SGK1 Inhibition Induces Fetal Hemoglobin in Erythroid Cells

Introduction: Novel and safe therapeutic targets to increase expression of fetal hemoglobin (HbF) have potential to treat b-hemoglobinopathies (Platt, Brambilla et al. 1994, Steinberg 2020), including sickle cell disease (SCD) in which red blood cell (RBC) hemoglobin S resulting from a mutation in the hemoglobin β-globin subunit causes RBC sickling and hemolysis triggering vascular inflammation (Piel, Steinberg et al. 2017, Kato, Piel et al. 2018). Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase in the AGK kinase family that controls physiological processes such as cell growth, proliferation, migration, and apoptosis (Hayashi, Tapping et al. 2001, Sang, Kong et al. 2020). SGK1 is regulated by multiple ligands (insulin, cAMP, IGF-1, steroids, IL-2 and TGF-β) and phosphorylation by SGK1 modulates the activity of downstream effectors including ion channels (ENaC), Na-Cl cotransporters (NCC), membrane transporters, cellular enzymes (GSK3B) and transcription factors (FOXO3a, β-catenin, NF-κB and SP1) (Brunet, Park et al. 2001, Snyder, Olson et al. 2002, Loffing, Flores et al. 2006, Bruhn, Pearson et al. 2010, Boccitto and Kalb 2011, Wang, Hu et al. 2017). Previous studies show that SGK1 mediates survival signals in HEK cells by inhibiting FOXO3a through phosphorylation at Ser-315 (Brunet, Park et al. 2001). Recently, metformin was shown to induce HbF in erythroid cells through FOXO3a activation and metformin prevents RBC sickling in SCD (Zhang, Paikari et al. 2018). Thus, we hypothesized that inhibition of SGK1 and subsequent alleviation of SGK1-induced FOXO3a inhibition, may induce expression of erythroid cell HbF.

Methods: We studied the ability of SGK1 to inhibit HbF induction in erythroid cells by culturing CD34+ hematopoietic progenitor stem cells from both healthy and SCD blood donors using a 21-day differentiation protocol. After confirming expression of SGK1 in CD34+ cells by Western blot, SGK1 activity was inhibited using the selective and potent SGK1 inhibitor RA04075215A (Halland, Schmidt et al. 2015). SGK1 is activated by phosphorylation at Thr256 and we confirmed target engagement through measurement of Thr256 phosphorylation on Western blots. To decipher the effect of SGK1 inhibition on the SGK1 downstream pathway, we assessed the inhibition of FOXO3a triggered by SGK1 through evaluation of FOXO3a phosphorylation Ser315. In parallel, we quantified HbF gene transcripts by qPCR, determined the level of HbF protein by Western blot, and quantified F-cells by flow cytometry. Finally, to evaluate the effect of SGK1 inhibition on RBC sickling, we performed a cell sickling assay upon completion of erythroid differentiation in culture. Fully differentiated CD34+ cells from SCD blood donors were incubated under in hypoxia (2% O ₂) for 4 hours and then abnormal shaped cells were analyzed using the Amnis® ImageStream® flow cytometer.

Results: By day 21 of differentiation, HbF protein expression in CD34+ cells increases significantly in RA04075215A-treated cells versus untreated controls. In addition, a combination of SGK1 inhibition and hydroxyurea treatment reveals a potential synergistic induction of HbF. Western blot analysis shows a decrease in phospho-SGK1 phosphorylated at Thr-256 with SGK1 inhibition, confirming target engagement and loss of SGK1 activity. Downstream of SGK1, phospho-FOXO3a phosphorylated at Ser-315 was also decreased significantly following SGK1 inhibition, demonstrating alleviation of FOXO3a inhibition. Finally, in the RBC sickling assay, RA04075215A-treated cells were significantly protected from sickling under hypoxia compared to controls.

Conclusion: In summary, this study establishes SGK1 as a potential new therapeutic target in SCD. We demonstrate that SGK1 inhibition induces HbF in CD34+ cells through FOXO3a transcription factor activation and prevents CD34+ cells from sickling. In the future, in vivo studies are necessary to confirm the role of SGK1 in HbF induction and to assess the efficacy of SGK1 inhibition in improving markers of SCD.