ABSTRACT
Elevated levels of Fetal Hemoglobin interfere with polymerization of sickle hemoglobin thereby reducing anemia, lessening the severity of symptoms, and increasing life span of patients with sickle cell disease. An affordable, small molecule drug that stimulates HbF expression in vivo would be ideally suited to treat the large numbers of SCD patients that exist worldwide. Our previous work showed that administration of the LSD1 (KDM1A) inhibitor RN-1 to normal baboons increased Fetal Hemoglobin (HbF) and was tolerated over a prolonged treatment period. HbF elevations were associated with changes in epigenetic modifications that included increased levels of H3K4 di-and tri-methyl lysine at the γ-globin promoter. While dramatic effects of the loss of LSD1 on hematopoietic differentiation have been observed in murine LSD1 gene deletion and silencing models, the effect of pharmacological inhibition of LSD1 in vivo on hematopoietic differentiation is unknown. The goal of these experiments was to investigate the in vivo mechanism of action of the LSD1 inhibitor RN-1 by determining its effect on γ-globin expression in highly purified subpopulations of bone marrow erythroid cells enriched for varying stages of erythroid differentiation isolated directly from baboons treated with RN-1 and also by investigating the effect of RN1 on the global transcriptome in a highly purified population of proerythroblasts. Our results show that RN-1 administered to baboons targets an early event during erythroid differentiation responsible for γ-globin repression and increases the expression of a limited number of genes including genes involved in erythroid differentiation such as GATA2, GFi-1B, and LYN.
Subject(s)
Anemia, Sickle Cell , Histone Demethylases , Animals , Humans , Mice , Anemia, Sickle Cell/genetics , Fetal Hemoglobin/genetics , gamma-Globins/genetics , Gene Expression , Histone Demethylases/antagonists & inhibitors , Papio anubis/geneticsABSTRACT
Increased fetal hemoglobin (HbF) levels reduce the symptoms of sickle cell disease (SCD) and increase the lifespan of patients. Because curative strategies for bone marrow transplantation and gene therapy technologies remain unavailable to a large number of patients, the development of a safe and effective pharmacological therapy that increases HbF offers the greatest potential for disease intervention. Although hydroxyurea increases HbF, a substantial proportion of patients fail to demonstrate an adequate response. Pharmacological inhibitors of DNA methyltransferase (DNMT1) and lysine-specific demethylase 1A (LSD1), 2 epigenome-modifying enzymes associated with the multiprotein corepressor complex recruited to the repressed γ-globin gene, are powerful in vivo inducers of HbF. The hematological side effects of these inhibitors limit feasible clinical exposures. We evaluated whether administering these drugs in combination could reduce the dose and/or time of exposure to any single agent to minimize adverse effects, while achieving additive or synergistic increases in HbF. The DNMT1 inhibitor decitabine (0.5 mg/kg per day) and the LSD1 inhibitor RN-1 (0.25 mg/kg per day) administered in combination 2 days per week produced synergistic increases in F-cells, F-reticulocytes, and γ-globin messenger RNA in healthy baboons. Large increases in HbF and F-cells were observed in healthy, nonanemic, and anemic (phlebotomized) baboons. Combinatorial therapy targeting epigenome-modifying enzymes could thus be a useful strategy for producing larger increases in HbF to modify the clinical course of SCD.
