Activation Of Glycolytic Pathway Research Articles

Spermatogenic cell-specific type 1 hexokinase (HK1S) is essential for capacitation-associated increase in tyrosine phosphorylation and male fertility in mice.

Hexokinase (HK) catalyzes the first irreversible rate-limiting step in glycolysis that converts glucose to glucose-6-phosphate. HK1 is ubiquitously expressed in the brain, erythrocytes, and other tissues where glycolysis serves as the major source of ATP production. Spermatogenic cell-specific type 1 hexokinase (HK1S) is expressed in sperm but its physiological role in male mice is still unknown. In this study, we generate Hk1s knockout mice using the CRISPR/Cas9 system to study the gene function in vivo. Hk1s mRNA is exclusively expressed in testes starting from postnatal day 18 and continuing to adulthood. HK1S protein is specifically localized in the outer surface of the sperm fibrous sheath (FS). Depletion of Hk1s leads to infertility in male mice and reduces sperm glycolytic pathway activity, yet they have normal motile parameters and ATP levels. In addition, by using in vitro fertilization (IVF), Hk1s deficient sperms are unable to fertilize cumulus-intact or cumulus-free oocytes, but can normally fertilize zona pellucida-free oocytes. Moreover, Hk1s deficiency impairs sperm migration into the oviduct, reduces acrosome reaction, and prevents capacitation-associated increases in tyrosine phosphorylation, which are probable causes of infertility. Taken together, our results reveal that HK1S plays a critical role in sperm function and male fertility in mice.

Abstract 4421: Intracellular cytomatrix actin and the Warburg effect

Abstract Introduction: The cytoplasm of living cells is dynamic and the motion arising from fluctuating forces in cells is an active process, rather than being thermally induced. Motor proteins could be the primary causative force of intracellular movement. However, which motor protein is involved in the intracellular dynamics, what process requires fluctuational intracellular activity, and where the energy source comes from is unknown. To address the impact of intracellular motion in cellular processes, we chemically separated the viscous fluid (cytosol) and elastic solid (cytomatrix) phases of cytoplasm. The multiomic analysis was used to dissect the compositional differences between the liquid and solid phases. LC-MS/MS-based proteome analysis was used to identify the composition of the HCT-15 colon cancer cell cytomatrix. The mRNA translation was measured by Ribosome footprinting. Results: Since the kinesis of the solid phase can trigger liquid phase flux, the composition of the cytomatrix was the primary focus of the study of cytoplasmic motion. The systemic analysis of the cytomatrix constituency allowed the identification of a diverse functional group of proteomes that included cytoskeletal and structural elements of organoids, metabolic enzymes, and normal- and proto-oncogenic signaling pathways. The two-phase system elucidated how diverse chemical reactions can occur simultaneously within the cell cytoplasm. Namely, the immobilization of catalytic complexes on the solid cytomatrix physically segregates biochemical reactants, thereby overcoming spatial impediments and allowing diverse metabolic reactions to coincide. Actomyosin and other actin-binding proteins of cytomatrix were the most abundant and diverse proteome, indicating that the primary machinery driving cytomatrix mobility consists of actomyosin, actin-binding, and actin-regulating proteins of cytomatrix. The cytomatrix dynamics trigger the cytosolic motion that carries substrates to immobilized catalytic complexes of the cytomatrix, thereby increasing product output, which appears to be a more intense process in cancer cells due to genetic and epigenetic aberrations. Under normal physiological conditions, mitochondrial respiration can deliver the energy requirements of cytomatrix micromechanics, but in cancer cells, additional energy of ATP is supplied by the aerobic glycolysis known as the Warburg effect to provide the ATP surplus for actomyosin mechanics. Conclusion: The intracellular motion is supported through non-muscle actomyosin action of the solid phase (cytomatrix), permitting increased glycolytic pathway activity in tumor cells. This novel concept of cancer links the Warburg effect with the bioenergetics of cytomatrix micromechanics and the dynamics of metabolic processes in tumors. Thus, two of the mysteries in cancer cell biology, how chemical reactions occur in the cytoplasm and energy is generated by tumor cells, have now been linked. Citation Format: Tattym E. Shaiken. Intracellular cytomatrix actin and the Warburg effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4421.

