Gamma-globin Gene Expression Research Articles

The Effect of Resveratrol on Gamma Globin Gene Expression in Patients with Beta Thalassemia: The Role of Adaptation to Cellular Stress

HbF induction is an appropriate strategy to ameliorate the severity of β-thalassemia symptoms. Hydroxyurea (HU) is the most common chemical agent introduced as an HbF inducer but responsiveness to HU is variable and the introduction of HbF inducers alternative to HU with low cytotoxicity has been a crucial challenge. Resveratrol is an HbF inducer agent that may have favorable effects on the differentiation of hematopoietic erythroid progenitors (HEPs). The present study aimed to investigate the effect of resveratrol on γ-globin, stress response, and anti-apoptotic gene expression among hydroxyurea (HU)-responders and HU-nonresponders (HU-NR). Four cases of HU-R and four cases of HU-NR were studied. HEPs of the patients were cultured, and the expression of γ-globin, Foxo3, and Bclxl was assessed. Moreover, the differentiation and apoptotic rate of the cells were investigated using flow cytometry analysis. In three cases, the γ-globin gene expression increased after resveratrol treatment. All of the HU-NR patients were also non-responders to resveratrol (Res-NR). The expression of Foxo3 and Bclxl genes was higher in responders to resveratrol (Res-R) compared to non-responders (Res-NR). The rate of apoptosis in Res-R patients was also lower than in Res-NR. Responders to resveratrol also had a higher rate of HEP maturation. The cells of both HU–NR and Res-NR patients could not adapt to stress conditions and proceed to the erythroid differentiation. In conclusion, resveratrol increased the γ-globin expression in HEPs of β-thalassemia patients. The response was observed only in R-HU patients with similar cellular pathways.

Hydroxyurea's Impact on Erythropoiesis, Cell Death and HbF Expression in the Hudep-2 Model

Background : Sickle cell disease (SCD) is an inherited disorder caused by a mutation in the beta-globin gene, leading to the synthesis of sickle hemoglobin called HbS. Under deoxygenated conditions, HbS polymerizes and forms fibers that weakens red blood cells (RBC) resulting in hemolysis and vaso-occlusion. Recently, ineffective erythropoiesis in SCD has been reported, characterized by higher apoptosis levels during late erythropoiesis. Interestingly, fetal hemoglobin (HbF)-expressing erythroblasts during erythroid differentiation seem to be protected from the apoptosis. Hydroxyurea (HU) is the first major approved drug for SCD in US and Europe; its proven efficacy in decreasing SCD morbidity and mortality is mainly mediated by the induction of HbF expression that is well-known as a modulator of disease severity through its ability to interfere with HbS polymerization. Despite being used for decades, HU action mechanism still remains to be fully elucidated. More precisely, HU impact on RBC maturation process is poorly documented as well as its mode of action on gamma-globin gene expression. Aim : Our aim is to investigate the effect of HU on erythropoiesis kinetics, HbF induction and apoptosis. Methods : We used HUDEP-2 (Human Umbilical cord Derived Erythroid Progenitors-2) cell line as a model for erythropoiesis. Cells were differentiated in normoxia (20% oxygen) or hypoxia (5% oxygen) - to mimic bone marrow environment, and were treated or not with HU allowing the comparison of HU effect both in normoxia and hypoxia. Analysis was performed at day D1, D3, D6, D8 and D10 of differentiation using flow cytometry, microscopy (MGG staining) and High-Performance Liquid Chromatography (HPLC). Kinetics of differentiation were followed by microscopy with MGG coloration and by flow cytometry using GPA, CD49d, Band3 and HbF antibodies. Apoptosis was assessed using annexin V staining. Results : Wedid first assess HU effect on HbF expression during erythropoiesis by flow cytometry and HPLC. Both methods showed that HU induced HbF expression from D1 to D6 of differentiation and that this induction is higher when the cells are in hypoxic conditions. We also investigated the apoptotic rate during erythropoiesis. Interestingly, HU treatment does not induce apoptosis in differentiating erythroblast, but decreased proliferation rates by 2-fold change in normoxia. Hypoxic conditions also decreased proliferative rate similarly to HU in normoxia but interestingly, in hypoxic conditions, HU did not furthermore decrease cell proliferation. Then, we investigated HU effect on the differentiation kinetics. Based on the differential expression of CD49d and Band3 along the differentiation, the percentage of each stage of differentiation (proerythroblast, basophilic, polychromatic and orthochromatic) was estimated by flow cytometry in GPA + cells at D1, D3, D6, D8 and D10 of the differentiation. Figure 1 depicted flow cytometry representative results from our 4 culture conditions at D1. Our results did show that HU accelerates HUDEP-2 differentiation as soon as D1 of culture (normoxia D1 NT vs HU: ProE 53.3% vs 44%, Baso 29.9% vs 33.1%, Poly 15.2% vs 18% and Ortho 3.3% vs 3.8%) until the end of the differentiation (D8 and 10) in normoxia. This acceleration is accentuated in hypoxic conditions (Hypoxia D1 NT vs HU: ProE 34.7% vs 17.9%, Baso 42.2% vs 50.3%, Poly 17.7% vs 25.8% and Ortho 3.8% vs 4.7%) with a cumulative effect of hypoxia and HU. All the flow cytometry results were confirmed by MGG staining and quantification by microscopy. For the same day of differentiation, HU-treated HUDEP-2 are in later stages than HU-non treated cells. Conclusion : Our study is the first to investigate the impact of HU on RBC maturation - erythropoiesis. HU treatment increases HbF expression without impacting apoptosis. The treatment tends to promote erythroid differentiation by accelerating maturation processes with stronger effect in hypoxic conditions that mimic the in vivo low oxygen environment of the bone marrow, strongly suggesting that HU has other molecular targets than inducing HbF. All these results will be confirmed in CD34 + erythroid progenitors from patients with SCD. Our results give new insights on the effect of HU on erythropoiesis that would ultimately lead to a better understanding of its mechanism of action and to the identification of new therapeutical targets to counteract SCD ineffective erythropoiesis.

