Selective silencing of α-globin by the histone demethylase inhibitor IOX 1 : a

Thalassemia is the world’s most common form of inherited anemia, and in economically undeveloped countries still accounts for tens of thousands of premature deaths every year. The accumulation of free excess α-globin chains in red blood cells and their precursors, as a result of the decreased production of b-globin, is believed to be the main pathophysiological mechanism leading to hemolytic anemia and ineffective erythropoiesis in b-thalassemia. Clinical genetic data accumulated over the last 30 years indicate that a natural reduction in α-globin chain output by 25-50%, resulting from coinherited α-thalassemia, ameliorates the disease phenotype in patients with b-thalassemia. Herein, we have developed and performed a targeted small molecule screen to identify compounds which downregulate α-globin expression. This identified IOX1, a pan-histone demethylase inhibitor, which selectively downregulates α-globin expression without perturbing erythroid differentiation or general gene expression, more specifically b-like globin expression. Our data show that selective silencing of α-globin expression in erythroid cells is pharmacologically feasible, and IOX1 is a lead compound to developing new therapy to treat b-thalassemia through the novel pathway of downregulating α-globin expression. We first optimized a serum-free, miniature erythroid differentiation system starting from primary human CD34 cells, the exact type of cells we would ultimately like to target in vivo (Figure 1). This culture system produced a sufficient number of viable, relatively pure, and synchronous populations of human erythroid cells in vitro to enable us to perform high throughput screens (Figure 1A,B). CD34 cells were differentiated in 96-well plates over 21 days along the erythroid lineage, and the morphology and immunophenotypical characteristics of the resultant cells faithfully recapitulated normal erythropoiesis (Figure 1C,D). These cells demonstrated a gradual increase in expression of the globin genes (Figure 1E) and other erythroid-specific genes (Online Supplementary Figure S1), and the hemoglobin protein analysis confirmed the higher proportion of fetal hemoglobin (HbF) and adult hemoglobin (HbA) in cells differentiated from umbilical cord and adult CD34 cells, respectively (Figure 1F). We then validated the culture system using hydroxyurea and sodium butyrate, which were previously shown to alter globin gene expression. Erythroid cells incubated with these compounds demonstrated a dose dependent increase in the g/b messenger ribonucleic acid (mRNA) ratio, consistent with previously reported data (Figure 1G,H). Next, we transfected erythroid cells with two validated small interfering RNAs targeting human α-globin RNA, which resulted in the expected knockdown of α-globin expression (Online Supplementary Figure S2). These observations confirm that the small-scale erythroid differentiation system which we have optimized is a valid tool to examine changes in globin gene expression in vitro. Previous studies have revealed contrasting epigenetic environments containing the human αand b-globin genes. The human α-globin gene cluster is located on chromosome 16, in a gene dense, early replicating, open chromatin environment and its promoter is associated with unmethylated CpG islands and, in non-erythroid cells, is enriched for H3K27me3 which signals transcriptional silencing. By contrast, in non-erythroid cells the b-globin gene is situated in a relatively gene sparse, late replicating, closed heterochromatic environment on chromosome 11, and the promoter of the b-globin gene is methylated rather than enriched for H3K27me3. Therefore, in the search for drugs which specifically alter expression of α-globin, we performed a selective screen, using a small molecule library of epigenetically active cell permeable compounds, potentially targeting these different epigenetic environments. This library contains a collection of 37 compounds that were designed to inhibit a wide range of epigenetic pathways (Online Supplementary Table S1). Erythroid cells were incubated for 72 hours with these compounds, and gene expression levels were obtained using Fluidigm high throughput quantitative polymerase chain reaction (qPCR) system. The primary screening criterion was downregulation of α-globin expression without altering b-globin expression, and an α/b globin mRNA ratio of less than 0.75 was considered as the cutoff for identifying high-scoring compounds. This screen identified four compounds that downregulate α-globin expression: histone demethylase (KDM) inhibitor, IOX1; histone deacetylase inhibitor, vorinostat; histone methyltransferase inhibitor, chaetocin and lysinespecific histone demethylase 1 inhibitor, tranylcypromine (Figure 2B and Online Supplementary Figures S3-S5). Of these compounds, the novel KDM inhibitor IOX1 provided the most promising results with the desired effects on globin gene expression. Chaetocin decreased the viability of erythroid cells at low concentrations and tranylcypromine markedly retarded erythroid differentiation, as evidenced by immature cell morphology and lack of expression of erythroid-specific cell surface proteins. Therefore these two compounds were not followed up further (Online Supplementary Figure S6). Vorinostat downregulated α-globin expression whilst inducing gglobin expression (Online Supplementary Figure S7) and is currently under further investigation. To further examine the effect of IOX1 on globin gene expression, we titrated the concentration of IOX1 with the developing erythroid cells. This confirmed initial observations: IOX1 caused a dose-dependent decrease in α-globin expression, whereas the expression of b-globin was largely unaffected (Online Supplementary Figures S8 and S9). The decrease in α/b-globin mRNA ratios was statistically significant at all doses tested (Figure 2C). We then analyzed the mRNA levels of all globin genes in erythroid cells treated with IOX1 using the nCounter Digital Analyzer (NanoString Technologies), which found that IOX1 significantly downregulated α-, g-, mand z-globin expression (Figure 2D). Interestingly, with the exception of g-globin, IOX1 downregulated αand other α-like globin genes (m and z) situated in the α-globin locus, whereas the expression levels of b-like globin genes (b, d and e) were unaffected, suggesting that IOX1 acts selectively on the α-globin locus. IOX1 reduced cell expansion by about 40% (fold expansion dropped from 18-fold to 11-fold) at 40mM concentration, but the proportion of viable cells remained unchanged over all dose levels (Figure 3A,B). This suggests that IOX1 has a mild inhibitory action on erythroid cell proliferation in vitro, although it does not adversely affect cellular viability. Morphologically, erythroid cells treated with a dose range of IOX1 differentiated in a sim-