Stable isotope-resolved metabolomics analyses of metabolic phenotypes reveal variable glutamine metabolism in different patient-derived models of non-small cell lung cancer from a single patient.

INTRODUCTION: Stable isotope tracers have been increasingly used in preclinical cancer model systems, including cell culture and mouse xenografts, to probe the altered metabolism of a variety of cancers, such as accelerated glycolysis and glutaminolysis and generation of oncometabolites. Comparatively little has been reported on the fidelity of the different preclinical model systems in recapitulating the aberrant metabolism of tumors. OBJECTIVES: We have been developing several different experimental model systems for systems biochemistry analyses of non-small cell lung cancer (NSCLC1) using patient-derived tissues to evaluate appropriate models for metabolic and phenotypic analyses. METHODS: To address the issue of fidelity, we have carried out a detailed Stable Isotope-Resolved Metabolomics study of freshly resected tissue slices, mouse patient derived xenografts (PDXs), and cells derived from a single patient using both 13C6-glucose and 13C5,15N2-glutamine tracers. RESULTS: Although we found similar glucose metabolism in the three models, glutamine utilization was markedly higher in the isolated cell culture and in cell culture-derived xenografts compared with the primary cancer tissue or direct tissue xenografts (PDX). CONCLUSIONS: This suggests that caution is needed in interpreting cancer biochemistry using patient-derived cancer cells in vitro or in xenografts, even at very early passage, and that direct analysis of patient derived tissue slices provides the optimal model for ex vivo metabolomics. Further research is needed to determine the generality of these observations.

Metabolic Reprogramming in Human Cancer Patients and Patient-Derived Models.

Stable isotope-resolved metabolomics delineates reprogrammed intersecting metabolic networks in human cancers. Knowledge gained from in vivo patient studies provides the "benchmark" for cancer models to recapitulate. It is particularly difficult to model patients' tumor microenvironment (TME) with its complex cell-cell/cell-matrix interactions, which shapes metabolic reprogramming crucial to cancer development/drug resistance. Patient-derived organotypic tissue cultures (PD-OTCs) represent a unique model that retains an individual patient's TME. PD-OTCs of non-small-cell lung cancer better recapitulated the in vivo metabolic reprogramming of patient tumors than the patient-derived tumor xenograft (PDTX), while enabling interrogation of immunometabolic response to modulators and TME-dependent resistance development. Patient-derived organoids (PDOs) are also good models for reconstituting TME-dependent metabolic reprogramming and for evaluating therapeutic responses. Single-cell based 'omics on combinations of PD-OTC and PDO models will afford an unprecedented understanding on TME dependence of human cancer metabolic reprogramming, which should translate into the identification of novel metabolic targets for regulating TME interactions and drug resistance.

Challenges of Spatially Resolved Metabolism in Cancer Research.

Stable isotope-resolved metabolomics comprises a critical set of technologies that can be applied to a wide variety of systems, from isolated cells to whole organisms, to define metabolic pathway usage and responses to perturbations such as drugs or mutations, as well as providing the basis for flux analysis. As the diversity of stable isotope-enriched compounds is very high, and with newer approaches to multiplexing, the coverage of metabolism is now very extensive. However, as the complexity of the model increases, including more kinds of interacting cell types and interorgan communication, the analytical complexity also increases. Further, as studies move further into spatially resolved biology, new technical problems have to be overcome owing to the small number of analytes present in the confines of a single cell or cell compartment. Here, we review the overall goals and solutions made possible by stable isotope tracing and their applications to models of increasing complexity. Finally, we discuss progress and outstanding difficulties in high-resolution spatially resolved tracer-based metabolic studies.

Exercise-induced changes in myocardial glucose utilization during periods of active cardiac growth.

