Gamma-globin Gene Research Articles

LIN28A-Mediated Expression Of Fetal Hemoglobin Ameliorates Erythrocyte Sickling

Highly-conserved LIN28 proteins regulate certain developmentally-timed events in multicellular organisms by decreasing the levels of let-7 miRNAs. It was recently reported that increased expression of LIN28 proteins or decreased expression of the target let-7 miRNAs in human erythroblasts cultured from healthy adult humans causes increased fetal hemoglobin expression. Here LIN28A expression in sickle cell donors' cells was explored for its potential to regulate fetal and sickle hemoglobin, and to affect the morphological sickling of the mature erythrocytes.After obtaining consent and assent, CD34(+) cells from five pediatric research subjects with HbSS genotype (ages 9-16 years old) were harvested from discarded whole blood following partial manual exchange transfusions. Transgenic expression of LIN28A was accomplished using lentiviral transduction of human CD34(+) sickle cells cultivated ex vivo in serum-free medium for a total of 21 days. On culture day 14, LIN28A over-expression (LIN28A-OE) was confirmed by Q-RT-PCR (control: 8.6E+00 ± 8.1E+00 copies/ng, LIN28A-OE: 2.3E+05 ± 2.1E+05 copies/ng) and Western blot analyses. Erythroblast differentiation and terminal maturation were not affected by LIN28A-OE. Enucleation, as assessed by thiazole orange (TO) staining, was equivalent between the LIN28A-OE cells and control transductions (LIN28A-OE enucleation 40.8 ± 17.0% compared to control 49.9 ± 23.4%, p=0.19). LIN28A-OE strongly suppressed all members of the let-7 family of miRNAs, with average reductions from 66% to 96% for let-7a, let-7b, let-7c, let-7d, let-7e, let-7f-2, let-7g and let-7i. LIN28A-OE caused reduced expression of BCL11A, a known repressor of gamma-globin gene expression. Gamma-, beta (sickle)- and alpha-globin mRNA levels were also investigated by Q-RT-PCR. Gamma-globin mRNA expression levels were significantly increased in LIN28A-OE samples (control: 2.0E+06 ± 7.0E+05 copies/ng, LIN28A-OE: 2.0E+07 ± 6.0E+06 copies/ng, p=0.006), and beta (sickle)-globin mRNA significantly decreased in LIN28A-OE samples (control: 2.0E+07 ± 5.2E+06 copies/ng, LIN28A-OE: 1.6E+07 ± 6.3E+06 copies/ng, p=0.024). Differences in alpha-globin mRNA expression were not statistically significant. Hemoglobin chromatography (HPLC) demonstrated that LIN28A-OE significantly increased the proportion of fetal hemoglobin (HbF control: 10.8 ± 7.1%, LIN28A-OE: 40.1 ± 14.0%; p=0.003) that was balanced by a significant decrease in the proportion of sickle hemoglobin. HbA was not detected.For investigation of the sickling phenotype, enucleated [TO(-)] sickle erythrocytes from LIN28A-OE and control transductions of two subjects' cells were sorted at the end of the culture period into duplicate tissue culture wells. The sorted erythrocytes were incubated in hypoxia (2% oxygen) for 16 hours, and imaged using inverted microscopy within three minutes after removal from the hypoxia incubator. Four random microscopic field images from each well were acquired. Blinded observers then scored the images from the control and LIN28A-OE transductions according to non-sickled versus sickled morphologies. Cultured erythrocytes from the control transductions demonstrated 86.3 ± 9.5% with sickled morphologies. By comparison, a significant reduction in sickling morphology was observed in the LIN28A-OE cells (56.2 ± 23.1% sickled morphologies; p=0.000009).These results demonstrate that transgenic expression of LIN28A during ex vivo erythropoiesis causes increased gamma-globin gene and protein expression balanced with decreased beta (sickle)-globin at levels that are sufficient to ameliorate hypoxia-related sickling of mature erythrocytes. Disclosures:No relevant conflicts of interest to declare.

Cord Blood CD34+ Stem Cell Sialyl Lewis X Levels and E-Selectin Binding Are Predictive Of Engraftment In Mice: Functional Separation Of Stemness and Homing To Improve Engraftment

BackgroundCord blood (CB) is an increasingly used alternative to bone marrow and mobilized peripheral blood as a source of hematopoietic tissue for transplantation. However, the relatively low cell dose and significantly delayed engraftment when compared to BM and mPB remain significant hurdles. A deficit in the homing of CB hematopoietic progenitor and stem cells (HPSC) to the hematopoietic microenvironment due to suboptimal expression and/or activity of homing molecules is thought to be responsible in part for the delayed engraftment seen with CB in patients. Sialyl Lewis X (sLeX) bearing cells can bind E-selectin, and we have previously reported that ex vivo fucosylation of CB HPSCs with fucosyltransferase VI or VII enhance the rapidity and magnitude of engraftment in mice. Here we explore the engraftment potential of endogenous sLeX bearing CD34+ CB HPSCs to determine if physiologic levels of E-selectin binding predicts engraftment in murine xenografts. ApproachCB cells were sorted with CD34 and HECA-452 (anti-sLex) antibodies. sLeX-bearing CD34+ HPSCs (CD34+HECA+) and CD34+ HPSCs lacking sLeX (CD34+HECA-) were compared phenotypically for stem cell and differentiation markers, by gene expression profiling, E/P/L-selectin binding, colony-forming assays, and for multi-lineage engraftment into NOD-SCID-IL2Rg immune-deficient mice. ResultsCord blood CD34+HECA+ cells represent 10-20% of CB MNCs and show no significant phenotypic differences from the CD34+HECA- cells in stem cell (CD133, CD90 (Thy-1), CD117 (c-kit), CD143/BB9) and differentiation (CD38, CD33, CD14, CD3, CD19) markers. In agreement, similar percentages of CD34+CD38- and CD34+CD38+ CB cells were found to be HECA+ (18% and 15% respectively, p=0.38), showing no significant bias toward the more immature CD34+CD38- phenotype. mRNA-seq expression analysis revealed relatively few differences in gene expression patterns, although CD34+HECA+ cells express higher levels of the gamma globin genes HBG1 and HBG2, the components of fetal hemoglobin. As predicted, CD34+HECA+ cells demonstrated significantly increased ability to bind recombinant E-selectin in vitro, with no differences in P- and L-selectin binding. Importantly, colony forming assays revealed a small (30%) disadvantage to the CD34+HECA+ cells revealing that the CD34+HECA+ CB cells do not have enriched stem cells activity by CFU assay.However, CD34+HECA+ cells demonstrated significantly higher rate and magnitude of engraftment when compared to CD34+HECA- cells in three independent NSG experiments (Figure 1). Indeed bone marrow, peripheral blood and splenic levels of human hematopoietic cells were consistently 3-5-fold higher in CD34+HECA+ injected mice than in CD34+HECA- injected controls (Figure 2). Multi-lineage engraftment data reveals higher levels of myeloid (CD33+/CD14+), B-lymphocytes (CD19+/CD20+) and platelets (6-14-fold, CD41a+/CD61+) in CD34+HECA+ cells, but interestingly lower levels of T-lymphocytes (CD3+). Finally, secondary transplants had equal magnitude of engraftment, indicating no bias in short- versus long-term HSPCs. ConclusionsData presented here supports the hypothesis that endogenous sLex levels on CD34+ cells is associated with enhanced engraftment rapidity and magnitude but that this is not reflective of an enriched stem cell fraction. Rather it appears to be an indicator of homing to the bone marrow through E-selectin binding. Functional separation of stemness and homing supports the approach to improve CB transplantation through decoration of CB cells with sLex via ex vivo fucosylation (see clinical trial abstract by Popat and Shpall)▪ Disclosures:No relevant conflicts of interest to declare.

