The LSD1 Inhibitor RN-1 Recapitulates the Fetal Pattern of Hemoglobin Synthesis in Anemic and Normal, Non-Anemic Baboons

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 642-642
Author(s):  
Angela Rivers ◽  
Kestis Vaitkus ◽  
Vinzon Ibanez ◽  
Maria Armila Ruiz ◽  
Ramaswamy Jagadeeswaran ◽  
...  
Keyword(s):  
Post Treatment ◽  
Globin Genes ◽  
Globin Chain ◽  
Cell Disease ◽  
Globin Mrna ◽  
F Cells ◽  
Globin Promoter ◽  
Chain Synthesis ◽  
Globin Chain Synthesis ◽  
Dose Dependent

Abstract Increased fetal hemoglobin (HbF) levels lessen the severity of symptoms and increase the lifespan of patients with sickle cell disease (SCD). Hydroxyurea (HU), the only drug approved for the treatment of sickle cell disease, increases HbF levels, reduces pain crises, and increases the lifespan of patients. Because HU is not effective in a large proportion of patients, new pharmacological agents that increase HbF 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 SCD mice. As the arrangement and developmental stage-specific expression pattern of the β-like globin genes is highly conserved between man and baboon, the baboon remains the best predictor of the activity of HbF-inducing agents in man. Therefore, experiments were performed to test the effect of RN-1 on HbF in anemic baboons. Anemia (Hct=20) was induced by repeated phlebotomies for 14 days prior to administration of RN-1. Five anemic baboons were treated with varying doses of RN-1(0.125-2.5 mg/kg/d; 5d; sc). Dose-dependent increases in HbF, F cells, F retics, and γ-globin chain synthesis in reticulocytes were associated with decreased neutrophils and increased monocytes. Globin chain synthesis analysis following RN-1 treatment showed that the 5' Iγ and 3' Vγ-globin genes were expressed in the ratio characteristic of fetal development (5'Iγ/3'Vγ>2) rather than that induced in adults by erythropoietic stress (5'Iγ/3'Vγ<0.5) or decitabine treatment where changes in the Iγ/Vγ ratio are observed but the fetal ratio is not attained. RT-PCR analysis of pre-and post-treatment BM showed that RN-1 increased γ-globin mRNA nearly 5 fold (p<0.05) with no effect on ε-globin mRNA. ChIP analysis of pre- and post-treatment BM erythroid cells showed increased levels of pol II, H3K9ac, H3K4me2 and H3K4me3 associated with the γ-globin but not with the ε- or β-globin promoter and IVS II regions consistent with increased γ-globin transcription. Levels of H3K9me2 were increased at the β-globin promoter and IVSII regions. Bisulfite sequence analysis showed a small decrease in level of DNA methylation of the γ-globin promoter in post-treatment (0.68+0.07% total cytosine) compared to pre-treatment (0.79+0.05%; p<0.05). To investigate whether induction of γ-globin expression was dependent on erythropoietic stress, four normal, non-anemic baboons were treated with varying doses and schedules of RN-1 (0.2-0.5mg/kg/d; 5d/wk; 1-10wks; sc). The effect of RN-1 was measured by analysis of F cells and globin chain synthesis in peripheral blood. The results (Table) show that RN-1 increases γ-globin expression and F cells in normal, non-anemic baboons in a dose-dependent manner. Analysis of globin chain synthesis showed predominant synthesis of the Iγ-globin chain with an Iγ/Vγ ratio exceeding the fetal ratio in all individuals. Effect of RN-1 in Normal, Non-Anemic Baboons Table.AnimalRN-1 Dose (mg/kg/d)ScheduleGlobin chain synthesis (γ/γ+β)Iγ/Vγ chain synthesisF cells (%)ANC (/μl)Plt(X103/μl)Mono(%)PrePostPrePostNadirNadirPeak85490.55d0.030.714.63NDND191016921.685490.255d0.010.264.47NDND191021914.586980.255d/wk/2 wks0.010.2020.12.09.21210687.986960.2-0.255d/wk/10 wksND0.106.633.526.81440646.1 Neutropenia following RN-1 treatment of anemic baboons (mean ANC nadir=746±156) was reduced in non-anemic animals (2 of 4 baboons >1500; mean ANC nadir=1617±350; p<0.02). Increased monocytes and decreased platelet counts were observed. Differences between anemic and non-anemic baboons may reflect perturbation of hematopoietic differentiation by phlebotomy. We conclude that RN-1 is a powerful in vivo inducer of HbF in both anemic and non-anemic baboons that preferentially increases synthesis of the 5' Iγ-globin gene and recapitulates the fetal pattern of hemoglobin synthesis. Our data predicts that RN-1 treatment will induce clinically relevant levels of HbF in SCD patients, although careful titration of dose may be required to minimize effects on hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Combinatorial Effect of Pharmacological Inhibitors of Lsd-1 and DNMT on γ-Globin Synthesis in Cultured Baboon Bone Marrow Erythroid Progenitors

