Ex Vivo Treatment By Mitapivat, an Allosteric Pyruvate Kinase Activator, Reduced Oxidative Stress and Promoted Terminal Erythropoiesis in a Severe Hemolytic Anemia Patient Due to Krϋppel-like Factor 1 Mutations

Introduction: Human erythroid Krüppel-like Factor 1 (EKLF or KLF1), a hematopoietic-specific transcription factor encoded from KLF1 plays roles as the master regulator of erythroid development, including globin switching and expression, pyruvate kinase ( PKLR) function, heme synthesis, structural membrane, minor blood group, and cell cycle regulation. Most patients carrying KLF1 mutations present with severe chronic hemolytic anemia with low pyruvate kinase-red blood cell (PKR) activity and require regular transfusion. Previously, our group described the effects of ex vivo mitapivat (AG-348), an allosteric activator of PKR, treatment on the peripheral RBCs of two non-regular transfused chronic hemolytic anemia patients due to KLF1 mutations by increased PKR activity and ATP production of treated RBCs (Suksangpleng et al, Blood 2019; 134 (Supplement_1): 2220). We are currently studying the erythropoietic characteristics and investigating the effects of treatment with mitapivat to improve the severe anemia observed in these KLF1 patients.

Objective: To evaluate the effects of ex vivo mitapivat treatment on the terminal erythropoiesis characteristics, including cell viability, oxidative stress, necrosis, and erythroid differentiation, of one severe hemolytic anemia patient due to KLF1 mutations.

Methods: The 20-mL EDTA whole blood sample was collected from one regular transfused severe hemolytic anemia patient who was identified with KLF1 alleles mutations. The CD34 ⁺-peripheral blood mononuclear cells (PBMCs) were separated by the specific anti-CD34 antibody-labelled magnetic beads and cultured by 3 phases of primary erythroid culture system, including phase 1: erythroblast transformation, phase 2: erythroblast expansion, and phase 3: erythroid differentiation. Cell viability was monitored in all 3 phases. The erythroid differentiation was monitored by stage-specific erythroid surface marker antibodies using flow cytometry. The ex vivo treatment was performed in phase 3 by incubating the cultured erythroid cells with the completed tissue cultured medium containing 40 nM mitapivat (Agios Pharmaceuticals, Inc.). Intracellular reactive oxygen species (ROS) were measured by fluorescent detection and necrotic cells were stained with Annexin V/PI and numbered by flow cytometry.

Results: One index patient with regular transfused chronic hemolytic anemia showed low level of Hb (< 11 g/dL), high reticulocyte count (> 2%), high Hb F level (> 2%), and no PKLR mutation identified. The allele genotypes of KLF1 were identified as compound heterozygous mutation including unusual amino acid (Q58X) and missense mutation (A298P) on allele 1 and 2, respectively (Table 1). Proerythroblasts were transformed from the separated CD34 ⁺-PBMCs in the Phase 1 culture period (Day 1 - Day 6) and highly expanded in Phase 2 (Day 7 - Day 13). After being induced to erythroid differentiation in Phase 3, the viability of erythroblasts gradually decreased while increased intracellular ROS was found. These phenomena corresponded to the increased numbers of necrotic cells during erythroid stage differentiation. Interestingly, the ex vivo treatment with mitapivat decreased intracellular ROS and necrotic cell numbers of the stage-differentiated erythroblasts, with more viable cell counts compared with the untreated condition in terminal erythropoiesis (Figure 1A, 1B, 1C). Moreover, the ex vivo treatment with mitapivat supported the effective stage differentiation shown by higher FITC- and lower PE-mean fluorescent intensity (MFI) that represented two stage-specific erythroid markers, including CD235a (glycophorin A) and CD71 (transferrin receptor), respectively, than those of untreated differentiated erythroblasts (Figure 1D).

Conclusion: This study showed the effects of ex vivo treatment with mitapivat which could reduce oxidative stress, promote cell viability and stage differentiation in terminal erythropoiesis of one regular transfused hemolytic anemia patient due to KLF1 mutations. Mitapivat is being studied in clinical trials in PK deficiency (NCT03853798, NCT05144256, and NCT05175105), thalassemia (NCT04770753 and NCT04770779) and sickle cell disease (NCT05031780). Further evaluation of mitapivat in patients with severe hemolytic anemia due to KLF1 mutations is warranted as a potential therapeutic option for patients with KLF1 disease.