Abstract MP213: Activation Of Biosynthetic Pathways Regulates Cardiomyocyte Cell Cycle Induction In Human Cardiomyocyte

Induction of cardiomyocyte proliferation is a promising therapeutic approach to treat heart failure. Several studies have identified metabolism as an important regulator of myocyte proliferation; however, the changes in metabolism during cardiomyocyte division remain unclear. Here, we use ectopic expression of cyclin B1, Cyclin D1, CDK1, and CDK4 (termed 4F) as a tool for understanding how metabolism influences cardiomyocyte proliferation. Mature hiPS-CMs stimulated to proliferate by 4F expression showed significant downregulation of oxidative phosphorylation genes, decreased glucose oxidation, and upregulation of genes that regulate biosynthetic pathways of glucose metabolism such as those involved in NAD(P) + synthesis ( NAMPT, NADK1, NNT ), the hexosamine biosynthetic pathway (HBP) and protein O-GlcNAcylation ( GFPT1 , OGT, OGA ), and the serine biosynthesis pathway (SBP; PHGDH , PSAT1 , SHMT2 ). In 4F-expressing hiPSC-CMs, stable isotope tracing indicated higher enrichment of glucose-derived 13 C in pentose phosphate intermediates, UDP-hexose, phospholipid precursors, NAD + , pyrimidines, UDP-HexNAc, and products of the serine biosynthesis pathway and one-carbon metabolism, suggesting that cell cycle induction activates biosynthetic pathways in cardiomyocytes. Knocking down nicotinamide phosphoribosyltransferase (NAMPT), a critical enzyme in the NAD + salvage pathway, 2 days before 4F overexpression significantly inhibited cell cycle progression in 4F-transduced hiPS-CMs. OGA overexpression, which catalyzes the hydrolytic cleavage of O-GlcNAc from post-transitionally modified proteins, completely abolished 4F-mediated cell cycle induction. Furthermore, NCT503, an inhibitor of the rate-limiting step in the serine biosynthesis pathway, abolished 4F-mediated increases in cell cycle markers. In a gain-of-function approach, we overexpressed phosphoenolpyruvate carboxykinase 2 (PCK2), which can drive carbon from the Krebs cycle to the glycolytic intermediate pool. PCK2 overexpression significantly augmented 4F-mediated cell cycle entry. These findings suggest that a metabolic shift from catabolic to anabolic activity is a critical step for cardiomyocyte cell cycle entry and is required to facilitate proliferation.

Glucose limitation activates AMPK coupled SENP1-Sirt3 signalling in mitochondria for T cell memory development

Metabolic programming and mitochondrial dynamics along with T cell differentiation affect T cell fate and memory development; however, how to control metabolic reprogramming and mitochondrial dynamics in T cell memory development is unclear. Here, we provide evidence that the SUMO protease SENP1 promotes T cell memory development via Sirt3 deSUMOylation. SENP1-Sirt3 signalling augments the deacetylase activity of Sirt3, promoting both OXPHOS and mitochondrial fusion. Mechanistically, SENP1 activates Sirt3 deacetylase activity in T cell mitochondria, leading to reduction of the acetylation of mitochondrial metalloprotease YME1L1. Consequently, deacetylation of YME1L1 suppresses its activity on OPA1 cleavage to facilitate mitochondrial fusion, which results in T cell survival and promotes T cell memory development. We also show that the glycolytic intermediate fructose-1,6-bisphosphate (FBP) as a negative regulator suppresses AMPK-mediated activation of the SENP1-Sirt3 axis and reduces memory development. Moreover, glucose limitation reduces FBP production and activates AMPK during T cell memory development. These data show that glucose limitation activates AMPK and the subsequent SENP1-Sirt3 signalling for T cell memory development.

Induced expression of the zwf gene in the presence of glucose contributes to lowering of glucose 6-phosphate level and consequently reduction of growth rate of Mycobacterium smegmatis

In Mycobacterium smegmatis (renamed Mycolicibacterium smegmatis ), glucose 6-phosphate (G6P) level is exceptionally high as compared to other bacteria, E. coli for example. Earlier investigations have indicated that G6P protects M. smegmatis (Msm) against oxidative stress-inducing agents. G6P is a glycolytic intermediate formed either directly through the phosphorylation of glucose or indirectly via the gluconeogenic pathway. Its consumption is catalysed by several enzymes, one of which being the NADPH dependent G6P dehydrogenase (G6PDH) encoded by zwf (msmeg_0314). While investigating the extent to which the carbon sources glucose and glycerol influence Msm growth, we observed that intracellular concentration of G6P was lower in the former’s presence than the latter. We could correlate this difference with that in the growth rate, which was higher in glycerol than glucose. We also found that lowering of G6P content in glucose-grown cells was triggered by the induced expression of zwf and the resultant increase in G6PDH activity. When we silenced zwf using CRISPR-Cas9 technology, we observed a significant rise in the growth rate of Msm. Therefore, we have found that depletion of G6P in glucose-grown cells due to increased G6PDH activity is at least one reason why the growth rate of Msm in glucose is less than glycerol. However, we could not establish a similar link-up between slow growth in glucose and lowering of G6P level in the case of Mycobacterium tuberculosis (Mtb). Mycobacteria, therefore, may have evolved diverse mechanisms to ensure that they use glycerol preferentially over glucose for their growth.