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 249-249
Author(s):  
Donald Lavelle ◽  
Kestis Vaitkus ◽  
Maria Armila Ruiz ◽  
Angela Rivers ◽  
James D. Engel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Sequence Analysis ◽  
Dna Methyltransferase ◽  
Erythroid Differentiation ◽  
Globin Chain ◽  
Erythroid Progenitors ◽  
Globin Promoter ◽  
Chain Synthesis ◽  
Globin Chain Synthesis ◽  
Dose Dependent

Abstract Abstract 249 Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). New therapies to increase HbF levels in patients are urgently needed. Previous studies from our laboratory have shown that decitabine, an inhibitor of DNA methyltransferase (DNMT), increases HbF to therapeutic levels in non-human experimental primates and SCD patients, thus demonstrating that drugs targeting repressive epigenetic modifications can be effective HbF-inducing agents. Both DNMT1 and Lysine Specific Demethylase-1, (LSD-1), a mono- and dimethyl- histone H3 K4 demethylating enzyme, are components of the DRED multiprotein complex, a repressor of γ-globin expression (Cui et al Mol Cell Biol 31:3298–3311, 2011). Experiments were performed to test the effect of the pharmacological LSD-1 inhibitor, tranylcypromine (TC), alone and in combination with the DNA methyltransferase inhibitor decitabine (DAC), on γ-globin expression. Baboon bone marrow (BM) CD34+ erythroid progenitors were isolated from BM aspirates of normal baboons and grown in a co-culture system with the AFT024 murine fetal liver cell line in Iscove's media containing 30% fetal bovine serum, 200ng/ml SCF, 2U/ml epo, and 1μM dexamethasone. TC (0.5 to 5.0μM) was added to cultures on d7 and d10, while decitabine (0.1μM) was added on d7. On d14 erythroid cells were purified by immunomagnetic column chromatograpy using a baboon-specific anti-red blood cell antibody (BD Bioscience). Effects on globin expression were measured by biosynthetic radiolabelling of nascent globin chain synthesis in the presence of [3H] leucine followed by HPLC separation of individual globin chains. DNA was isolated and effect of the drugs on DNA methylation of the γ-globin promoter determined by bisulfite sequence analysis. Effects on erythroid differentiation were investigated by examination of Wright's stained cytospin preparations. In three independent cultures TC induced dose-dependent increases in γ-globin chain synthesis (γ/γ+β). In addition TC and DAC in combination increased γ-globin chain synthesis in an additive manner (see Table). CD34+ cell source TC (μM) DAC (μM) γ/γ+β PA 7680 0 0 0.06 0 0.1 0.32 5.0 0 0.30 5.0 0.1 0.62 PA 7482 0 0 0.09 0 0.1 0.35 2.5 0 0.16 5.0 0 0.30 2.5 0.1 0.54 5.0 0.1 0.68 Bisulfite sequence analysis showed that increased γ-globin chain synthesis in TC-treated cultures was not associated with increased DNA hypomethylaton of the γ-globin promoter. Examination of Wright's stained cytospin preparations of d14 cultures showed increased numbers of basophilic and polychromatic erythroblasts in cultures exposed to TC compared to untreated controls suggesting that TC reduced the rate of erythroid differentiation. In contrast, numbers of basophilic and polychromatic cells were reduced and orthochromatic cells were increased in cultures treated with TC and DAC compared to TC alone. We conclude that TC increased γ-globin chain synthesis in a dose-dependent manner in baboon erythroid progenitor cell cultures while TC and DAC, in combination, produced additive effects on γ-globin chain synthesis with minimal effects on erythroid differentiation. These results suggest that combinatorial use of epigenetic modifiers may be superior to single agents and support further studies to determine whether TC and other LSD-1 pharmacological inhibitors can effectively be used in combination with DNMT inhibition to increase HbF levels in vivo. Disclosures: No relevant conflicts of interest to declare.