Glutamine addiction and therapeutic strategies in pancreatic cancer

Pancreatic cancer remains one of the most lethal diseases worldwide owing to its late diagnosis, early metastasis, and poor prognosis. Because current therapeutic options are limited, there is an urgent need to investigate novel targeted treatment strategies. Pancreatic cancer faces significant metabolic challenges, principally hypoxia and nutrient deprivation, due to specific microenvironmental constraints, including an extensive desmoplastic stromal reaction. Pancreatic cancer cells have been shown to rewire their metabolism and energy production networks to support rapid survival and proliferation. Increased glucose uptake and glycolytic pathway activity during this process have been extensively described. However, growing evidence suggests that pancreatic cancer cells are glutamine addicted. As a nitrogen source, glutamine directly (or indirectly via glutamate conversion) contributes to many anabolic processes in pancreatic cancer, including amino acids, nucleobases, and hexosamine biosynthesis. It also plays an important role in redox homeostasis, and when converted to α-ketoglutarate, glutamine serves as an energy and anaplerotic carbon source, replenishing the tricarboxylic acid cycle intermediates. The present study aims to provide a comprehensive overview of glutamine metabolic reprogramming in pancreatic cancer, focusing on potential therapeutic approaches targeting glutamine metabolism in pancreatic cancer.

Nicotinamide Mononucleotide Improves Spermatogenic Disorders in Aluminum-Exposed Rats by Modulating the Glycolytic Pathway.

This study aimed to investigate the protective effect of nicotinamide mononucleotide (NMN) on testicular spermatogenesis in aluminum chloride (AlCl3)-exposed rats and to elucidate the potential underlying mechanism. The results indicated that AlCl3-induced testicular damage, leading to reduced sperm quality, increased apoptosis, decreased cell proliferation, and impaired Sertoli cell function in rats. Additionally, glycolytic metabolism was observed to be hindered. However, after NMN treatment, there was a noticeable improvement in testicular damage among the rats, marked by increased sperm quality, reduced apoptosis, enhanced cell proliferation, improved Sertoli cell function, and an activated glycolytic metabolism. The findings of this study suggest that NMN alleviates testicular spermatogenesis impairment induced by AlCl3 exposure through the inhibition of spermatogenic cell apoptosis, promotion of spermatogenic cell proliferation, and activation of glycolytic pathways. The study contributes an experimental foundation for potential future clinical applications of NMN in cases of AlCl3-exposed spermatogenic dysfunction.

Single-cell characterization revealed hypoxia-induced metabolic reprogramming of gastric cancer

IntroductionGastric cancer (GC) is one of the most common malignant tumors worldwide. Single-cell sequencing technology can achieve an accurate and unbiased assessment of cell heterogeneity. Therefore, it is necessary to explore the molecular characteristics of GC-related malignant cells at a single-cell resolution. MethodsSingle-cell RNA sequencing GC profiles were collected from the Gene Expression Omnibus database. Moreover, feature gene sets of metabolic pathways and hypoxia signals were collected from the Molecular Signatures Database. The marker genes of specific cell types were collected from the published literature and CellMarker database. The R package InferCNV was used to calculate the copy-number variations of cells and to identify real cancer cells. The weighted relative pathway activity algorithm was used to evaluate the differences in metabolic activity between cell types. ResultsOur study found that cancer epithelial cells exhibited individual differences in molecular features and showed metabolic heterogeneity. Oxidative phosphorylation and glycolytic pathway activity were the major contributors to the metabolic heterogeneity of cancer epithelial cells. Furthermore, we used the hypoxia signaling pathway to indirectly evaluate the oxygen content of cells and found that hypoxia contributed to the heterogeneity of cancer epithelial cells. Finally, functional identification of genes co-expressed with HIF1A showed that the reprogramming of the oxidative stress response contributed to the tumor malignant progression. ConclusionsThis study described hypoxia-induced metabolic reprogramming of GC at a single-cell level, partially addressing the lack of insight into the heterogeneity of cancer cell metabolism when using traditional sequencing technology.

Identification of genomic determinants contributing to cytokine release in immunotherapies and human diseases

BackgroundCytokine release syndrome (CRS) is a strong immune system response that can occur as a result of the reaction of a cellular immunotherapy with malignant cells. While the frequency and management of CRS in CAR T-cell therapy has been well documented, there is emerging interest in pre-emptive treatment to reduce CRS severity and improve overall outcomes. Accordingly, identification of genomic determinants that contribute to cytokine release may lead to the development of targeted therapies to prevent or abrogate the severity of CRS.MethodsForty three clinical CD22 CAR T-cell products were collected for RNA extraction. 100 ng of mRNA was used for Nanostring assay analysis which is based on the nCounter platform. Several public datasets were used for validation purposes.ResultsWe found the expression of the PFKFB4 gene and glycolytic pathway activity were upregulated in CD22 CAR T-cells given to patients who developed CRS compared to those who did not experience CRS. Moreover, these results were further validated in cohorts with COVID-19, influenza infections and autoimmune diseases, and in tumor tissues. The findings were similar, except that glycolytic pathway activity was not increased in patients with influenza infections and systemic lupus erythematosus (SLE).ConclusionOur data strongly suggests that PFKFB4 acts as a driving factor in mediating cytokine release in vivo by regulating glycolytic activity. Our results suggest that it would beneficial to develop drugs targeting PFKFB4 and the glycolytic pathway for the treatment of CRS.