Molecular Docking of Andrographoilde and HDAC2 inhibitor an approach to manage for Beta Thalassemia

Beta thalassemia is a disorder of globin gene synthesis resulting in the absent production of beta globin chain in RBC. The property of inducing gamma globin gene expression by the search for bioactive compounds exhibiting is of great interest. Regulation of HDAC activity by dietary flavones couldhave important implication in developing epigenetic therapy to regulate the cell geneexpression. Several pharmacological agents have been identified overthe past two decades that reactivate gamma globin gene transcription through differentcellular system. We will search for the complementary and alternative medicine (CAM)which regulated switch from fetal to adult globin gene expression could be prevented hasresult in the development of new class of therapeutic agent consisting of some bioactivecompounds such as flavanone for the treatment of beta thalassemia. Flavones one suchmolecule of plant origin are known to interfere with HDAC2 enzyme and to enhance theacetylation restoring cell homeostasis, p38 MAPK pathway. Induction of HbF expression inerythroid cells is an important and another therapeutic approach in beta thalassemia. We used Schrodinger suites to develop computational approaches for estimating drug and target binding affinities. The target enzyme's sequence alignments contain 539 amino acid residues, and bioactive compounds that act as ligand molecules were acquired from the plant Andrographis paniculata. We used molecular docking, and ADME analysis. The current study explores the binding pattern of Andrographoilde and Vorinostat (SAHA) against their target protein (PDBIDs: 6G3O), respectively using molecular docking approaches. The molecular docking results show that Andrographoilde binds within the active region of HDAC2 inhibitors by forming hydrogen bonds against ASP104 and TYR 308 respectively. However, the Vorinostat interacts with TYR 308 and GLY 142respectively. Our comparative results show that andrographoilde may be a similar therapeutic agent for managing beta thalassemia as compare to vorinostat. However, functional studies are needed to confirm their proposed relevance in beta thalassemia in vitro and in vivo evaluation studies are required.

Whole Blood Transcriptome Analysis in Children with Sickle Cell Anemia.

Whole transcriptome RNA-sequencing was performed to quantify RNA expression changes in whole blood samples collected from steady state sickle cell anemia (SCA) and control subjects. Pediatric SCA and control subjects were recruited from Atlanta (GA)—based hospital(s) systems and consented for RNA sequencing. RNA sequencing was performed on an Ion Torrent S5 sequencer, using the Ion Total RNA-seq v2 protocol. Data were aligned to the hg19 reference genome and analyzed in the Partek Genomics studio package (v7.0). 223 genes were differentially expressed between SCA and controls (± 1.5 fold change FDR p < 0.001) and 441 genes show differential transcript expression (± 1.5 fold FDR p < 0.001). Differentially expressed RNA are enriched for hemoglobin associated genes and ubiquitin-proteasome pathway genes. Further analysis shows higher gamma globin gene expression in SCA (33-fold HBG1 and 49-fold HBG2, both FDR p < 0.05), which did not correlate with hemoglobin F protein levels. eQTL analysis identified SNPs in novel non-coding RNA RYR2 gene as having a potential regulatory role in HBG1 and HBG2 expression levels. Gene expression correlation identified JHDM1D-AS1(KDM7A-DT), a non-coding RNA associated with angiogenesis, enhanced GATA1 and decreased JAK-STAT signaling to correlate with HBG1 and HBG2 mRNA levels. These data suggest novel regulatory mechanisms for fetal hemoglobin regulation, which may offer innovative therapeutic approaches for SCA.

Cell erythroid maturation approach: a new paradigm in the road map towards a cure for β-thalassaemia syndromes.

Cell erythroid maturation approach: a new paradigm in the road map towards a cure for β-thalassaemia syndromes.

Fetal Hemoglobin (HbF) Silencer BCL11A Acts through a Novel DNA-Motif in the Gamma-Globin Promoters, Simplifying the Model for Hemoglobin Switching

Fetal hemoglobin (HbF) level is under strong genetic control. Common genetic variation is associated with modest elevation in HbF levels in adults. Genome-wide association studies led to identification of the regulatory factor BCL11A, which is a major contributor to HbF silencing. Despite clear evidence for its involvement, how BCL11A acts to repress gamma-globin gene expression has remained unknown. Conflicting data exist in the literature with regard to whether BCL11A binds DNA directly and, if so, the sequence(s) it may recognize. Prior experiments from our laboratory suggested that BCL11A acts at a distance from the gamma-globin gene promoters, but failed to define with confidence sites of occupancy within the beta globin locus. To explore mechanisms by which BCL11A functions, we first devised a cellular assay for rescue of BCL11A-deficient erythroid cells with BCL11A cDNA constructs. This approach established that of the two longer BCL11A isoforms (L and XL), only XL (“eXtraLong”) is competent to repress globin expression in cells. XL differs from L by the presence of an alternatively spliced terminal exon encoding three closely spaced zinc-fingers (ZF4-6), implicating this domain in an essential function of the protein. Given that ZFs often mediate sequence-specific DNA-binding, we employed a Protein Binding Microarray platform (PBM) that allows for high-throughput determination of transcription factor binding specificities at high-throughput on an oligonucleotide matrix. We determined that recombinant ZF4-6, as well as BCL11A-XL, binds sequence-specifically to a novel consensus motif with a central core, TGACCA. Two biophysical methods confirmed recognition of this motif by recombinant ZF4-6 with an affinity of ~ 25nM. The motif is present in promoters of all fetal or embryonic-expressed globin genes in human and mouse, and in two copies in each human gamma-globin gene promoter (-119 to -113 and -92 to -86). The motif is altered by single base substitutions in rare HPFH alleles (-117 and -114) and by a microdeletion HPFH (-114 to -102). HPFH-associated single base substitutions greatly impair ZF4-6 binding, therefore strongly implicating a direct role for BCL11A at the gamma-globin gene promoters. However, numerous attempts to localize endogenous BCL11A protein in chromatin of erythroid cells by conventional chromatin immunoprecipitation sequencing (ChIP-seq) proved unsuccessful. To determine if the gamma-globin gene promoters are indeed bound by BCL11A in vivo in erythroid cells, we adapted a newly described method for detection of protein occupancy in chromatin (CUT&RUN) at higher resolution and without crosslinking that is commonly used in ChIP-seq. With this method, we detected BCL11A occupancy in the gamma-globin gene promoters in adult erythroid cells, including CD34 cells, and moreover observed that the distal one of the repeated motifs is the predominant binding site in vivo . Taken together, these experiments provide persuasive evidence that BCL11A acts locally at the gamma-globin gene promoters to repress transcription and that classical HPFH alleles in the (-117 to -114) region lead to increased HbF due to failure of BCL11A binding. These findings are consistent with a revised model of HbF silencing in which BCL11A acts through direct gamma-globin promoter binding, but do not exclude additional architectural or long-distance roles of BCL11A in HbF silencing. The critical role of the BCL11A DNA-binding motif in mediating HbF repression further supports the rationale for use of genome editing to target this region as therapy of the hemoglobinopathies. DisclosuresOrkin:Bioverativ: Consultancy; Epizyme Inc.: Consultancy.

