Heme-Regulated eIF2α Kinase (HRI) Inhibition Decreases PKR Activation in HUDEP2 Cells
Authors: Hara Yannis , Gupta Dipti , Mercadante Courtney , Alving Kim , Nakamura Yukio , Krishnamoorthy Sriram , Hicks Alexandra , Demers Melanie 
Published: November 2021

Abstract
Introduction: Sickle Cell Disease (SCD) is a group of inherited disorders caused by mutations in the β-globin gene which encodes the hemoglobin subunit β in erythrocytes [1]. Hemoglobin containing the mutant β-globin polymerizes and causes sickling of erythrocytes, which subsequently leads to vaso-occlusion, hemolysis, and activation of the immune system by release of free heme [2]. Heme-regulated eIF2α kinase, also known as heme-regulated inhibitor (HRI/EIF2AK1), the dsRNA-activated kinase Protein Kinase R (PKR/EIF2AK2), PKR-like endoplasmic reticulum kinase (PERK/EIF2AK3) and General Control Non-Depressible 2 (GCN2/EIF2AK4) are four kinases belonging to the eIF2α kinases family and play key functions in the Integrated Stress Response-ATF4 pathway, which is critical for translational control in response to various stress conditions [3]. These kinases are activated respectively by heme depletion, viral infection, endoplasmic reticulum stress, and amino acid starvation, and they phosphorylate eukaryotic initiation factor-2α (eIF2α). Recently, it has been shown that HRI inhibition induces fetal hemoglobin in HUDEP-2 cells and CD34+ hematopoietic progenitor stem cells and prevents sickling, suggesting HRI as a potential therapeutic target for SCD [4, 5]. Moreover, it has been well documented that kinase inhibition can activate compensatory loops (bypass signaling) to circumvent the inhibited target, in particular by overexpression and activation of kinases having the same substrate [6, 7]. Thus, in this study, we investigated if the inhibition of HRI in HUDEP-2 cells lead to compensatory mechanisms by modulation of the expression and activation of the other eIF2α kinases.
Methods: To inhibit HRI, we generated HUDEP-2 [8] HRI Knock-Out cells (HRI-KO) and HUDEP-2 HRI Kinase Dead cells (K196R) (clones HRI-KD1 and HRI-KD2) by using CRISPR-Cas9 gene editing technology [9]. We confirmed the induction of fetal hemoglobin for each clone by flow cytometry. To evaluate a potential compensatory mechanism, we measured the effects of HRI inhibition on the expression and activation of the other eIF2α kinases by western blot (WB) and the regulation at the transcriptomic level by qPCR. Based on the results of preliminary studies, we generated HUDEP-2 PKR Knock-Out cells (PKR-KO) by CRISPR-Cas9. We differentiated them for 7 days and we quantified the level of fetal hemoglobin by flow cytometry, AlphaLISA® and WB.
Results: HRI-KO, HRI-KD1 and HRI-KD2 clones expressed fetal hemoglobin after 7-day of differentiation consistent with published data [4]. HRI inhibition did not result in any modulation of PKR protein expression, but the activation of PKR, measured by phosphorylation at its residue Threonine 446, was decreased in HRI-KD1, HRI-KD2, and in HRI-KO cells at day 0 and day 7 of differentiation. As PKR and HRI have the same downstream target eIF2α and HRI inhibition induces fetal hemoglobin through eIF2α-ATF4-Bcl11a axis, we verified if fetal hemoglobin induction is due to HRI inhibition exclusively and is not a consequence of the decrease in PKR activation when HRI is inhibited. We measured the protein expression level of fetal hemoglobin in PKR-KO cells and results obtained by flow cytometry, western blot and AlphaLISA® did not show any regulation in fetal hemoglobin in PKR-KO cells after 7 days of differentiation. Finally, HRI inhibition did not result in any regulation of kinases PERK and GCN2 activation and expression, at the RNA and protein level. The expression of these two eIF2α kinases was low compared to HRI and PKR.
Conclusion: HRI inhibition does not cause any modulation in the expression and activation of GCN2 and PERK in HUDEP-2 cells but results in a decrease in PKR activation. This outcome could be explained by a possible increase in the expression of proteins that inhibit PKR, such as TRBP or Hsp40, and are induced by HRI silencing (Hsp40) [10-12]. Nonetheless PKR inhibition does not induce fetal hemoglobin in HUDEP-2 cells after 7 days of differentiation. Overall, this study provides evidence that fetal hemoglobin induction by HRI inhibition in HUDEP-2 cells is independent from the other eIF2α kinases and supports HRI as a potential therapeutic target in SCD. However, the biological implications of a potential compensatory effect on PKR signaling in HRI-expressing tissues warrant further investigation.

Disclosures
Krishnamoorthy: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company.