Structure-Activity Relationships and Transcriptomic Analysis of Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitors.

To evaluate the differences in action of commercially available 2-oxoglutarate mimetics and “branched-tail” oxyquinoline inhibitors of hypoxia-inducible factor prolyl hydroxylase (HIF PHD), the inhibitors’ IC50 values in the activation of HIF1 ODD-luciferase reporter were selected for comparative transcriptomics. Structure–activity relationship and computer modeling for the oxyquinoline series of inhibitors led to the identification of novel inhibitors, which were an order of magnitude more active in the reporter assay than roxadustat and vadadustat. Unexpectedly, 2-methyl-substitution in the oxyquinoline core of the best HIF PHD inhibitor was found to be active in the reporter assay and almost equally effective in the pretreatment paradigm of the oxygen-glucose deprivation in vitro model. Comparative transcriptomic analysis of the signaling pathways induced by HIF PHD inhibitors showed high potency of the two novel oxyquinoline inhibitors (#4896-3249 and #5704-0720) at 2 μM concentrations matching the effect of 30 μM roxadustat and 500 μM dimethyl oxalyl glycine in inducing HIF1 and HIF2-linked pathways. The two oxyquinoline inhibitors exerted the same activation of HIF-triggered glycolytic pathways but opposite effects on signaling pathways linked to alternative substrates of HIF PHD 1 and 3, such as p53, NF-κB, and ATF4. This finding can be interpreted as the specificity of the 2-methyl-substitute variant for HIF PHD2.

Characterizing the Metabolic and Immune Landscape of Non-small Cell Lung Cancer Reveals Prognostic Biomarkers Through Omics Data Integration.

Non-small cell lung cancer (NSCLC) is one of the most common malignancies worldwide. The development of high-throughput single-cell RNA-sequencing (RNA-seq) technology and the advent of multi-omics have provided a solid basis for a systematic understanding of the heterogeneity in cancers. Although numerous studies have revealed the molecular features of NSCLC, it is important to identify and validate the molecular biomarkers related to specific NSCLC phenotypes at single-cell resolution. In this study, we analyzed and validated single-cell RNA-seq data by integrating multi-level omics data to identify key metabolic features and prognostic biomarkers in NSCLC. High-throughput single-cell RNA-seq data, including 4887 cellular gene expression profiles from NSCLC tissues, were analyzed. After pre-processing, the cells were clustered into 12 clusters using the t-SNE clustering algorithm, and the cell types were defined according to the marker genes. Malignant epithelial cells exhibit individual differences in molecular features and intra-tissue metabolic heterogeneity. We found that oxidative phosphorylation (OXPHOS) and glycolytic pathway activity are major contributors to intra-tissue metabolic heterogeneity of malignant epithelial cells and T cells. Furthermore, we constructed T-cell differentiation trajectories and identified several key genes that regulate the cellular phenotype. By screening for genes associated with T-cell differentiation using the Lasso algorithm and Cox risk regression, we identified four prognostic marker genes for NSCLC. In summary, our study revealed metabolic features and prognostic markers of NSCLC at single-cell resolution, which provides novel findings on molecular biomarkers and signatures of cancers.

498-P: Resistance Exercise Rapidly Alters Citric Acid Cycle and Amino Acid Metabolism in Skeletal Muscle

Muscle glucose metabolism is improved by either aerobic or resistance exercise (RE) training, but the mechanisms leading to improved metabolism for each of these exercise modes are not identical. Much work has been done onimproved oxidative metabolism by aerobic exercise, but far less is known about how RE impacts fuel metabolism. Here, TCA and amino acid metabolites are measured in skeletal muscle after a single bout of RE. Twenty one healthy subjects performed a session of one-legged knee extension RE that consisted of 3 sets of 10 reps at 70% of their 1 rep max. Two muscle biopsies were obtained from the vastus lateralis of each leg. From the RE leg, biopsies were obtained ≤10min post-RE (0hr PEX) and approximately 1hr post-RE (1hr PEX), respectively. From the non-RE leg, biopsies were obtained before and 1hr PEX, respectively. Muscle samples were frozen and stored until mass spectrometry analysis of metabolites was performed. A 2-fold increase in plasma lactate (P<0.05) and a 30-40% increase in muscle glycolytic enzyme activity (P<0.05) at 0hr PEX suggest increased activation of the glycolytic pathway. Also at 0hr PEX, TCA metabolites citrate and isocitrate were increased (P<0.05) compared to the pre-RE biopsy (0.35 and 0.01 mmol/μg, respectively). Further, multiple amino acid metabolites Arg, Asn, Lys, Thr, and Val were increased (P<0.05) in the REmuscle at 0hr PEX. These amino acids can enter the TCA cycle at various steps, indicating increased flux through the TCA cycle immediately after RE. By 1hr PEX, TCA intermediates malate and fumarate were reduced (P<0.05)compared to the non-RE leg (-0.65 and -0.35 mmol/μg, respectively), and multiple amino acid metabolites were also reduced (P<0.05). The decrease in these metabolites at 1hr PEX suggests increased use of metabolites for mitochondrial respiration. These results suggest activation of glycolytic and amino metabolism pathways, contributing to citric acid flux immediately following RE in order to meet energy needs in skeletal muscle. Disclosure M. W. Pataky: None. A. Prabha kumar: None. K. Klaus: None. K. Nair: None. Funding National Institutes of Health (R01AG062859)

