Metabolomic and epigenetic signatures of hemin-induced stress in sickle cell erythroblasts

Abstract Introduction: Patients with sickle cell disease (SCD) endure systemic hypoxia and chronic hemolysis, resulting in excessive heme exposure and disrupted metabolomic profiles. These disruptions involve key pathways such as glycolysis, the tricarboxylic acid (TCA) cycle, nucleotide catabolism, and glutathione metabolism. Since these metabolic pathways are essential to the hypoxic response, red blood cell (RBC) sickling, inflammation, and oxidative stress, it is critical to elucidate the mechanisms by which heme alters cellular metabolism and contributes to SCD progression and severity. Objectives: This study aims to determine the mechanism by which hemin affects the metabolism of SCD erythroblasts, particularly in the regulation of redox homeostasis. We seek to uncover the mechanisms underlying hemin-integrated metabolic, transcriptomic, and epigenetic alterations. Methods: CD34⁺ hematopoietic stem cells were isolated from the peripheral blood mononuclear cells of SCD patients and cultured in a two-phase erythroid differentiation system under hypoxic conditions. The impact of hemin on redox homeostasis was assessed by measuring oxidative stress and ferroptosis markers. Metabolomic profiling, global RNA sequencing, and ATAC-seq analyses were performed to investigate the underlying mechanisms. Results: Under hypoxic conditions, hemin induces dysregulation of redox homeostasis and ferroptosis signaling in SCD erythroblasts. Transcriptomic analysis showed hemin upregulates the expression of cystine transporter xCT (SLC7A11 and SLC3A2), and glutamate-cysteine ligase subunits (GCLC and GCLM), but downregulates GPX4, glutaminase (GLS), and glutamine synthetase (GLUL). These expression changes impaired glutathione (GSH) utilization and glutamine/glutamate metabolism. In addition, metabolomic analysis showed that hemin interferes with TCA cycle activity, resulting in accumulation of 2-oxoglutarate (2OG), which is redirected to L-2-hydroxyglutarate (L2HG). Both 2OG and L2HG act as epigenetic modifiers to influence the activity of 2OG-dependent histone demethylases, thereby promoting global histone hypermethylation. Subsequent chromatin immunoprecipitation analysis showed increased association of histone methylation codes on the genes involved in heme/iron metabolism and ferroptosis response. Supplementation with TCA cycle derivatives, such as dimethyl ketoglutarate and dimethyl fumarate, reversed the histone hypermethylation and mitigated the hemin-induced ferroptosis. Further, hemin was found to induce nuclear factor erythroid 2-related factor 2 (NRF2) expression as a compensatory response to protect against ferroptosis. NRF2 silencing abrogated the expression of GPX4 and SLC7A11, along with elevated ferroptosis. Conclusions: By suppressing TCA cycle activity and promoting L2HG accumulation, hemin induces ferroptosis through altered redox homeostasis and epigenetic modifications. These deleterious effects can be partially reversed by TCA cycle metabolite derivatives. NRF2 plays a critical compensatory role in regulating the ferroptosis induced by hemin. Collectively, our findings define a heme/NRF2/L2HG axis that integrates metabolic, epigenetic, and ferroptotic pathways in the response of SCD erythroblasts to excess heme exposure.