SALUBRINAL MEDIATES FETAL HEMOGLOBIN INDUCTION VIA THE EIF2α-ATF4 SIGNALING PATHWAY FOR THE TREATMENT OF SICKLE CELL DISEASE)

Abstract

Sickle cell disease (SCD) is an inherited disorder caused by the βS-globin mutation leading to hemoglobin polymerization, vaso-occlusion, chronic hemolysis and progressive organ damage. SCD affects ~100,000 people of African descent in the United States and millions worldwide. An effective therapy for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents such as Hydroxyurea (HU), the only drug with FDA-approval that works via this mechanism. The goal of our study was to determine whether Salubrinal (SAL), a selective protein phosphatase 1 inhibitor, induces HbF expression by the activation of p-eIF2α (phosphorylated eukaryotic initiation factor 2α) and ATF4 (activating transcription factor 4). ChIP analysis in K562 cells showed ATF4 binding in the locus control region along with the predicted ATF4 binding sites in the y-globin promoter and HBB locus. Sickle erythroid progenitors treated with SAL 9, 18 and 24 µM increased F-cells from 5.2%, 7.7% and 9% (p<0.05) respectively compared to untreated cells and decreased oxidative stress. Western blot analysis showed SAL 24 µM induce HbF by 1.5-fold and mediated dose-dependent increases of p-eIF2α and ATF4 up to 11.1%. In preparation for preclinical studies, pharmacokinetic studies showed plasma concentration of SAL (5mg/kg) peaked 6 hours post IP injection. Subsequent treatments of SCD mice (n=10 per group) were conducted with SAL (3 and 5mg/kg), HU (100mg/kg) and water control (vehicle), 5 days a week for 4 weeks. Flow cytometry showed SAL produced a significant 2.3-fold increase in F-cells compared to a 2.6-fold increase by HU on week 4 (p<0.05); SAL did not produce significant changes in peripheral blood counts. Our findings, supports HbF induction and decrease sickle cell formation by SAL in vivo and the potential this agent might be developed as a novel treatment option for SCD.