Krüppel-like factor 1 (KLF1) gene single nucleotide polymorphisms in sickle cell disease and its association with disease-related morbidities.

Sickle cell disease has varied clinical symptoms, and patients having high fetal hemoglobin (HbF) have milder symptoms. Various genetic factors are known to modulate the HbF levels. Krüppel-like factor 1 (KLF1) is a transcription factor that regulates the beta-like globin gene expression. Any variation in KLF1 gene may alter the sickle cell disease phenotype. Xmn-I polymorphism is also known to regulate the gamma globin gene expression. Present studies were carried out to investigate the effect of KLF1 gene mutations and Xmn-I polymorphism on the sickle cell disease severity and to ascertain the genotype-phenotype correlation. One hundred and eighteen sickle cell disease patients having a median follow-up of 5years (3-10years) were recruited. Clinical details were recorded from their retrospective medical records. Xmn-I polymorphism were analyzed using PCR-RFLP method. Variations in KLF1 gene were identified using Sanger sequencing. Out of 118 patients, 24 had acute chest syndrome and 21 patients had more than 2 pain episodes per year. There were no significant differences in sickle cell disease-related morbidities in male and females barring leg ulcers. A total of 6 polymorphism were observed in KLF1 gene, out of which 3 are novel (c.-304G > C, c.*141A > G and c.*178A > G). No statistically significant association of any of SNPs identified in KLF1 gene or Xmn-I polymorphism was seen with HbF levels as well as the sickle cell disease-related morbidities. No association exists between fetal hemoglobin or sickle cell disease-related morbidities and Xmn-I polymorphism or with SNPs identified in KLF1 gene in the studied cohort.

Manipulation of Developmental Gamma-Globin Gene Expression: an Approach for Healing Hemoglobinopathies.

β-Hemoglobinopathies are the most common monogenic disorders, and a century of research has provided us with a better understanding of the attributes of these diseases. Allogenic stem cell transplantation was the only potentially curative option available for these diseases until the discovery of gene therapy. The findings on the protective nature of fetal hemoglobin in sickle cell disease (SCD) and thalassemia patients carrying hereditary persistence of fetal hemoglobin (HPFH) mutations has given us the best evidence that the cure for β-hemoglobinopathies remains hidden in the hemoglobin locus. The detailed understanding of the developmental gene regulation of gamma-globin (γ-globin) and the emergence of gene manipulation strategies offer us the opportunity for developing a γ-globin gene-modified autologous stem cell transplantation therapy. In this review, we summarize different therapeutic strategies that reactivate fetal hemoglobin for the gene therapy of β-hemoglobinopathies.

Establishing a Link between Zinc Homeostasis and Globin Gene Expression

Sickle cell disease (SCD) affects millions of people around the world and is the most common inherited disease in the United States, affecting 1 of 1800 births and 1 of 400 African-American births. Patients with SCD have an improved clinical course when fetal hemoglobin (HbF) levels are increased, and this is the basis for treatment with hydroxyurea as a disease modifying therapy. Hydroxyurea has been shown to decrease the frequency of pain episodes and decrease the incidence of stroke in patients who are at risk based on transcranial doppler velocities. Hydroxyurea is one of two FDA approved treatments for SCD patients, so there is need for new therapies, including the identification of druggable transcriptional activators that specifically up-regulate the gamma-globin genes (HbF). Understanding the mechanisms underlying control of globin gene expression, particularly those involved in activation of gamma-globin gene expression, is important for developing new treatments for SCD. Metal-responsive transcription factor-1 (MTF-1) is a key regulator of zinc metabolism in higher eukaryotes that controls the metal-inducible expression of metallothioneins and a number of other genes directly involved in the intracellular sequestration and efflux transport of zinc. Bao et al published that adults with SCD showed increased red blood cells (RBCs), hemoglobin (Hb) and hematocrit levels after 3 months of zinc supplementation compared to the placebo group. Furthermore, previous studies demonstrated that MTF-1 plays an essential role in liver development and that MTF-1-deficient mice display an anemic phenotype, suggesting a role for MTF-1 in hematopoiesis. In our study, we observed a 2.4-fold increase in gamma-globin expression in K562 cells at 4 hours. We also demonstrated increased gamma-globin expression in adult blood from MTF-1 overexpression, human beta-globin locus yeast artificial chromosome (beta-YAC) bi-transgenic (bigenic) mouse lines at the mRNA level by quantitative real-time RT-PCR (qPCR) and at the protein level by FACS analysis. Lastly, gamma-globin gene expression was induced 12-fold in bone marrow cells (BMCs) derived from these bigenic mice compared to BMCs derived from beta-YAC-only mice, and 3-fold after 6 hours of zinc treatment in beta-YAC-only BMCs. Co-immunoprecipitation (Co-IP) analysis showed that GATA-2 associated with MTF-1 in MTF-1 overexpression, beta-YAC BMCs, but not in beta-YAC-only BMCs, suggesting that reactivation of gamma-globin expression by MTF-1 might be mediated by a MTF-1-GATA-2 protein complex. Chromatin immunoprecipitation (ChIP) experiments indicated that MTF-1 and GATA-2 co-occupy the same sites in the gamma-globin promoter. Our data suggest that activation of gamma globin by MTF-1 is mediated by protein-protein interaction with GATA-2 and that this multi-protein complex is targeted to GATA sites located in the gamma-globin gene-promoters via binding of the GATA-2 protein. Based on our data, we hypothesize that zinc supplementation will increase plasma HbF by inducing MTF-1 thus activating gamma-globin gene expression. This protein represents a potential new target in strategies to reactivate gamma-globin in hemoglobinopathies where higher levels of HbF would have beneficial effects. More clinical and basic science studies are needed to further characterize the role of zinc in globin gene expression and hemoglobin F levels in the clinical setting. Disclosures No relevant conflicts of interest to declare.