Haemoglobinopathies

2013 ◽  
pp. 1466-1470
Author(s):  
David Rees
Keyword(s):  
Bone Marrow ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Lupus Erythematosus ◽  
Single Gene ◽  
Globin Genes ◽  
Globin Chain ◽  
Cell Disease ◽  
Chain Synthesis ◽  
Marrow Expansion

Inherited abnormalities of the globin genes are the commonest single-gene disorders in the world and fall into two main groups: thalassaemias and sickle cell disease. Thalassaemias are due to quantitative defects in globin chain synthesis which cause variable anaemia and ineffective erythropoiesis. Thalassaemia was initially thought to be a disease of the bones due to uncontrolled bone marrow expansion causing bony distortion, although this is now unusual with appropriate blood transfusions. Osteopenia, often severe, is a feature of most patients with thalassaemia major and intermedia, caused by bone marrow expansion, iron overload, endocrinopathy, and iron chelation. Treatment with bisphosphonates is generally recommended. Other rheumatological manifestations include arthropathy associated with the use of the iron chelator deferiprone. Sickle cell disease involves a group of conditions caused by polymerization of the abnormal -globin chain, resulting in abnormal erythrocytes which cause vaso-occlusion, vasculopathy, and ischaemic tissue damage. The characteristic symptom is acute bone pain caused by vaso-occlusion; typical episodes require treatment with opiate analgesia and resolve spontaneously by 5 days with no lasting bone damage. The frequency of acute episodes varies widely between patients. The incidence of osteomyelitis is increased, particularly with salmonella, although it is much rarer than acute vaso-occlusion. Avascular necrosis can affect the hips, and less commonly the shoulders and knees. Coincidental rheumatological disease sometimes complicates the condition, particularly systemic lupus erythematosus (SLE) which is more prevalent in populations at increased risk of sickle cell disease.

Studies of Globin Chain Synthesis and Globin mRNA Content in a Patient Homozygous for Hemoglobin Lepore

Hemoglobin ◽  
1978 ◽  
Vol 2 (2) ◽  
pp. 117-128 ◽  
Author(s):  
B. G. Forget ◽  
C. Cavallesco ◽  
E. J. Benz ◽  
P. D. McClure ◽  
D. G. Hillman ◽  
...  
Keyword(s):  
Globin Chain ◽  
Globin Mrna ◽  
Mrna Content ◽  
Chain Synthesis ◽  
Globin Chain Synthesis

scholarly journals Butyrate increases the efficiency of translation of γ-globin mRNA

Blood ◽  
2005 ◽  
Vol 105 (4) ◽  
pp. 1807-1809 ◽  
Author(s):  
Rona S. Weinberg ◽  
Xinjun Ji ◽  
Millicent Sutton ◽  
Susan Perrine ◽  
Yelena Galperin ◽  
...  
Keyword(s):  
Fetal Hemoglobin ◽  
Peak Level ◽  
Mrna Levels ◽  
Globin Genes ◽  
Globin Mrna ◽  
Multiple Treatment ◽  
Rapid Induction ◽  
Polysomal Fraction ◽  
Chain Synthesis ◽  
Globin Chain Synthesis