CNTO 530 Increases Expression of HbA and HbF in Murine Models of β-Thalassemia and Sickle Cell Anemia

CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia). We investigated the efficacy of CNTO 530 in murine models of β-thalassemia and sickle cell anemia (Berkeley mice). β- thalassemic mice are deficient in expression of α-globin chain and heterozygous mice are characterized by a clinical syndrome similar to the human β-thalassemia intermedia. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Berkeley mice thus express human sickle hemoglobin A (HbS) and can also express human fetal hemoglobin. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. To test the effectiveness of CNTO 530, mice from both genotypes received a single subcutaneous (s.c.) dose of CNTO 530 or darbepoetin-α (as a comparator) at 10,000 U/kg, a dose shown to cause a similar increase in reticulocytes and hemoglobin in normal mice. Hematologic parameters were evaluated over time. CNTO 530, but not darbepoetin-α, increased reticulocytes, red blood cells and total hemoglobin in β- thalassemic mice. In Berkeley mice CNTO 530 showed an increase in reticulocytes, red blood cells, F-cells, total hemoglobin and fetal hemoglobin. In conclusion, CNTO 530 is effective in murine models of β-thalassemia and sickle cell anemia. These data suggest that CNTO 530 may have beneficial effects in patients with genetically mediated hemoglobinopathies.

CNTO 530 Increases Expression of HbA and HbF in Murine Models of β-Thalassemia and Sickle Cell Anemia

CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia). We investigated the efficacy of CNTO 530 in murine models of β-thalassemia and sickle cell anemia (Berkeley mice). β- thalassemic mice are deficient in expression of α-globin chain and heterozygous mice are characterized by a clinical syndrome similar to the human β-thalassemia intermedia. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Berkeley mice thus express human sickle hemoglobin A (HbS) and can also express human fetal hemoglobin. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. To test the effectiveness of CNTO 530, mice from both genotypes received a single subcutaneous (s.c.) dose of CNTO 530 or darbepoetin-α (as a comparator) at 10,000 U/kg, a dose shown to cause a similar increase in reticulocytes and hemoglobin in normal mice. Hematologic parameters were evaluated over time. CNTO 530, but not darbepoetin-α, increased reticulocytes, red blood cells and total hemoglobin in β- thalassemic mice. In Berkeley mice CNTO 530 showed an increase in reticulocytes, red blood cells, F-cells, total hemoglobin and fetal hemoglobin. In conclusion, CNTO 530 is effective in murine models of β-thalassemia and sickle cell anemia. These data suggest that CNTO 530 may have beneficial effects in patients with genetically mediated hemoglobinopathies. Keywords: Erythropoiesis, Flow Cytometry, Fusion protein, Hematology, Ion Exchange Chromatography, Pharmacodynamics

Induction of human fetal hemoglobin expression by adenosine-2’,3’-dialdehyde

BackgroundPharmacologic reactivation of fetal hemoglobin expression is a promising strategy for treatment of sickle cell disease and β-thalassemia. The objective of this study was to investigate the effect of the methyl transferase inhibitor adenosine-2’,3’-dialdehyde (Adox) on induction of human fetal hemoglobin (HbF) in K562 cells and human hematopoietic progenitor cells.MethodsExpression levels of human fetal hemoglobin were assessed by northern blot analysis and Real-time PCR. HbF and adult hemoglobin (HbA) content were analyzed using high-performance liquid chromatography (HPLC). DNA methylation levels on human gamma-globin gene promoters were determined using Bisulfite sequence analysis. Enrichment of histone marks on genes was assessed by chromosome immunoprecipitation (ChIP).ResultsAdox induced γ-globin gene expression in both K562 cells and in human bone marrow erythroid progenitor cells through a mechanism potentially involving inhibition of protein arginine methyltransferase 5 (PRMT5).ConclusionsThe ability of methyl transferase inhibitors such as Adox to efficiently reactivate fetal hemoglobin expression suggests that these agents may provide a means of reactivating fetal globin expression as a therapeutic option for treating sickle cell disease and β-thalassemia.