Subject(s)
Anemia, Sickle Cell , gamma-Globins , Humans , Animals , Decitabine/pharmacology , Decitabine/therapeutic use , gamma-Globins/genetics , Epigenome , Fetal Hemoglobin/genetics , Anemia, Sickle Cell/genetics , Papio/genetics , Histone Demethylases/genetics , Histone Demethylases/therapeutic useABSTRACT
Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). More effective strategies to increase HbF are needed because the current standard of care, hydroxyurea, is not effective in a significant proportion of patients. Treatment of the millions of patients projected worldwide would best be accomplished with an orally administered drug therapy that increased HbF. LSD1 is a component of corepressor complexes that repress γ-globin gene expression and are a therapeutic target for HbF reactivation. We have shown that subcutaneous administration of RN-1, a pharmacological LSD1 inhibitor, increased γ-globin expression in SCD mice and baboons, which are widely acknowledged as the best animal model in which to test the activity of HbF-inducing drugs. The objective of this investigation was to test the effect of oral administration of a new LSD1 inhibitor, ORY-3001. Oral administration of ORY-3001 to SCD mice (nâ¯=â¯3 groups) increased γ-globin expression, Fetal Hemoglobin (HbF)-containing (F) cells, and F reticulocytes (retics). In normal baboons (nâ¯=â¯7 experiments) treated with ORY-3001, increased F retics, γ-globin chain synthesis, and γ-globin mRNA were observed. Experiments in anemic baboons (nâ¯=â¯2) showed that ORY-3001 increased F retics (PA8695, predoseâ¯=â¯24%, postdoseâ¯=â¯66.8%; PA8698: predoseâ¯=â¯13%, postdoseâ¯=â¯93.6%), γ-globin chain synthesis (PA8695: predoseâ¯=â¯0.07 γ/γ+ß, postdoseâ¯=â¯0.20 γ/γ+ß; PA8698: predoseâ¯=â¯0.02 γ/γ+ß, postdoseâ¯=â¯0.44 γ/γ+ß), and γ-globin mRNA (PA8695: predoseâ¯=â¯0.06 γ/γ+ß, postdoseâ¯=â¯0.18 γ/γ+ß; PA8698: predoseâ¯=â¯0.03 γ/γ+ß, postdoseâ¯=â¯0.33 γ/γ+ß). We conclude that oral administration of ORY-3001 increases F retics, γ-globin chain synthesis, and γ-globin mRNA in baboons and SCD mice, supporting further efforts toward the development of this drug for SCD therapy.
Subject(s)
Anemia, Sickle Cell/drug therapy , Enzyme Inhibitors/therapeutic use , Fetal Hemoglobin/biosynthesis , Histone Demethylases/antagonists & inhibitors , gamma-Globins/biosynthesis , Administration, Oral , Anemia/blood , Anemia/drug therapy , Anemia, Sickle Cell/blood , Animals , Blood Cell Count , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Enzyme Inhibitors/administration & dosage , Female , Fetal Hemoglobin/genetics , Gene Expression Regulation/drug effects , Mice , Papio , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reticulocytes/metabolism , gamma-Globins/geneticsABSTRACT
BACKGROUND: Sickle cell disease (SCD), a congenital hemolytic anemia that exacts terrible global morbidity and mortality, is driven by polymerization of mutated sickle hemoglobin (HbS) in red blood cells (RBCs). Fetal hemoglobin (HbF) interferes with this polymerization, but HbF is epigenetically silenced from infancy onward by DNA methyltransferase 1 (DNMT1). METHODS AND FINDINGS: To pharmacologically re-induce HbF by DNMT1 inhibition, this first-in-human clinical trial (NCT01685515) combined 2 small molecules-decitabine to deplete DNMT1 and tetrahydrouridine (THU) to inhibit cytidine deaminase (CDA), the enzyme that otherwise rapidly deaminates/inactivates decitabine, severely limiting its half-life, tissue distribution, and oral bioavailability. Oral decitabine doses, administered after oral THU 10 mg/kg, were escalated from a very low starting level (0.01, 0.02, 0.04, 0.08, or 0.16 mg/kg) to identify minimal doses active in depleting DNMT1 without cytotoxicity. Patients were SCD adults at risk of early death despite standard-of-care, randomized 3:2 to THU-decitabine versus placebo in 5 cohorts of 5 patients treated 2X/week for 8 weeks, with 4 weeks of follow-up. The primary endpoint was ≥ grade 3 non-hematologic toxicity. This endpoint was not triggered, and adverse events (AEs) were not significantly different in THU-decitabine-versus placebo-treated patients. At the decitabine 0.16 mg/kg dose, plasma concentrations peaked at approximately 50 nM (Cmax) and remained elevated for several hours. This dose decreased DNMT1 protein in peripheral blood mononuclear cells by >75% and repetitive element CpG methylation by approximately 10%, and increased HbF by 4%-9% (P < 0.001), doubling fetal hemoglobin-enriched red blood cells (F-cells) up to approximately 80% of total RBCs. Total hemoglobin increased by 1.2-1.9 g/dL (P = 0.01) as reticulocytes simultaneously decreased; that is, better quality and efficiency of HbF-enriched erythropoiesis elevated hemoglobin using fewer reticulocytes. Also indicating better RBC quality, biomarkers of hemolysis, thrombophilia, and inflammation (LDH, bilirubin, D-dimer, C-reactive protein [CRP]) improved. As expected with non-cytotoxic DNMT1-depletion, platelets increased and neutrophils concurrently decreased, but not to an extent requiring treatment holds. As an early phase study, limitations include small patient numbers at each dose level and narrow capacity to evaluate clinical benefits. CONCLUSION: Administration of oral THU-decitabine to patients with SCD was safe in this study and, by targeting DNMT1, upregulated HbF in RBCs. Further studies should investigate clinical benefits and potential harms not identified to date. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01685515.