AbstractFetal hemoglobin (Hb F) levels increase in most patients with sickle cell disease following intermittent butyrate therapy. Although the full effects of butyrate on Hb F levels usually require multiple treatment cycles, in some patients a peak level is achieved after a few days of butyrate therapy. Our investigation of the mechanism(s) responsible for this rapid induction of Hb F by butyrate showed that reticulocyte γ-globin chain synthesis markedly increased within 24 hours of butyrate exposure, without concomitant changes in reticulocyte γ-globin mRNA levels. This suggests that butyrate might induce Hb F by increasing the efficiency of translation of γ-globin mRNA. This hypothesis was confirmed by ribosome loading studies that demonstrated enrichment of the polysomal fraction of reticulocytes with γ-globin mRNA following butyrate exposure. Thus, the induction of Hb F by butyrate may be mediated by translational effects in addition to its well-known effects on transcription of the γ-globin genes. (Blood. 2005;105:1807-1809)

scholarly journals Reticulocytosis, hypochromia, and microcytosis: an unusual presentation of the preleukemic syndrome

Blood ◽  
1982 ◽  
Vol 59 (2) ◽  
pp. 293-299 ◽  
Author(s):  
M Tulliez ◽  
U Testa ◽  
H Rochant ◽  
A Henri ◽  
W Vainchenker ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Cell Phenotype ◽  
Globin Chain ◽  
F Cells ◽  
Chain Synthesis ◽  
Antigen Activity ◽  
Immunofluorescence Labeling ◽  
Globin Chain Synthesis

Abstract Two patients exhibiting a highly unusual preleukemic syndrome with marked reticulocytosis, hypochromia, and microcytosis are reported. This red cell phenotype has been investigated by means of HbF, HbA2, and i-antigen activity dosages, immunofluorescence labeling of F cells, reticulocyte survival, globin chain synthesis, and electron microscopy study. The marked reticulocytosis is explained by a delayed disappearance of the reticulum. Serum iron is normal, and a thalassemic syndrome is excluded because of a balanced alpha/non-alpha globin chain synthesis. Electron microscopy studies are consistent with a defect in iron uptake by erythroid cells. All the hematologic data and investigations are similar to those observed for the Belgrade laboratory rat. It is hypothesized that the low expression of HbF and i- Ag associated with microcytosis are related to a prolongation of erythroid maturation as reflected by abnormal reticulocyte survival.

βMinor-globin messenger RNA accumulation in reticulocytes governs improved erythropoiesis in β thalassemic mice after erythropoietin complementary DNA electrotransfer in muscles

Blood ◽  
2001 ◽  
Vol 97 (8) ◽  
pp. 2213-2220 ◽  
Author(s):  
Selda Samakoglu ◽  
Elena Fattori ◽  
Stefania Lamartina ◽  
Carlo Toniatti ◽  
Daniel Stockholm ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Quantitative Polymerase Chain Reaction ◽  
Mrna Levels ◽  
Globin Chain ◽  
Dna Encoding ◽  
Globin Mrna ◽  
Naked Dna ◽  
Chain Synthesis ◽  
Globin Chain Synthesis ◽  
Fetal Erythropoiesis