Globin gene expression in correlation with G protein-related genes during erythroid differentiation

BackgroundThe guanine nucleotide binding protein (G protein)-coupled receptors (GPCRs) regulate cell growth, proliferation and differentiation. G proteins are also implicated in erythroid differentiation, and some of them are expressed principally in hematopoietic cells. GPCRs-linked NO/cGMP and p38 MAPK signaling pathways already demonstrated potency for globin gene stimulation. By analyzing erythroid progenitors, derived from hematopoietic cells through in vitro ontogeny, our study intends to determine early markers and signaling pathways of globin gene regulation and their relation to GPCR expression.ResultsHuman hematopoietic CD34+ progenitors are isolated from fetal liver (FL), cord blood (CB), adult bone marrow (BM), peripheral blood (PB) and G-CSF stimulated mobilized PB (mPB), and then differentiated in vitro into erythroid progenitors. We find that growth capacity is most abundant in FL- and CB-derived erythroid cells. The erythroid progenitor cells are sorted as 100% CD71+, but we did not find statistical significance in the variations of CD34, CD36 and GlyA antigens and that confirms similarity in maturation of studied ontogenic periods. During ontogeny, beta-globin gene expression reaches maximum levels in cells of adult blood origin (176 fmol/μg), while gamma-globin gene expression is consistently up-regulated in CB-derived cells (60 fmol/μg). During gamma-globin induction by hydroxycarbamide, we identify stimulated GPCRs (PTGDR, PTGER1) and GPCRs-coupled genes known to be activated via the cAMP/PKA (ADIPOQ), MAPK pathway (JUN) and NO/cGMP (PRPF18) signaling pathways. During ontogeny, GPR45 and ARRDC1 genes have the most prominent expression in FL-derived erythroid progenitor cells, GNL3 and GRP65 genes in CB-derived cells (high gamma-globin gene expression), GPR110 and GNG10 in BM-derived cells, GPR89C and GPR172A in PB-derived cells, and GPR44 and GNAQ genes in mPB-derived cells (high beta-globin gene expression).ConclusionsThese results demonstrate the concomitant activity of GPCR-coupled genes and related signaling pathways during erythropoietic stimulation of globin genes. In accordance with previous reports, the stimulation of GPCRs supports the postulated connection between cAMP/PKA and NO/cGMP pathways in activation of γ-globin expression, via JUN and p38 MAPK signaling.

Blood group phenotypes resulting from mutations in erythroid transcription factors

This review describes the genetics of unusual blood group phenotypes, particularly those with altered expression of Lutheran antigens, and how this area of study has informed our understanding of erythropoiesis in general and haemoglobin switching in particular. Mutations in erythroid transcription factors GATA1 (GATA1 binding protein 1) and KLF1 (Kruppel-like factor 1) cause benign and disease phenotypes in humans [X-linked Lu(a-b-) phenotype, In(Lu) blood group phenotype, hereditary persistence of foetal haemoglobin, borderline HbA(2), and congenital dyserythropoietic anaemia (CDA)]. These studies explain the occurrence of rare blood group phenotypes with simultaneous altered expression of antigens from several blood group systems and illuminate the role of KLF1 in gamma and delta globin gene regulation. The study of rare blood group phenotypes is a potent tool for discovery of mutations in human genes. Elucidation of the molecular basis of the rare In(Lu) phenotype revealed the first mutations in human KLF1. Subsequently, numerous additional mutations have been described, one of which causes a rare form of CDA. Analysis of the X-linked Lu(a-b-) phenotype revealed a mutation in the C-terminal domain of human GATA1. The apparent sensitivity of the Lutheran glycoprotein to alterations in GATA1 and KLF1 activity suggest that it could be a useful biomarker of erythroid transcription factor mutation.

Anemia falciforme: desafios e avanços na busca de novos fármacos

Sickle Cell Disease (SCD) is a disease characterized by a punctual mutation (GTG - GAG) in the sixth codon of the gamma globin gene leading to a substitution of glutamic acid by a valine in the β chain of hemoglobin. Despite the huge progress on the molecular knowledge of the disease in recent years, few therapeutic resources were developed. Currently, the treatment is mainly done with the anticancer agent hydroxyurea. This review summarizes current knowledge about possible targets and new approaches to the discovery new compounds to treat the symptoms of SCD.

Expressions of KLF1, FOXO3a, HOOK3 and MIER1 genes Are Associated with Fetal Hemoglobin Levels in Hereditary Persistence of Fetal Hemoglobin,

Abstract 3195The hereditary persistence of fetal hemoglobin (HPFH) is the consequence of impaired switching in adult life, which results in the continued expression of gamma globin gene. The Brazilian HPFH type is characterized by a C → G substitution at the –195 position of the A gamma globin gene promoter, and associated with HbF levels ranging from 6 to 16% in the heterozygote state. This study was undertaken to identify genes that may be involved in hemoglobin switching and/or maintenance of elevated HbF levels in Brazilian HPHF subjects. The Suppressive Subtractive Hybridization libraries were constructed using a pool of RNA extracted from reticulocytes of peripheral blood in individuals with normal hematological data and from subjects with Brazilian and 55 and 57 overexpressed genes were identified in the normal and Brazilian HPFH library, respectively; findings were validated by qRT-PCR and Western blotting. One transcription factor identified was FOXO3a, whose expression was increased in Brazilian HPFH subjects, compared to control subjects. Moreover, the non-phosphorylated Foxo3a protein that interacts with DNA, was detected only in Brazilian HPFH when analyzed by Western blotting. FOXO3a binds to PAX1 promoter, a transcription factor whose activity was higher in Brazilian HPFH. The FOXO3a/PAX1 complex may be important in the mechanism responsible for increasing HbF levels in this genetic disorder. Another gene analyzed was KLF1, a transcription factor known as a regulator of switching from the gamma to beta globin gene. This gene was underexpressed in the reticulocytes of HPFH subjects. Klf1 protein activity in erythroid cells of healthy donors was also increased compared to Brazilian HPFH, when analyzed by DNA/protein array. Results suggest that the decreased KLF1 levels in Brazilian HPFH favors the interaction between the A gamma globin gene and the Locus Control Region, agreeing with previous studies that demonstrated that knockdown of KLF1 in adult erythroid progenitors favors the formation of complexes that bind to the gamma globin gene promoter. MIER1 expression was also found to be decreased in Brazilian HPFH reticulocytes, compared to controls. The MIER1 gene is able to recruit chromatin remodeling-enzymes leading, to the formation of heterochromatin and, consequently, silencing genes. This MIER1 may be an important gene in gamma to beta globin gene switching, where it could help in the maintenance of a closed chromatin structure in the gamma globin gene in individuals with low HbF levels. The expression of the HOOK3 gene was decreased in Brazilian HPFH reticulocytes, compared to controls. The HOOK3 encodes a protein that interacts with a GTPase protein, stimulating INF-gamma, responsible for the phosphorylation of p65/p50 and RXR beta like proteins which regulate the transcription of the beta globin gene. The reduced beta globin gene expression in Brazilian HPFH resulting from the reactivation of gamma globin gene may be responsible for a decrease in the HOOK3 expression. These results suggest that, in the HPFH Brazilian type, the FOXO3a/PAX1 complex could contribute to the continued expression of the A gamma globin gene. Additionally, some cellular modifications, such as low Klf1 activity and decreased HOOK3 and MIER1 expression, could participate in the maintenance of HbF levels. These genes could be important in therapeutic approaches for the development of new HbF induction agents for the hemoglobinopathies.Support by FAPESP, CNPq.and INCTS Disclosures:No relevant conflicts of interest to declare.

