Sickle cell disease and β-Thalassemia are two of the most prevalent hemoglobinopathies worldwide. Both occur due to genetic mutations within the HBB gene and are characterized by red blood cell dysfunction, anemia, and end-organ injury. The spleen and liver are the primary organs where erythrophagocytosis, engulfing the red blood cells, occurs in these diseases. Understanding metabolism and protein composition within these tissues can therefore inform the extent of hemolysis and disease progression. We utilized a multiomics approach to highlight metabolomic and proteomic differences in the spleen and liver. The Berkley sickle cell disease (Berk-SS), heterozygous B1/B2 globin gene deletion (HbbTh3/+) a known β-Thalassemia model, and wildtype (WT, C57/Bl6) murine models were evaluated in this report. This analysis showed Berk-SS and HbbTh3/+ shared distinct antioxidant and immunosuppressive splenic phenotypes compared to WT mice with divergence in purine metabolism, gluconeogenesis, and glycolysis. In contrast, Berk-SS mice have a distinct liver pro-inflammatory phenotype not shared by HbbTh3/+ or WT mice. Together, these data emphasize that metabolic and proteomic reprogramming of the spleen and livers in Berk-SS and HbbTh3/+mice may be relevant to the individual disease processes.

Mitapivat, a pyruvate kinase activator, shows great potential as a sickle cell disease (SCD)-modifying therapy. The safety and efficacy of mitapivat as a long-term maintenance therapy are currently being evaluated in two open-label studies. Here we applied a comprehensive multi-omics approach to investigate the impact of activating pyruvate kinase on red blood cells (RBC) from 15 SCD patients. HbSS patients were enrolled in one of the open-label, extended studies (NCT04610866). Leukodepleted RBC obtained from fresh whole blood at baseline, prior to drug initiation, and at longitudinal timepoints over the course of the study were processed for multi-omics through a stepwise extraction of metabolites, lipids and proteins. Mitapivat therapy had significant effects on the metabolome, lipidome and proteome of SCD RBC. Mitapivat decreased 2,3-diphosphoglycerate levels, increased adenosine triphosphate levels, and improved hematologic and sickling parameters in patients with SCD. Agreement between omics measurements and clinical measurements confirmed the specificity of mitapivat on targeting late glycolysis, with glycolytic metabolites ranking as the top correlates to parameters of hemoglobin S oxygen affinity (p50) and sickling kinetics (t50) during treatment. Mitapivat markedly reduced levels of proteins of mitochondrial origin within 2 weeks of initiation of treatment, with minimal changes in reticulocyte counts. In the first 6 months of treatment there were also transient elevations of lysophosphatidylcholines and oxylipins with depletion of free fatty acids, suggestive of an effect on membrane lipid remodeling. Multi-omics analysis of RBC identified benefits for glycolysis, as well as activation of the Lands cycle.

Introduction: Band 3, or anion exchanger 1 (AE1), facilitates chloride-bicarbonate exchange across the membrane of red blood cells (RBCs), where it acts as critical regulator of oxygen delivery and red blood cell metabolism. The N-terminal domain of AE1 (residues 12-23) also serves as a binding site to deoxygenated hemoglobin, while AE1 residues 1-11 bind glycolytic enzymes (PFK, ALDOA, GAPDH) at high oxygen saturation. Therefore, the AE1-hemoglobin interaction acts as an oxygen sensor for metabolic regulation, a mechanism that ensures RBCs prioritize generation of energy and allosteric regulators that favor oxygen off-loading under hypoxia (e.g., ATP, 2,3-bisphosphoglycerate (BPG)), while favoring NADPH production by redirecting metabolism to the Pentose Phosphate Pathway during high oxidative stress. This allows RBC function to adapt to varied physiological conditions, such as high altitude or high oxygen demand during exercise. Under these hypoxic conditions, increasing the glycolytic rate fuels the Rapoport-Luebering shunt and BPG synthesis to favor O2 off-loading to tissues in the face of exercise-induced increase in oxygen demand. Dysregulation of AE1 activity in the setting of aging, exercise, or cell damage compromises metabolic flexibility and leads to RBC degradation, emphasizing AE1's role in maintaining RBC health. Methods: Here we generated novel humanized mouse models carrying either (i) human canonical band3 (HUB3) or two different band3 knock out variations: (ii) high affinity deletion (first 11 amino acids deleted, HA-Del) or (iii) binding site knock out (amino acid residues 12-23, BS-KO). The original AE1 mice were generated by the Low lab on a 129 background, prior to backcrossing for 6-7 generations to C57BL6. As we recently noted, some residual 129 mouse polymorphisms were carried over, particularly on chromosome 1, coding for genes like Steap3 that affect RBC lipid peroxidation. To overcome these limitations, we generated new mouse models by introducing identical AE1 (HUB3, HA-DEL and BS-KO) on a clean C57BL6/J background. We then leveraged these mouse models to assess the effects of AE1 regulatory activity on critical speed (CS), a functional measurement of exercise tolerance to exhaustion. To expand the molecular resolution of genetically associated adaptations to exercise, CS tests were combined with hemodynamic measurements, imaging (e.g., scanning electron microscopy of RBCs before and after exercise) and multi-omics (mass spectrometry-based metabolomics, lipidomics and proteomics) characterization of plasma and RBCs pre and post run to exhaustion. Results: The BS-KO mice had a significantly slower median CS compared to both HA-Del and HUB3 (20.9% and 11.2%). Complete blood count and hemodynamic data showed no significant difference between the three groups, aside from significantly decreased cardiovascular efficiency in the BS-KO mice (p=0.004). SO2 was significantly decreased in BS-KO mice at 85%. RBC metabolomics analysis highlighted changes pre and post exercise, showing increased Land's cycle activity and pantothenate levels in BS-KO mice, which are critical for membrane repair. Plasma metabolomics reflected the systemic hypoxia the KO animals experienced during exercise, showing significant increases of Kreb's cycle metabolites such as malate, fumarate, and succinate (p=0.001). Conclusion: Using novel humanized mouse models with modification of Band 3 regulatory protein binding sites, we observed that the deoxyhemoglobin BS-KO mice had significantly slower CS and cardiovascular efficiency compared to other models, indicating impaired metabolic and physiological adaptability to the high physiological demand of exercise. Metabolomics and proteomics analyses revealed that BS-KO mice experienced systemic hypoxia and membrane damage, with significant changes in metabolites linked to hypoxia and stress responses. These findings underscore the importance of AE1 in maintaining RBC metabolic flexibility particularly under stressful conditions such as exercise. The results suggest potential therapeutic targets for mitigating RBC-related pathologies in conditions of oxidative stress and metabolic dysregulation.