Abstract Mechanisms governing the induction of effective erythropoiesis in response to erythropoietin (Epo) oversecretion have been investigated in β thalassemic C57Bl/6Hbbth mice. Naked DNA encoding an expression vector for mouse Epo was introduced into skeletal muscles by electrotransfer. A transient increase of serum Epo concentrations with a proportional augmentation of hematocrit values was observed. Various parameters relevant to β thalassemia were surveyed in blood samples taken before treatment, at the peak of Epo secretion, and when the phenotype reverted to anemia. We measured globin messenger RNA (mRNA) levels in reticulocytes by real-time quantitative polymerase chain reaction, globin chain synthesis levels, and several indicators of erythrocyte membrane quality, including bound α chains, bound immunoglobulins, main protein components, and iron compartmentalization. Data indicated that high serum Epo levels primarily affect βminor-globin mRNA accumulation in reticulocytes. Other changes subsequent to intense Epo stimulation, like increased βminor/α-globin chain synthesis ratio, reduced levels of α chains and immunoglobulins bound to membranes, improved spectrin/band 3 ratio, increased red blood cell survival, and improved erythropoiesis appeared as consequences of increased βminor-globin mRNA levels. This conclusion is consistent with models postulating that intense Epo stimulation induces the expansion and differentiation of erythroid progenitors committed to fetal erythropoiesis. Although phenotypic correction was partial in mice, and comparable achievements will probably be more difficult to obtain in humans, naked DNA electrotransfer may provide a safe and low-cost method for reassessing the potentials of Epo as an inducer of fetal erythropoiesis reactivation in patients with β thalassemia.

scholarly journals Absence of functional messenger RNA activity for beta globin chain synthesis in beta 0-thalassemia

Blood ◽  
1975 ◽  
Vol 45 (1) ◽  
pp. 1-10
Author(s):  
EJ Jr Benz ◽  
PS Swerdlow ◽  
BG Forget
Keyword(s):  
Messenger Rna ◽  
Globin Chain ◽  
Globin Mrna ◽  
Free System ◽  
Gamma Globin ◽  
Globin Chains ◽  
Mouse Ascites ◽  
Chain Synthesis ◽  
A Chain ◽  
Globin Chain Synthesis

Functional human globin messenger RNA was isolated from reticulocytes of two patients with homozygous beta 0-thalassemia, three patients with sickle cell beta 0-thalassemia, andone patient doubly heterozygous for beta 0-thalassemia and hemoglobin Lepore. When incubated in the Krebs type II mouse ascites tumor-cell-free system, messenger RNA from these patients actively directed the synthesis of human beta s and/or alpha- and gamma-globin chains but failed to stimulate the synthesis of any beta A-chains, even though nonthalassemic human globin mRNA preparations consistently stimulated two to four times as much beta A- or beta S-globin chain synthesis as alpha-chain synthesis when incubated in the same system under the same conditions. These results strongly suggest that functional beta A-chain-specific globin mRNA is absent in beta 0-thalassemia.

scholarly journals Absence of functional messenger RNA activity for beta globin chain synthesis in beta 0-thalassemia

Blood ◽  
1975 ◽  
Vol 45 (1) ◽  
pp. 1-10 ◽  
Author(s):  
EJ Jr Benz ◽  
PS Swerdlow ◽  
BG Forget
Keyword(s):  
Messenger Rna ◽  
Globin Chain ◽  
Globin Mrna ◽  
Free System ◽  
Gamma Globin ◽  
Globin Chains ◽  
Mouse Ascites ◽  
Chain Synthesis ◽  
A Chain ◽  
Globin Chain Synthesis

Abstract Functional human globin messenger RNA was isolated from reticulocytes of two patients with homozygous beta 0-thalassemia, three patients with sickle cell beta 0-thalassemia, andone patient doubly heterozygous for beta 0-thalassemia and hemoglobin Lepore. When incubated in the Krebs type II mouse ascites tumor-cell-free system, messenger RNA from these patients actively directed the synthesis of human beta s and/or alpha- and gamma-globin chains but failed to stimulate the synthesis of any beta A-chains, even though nonthalassemic human globin mRNA preparations consistently stimulated two to four times as much beta A- or beta S-globin chain synthesis as alpha-chain synthesis when incubated in the same system under the same conditions. These results strongly suggest that functional beta A-chain-specific globin mRNA is absent in beta 0-thalassemia.

