Salubrinal Mediated Fetal Hemoglobin Induction Through The PERK-eIF2α-ATF4 Signaling Pathway

Abstract

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene affecting ~100,000 people in the United States. These individuals suffer from hemolytic anemia, pain, and progressive organ damage. The best therapeutic intervention in SCD is fetal hemoglobin (HbF) induction by pharmacologic agents, however, Hydroxyurea is the only FDA-approved drug with proven efficacy. The goal of this project is to discover drugs that induce HbF by novel mechanisms for SCD treatment. Salubrinal (SAL), a selective inhibitor of eukaryotic initiation factor 2α (eIF2α), was shown to increase HbF levels by enhancing γ-globin mRNA translation. These findings lead us to test the hypothesis that SAL activates the PERK-eIF2α-ATF4 stress response, as a mechanism of HbF induction in erythroid progenitors. Studies were conducted in K562 and erythroid progenitor generated from CD34+ stem cells treated with SAL (5, 12, and 18µM) for 48hr. RT-qPCR and western blot were used to measure γ-globin mRNA and HbF protein levels respectively. Preliminary data revealed a dose-dependent increase for HbF levels in K562 and erythroid progenitors treated with SAL. Flow cytometry showed an increase in the number of cells producing HbF (%F-cells). Furthermore, eIF2α and ATF4 levels were increased by SAL in K562 cells. These findings suggest SAL mediates HbF induction through eIF2α/ATF-4 signaling; future studies using the preclinical sickle cell mouse model will be investigated.