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
Human hemoglobin switching describes the highly regulated, sequential expression of the 5 ß-like globin genes (HBE, HBG2, HBG1, HBD and HBB) of the human ß-globin gene complex. The sequential activation of these ß or ß-like globin genes during human development from early embryonic through late fetal ('adult') stages, and during erythroid maturation, occurs in an order corresponding to their 5' to 3' location on chromosome 11. The ß-hemoglobinopathies are the most common inherited diseases in humanity, and are diseases of mutated HBB or its altered regulation. Since the other ß-like globin genes can potentially substitute for defective HBB, much translational research is directed toward understanding and manipulating sequential activation at the human ß-globin gene complex to treat ß-hemoglobinopathies. Non-human primates provide a vital contribution to such efforts because of their recapitulation of the developmental/maturational switch in hemoglobin production as observed in humans (mice do not model this switch). Valuable insights into druggable epigenetic forces that mediate the switch have been thereby gained. We review important lessons learned in non-human primates, complemented by other studies, and suggest rational next steps.
Subject(s)
Epigenesis, Genetic , Hemoglobinopathies , Animals , Hemoglobinopathies/genetics , Humans , Mice , Primates/genetics , beta-Globins/geneticsABSTRACT
Sickle cell disease (SCD) and ß-thalassemia are caused by structural abnormality or inadequate production of adult hemoglobin (HbA, α2ß2), respectively. Individuals with either disorder are asymptomatic before birth because fetal hemoglobin (HbF, α2γ2) is unaffected. Thus, reversal of the switch from HbF to HbA could reduce or even prevent symptoms these disorders. In this study, we show that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is one factor that could accomplish this goal. IGF2BP1 is a fetal factor that undergoes a transcriptional switch consistent with the transition from HbF to HbA. Lentivirus delivery of IGF2BP1 to CD34+ cells of healthy adult donors reversed hemoglobin production toward the fetal type in culture-differentiated erythroid cells. Analogous studies using patient-derived CD34+ cells revealed that IGF2BP1-dependent HbF induction could ameliorate the chain imbalance in ß-thalassemia or potently suppress expression of sickle ß-globin in SCD. In all cases, fetal γ-globin mRNA increased and adult ß-globin decreased due, in part, to formation of contacts between the locus control region (LCR) and γ-globin genes. We conclude that expression of IGF2BP1 in adult erythroid cells has the potential to maximize HbF expression in patients with severe ß-hemoglobin disorders by reversing the developmental γ- to ß-globin switch.
ABSTRACT
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
Sickle cell disease (SCD) is caused by a mutation of the ß-globin gene (Ingram VM. Nature 180: 326-328, 1957), which triggers the polymerization of deoxygenated sickle hemoglobin (HbS). Approximately 100,000 SCD patients in the United States and millions worldwide (Piel FB, et al. PLoS Med 10: e1001484, 2013) suffer from chronic hemolytic anemia, painful crises, multisystem organ damage, and reduced life expectancy (Rees DC, et al. Lancet 376: 2018-2031, 2010; Serjeant GR. Cold Spring Harb Perspect Med 3: a011783, 2013). Hematopoietic stem cell transplantation can be curative, but the majority of patients do not have a suitable donor (Talano JA, Cairo MS. Eur J Haematol 94: 391-399, 2015). Advanced gene-editing technologies also offer the possibility of a cure (Goodman MA, Malik P. Ther Adv Hematol 7: 302-315, 2016; Lettre G, Bauer DE. Lancet 387: 2554-2564, 2016), but the likelihood that these strategies can be mobilized to treat the large numbers of patients residing in developing countries is remote. A pharmacological treatment to increase fetal hemoglobin (HbF) as a therapy for SCD has been a long-sought goal, because increased levels of HbF (α2γ2) inhibit the polymerization of HbS (Poillin WN, et al. Proc Natl Acad Sci USA 90: 5039-5043, 1993; Sunshine HR, et al. J Mol Biol 133: 435-467, 1979) and are associated with reduced symptoms and increased lifespan of SCD patients (Platt OS, et al. N Engl J Med 330: 1639-1644, 1994; Platt OS, et al. N Engl J Med 325: 11-16, 1991). Only two drugs, hydroxyurea and l-glutamine, are approved by the US Food and Drug Administration for treatment of SCD. Hydroxyurea is ineffective at HbF induction in ~50% of patients (Charache S, et al. N Engl J Med 332: 1317-1322, 1995). While polymerization of HbS has been traditionally considered the driving force in the hemolysis of SCD, the excessive reactive oxygen species generated from red blood cells, with further amplification by intravascular hemolysis, also are a major contributor to SCD pathology. This review highlights a new class of drugs, lysine-specific demethylase (LSD1) inhibitors, that induce HbF and reduce reactive oxygen species.