Knockdown of HNF4A Gene Increases Fetal Hemoglobin Synthesis in Hudep-2

Sickle cell anemia is a recessive inherited disease caused by a single nucleotide polymorphism in the β-globin gene the resulting substitution of glutamic acid by valine causes red blood cell sickling when deoxygenated. Some hypomethylating agents are able to induce the expression of γ-globin by inhibiting DNA methylation at the gene promoter. However, it is not completely understood how this regulation occurs and which genes are involved in this process. To understand fetal hemoglobin (HbF) regulation, CD34+ cells were treated with 1µM of decitabine on the 9th day of culture to induce HbF production. The expression levels of transcription factors and chromatin modifiers were evaluated through the PCR Array platform (Qiagen™ Germany). The transcription factor, HNF4A (Hepatocyte Nuclear Factor 4 Alpha), was highly upregulated in cells treated with decitabine, compared to the control cells, and was chosen as a candidate for CRISPR/Cas9 knockout in HUDEP-2 cells (immortalized human erythroid progenitor cells). The HNF4A gene has been reported as a transcription factor, which regulates the expression of several hepatic genes, and is able to play a role in the development of the liver, kidney, and intestines. Moreover, HNF4A is expressed in the hematopoietic tissue. To the best of our knowledge, the association between HNF4A and gamma globin gene synthesis has not been previously described. HUDEP-2 cells were cultured and then treated with 50nM decitabine. After 72 hours, HbF levels were measured with anti-HbF antibody by flow cytometry in three biological replicates. The percentage of cells positive for HbF in decitabine-treated HUDEP-2 were 12.27 ± 0.7%, N=3, while in control cells the percentage was 1.0 ± 0.06%, N=3 (p&amp;lt;0.0001). These results corroborate the increased expression measured in CD34+ cells. To knock out HNF4A in HUDEP-2 cells, we used the CRISPR/Cas9 system. We generated INDELs in heterozygosity for HNF4A. Briefly, HUDEP-2 cells were nucleofected with Cas9 high fidelity ribonucleoprotein (104 pmol), crRNA:tracrRNA (120 pmol) complex and 1µM of gRNA HNF4A using a CD34+ human cell kit and the E-001 program in an AMAXA Nucleofector 4D-device (Lonza). Two days after nucleofection, edited HUDEP-2 cells were submitted to clonal selection and expanded for approximately 28 days. Genomic DNA from clones was analyzed by a Sanger Sequencer, and four edited HNF4A-HUDEP-2 clones were selected with INDELs in the fourth exon of HNF4A gene. These clones were expanded in culture with controls, and the HbF levels were quantified by flow cytometry. HbF levels in these four clones were 8.2 ± 2.4%, while in the wild type HUDEP-2, HbF was 0.9 ± 0.02% (p&amp;lt; 0.05). The edited clones expressed significantly more HbF than the controls, although this expression was not homogeneous. Western blotting of edited HNF4A-HUDEP-2 clones demonstrated decreased HNF4A at the protein level (0.35 ± 0.9 AU N=8), compared to controls (2.02± 1.1 AU N=3, p&amp;lt; 0.05). Results suggest that HNF4A may play a role in the gamma globin gene transcription in HUPED-2 cells. The mechanism of how HNF4A may regulate gamma globin gene expression remains to be clarified. Disclosures No relevant conflicts of interest to declare.

PB2402 BETA‐GLOBIN GENE CLUSTER HAPLOTYPES ASSOCIATED WITH HBF VARIATION IN β‐THALASSEMIA CARRIERS OF PORTUGUESE ORIGIN

Background:Genetic association studies showed that HbF levels variation in haemoglobinopathies, such as sickle cell disease and β‐thalassemia (β‐thal), are under the influence of single nucleotide polymorphisms (SNPs) in several genomic loci. The first described polymorphism influencing gamma globin gene expression was XmnI in HBG2 promoter −158C &gt; T (rs7482144). Several reports showed that β‐thal carriers with HbF raised levels have a significantly higher rs7482144 minor allele frequency than patients with low HbF levels. The functional significance of HBG2‐XmnI mutation has never been clearly defined and it is possible that it reflects a marker in linkage disequilibrium (LD) with other functional polymorphism elsewhere in the β‐globin gene cluster. It has been suggested that the role assigned to XmnI could be played by the rs16912979 A &gt; G polymorphism in the palindromic region of 5′HS4 β‐globin locus control region (Neishabury et al., Blood Cells Mol Dis. 2012;48:1‐5).Aims:The main goal of this work was to define haplotype backgrounds in β‐globin gene cluster that could explain HbF variation in a population sample of β‐thal carriers.Methods:We studied 79 Portuguese β‐thal carriers (39 males, 40 females), aged between 2 to 70 years old, with HbF levels ranging from 0.2% to 5.1%. HbA2 levels were 3.4 to 5.8% (mean 4.71). Six different HBB mutations were identified at heterozygous state, the two most commons were c.92+6T &gt; C [IVSI‐6 (T &gt; C)] (n = 30) and c.118C &gt; T [CD39 CAG &gt; TAG)] (n = 23). The remaining mutations were c.48G &gt; A [CD15 TGG &gt; TGA] (n = 11), c.92 + 1G &gt; A [IVS‐I‐1 (G &gt; A)] (n = 10), c.93‐21G &gt; A [IVSI‐110 G &gt; A] (n = 3) and c.20delA [CD 6 ‐A] (n = 2). HbA2 and HbF levels were determined by HPLC. The two SNPs rs16912979 and rs7482144 were genotyped by PCR‐RFLP and the HBB gene mutations were identified by Sanger sequencing. Associations of SNPs with HbF levels were assessed by linear regression models using PLINK. Informed consent was provided by all the participants.Results:The two SNPs HBG2‐XmnI rs7482144 and 5′HS4 rs16912979 were in moderate LD (r2 = 0.65): from a total of 41 rs16912979 G alleles, 73.2% were linked to an rs7482144 T‐allele. Linear regression, in the additive model, showed significant association between the XmnI rs7482144 minor T‐allele and increased levels of HbF (β = 0.648; P = 0.0004). A marginal association was found between the 5’HS4 rs16912979 minor G‐allele and HbF (β = 0.648; P = 0.039), however, no significant association was found after conditioning on XmnI rs7482144 (P = 0.192). The common mutation HBB: c.118C &gt; T [CD39] was found strongly associated with increased levels of HbF (β = 0.948; P = 6.4e‐5), remaining significant after conditional analysis on XmnI rs7482144 (P = 0.0002). Haplotypes combining the HbF‐associated alleles on SNPs rs16912979 and rs7482144 (haplotype GT) showed a significant association with raised levels of HbF (P = 0.0008), explaining 13.7% of HbF levels variation. Combining rs16912979, rs7482144 and mutation HBB: c.118T, three haplotypes were found significantly associated with HbF: GTT (β = 1.346; P = 0.00089), ACT (β = 0.784; P = 0.017) and GTC (β = 0.492; P = 0.032), explaining 13.4%, 7.2% and 5.9% of HbF levels variance, respectively.Summary/Conclusion:Our data suggest that in Portuguese β‐thal carriers the HBG2‐XmnI polymorphism and HBB: c.118C &gt; T [CD39] mutation are associated with HbF levels, showing independent effects. The marginally significant effect on HbF levels variation of SNP 5’HS4 rs16912979 appear to be mediated by the effect of XmnI polymorphism.

