Therapeutic fetal-globin inducers reduce transcriptional repression in hemoglobinopathy erythroid progenitors through distinct mechanisms

Abstract

Pharmacologic augmentation of γ-globin expression sufficient to reduce anemia and clinical severity in patients with diverse hemoglobinopathies has been challenging. In studies here, representative molecules from four chemical classes, representing several distinct primary mechanisms of action, were investigated for effects on γ-globin transcriptional repressors, including components of the NuRD complex (LSD1 and HDACs 2-3), and the downstream repressor BCL11A, in erythroid progenitors from hemoglobinopathy patients. Two HDAC inhibitors (MS-275 and SB939), a short-chain fatty acid derivative (sodium dimethylbutyrate [SDMB]), and an agent identified in high-throughput screening, Benserazide, were studied. These therapeutics induced γ-globin mRNA in progenitors above same subject controls up to 20-fold, and increased F-reticulocytes up to 20%. Cellular protein levels of BCL11A, LSD-1, and KLF1 were suppressed by the compounds. Chromatin immunoprecipitation assays demonstrated a 3.6-fold reduction in LSD1 and HDAC3 occupancy in the γ-globin gene promoter with Benserazide exposure, 3-fold reduction in LSD-1 and HDAC2 occupancy in the γ-globin gene promoter with SDMB exposure, while markers of gene activation (histone H3K9 acetylation and H3K4 demethylation), were enriched 5.7-fold. These findings identify clinical-stage oral therapeutics which inhibit or displace major co-repressors of γ-globin gene transcription and may suggest a rationale for combination therapy to produce enhanced efficacy.

Keywords: Chromatin immunoprecipitation;; Erythroid progenitors; Fetal globin gene expression;; Hemoglobinopathies;; Repressors;.

Copyright © 2015 Elsevier Inc. All rights reserved.

Figures

Figure 1. Differentiation of erythroid progenitors

Photomicrographs of Wright-Giemsa stained erythroid progenitors in Phase 2 of culture, showing glycophorin-positive cells on day 5 (panel A), day 10 (panel B), and day 15 (panel C). Scale, 10 μm.

Figure 2. γ-globin mRNA and F-reticulocytes induced by therapeutic candidates

A: Mean fold change in γ-globin mRNA in erythroid progenitor cells treated with the designated therapeutic candidates (MS-275, SDMB, SB939, or Benserazide), compared to vehicle-treated cells from the same subject. All changes are significant, p <0.001. Error bars indicate SD. B. Mean change in proportions of cells expressing HbF protein (F-reticulocytes) in erythroid progenitor cells treated with therapeutic candidates (SDMB, Benserazide, MS275, SB939), compared to control cells from the same subject. Error bars indicate SD. * Asterisks indicate statistically significant differences (* p<0.05, ** p<0.01. *** p<0.001) C. Western blot demonstrating increased total HbF in thalassemic or cord blood erythroid progenitors cultured with Benserazide, compared to control cells from the same sources. Fetal globin protein increased by 2.6-fold and 1.6 fold above control levels.

Figure 3. Mean mRNA levels of repressor components in erythroid cells treated with therapeutic candidates

3A) Mean transcript levels of LSD1, 3B) BCL11A and 3C) KLF1 assayed by quantitative RT-PCR relative to levels in control cells are shown; 18S transcripts were used as internal controls. The mean value represents data from at least three independent experiments. The error bars represent the SD. Asterisks indicate significant differences between the treatment relative to the controls (* p<0.05).

Figure 4. Effects of therapeutic candidates on LSD1, BCL11A and KLF1 protein levels in hemoglobinopathy erythroid cells from hemoglobinopathy patients

A. LSD1 protein in HbE β-thalassemia cells treated with Benserazide, MS275 or SDMB. The ratio of LSD1:β-actin protein is shown below each panel. Each panel designates an immunoblot performed from at least two different patients. Control designates protein in untreated cells from the same subject. B. BCL11A protein in sickle cell progenitors treated with SB939, in HbE β-thalassemia cells treated with Benserazide or SDMB (left panel). BCL11A protein in cord blood erythroid progenitor cells treated with MS275, SB939, or Benserazide (right panel). C. KLF-1 protein in erythroid progenitor cells treated with MS275.

Figure 5. Chromatin immunoprecipitation assays of binding of co-repressors in the γ-globin gene promoter

A) LSD1 protein binding in γ-globin gene promoter in HbE β-thalassemia progenitor cells treated with Benserazide. The bars represent the standard deviation (* p< 0.05). B) HDAC3 protein binding in the γ-globin gene promoter in HbE β-thalassemia cells progenitors treated with Benserazide. The bars represent the standard deviation (* p< 0.05). C) LSD1 and HDAC2 protein binding in γ-globin gene promoter in K562 cells treated with SDMB. The error bars represent the standard deviation. Asterisks indicate significant differences between the treated and vehicle-treated cells (* p<0.05).

Figure 6. Activation markers of histone modification (H3AcK9 & H3K4me2) in the γ-globin gene promoter in HbE β-thalassemia progenitors treated with Benserazide (BEN)

H3K4me2 and H3AcK9 enrichment in the γ-globin gene promoter is shown in Benserazide-treated HbE β-thalassemia progenitors and vehicle-treated control. The error bars represent the SEM. Asterisks indicate significant differences between the vehicle controls and Benserazide-treated cells (*p

Figure 7

Schematic diagram of sites of molecular mechanisms of γ-globin induction by Benserazide (BEN), MS275 (Etinostat), SDMB (dimethylbutyrate), and SB939 (Pracinostat).