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Forced chromatin looping raises fetal hemoglobin in adult sickle cells to higher levels than pharmacologic inducers.

ABSTRACT: Overcoming the silencing of the fetal γ-globin gene has been a long-standing goal in the treatment of sickle cell disease (SCD). The major transcriptional enhancer of the β-globin locus, called the locus control region (LCR), dynamically interacts with the developmental stage-appropriate β-type globin genes via chromatin looping, a process requiring the protein Ldb1. In adult erythroid cells, the LCR can be redirected from the adult β- to the fetal γ-globin promoter by tethering Ldb1 to the human γ-globin promoter with custom-designed zinc finger (ZF) proteins (ZF-Ldb1), leading to reactivation of γ-globin gene expression. To compare this approach to pharmacologic reactivation of fetal hemoglobin (HbF), hematopoietic cells from patients with SCD were treated with a lentivirus expressing the ZF-Ldb1 or with chemical HbF inducers. The HbF increase in cells treated with ZF-Ldb1 was more than double that observed with decitabine and pomalidomide; butyrate had an intermediate effect whereas tranylcypromine and hydroxyurea showed relatively low HbF reactivation. ZF-Ldb1 showed comparatively little toxicity, and reduced sickle hemoglobin (HbS) synthesis as well as sickling of SCD erythroid cells under hypoxic conditions. The efficacy and low cytotoxicity of lentiviral-mediated ZF-Ldb1 gene transfer compared with the drug regimens support its therapeutic potential for the treatment of SCD.

SUBMITTER: Breda L

PROVIDER: S-EPMC5000846 | biostudies-other | 2016 Aug

REPOSITORIES: biostudies-other

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HRI depletion cooperates with pharmacologic inducers to elevate fetal hemoglobin and reduce sickle cell formation.

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Project description:Increasing fetal hemoglobin (HbF) provides clinical benefit in patients with sickle cell disease (SCD). We recently identified heme-regulated inhibitor (HRI, EIF2AK1) as a novel HbF regulator. Since HRI is an erythroid-specific protein kinase it presents a potential target for pharmacologic intervention. We find that maximal HbF induction requires >80-85% HRI depletion. As it remains unclear whether this degree of HRI inhibition can be achieved pharmacologically, we explored whether HRI knockdown can be combined with pharmacologic HbF inducers to achieve greater HbF production and minimize potential adverse effects associated with treatments. Strongly cooperative HbF induction was observed when HRI depletion was combined with exposure to pomalidomide or the EHMT1/2 inhibitor UNC0638, but not hydroxyurea. Mechanistically, reduction in the levels of the HbF repressor BCL11A reflected the cooperativity of HRI loss and pomalidomide treatment, whereas UNC0638 did not modulate BCL11A levels. In conjunction with HRI loss, pomalidomide maintained its HbF inducing activity at 10-fold lower concentrations, at which condition there were minimal observed detrimental effects on erythroid cell maturation and viability as well as fewer alterations in the erythroid transcriptome. In sum, this study provides a foundation for the exploration of combining future small molecule HRI inhibitors with additional pharmacologic HbF inducers to maximize HbF production and preserve erythroid cell functionality for the treatment of SCD and other hemoglobinopathies.

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