Targeting a Novel Epigenetic Silencing Mechanism to Efficiently Upregulate Fetal Globin Gene Expression

Abstract 352L3MBTL1 is a Polycomb group protein, commonly deleted in patients with myeloid disorders associated with the 20q- chromosomal abnormality. After crystallizing the MBT repeat domain, we demonstrated that L3MBTL1 compacts chromatin by binding mono- and di-methylated lysine residues in histones H1 (H1K26) and H4 (H4K20), ultimately leading to gene repression. Despite its role in affecting the chromatin structure, the role of L3MBTL1 in hematopoiesis has remained largely unknown.We recently demonstrated that lack of L3MBTL1 accelerates the erythroid differentiation of human hematopoietic stem cells and here we reveal that L3MBTL1 represses the expression of the fetal gamma globin gene. We lentivirally expressed shRNAs targeting L3MBTL1 in human cord blood (CB) CD34+ cells and in K562 erythroleukemia cells, and consistently observed upregulation of gamma globin gene expression, while beta globin gene expression decreased. Remarkably, we observed similar findings in human embryonic stem (hES) cells, where knock-down of L3MBTL1 triggered a BMP4-like spontaneous differentiation. Given the potential impact of therapeutically increasing fetal hemoglobin expression in patients with hemoglobinopathies, we targeted L3MBTL1 in induced pluripotent stem (iPS) cells derived from patients with β-thalassemia. The gene expression profile of L3MBTL1-KD normal and thalassemic iPS cells indicated clear activation of fetal hemoglobin (HbF) expression, activation of BMP4 signaling and upregulation of specific smad5 target genes (e.g. EKLF, HHEX, ID2/3). We generated and utilized a model of “stress erythropoiesis” in L3MBTL1 KO mice and observed in vivo BMP4-mediated expansion of spleen immature erythroid progenitors, as indicated by increased spleen weight and splenic BFU-E colonies in KO mice compared to controls.We also examined K562 cells, human CB CD34+ cells and hES cells, using chromatin immunoprecipitation assays, and found that L3MBTL1 directly associates with the human β-globin locus, occupying discrete regions within the human β-globin cluster. Furthermore, L3MBTL1 colocalized with H4K20me within the Locus Control Region (LCR), a primary attachment site for chromatin modifiers. We observed clearance of L3MBTL1 and its associated histone marks (H4K20me1/2) from the LCR upon treatment with hemin, erythropoietin or TGFβ, three agents that potently induce erythroid differentiation. This suggests that this polycomb repressor complex responds to cytokine signaling.In summary, we have identified a novel epigenetic regulatory mechanism to control fetal globin gene expression; the Polycomb protein L3MBTL1 regulates BMP4 signaling and the chromatin structure of globin genes. Targeting this regulatory system represents a means to efficiently increase HbF in a human model of β-thalassemia (i.e. with the use of patient-derived iPS cells) and to potentially ameliorate hematological and clinical symptoms of patients with red cell disorders. Disclosures:No relevant conflicts of interest to declare.

Novel 1,2,5-Oxadiazole 2-Oxide Derivatives with Analgesic and Fetal Hemoglobin Induced Properties Designed As Drug Candidate to Treat Sickle Cell Disease Symptoms

Abstract 2137 Introduction.The combination of multiple activities in the same structure using molecular modification is a powerful tool to discover more effective and safe compounds to treat sickle cell disease (SCD) symptoms. The only FDA approved drug for SCD, at present, is hydroxyurea (HU). The release of NO by HU is an important mechanism of its HbF-inducing properties. Thalidomide and some analogues are known to inhibit cytokines release, presenting important analgesic activity. Recently the drug has also been reported to induce gamma-globin expression. Our rational drug design used molecular hybridization between thalidomide and NO donor subunits, represented by 1,2,5-oxadiazole 2-oxide. The aim of this study was to evaluate the effects of eight novel hybrid compounds (3a-h) on NO donor activity, analgesic activity and γ-globin gene expression. Methods. 1. Detection of nitrite. A solution of the appropriate compound (20 μL) in DMSO was added to 2 mL of a mixture of 50 mM phosphate buffer (pH 7.4) and methanol (1:1, v:v), containing 5 mM of L-cysteine. The final concentration of the compound was 10−4 M. After 1 h at 37 °C, 1 mL of the reaction mixture was treated with 250 μL of Griess reagent. After 10 min at room temperature, the absorbance was measured at 540 nm using a spectrophotometer. Standard sodium nitrite solutions (10–80 nmol/mL) were used to construct the calibration curve. The yields of nitrite are expressed as % NO2∓ (mol/mol). 2. Antinociceptive activity. Analgesic activity was determined in vivo with the acetic-acid-induced (0.6%, 0.1 mL/10 g) abdominal constriction test in mice. Swiss mice of both sexes (18–23 g) were used. The compounds were administered orally (100 μmol/kg) as a suspension in 5% arabic gum in saline (vehicle). Dypirone (100 μmol/kg) was used as the standard drug. Acetic acid solution was administered i.p. 1 h after the administration of the compounds. Ten minutes after the i.p. acetic acid injection, the number of constrictions per animal was recorded for 20 min. The control animals received an equal volume of vehicle. Antinociceptive activity was expressed as percentage inhibition of the constrictions compared with those in the vehicle-treated control group. The data were analyzed statistically with Student’s t test at a significance level of P < 0.05. 3. Gamma-globin gene expression. Human K562 cells were maintained in DMEM with 10% FBS, Pen/Strep, in humidified air (5% CO2, 37°C). Cells (1×107cells/100mL) were incubated with compounds at different concentrations (5, 30, 60, and 100μM) for 24, 48, 72 and 96h. g-Globin gene expression was analyzed by qRT-PCR and quantified using the Gnorm program. Results are expressed as arbitrary units. Results. 1. Detection of nitrite: The quantification of the nitrite produced resulted from the oxidative reaction of NO, oxygen and water. All eight compounds were capable of inducing nitrite formation at concentrations of between 9.5% and 28%. Isosorbide dinitrate (DNS), used as the control, induced 11.7% nitrite formation. 2.Antinociceptive activity: Compounds 3c and 3d were the most active antinociceptive compounds, significantly reducing the acetic-acid-induced abdominal constrictions by 43% and 38%, respectively, while dypirone used as a control inhibited constrictions by 34%. 3. Gamma-globin gene expression: Compound 3c demonstrated inverse dose-response relationships, achieving the highest levels of γ-globin induction at 5 and 30 μM. Compound 3c achieved maximal γ-globin induction as early on as 48h after treatment ([48h; 5 μM]: 1,88±0.06 AU; control 0.78±0.1 AU; P<0.05). Testing of compound 3d is currently underway. Conclusions. Results demonstrate that molecular hybridization between a thalidomide derivative and nitric oxide donors may be an important tool to treat SCD symptoms. These compounds demonstrated NO donor and analgesic activity. Specifically, the compound 3c was capable of inducing gamma-globin expression. Testing of our lead compound 3c in a preclinical mouse model of SCD will be performed in order to evaluate its efficacy in the treatment of this hemoglobinopathy. Disclosures:No relevant conflicts of interest to declare.