Flexibility of equine bioenergetics and muscle plasticity in response to different types of training: An integrative approach, questioning existing paradigms.

Equine bioenergetics have predominantly been studied focusing on glycogen and fatty acids. Combining omics with conventional techniques allows for an integrative approach to broadly explore and identify important biomolecules. Friesian horses were aquatrained (n = 5) or dry treadmill trained (n = 7) (8 weeks) and monitored for: evolution of muscle diameter in response to aquatraining and dry treadmill training, fiber type composition and fiber cross-sectional area of the M. pectoralis, M. vastus lateralis and M. semitendinosus and untargeted metabolomics of the M. pectoralis and M. vastus lateralis in response to dry treadmill training. Aquatraining was superior to dry treadmill training to increase muscle diameter in the hindquarters, with maximum effect after 4 weeks. After dry treadmill training, the M. pectoralis showed increased muscle diameter, more type I fibers, decreased fiber mean cross sectional area, and an upregulated oxidative metabolic profile: increased β-oxidation (key metabolites: decreased long chain fatty acids and increased long chain acylcarnitines), TCA activity (intermediates including succinyl-carnitine and 2-methylcitrate), amino acid metabolism (glutamine, aromatic amino acids, serine, urea cycle metabolites such as proline, arginine and ornithine) and xenobiotic metabolism (especially p-cresol glucuronide). The M. vastus lateralis expanded its fast twitch profile, with decreased muscle diameter, type I fibers and an upregulation of glycolytic and pentose phosphate pathway activity, and increased branched-chain and aromatic amino acid metabolism (cis-urocanate, carnosine, homocarnosine, tyrosine, tryptophan, p-cresol-glucuronide, serine, methionine, cysteine, proline and ornithine). Trained Friesians showed increased collagen and elastin turn-over. Results show that branched-chain amino acids, aromatic amino acids and microbiome-derived xenobiotics need further study in horses. They feed the TCA cycle at steps further downstream from acetyl CoA and most likely, they are oxidized in type IIA fibers, the predominant fiber type of the horse. These study results underline the importance of reviewing existing paradigms on equine bioenergetics.

Progress in studies on production of chemicals from xylose by Saccharomyces cerevisiae

Effective utilization of xylose is a basis for economic production of biofuels or chemicals from lignocellulose biomass. Over the past 30 years, through metabolic engineering, evolutionary engineering and other strategies, the metabolic capacity of xylose of the traditional ethanol-producing microorganism Saccharomyces cerevisiae has been significantly improved. In recent years, the reported results showed that the transcriptome and metabolome profiles between xylose and glucose metabolism existed significant difference in recombinant yeast strains. Compared with glucose, the overall process of xylose metabolism exhibits Crabtree-negative characteristics, including the limited glycolytic pathway activity, which reduces the metabolic flux of pyruvate to ethanol, and the enhanced cytosolic acetyl-CoA synthesis and respiratory energy metabolism. These traits are helpful to achieve efficient synthesis of downstream products using pyruvate or acetyl-CoA as precursors. This review provides a detailed overview on the modification and optimization of xylose metabolic pathways in S. cerevisiae, the characteristics of xylose metabolism, and the construction of cell factories for production of chemicals using xylose as a carbon source. Meanwhile, the existed difficulties and challenges, and future studies on biosynthesis of bulk chemicals using xylose as an important carbon source are proposed.

