Salubrinal Mediated Fetal Hemoglobin Induction through the eIF2α-ATF4 Signaling Pathway

Sickle cell disease (SCD) is a genetic disorder caused by a point mutation in the adult β-globin gene affecting ~100,000 people in the United States and millions worldwide. The clinical symptoms of SCD include chronic hemolytic anemia, pain, and progressive organ damage creating a great burden to annual healthcare costs. An effective therapeutic intervention for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents to ameliorate clinical symptoms. Hydroxyurea (HU) is the only FDA-approved drug used to induce HbF in SCD, however, it is not effective in all people. Moreover, a critical barrier to the development of additional effective HbF inducers is the limited knowledge of mechanisms involved in γ-globin gene regulation. The goal of this project is to determine if Salubrinal (SAL) can be used as a therapeutic HbF inducer in SCD. Salubrinal is a selective inhibitor of protein phosphatase 1 leading to increased levels of p-eIF2α (phosphorylated eukaryotic initiation factor 2α) and a block in protein synthesis. It is known that ATF4 (activating transcription factor 4) is a downstream target of p-eIF2α under oxidant stress conditions. Previously, we identified a Gγ-globin cAMP response element (G-CRE) that binds ATF2, a known binding partner of ATF4 (Sangerman et al. Blood 2006). Furthermore, ENCODE in silico analysis showed predicted ATF4 binding sites at -822Gγ and in the second intron of the β-globin gene. Therefore, studies were conducted to determine if ATF4 is involved in the mechanism of HbF induction by SAL.

Initial experiments were conducted with day 8 erythroid progenitors generated from human CD34⁺ stem cells isolated from healthy donors. Cells were treated with SAL (5 µM and 12 µM), HU (100 μM), and 1% DMSO (vehicle control) for 48 h; cell viability remained >90% for all drug conditions. SAL produced a 2.2-fold increase in γ-globin mRNA and a similar increase in HbF levels by western blot analysis; flow cytometry revealed a 2.1-fold increase (p<0.05) in F-cells (HbF positive cells). Subsequently, similar studies were completed in sickle erythroid progenitors generated from peripheral blood mononuclear cells where SAL (18 μM) increased γ-globin mRNA 3.2-fold and F-cells 2.5-fold (p<0.05). To gain insights into mechanisms of HbF induction, K562 cells were treated with SAL (12 µM and 18µM), Hemin (50 μM), and 1% DMSO for 48 h. Western blot analysis showed a parallel increase of HbF levels, p-eIF2α and p-ATF4 mediated by SAL. To evaluate whether SAL protects against oxidative stress, K562 cells were treated for 48 h and then 10 µM dichlorofluorescin diacetate was added to measure reactive oxygen species (ROS). SAL decreased ROS by 50% compared to DMSO controls; in contrast hemin known to mediate oxidative stress, produced a 3.7-fold increase in ROS levels.

To determine if SAL activates HbF expression in vivo ,the Townes knock-in SCD mice were treated with SAL (1mg/kg) and 0.5% DMSO (vehicle control), 5 days a week for 2 weeks; Dectiabine (1.25mg/kg), a known HbF inducer, was given once a week for 2 weeks. Blood was drawn at week 0 (baseline) and week 2 after completion of treatment. The SAL treatment was well tolerated and did not cause a significant change in the complete blood count and differential or reticulocyte count. Flow cytometry analysis of peripheral blood showed that SAL produced a 4.5-fold increase in F-cells by 2 weeks of treatment compared to the DMSO treated mice (p<0.05). At the dose of Decitabine given, there was not a significant change in F-cells.

In summary, our in vitro data supports HbF induction by SAL involves p-eIF2α mediated activation of ATF4. The interaction of ATF4 in the G-CRE and/or other predicted binding sites to activate γ-globin expression will be investigated. To support clinical development, studies in the SCD mouse model support the ability of SAL to induce HbF in vivo . Additional studies are underway to determine if longer treatment periods will be more effective. Defining the mechanism of HbF induction by SAL has the potential to impact treatment for SCD and other β-hemoglobinopathies.