Subject(s)
Anemia, Sickle Cell/drug therapy , Azacitidine/analogs & derivatives , Enzyme Inhibitors/administration & dosage , Epigenesis, Genetic/drug effects , Gene Expression Regulation/drug effects , Tetrahydrouridine/administration & dosage , Adult , Anemia, Sickle Cell/genetics , Azacitidine/administration & dosage , Azacitidine/pharmacology , Decitabine , Drug Therapy, Combination , Enzyme Inhibitors/pharmacology , Female , Fetal Hemoglobin/drug effects , Gene Silencing/drug effects , Humans , Male , Middle Aged , Tetrahydrouridine/pharmacology , Treatment Outcome , Young AdultABSTRACT
Sickle cell disease (SCD), an inherited blood disorder caused by a point mutation that renders hemoglobin susceptible to polymerization when deoxygenated, affects millions of people worldwide. Manifestations of SCD include chronic hemolytic anemia, inflammation, painful vaso-occlusive crises, multisystem organ damage, and reduced life expectancy. Part of SCD pathophysiology is the excessive formation of intracellular reactive oxygen species (ROS) in SCD red blood cells (RBCs), which accelerates their hemolysis. Normal RBC precursors eliminate their mitochondria during the terminal differentiation process. Strikingly, we observed an increased percentage of RBCs retaining mitochondria in SCD patient blood samples compared with healthy individuals. In addition, using an experimental SCD mouse model, we demonstrate that excessive levels of ROS in SCD are associated with this abnormal mitochondrial retention. Interestingly, the LSD1 inhibitor, RN-1, and the mitophagy-inducing agent mammalian target of rapamycin (mTOR) inhibitor, sirolimus, increased RBC lifespan and reduced ROS accumulation in parallel with reducing mitochondria-retaining RBCs in the SCD mouse model. Furthermore, gene expression analysis of SCD mice treated with RN-1 showed increased expression of mitophagy genes. Our findings suggest that reduction of mitochondria-retaining RBCs may provide a new therapeutic approach to preventing excessive ROS in SCD.