Métabolomique et imagerie TEP-FDG des cancers du sein

Cancer metabolism is an essential aspect of tumorogenesis, as cancer cells have increased energy requirements in comparison to normal cells. Metabolomic techniques can provide quantitative data for a large number of small molecules in tissues and enable the analysis of multiple intricate metabolic pathways. Positron emission tomography (PET) using 18F-Fluorodeoxyglucose (FDG) enables in vivo analysis of glycolysis and is widely used in oncology. High tumor FDG uptake is a prognostic factor in breast cancer and has been associated with tumor aggressively. Seventy breast cancer samples obtained from untreated patients who had underwent FDG-PET imagery were analyzed through an untargeted metabolomic approach using liquid chromatography-mass spectroscopy (LC-MS) to study possible correlations between metabolomic data and FDG uptake. Tumors were split into two groups depending on avidity for FDG as measured with PET. The Compound Discoverer 4.0 software enabled identification of 854 metabolites. PLSDA based models predicted FDG uptake with an accuracy ranging from 0,73 to 0,77. Selected metabolites varied depending on the use of scaling or log transformation. Metabolites correlated with tumor FDG uptake were, among others, glutathione, amino-acids such as glutamate, proline or tyrosine, L-acetyl-carnitine, metabolites from the kynurenine pathway such as L-kynurenine or formyl-kynurenine and polyamines such as N1,N12-diacetylspermine or N1-acetylspermine. These metabolites have been previously shown to reflect cancer aggressivity. The correlation between the glycolytic pathway activation and tumor FDG uptake could not be directly assessed but indirect signs showed a higher glycolytic activity in tumours presenting a higher FDG uptake. Studying new metabolites identified through this process could enable a better understanding of tumor metabolism and identification of new biomarkers.

Overexpression of fructose-1,6-bisphosphate aldolase 1 enhances accumulation of fatty acids in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtti is a promising source of bioenergy and understanding its carbon metabolism is important. In this organism, carbon accumulation can be manipulated with nitrogen supply. Chlamydomonas reinhardtii is a mixotroph that can act as an autotroph or heterotroph, depending on conditions. Fructose-1,6-bisphosphate aldolases (FBAs) are important enzymes in energy pathways of algae; they convert fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Expression of both CrFBA1 and CrFBA2 was less under nitrogen-deplete conditions than under nitrogen-replete conditions in Chlamydomonas cells. To explore CrFBA1 function, CrFBA1-overexpressing Chlamydomonas lines were generated. Compared to wild-type, transformed cells grew more slowly in photoautotrophic conditions, but at the same rate in mixotrophic condition. We suggest that overexpression of CrFBA1 affected photosynthetic carbon fixation in the Calvin-Benson cycle. The CrFBA1-overexpressing lines had less starch than the wild-type under mixotrophic, low-nitrogen conditions, but more fatty acids. In the transgenic cells, expression of granule bound starch synthase 1 (GBSS1) determined by quantitative reverse transcription-polymerase chain reaction was down-regulated, but acetyl-CoA synthetases (ACS1 and ACS2) and phosphoglucomutase (PGM) were up-regulated compared to wild-type. We propose three possible explanations for the observed low starch and high fatty acid content of the transgenic cells. Firstly, abnormal carbon flux by CrFBA1 overexpression leads to GBSS1 down-regulation in a feed-forward regulation, which resulted in the low starch accumulation. Secondly, increase of exogenous acetate assimilation by ACS1 and ACS2 up-regulation results in high accumulation of total fatty acid in transformants, perhaps due to compensation for reduced-carbon fixation. Lastly, activation of glycolytic pathway by CrFBA1 overexpression enhances conversion of starch to fatty acid. We conclude that overexpression of CrFBA1 deregulates carbon metabolism in Chlamydomonas reinhardtii.

AG-348 (Mitapivat), an allosteric activator of red blood cell pyruvate kinase, increases enzymatic activity, protein stability, and ATP levels over a broad range of PKLR genotypes.

Pyruvate kinase (PK) deficiency is a rare hereditary disorder affecting red blood cell (RBC) glycolysis, causing changes in metabolism including a deficiency in adenosine triphosphate (ATP). This affects red cell homeostasis, promoting premature removal of RBC from the circulation. In this study, we characterized and evaluated the effect of AG-348, an allosteric activator of PK that is currently in clinical trials for treatment of PK deficiency, on RBC and erythroid precursors from PK-deficient patients. In 15 patients, ex vivo treatment with AG-348 resulted in increased enzymatic activity in all patients' cells after 24 hours (h) (mean increase: 1.8-fold; range: 1.2-3.4). ATP levels increased (mean increase: 1.5-fold; range: 1.0-2.2) similar to control cells (mean increase: 1.6-fold; range: 1.4-1.8). Generally, PK thermostability was strongly reduced in PK-deficient RBC. Ex vivo treatment with AG-348 increased residual activity from 1.4- to >10-fold more than residual activity of vehicle-treated samples. Protein analyses suggest that a sufficient level of PK protein is required for cells to respond to AG- 348 treatment ex vivo, as treatment effects were minimal in patient cells with very low or undetectable levels of PK-R. In half of the patients, ex vivo treatment with AG-348 was associated with an increase in RBC deformability. These data support the hypothesis that drug intervention with AG- 348 effectively up-regulates PK enzymatic activity and increases stability in PK-deficient RBC over a broad range of PKLR genotypes. The concomitant increase in ATP levels suggests that glycolytic pathway activity may be restored. AG-348 treatment may represent an attractive way to correct the underlying pathologies of PK deficiency. (AG-348 is currently in clinical trials for the treatment of PK deficiency. Registered at clinicaltrials.gov identifiers: NCT02476916, NCT03853798, NCT03548220, NCT03559699).

