scholarly journals Epigenetic and molecular profiles of erythroid cells after hydroxyurea treatment in sickle cell anemia

Blood ◽  
2011 ◽  
Vol 118 (20) ◽  
pp. 5664-5670 ◽  
Author(s):  
Aisha L. Walker ◽  
Shirley Steward ◽  
Thad A. Howard ◽  
Nicole Mortier ◽  
Matthew Smeltzer ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Mirna Expression ◽  
Molecular Mechanisms ◽  
Fetal Hemoglobin ◽  
Maximum Tolerated Dose ◽  
Cross Sectional ◽  
Hydroxyurea Treatment ◽  
Hbf Induction ◽  
Globin Promoter

Abstract Hydroxyurea has been shown to be efficacious for the treatment of sickle cell anemia (SCA), primarily through the induction of fetal hemoglobin (HbF). However, the exact mechanisms by which hydroxyurea can induce HbF remain incompletely defined, although direct transcriptional effects and altered cell cycle kinetics have been proposed. In this study, we investigated potential epigenetic and alternative molecular mechanisms of hydroxyurea-mediated HbF induction by examining methylation patterns within the Gγ-globin promoter and miRNA expression within primary CD71+ erythrocytes of patients with SCA, both at baseline before beginning hydroxyurea therapy and after reaching maximum tolerated dose (MTD). Using both cross-sectional analysis and paired-sample analysis, we found that the highly methylated Gγ-globin promoter was inversely correlated to baseline HbF levels, but only slightly altered by hydroxyurea treatment. Conversely, expression of several specific miRNAs was significantly increased after hydroxyurea treatment, and expression of miR-26b and miR-151-3p were both associated with HbF levels at MTD. The significant associations identified in these studies suggest that methylation may be important for regulation of baseline HbF, but not after hydroxyurea treatment, whereas changes in miRNA expression may be associated with hydroxyurea-mediated HbF induction. This study was registered at ClinicalTrials.gov (NCT00305175).

Modulation of MicroRNA Expression In Sickle Reticulocytes Is Associated with Hydroxyurea Treatment and Fetal Hemoglobin Induction

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2650-2650
Author(s):  
Aisha L Walker ◽  
Shirley Steward ◽  
Michael Wang ◽  
Matthew P Smeltzer ◽  
Russell E. Ware
Keyword(s):  
Real Time ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Mirna Expression ◽  
Real Time Pcr ◽  
Mixed Model ◽  
Cross Sectional ◽  
Mixed Model Analysis ◽  
Hydroxyurea Treatment ◽  
Hbf Induction

Abstract Abstract 2650 Background: Hydroxyurea has both laboratory and clinical efficacy for children, adolescents, and adults with Sickle Cell Anemia (SCA), and its benefits are primarily due to its ability to increase fetal hemoglobin (HbF). However, HbF induction by hydroxyurea is highly variable among patients, and its mechanism of HbF reactivation remains unclear. MicroRNAs (miRNA) are small non-coding RNAs that can regulate gene expression by inhibiting transcription or translation of targeted proteins. Recently, miRNAs have been implicated in cellular regulation and differentiation including hematopoiesis and hemoglobin switching. In an effort to elucidate the mechanisms behind hydroxyurea-mediated HbF induction, we tested the hypothesis that hydroxyurea modulates miRNA expression in sickle reticulocytes in vivo and this modulation is associated with changes in HbF levels. Methods: As part of the prospective Hydroxyurea Study of Long-term Effects (HUSTLE, NCT00305175), total RNA was purified from CD71+ reticulocytes isolated from the peripheral blood of SCA patients, either prior to hydroxyurea treatment or after reaching stable maximum tolerated dose (MTD); non-SCA adults were included as controls. Initially, differential miRNA expression associated with disease and/or hydroxyurea exposure was determined by microarray and then confirmed by real-time PCR in a cross-sectional analysis of hydroxyurea-treated (n=13) and untreated (n=22) SCA patients, plus controls (n=8). Subsequently to identify hydroxyurea-mediated changes in miRNA expression and its association with HbF induction, miRNA levels were measured by real-time PCR in paired samples collected from patient reticulocytes at baseline and at hydroxyurea MTD (n=41). Statistical correlation to HbF levels used Spearman correlation coefficient and mixed model analysis. Results: Microarray analysis identified 108 miRNAs expressed in CD71+ reticulocytes, 10 of which were significantly different in SCA patients with or without hydroxyurea exposure and normal controls. Real-time PCR confirmed that miRNA expression of human miRNA (hsa-mir) 29a, 130b, 215, and 494 were upregulated, while hsa-mir-223 was downregulated, in untreated SCA patients compared to non SCA controls. Additional analysis of 41 paired samples showed that 3 miRNAs, hsa-mir 148a, 151-3p, and 494, were significantly upregulated with hydroxyurea treatment (Table 1). At MTD, HbF levels increased an average of 17% from a mean of 8.9 ± 6.1% at baseline (range 0.0–22.9%) to a mean of 25.9 ± 9.0% at MTD (range 9.4–55.9%).Significant associations between hsa-mir 26b miRNA expression and HbF levels were identified, both at baseline (rs= -.34; p=0.03) and at MTD (rs= -.32; p=0.04). Using mixed model analysis, change in hsa-mir 151-3p expression was significantly associated with the change in HbF (p=0.047) from baseline to MTD. Conclusion: We identified specific miRNAs that are significantly associated with sickle cell anemia, hydroxyurea treatment, and hydroxyurea-mediated HbF induction. These studies suggest that miRNA regulation, specifically hsa-mir 26b and 151-3p, may be involved in hydroxyurea-mediated HbF induction in patients with sickle cell anemia. Future studies to identify the relevant protein targets may lead to a better understanding of hydroxyurea's mechanisms of action and patient response to the drug including the observed inter-patient variability in HbF response. Results from real-time PCR shows differential miRNA expression in non-SCA controls compared to untreated SCA patients from cross-sectional analysis, and upregulation of 3 miRNAs in SCA patients at baseline compared to MTD from paired-sample analysis. Negative fold change indicates a downregulation. Disclosure: Off Label Use: Hydroxyurea used to treat sickle cell anemia in children. Disclosures: Off Label Use: Hydroxyurea used to treat sickle cell anemia in children.