Subject(s)
Anemia, Sickle Cell/metabolism , Erythrocytes/drug effects , Erythrocytes/metabolism , Histone Demethylases/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , TOR Serine-Threonine Kinases/metabolism , Animals , Disease Models, Animal , Humans , Mice , Models, Biological , Rhodamines/pharmacology , Sirolimus/pharmacology , Spiro Compounds/pharmacology , Thiophenes/pharmacologySubject(s)
Anemia, Sickle Cell/blood , Anemia, Sickle Cell/drug therapy , Enzyme Inhibitors/pharmacology , Fetal Hemoglobin/metabolism , Histone Demethylases/antagonists & inhibitors , Rhodamines/pharmacology , Spiro Compounds/pharmacology , Thiophenes/pharmacology , Animals , Drug Evaluation, Preclinical , Enzyme Inhibitors/adverse effects , Papio , Rhodamines/adverse effects , Spiro Compounds/adverse effects , Thiophenes/adverse effectsABSTRACT
Increased fetal hemoglobin levels lessen the severity of symptoms and increase the lifespan of patients with sickle cell disease. Hydroxyurea, the only drug currently approved for the treatment of sickle cell disease, is not effective in a large proportion of patients and therefore new pharmacological agents that increase fetal hemoglobin levels have long been sought. Recent studies identifying LSD-1 as a repressor of γ-globin expression led to experiments demonstrating that the LSD-1 inhibitor RN-1 increased γ-globin expression in the sickle cell mouse model. Because the arrangement and developmental stage-specific expression pattern of the ß-like globin genes is highly conserved between man and baboon, the baboon model remains the best predictor of activity of fetal hemoglobin-inducing agents in man. In this report, we demonstrate that RN-1 increases γ-globin synthesis, fetal hemoglobin, and F cells to high levels in both anemic and non-anemic baboons with activity comparable to decitabine, the most potent fetal hemoglobin-inducing agent known. RN-1 not only restores high levels of fetal hemoglobin but causes the individual 5' Iγ- and 3' Vγ-globin chains to be synthesized in the ratio characteristic of fetal development. Increased fetal hemoglobin was associated with increased levels of acetylated Histone H3, H3K4Me2, H3K4Me3, and RNA polymerase II at the γ-globin gene, and diminished γ-globin promoter DNA methylation. RN-1 is likely to induce clinically relevant levels of fetal hemoglobin in patients with sickle cell disease, although careful titration of the dose may be required to minimize myelotoxicity.
Subject(s)
Enzyme Inhibitors/pharmacology , Fetal Hemoglobin/biosynthesis , Fetal Hemoglobin/genetics , Histone Demethylases/antagonists & inhibitors , Anemia/blood , Anemia/drug therapy , Anemia/etiology , Animals , Blood Cell Count , DNA Methylation/drug effects , Disease Models, Animal , Gene Expression Regulation/drug effects , Histones/metabolism , Papio , Reticulocytes/drug effects , Reticulocytes/metabolism , gamma-Globins/biosynthesis , gamma-Globins/geneticsABSTRACT
The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5 hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5 mC) and 5 hmC at a CCGG site within the 5' γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5 mC and 5 hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5 hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5 hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5 hmC and negatively correlated with 5 mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5 hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter.
Subject(s)
Cell Differentiation/drug effects , Cytosine/analogs & derivatives , DNA Methylation/drug effects , Erythroid Cells/cytology , Promoter Regions, Genetic/drug effects , gamma-Globins/metabolism , 5-Methylcytosine/metabolism , Animals , Animals, Newborn , Antineoplastic Agents/pharmacology , Ascorbic Acid/pharmacology , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Bone Marrow Cells , Cells, Cultured , Cytosine/metabolism , Cytosine/pharmacology , Decitabine , Dioxygenases/metabolism , Erythroid Cells/drug effects , Erythroid Cells/metabolism , Histone Demethylases/metabolism , Humans , Hydroxyurea/pharmacology , Liver/cytology , Liver/drug effects , Papio anubis , Tranylcypromine/pharmacologyABSTRACT
Increased levels of fetal hemoglobin are associated with decreased symptoms and increased lifespan in patients with sickle cell disease (SCD). Hydroxyurea, the only drug currently approved for SCD, is not effective in a large fraction of patients, and therefore, new agents are urgently needed. Recently it was found that lysine demethylase 1, an enzyme that removes monomethyl and dimethyl residues from the lysine 4 residue of histone H3, is a repressor of γ-globin gene expression. In this article, we have compared the ability of tranylcypromine (TCP) and a more potent TCP derivative, RN-1, to increase γ-globin expression in cultured baboon erythroid progenitor cells and in the SCD mouse model. The results indicate that the ability of RN-1 to induce F cells and γ-globin mRNA in SCD mice is similar to that of decitabine, the most powerful fetal hemoglobin-inducing drug known, and greater than that of either TCP or hydroxyurea. We conclude that RN-1 and other lysine demethylase 1 inhibitors may be promising new γ-globin-inducing agents for the treatment of SCD that warrant further studies in other preclinical models, such as nonhuman primates.