Monocyte glycolysis determines CD8+ T cell functionality in human Chagas disease.

Chagas disease is a lifelong pathology resulting from Trypanosoma cruzi infection. It represents one of the most frequent causes of heart failure and sudden death in Latin America. Herein, we provide evidence that aerobic glycolytic pathway activation in monocytes drives nitric oxide (NO) production, triggering tyrosine nitration (TN) on CD8+ T cells and dysfunction in patients with chronic Chagas disease. Monocytes from patients exhibited a higher frequency of hypoxia-inducible factor 1α and increased expression of its target genes/proteins. Nonclassical monocytes are expanded in patients' peripheral blood and represent an important source of NO. Monocytes entail CD8+ T cell surface nitration because both the frequency of nonclassical monocytes and that of NO-producing monocytes positively correlated with the percentage of TN+ lymphocytes. Inhibition of glycolysis in in vitro-infected peripheral blood mononuclear cells decreased the inflammatory properties of monocytes/macrophages, diminishing the frequency of IL-1β- and NO-producing cells. In agreement, glycolysis inhibition reduced the percentage of TN+CD8+ T cells, improving their functionality. Altogether, these results clearly show that glycolysis governs oxidative stress on monocytes and modulates monocyte-T cell interplay in human chronic Chagas disease. Understanding the pathological immune mechanisms that sustain an inflammatory environment in human pathology is key to designing improved therapies.

PF426 GENOTYPE‐RESPONSE CORRELATION IN DRIVE PK, A PHASE 2 STUDY OF MITAPIVAT (AG‐348) IN PATIENTS WITH PYRUVATE KINASE DEFICIENCY

Background:Pyruvate kinase (PK) deficiency is a congenital hemolytic anemia caused by mutations in the PKLR gene, leading to a deficiency of the glycolytic enzyme red cell PK (PK‐R). Mitapivat (AG‐348) is an oral, small‐molecule, allosteric activator of PK‐R that activates the wild type and a range of mutant PK‐R enzymes associated with PK deficiency. In a phase 2 study in 52 patients (pts) with PK deficiency (DRIVE PK, NCT02476916), 50% of pts experienced a maximum hemoglobin (Hb) increase of >1.0 g/dL (mean maximum increase, 3.4 g/dL; range, 1.1–5.8 g/dL). Hb increases were observed in patients with a variety of PKLR mutations and were associated with the presence of ≥1 missense mutation.As PK deficiency is genetically heterogeneous (>200 described mutations), we sought to better understand the molecular parameters associated with Hb increases in mitapivat‐treated pts.Aims:To analyze the relationship between Hb increase and pt genotype, biochemical response to mitapivat, and baseline PK‐R protein level.Methods:Whole blood samples were collected from pts enrolled in DRIVE PK. Genotypes were determined by Centogene AG. Baseline PK‐R protein was quantitated by antibody‐based capture and detection using a Meso Scale assay and the signal normalized to a control sample from a subject without PK deficiency.Results:Of 106 PKLR mutations in the 52 pts (2 pts carried 3 mutations), there were 47 unique mutations, including 28 missense and 19 non‐missense. Of these, only 10 were present in ≥2 pts. Of these 10 mutations, some were more likely to be associated with Hb increases (e.g. increases were observed in 5/5 pts with ≥1 R486W mutation and 4/4 pts with ≥1 T384 M mutation). Other mutations, including V134D (0/3), E241stop (1/6), and R510Q (5/14), were more likely to be associated with a lack of Hb increase. Notably, 1/10 pts who had 2 non‐missense mutations and 0/5 pts homozygous for the R479H “Amish” mutation had Hb increases.We have previously reported that, in biochemical assays, mitapivat could efficiently activate recombinantly expressed R479H and R510Q mutant enzymes. Although both of these are technically missense mutations, it is well understood that R510Q results in severely decreased protein stability, and the R479H mutation, occurring at the splice junction between 2 exons, can deleteriously affect mRNA splicing. Therefore, as mitapivat works by directly binding and stimulating the activity of residual mutant PK‐R enzyme, we explored the hypothesis that PK‐R protein levels in pts with these mutations might be too low to allow for adequate target engagement at clinically tested doses.Pts with Hb increases had, on average, higher baseline PK‐R protein levels (49% of control vs 13% in pts without Hb increases). All 10 pts with non‐missense/non‐missense mutations had nearly undetectable PK‐R protein levels (average of 3.7% of control), consistent with and confirming the predicted effect of these mutations on protein expression. Baseline PK‐R protein levels were also lower in pts with ≥1 R510Q (18% of control) or R479H (19% of control) mutation than in pts with other missense mutations (59% of control).Summary/Conclusion:These analyses demonstrate that Hb increases upon mitapivat treatment are associated with the presence of ≥1 missense PK‐R mutant enzyme with residual protein for mitapivat to bind and activate. These data strongly suggest that the hematologic effects of mitapivat come from the proposed mechanism of action of restoration of glycolytic pathway activity in PK‐deficient red cells.image