Delayed fetal hemoglobin switching in subjects with KLF1 gene mutation

Variations at the KLF1 gene have been associated with a series of human erythroid phenotypes including the In-(Lu) phenotype, hereditary persistence of fetal hemoglobin, congenital dyserythropoietic anemia, borderline HbA 2 and increased red blood cell protoporphyrin. Natural mutations have shown that KLF1 regulates gamma globin gene expression and its role in the switching from fetal to adult globin expression has been suggested by experimental studies. In this paper we report that subjects with S270X KLF1 mutations show a decrease of HbF levels with increasing age, supporting in vivo the role of KLF1 in hemoglobin switching in humans.

C(5) modified uracil derivatives showing antiproliferative and erythroid differentiation inducing activities on human chronic myelogenous leukemia K562 cells

The K562 cell line has been proposed as a useful experimental system to identify anti-tumor compounds acting by inducing terminal erythroid differentiation. K562 cells exhibit a low proportion of hemoglobin-synthesizing cells under standard cell growth conditions, but are able to undergo terminal erythroid differentiation when treated with a variety of anti-tumor compounds. In this paper we report a screening study on a set of different modified C(5) uracil derivatives for the evaluation of their antiproliferative effect in connection with erythroid differentiation pathways, and for defining a new class of drug candidates for the treatment of chronic myelogenous leukemia. Activity of the derivatives tested can be classified in two effect: an antiproliferative effect linked to a high level of erythroid differentiation activity and an antiproliferative effect without activation of gamma globin genes The highest antiproliferative effect and erythroid induction was shown by compound 9, a thymine derivative bearing a n-octyl chain on nitrogen N(1), whereas thymine did not show any effect, suggesting the importance of the linear alkyl chain in position N(1). To our knowledge this compound should be considered among the most efficient inducers of erythroid differentiation of K562 cells. This work is the starting point for the quest of more effective and specific drugs for the induction of terminal erythroid differentiation, for leading new insights in the treatment of neoplastic diseases with molecules acting by inducing differentiation rather than by simply exerting cytotoxic effects.

The -195C→G Substitution In Brazilian Hereditary Persistence of Fetal Hemoglobin Decreases NF-E1/YY1 Binding and Increases PAX1 Binding to the A Gamma Globin Promoter Region

AbstractAbstract 2066Hereditary persistence of fetal hemoglobin (HPFH) is a condition that prevents hemoglobin switching and the consequent silencing of the gamma globin genes, resulting in continued hemoglobin (Hb) F synthesis in adults. Two types of HPFH are responsible for this phenotype: deletional HPFH – deletions in the end of the beta globin locus – and non-deletional HPFH (ndHPFH) – single point mutations in the proximal promoter of both gamma globin genes. Sickle cell anemia patients or beta-thalassemia patients that present HPFH show high levels of HbF that are associated with less severe clinical course in these diseases. The development of new therapies based on the reactivation of gamma globin expression may be important for the treatment of these patients. The Brazilian ndHPFH type is characterized as a C→G substitution in the A gamma globin promoter at position –195 and the molecular mechanism responsible for the reactivation of this gene in the Brazilian ndHPFH type remains unclear. In contrast to the British ndHPFH type (-198), where the mechanism responsible for the increase of HbF levels is mediated by the raising in the affinity for the Sp1 transcription factor (TF), the Brazilian ndHPFH mutation does not affect Sp1 binding. Thus, other TF may be involved in the reactivation of the A gamma globin gene in the Brazilian ndHPFH type. The aim of this study was to investigate the mechanism involved in the reactivation or repression of the A gamma globin gene in the Brazilian ndHPFH type and identify possible TF responsible for this phenotype. In vitro primary human erythroblast cultures, derived from human CD34+ hematopoietic cells from 4 Brazilian ndHPFH type subjects and 4 control subjects, were proliferated and differentiated into late stage erythroblasts. The nuclear extracts from predominantly basophilic and polychromatic erythroblasts were used to profile TF activity using Protein-DNA Array method. The analysis of the array densitometry identified a number of TF whose DNA binding activities were either enhanced or repressed in the Brazilian ndHPFH cultures. Among the TF analyzed, the NF-E1/YY1 and the PAX-1 were selected for this study. Since this assay requires a secondary method to confirm these results, nuclear extracts were used to conduct chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). ChIP was carried out using antibodies against NF-E1/YY1 and PAX-1 to quantify the binding to these TF to the –195 A gamma globin promoter region. EMSA was performed using probes with the same sequence spotted on the array membrane to analyze the activity of NF-E1/YY1 and PAX-1. Both methods confirmed and validated the previous array results. NF-E1/YY1 is a transcription factor that represses embryonic (epsilon) and fetal (gamma) globin genes. Protein-DNA array and EMSA showed a decreased binding of NF-E1/YY1 in Brazilian ndHPFH nuclear extracts and ChIP analysis revealed diminished NF-E1/YY1 occupancy at the –195 A gamma globin promoter region of Brazilian ndHPFH. The consensus binding site for NF-E1/YY1 is a CCAN motif that is observed between the –195 and –192 position in the A gamma globin promoter region. The C→G substitution at –195 position may disrupt this DNA binding site, cause decreased NF-E1/YY1 interaction and probably allows the binding of PAX-1, a transcriptional activator with a paired box DNA-binding domain that has as a DNA binding core motif, the sequence TTCCGC. This sequence, located between the –199 and –194 position in the A gamma globin promoter, is only presente in the Brazilian type of ndHPFH. Our protein-DNA array and EMSA results showed an increased binding of PAX-1 in the Brazilian ndHPFH nuclear extracts and quantitative ChIP analysis with anti-PAX-1 antibody showed that PAX-1 binds to the –195 A gamma globin promoter region only in the presence of this C→G substitution. These results suggest that the –195 site (C→G) in the A gamma globin promoter region may decrease NF-E1/YY1 binding and increase PAX-1 binding in this DNA region, probably resulting in the reactivation of the A gamma globin gene. The increase in the HbF levels in the Brazilian ndHPFH occurs differently from the British ndHPFH type and represents a novel mechanism of A gamma globin reactivation. Such findings may lead to the development of future therapeutic strategies for HbF induction in the treatment of other hemoglobinopathies.Support by FAPESP and CNPq. Disclosures:No relevant conflicts of interest to declare.