Subject(s)
Anemia, Sickle Cell/genetics , Fetal Hemoglobin/biosynthesis , Histone Demethylases/antagonists & inhibitors , Reticulocytes/drug effects , Tranylcypromine/pharmacology , gamma-Globins/biosynthesis , Anemia, Sickle Cell/drug therapy , Anemia, Sickle Cell/enzymology , Animals , Disease Models, Animal , Drug Evaluation, Preclinical , Enzyme Inhibitors/pharmacology , Erythroid Precursor Cells/drug effects , Erythroid Precursor Cells/metabolism , Erythropoiesis/drug effects , Fetal Hemoglobin/genetics , Gene Expression Regulation/drug effects , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Histones/metabolism , Humans , Hydroxyurea/pharmacology , Methylation , Mice , Mice, Transgenic , Papio , Protein Processing, Post-Translational/drug effects , Transgenes/drug effects , Tranylcypromine/analogs & derivatives , Tretinoin/pharmacology , U937 Cells , gamma-Globins/geneticsABSTRACT
This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction.
Subject(s)
Bone Marrow Cells/cytology , Erythroid Precursor Cells/metabolism , Fetus/cytology , Gene Expression Regulation, Developmental , Globins/genetics , Liver/cytology , Stromal Cells/cytology , Animals , Antigens, CD34/metabolism , Aorta/cytology , Aorta/embryology , Cell Line , DNA Methylation , Erythroid Precursor Cells/cytology , Gonads/cytology , Gonads/embryology , Mesonephros/cytology , Mesonephros/embryology , PapioABSTRACT
The transcription factor (TF) RUNX1 cooperates with lineage-specifying TFs (eg, PU.1/SPI1) to activate myeloid differentiation genes, such as macrophage and granulocyte macrophage colony-stimulating factor receptors (MCSFR and GMCSFR). Disruption of cooperative gene activation could contribute to aberrant repression of differentiation genes and leukemogenesis initiated by mutations and translocations of RUNX1. To investigate the mechanisms underlying cooperative gene activation, the effects of Runx1 deficiency were examined in an in vitro model of Pu.1-driven macrophage differentiation and in primary cells. Runx1 deficiency decreased Pu.1-mediated activation of Mcsfr and Gmcsfr, accompanied by decreased histone acetylation at the Mcsfr and Gmcsfr promoters, and increased endogenous corepressor (Eto2, Sin3A, and Hdac2) coimmunoprecipitation with Pu.1. In cotransfection experiments, corepressors were excluded from a multiprotein complex containing full-length RUNX1 and PU.1. However, corepressors interacted with PU.1 if wild-type RUNX1 was replaced with truncated variants associated with leukemia. Histone deacetylase (HDAC) enzyme activity is a major component of corepressor function. HDAC inhibition using suberoylanilide hydroxamic acid or MS-275 significantly increased MCSFR and GMCSFR expression in leukemia cell lines that express PU.1 and mutated or translocated RUNX1. RUNX1 deficiency is associated with persistent corepressor interaction with PU.1. Thus, inhibiting HDAC can partly compensate for the functional consequences of RUNX1 deficiency.