HIF1α-Induced Glycolysis in Macrophage Is Essential for the Protective Effect of Ouabain during Endotoxemia

Ouabain, a steroid binding to the Na+/K+-ATPase, has several pharmacological effects. In addition to the recognized effects of blood pressure, there is more convincing evidence suggesting that ouabain is involved in immunologic functions and inflammation. Hypoxia-inducible factor 1α (HIF-1α) is a metabolic regulator which plays a considerable role in immune responses. Previous studies had shown that HIF-1α-induced glycolysis results in functional reshaping in macrophages. In this study, we investigated the role of glycolytic pathway activation in the anti-inflammatory effect of ouabain. We found that ouabain is involved in anti-inflammatory effects both in vivo and in vitro. Additionally, ouabain can inhibit LPS-induced upregulation of GLUT1 and HK2 at the transcriptional level. GM-CSF pretreatment almost completely reversed the inhibitory effect of ouabain on LPS-induced release of proinflammatory cytokines. Alterations in glycolytic pathway activation were required for the anti-inflammatory effect of ouabain. Ouabain can significantly inhibit the upregulation of HIF-1α at the protein level. Our results also revealed that the overexpression of HIF-1α can reverse the anti-inflammatory effect of ouabain. Thus, we conclude that the HIF-1α-dependent glycolytic pathway is essential for the anti-inflammatory effect of ouabain.

Rodent Skeletal Muscle Metabolomic Changes Associated With Static Cold Storage

ObjectiveThe aim of this study was to evaluate the impact of static cold storage preservation on skeletal muscle metabolism using a rodent model. MethodsSixteen male Lewis rats (250 ± 25 g) were distributed into 4 groups, including naive control, warm ischemia for 2 hours, static warm storage for 6 hours, and static cold storage for 6 hours. Energy status, metabolomics profiling, and histopathology of the muscle were analyzed. ResultsIn the warm ischemia and static warm storage groups, glycolytic pathway metabolites decreased, but the Krebs cycle metabolite of succinate and the purine degradation product of hypoxanthine accumulated. Increased succinate and hypoxanthine levels were associated with increased injury severity scores. During static cold storage, the glycolytic pathway activity and the energy status were preserved. Succinate and hypoxanthine levels showed no significant difference from the naive group. ConclusionWarm ischemia results in reduced glycolysis and Krebs cycle metabolites. Static cold storage preserves the glycolytic pathway and represents a favorable contribution to cellular energy demand. Succinate and hypoxanthine might be used as novel potential biomarkers for the assessment of viability and injury severity.

Genotype-Response Correlation in DRIVE PK, a Phase 2 Study of AG-348 in Patients with Pyruvate Kinase Deficiency

