Mitapivat reprograms the RBC metabolome and improves anemia in a mouse model of hereditary spherocytosis

Hereditary spherocytosis (HS) is the most common, nonimmune, hereditary, chronic hemolytic anemia after hemoglobinopathies. The genetic defects in membrane function causing HS lead to perturbation of the RBC metabolome, with altered glycolysis. In mice genetically lacking protein 4.2 (4.2–/–; Epb42), a murine model of HS, we showed increased expression of pyruvate kinase (PK) isoforms in whole and fractioned RBCs in conjunction with abnormalities in the glycolytic pathway and in the glutathione (GSH) system. Mitapivat, a PK activator, metabolically reprogrammed 4.2–/– mouse RBCs with amelioration of glycolysis and the GSH cycle. This resulted in improved osmotic fragility, reduced phosphatidylserine positivity, amelioration of RBC cation content, reduction of Na/K/Cl cotransport and Na/H-exchange overactivation, and decrease in erythroid vesicles release in vitro. Mitapivat treatment significantly decreased erythrophagocytosis and beneficially affected iron homeostasis. In mild-to-moderate HS, the beneficial effect of splenectomy is still controversial. Here, we showed that splenectomy improves anemia in 4.2–/– mice and that mitapivat is noninferior to splenectomy. An additional benefit of mitapivat treatment was lower expression of markers of inflammatory vasculopathy in 4.2–/– mice with or without splenectomy, indicating a multisystemic action of mitapivat. These findings support the notion that mitapivat treatment should be considered for symptomatic HS.

min and a 1-min post-time solvent A hold.The UHPLC system was coupled online with a mass spectrometer Q-Exactive (Thermo) scanning in full MS mode (2 μscans) at 70,000 resolution in the 60-1000 m/z range, target of 1 × 106 ions and a maximum ion injection time (IT) of 35 ms.Source ionization parameters were: spray voltage, 3.8 kV; capillary temperature, 300 °C; sheath gas, 40; auxiliary gas, 25; S-Lens level, 45.Calibration was performed before each analysis against positive ion mode calibration mixes (Piercenet, Thermo Fisher, Rockford, IL) to ensure sub ppm error of the intact mass.Data files were processed by MAVEN.8.1 (http://genomicspubs.princeton.edu/mzroll/)upon conversion of raw files into mzXML format through MassMatrix (Cleveland, OH) and then processed by MAVEN 5.2 (available at http://genomics-pubs.princeton.edu/mzroll/);spectrometry chromatograms were assessed for peak alignment, matching and the comparison of parent and fragment ions, and tentative metabolite identification (within a 2 ppm mass-deviation range between observed and expected results against the imported KEGG database).Furthermore, t-test analysis of variance was performed using GraphPad Prism (version 9.0.0 (121), GraphPad Software, San Diego, CA, USA, www.graphpad.com)with p ≤ 0.05 considered as being statistically significant.The results are expressed as mean ± standard deviation (SD) in the tables, and box and whisker plots were generated to show all points as well as median and ranges (whiskers = min.and max.) (47)(48).

