BACH1 Inhibitor Hpp-D Mediates γ-Globin Gene Activation in Sickle Erythroid Progenitors

Abstract

Introduction: Sickle cell disease (SCD) is one of the most common β-hemoglobinopathy caused by a point mutations in the adult β-globin gene. These individuals are affected with clinical symptoms such as anemia, pain, and progressive organ damage creating a great burden to annual healthcare costs. Induction of fetal hemoglobin (HbF) using pharmacologic agents is considered as one of the best therapeutic strategies to manage clinical symptoms of SCD. At present, hydroxyurea (HU) is the only FDA-approved drug used to induce HbF in SCD patients, however, it is not effective in all patients. In our previous studies we confirmed a role of NRF2 in γ-globin gene regulation and established molecular mechanisms involving competitive binding of the transcription factors NRF2 and BACH1 in the γ-globin gene promoter, antioxidant response elements (ARE) (Zhu X et al., Haematologica. 2017). Moreover, Attucks and colleagues, our collaborators at vTv Therapeutics LLC showed that HPP-D, a BACH1 inhibitor promotes nuclear export of BACH1 and translocation of NRF2 to regulate antioxidant gene expression (Attucks OC et al., PLoS One. 2014). Therefore, we tested the ability of the novel small molecule BACH1 inhibitor, HPP-D to induce HbF expression by increasing competitive binding of NRF2 in the γ-globin ARE in KU812 cells and human sickle erythroid progenitors. Methods: Human KU812 cells were cultured in Iscove's Modified Dulbecco's medium (IMDM) along with 10% fetal bovine serum, penicillin (100 unit/ml), streptomycin (0.1 mg/ml) and 4mM L-Glutamine at 37oC with 5% CO2. Human sickle cell erythroid progenitors generated from peripheral blood mononuclear cells isolated from SCD patients, were cultured using a two-phase erythroid culturing system (Li et al., Haematologica. 2018). Both cell types were treated with 0.5, 2.5, 5, 10, and 20µM of HPPD, 100µM HU or 50µM hemin for 48 hours. The HbF positive cells (F cells) were measured by flow cytometry analysis, γ-globin, β -globin gene expressions were quantified by Real Time - qPCR and BACH1, α-tubulin, and HbF protein levels were measured by Western blot analysis. ChIP assay was performed to investigate binding competition between Bach1 and NRF2 at the locus control region-hypersensitive site 2 (LCR-HS2) and γ-globin ARE in KU812 cells. The effects of HPPD on sickling of erythroid progenitors were measured by in vitro sickling assay at 1% oxygen for 6 hrs in a hypoxic chamber. Results: The KU812 cells treated with lower concentrations of HPPD (0.5, 2.5 and 5 µM) did not show toxicity to cell growth and viability. Moreover, we observed a significant increase in HbF positive cells (F-cells) by 2.5-fold (0.5μM) and 1.6-fold (5μM) when compared to the vehicle control, DMSO. In addition, Western blot analysis showed a maximal 2.7-fold induction of HbF (p<0.05) by 0.5μM HPP-D. While KU812 cells treated with 0.5µM HPP-D alone showed 20% F-cells increase, when combined with 100µM HU the F-cells increased to 46%; similar findings were observed for mean fluorescence intensity analysis by flow cytometry analysis. Simillarly, when quantifying HbF protein levels by Western blot, 0.5 μM HPP-D alone produced a 1.4-fold increase (p<0.05) of HbF and when combined with 100µM HU showed 1.7-fold HbF increase. Remarkably, enhanced binding of NRF2 were observed in the γ-globin ARE and LCR-HS2 while BACH1 binding affinity reduced in HPP-D treated cells. By contrast, no significant change was observed for NRF2 binding in the β-globin promoter by HPP-D treatment by ChIP assay. To confirm HbF induction by HPP-D, we performed similar experiments conducted at low concentrations (0.5μM and 2.5μM) in sickle erythroid progenitors without affecting cell growth and viability; HPP-D induced HbF levels by 1.6- and 1.9-fold (p<0.05) respectively, without affecting HbS protein level. Moreover, under hypoxic conditions HPP-D treatment reduced erythroid cells sickling by 50% when compared to DMSO control. Conclusions: Overall, our study findings support the ability of HPP-D to induce γ-globin protein as a single agent or in combination with HU through BACH1 silencing and enhancing NRF2 binding in the ARE region. Additionally, HPP-D induced HbF expression and mediated an anti-sickling effect in sickle erythroid progenitors. Future in vivo studies in the SCD mouse model will be conducted confirm our in vitro findings.