Pyruvate kinase (PK) deficiency is a congenital hemolytic anemia caused by mutations in the PKLR gene, leading to a deficiency of the glycolytic enzyme red cell PK (PK-R). AG-348 is an orally available small-molecule allosteric activator of PK-R that activates the wild type and a range of mutant PK-R enzymes associated with PK deficiency (Kung et al. Blood 2017;130:1347-56).In a phase 2 clinical study in patients with PK deficiency (DRIVE PK, NCT02476916), 26 of 52 patients (50%) experienced a maximum hemoglobin (Hb) increase of >1.0 g/dL (mean maximum increase, 3.4 g/dL; range, 1.1-5.8 g/dL). In most cases, Hb increases were rapid and sustained, and seen across a wide dose range from 5 to 300 mg twice daily. Hemolysis markers (reticulocytes, indirect bilirubin, haptoglobin) improved in patients who experienced a maximum Hb increase of >1.0 g/dL. Hb increases were observed in patients with a variety of PKLR mutations and were associated with the presence of at least 1 missense mutation.As PK deficiency is a genetically heterogeneous disease, with over 200 described mutations, we sought to understand in greater detail the molecular parameters that were associated with Hb increases in AG-348-treated patients. Here, we have analyzed the relationship between Hb increase and patient genotype, biochemical response to AG-348 treatment, and baseline PK-R protein level. The results are consistent with the proposed mechanism of action of AG-348 through binding and activation of residual mutant PK-R enzyme.Of the 106 PKLR mutations in the 52 subjects (2 subjects carried 3 mutations), there were 47 unique mutations, including 28 missense and 19 non-missense. Of these mutations, only 10 were present in at least 2 patients, highlighting the intrinsic limitations of findings from this small sample size. That notwithstanding, among these 10 mutations, we observed that some were more likely to be associated with Hb increases (e.g. increases were observed in 5/5 patients with at least 1 R486W mutation and 4/4 patients with at least 1 T384M mutation). Other mutations, including V134D (0/3), E241stop (1/6), and R510Q (5/14), were associated with a lack of Hb increase. Notably, 1/10 patients who had 2 non-missense mutations and 0/5 patients who were homozygous for the R479H “Amish” mutation had Hb increases.We have previously reported that, in biochemical assays, AG-348 could efficiently activate recombinantly expressed R479H and R510Q mutant enzymes. Although both of these are technically missense mutations, it is well understood that R510Q results in severely decreased protein stability (Wang et al. Blood 2001;98:3113-20), and the R479H mutation, occurring at the splice junction between 2 exons, can deleteriously affect mRNA splicing (van Wijk et al. Br J Haematol 2004;125:253-63). Therefore, as AG-348 works by directly binding and stimulating the activity of residual mutant PK-R enzyme, we explored the hypothesis that PK-R protein levels in patients with these mutations might be too low to allow for adequate target engagement at clinically tested doses.Using antibody-based capture and detection, we quantitated baseline PK-R protein levels of DRIVE PK subjects. Analysis of these data revealed that patients with Hb increases had, on average, higher PK-R protein levels (49% of reference control compared to 13% in patients without Hb increases). All 10 patients with non-missense/non-missense mutations had nearly undetectable PK-R protein levels (average of 3.7% of reference control), consistent with and confirming the predicted effect of these mutations on protein expression. Baseline PK-R protein levels were also lower in patients with at least 1 R510Q (18% of reference control) or R479H (19% of reference control) mutation compared to patients with other missense mutations (59% of reference control).Taken together, these analyses demonstrate that Hb increases upon AG-348 treatment are associated with the presence of at least 1 missense PK-R mutant enzyme with residual protein for AG-348 to bind and activate. These data strongly suggest that the hematological effects of AG-348 come from the proposed mechanism of action of restoration of glycolytic pathway activity in PK-deficient red cells. The genetic complexity of PK deficiency, combined with the relatively small sample size, limit extrapolation of these findings to the entire PK deficiency patient population, but may help to inform future studies. DisclosuresKung:Agios: Employment, Equity Ownership. Kosinski:Agios: Employment, Equity Ownership. Mangus:Agios: Employment, Equity Ownership. Hua:Agios: Employment, Equity Ownership. Connor:Agios: Employment, Equity Ownership. Mobilia:Agios: Employment, Equity Ownership. Sullivan:Agios: Employment, Equity Ownership. Frye:Agios: Employment, Equity Ownership. Jouvin:Agios: Employment, Equity Ownership. Grace:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Consultancy. Glader:Agios: Consultancy, Research Funding. Bowden:Agios: Employment, Equity Ownership.

N-3 Polyunsaturated fatty acids induce acute myeloid leukemia cell death associated with mitochondrial glycolytic switch and Nrf2 pathway activation.

Acute Myeloid Leukemia (AML) remains a therapeutic challenge and improvements in chemotherapy are needed. n-3 polyunsaturated fatty acids (PUFAs), present in fish oil (FO) at high concentrations, have antitumoral properties in various cancer models. We investigated the effects of two n-3 PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), in AML cell lines and primary AML blasts. EPA and DHA induced a dose-dependent decrease in cell viability in five AML cell lines, which was also observed with FO, but not SO (devoid of n-3 PUFAs) in cell lines and primary leucoblasts. Mitochondrial energy metabolism shifted from oxidative respiration to glycolytic metabolism in the U937, MOLM-13, and HL-60 cell lines. This phenomenon was associated with major disorganization of the mitochondrial network and mitochondrial swelling. Transcriptomic analysis after 6 h and 24 h of exposure to FO revealed a Nrf2 activation signature, which was confirmed by evidence of Nrf2 nuclear translocation in response to oxidative stress, but insufficient to prevent cell death following prolonged exposure. Apoptosis studies showed consistent phosphatidylserine exposition among the AML cell lines tested and a reduced mitochondrial membrane potential. The cell-killing effect of FO was additive with that of cytarabine (AraC), by the Chou and Talalay method, and this combination effect could be reproduced in primary AML blasts. Altogether, our results show deleterious effects of n-3 PUFAs on mitochondrial metabolism of AML cells, associated with oxidative stress and Nrf2 response, leading to cell death. These observations support further investigation of n-3 PUFA addition to standard chemotherapy in AML.