Identification of novel γ-globin inducers among all potential erythroid druggable targets

Abstract

Human γ-globin is predominantly expressed in fetal liver erythroid cells during gestation from 2 nearly identical genes, HBG1 and HBG2, that are both perinatally silenced. Reactivation of these fetal genes in adult red blood cells can ameliorate many symptoms associated with the inherited β-globinopathies, sickle cell disease, and Cooley anemia. Although promising genetic strategies to reactivate the γ-globin genes to treat these diseases have been explored, there are significant barriers to their effective implementation worldwide; alternatively, pharmacological induction of γ-globin synthesis could readily reach the majority of affected individuals. In this study, we generated a CRISPR knockout library that targeted all erythroid genes for which prospective or actual therapeutic compounds already exist. By probing this library for genes that repress fetal hemoglobin (HbF), we identified several novel, potentially druggable, γ-globin repressors, including VHL and PTEN. We demonstrate that deletion of VHL induces HbF through activation of the HIF1α pathway and that deletion of PTEN induces HbF through AKT pathway stimulation. Finally, we show that small-molecule inhibitors of PTEN and EZH induce HbF in both healthy and β-thalassemic human primary erythroid cells.

Graphical abstract

Figure 1.

VHL deletion induces γ-globin through the activation of HIF1α. (A-B) Deletion of VHL in HuDEP2 cells using sgRNA-1 (g1) or sgRNA-4 (g4) induces HbF, a finding that is abolished by concomitant deletion of HIF1α. (C) γ-globin, VHL, and HIF1α mRNA levels were analyzed by quantitative reverse transcriptase-polymerase chain reaction. Data are shown as means ± standard deviation from 3 experiments (**P < .01; ***P < .001; n.s., not significant; unpaired Student t test).

Figure 2.

Deletion and pharmacological inhibition of PTEN induces γ-globin. (A) HbF induction in 2 independent PTEN-deficient clonal HuDEP2 cell lines (PTEN^−/− #35 and #54) compared with control cells (nontargeting-4), as demonstrated by HbF staining and flow cytometry (n = 3). (B) Analysis of γ-globin and β-globin mRNA levels in PTEN-deleted clonal cells (n = 3). (C-F) CD34⁺ cells isolated from healthy donors were differentiated into erythroid cells and treated with LSD1 (GSK690), EZH (Taze), or PTEN inhibitors (bpV), showing induction of HbF with all 3 compounds, as demonstrated by HbF staining and flow cytometry (C-D; n = 5) and mRNA analysis (E-F; n = 4). (G-H) CD34⁺ cells isolated from β-thalassemia patients were differentiated into erythroid cells and treated with EZH (Taze, n = 3) or PTEN (bpV, n = 6) inhibitors. Both compounds result in HbF induction by high-performance liquid chromatography analysis. Data are shown are means ± standard deviation (A-B,E-F: **P < .01; ***P < .001; n.s., not significant; unpaired Student t test; D,H: *P < .05; **P < .01; paired Student t test).