scholarly journals Optimizing hydroxyurea therapy for sickle cell anemia

Hematology ◽  
2015 ◽  
Vol 2015 (1) ◽  
pp. 436-443 ◽  
Author(s):  
Russell E. Ware
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Fetal Hemoglobin ◽  
Maximum Tolerated Dose ◽  
Sub Saharan Africa ◽  
Future Research ◽  
Published Evidence ◽  
Treatment Indications ◽  
Hydroxyurea Treatment ◽  
Hydroxyurea Therapy

Abstract Hydroxyurea has proven efficacy in numerous clinical trials as a disease-modifying treatment for patients with sickle cell anemia (SCA) but is currently under-used in clinical practice. To improve the effectiveness of hydroxyurea therapy, efforts should be directed toward broadening the clinical treatment indications, optimizing the daily dosage, and emphasizing the benefits of early and extended treatment. Here, various issues related to hydroxyurea treatment are discussed, focusing on both published evidence and clinical experience. Specific guidance is provided regarding important but potentially unfamiliar aspects of hydroxyurea treatment for SCA, such as escalating to maximum tolerated dose, treating in the setting of cerebrovascular disease, switching from chronic transfusions to hydroxyurea, and using serial phlebotomy to alleviate iron overload. Future research directions to optimize hydroxyurea therapy are also discussed, including personalized dosing based on pharmacokinetic modeling, prediction of fetal hemoglobin responses based on pharmacogenomics, and the risks and benefits of hydroxyurea for non-SCA genotypes and during pregnancy/lactation. Another critical initiative is the introduction of hydroxyurea safely and effectively into global regions that have a high disease burden of SCA but limited resources, such as sub-Saharan Africa, the Caribbean, and India. Final considerations emphasize the long-term goal of optimizing hydroxyurea therapy, which is to help treatment become accepted as standard of care for all patients with SCA.

Optimizing hydroxyurea therapy for sickle cell anemia

Hematology ◽  
2015 ◽  
Vol 2015 (1) ◽  
pp. 436-443 ◽  
Author(s):  
Russell E. Ware
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Fetal Hemoglobin ◽  
Maximum Tolerated Dose ◽  
Sub Saharan Africa ◽  
Future Research ◽  
Published Evidence ◽  
Treatment Indications ◽  
Hydroxyurea Treatment ◽  
Hydroxyurea Therapy

