Nrf2 Activation and Fetal Hemoglobin Induction in Sickle Cell Disease

Sickle Cell Disease (SCD) is caused by a point mutation in the beta- chain of hemoglobin, triggering a complex pathophysiology resulting in recurrent, painful vaso-occlusive events (VOCs) and chronic hemolytic anemia. Induction of fetal hemoglobin (HbF), is a well-established approach towards the treatment and potentially cure for SCD. This is exemplified by the condition known as Hereditary Persistence of Fetal Hemoglobin (HPFH), which when present in SCD patients results in asymptomatic disease. Reactivation of fetal hemoglobin can be achieved by different methodologies both genetic manipulation and pharmacological agents. The mechanism of action of hydroxyurea, the only FDA approved drug for the treatment of SCD, is by activating HbF. We have investigated the role of Nrf2 in activating fetal hemoglobin and it potential to ameliorate the symptoms of SCD. Nrf2 is a basic leucine zipper transcription factor that is bound to a cytosolic protein Keap1, a Kelch domain containing protein, which targets the factor to the proteasome. Previous studies have shown that chemical activation of Nrf2 leads to induction of HbF. Here we show that siRNA based knockdown of Keap1 led to ~70 % reduction in Keap1 mRNA levels in Human Umbilical cord Derived Eryrthroid Progenitor cells (HUDEP), and concomitantly mediated a 2-fold induction of g-globin mRNA levels. Peripheral blood derived mononuclear cells differentiated into erythroid progenitors treated with bardoxolone-Me, a potent Nrf2 activator, resulted in a significant 4-fold induction in γ-globin mRNA, 2.5 fold induction in F-cells and up to 2.5-fold up-regulation in both Aγ and Gγ globin protein. Bardoxolone-Me treated erythroid progenitors also demonstrated a significant reduction in hypoxia-induced shape changes of sickle cells - about 42 % decrease at 100 nM and 55 % decrease at 250 nM. Bardoxolone-Me was also evaluated in a Townes SCD mouse model carrying an inducible γ-globin gene. A single oral dose of bardoxolone-Me at 30mg/kg, showed 9-fold induction in γ-globin mRNA levels at 6 h compared to untreated mice. Based on these findings, Nrf2 activation, can potentially be utilized for HbF induction in the treatment of SCD.