Chemical genetic strategy identifies histone deacetylase 1 (HDAC1) and HDAC2 as therapeutic targets in sickle cell disease

Abstract

The worldwide burden of sickle cell disease is enormous, with over 200,000 infants born with the disease each year in Africa alone. Induction of fetal hemoglobin is a validated strategy to improve symptoms and complications of this disease. The development of targeted therapies has been limited by the absence of discrete druggable targets. We developed a unique bead-based strategy for the identification of inducers of fetal hemoglobin transcripts in primary human erythroid cells. A small-molecule screen of bioactive compounds identified remarkable class-associated activity among histone deacetylase (HDAC) inhibitors. Using a chemical genetic strategy combining focused libraries of biased chemical probes and reverse genetics by RNA interference, we have identified HDAC1 and HDAC2 as molecular targets mediating fetal hemoglobin induction. Our findings suggest the potential of isoform-selective inhibitors of HDAC1 and HDAC2 for the treatment of sickle cell disease.

Fig. 1.

A unique multiplexed globin screening assay. (A) A multiplexed assay was designed to detect expression of each globin gene. The β-globin locus contains embryonic (ε), fetal (γ), and adult (δ and β) globin genes on Chromosome (Chr) 11. The α-globin locus is comprised of embryonic (ζ) and adult (α and θ) globin genes. (B) Primary human erythroid progenitor cells were generated from in vitro differentiation of CD34⁺ bone-marrow cells for 7 d. Cells were treated with compounds for 3 d, and poly(A) mRNA was purified on oligo-dT coated plates and reverse transcribed. Following ligation-mediated amplification of globin cDNAs, the bar-coded amplicons were detected by fluorescent microspheres linked to capture probes complementary to the bar codes. (C) Expression of α- and β-globin increased during in vitro erythroid differentiation. The expression of γ-globin was higher in cells from umbilical cord blood than adult bone marrow. Mean fluorescence intensity for each globin gene was normalized to GAPDH expression. (D) Known small molecules induce the relative expression of γ-globin relative to β-globin in dose-ranging studies. The γ/β-globin ratio is shown in red, and the ε/β-globin ratio is shown in black.

Fig. 2.

HDAC inhibitors induce γ and ε hemoglobin in adult progenitors and exert long-range conformational effects on chromatin. (A) Known and novel HDAC inhibitors increase the relative expression of γ-globin RNA, as assayed by quantitative RT-PCR. Fold induction was calculated as the γ/β-globin ratio in compound-treated cells relative to untreated cells using the ΔΔCt method. To validate changes in globin protein expression, erythroid progenitor cells were treated with 0.5 mM butyrate, 2 μM NK57, 0.5 μM SAHA, 1 μM NK125, 0.5 μM scriptaid. The ratio of γ/β-globin protein was assayed by HPLC (B), and F-cell percentage was assayed by flow cytometry (C). HPLC traces are shown in Fig. S3. The HDAC inhibitors also induce the expression of ε- and ζ-globin gene expression (D and E), as assayed by quantitative RT-PCR. (F) NK57 and SAHA alter the chromatin structure of the β-globin locus, increasing contact between the LCR and the ε- and γ-globin promoters using 3C. Quantitative PCR was performed using one primer in the LCR (HS5) paired with primers located throughout the β-globin locus (Fig. S5). Hypersensitive sites 1 to 5 in the LCR and the β-globin locus genes are noted in the schema at the top. The signal-to-noise ratio of umbilical cord blood or compound-treated cells was calculated relative to untreated bone-marrow cells. Peaks at the ε- and γ-globin promoters indicate increased contact with the LCR.

Fig. 3.

A chemical genetic approach to HDAC selectivity for γ globin gene induction. (A) The ability of 14 compounds to inhibit the activity of HDAC1-9 was tested using in vitro homogeneous assays of deacetylase activity (30). For each compound, the IC₅₀ was calculated for HDAC1-9 (Table S2). The data are summarized in a heat map in which the colors range from deepest blue (lowest IC₅₀) to red (highest IC₅₀). Effects on globin gene expression for each compound, at each concentration, are shown in Fig. S6. (B) HDAC biochemical profiling data presented for ortho-amino anilides CI-994, MGCD-0103, and MS-275. (C) Dose-dependent induction of γ-globin transcription by CI-994, MGCD-0103, and MS-275. Doses, in micromolars, are shown on the x axis. The γ/β-globin ratio is shown in red, at each compound concentration. Error bars represent the mean ± SE of three measurements. (D) Lentiviruses expressing shRNAs targeting HDAC1 or HDAC2 increased the expression of γ-globin relative to β-globin in primary human erythroid progenitor cells, assayed by quantitative RT-PCR, and calculated relative to a control shRNA; shRNAs targeting HDAC3 had no effect. The knockdown efficiency for each shRNA is shown in Table S3. (E) HDAC1 and HDAC2 shRNAs also increased the ε/β-globin ratio.

Fig. 4.

HDAC1 and HDAC2 regulate unique and overlapping sets of genes in human erythroid progenitor cells. (A) Primary human erythroid cells expressing shRNAs targeting HDAC1 or HDAC2 did not increase total nuclear histone acetylation in a quantitative, cell-based, immunofluorescence assay, but treatment with SAHA or LBH-589 did increase nuclear histone acetylation. The shRNAs and compounds induced γ-globin equivalently in this experiment (data not shown). (B) Gene expression profiling was performed on primary human erythroid progenitor cells expressing a control shRNA (luciferase, n = 3), three different HDAC1 shRNAs (n = 2 per shRNA), and three different HDAC2 shRNAs (n = 2 per shRNA). The heat map shows the top 20 genes that discriminate between treatment condition and control. Genes with high expression are portrayed in red, and genes with low expression are shown in blue. (C) Pie chart illustrating the proportion of genes that are targets of HDAC1, HDAC2, or both, among all genes regulated by compound treatment (greater than 1.5-fold).

Fig. 5.

A potent, orally-bioavailable inhibitor of HDAC1 and HDAC2 induces fetal hemoglobin in human subjects. (A) Inhibition of HDAC1 (red) and HDAC2 (blue) by sodium butyrate, valproate, and LBH-589. (B) Biochemical profiling data (IC₅₀) of known carboxylic acid inducers of fetal hemoglobin against HDAC1-9. (C) Correlative biomarker data collected from a prospective, open-label study of oral LBH-589 in hematologic malignancies. Relative fetal hemoglobin content (circles; HbF; %) was measured by hemoglobin electrophoresis and total hemoglobin (squares; Hgb; g/dL) was measured as cyanide-reacted cyanmethemoglobin by absorbance (540 nm) in the clinical laboratory at the Dana-Farber Cancer Institute. Size of circles and squares corresponds to dose of LBH-589 (60, 40, or 30 mg; each three times weekly). Open circles and squares indicate weeks in which doses were held.