Functional and multi-omics signatures of mitapivat efficacy upon activation of pyruvate kinase in red blood cells from patients with sickle cell disease.

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.

Peritoneal Infusion of Oxygen Microbubbles Alters the Metabolomic Profile of the Lung and Spleen in Acute Hypoxic Exposure.

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.

Simultaneous targeting of PD-1 and IL-2Rßγ with radiation therapy inhibits pancreatic cancer growth and metastasis.

In pancreatic ductal adenocarcinoma (PDAC) patients, we show that response to radiation therapy (RT) is characterized by increased IL-2Rß 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.

Metabolic rewiring induced by ranolazine improves melanoma responses to targeted therapy and immunotherapy.

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 NGFRhi 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.