β-thalassemia (β-thal) is a genetic red cell disorder characterized by chronic hemolytic anemia due to ineffective erythropoiesis and reduced red cell survival. 1-3 Chronic transfusion and intensive iron chelation are standard treatments for β-thalassemic syndromes, 1 but new therapeutic options are being developed, including gene therapy4 and novel pharmacologic approaches. We have shown that mitapivat, a pyruvate kinase activator, improves anemia and ineffective erythropoiesis in Hbbth3/+ mice, a widely used model for β-thal. 5 The effects of mitapivat are not limited to the erythroid compartment: mitapivat also modulates DMT1 expression, controlling iron absorption in the duodenum in Hbbth3/+ mice, with an increase of hepcidin related to the improvement in ineffective β-thalassemic erythropoiesis. 5 Results from a phase II trial of mitapivat in non–transfusion-dependent β-thal patients previously demonstrated a sustained long-term increase in hemoglobin (Hb≥ 1 g/dL) with improvement of hemolysis and ineffective erythropoiesis. 6 Here, we asked whether mitapivat might be a potential therapeutic option also for β-thal patients under chronic transfusion regimen. In order to address this question, we exposed female Hbbth3/+ mice (3-4 months of age) to chronic transfusion with or without mitapivat (50 mg/kg twice daily [BID]). Hbbth3/+ mice were treated by oral gavage with mitapivat (50 mg/kg BID) or vehicle for 10 days, and then transfused with 400 µL washed red blood cells at 40-45% hematocrit (Hct) 7 (Figure 1A). We defined Hb values≤ 10.5 g/dL as the transfusion threshold, corresponding to the reduction of~ 50% of post-transfusion Hb values. Normality of data was assessed with the Shapiro-Wilk test. Two-tailed unpaired Student t-test or two-way analysis of variance with Tukey’s multiple comparisons were used for data analyses. Data show values from individual mice and are presented with mean±standard error of the mean (differences with P< 0.05 were considered significant). As shown in Figure 1B, mitapivat-treated β-thal mice exposed to chronic transfusion displayed a greater sustained rise in Hb from baseline compared to vehicle-treated transfused β-thal mice. This resulted in a longer interval between transfusions (13.8±1.0 days in mitapivat-treated β-thal mice vs. 10.5±1.0 days in vehicletreated β-thal mice; Figure 1C). Chronic transfusion resulted in a significant reduction of splenomegaly in both mitapivat-and vehicle-treated β-thal mice (Online Supplementary Figure 1SA) compared to untreated β-thal mice, but spleen iron accumulation was significantly lower in mitapivat-treated β-thal mice when compared to vehicle-treated β-thal mice (Figure 1D). A significant reduction of both bone marrow and spleen ineffective erythropoiesis was observed in all transfused β-thal mice (Figure 1E; Online Supplementary Figure S1B). Of note, mitapivat-treated transfused β-thal mice showed a slight increase of bone marrow erythropoiesis with a trend towards an improvement of maturation index compared to vehicle-treated transfused β-thal mice evaluated at the end of the study. 5 This is most likely related to a protective effect of mitapivat on residual bone marrow and spleen erythropoiesis (Figure 1F). Indeed, plasma erythropoietin was lower in mitapivat-treated transfused β-thal mice than in vehicle-treated transfused β-thal mice (Online Supplementary Figure S1C). Since splenic macrophages contribute to both erythrophagocytosis and iron recycling, we evaluated the functional profile of spleen macrophages in the different mouse groups. 8 As shown in Figure 1G, flow cytometric analysis of the surface expression of the M1 marker CD80 and the …