Hydroxyurea has proven efficacy in numerous clinical trials as a disease-modifying treatment for patients with sickle cell anemia (SCA) but is currently under-used in clinical practice. To improve the effectiveness of hydroxyurea therapy, efforts should be directed toward broadening the clinical treatment indications, optimizing the daily dosage, and emphasizing the benefits of early and extended treatment. Here, various issues related to hydroxyurea treatment are discussed, focusing on both published evidence and clinical experience. Specific guidance is provided regarding important but potentially unfamiliar aspects of hydroxyurea treatment for SCA, such as escalating to maximum tolerated dose, treating in the setting of cerebrovascular disease, switching from chronic transfusions to hydroxyurea, and using serial phlebotomy to alleviate iron overload. Future research directions to optimize hydroxyurea therapy are also discussed, including personalized dosing based on pharmacokinetic modeling, prediction of fetal hemoglobin responses based on pharmacogenomics, and the risks and benefits of hydroxyurea for non-SCA genotypes and during pregnancy/lactation. Another critical initiative is the introduction of hydroxyurea safely and effectively into global regions that have a high disease burden of SCA but limited resources, such as sub-Saharan Africa, the Caribbean, and India. Final considerations emphasize the long-term goal of optimizing hydroxyurea therapy, which is to help treatment become accepted as standard of care for all patients with SCA.

scholarly journals Pharmacokinetics, pharmacodynamics, and pharmacogenetics of hydroxyurea treatment for children with sickle cell anemia

Blood ◽  
2011 ◽  
Vol 118 (18) ◽  
pp. 4985-4991 ◽  
Author(s):  
Russell E. Ware ◽  
Jenny M. Despotovic ◽  
Nicole A. Mortier ◽  
Jonathan M. Flanagan ◽  
Jin He ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Oral Absorption ◽  
Fetal Hemoglobin ◽  
Maximum Tolerated Dose ◽  
Interpatient Variability ◽  
Nucleotide Polymorphisms ◽  
Hydroxyurea Treatment ◽  
Treatment Responses ◽  
Hydroxyurea Therapy

Abstract Hydroxyurea therapy has proven laboratory and clinical efficacies for children with sickle cell anemia (SCA). When administered at maximum tolerated dose (MTD), hydroxyurea increases fetal hemoglobin (HbF) to levels ranging from 10% to 40%. However, interpatient variability of percentage of HbF (%HbF) response is high, MTD itself is variable, and accurate predictors of hydroxyurea responses do not currently exist. HUSTLE (NCT00305175) was designed to provide first-dose pharmacokinetics (PK) data for children with SCA initiating hydroxyurea therapy, to investigate pharmacodynamics (PD) parameters, including HbF response and MTD after standardized dose escalation, and to evaluate pharmacogenetics influences on PK and PD parameters. For 87 children with first-dose PK studies, substantial interpatient variability was observed, plus a novel oral absorption phenotype (rapid or slow) that influenced serum hydroxyurea levels and total hydroxyurea exposure. PD responses in 174 subjects were robust and similar to previous cohorts; %HbF at MTD was best predicted by 5 variables, including baseline %HbF, whereas MTD was best predicted by 5 variables, including serum creatinine. Pharmacogenetics analysis showed single nucleotide polymorphisms influencing baseline %HbF, including 5 within BCL11A, but none influencing MTD %HbF or dose. Accurate prediction of hydroxyurea treatment responses for SCA remains a worthy but elusive goal.

Assessment of Genotoxicity Associated with Hydroxyurea Therapy in Children with Sickle Cell Anemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2554-2554
Author(s):  
Jonathan M Flanagan ◽  
Thad A. Howard ◽  
Nicole Mortier ◽  
Svetlana Avlasevich ◽  
Matthew Smeltzer ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Drug Exposure ◽  
Fold Increase ◽  
Cross Sectional ◽  
Flow Cytometric ◽  
Hydroxyurea Treatment ◽  
Serial Measurements ◽  
Hydroxyurea Therapy