The PcG Protein L3MBTL1 Transcriptionally Represses Human Embryonic and Fetal Globin Genes: a Novel Prospect for HbF Activation

AbstractAbstract 1014L3MBTL1 is the human homolog of the Drosophila Polycomb Group tumor suppressor gene, lethal(3)malignant brain tumor. We demonstrated that human L3MBTL1 functions as a transcriptional repressor and after crystallizing the MBT repeat domain determined that L3MBTL1 compacts chromatin by binding mono- and di-methylated lysine residues in histones H1 (H1K26) and H4 (H4K20).Despite the known role of L3MBTL1 in affecting chromatin structure, the function of L3MBTL1 in human hematopoiesis has remained largely unknown. We recently demonstrated that L3MBTL1 enforces cell fate decision toward the erythroid lineage and that knockdown of L3MBTL1 accelerates the erythroid differentiation of human hematopoietic stem/progenitor cells, suggesting that its deletion contributes to the pathogenesis of 20q- erythroid malignancies. Consistently with its role in erythropoiesis, here we reveal that L3MBTL1 is a novel transcriptional repressor of fetal globin genes and it may work in concert with BCL11A and EKLF to control globin gene expression.By utilizing RNA interference to reduce L3MBTL1 expression, we have found that knockdown of L3MBTL1 in human cord blood hematopoietic stem/progenitor cells consistently upregulates the expression of the epsilon, gamma, and zeta globin genes, but not the beta globin gene. Similar effects were seen following knockdown of L3MBTL1 in the human erythroleukemia cell line K562, and knockdown of L3MBTL1 in human embryonic stem cells (ESCs) led to the inappropriate expression of fetal and embryonic globin genes (which increases more than 50-fold after the L3MBTL1-KD). These data suggest a role for L3MBTL1 in regulating the globin switch.To investigate the mechanism by which L3MBTL1 silences embryonic and fetal globin gene expression, we used chromatin immunoprecipitation (ChIP) assays to show that L3MBTL1 directly associates with the human β-globin locus. L3MBTL1 occupies several discrete regions within the human β-globin cluster and colocalizes with H4K20me within the Locus Control Region (LCR), a primary attachment site for chromatin modifiers. As confirmation, we found that treatment of K562 cells with hemin, which broadly increases H3K9 acetylation over the β-globin locus and activates the transcription of globin genes, leads to decreases in expression of the repressive H4K20me2 methylmark and L3MBTL1 to the beta-globin cluster.Given the recent identification of the repressor of gamma globin gene expression, BCL11A, we investigated a potential relationship between L3MBTL1 and BCL11A. We found that knockdown of L3MBTL1 led to downregulation of BCL11A mRNA. Accordingly, we have also found that overexpression of L3MBTL1 is associated with an upregulation of BCL11A mRNA, suggesting that L3MBTL1 and BCL11A may function cooperatively to silence globin gene expression. Knockdown of L3MBTL1 also upregulated EKLF mRNA levels which could relate to the decreased BCL11A expression.In summary our data demonstrate that knock-down of L3MBTL1 upregulates embryonic and fetal globin genes in cell contexts where they are usually silenced, indicating the functional importance of this Polycomb protein for repressing the globin gene locus. The clearance of L3MBTL1 and its associated histone mark (H4K20me2) during treatments that induce potent transcriptional activation of globin genes suggest that repression induced by L3MBTL1 is dynamic and may be involved in the fetal-to-adult globin switch. L3MBTL1 therefore emerges as a novel transcriptional repressor of fetal globin genes whose expression may be coordinated with that of BCL11A and EKLF. Understanding the role of L3MBTL1 and the H4K20 methylmark in globin gene switching offers the prospect of the targeted activation of HbF in erythroid cells of patients with hemoglobin disorders. Disclosures:No relevant conflicts of interest to declare.

Analysis of Fetal Hemoglobin Expression within Humanized Sickle Cell Disease Mice Overexpressing the TR2/4 Transgene.