Selection for rapid uptake of scarce or fluctuating resource explains vulnerability of glycolysis to imbalance

Glycolysis is a conserved central pathway in energy metabolism that converts glucose to pyruvate with net production of two ATP molecules. Because ATP is produced only in the lower part of glycolysis (LG), preceded by an initial investment of ATP in the upper glycolysis (UG), achieving robust start-up of the pathway upon activation presents a challenge: a sudden increase in glucose concentration can throw a cell into a self-sustaining imbalanced state in which UG outpaces LG, glycolytic intermediates accumulate and the cell is unable to maintain high ATP concentration needed to support cellular functions. Such metabolic imbalance can result in “substrate-accelerated death”, a phenomenon observed in prokaryotes and eukaryotes when cells are exposed to an excess of substrate that previously limited growth. Here, we address why evolution has apparently not eliminated such a costly vulnerability and propose that it is a manifestation of an evolutionary trade-off, whereby the glycolysis pathway is adapted to quickly secure scarce or fluctuating resource at the expense of vulnerability in an environment with ample resource. To corroborate this idea, we perform individual-based eco-evolutionary simulations of a simplified yeast glycolysis pathway consisting of UG, LG, phosphate transport between a vacuole and a cytosol, and a general ATP demand reaction. The pathway is evolved in constant or fluctuating resource environments by allowing mutations that affect the (maximum) reaction rate constants, reflecting changing expression levels of different glycolytic enzymes. We demonstrate that under limited constant resource, populations evolve to a genotype that exhibits balanced dynamics in the environment it evolved in, but strongly imbalanced dynamics under ample resource conditions. Furthermore, when resource availability is fluctuating, imbalanced dynamics confers a fitness advantage over balanced dynamics: when glucose is abundant, imbalanced pathways can quickly accumulate the glycolytic intermediate FBP as intracellular storage that is used during periods of starvation to maintain high ATP concentration needed for growth. Our model further predicts that in fluctuating environments, competition for glucose can result in stable coexistence of balanced and imbalanced cells, as well as repeated cycles of population crashes and recoveries that depend on such polymorphism. Overall, we demonstrate the importance of ecological and evolutionary arguments for understanding seemingly maladaptive aspects of cellular metabolism.

Chemoproteomics Maps Glycolytic Targetome in Cancer Cells

Hyperactivated glycolysis, favoring uncontrolled growth and metastasis by producing essential metabolic intermediates engaging bioenergetics and biosynthesis, is a metabolic hallmark of most cancer cells. Although sporadic information has revealed glycolytic metabolites also possess non-metabolic function as signaling molecules, it remains largely elusive how these metabolites interact with and functionally regulate their binding targets. Here we introduce a Target Responsive Accessibility Profiling (TRAP) approach that measures ligand binding-induced steric hindrance in protein targets via global profiling accessibility changes in reactive lysines, and mapped 913 target candidates and 2,487 interactions for 10 major glycolytic metabolites in cancer cells via TRAP. The elucidated targetome uncovers diverse regulatory modalities of glycolytic metabolites involving the direct perturbation of carbohydrate metabolism enzymes, intervention of transcriptional control, modulation of proteome-level acetylation and protein complex assemblies. The advantages gained from glycolysis by cancer cells are expanded by discovering lactate as a ligand for an orphan transcriptional regulator TRIM 28 that promotes p53 degradation, and by identifying pyruvate acting against a cell apoptosis inducer trichostatin A via attenuating protein acetylation. Lastly, the inhibition of glycolytic key enzymes led to identify an intrinsically active glycolytic intermediate glyceraldehyde 3-phosphate that elicits its cytotoxicity by engaging with ENO1 and MTHFD1. Collectively, the glycolytic targetome depicted by TRAP constitutes a fertile resource for understanding how glycolysis finely tunes metabolism and signaling in support of cancer cells, and fostering the exploitation of glycolytic targetome as promising nodes for anti-cancer therapeutics development.