Abstract Abstract 2554 Poster Board II-531 Introduction: Hydroxyurea induces fetal hemoglobin (HbF), improves laboratory parameters, and reduces acute clinical complications of sickle cell anemia (SCA), but its long-term efficacy and safety remain incompletely defined. One long-term safety concern is that hydroxyurea may elicit DNA alterations via genotoxic damage. During normal erythropoiesis, red blood cells (RBC) extrude their nucleus as they develop into functional reticulocytes. Occasionally, membrane bound DNA remains in the cell after erythrocyte maturation and these inclusion bodies are known as micronuclei (MN) or Howell-Jolly Bodies. MN-containing reticulocytes are formed at higher frequency upon exposure to genotoxic agents. Patients with SCA have increased basal MN production while also having decreased MN clearance due to diminished splenic filtrative function. In a previous small cross sectional study, we showed that hydroxyurea exposure further increases MN production in SCA patients. To better address this long-term safety issue of hydroxyurea, we evaluated MN production and clearance both in a large cross-sectional and prospective study of children with SCA on hydroxyurea therapy. Patients and Methods: A high-throughput flow cytometric technique was used to detect and quantitate MN within circulating erythrocyte subpopulations. After written informed consent, venous blood samples were collected from children with SCA enrolled in the Hydroxyurea Study of Long-term Effects (HUSTLE, ClinicalTrials.gov NCT00305175). A total of 105 subjects had at least 1 MN measurement, including 37 subjects with serial measurements at baseline and at follow-up time points up to 24 months of hydroxyurea exposure. MN were quantified in both reticulocytes (MN-CD71(+)) and mature RBC (MN-RBC), and then tested for associations with individual subject laboratory and clinical data. Results: In cross-sectional analysis of 293 samples from 105 children with SCA and a median of 2 years hydroxyurea exposure (range 3 months – 12 years), hydroxyurea therapy significantly lowered %CD71(+) reticulocytes (mean fold reduction 0.53 ± 0.44, p < 0.001). Compared to baseline values, hydroxyurea treatment increased levels of MN in both reticulocytes (%MN-CD71(+), mean fold increase 1.80 ± 0.91, p < 0.05) and mature RBC (%MN-RBC, mean fold increase 1.89 ± 1.39, p<0.01). The increase in MN-CD71(+) was evident by 6 months of hydroxyurea treatment, but did not significantly escalate further with up to 12 years of continued drug exposure (Fig. 1). To prospectively determine the genotoxic effect of hydroxyurea, serial measurements over 2 years were performed on 37 patients. After 9 months on hydroxyurea therapy all subjects were on a stable maximum tolerated dose (MTD, average 25.1 mg/kg/day); 15 of 37 children had > 3.0 fold increase in %MN-CD71(+) while 22 of 37 had < 3.0 fold increase (Mean 3.68 ± 0.65 vs. 1.52 ± 0.52, p < 0.001). The observed inter-individual variation was associated with the predicted laboratory effects of hydroxyurea; increases in %MN-CD71(+) were positively correlated with MTD values of HbF (r2=0.22, p=0.005), mean corpuscular volume (r2=0.35, p=0.002), and mean corpuscular hemoglobin (r2=0.29, p=0.006) but negatively correlated with absolute neutrophil count (r2=0.14, p=0.02) and bilirubin levels (r2=0.20, p=0.008). There were no significant associations between %MN-CD71(+) and gender, age, or hydroxyurea dosage although %MN-RBC clearance decreased with age. Conclusions: A highly sensitive and quantitative flow cytometric technique can detect circulating MN-containing erythrocytes and this technique may be used to assess the in vivo genotoxic effect of any drug. Children with SCA have high basal MN production that is probably related to the degree of erythropoiesis in these patients. Hydroxyurea therapy was associated with genotoxicity but with substantial inter-patient variability in hydroxyurea induced %MN-CD71(+) levels. The increases in %MN-CD71(+) are observed within 6 months of starting hydroxyurea therapy but persist at the same level in patients with up to 12 years of continued drug exposure. Correlations between increased %MN-CD71(+) and predicted hydroxyurea effects on other laboratory parameters suggest that hydroxyurea induces measurable genotoxicity that may be related to individual patient sensitivity and efficacy of hydroxyurea within the bone marrow. These patients will be monitored further to confirm that hydroxyurea does not pose any long term safety issues. Disclosures: Off Label Use: Off label use of hydroxyurea in children with sickle cell anemia. Avlasevich:Litron Laboratories: Employment. Dertinger:Litron Laboratories: Employment.

scholarly journals Soluble Guanylate Cyclase Modulators Effect Fetal Hemoglobin Levels through a Non-cGMP-Protein Kinase G Signaling Pathway

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1060-1060
Author(s):  
Bin Sun ◽  
Valerie Miller ◽  
Karin P Potoka ◽  
Yukio Nakamura ◽  
Mark T Gladwin ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Active Site ◽  
Soluble Guanylate Cyclase ◽  
Fetal Hemoglobin ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Hbf Induction