Subject(s)
Anemia, Sickle Cell/drug therapy , Enzyme Inhibitors/pharmacology , Erythrocytes/drug effects , Histone Demethylases/antagonists & inhibitors , Anemia, Sickle Cell/blood , Anemia, Sickle Cell/enzymology , Animals , Biomarkers/blood , Disease Models, Animal , Drug Design , Erythrocytes/enzymology , Fetal Hemoglobin/metabolism , Histone Demethylases/metabolism , Humans , Mice , Molecular Targeted Therapy , Papio , Reactive Oxygen Species/blood , Risk AssessmentABSTRACT
Fetal hemoglobin (HbF) inhibits the root cause of sickle pathophysiology, sickle hemoglobin polymerization. Individuals who naturally express high levels of HbF beyond infancy thus receive some protection from sickle complications. To mimic this natural genetic experiment using drugs, one guiding observation was that HbF is increased during recovery of bone marrow from extreme stress. This led to evaluation and approval of the cytotoxic (cell killing) drug hydroxyurea to treat sickle cell disease. Cytotoxic approaches are limited in potency and sustainability, however, since they require hematopoietic reserves sufficient to repeatedly mount recoveries from stress that destroys their counterparts, and such reserves are finite. HbF induction even by stress ultimately involves chromatin remodeling of the gene for HbF (HBG), therefore, a logical alternative approach is to directly inhibit epigenetic enzymes that repress HBG-implicated enzymes include DNA methyltransferase 1, histone deacetylases, lysine demethylase 1, protein arginine methyltransferase 5, euchromatic histone lysine methyltransferase 2 and chromodomain helicase DNA-binding protein 4. Clinical proof-of-principle that this alternative, noncytotoxic approach can generate substantial HbF and total hemoglobin increases has already been generated. Thus, with continued careful attention to fundamental biological and pharmacologic considerations (reviewed herein), there is potential that rational, molecular-targeted, safe and highly potent disease-modifying therapy can be realized for patients with sickle cell disease, with the accessibility and cost-effective properties needed for world-wide effect.
Subject(s)
Antineoplastic Agents/therapeutic use , Epigenomics/methods , Fetal Hemoglobin/drug effects , Hydroxyurea/therapeutic use , Transcription Factors/metabolism , Antineoplastic Agents/pharmacology , Fetal Hemoglobin/metabolism , Humans , Hydroxyurea/pharmacologyABSTRACT
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
Inhibition of lysine-specific demethylase 1 (LSD1) has been shown to induce fetal hemoglobin (HbF) levels in cultured human erythroid cells in vitro. Here we report the in vivo effects of LSD1 inactivation by a selective and more potent inhibitor, RN-1, in a sickle cell disease (SCD) mouse model. Compared with untreated animals, RN-1 administration leads to induced HbF synthesis and to increased frequencies of HbF-positive cells and mature erythrocytes, as well as fewer reticulocytes and sickle cells, in the peripheral blood of treated SCD mice. In keeping with these observations, histologic analyses of the liver and spleen of treated SCD mice verified that they do not exhibit the necrotic lesions that are usually associated with SCD. These data indicate that RN-1 can effectively induce HbF levels in red blood cells and reduce disease pathology in SCD mice, and may therefore offer new therapeutic possibilities for treating SCD.
Subject(s)
Anemia, Sickle Cell/prevention & control , Fetal Hemoglobin/biosynthesis , Histone Demethylases/antagonists & inhibitors , Rhodamines/pharmacology , Spiro Compounds/pharmacology , Splenomegaly/prevention & control , Thiophenes/pharmacology , Anemia, Sickle Cell/blood , Anemia, Sickle Cell/pathology , Animals , Blotting, Western , Cells, Cultured , Disease Models, Animal , Fetal Hemoglobin/drug effects , Flow Cytometry , Humans , Immunoenzyme Techniques , Mice , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Splenomegaly/blood , Splenomegaly/pathology , beta-Globins/genetics , beta-Globins/metabolismABSTRACT
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
Epigenetic therapies, including DNA methyltransferase and histone deacetylase (HDAC) inhibitors, are increasingly being considered to treat hematological malignancies, but their effects on normal hematopoietic stem cells (HSCs) remain largely unexplored. We compared the effects of several HDAC inhibitors, including valproic acid (VPA) and trichostatin A (TSA), alone or in combination with 5-aza-2'-deoxycytidine (5azaD) on the expansion of HSCs. VPA induced the highest expansion of CD34+CD90+ cells and progenitor cells compared with other HDAC inhibitors or the sequential addition of 5azaD/TSA in culture. Xenotransplantation studies demonstrated that VPA prevents HSC loss, whereas 5azaD/TSA treatment leads to a net expansion of HSCs that retain serial transplantation ability. 5azaD/TSA-mediated HSC expansion was associated with increased histone acetylation and transient DNA demethylation, which corresponded with higher gene transcript levels. However, some genes with increased transcript levels lacked changes in methylation. Importantly, a global microarray analysis revealed a set of differentially expressed genes in 5azaD/TSA- and VPA-expanded CD34+ cells that might be involved in the expansion and maintenance of transplantable HSCs, respectively. In summary, our data indicate that treatment of HSCs with different chromatin-modifying agents results in either the expansion or maintenance of HSCs, an observation of potential therapeutic importance.
