The pyruvate kinase activator mitapivat reduces hemolysis and improves anemia in a β-thalassemia mouse model
Alessandro Matte, … , Carlo Brugnara, Lucia De Franceschi
Alessandro Matte, … , Carlo Brugnara, Lucia De Franceschi
Published April 6, 2021
Citation Information: J Clin Invest. 2021;131(10):e144206. https://doi.org/10.1172/JCI144206.
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Research Article Hematology

The pyruvate kinase activator mitapivat reduces hemolysis and improves anemia in a β-thalassemia mouse model

Abstract

Anemia in β-thalassemia is related to ineffective erythropoiesis and reduced red cell survival. Excess free heme and accumulation of unpaired α-globin chains impose substantial oxidative stress on β-thalassemic erythroblasts and erythrocytes, impacting cell metabolism. We hypothesized that increased pyruvate kinase activity induced by mitapivat (AG-348) in the Hbbth3/+ mouse model for β-thalassemia would reduce chronic hemolysis and ineffective erythropoiesis through stimulation of red cell glycolytic metabolism. Oral mitapivat administration ameliorated ineffective erythropoiesis and anemia in Hbbth3/+ mice. Increased ATP, reduced reactive oxygen species production, and reduced markers of mitochondrial dysfunction associated with improved mitochondrial clearance suggested enhanced metabolism following mitapivat administration in β-thalassemia. The amelioration of responsiveness to erythropoietin resulted in reduced soluble erythroferrone, increased liver Hamp expression, and diminished liver iron overload. Mitapivat reduced duodenal Dmt1 expression potentially by activating the pyruvate kinase M2-HIF2α axis, representing a mechanism additional to Hamp in controlling iron absorption and preventing β-thalassemia–related liver iron overload. In ex vivo studies on erythroid precursors from patients with β-thalassemia, mitapivat enhanced erythropoiesis, promoted erythroid maturation, and decreased apoptosis. Overall, pyruvate kinase activation as a treatment modality for β-thalassemia in preclinical model systems had multiple beneficial effects in the erythropoietic compartment and beyond, providing a strong scientific basis for further clinical trials.

Authors

Alessandro Matte, Enrica Federti, Charles Kung, Penelope A. Kosinski, Rohini Narayanaswamy, Roberta Russo, Giorgia Federico, Francesca Carlomagno, Maria Andrea Desbats, Leonardo Salviati, Christophe Leboeuf, Maria Teresa Valenti, Francesco Turrini, Anne Janin, Shaoxia Yu, Elisabetta Beneduce, Sebastien Ronseaux, Iana Iatcenko, Lenny Dang, Tomas Ganz, Chun-Ling Jung, Achille Iolascon, Carlo Brugnara, Lucia De Franceschi

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Figure 4

In Hbbth3/+ mice, mitapivat reduced liver iron overload and oxidative stress and increased hepcidin.

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In Hbbth3/+ mice, mitapivat reduced liver iron overload and oxidative st...
(A, left panel) Iron staining (Perl’s Prussian blue) in liver from WT and Hbbth3/+ mice treated with vehicle or mitapivat. Original magnification ×200. Scale bars: 100 μM. One representative image from 4 with similar results. Black arrows indicate liver iron deposits. (A, right panel) Quantification of iron staining in liver. Data are mean ± SD (n = 6). #P < 0.05 compared with WT mice and *P < 0.05 compared with vehicle-treated mice by 2-way ANOVA with Bonferroni multiple comparison correction. (B) Soluble fractions of liver from WT and Hbbth3/+ mice with and without mitapivat were analyzed with 12% SDS-PAGE and OxyBlot. Quantification of band area was performed by densitometry and expressed as a percentage of WT. Data are mean ± SD, n = 6. (C and D) mRNA expression of hepcidin (Hamp) and Id1 by qRT-PCR on liver from WT and Hbbth3/+ mice treated with vehicle or mitapivat. Experiments were performed in triplicate. Data are mean ± SD. #P < 0.05 compared with WT mice and *P < 0.05 compared with vehicle-treated mice by multiple comparisons using ANOVA; internal comparisons were calculated by unpaired 2-tailed Student t test. ##P < 0.05 compared with WT mice, ###P < 0.02 compared with WT mice, *P < 0.05 compared with HBBth3/+ mice. A 2-sided P < 0.05 was considered statistically significant. (E, left panel) Western blot analysis with specific antibodies against phospho (p) NF-κB p65, NF-κB p65, pSTAT3, STAT3, pNRF2, and NRF2 of liver from WT and Hbbth3/+ mice with vehicle or mitapivat treatment, GAPDH as loading control. One representative gel from 6 with similar results. (E, right panel) Densitometric analyses of the Western blots. Data are mean ± SD (n = 6). (B and E) #P < 0.05 compared with WT mice and *P < 0.05 compared with vehicle-treated mice by 2-way ANOVA with Bonferroni multiple comparison correction.