γ-Globin Gene (HBG) Promoter Methylation and Gene Expression Is Affected by the Micro-Environment In Adult but Not Fetal Erythroid Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4271-4271
Author(s):  
Donald Lavelle ◽  
Yogenthiran Saunthararajah ◽  
Nadim Mahmud ◽  
Vinzon Ibanez ◽  
Maria Armila Ruiz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Globin Gene ◽  
Feeder Layer ◽  
Erythroid Cells ◽  
Globin Chain ◽  
Globin Mrna ◽  
Feeder Layers ◽  
Chain Synthesis ◽  
Globin Chain Synthesis

Abstract Abstract 4271 Background: During adult erythroid differentiation, there is a period of transient γ-globin (HBG) gene expression that coincides with transient DNA hypomethylation of the HBG gene promoter. This precedes the final pattern of high β-globin (HBB) gene expression in terminally differentiated adult erythroid cells (the ‘maturational switch’). Possibly, the micro-environment can modulate the period of transient HBG promoter hypomethylation and gene expression, which could have implications for efforts to sustain HBG expression for therapeutic objectives. To evaluate the responsiveness of the maturational switch to external factors, CD34+ hematopoietic progenitor cells from adult baboon bone marrow were cultured with and without AFT024 murine fetal liver stromal cell line feeder layers, as the micro-environmental variable. Methods: All cultures contained Iscove's media with 30% fetal bovine serum, 200ng/ml stem cell factor, 2U/ml erythropoietin, and 1μM dexamethasone. Globin chain synthesis was measured by biosynthetic radiolabelling of globin chains on d11 and d14 followed by HPLC separation, HBG transcript levels were determined by real time PCR, and F cells were analyzed by flow cytometry. Results: γ-globin chain synthesis was significantly lower in the presence of AFT024 feeder layers (0.07±0.05 γ/γ+β, n=4 versus 0.34±0.09 γ/γ+β, n=4, mean±SD, p<0.01). HBG mRNA was also significantly lower in the presence of AFT024 feeder layers (0.09±0.05 γ/total β-like globin mRNA, n=3, versus 0.32±0.10 γ/total β-like globin mRNA, n=3, p<0.025). Accordingly, F cell numbers were substantially decreased in the presence of feeder layers (38 versus 64%). Cell growth was similar in the presence and absence of feeder layers (115±48 versus 86±8.5 fold expansion, n=3). To test whether decreased γ-globin expression in the presence of ATF024 feeder cells is secondary to preferential expansion of more differentiated progenitors less capable of HBG expression, BM cells were fractionated into CD34+ CD36+ and CD34+ CD36- subpopulations prior to culture with and without feeder layers. Expression of HBG in both subpopulations was similarly reduced by the feeder layers, suggesting that decreased HBG expression was due to a direct effect of the feeder layer rather than to selective expansion of a more differentiated subpopulation. Bisulfite sequence analysis was performed to determine if differences in HBG expression were associated with differences in DNA methylation. DNA methylation of 5 CpG residues in the HBG promoter in purified erythroid cells from two independent cultures was 97.6 and 97.7% in the presence of feeder layers and 68.6 and 69.7% without feeder layers. Addition of the DNA methyltransferase inhibitor decitabine (0.5 × 10-6 M), to cells cultured in the presence of feeder layers increased γ-globin synthesis nearly tenfold (0.60 γ/γ+β) compared to untreated controls (0.063 γ/γ+β), consistent with mechanistic role for DNA methylation in HBG repression associated with feeder layer culture. In contrast to the results with adult-derived CD34+ BM cells, cord blood derived CD34+ cells sustained high levels of HBG expression (0.84 γ/γ+β)in the presence of feeder layers. Conclusion: The switch from HBG to HBB gene expression that occurs during the differentiation of adult erythroid cells is responsive to the micro-environment. Furthermore, this switch depends on DNA methylation, and depleting DNMT1 counteracts the micro-environmentally induced switch to sustain γ-globin expression in adult erythroid cells. In contrast to adult erythroid cells, fetal erythroid cells were resistant to micro-environmental induction of a HBG to HBB switch, consistent with previous studies showing that intrinsic differences between adult and fetal erythroid cells are a critical component in developmental stage-specific differences in globin gene expression. Disclosures: No relevant conflicts of interest to declare.