The Role of Adenosine Monophosphate Activated Protein Kinase Alpha 1 (AMPK) in Gamma-Globin Regulation

Background: Fetal hemoglobin (HbF, α2g2) induction has long been an area of investigation, as it is known to reduce the clinical complications of sickle cell disease (SCD) and beta thalassemia. Progress in identifying novel HbF inducing strategies has been stymied by an incomplete understanding of gamma-globin regulation. We used natural genetic variation to identify novel genes and pathways associated with HbF levels in patients with SCD. Our whole exome sequencing analysis of 1290 samples from patients with SCD identified the insulin signaling pathway to be related to HbF regulation. Functional studies performed in hematopoietic stem and progenitor cells (HSPCs) from patients with SCD established that FOXO3 is a positive regulator of HbF, and that metformin, a FOXO3 and AMPK activator, can induce HbF (Zhang et al, Blood 2018). We hypothesized that other proteins in the insulin signaling pathway, particularly AMPK, a direct activator of FOXO3, may contribute to HbF regulation and be a potential target for pharmacologic induction of HbF.Objectives: We now seek to determine the role of AMPK and AMPK activators such as piceatannol in HbF regulation through functional studies in HSPCs from patients with SCD.Methods: HSPCs from 3 unique patients with SCD were transduced with AMPK shRNA on day 5 of two phase primary erythroid culture. AMPK, FOXO3, gamma and beta globin gene expression were measured by RT-qPCR and HbF by HPLC respectively on day 14 of culture. HSPCs from 3 unique patients with SCD were treated with AICAR, piceatannol at 12.5µM and metformin at 100 µM on day 7 of erythroid culture. Cell lysate was collected on day 14, and AMPK, FOXO3, gamma and beta globin gene expression and protein levels measured by RT-qPCR and western blot respectively. Levels of pAMPK, at Thr172, were quantified by western blot. 1 µM Compound C was added with piceatannol and with metformin in separate erythroid cultures on day 7, and the effect on gamma globin and phosphorylation of AMPK at Thr172 was measured on day 14 by RT-qPCR and western blot respectively.Results: 70% knockdown of AMPK resulted in a 50% decrease in HbF (p<0.01) and a three-fold reduction in gamma-globin expression (p<0.001). HSPCs treated with metformin or piceatannol exhibited a 2-3 fold rise in AMPK, FOXO3 and gamma globin gene expression (p<0.001). HSPCs treated with piceatannol and metformin showed an increase in pAMPK at Thr172, the activated form of AMPK. In the presence of a specific AMPK inhibitor, Compound C, metformin and piceatannol, no induction of gamma globin was observed (Figure 1), and pAMPK was reduced to untreated levels.Conclusions: Knockdown of AMPK in HSPCs reduces gamma globin expression and %HbF, supporting the role of AMPK in gamma globin regulation. Drugs known to activate AMPK, metformin and piceatannol, increase gamma globin in SCD patient derived HSPCs. Pharmacologic blockage of AMPK activity with Compound C results in reduction of HbF induction, and reduces the gamma globin induction of metformin and piceatannol to untreated levels. We therefore conclude that AMPK is a positive regulator of HbF, and that pharmacologic induction of HbF with metformin and piceatannol requires AMPK activity. Further work is needed to establish if FOXO3 and AMPK alone are instrumental in HbF regulation, or if other proteins in the insulin signaling pathway may play a role in HbF regulation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Synergistic Effect of Simvastatin and Romidepsin on Gamma-globin Gene Induction.

Objective Hemoglobinopathies such as beta-thalassemia and sickle cell disease (SCD) are inherited disorders that are caused by mutations in beta-globin chain. Gamma-globin gene reactivation can ameliorate clinical manifestations of beta- thalassemia and SCD. Drugs that induce fetal hemoglobin (HbF) can be promising tools for treatment of beta-thalassemia and SCD patients. Recently, it has been shown that Simvastatin (SIM) and Romidepsin (ROM) induce HbF. SIM is a BCL11a inhibitor and ROM is a HDAC inhibitor and both of these drugs are Food and Drug Administration (FDA)-approved for hypercholesterolemia and cutaneous T-cell lymphoma respectively. Our aim was to evaluate the synergistic effects of these drugs in inducing HbF. Materials and Methods In our experimental study, we isolated CD34+ cells from five cord blood samples that were cultured in erythroid differentiation medium containing ROM and Simvastatin. Then Gamma-globin, BCL11a and HDAC gene expression were evaluated on the 7thand 14thday of erythroid differentiation by real-time polymerase chain reaction (PCR) and immunocytochemistry. Results Our results showed that combination of SIM and ROM significantly increased Gamma-globin gene expression and inhibit BCL11a and HDAC expression compared to results of using each of them alone. SIM and ROM lead to 3.09- fold increase in HbF production compared to the control group. Also, SIM inhibited BCL11a expression (0.065-fold) and ROM inhibited HDAC1 expression (0.47-fold) as two important inhibitors of HbF production after birth. ConclusionWe propose combination therapy of these drugs may be ameliorate clinical manifestation in beta-thalassemia and SCD with at least side effects and reduce the need for blood transfusion.