Pyruvate Kinase Assay
The pyruvate kinase activity was measured by using a commercial pyruvate kinase assay kit (Sigma-Aldrich, MAK072).Following the manufacturer's instructions, red blood cells were homogenized with pyruvate kinase assay buffer and spun at 15,000xg for 10 minutes to remove cell debris.Aliquots of the supernatant were transferred to a clear-bottom 96-well plate where the appropriate reaction mixtures were added to a final volume of 100 μl per well.All measurements were performed by measuring the absorbance of each sample at 570 nm using a TECAN Infinite M200 microplate reader (Tecan, Männedorf, Switzerland).
Cells were centrifuged at 3000 rpm for 5 min and washed with PBS BSA 1%.The same number of RBCs were incubated in osmotic test solutions (NaCl solution at 192 mOsm) or isosmotic solution (320 mOsm) for 10 minutes at 37°C.Lysis was immediately stopped with 4x volumes of quenching solution (PBS at 320 mOsm) added of Count Bright Absolute Counting Beads (#C36950, eBiosciences, Thermo Fisher Scientific).Cells with normal flow cytometric FSC/SSC profiles were considered to be intact.Flow cytometric analysis was carried out with the FACSCanto I flow cytometer (Becton Dickinson).The biparametric scatter plots were analyzed with FlowJo software version 10 (Tree Star).The number of acquired counting beads was used to calculate the absolute number of intact red cells and percent of lysis was determined by the normalization to the 320 mOsm control condition (0% of lysis).
Erythroid vesicles were evaluated as previously described (23).Briefly, platelet pure plasma (PPP) was prepared from heparinized blood collected from wild-type and, 4.2 -/-mice treated with either vehicle or mitapivat (100 mg/Kg/day) for 5 months.
Blood was centrifuged at 3000 rpm for 10 min.red cells were removed, and plasma was collected and centrifuged again at 3500 rpm for 15 min to obtain PPP. 5 µl of PPP were incubated in 50 µl Ringer buffer (32 mM Hepes-HCl pH 7.4, 125 mM NaCl, 5 mM glucose, 5 mM KCl, 1 mM MgSO4, 2.5 mM CaCl2), 0.2% BSA added of 0.5 µl of anti Ter119-APC ((# 17-5921-82, eBiosciences), 0.5 ul of AnnexinV-PE (# 128102-69, eBiosciences), 0.05 µl Phalloidin FITC (# F432, ThermoFisher Scientific, to remove RBC debris and ghosts).After 30 min of incubation at 4°C wash with 100 µl ringer buffer, 0.2% BSA added of Count Bright Absolute Counting Beads.Flow cytometric analysis was carried out with the FACSCanto I flow cytometer (Becton Dickinson).The biparametric scatter plots were analyzed with FlowJo software version 10 (Tree Star).The amounts of plasmatic vesicles was determined using the formula: Analysis of in vitro release of erythroid vesicles was carried out as previously reported (Ferru E et al. Blood 2011).Briefly, red cells from wild-type (WT) and 4.2 -/- mice were washed 3 times with PBS, 5 mM Glucose and incubated, at 3% Hct, in preservation buffer (155 mM KCl, 1 mM NaCl, 0.25 mM KPO4 pH 7.4, 1 mM glucose) added of vehicle or mitapivat (2 µM) for 1h at 37°C.After 3 washes with PBS, 5 mM glucose at 4°C, treated red cells were incubated under shacking conditions, 1200 rpm, to mimic in vivo share stress at 42°C, for 50 min in presence of vehicle or mitapivat (2 µM).The suspension was then centrifuged at 3000 rpm 5 min at 4°C, RBC were removed, and the supernatant was used either for flow cytometric analysis of erythroid microparticles or centrifuged at 13000 rpm 5 min to remove ghosts, ultracentrifuged at 100,000 g for 2 h, to collect MP for western blot analysis.
The following antibodies were used: anti Band-3 (Clone IVF12, Developmental Studies Hybridoma Bank, Iowa City, IA, USA, dilution 1:1000) and antiperoxiredoxin-2 C-terminal (kindly gift of Prof. Chae HZ, Chonnam National University, South Korea).Coomassie staining was used to visualize the general protein pattern of erythroid vesicles.

Flow cytometric analysis of mouse erythroid precursors
Briefly, 500,000 cells from bone marrow were incubated with the following antibodies from eBiosciences (ThermoFisher Scientific): anti-CD16/CD32 blocking agent (clone

Liver and duodenum molecular analysis
Western-blot analysis.Frozen livers from WT and Hbb th3/+ mice were homogenized and lysed with ice cold lysis buffer (150 mM NaCl, 25 mM bicine, 0.1% SDS, 2% Triton X-100, 1 mM EDTA, protease inhibitor cocktail tablets, 1 mM Na3VO4 final concentration) followed by centrifugation for 30 minutes at 4°C at 12,000g.Proteins RT-PCR analysis.Total RNA was extracted from mouse tissues (liver and Cduodenum) using TRIzol reagent (Life Technologies).Synthesis of cDNA from total RNA (2 μg) was performed using SuperScript II First Strand kits (Life Technologies) and qRT-PCR was performed with SYBR Green PCR Master Mix (Applied Biosystems) using Applied Biosystems Model 7900HT Sequence Detection System.Detailed primer sequences are available in Supplemental Table 1.All PCR reactions were performed in triplicate.Relative gene expression was calculated using the 2 -ΔCt method, in which Ct indicates cycle threshold, the cycle number where the fluorescent signal reaches the detection threshold.ΔCt was computed by calculating the difference of the average Ct between the test gene and the internal control gene, Gapdh (17).
Liver and spleen iron content.Spleen and liver samples were dried at 90°C overnight and weighed.About 10-20 mg of dry tissue was digested in 1 mL of acid solution (3 M HCl, 0.6 M trichloroacetic acid) at 65°C for 16 hours.Next, 20 µL of the acid extract were analyzed using 1 mL of chromogen solution (0.1% bathophenanthroline sulfate, 1% idrossilammine, and 10% sodium acetate pH 6.5), evaluating the absorbance at 535 nm.A standard curve was generated using an acid solution containing increasing amounts of iron sulfate (17).