Subject(s)
Epigenesis, Genetic , Fetal Blood/drug effects , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/drug effects , Histone Deacetylase Inhibitors/pharmacology , Histones/genetics , Animals , Antigens, CD34/genetics , Antigens, CD34/immunology , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Cell Proliferation/drug effects , Cells, Cultured , DNA Methylation , Decitabine , Fetal Blood/cytology , Fetal Blood/immunology , Gene Expression , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/immunology , Histones/metabolism , Humans , Hydroxamic Acids/pharmacology , Mice , Mice, Inbred NOD , Mice, SCID , Thy-1 Antigens/genetics , Thy-1 Antigens/immunology , Transplantation, Heterologous , Valproic Acid/pharmacologyABSTRACT
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 deoxycytidine analog decitabine (DAC) can deplete DNA methyl-transferase 1 (DNMT1) and thereby modify cellular epigenetics, gene expression, and differentiation. However, a barrier to efficacious and accessible DNMT1-targeted therapy is cytidine deaminase, an enzyme highly expressed in the intestine and liver that rapidly metabolizes DAC into inactive uridine counterparts, severely limiting exposure time and oral bioavailability. In the present study, the effects of tetrahydrouridine (THU), a competitive inhibitor of cytidine deaminase, on the pharmacokinetics and pharmacodynamics of oral DAC were evaluated in mice and nonhuman primates. Oral administration of THU before oral DAC extended DAC absorption time and widened the concentration-time profile, increasing the exposure time for S-phase-specific depletion of DNMT1 without the high peak DAC levels that can cause DNA damage and cytotoxicity. THU also decreased interindividual variability in pharmacokinetics seen with DAC alone. One potential clinical application of DNMT1-targeted therapy is to increase fetal hemoglobin and treat hemoglobinopathy. Oral THU-DAC at a dose that would produce peak DAC concentrations of less than 0.2µM administered 2×/wk for 8 weeks to nonhuman primates was not myelotoxic, hypomethylated DNA in the γ-globin gene promoter, and produced large cumulative increases in fetal hemoglobin. Combining oral THU with oral DAC changes DAC pharmacology in a manner that may facilitate accessible noncytotoxic DNMT1-targeted therapy.
Subject(s)
Azacitidine/analogs & derivatives , Tetrahydrouridine/pharmacology , Administration, Oral , Animals , Antimetabolites/pharmacology , Antimetabolites, Antineoplastic/adverse effects , Antimetabolites, Antineoplastic/metabolism , Antimetabolites, Antineoplastic/pharmacokinetics , Area Under Curve , Azacitidine/administration & dosage , Azacitidine/adverse effects , Azacitidine/metabolism , Azacitidine/pharmacokinetics , Biological Availability , DNA Damage/drug effects , DNA Methylation/drug effects , Decitabine , Drug Interactions , Female , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Inactivation, Metabolic , Injections, Intravenous , Injections, Subcutaneous , Mice , Papio anubisABSTRACT
In sickle cell disease (SCD), vigorous reticulocytosis is required to partially compensate for chronic hemolytic anaemia. Consequently, early renal damage, insufficient to cause azotemia but sufficient to cause erythropoietin deficiency and chronic relative reticulocytopenia (chRR), could have severe clinical consequences. chRR was defined as reticulocytes <250×10(9) /l despite haemoglobin <9 g/dl on ≥ two occasions ≥4 weeks apart. The influence of multiple variables including chRR on time from first clinic visit to death was evaluated in 306 SCD patients. In univariate analyses, fetal haemoglobin, indices of renal damage (serum creatinine, proteinuria), chRR and age, were associated with rate of death. In multivariate analysis, only age and chRR (Hazard ratio 3·6, 95% CI 2·049-6·327, P<0·0001) were significant, underlining that chRR could be an early and important clinical consequence of renal damage. Even in chRR patients with normal serum creatinine levels, low haemoglobin and low reticulocyte counts were associated with low erythropoietin levels. In the general population, evaluation of erythropoietin levels is prompted by the combination of anaemia and abnormal serum creatinine. In SCD patients, this standard approach can miss a substantial risk factor for early death. chRR could be a practical and important criterion for diagnosis of erythropoietin deficiency in SCD.