Nrf2 Activation Attenuates Hemolysis but Promotes Pulmonary Vascular Dysfunction in Sickle Mice

Nuclear factor erythroid 2-related factor 2 (Nrf2) is the major transcription factor that coordinates the body’s antioxidant and cytoprotective defense against a variety of toxins. Several Nrf2 activators can reactivate gamma globin gene expression and augment fetal hemoglobin production. More recently, genetic and pharmacologic evidence have shown that Nrf2 activation can specifically mitigate the severity of hemolytic anemia, and systemic and local inflammation in transgenic sickle cell disease (SCD) mice. Based on these encouraging results Nrf2 activation has emerged as an attractive therapeutic strategy in SCD. However, the BEACON trial of the Nrf2 activator CDDO-Methyl Ester (CDDO-Me) showed that this therapeutic approach can cause adverse cardiovascular events in patients with chronic kidney disease with comorbid diabetes. Hitherto, the efficacy-toxicity profile generated by individual Nrf2 activating drugs has not been investigated in SCD. There are hundreds of synthetic and naturally occurring Nrf2 activating compounds, and each class of Nrf2 activating compound has a unique pharmacokinetic, pharmacodynamic, toxicokinetic and toxicodynamic profile. We have recently demonstrated that intravascular hemolysis deteriorates with aging in transgenic sickle (SS) mice in a process that can be mitigated by the Nrf2 activator 3H-1,2-dithiole-3-thione (D3T) (Ghosh et al., JCI Insight, 2016). In this study, an in vitro screen of five Nrf2 activating compounds revealed CDDO-Me to be the most potent inducer of cytoprotective enzymes in human pulmonary microvascular endothelial cells. Thus, we performed a long-term prophylactic CDDO-Me treatment of SS mice and examined the effect of the drug on intravascular hemolysis and vascular dysfunction. A cohort of newly weaned SS mice aged ~4 weeks were randomly assigned to receive CDDO-Me (20µmoles/kg/TIW, n=6) or Vehicle (DMSO/TIW, n=10) by oral gavage for 4 months. After the treatment, the total hemoglobin increased by 10% in the CDDO-Me group while it decreased by 5% in the vehicle-treated group (p<0.05). Plasma concentration of VCAM-1 increased in both groups compared to their respective baseline (vehicle p<0.001; CDDO-Me p<0.05). The lack of impact on VCAM-1 by CDDO-Me was contrary to our results with D3T (Ghosh et al., JCI Insight, 2016). To examine this further, we performed vascular reactivity studies on isolated pulmonary arteries. Second order pulmonary arteries were isolated, cut into 2mm rings, and mounted using 40 μm wire. Rings were set at a resting tension equivalent to 100 mmHg transmural pressure. Cumulative dose response curves of endothelin-1 (100 pM-10 nM) and acetylcholine (10nM-100 μM) were used to study vasoconstriction and vasodilation, respectively. Pulmonary arteries isolated from the CDDO-Me treated SS mice showed a 15% reduction in acetylcholine stimulated vasodilation compared to the arteries isolated from the vehicle treated littermates (vehicle n=5, CDDO-Me n=5; p<0.001). In vitro studies of rat smooth muscle cells revealed significant up-regulation of endothelin receptor subtype A (ETA) mRNA by CDDO-Me (p < 0.01, n=6). We obtained similar results of increased ETA expression in rat smooth muscle cells treated with another Nrf2 activator, Dimethyl Fumarate (DMF) (p < 0.01, n=6). Upregulation of endothelin or the ETA is frequently found in pulmonary hypertension. The hypertensive phenotype that we report here in SS mice treated with CDDO-Me is consistent with the adverse cardiovascular events found in the BEACON trial. Together our results show that while CDDO-Me can stabilize and potentially improve hemolytic anemia in SCD, this drug and potentially other Nrf2 activators may promote the development of pulmonary hypertension, which is a major adult complication of SCD. DisclosuresNo relevant conflicts of interest to declare.

HMGA2 Moderately Increases Fetal Hemoglobin Expression in Human Adult Erythroblasts

Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with beta-hemoglobin disorders. Previous studies showed that let-7 microRNAs (miRNAs) are highly regulated in erythroid cells during the fetal-to-adult developmental transition, and that targeting let-7 mediated the up-regulation of HbF to greater than 30% of the total globin levels in human adult cultured erythroblasts. HMGA2 is a member of the high-mobility group A family of proteins and a validated target of the let-7 family of miRNAs. Here we investigate whether expression of HMGA2 directly regulates fetal hemoglobin in adult erythroblasts. Let-7 resistant HMGA2 expression was studied after lentiviral transduction of CD34(+) cells. The transgene was regulated by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2-OE). HMGA2-OE caused significant increases in gamma-globin mRNA expression and HbF to around 16% of the total hemoglobin levels compared to matched control transductions. Interestingly, no significant changes in KLF1, SOX6, GATA1, ZBTB7A and BCL11A mRNA levels were observed. Overall, our data suggest that expression of HMGA2, a downstream target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in adult human erythroblasts.

Erythroid-Specific Expression of LIN28A Is Sufficient for Robust Gamma-Globin Gene and Protein Expression in Adult Erythroblasts.

Increasing fetal hemoglobin (HbF) levels in adult humans remains an active area in hematologic research. Here we explored erythroid-specific LIN28A expression for its effect in regulating gamma-globin gene expression and HbF levels in cultured adult erythroblasts. For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes. Transgene expression of LIN28A with a linked puromycin resistance marker was restricted to the erythroid lineage as demonstrated by selective survival of erythroid colonies (greater than 95% of all colonies). Erythroblast LIN28A over-expression (LIN28A-OE) did not significantly affect proliferation or inhibit differentiation. Greater than 70% suppression of total let-7 microRNA levels was confirmed in LIN28A-OE cells. Increases in gamma-globin mRNA and protein expression with HbF levels reaching 30–40% were achieved. These data suggest that erythroblast targeting of LIN28A expression is sufficient for increasing fetal hemoglobin expression in adult human erythroblasts.