Analysis of erythrophagocytosis and macrophage receptors
Spleens were gently dissociated into single cells using GentleMACS dissociator (Miltenyi Biotec, Germany) and stained with F4/80 PE-Cy7 (Biolegend, CA, United States).Following staining cells were fixed, permeabilized, and counter-labeled with anti-Ter-119 FITC (Biolegend) to measure macrophage intracellular fluorescence associated with phagocytosed red cells.Cells stained as above without permeabilization served as negative controls of intracellular staining, as previously reported ( 24).Anti-CD80 PerCP-Cy5.5 (Biolegend) was used to determine surface expression of phagocytic receptors on spleen and lungs macrophages identified using an anti-F4/80 PE-Cy7 antibody.
Flow cytometry was carried out on a BD FACS Canto II (BD Biosciences, NJ, United States) and results were analyzed with the FACS DIVA software (BD Biosciences).1S.List of primers used in quantitative real-time PCR

Figure 1S .
Figure 1S.Pyruvate kinase (PK) activity of red blood cell samples from wild-type (WT) and 4.2 -/-mice was recorded by measuring A570 every 5 minutes for 60 minutes.The absorbance values were converted to nmol of pyruvate produced by one microliter of red cell lysate based on the standard curve generated by serial dilutions of the pyruvate standard solution following the instructions of the manufacturer of the commercial kit (MAK072) and plotted as the means ± S.E.M. 4 experiments were performed in triplicate comparing the PK activity of WT mice with that of 4.2 -/-mice.The inset shows the PK activity recorded after 30 minutes' incubation and converted to nmol/min/ml following the manufacturer's instructions.The statistical significance of the experimental results was determined by unpaired t-test with Welch's correction *, p <0.05.

Figure
Figure 4S. A. Spleen weight:mouse weight ratio (mg/g) in wild-type (WT) and 4.2 -/-mice treated with vehicle or mitapivat (100 mg/Kg/day) for 6 months.Data are mean ± SEM (n = 6-12).* P < 0.05 compared with WT mice and ° P < 0.05 compared with vehicle-treated mice by one-way ANOVA.Quantification of the splenic Perl's iron staining (upper panel) and the non-Heme splenic iron content determined using the bathophenanthroline staining method (lower panel), in WT and 4.2 -/-mice treated with vehicle or mitapivat (100 mg/Kg/day) for 6 months.Data are mean ± SEM (n = 3-7).* P < 0.05 compared with WT mice and ° P < 0.05 compared with vehicle-treated mice by t-test.C-E.Representative scatter plots of the gating strategy used to analyze erythropoiesis and total amount of erythropoietic cells in the bone marrow (BM, D-upper panel bar graph) and spleen (D-lower panel bar graph) and in WT and 4.2 -/-mice treated with either vehicle or mitapivat (100 mg/Kg/day) for 6 months.Data are mean ± SEM (n = 6-12).* P < 0.05 compared with WT mice and ° P < 0.05 compared with vehicle-treated mice by one-way ANOVA.E. Erythroblast populations in bone marrow and spleen from 4.2 -/-mice treated with vehicle or (100 mg/Kg/day) for 6 months.Population I (Pop I) corresponding to proerythroblasts, population II (Pop II), corresponding to basophilic erythroblasts; population III (Pop III), corresponding to polychromatic erythroblasts and population IV (Pop IV), corresponding to orthochromatic erythroblasts.Data are shown as mean ± SEM (n=6-12).° P < 0.05 compared with vehicle-treated mice by two-way ANOVA.

