the absence of AHSP in EKLF-deficient cells. Chromatin at the AHSP promoter from EKLF-deficient cells lacked a DNase I hypersensitive site and exhibited histone hypoacetylation across the locus compared to hyperacetylation of wild-type chromatin. Wild-type chromatin demonstrated a peak of EKLF binding over a promoter region CACCC box that differs from the EKLF consensus by a nucleotide. In mobility shift assays, the AHSP promoter CACCC site bound EKLF in a manner comparable to the beta-globin promoter CACCC site, indicating a broader recognition sequence for the EKLF consensus binding site. The AHSP promoter was transactivated by EKLF in K562 cells, which lack EKLF. These results support the hypothesis that EKLF acts as a transcription factor and a chromatin modulator for the AHSP and beta-globin genes and indicate that EKLF may play similar roles for other erythroid genes.

p38 MAP kinase activation mediates gamma-globin gene induction in erythroid progenitors.

OBJECTIVE: Our goal was to determine the role of p38 mitogen-activated protein kinase (MAPK) signaling in fetal hemoglobin (HbF) induction. Two histone deacetylase inhibitors (HDAIs), sodium butyrate (NB), and trichostatin (TSA) and hemin were analyzed. In addition, the effect of direct activation of p38 MAPK on gamma-globin gene activity was studied. METHOD: Primary erythroid progenitors derived from peripheral blood mononuclear cell and K562 erythroleukemia cells were analyzed. Cells were grown in NB, TSA, hemin, or anisomycin either alone or in the presence of the p38 MAPK inhibitor SB203580. The effects of the various treatments on gamma-globin RNA, HbF, and phosphorylated p38 MAPK levels were measured by RNase protection assay, alkaline denaturation, and Western blot analysis, respectively. A K562 stable line overexpressing constitutively active p38 MAPK was established using MAPK kinase kinase 3 (MKK3) and MKK6, the immediate upstream activators of p38. The direct effect of p38 MAPK overexpression on gamma-globin mRNA synthesis was analyzed. RESULTS: NB and TSA activated p38 MAPK and increased gamma-globin mRNA levels in K562 cells and primary erythroid progenitors. Pretreatment with SB203580 blocked p38 MAPK and gamma-globin gene activation. In contrast, no change in p38 activity was observed with hemin inductions. Direct activation of p38 by anisomycin or constitutive overexpression also increased gamma-globin mRNA in the absence of HbF inducers in wild-type K562 cells and in the MKK stable lines. CONCLUSION: This study supports a novel role for p38 MAPK in gamma-globin regulation in human erythroid progenitors.