Increased Fetal Hemoglobin Levels Following Decitabine Treatment of Normal Baboons (P. anubis) are Achieved by Transcriptional Activation of the γ-Globin Gene.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4066-4066
Author(s):  
Imo Akpan ◽  
Virryan Banzon ◽  
Vinzon Ibanez ◽  
Kestis Vaitkus ◽  
Joseph DeSimone ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase Ii ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Post Treatment ◽  
Globin Chain ◽  
Pre Treatment ◽  
Chain Synthesis ◽  
Globin Chain Synthesis

Abstract Abstract 4066 Poster Board III-1001 Increased fetal hemoglobin (HbF) levels can alleviate symptoms associated with sickle cell disease (SCD) and β-thalassemia. The development of pharmacological inducers of HbF is a desirable goal expected to impact the clinical course of these diseases. Pharmacological inhibitors of DNA methyltransferase, 5-azacytidine and decitabine (2'-deoxy-5-azacytidine), induce high levels of HbF in vivo in the baboon model and in patients with SCD and β-thalassemia Bisulfite sequence analysis has shown that increased HbF is associated with a decrease in DNA methylation of 5 CpG residues within the 5' γ-globin gene region in both baboons and SCD patients treated with decitabine. Chromatin immunoprecipitation experiments have also shown increased association of RNA polymerase II, histone acetylation, and histone H3 (lys4) trimethyl with the γ-globin gene in bone marrow erythroid cells following decitabine treatment of experimental baboons. These results are consistent with the hypothesis that γ-globin expression is repressed by DNA methylation and that DNMT inhibitors increase HbF by reducing DNA methylation of the γ-globin promoter, thus allowing increased transcription of the γ-globin gene. The mechanism of action of these drugs remains controversial, however. A recent model based on experiments conducted in a primary human erythroid cell culture system suggested that DNMT inhibitors increase HbF by post-transcriptional or translational mechanisms mediated by stress signal transduction pathways (Mabaera et al, Exp Hematol 36:1057, 2008). Therefore we have conducted experiments in the baboon model to determine to what extent transcriptional versus translational and post-transcriptional mechanisms are responsible for increased HbF following decitabine treatment in vivo. Three normal baboons were treated with decitabine (0.5mg/kg/d) for ten days. The baboons were not phlebotomized to test whether the ability of decitabine to increase HbF levels required erythropoietic stress. Levels of γ-globin chain synthesis (γ/γ+β ratio) in pre-treatment and post-treatment bone marrow (BM) aspirates were determined by biosynthetic radiolabelling with [3H] leucine followed by separation of globin chains by HPLC. The effect of decitabine treatment on the level of γ-globin transcripts was determined by real time PCR using the ΔΔCT method. Results showed that the γ/γ+β chain ratios in pre-treatment BM aspirates of 3 normal baboons were 0.01, 0.01, and 0.1 and increased to 0.51, 0.24, and 0.49, respectively, in post-treatment BM. Decitabine treatment increased the level of γ-globin transcripts 55, 45, and 5.9 fold in these three baboons (see table). Animal Pre-treatment γ-globin chain synthesis (γ/γ+β) Post-treatment γ-globin chain synthesis (γ/γ+β) Fold change γ-globin chain synthesis Fold change γ-globin mRNA 7482 0.01 0.51 51 55 7470 0.01 0.24 24 45 7472 0.10 0.49 4.9 5.9 These results show that decitabine treatment induces similar fold increases in γ-globin chain synthesis and γ-globin transcripts. Therefore we conclude that decitabine does not increase HbF levels through a translational mechanism. Previous results from our laboratory showed that association of RNA polymerase II and histone H3 (lys4) trimethyl with the γ-globin gene were increased following decitabine treatment to levels similar to those associated with the β-globin gene. Therefore we conclude that decitabine increases HbF mainly by increasing γ-globin gene transcription rather than through post-transcriptional or translational effects. Because our experiments were conducted in normal unbled baboons, we also conclude that the ability of decitabine to increase HbF levels does not require erythropoietic stress. Disclosures: No relevant conflicts of interest to declare.

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