Protein synthesis inhibitors stimulate MondoA transcriptional activity by driving an accumulation of glucose 6-phosphate

Background Protein synthesis is regulated by the availability of amino acids, the engagement of growth factor signaling pathways, and adenosine triphosphate (ATP) levels sufficient to support translation. Crosstalk between these inputs is extensive, yet other regulatory mechanisms remain to be characterized. For example, the translation initiation inhibitor rocaglamide A (RocA) induces thioredoxin-interacting protein (TXNIP). TXNIP is a negative regulator of glucose uptake; thus, its induction by RocA links translation to the availability of glucose. MondoA is the principal regulator of glucose-induced transcription, and its activity is triggered by the glycolytic intermediate, glucose 6-phosphate (G6P). MondoA responds to G6P generated by cytoplasmic glucose and mitochondrial ATP (mtATP), suggesting a critical role in the cellular response to these energy sources. TXNIP expression is entirely dependent on MondoA; therefore, we investigated how protein synthesis inhibitors impact its transcriptional activity. Methods We investigated how translation regulates MondoA activity using cell line models and loss-of-function approaches. We examined how protein synthesis inhibitors effect gene expression and metabolism using RNA-sequencing and metabolomics, respectively. The biological impact of RocA was evaluated using cell lines and patient-derived xenograft organoid (PDxO) models. Results We discovered that multiple protein synthesis inhibitors, including RocA, increase TXNIP expression in a manner that depends on MondoA, a functional electron transport chain and mtATP synthesis. Furthermore, RocA and cycloheximide increase mtATP and G6P levels, respectively, and TXNIP induction depends on interactions between the voltage-dependent anion channel (VDAC) and hexokinase (HK), which generates G6P. RocA treatment impacts the regulation of ~ 1200 genes, and ~ 250 of those genes are MondoA-dependent. RocA treatment is cytotoxic to triple negative breast cancer (TNBC) cell lines and shows preferential cytotoxicity against estrogen receptor negative (ER−) PDxO breast cancer models. Finally, RocA-driven cytotoxicity is partially dependent on MondoA or TXNIP. Conclusions Our data suggest that protein synthesis inhibitors rewire metabolism, resulting in an increase in mtATP and G6P, the latter driving MondoA-dependent transcriptional activity. Further, MondoA is a critical component of the cellular transcriptional response to RocA. Our functional assays suggest that RocA or similar translation inhibitors may show efficacy against ER− breast tumors and that the levels of MondoA and TXNIP should be considered when exploring these potential treatment options.

Bioenergetic Profiling of the Differentiating Human MDS Myeloid Lineage with Low and High Bone Marrow Blast Counts

Myelodysplastic syndromes (MDS) encompass a very heterogeneous group of clonal hematopoietic stem cell differentiation disorders with malignant potential and an elusive pathobiology. Given the central role of metabolism in effective differentiation, we performed an untargeted metabolomic analysis of differentiating myeloid lineage cells from MDS bone marrow aspirates that exhibited <5% (G1) or ≥5% (G2) blasts, in order to delineate its role in MDS severity and malignant potential. Bone marrow aspirates were collected from 14 previously untreated MDS patients (G1, n = 10 and G2, n = 4) and age matched controls (n = 5). Following myeloid lineage cell isolation, untargeted mass spectrometry-based metabolomics analysis was performed. Data were processed and analyzed using Metabokit. Enrichment analysis was performed using Metaboanalyst v4 employing pathway-associated metabolite sets. We established a bioenergetic profile coordinated by the Warburg phenomenon in both groups, but with a massively different outcome that mainly depended upon its group mitochondrial function and redox state. G1 cells are overwhelmed by glycolytic intermediate accumulation due to failing mitochondria, while the functional electron transport chain and improved redox in G2 compensate for Warburg disruption. Both metabolomes reveal the production and abundance of epigenetic modifiers. G1 and G2 metabolomes differ and eventually determine the MDS clinical phenotype, as well as the potential for malignant transformation.

Chemoproteomics Maps Glycolytic Targetome in Cancer Cells

ABSTRACTHyperactivated glycolysis, favoring uncontrolled growth and metastasis by producing essential metabolic intermediates engaging bioenergetics and biosynthesis, is a metabolic hallmark of most cancer cells. Although sporadic information has revealed glycolytic metabolites also possess non-metabolic function as signaling molecules, it remains largely elusive how these metabolites interact with and functionally regulate their binding targets. Here we introduce a Target Responsive Accessibility Profiling (TRAP) approach that measures ligand binding-induced steric hindrance in protein targets via global profiling accessibility changes in reactive lysines, and mapped 913 target candidates and 2,487 interactions for 10 major glycolytic metabolites in cancer cells via TRAP. The elucidated targetome uncovers diverse regulatory modalities of glycolytic metabolites involving the direct perturbation of carbohydrate metabolism enzymes, intervention of transcriptional control, modulation of proteome-level acetylation and protein complex assemblies. The advantages gained from glycolysis by cancer cells are expanded by discovering lactate as a ligand for an orphan transcriptional regulator TRIM 28 that promotes p53 degradation, and by identifying pyruvate acting against a cell apoptosis inducer trichostatin A via attenuating protein acetylation. Lastly, the inhibition of glycolytic key enzymes led to identify an intrinsically active glycolytic intermediate glyceraldehyde 3-phosphate that elicits its cytotoxicity by engaging with ENO1 and MTHFD1. Collectively, the glycolytic targetome depicted by TRAP constitutes a fertile resource for understanding how glycolysis finely tunes metabolism and signaling in support of cancer cells, and fostering the exploitation of glycolytic targetome as promising nodes for anti-cancer therapeutics development.