Study on the Efficacy of a JAK Inhibitor Pharmacological Agent As Inducer of Fetal Hemoglobin Production in Cultured Erythroid Precursors from Sickle Cell and Beta-Thalassemia Patients

Phenotypic improvement of hemoglobinopathies such as sickle cell disease and beta-thalassemia (beta-thal) has been shown in patients with high levels of fetal hemoglobin (HbF). In sickle cell disease (SCD) the beneficial effects of HbF are due to the inhibition of HbS polymerization and to the dilution of HbS determining the reduction of sickling and vascular occlusion. Moreover, in beta-thal, high levels of gamma-chains combined with the redundant alpha-chains, lead to a reduction of dyserythropoiesis and of the requirement for blood transfusions. The only drug approved for the treatment of adult patients with SCD and that has been entered in clinical practice of patients affected by beta-thal is hydroxyurea (HU); however there is a great variability in the responses of patients to HU, in fact some patients are good responder, while others exhibit little or no change in HbF levels after HU treatment; moreover a decrease in the efficacy during long term treatment was observed. Other pharmacological compounds, including 5-azacytidine and thalidomide have been shown to increase HbF production. Due to concerns about the safety of this agents, their use was limited to severe cases for whom conventional therapy was unfeasible. For this reason the search of new inducers of HbF production is important. Ruxolitinib is a JAK inhibitor and decreases the phosphorilation of STAT (Signal transducers and activators of transcription) family proteins, in particular STAT5 and STAT3. Phosphorylation of STAT5 is essential for basal erythropoiesis and for its acceleration during stress erythropoiesis. STAT3 plays an essential role in regulating gene expression of several genes involved in cell growth and apoptosis, in particular it was demonstrated to inhibit gamma-globin gene expression. The decrease of STAT3 phoshorilation could decrease the inhibition of gamma-globin gene expression; for this reason we considered ruxolitinib a candidate as inducer of HbF production.In our laboratory an ex vivo system was developed predictive of the in vivo response to hydroxyurea treatment by using liquid erythroid cultures, an in vitro culture system that recapitulates the process of human erythropoiesis.To evaluate the efficacy of ruxolitinib in increasing gamma-globin gene expression we carried out a study in vitro using liquid erythroid cultures. In this study we developed and exposed to ruxolitinib liquid erythroid precursors from 4 SCD and 17 beta-thal intermedia (beta-TI) patients. The use of quantitative Real-Time-polymerase chain reaction allowed us to determine the increase in gamma-globin mRNA expression in human erythroid cells treated with ruxolitinib compared to untreated cells. The results are summarized in Table 1 and showed that ruxolitinib at 200nM is able to determine a significant increase of gamma-globin gene expression (3.4±0.1)compared to HU (2.0± 0.2).In conclusion our study suggests that ruxolitinib could be considered an inducer of HbF and could be used in vivo for the treatment of hemoglobinopathies, particularly in patients who do not respond to HU therapy or who show a decreased response after long-term treatment.Table 1Fold increase of Gamma-globin gene expression in presence of Ruxolitinib in erythroid cultured cells.PatientSexGenotypegamma-globin mRNA fold increasein the presence of ruxolitinib#1Mb039/aaa+1#2Fb039/aaa+1.65#3Fb039/b039+1.9#4Fb039/IVS1,110+1.5#5MIVS1,1/aaa+2.5#6MIVS1,110/IVS1,1+9.2#7Mb039/bs+6#8Fbs/b039+1.6#9Fb039/IVS1,6+1.7#10MIVS1,6/frcd6+3#11MIVS1,6/bs+2.5#12MIVS1,6/frcd6+8#13FIVS1,6/b039+9#14MIVS1,1/b039+2.2#15Mdb/IVS1,110+8#16Fdb/IVS1,110+1.8#17FIVS2,1/aaa+3.9#18Mb039/-101+1.4#19MIVS1,6/b039+1#20Mbs/IVS1,110+1.4#21MIVS1,6/IVS1,6+1.9 DisclosuresNo relevant conflicts of interest to declare.

Erythroid-Expression of HMGA2 Increases Fetal Hemoglobin in Human Adult Erythroblasts

