Ex vivo treatment by mitapivat, an allosteric pyruvate kinase activator, reduced oxidative stress to support terminal erythropoiesis of non-transfusion dependent thalassemia patients due to β-thalassemia/hb e disease

Abstract Introduction: Ineffective erythropoiesis is one of the pathophysiological characteristics in the bone marrow of severely anemic patients due to β-globin gene mutations. The intracellular reactive oxygen species (ROS) generated by free α-globin chain aggregation could affect the lifespan of erythroid cells during erythropoiesis. Previously, our group described the effects of mitapivat, an allosteric activator of pyruvate kinase (PK), on reducing intracellular ROS in red blood cells of non-transfusion-dependent thalassemia (NTDT) patients with β-thalassemia/hemoglobin E (β-thal/Hb E) disease in an ex vivo study (Suksangpleng et al. , Blood 2024; 144 (Supplement₁): 2479). We are currently studying the effects of mitapivat on oxidative stress during the terminal erythropoiesis of β-thal/Hb E patients. Objective: To study the effects of mitapivat on oxidative stress, erythroid cell viability, ATP production, and erythroid differentiation during terminal erythropoiesis of NTDT patients with β-thal/Hb E disease in an ex vivo setting. Methods: A 6-mL EDTA whole blood sample was collected from five NTDT patients diagnosed as β-thal/Hb E disease using multiplex PCR methods to specifically detect 9 common β-globin gene mutations found in the Thai population. The CD34+ peripheral blood mononuclear cells (PBMCs) were separated by anti-CD34 antibody-labeled magnetic beads. The three-phase erythroid culture system, which consisted of proerythroblast transformation, proerythroblast proliferation, and erythroid differentiation, was used to culture the CD34+ PBMCs for 18 days. The ex vivo treatment was performed in phase three by incubating the cultured erythroid cells with the completed hematopoietic cultured medium containing 10 nM mitapivat. The intracellular ROS, erythroid cell viability, ATP production, and erythroid differentiation of mitapivat-treated erythroid cells were measured during phase three. Results: Five NTDT patients, consisting of 3 and 2 cases with βoβE and β+βE genotypes, respectively, had chronic anemia with a mean (± standard deviation SD) Hb concentration of 7. 9 ± 1. 4 and 8. 7 ± 0. 6 gm/dL, respectively. The CD34+ PBMCs of NTDT patients were able to differentiate the proerythroblasts in phase one of the erythroid culture system (Day 1 to Day 10), followed by proliferating in large expansion similar to those of healthy controls in phase two (Day 11 to Day 13). After performing the ex vivo mitapivat treatment in phase three (Day 14 to Day 18), the intracellular ROS represented by mean fluorescent intensity (MFI) gradually decreased from Day 14, Day 16, and Day 18, respectively, compared to the baseline MFI of the erythroid cultured with 0. 1% DMSO (v/v) used as the solvent control. The decrease in intracellular ROS corresponded to an increase in the % of viable cells in the culture system. The ATP levels detected in the mitapivat-treated erythroid cells significantly increased at the late stages of phase three, including Day 16 and Day 18, compared to the baseline ATP levels of the erythroid cells cultured with solvent control. Finally, the erythroid differentiated cells, including proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts, reticulocytes, and RBCs, were detected by microscopic examination using a cytospin and Wright's stain at Day 14, 16, and 18. The mitapivat-treated erythroid cells showed more effective differentiation and gained a greater percentage of orthochromatic erythroblasts, reticulocytes, and RBCs than those of the erythroid cells cultured with solvent control. Conclusion: This ex vivo study showed that the intracellular ROS decreased in the mitapivat-treated erythroid cells during the erythroid differentiation stages. The reduced oxidative stress and increased ATP production accompanied erythroid cell viability during the erythroid differentiation stages. Moreover, mitapivat-treated erythroid cells could differentiate to the late stages of erythropoiesis compared to the vehicle. This study provides further mechanistic insight into clinical benefits that have been demonstrated for mitapivat in patients with thalassemia.