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

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 G ␥-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 G ␥-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) . IntroductionHydroxyurea is a potent ribonucleotide reductase inhibitor that was initially developed as an antineoplastic agent, but was later found to be beneficial for the treatment of sickle cell anemia (SCA) primarily through its ability to induce fetal hemoglobin (HbF). [1][2][3][4] Clinical trials have proven hydroxyurea to be efficacious in children, adolescents, and adults with SCA for increasing HbF. 2,[5][6][7] Hydroxyurea decreases the number and frequency of vasoocclusive pain crises, episodes of acute chest syndrome, hospitalizations, 6,8-10 and more recently has been shown to also reduce mortality. 11,12 Although highly efficacious for most patients with SCA, there is considerable inter-patient variability creating a broad spectrum of HbF induction. 13,14 HbF induction by cytotoxic drugs, such as hydroxyurea, has been correlated to cell cycle inhibition leading to activation of stress erythropoiesis. [15][16][17] Other studies have suggested that HbF induction by hydroxyurea is mediated more specifically via nitric oxide-dependent transcriptional mechanisms 18,19 and cyclic nucleotides. 20,21 However, the precise mechanisms by which hydroxyurea can induce HbF in patients with SCA remain incompletely defined.Expression of erythroid genes at the ␤-globin locus is developmentally regulated by a complex series of epigenetic and molecular processes. Proposed epigenetic mechanisms of HbF regulation in hemoglobinopathies include methylation, histone deacetylation, and chromosomal looping (reviewed in Sankaran et al, 22 Bank et al, 23 and Stamatoyannopoulos et al 24 ). Methylation i...

A systematic review of known mechanisms of hydroxyurea-induced fetal hemoglobin for treatment of sickle cell disease

“…It has also been shown that BCL11A occupies the LCR HS3 and the intergenic region between G γA and δ-globin genes [31]. Acetylation-based transcriptional activity has been observed in the LCR, δ- and β-globin genes as well as the intergenic region between A γ and δ-globin genes, whereas methylation was most observed in the CpG islands of the γ-globin proximal promoter [38,84]. Furthermore, HU-induced HbF has previously been inversely correlated to Gγ-globin methylation in SCD patients [84].…”

“…Acetylation-based transcriptional activity has been observed in the LCR, δ- and β-globin genes as well as the intergenic region between A γ and δ-globin genes, whereas methylation was most observed in the CpG islands of the γ-globin proximal promoter [38,84]. Furthermore, HU-induced HbF has previously been inversely correlated to Gγ-globin methylation in SCD patients [84]. Albeit, the changes in methylation did not reach statistical significance, decreases in methylated CpG islands in the Gγ-globin promoter were identified and associated with increased γ-globin expression.…”

Original Research: Stable expression of miR-34a mediates fetal hemoglobin induction in K562 cells

“…40 The role of miRNAs in drug-mediated g-globin activation has also been investigated. Walker et al 41 verified that miR-26b was correlated with steady-state HbF levels in children with sickle cell disease, and miR-151-3p expression was associated with the maximal tolerated dose of hydroxyurea.…”

Reactivation of Fetal Hemoglobin in Thalassemia and Sickle Cell Disease

“…48 There is however, even in the case of HU-induced expression of Hb, some evidence for an epigenetic regulation, perhaps mediated by micro-RNA's (miRNAs). 49 The role of inactivation of repressor genes has also been recently documented as before said.…”