Figure
Figure 5S. A. Cation content (Na + , K + ) in red cells from wild-type (WT) and 4.2 -/- mice treated with vehicle or mitapivat (100 mg/Kg/day) for 6 months.Data are mean ± SEM (n = 5).* P < 0.05 compared with WT mice and ° P < 0.05 compared with vehicle-treated mice by one-way ANOVA.B. Bumetanide (10 uM) sensitive Na/K/2Cl cotransport activity as Na+ efflux in red cells from wild-type (WT) and 4.2 -/-mice treated with vehicle or mitapivat.Data are mean ± SEM (n = 5).* P < 0.05 compared with WT mice and ° P < 0.05 compared with vehicle-treated mice by one-way ANOVA.C. Amiloride (10 uM) sensitive Na/H exchange activity as Na+ efflux in red cells from wild-type (WT) and 4.2 -/-mice treated with vehicle or mitapivat.Data are mean ± SEM (n = 5).* P < 0.05 compared with WT mice and ° P < 0.05 compared with vehicle-treated mice by one-way ANOVA.

Figure 6S .
Figure 6S.Representative scatter plots of the gating strategy used to analyze (A) red blood cells (RBCs) osmotic fragility, (B) Erythroid plasmatic vesicles, (C) in vitro share stress induced erythroid vesicles.

Figure
Figure 8S. A. mRNA expression of hepcidin (Hamp) and Id1 by qRT-PCR on the liver from WT and 4.2 -/-mice treated with vehicle or mitapivat (100 mg/Kg/day) for 5 months.Experiments were performed in triplicate.Data are mean ± SD (n=6-8).°°°P < 4.2 -/-vehicle vs WT mice; ***P < 0.01 vehicle vs mitapivat treated mice.P value was calculated by ANOVA, internal comparisons by post-hoc correction by Tukey's multiple comparisons test.B-D.Densitometric analysis of Western-blots as in Figure 5C-E.Data are presented as mean ±SEM (n=4), * P < 0.05 compared with WT mice and ° P < 0.05 compared with vehicle-treated mice by t-test.

Figure
Figure 9S. A. Iron staining (Perl's Prussian blue is a semi-quantitative method to assess organ iron accumulation) in duodenum from WT and 4.2 -/-mice treated with either vehicle or mitapivat (100 mg/Kg/day) for 6 months.One representative image for each group is shown.Data are mean ± SEM. * P < 0.05 compared with WT mice and ° P < 0.05 compared with vehicle-treated mice by t-test.B. Western-analysis (Wb) using specific antibodies against PKM2 and PKLR in duodenum from WT and 4.2 -/-mice treated as in A. 50 µgr of protein loaded on an 8% T, 2.5%C polyacrylamide gel.Gapdh serves as protein loading control.One representative gel from 4 with similar results is shown.Lower panel.Densitometric analysis of Western-blot (Wb).Data are presented as mean ±SEM (n=4), * P < 0.05 compared with WT mice and ° P < 0.05 compared with vehicletreated mice by t-test.C. OxyBlot analysis of the soluble fractions of duodenum from WT and 4.2 -/-mice treated as in A. The carbonylated proteins (1 mg) were detected by treating with 2,4-dinitrophenylhydrazine and blotted with anti-DNP antibody.Gapdh serves as protein loading control.Quantification of band area is shown in the right panel.Data are presented as mean ±SEM (n=3), ° P < 0.05 compared with vehicle-treated mice by t-test.D. Western-blot (Wb)analysis using specific antibodies against phosphorylated (p-)NF-κB p65, NF-κB p65 and HIF 2 α in duodenum from WT and 4.2 -/-mice treated as in A. 75 µgr of protein loaded on an 8% T, 2.5%C polyacrylamide gel.Gapdh serves as protein loading control.One representative gel from 4 with similar results is shown.Densitometric analysis of immunoblots is shown in the lower panel.Data are presented as mean ±SEM (n=4), * P < 0.05 compared with WT mice and ° P < 0.05 compared with vehicle-treated mice by t-test.E. mRNA expression of Dmt1 (IRE) by qRT-PCR of duodenum from WT and 4.2 -/-mice treated as in A. Experiments were performed in triplicate.Data are mean ± SD (n=3).°°°P < 0.01, 4.2 -/-vehicle vs WT mice; ***P < 0.01 4.2 -/-vehicle vs mitapivat treated mice.P value was calculated by ANOVA, internal comparisons by post-hoc correction by Tukey's multiple comparisons test.