Subject(s)
Co-Repressor Proteins/metabolism , Core Binding Factor Alpha 2 Subunit/physiology , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Animals , Cell Line, Tumor , Co-Repressor Proteins/genetics , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Gene Expression Profiling , Gene Expression Regulation, Leukemic , Humans , Mice , Mice, Transgenic , Microarray Analysis , NIH 3T3 Cells , Protein Binding/genetics , Proto-Oncogene Proteins/genetics , Trans-Activators/genetics , TransfectionABSTRACT
OBJECTIVE: These studies were performed to test the hypothesis that DNMT1 is required for maintenance of DNA methylation and repression of the γ-globin gene in adult-stage erythroid cells. MATERIALS AND METHODS: DNMT1 levels were reduced by nucleofection of small interfering RNA targeting DNMT1 in chemical inducer of dimerization-dependent multipotential mouse bone marrow cells containing the human ß-globin gene locus in the context of a yeast artificial chromosome and in primary cultures of erythroid progenitor cells derived from CD34(+) baboon bone marrow cells. The effect of reduced DNMT1 levels on globin gene expression was measured by real-time polymerase chain reaction and the effect on globin chain synthesis in primary erythroid progenitor cell cultures was determined by biosynthetic radiolabeling of globin chains followed by high-performance liquid chromatography analysis. The effect on DNA methylation was determined by bisulfite sequence analysis. RESULTS: Reduced DNMT1 levels in cells treated with siDNMT1 were associated with increased expression of γ-globin messenger RNA, an increased γ/γ+ß chain ratio in cultured erythroid progenitors, and decreased DNA methylation of the γ-globin promoter. Similar effects were observed in cells treated with decitabine, a pharmacological inhibitor of DNA methyltransferase inhibitor. CONCLUSIONS: DNMT1 is required to maintain DNA methylation of the γ-globin gene promoter and repress γ-globin gene expression in adult-stage erythroid cells.
Subject(s)
Bone Marrow Cells/metabolism , DNA (Cytosine-5-)-Methyltransferases/physiology , Erythroid Precursor Cells/metabolism , gamma-Globins/genetics , Animals , Antigens, CD34/immunology , Blotting, Western , Cells, Cultured , Chromosomes, Artificial, Yeast , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methylation , Dimerization , Erythroid Precursor Cells/immunology , Humans , Mice , Papio , RNA, Small Interfering/genetics , TransfectionABSTRACT
OBJECTIVE: The mechanism responsible for increased fetal hemoglobin levels following decitabine treatment remains controversial. These experiments were performed to evaluate the role of transcriptional vs. translational mechanisms in the ability of decitabine to increase fetal hemoglobin levels in vivo. MATERIALS AND METHODS: Three normal, nonanemic baboons were treated with decitabine subcutaneously (0.5 mg/kg/d) for 10 days. The effect of decitabine on globin chain synthesis and globin messenger RNA levels was measured in pre- and posttreatment bone marrow aspirates by biosynthetic radiolabeling with [(3)H] leucine followed by separation of globin chains by high-performance liquid chromatography, and real-time polymerase chain reaction, respectively. The effect on DNA methylation of the É- and γ-globin gene promoters was determined by bisulfite sequence analysis. RESULTS: Decitabine treatment of normal, nonanemic baboons induced similar increases in the γ/γ+ß chain synthetic ratio and the γ/total ß-like globin RNA ratio and also increased expression of É-globin transcripts. Increased expression of É- and γ-globin was associated with decreased DNA methylation of the É- and γ-globin gene promoters. CONCLUSIONS: Decitabine increases fetal hemoglobin in vivo by transcriptional activation of the γ-globin gene.