AbstractAbstract 1619 Background:Sickle Cell Disease (SCD) is a chronic debilitating hematologic condition caused by a missense mutation within the adult beta globin gene leading to significant morbidity and mortality. Increased Fetal Hemoglobin production has been shown to significantly ameliorate SCD symptoms and improve survival. A novel specific DNA-binding factor DRED (direct repeat erythroid definitive) was recently identified that regulated epsilon and gamma globin gene expression (Tanimoto et al Genes Dev 2000). Purification of DRED revealed that it harbored the nuclear orphan hormone receptors, TR2/TR4, as its DNA binding core (Tanabe et al EMBO 2002). Overexpression of TR2/TR4 Transgene within Human Beta Globin Yeast Artificial Chromosome Transgenic Mice resulted in 4-fold induction of the gamma globin mRNA levels (Tanabe et al EMBO 2007). Therefore, we wanted to determine if the overexpression of TR2/TR4 within a humanized sickle cell disease model would result in fetal hemoglobin induction. Methods:Humanized Homozygous Knock-In UAB-Sickle Cell (UAB-Hbahα/hα Hbbhβs/hβs) Mice (Wu et al Blood 2006) was mated to TR2/TR4 Overexpressing Mice (TgTR2/TR4) to generate homozygous SS-TR2/TR4 compound heterozygotes (UAB-Hba hα/hα Hbb hβs/hβs TgTR2/TR4). We generated four 2–3 month old homozygous SS-TR2/TR4 transgenic mice and compared hemoglobin F levels, complete blood cell counts and % body weight (liver, spleen, kidney) to six 2–3 month old homozygous SS mice (Hbahα/hα Hbb hβs/hβs)without the overexpressing TgTR2/TR4. Tail PCR genotyping of all sickle cell mice (with and without TgTR2/TR4) and Hemoglobin F(Hgb F) and Sickle (HgbS) levels were confirmed by HPLC Hemoglobin electrophoresis. Results:The mean Hgb F: 7.8% (n=6, sd 1.63+/−) in the homozygous SS control mice vs. 16.5% (n=4, sd 2.64+/−)in the homozygous SS-TR2/4 Mice (2 Fold higher). Hematologic profile revealed a mean Hct: 25.2 (n=6, sd 5.50 +/−) mean MCV: 75.4 (n=6, sd 10+/−) and a mean WBC: 22.6 (n= 6, sd 13.9 +/−) in the homozygous SS control mice vs. a mean Hct: 31.25(n=4, sd 6.89+/−), mean MCV: 61(n=4, sd 3.5+/−) mean WBC: 16.3(n= 4, sd 5.99+/−) in the homozygous SS-TR2/TR4 mice. Lastly, initial organ (spleen, liver, kidney) pathology evaluation revealed decreased % body weight (bw) in homozygous SS TR2/TR4 Mice vs. homozygous SS controls: 1) Spleen %bw: 4.3% vs. 3.5% TgTR2/TR4), 2) Liver % bw: 8.8% vs. 7.7% TgTR2/TR4), and 3) Kidney %bw: 1.14% vs. 1.02% TgTR2/TR4). Conclusions:Our preliminary analysis revealed that TR2/TR4 overexpression within a humanized sickle cell disease mouse model resulted in a 2-fold induction of fetal hemoglobin based on HPLC hemoglobin electrophoresis. Further, increased TR2/TR4 overexpression improved anemia and organomegaly within sickle cell disease mice. TR2/TR4 may be an attractive target for fetal hemoglobin induction for the treatment of sickle cell disease. Ongoing studies will determine if TR2/TR4 decreases organ specific disease pathology. We will also determine the cellular distribution of fetal hemoglobin in future studies. Disclosures:No relevant conflicts of interest to declare.

Butyrate Downregulates the Expression of a Number of Repressors of Fetal Hemoglobin In Definitive Erythroid Cells

AbstractAbstract 2080Elevated fetal hemoglobin (HbF) is ameliorative for beta-globin gene disorders. Butyrate, a short chain fatty acid, is a potent inducer of fetal hemoglobin with limited clinical applicability. We wanted to examine non-globin gene targets of butyrate that are regulated in definitive erythroid cells prior to the induction of embryonic/fetal beta-type globin genes. Mechanistic insights may improve clinical utility for short chain fatty acids by identifying novel molecular therapeutic targets.Induced embryonic/fetal globin gene expression is detectable in murine fetal liver-derived definitive erythroid cells (FL EryD) from wildtype and human beta-globin YAC transgenic mice after 19 hours in culture with butyrate & erythropoietin (EPO), but not in EPO alone. Differential regulation of non-globin gene targets in wildtype FL EryD was studied on a Mouse Gene 1.0ST Affymetrix Array after culture in EPO only or butyrate & EPO at 6 hours (when no embryonic globin gene expression is detectable, n=3). Data from biological replicates were normalized by robust multichip average and analyzed with expression console software. As shown in Table 1, several confirmed and putative repressors of embryonic/fetal beta-type globin gene expression, including SOX6, Bcl11A, and Ikaros 1 (but not cMyb) were significantly down regulated by Butyrate at 6 hours (n=3); this was confirmed by RT-PCR. The histone deacetylase inhibitor trichostatin A (TSA), which also induces embryonic globin gene expression in murine FL EryD, has a directionally similar effect (Table 1).Down regulation of some fetal/embryonic globin gene repressors, relative to identically handled EPO-only treated samples, was detectable by RT-PCR as early as 60 to 120 minutes after butyrate induction. These repressors included Bcl11A (60min: 0.66±0.005, p<.001, n=2; 120min: 0.4±0.24, p<.01, n=4), Sox6 (60min: 0.55± 0.18, p=0.08, n=2; 120min: 0.63± 0.06, p<.001, n=4) and Ikaros1 (60min: 0.63±0.45, p=0.36, n=2; 120min: 0.42±0.15, p<.001, n=4).The proximate molecular mechanisms through which butyrate act, while unknown in detail, have been posited to include ‘stress’ signaling via p38 and/or direct activation of gamma-globin gene expression through inhibited histone deacetylation. We found no evidence for butyrate-mediated enhancement of p38 phosphorylation in FL EryD at 0–120 minutes in culture. However, bulk histone acetylation measured by western for histone 3 (H3), was >1.5 fold greater with butyrate induction at 60–90 minutes relative to baseline, while less than baseline in EPO-only treated FL EryD (n=2).Cumulatively, these data suggest that the down regulation by butyrate of major molecular repressors of embryonic/fetal globin gene expression, likely mediated directly or indirectly through epigenetic modifications, is a key underlying mechanism for the induction of fetal hemoglobin in definitive erythroid cells by short chain fatty acids. Disclosures:No relevant conflicts of interest to declare.