HMGA2 is a member of the high-mobility group A family and plays a role in the regulation of gene transcription and chromatin structure. HMGA2 is a validated target of the let-7 family of miRNAs. Let-7 miRNAs are highly regulated in erythroid cells during the fetal-to-adult developmental transition (1). Recent studies demonstrated that the LIN28 -let-7 axis mediated up-regulation of fetal hemoglobin (HbF) expression to >30% of the total globin levels in cultured erythroblasts from adult humans (2) and the amelioration of hypoxia-related sickling of cultured mature erythrocytes from pediatric patients with sickle cell disease (3). Interestingly, increased expression of endogenous HbF in a patient receiving gene therapy was also associated with truncated HMGA2 protein expression after lentiviral integration and disruption of let-7 targeting at the HMGA2 gene locus (4). Therefore, we hypothesized that HMGA2 may be involved in fetal hemoglobin regulation as a downstream target of the let-7 miRNAs.To study the effects of HMGA2 upon erythropoiesis and globin expression, lentiviral constructs were designed for let-7 resistant expression of HMGA2 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2 -SPTA1-OE), with a matched empty vector control. Transductions were performed in CD34+ cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Overexpression of HMGA2 was confirmedby Q-RT-PCR (control: below detection limits; HMGA2 -SPTA1-OE: 2.51E+04 ± 3.44E+04 copies/ng) and Western blot analyses at culture day 14. Cell counting revealed no significant changes between HMGA2 -SPTA1-OE and control (empty vector) transductions at culture day 14. Terminal maturation with loss of CD71 from the erythroblast cell surface and enucleation assessed by thiazole orange staining were analyzed in the control and HMGA2 -SPTA1 -OE samples at the end of the culture period. Globin genes expression levels were evaluated for HMGA2 -SPTA1-OE by Q-RT-PCR. HMGA2 -SPTA1-OE caused a significant increase in gamma-globin mRNA expression levels compared to controls (control: 5.02E+05 ± 8.62E+04 copies/ng; HMGA2 -SPTA1-OE: 1.45E+06 ± 7.31E+05 copies/ng; p=0.037). Consistent with the increase in gamma-globin mRNA levels, HPLC analyses at culture day 21 demonstrated modest but significant increases in HbF levels in HMGA2 -SPTA1-OE compared to controls (HbF control: 5.41 ± 2.15%; HMGA2 -SPTA1-OE: 16.53 ± 4.43%; p=0.006).Possible effect(s) and downstream mechanism(s) triggered by HMGA2 -SPTA1-OE were investigated. Q-RT-PCR analyses demonstrated no significant changes in the let-7 family of miRNAs in HMGA2 -SPTA1-OE compared to controls. Expression patterns of several transcription factors such as BCL11A, KLF1, SOX6 and GATA1 were investigated by Q-RT-PCR and no significant changes were detected in HMGA2 -SPTA1-OE compared to controls. While BCL11A mRNA levels were decreased by HMGA2 -SPTA1 -OE, the differences did not reach statistical significance (control: 4.26E+02 ± 8.18E+01 copies/ng; HMGA2 -SPTA1 -OE: 2.84E+02 ± 1.48E+02 copies/ng; p=0.104). However, nuclear BCL11A protein levels assessed by Western analysis were suppressed in HMGA2 -SPTA1 -OE.In summary, these results demonstrate that HMGA2, a validated target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in human erythroblasts, and reduces levels of BCL11A protein. These data thus support the notion that suppression of let-7 miRNAs increases fetal hemoglobin, in part, by the targeting of erythroblast HMGA2 mRNA.(1) Noh SJ et al. J Transl Med. 7:98 (2009).(2) Lee YT et al. Blood. 122:1034-41 (2013).(3) Vasconcellos JF et al. PLoS One. 9:e106924 (2014).(4) Cavazzana-Calvo M et al. Nature. 467:318-22 (2010). DisclosuresNo relevant conflicts of interest to declare.

The investigation of resveratrol and analogs as potential inducers of fetal hemoglobin

Βeta-thalassemia, is a hemoglobinopathy characterized by reduced beta-globin chain synthesis, leading to imbalanced globin chain production, ineffective erythropoiesis and anemia. Increasing gamma-globin gene expression is a promising therapeutic approach as it reduces this imbalance by combining with the excess alpha globin chains and producing fetal hemoglobin (HbF). Furthermore, increased iron absorption and repeated blood transfusions lead to iron overload and tissue damage secondary to reactive oxygen species. Compounds exhibiting both antioxidant and HbF inducing activities are, therefore, highly desirable therapeutic agents. Resveratrol, a natural phytoalexin, combines these two activities but is also cytotoxic. Nine hydroxystilbenic resveratrol derivatives were synthesized in an attempt to identify compounds that retain the HbF-inducing and antioxidant activities of resveratrol but exhibit reduced cytotoxicity. Three derivatives (P1, P4 and P11) exhibited similar hemoglobin-inducing properties to resveratrol in K562 cells, however, only P11 showed reduced cytotoxicity. All three derivatives demonstrated variable HbF-inducing activity in primary erythroid progenitor cells from healthy donors. Resveratrol and P11 increased HbF induction significantly, with P11 having the highest activity. Additionally, P4 significantly increased progenitor numbers. A combinatorial treatment in K562 cells using resveratrol and decitabine resulted in a statistically significant increase in hemoglobin-inducing activity only above the level shown by resveratrol alone.

Multiple Oral Therapeutics Suppress Repressors (LSD-1, HDACs, and BCL11A) of Gamma Globin Gene Expression

Induction of gamma globin expression to certain target levels reduces clinical severity in sickle cell disease, globin imbalance, and anemia in the beta thalassemias. However, achieving high level expression in diverse patients with variable baseline HbF levels is clinically challenging. To determine whether different therapeutics have complimentary molecular actions that could be combined for higher efficacy, we evaluated a panel of oral fetal globin-inducing therapeutics in clinical use or trials for effects on transcriptional suppressors of HBG, including components of the NuRD complex, LSD1 and HDACs 1-3, and the downstream repressor BCL11A, in erythroid progenitors cultured from sickle cell and beta thalassemia patients. Using chromatin immunoprecipitation assays, immunoblot, and qRT-PCR, we investigated two pan-HDAC inhibitors, MS-275 and SB939, a short chain fatty acid derivative without global histone deacetylation effects, sodium dimethylbutyrate (ST20), and PB-04, a PK-enhancing drug identified through high throughput screening (HTS), which has a long-standing benign clinical safety profile. These therapeutics induce fetal globin expression by 1.5 to 12-fold in different individuals’ progentiors over untreated controls and are active in 60-90% of patients’ progenitors. BCL11A mRNA and protein were suppressed by 3-7 fold and 5-10 fold, respectively, over untreated cells from the same subject by all agents tested. PB-04 treatment decreased LSD1 mRNA by 5-fold. MS-275 and ST20 suppressed KLF-1 mRNA by 3- and 2.5-fold respectively. ChIP analyses demonstrated that PB-04 reduced LSD1 and HDAC-3 occupancy on the gamma globin gene promoter by 4-fold and 3-fold, respectively, coincident with gamma globin induction; ST20 decreased HDAC-2 and LSD1 binding by 6- and 2-fold respectively. Further, histone marks associated with gene activation were enriched at the gamma-globin promoter by treatment with PB-04, including acetylated Histone H3K9 (AcH3K9), the target of HDAC3, and dimethylated H3K4 (H3K4me2), a target of LSD1. Collectively, these results identify oral clinical stage therapeutics which disrupt two major mechanisms of transcriptional repression. The findings provide a basis for targeting therapies in individuals and combining pharmacologic agents which induce HbF expression through complimentary molecular mechanisms to provide high level induction. DisclosuresFaller:Phoenicia BioSciences: Employment, Membership on an entity’s Board of Directors or advisory committees. Perrine:Phoenicia BioSciences: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.