Subject(s)
Azacitidine/analogs & derivatives , Fetal Hemoglobin/genetics , gamma-Globins/genetics , Animals , Azacitidine/administration & dosage , Azacitidine/pharmacology , Chromatography, High Pressure Liquid , DNA Methylation/drug effects , Decitabine , Fetal Hemoglobin/metabolism , Papio , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Transcription, Genetic/drug effects , beta-Globins/genetics , beta-Globins/metabolism , epsilon-Globins/genetics , epsilon-Globins/metabolism , gamma-Globins/metabolismABSTRACT
OBJECTIVE: To investigate the mechanism(s) responsible for increased gamma-globin expression in vivo in decitabine-treated baboons and in vitro in cultured erythroid progenitor cells (EPC) from adult baboon bone marrow (BM). MATERIALS AND METHODS: Fetal liver, adult BM erythroid cells pre- and post-decitabine, and cultured EPCs were analyzed for distribution of RNA polymerase II, histone acetylation, and histone H3 (lys4) trimethyl throughout the gamma-globin gene complex by chromatin immunoprecipitation. DNA methylation of the gamma-globin promoter was determined by bisulfite sequencing. Expression of the baboon Igamma- and Vgamma-globin chains was determined by high performance liquid chromatography (HPLC). Expression of BCL11A, a recently identified repressor of gamma-globin expression, was analyzed by Western blot. RESULTS: Increased gamma-globin expression in decitabine-treated baboons and cultured EPC correlated with increased levels of RNA polymerase II, histone acetylation, and histone H3 (lys4) trimethyl associated with the gamma-globin gene consistent with a transcriptional activation mechanism. Cultured EPC expressed the Igamma- and Vgamma-globin chains in a pattern characteristic of fetal development. The level of DNA methylation of the gamma-globin gene promoter in EPC cultures was similar to BM erythroid cells from normal adult baboons. Different BCL11A isoforms were observed in BM erythroid cells and cultured EPC. CONCLUSION: The mechanism responsible for increased gamma-globin expression in cultured EPC was unexpectedly not associated with increased DNA hypomethylation of the gamma-globin gene promoter compared to normal BM erythroid cells, in contrast to BM erythroid cells of decitabine-treated baboons. Rather, increased fetal hemoglobin in EPC cultures was associated with a fetal Igamma/Vgamma chain ratio and a difference in the size of the BCL11A protein compared to normal BM erythroid cells.
Subject(s)
Azacitidine/analogs & derivatives , Erythroid Precursor Cells/metabolism , Papio anubis/genetics , Transcription, Genetic/drug effects , gamma-Globins/genetics , Age Factors , Animals , Azacitidine/pharmacology , Carrier Proteins/physiology , Cells, Cultured/metabolism , Chromatin Immunoprecipitation , DNA Methylation/drug effects , Decitabine , Fetal Blood/metabolism , Fetal Hemoglobin/biosynthesis , Fetal Hemoglobin/genetics , Gestational Age , Nuclear Proteins/physiology , Phlebotomy , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/genetics , Protein Isoforms/physiology , Protein Processing, Post-Translational , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , SUMO-1 Protein/metabolism , gamma-Globins/biosynthesisABSTRACT
The t(8;21) chromosome abnormality in acute myeloid leukemia targets the AML1 and ETO genes to produce the leukemia fusion protein AML1-ETO. Another member of the ETO family, ETO-2/MTG16, is highly expressed in murine and human hematopoietic cells, bears >75% homology to ETO, and like ETO, contains a conserved MYND domain that interacts with the nuclear receptor corepressor (N-CoR). AML1-ETO prevents granulocyte but not macrophage differentiation of murine 32Dcl3 granulocyte/macrophage progenitors. One possible mechanism is recruitment of N-CoR to aberrantly repress AML1 target genes. We wished to examine another mechanism by which AML1-ETO might impair granulocyte differentiation. We demonstrate that AML1-ETO decreases interactions between ETO-2 and N-CoR. Furthermore, overexpression of ETO-2 relieves AML1-ETO-induced granulocyte differentiation arrest. This suggests that decreased interactions between ETO-2 and N-CoR may contribute to granulocyte differentiation impairment. The MYND domain coimmunoprecipitates with N-CoR and inhibits interactions between ETO-2 and N-CoR, presumably by occupying the ETO-2 binding site on N-CoR. This inhibition of ETO-2 interactions with N-CoR is specific because the MYND domain does not inhibit retinoic acid receptor interactions with N-CoR. To examine the effect of decreasing interactions between ETO-2 and N-CoR in hematopoietic cells, without effects of AML1-ETO such as direct repression of AML1 target genes, the MYND domain was expressed in 32Dcl3 and human CD34+ cells. The MYND domain prevented granulocyte but not macrophage differentiation of both 32Dcl3 and human CD34+ cells, recapitulating this effect of AML1-ETO. In conclusion, decreasing interactions between ETO-2 and N-CoR, an effect of AML1-ETO, inhibits granulocyte differentiation.