Protein synthesis inhibitors stimulate MondoA transcriptional activity by driving an accumulation of glucose 6-phosphate

BACKGROUND Protein synthesis is regulated by the availability of amino acids, the engagement of growth factor signaling pathways and ATP levels sufficient to support translation. Crosstalk between these inputs is extensive, yet other regulatory mechanisms remain to be characterized. For example, the translation initiation inhibitor Rocaglamide A (RocA) induces Thioredoxin Interacting Protein (TXNIP). TXNIP is a negative regulator of glucose uptake, thus its induction by RocA links translation to the availability of glucose. MondoA is the principal regulator of glucose-induced transcription and its activity is triggered by the glycolytic intermediate, glucose 6-phosphate (G6P). MondoA responds to G6P generated by cytoplasmic glucose and mitochondrial ATP (mtATP), suggesting a critical role in the cellular response to these energy sources. TXNIP expression is entirely dependent on MondoA, therefore, we investigated how protein synthesis inhibitors impact its transcriptional activity.METHODS We investigated how translation regulates MondoA activity using cell line models and loss-of-function approaches. We examined how protein synthesis inhibitors effect gene expression and metabolism using RNA-sequencing and metabolomics, respectively. The biological impact of RocA was evaluated using cell lines and Patient-Derived xenograft Organoid (PDxO) models. RESULTS We discovered that multiple protein synthesis inhibitors, including RocA, increase TXNIP expression in a manner that depends on MondoA, a functional electron transport chain and mtATP synthesis. Furthermore, RocA increases mtATP and G6P levels and TXNIP induction depends on interactions between the Voltage-Dependent Anion Channel (VDAC) and hexokinase, which generates G6P. RocA treatment impacts the regulation of ~1200 genes and ~250 of those genes are MondoA-dependent. RocA treatment is cytotoxic to Triple Negative Breast Cancer cell lines and shows preferential cytotoxicity against ER- PDxO breast cancer models. Finally, RocA-driven cytotoxicity is partially-dependent on MondoA or TXNIP.CONCLUSIONS Our data suggest that protein synthesis inhibitors rewire metabolism, resulting in an increase in mtATP and G6P, the latter driving MondoA-dependent transcriptional activity. Further, MondoA is a critical component of the cellular transcriptional response to RocA. Our functional assays suggest that RocA or similar translation inhibitors may show efficacy against ER- breast tumors and that the levels of MondoA and TXNIP should be considered when exploring these potential treatment options.

β-Hydroxybutyrate Oxidation Promotes the Accumulation of Immunometabolites in Activated Microglia Cells

Metabolic regulation of immune cells has arisen as a critical set of processes required for appropriate response to immunological signals. While our knowledge in this area has rapidly expanded in leukocytes, much less is known about the metabolic regulation of brain-resident microglia. In particular, the role of alternative nutrients to glucose remains poorly understood. Here, we use stable-isotope (13C) tracing strategies and metabolomics to characterize the oxidative metabolism of β-hydroxybutyrate (BHB) in human (HMC3) and murine (BV2) microglia cells and the interplay with glucose in resting and LPS-activated BV2 cells. We found that BHB is imported and oxidised in the TCA cycle in both cell lines with a subsequent increase in the cytosolic NADH:NAD+ ratio. In BV2 cells, stimulation with LPS upregulated the glycolytic flux, increased the cytosolic NADH:NAD+ ratio and promoted the accumulation of the glycolytic intermediate dihydroxyacetone phosphate (DHAP). The addition of BHB enhanced LPS-induced accumulation of DHAP and promoted glucose-derived lactate export. BHB also synergistically increased LPS-induced accumulation of succinate and other key immunometabolites, such as α-ketoglutarate and fumarate generated by the TCA cycle. Finally, BHB upregulated the expression of a key pro-inflammatory (M1 polarisation) marker gene, NOS2, in BV2 cells activated with LPS. In conclusion, we identify BHB as a potentially immunomodulatory metabolic substrate for microglia that promotes metabolic reprogramming during pro-inflammatory response.