Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS) Are Active In Vivo in Anemic Baboons and Transgenic Mice

We thank Sarah Haigh, Ada Kane, Nicole Reuter, David Carey, and Marilyn Perry Carey for dedicated and expert technical assistance and Cloret Carl for assistance with preparation of the manuscript.This work was supported by grants from the National Institutes of Health, R01 DK-52962, (SPP, Boston University), R41 HL-105816 (SPP, Phoenicia BioSciences), and R42 HL-110727 (Phoenicia BioSciences), 2 P40 ODO010988-16 (GLW, University of Oklahoma) and UL1-TR000157 (RFW, University of Oklahoma). SMN was supported by P50 HL-118006. The funders had no role in study design, data collection or analysis, decision to publish, or preparation of the manuscript.High-level fetal (γ) globin expression ameliorates clinical severity of the beta (β) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.Yeshttp://www.plosone.org/static/editorial#pee

Failure of Terminal Erythroid Differentiation in EKLF-Deficient Mice Is Associated with Cell Cycle Perturbation and Reduced Expression of E2F2▿ †

Erythroid Krüppel-like factor (EKLF) is a Krüppel-like transcription factor identified as a transcriptional activator and chromatin modifier in erythroid cells. EKLF-deficient (Eklf−/−) mice die at day 14.5 of gestation from severe anemia. In this study, we demonstrate that early progenitor cells fail to undergo terminal erythroid differentiation in Eklf−/− embryos. To discover potential EKLF target genes responsible for the failure of erythropoiesis, transcriptional profiling was performed with RNA from wild-type and Eklf−/− early erythroid progenitor cells. These analyses identified significant perturbation of a network of genes involved in cell cycle regulation, with the critical regulator of the cell cycle, E2f2, at a hub. E2f2 mRNA and protein levels were markedly decreased in Eklf−/− early erythroid progenitor cells, which showed a delay in the G1-to-S-phase transition. Chromatin immunoprecipitation analysis demonstrated EKLF occupancy at the proximal E2f2 promoter in vivo. Consistent with the role of EKLF as a chromatin modifier, EKLF binding sites in the E2f2 promoter were located in a region of EKLF-dependent DNase I sensitivity in early erythroid progenitor cells. We propose a model in which EKLF-dependent activation and modification of the E2f2 locus is required for cell cycle progression preceding terminal erythroid differentiation

Fetal globin induction in an anemic baboon treated with MS-275 (class I HDAC inhibitor).

<p>(A.) Treatment with MS-275 (0.2 mg/kg/dose three times/week), shown by the black bars above the graph, resulted in a 2.7-fold induction of γ-globin mRNA, (B.) 7% increase in F-cells, (C.) 30% increase in F reticulocytes, and (D.) a 1 g/dl rise in total hemoglobin despite daily phlebotomy of 6 ml/kg.</p

γ-globin mRNA induction in human erythroid progenitors treated with therapeutic candidates.

<p>(A.) Mean fold-induction of γ-globin mRNA with addition of Ambroxol (ABX), resveratrol (RSV), Desloratadine (DLT), NSC-95397 (NSC), MS-275, or Benserazide (BEN), compared to vehicle-treated control (Con) in erythroid progenitors from the same subject and to arginine butyrate (AB)-treatment. The range in γ-globin mRNA induction from 2.4- to 4-fold, compared to the subject’s baseline was statistically significant for all candidates and exceeded induction of γ-globin mRNA compared by butyrate (AB) in the same cultures. Error bars indicate standard deviation (SD). (B.) Western blot demonstrating HbF protein induction in erythroid cells treated with therapeutic candidates. HbF protein relative to beta actin is shown below the bands. HbF increased above the ratios in control cells from the same source, by 3.2-fold with Benserazide treatment and by 2.5-fold with MS-275 treatment.</p

Structures of therapeutic candidates identified in high-throughput screening.

<p>Structures of the drug candidates which induce the fetal globin gene promoter are shown. Butyric acid, a known inducer of the fetal globin gene promoter, is shown for comparison.</p

Responses of transgenic mice to treatment with Benserazide or Hydroxyurea.

<p>(A. & C.) Mice containing the human non-alpha globin genes in a YAC were treated with vehicle (water) as a control, Hydroxyurea (HU), or Benserazide. % F-cells and mean fluorescent intensity are shown pretreatment <i>vs</i>. 2 weeks after initiation of treatment, and (B. & D) pretreatment <i>vs</i>. 5 weeks after initiation of treatment. Changes with treatment were significant, (p<0.001 for Benserazide and p< 0.05 for Hydroxyurea). (E.) Total hemoglobin levels are shown pre- treatment <i>vs</i>. 2 weeks and 5 weeks after initiation of each treatment.</p

Schema of the high-throughput screening assay (HTS).

<p>(A.) A construct containing the 1.4-kilobase (kb) KpnI-BglII fragment of HS2 of the locus control region (LCR) linked to the γ-globin gene promoter driving the enhanced green fluorescent protein (EGFP) reporter gene stably transfected in K562 cells. Transfected cells were treated with compounds from diverse chemical libraries in an HTS format. (B.) Example of a hit in the high-throughput system is shown for compound MS-275. Fluorescent microphotographs of untreated cells are shown in the left panel; cells treated with MS-275 are shown in the right panel. (C.) Relative activity (relative fluorescence units) of new candidates identified by the HTS screen: Ambroxol (ABX), Desloratadine (DLT), MS-275, Resveratrol (RSV), Benserazide (BEN), NSC-95397 (NSC), and Idarubicin (IDA) are shown.</p

Comparison of fetal globin induction in anemic baboons treated with lead and known candidates.

<p>(A.) The magnitude of γ-globin mRNA induction with treatment with Hydroxyurea (HU), MS-275, DLT (mean 7-fold), and Benserazide (27-fold) are shown, and (B.) % F-reticulocytes with the drug treatments are shown. Error bars indicate standard deviation (SD).</p

Fetal globin induction in an anemic baboon treated with Desloratidine (DLT).

<p>(A.) Treatment with DLT (once per day, 5 days/week), shown by the black bars above the graph, resulted in 5-to 11-fold induction in γ-globin mRNA, (B.) a 15% increase in F-reticulocytes, and (C.) a 1.0 gm/dL increase in total hemoglobin.</p