Novel Hybrids of Hydroxyurea and Thalidomide Based Pharmacophores Induce Fetal Hemoglobin and Block Monocyte Activation

AbstractAbstract 2673 Introduction:Fetal hemoglobin induction is an effective strategy for the treatment of sickle cell disease (SCD). In addition, targeting systemic inflammation, which consists of activated and injured vascular endothelium, elevated pro-inflammatory cytokines, and activated WBC, promises to reduce vaso-occlusion and disease severity. We therefore aimed to develop novel compounds that combine potent HbF-inducing and anti-inflammatory activities with the goal to improve treatment outcomes. In principle, our rational drug design used molecular hybridization to synthesize three compounds (Lapdesf 1, 2, 3) that link Hydroxyurea's nitric oxide-donating nitrate ester subunit and thalidomide's phthalimide ring with three different intermolecular spacers. Hydroxyurea is to date the only FDA approved drug for the treatment of adult patients with SCD that induces HbF and reduces clinical complications. The release of nitric oxide by HU is an important mechanism of its HbF-inducing properties. On the other hand, thalidomide and its recently developed IMiD derivatives potently inhibit cytokine release from activated monocytes and suppress adhesion molecule expression on vascular endothelium. These properties are critically linked to the phthalimide ring in the thalidomide molecule. The aim of this study was to evaluate the effects of three novel hybrid compounds Lapdesf 1 (2-[4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)phenyl]ethyl nitrate), Lapdesf 2 (4-[1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl]-N-hydroxybenzenesulfonamide), and Lapdesf 3 (3-[1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl]benzyl nitrate) on γ-globin gene expression and cytokine release from activated monocytes. Methods:1. Gamma-globin gene expression. Human K562 cells were maintained in DMEM with 10% FBS, Pen/Strep, in humidified air (5% CO2, 37°C). Cells (1×107cells/100mL) were incubated with compounds at different concentrations (5, 30, 60, and 100μM) for 24, 48, 72 and 96h. Gamma-globin gene expression was analyzed by qRT-PCR and quantified using the Gnorm program. Results were expressed as arbitrary units. 2. Monocyte stimulation. Animals: Adult knockout-transgenic sickle cell mice were anesthetized with Ketamine/Xylazine and blood collected by intracardiac puncture. Mononuclear cells were purified from peripheral blood using Ficoll gradient separation. Mononuclear cells were placed in plastic dishes with DMEM with 10% calf serum and incubated for 2 h in humidified air (5% CO2, at 37°C). Purity of monocytes was > 90% as determined by May-Giemsa staining. Lapdesf 1, 2, and 3 were added to the monocytes cultures at different concentrations (100μM, 300μM and 600μM) 30 minutes before LPS (1 μg/ml). After 20 h incubation supernatants were collected and stored at −80°C. Cytokines were determined by ELISA. Results:1. Gamma-globin gene expression. Lapdesf 1 and 2 were equipotent and demonstrated inverse dose-response relationships achieving the highest levels of γ-globin induction at 5 and 30 μM; in contrast, Lapdesf 2 achieved maximal γ-globin induction as early as 24h after treatment, whereas Lapdesf 1 required 72h of incubation (Lapdesf 2 [24h; 5 μM]: 1.48±0.06 AU; control 0.78±0.1 AU; P<0.05; Lapdesf 1 [72h; 5 μM] 1.78±0.5 AU; control 0.64±0.26; P<0.05). Testing of Lapdesf 3 is currently underway. 2. Monocyte stimulation. Monocytes from sickle mice produced significantly higher levels of TNF-α (1.9 fold), IL-6 (3.48 fold), IL-8 (1.95 fold) and IL-1β (3.42 fold) after LPS stimulation compared to hemizygous controls (P<0.05). Lapdesf 1 and 3 (300 μM) significantly suppressed cytokine production compared to vehicle in LPS-stimulated sickle monocytes and were more potent inhibitors than dexamethasone (Lapdesf 1; Lapdesf 3; Dex: TNF-α : −14.71 fold; −4.64 fold; −4.44 fold; n ≥5; P<0.001; IL-6: −35.68 fold; −12.46 fold, −6.13 fold; n ≥5; P<0.001; IL-8: −7.83 fold; −3.10 fold; −2.55 fold; n ≥5; P<0.001; IL-1β: −14.34 fold; −5.26 fold; −8.9 fold; n ≥5; P<0.01). Lapdesf 2 (300 μM) failed to block cytokine release. Conclusions:These results provide proof-of-concept that our hybrid compounds utilizing critical hydroxyurea and thalidomide based pharmacophores are capable of potent HbF stimulation and anti-inflammatory activities. Testing of our lead compound Lapdesf 1in a preclinical mouse model of SCD is currently underway to evaluate its efficacy in the treatment of this hemoglobinopathy. Disclosures:No relevant conflicts of interest to declare.

Therapy for β‐globinopathies: a brief review and determinants for successful and safe correction

Gene therapy for beta-globinopathies, particularly beta-thalassemia and sickle cell anemia, hold much promise for the future, as a one time cure for these common and debilitating disorders. Correction of the beta-globinopathies using lentivirus vectors (LV) carrying the beta- or gamma-globin genes and elements of the locus control region has been well established in murine models, and a good idea of "what it will take to cure these diseases" has been developed in the first decade of the twenty-first century. A clinical trial using one such vector has been initiated in France while other trials are in development. Vector improvements to enhance the safety and efficiency of LV are being explored, while newer strategies, like homologous recombination in induced pluripotent cells for correction of sickle cell anemia, has been shown as a proof-of-concept. Here we provide a review of current progress in genetic correction of beta-globin disorders.

KLF1 regulates BCL11A expression and γ- to β-globin gene switching

We show that knockdown of KLF1 in human and mouse adult erythroid progenitors markedly reduces BCL11A levels and increases human gamma-globin/beta-globin expression ratios. These results suggest that KLF1 controls globin gene switching by directly activating beta-globin and indirectly repressing gamma-globin gene expression. Controlled knockdown of KLF1 in adult erythroid progenitors may provide a method to activate fetal hemoglobin expression in individuals with beta-thalassemia or sickle cell disease.