Abstract Sickle cell anemia results from a point mutation in both alleles of the β-globin gene. This homozygous mutation ultimately leads to a structural alteration of the hemoglobin protein that promotes polymerization of the mutant sickle hemoglobin tetramer (HbS) upon deoxygenation. HbS polymerization results in rigid, sickle-shaped RBCs with increased cell-to-cell adhesion properties. These sticky and rigid RBCs are prone to become trapped in small capillary networks leading to ischemia-reperfusion injury, endothelial damage and the hallmark pain crisis of sickle cell anemia. Neonates are protected from deoxy-HbS polymerization by high levels of fetal hemoglobin (HbF). HbF is composed of two α-globin and two γ-globin subunits(α2γ2), The γ-globin molecule cannot interact with deoxy-βS-globin polymers, which makes HbF an effective inhibitor of deoxy-HbS polymerization. The level of HbF required to reduce the symptoms of sickle cell anemia is 20-25%, but levels as low as 9% can prolong red cell survival. Treatment with hydroxyurea (HU) induces HbF and reduces the hematologic and clinical consequences of sickle cell anemia. Basal and inducible HbF levels are important in predicting the severity of sickle cell anemia and are highly phenotypically variable among patients, leading to varied responses to treatment. Patients are also variably susceptible to HU-induced cytopenias, which limits the use of HU in certain patients. HU is currently the only FDA-approved HbF-inducer for the treatment of sickle cell anemia and there is clearly a need for alternative HbF-inducers. Understanding the signaling pathways that regulate HbF induction will lead to novel therapeutic targets for sickle cell anemia. The soluble guanylate cyclase/cyclic guanosine monophosphate-dependent protein kinase (sGC/PKG) signaling pathway potentially links HU to the induction of HbF expression. In this study we investigated the direct role of sGC in HbF induction using novel pharmacologic modulators of sGC. Nitric oxide (NO) activates sGC by binding to the ferrous iron (Fe2+) in the active site heme moiety. Once activated, sGC converts GTP to cGMP, which in-turn activates PKG. Reactive oxygen species (ROS) oxidize the active site heme of sGC leading to NO-insensitivity. We tested the ability of a novel sGC activator, BAY 58-2667, to induce γ-globinin primary and immortalized (HUDEP-2) human erythroid progenitor cells. BAY 54-6544 binds to heme-free inactivated sGC to restore its guanylyl cyclase activity independent of NO. We also tested the ability of the sGC stimulator, BAY 41-2272, to induce γ-globinin primary and HUDEP-2 human erythroid progenitor cells. BAY 41-2272, binds to the ferrous iron at the active site of non-oxidized sGC to stimulate guanylyl cyclase activity in a synergistic manner with NO. We compared g-globin mRNA and protein expressionin the primary and immortalized human erythroid progenitors after treatment with different concentrations and combinations of BAY 54-6544, BAY 41-2667 and HU. We also evaluated g-globin induction in cellstreated with the pan-phosphodiesterase inhibitor IBMX and a synthetic cGMP analog. Although we see robust induction of cGMP and activation of PKG with all treatments, we only see significant induction of g-globin expression in the HU treated cells. This data suggests that the induction of HbF occurs through a non-sGC/PKG-dependent signaling pathway. These data demonstrate a very limited induction of γ-globin by BAY 54-6544 and BAY 41-2667 that appears to be disproportionate to, and independent of, cGMP/PKG signaling. These data also demonstrate, for the first time, that HU treatment of the immortalized HUDEP-2 cell line induces γ-globin expression more consistently than in primary erythroid progenitors. Disclosures No relevant conflicts of interest to declare.

scholarly journals Hydroxyurea therapy of a murine model of sickle cell anemia inhibits the progression of pneumococcal disease by down-modulating E-selectin

Blood ◽  
2012 ◽  
Vol 119 (8) ◽  
pp. 1915-1921 ◽  
Author(s):  
Jeffrey D. Lebensburger ◽  
Thad Howard ◽  
Yunming Hu ◽  
Tamara I. Pestina ◽  
Geli Gao ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Vascular Inflammation ◽  
Pneumococcal Infection ◽  
Fetal Hemoglobin ◽  
Neutrophil Extravasation ◽  
Hydroxyurea Treatment ◽  
Inflammatory State ◽  
The Impact ◽  
Hydroxyurea Therapy

Abstract Sickle cell anemia is characterized by chronic hemolysis coupled with extensive vascular inflammation. This inflammatory state also mechanistically promotes a high risk of lethal, invasive pneumococcal infection. Current treatments to reduce vaso-occlusive complications include chronic hydroxyurea therapy to induce fetal hemoglobin. Because hydroxyurea also reduces leukocytosis, an understanding of the impact of this treatment on pneumococcal pathogenesis is needed. Using a sickle cell mouse model of pneumococcal pneumonia and sepsis, administration of hydroxyurea was found to significantly improve survival. Hydroxyurea treatment decreased neutrophil extravasation into the infected lung coincident with significantly reduced levels of E-selectin in serum and on pulmonary epithelia. The protective effect of hydroxyurea was abrogated in mice deficient in E-selectin. The decrease in E-selectin levels was also evident in human sickle cell patients receiving hydroxyurea therapy. These data indicate that in addition to induction of fetal hemoglobin, hydroxyurea attenuates leukocyte–endothelial interactions in sickle cell anemia, resulting in protection against lethal pneumococcal sepsis.

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