Administration of oxygen microbubbles (OMBs) has been shown to increase oxygen and decrease carbon dioxide in systemic circulation, as well as reduce lung inflammation and promote survival in preclinical models of hypoxia caused by lung injury. However, their impact on microenvironmental oxygenation remains unexplored. Herein, we investigated the effects of intraperitoneal administration of OMBs in anesthetized rats exposed to hypoxic ventilation (FiO2 = 0.14). Blood oxygenation and hemodynamics were evaluated over a 2 h time frame, and then organ and tissue samples were collected for hypoxic and metabolic analyses. Data showed that OMBs improved blood SaO2 (~14%) and alleviated tissue hypoxia within the microenvironment of the kidney and intestine at 2 h of hypoxia. Metabolomic analysis revealed OMBs induced metabolic differences in the cecum, liver, kidney, heart, red blood cells and plasma. Within the spleen and lung, principal component analysis showed a metabolic phenotype more comparable to the normoxic group than the hypoxic group. In the spleen, this shift was characterized by reduced levels of fatty acids and 2-hydroxygluterate, alongside increased expression of antioxidant enzymes such as glutathione and hypoxanthine. Interestingly, there was also a shuttle effect within the metabolism of the spleen from the tricarboxylic acid cycle to the glycolysis and pentose phosphate pathways. In the lung, metabolomic analysis revealed upregulation of phosphatidylethanolamine and phosphatidylcholine synthesis, indicating a potential indirect mechanism through which OMB administration may improve lung surfactant secretion and prevent alveolar collapse. In addition, cell-protective purine salvage was increased within the lung. In summary, oxygenation with intraperitoneal OMBs improves systemic blood and local tissue oxygenation, thereby shifting metabolomic profiles of the lung and spleen toward a healthier normoxic state.

Resistance of melanoma to targeted therapy and immunotherapy is linked to metabolic rewiring. Here, we show that increased fatty acid oxidation (FAO) during prolonged BRAF inhibitor (BRAFi) treatment contributes to acquired therapy resistance in mice. Targeting FAO using the US Food and Drug Administration-approved and European Medicines Agency-approved anti-anginal drug ranolazine (RANO) delays tumour recurrence with acquired BRAFi resistance. Single-cell RNA-sequencing analysis reveals that RANO diminishes the abundance of the therapy-resistant NGFR hi neural crest stem cell subpopulation. Moreover, by rewiring the methionine salvage pathway, RANO enhances melanoma immunogenicity through increased antigen presentation and interferon signalling. Combination of RANO with anti-PD-L1 antibodies strongly improves survival by increasing antitumour immune responses. Altogether, we show that RANO increases the efficacy of targeted melanoma therapy through its effects on FAO and the methionine salvage pathway. Importantly, our study suggests that RANO could sensitize BRAFi-resistant tumours to immunotherapy. Since RANO has very mild side-effects, it might constitute a therapeutic option to improve the two main strategies currently used to treat metastatic melanoma.

In pancreatic ductal adenocarcinoma (PDAC) patients, we show that response to radiation therapy (RT) is characterized by increased IL-2Rβ and IL-2Rγ along with decreased IL-2Rα expression. The bispecific PD1-IL2v is a PD-1-targeted IL-2 variant (IL-2v) immunocytokine with engineered IL-2 cis targeted to PD-1 and abolished IL-2Rα binding, which enhances tumor-antigen-specific T cell activation while reducing regulatory T cell (Treg) suppression. Using PD1-IL2v in orthotopic PDAC KPC-driven tumor models, we show marked improvement in local and metastatic survival, along with a profound increase in tumor-infiltrating CD8+ T cell subsets with a transcriptionally and metabolically active phenotype and preferential activation of antigen-specific CD8+ T cells. In combination with single-dose RT, PD1-IL2v treatment results in a robust, durable expansion of polyfunctional CD8+ T cells, T cell stemness, tumor-specific memory immune response, natural killer (NK) cell activation, and decreased Tregs. These data show that PD1-IL2v leads to profound local and distant response in PDAC.