Exercise training can promote physiological cardiac growth, which has been suggested to involve changes in glucose metabolism to facilitate hypertrophy of cardiomyocytes. In this study, we used a dietary, in vivo isotope labeling approach to examine how exercise training influences the metabolic fate of carbon derived from dietary glucose in the heart during acute, active, and established phases of exercise-induced cardiac growth. Male and female FVB/NJ mice were subjected to treadmill running for up to 4 weeks and cardiac growth was assessed by gravimetry. Cardiac metabolic responses to exercise were assessed via in vivo tracing of [13C6]-glucose via mass spectrometry and nuclear magnetic resonance. We found that the half-maximal cardiac growth response was achieved by approximately 1 week of daily exercise training, with near maximal growth observed in male mice with 2 weeks of training; however, female mice were recalcitrant to exercise-induced cardiac growth and required a higher daily intensity of exercise training to achieve significant, albeit modest, increases in cardiac mass. We also found that increases in the energy charge of adenylate and guanylate nucleotide pools precede exercise-induced changes in cardiac size and were associated with higher glucose tracer enrichment in the TCA pool and in amino acids (aspartate, glutamate) sourced by TCA intermediates. Our data also indicate that the activity of collateral biosynthetic pathways of glucose metabolism may not be markedly altered by exercise. Overall, this study provides evidence that metabolic remodeling in the form of heightened energy charge and increased TCA cycle activity and cataplerosis precedes cardiac growth caused by exercise training in male mice.

Myeloid-derived suppressor cell mitochondrial fitness governs chemotherapeutic efficacy in hematologic malignancies.

Myeloid derived suppressor cells (MDSCs) are key regulators of immune responses and correlate with poor outcomes in hematologic malignancies. Here, we identify that MDSC mitochondrial fitness controls the efficacy of doxorubicin chemotherapy in a preclinical lymphoma model. Mechanistically, we show that triggering STAT3 signaling via ß2-adrenergic receptor (ß2-AR) activation leads to improved MDSC function through metabolic reprograming, marked by sustained mitochondrial respiration and higher ATP generation which reduces AMPK signaling, altering energy metabolism. Furthermore, induced STAT3 signaling in MDSCs enhances glutamine consumption via the TCA cycle. Metabolized glutamine generates itaconate which downregulates mitochondrial reactive oxygen species via regulation of Nrf2 and the oxidative stress response, enhancing MDSC survival. Using ß2-AR blockade, we target the STAT3 pathway and ATP and itaconate metabolism, disrupting ATP generation by the electron transport chain and decreasing itaconate generation causing diminished MDSC mitochondrial fitness. This disruption increases the response to doxorubicin and could be tested clinically.

CD8+ T cell metabolic flexibility elicited by CD28-ARS2 axis-driven alternative splicing of PKM supports antitumor immunity.

Metabolic flexibility has emerged as a critical determinant of CD8+ T-cell antitumor activity, yet the mechanisms driving the metabolic flexibility of T cells have not been determined. In this study, we investigated the influence of the nuclear cap-binding complex (CBC) adaptor protein ARS2 on mature T cells. In doing so, we discovered a novel signaling axis that endows activated CD8+ T cells with flexibility of glucose catabolism. ARS2 upregulation driven by CD28 signaling reinforced splicing factor recruitment to pre-mRNAs and affected approximately one-third of T-cell activation-induced alternative splicing events. Among these effects, the CD28-ARS2 axis suppressed the expression of the M1 isoform of pyruvate kinase in favor of PKM2, a key determinant of CD8+ T-cell glucose utilization, interferon gamma production, and antitumor effector function. Importantly, PKM alternative splicing occurred independently of CD28-driven PI3K pathway activation, revealing a novel means by which costimulation reprograms glucose metabolism in CD8+ T cells.

Cyclin D1 extensively reprograms metabolism to support biosynthetic pathways in hepatocytes.

Cell proliferation requires metabolic reprogramming to accommodate biosynthesis of new cell components, and similar alterations occur in cancer cells. However, the mechanisms linking the cell cycle machinery to metabolism are not well defined. Cyclin D1, along with its main partner cyclin-dependent kinase 4 (Cdk4), is a pivotal cell cycle regulator and driver oncogene that is overexpressed in many cancers. Here, we examine hepatocyte proliferation to define novel effects of cyclin D1 on biosynthetic metabolism. Metabolomic studies reveal that cyclin D1 broadly promotes biosynthetic pathways including glycolysis, the pentose phosphate pathway, and the purine and pyrimidine nucleotide synthesis in hepatocytes. Proteomic analyses demonstrate that overexpressed cyclin D1 binds to numerous metabolic enzymes including those involved in glycolysis and pyrimidine synthesis. In the glycolysis pathway, cyclin D1 activates aldolase and GAPDH, and these proteins are phosphorylated by cyclin D1/Cdk4 in vitro. De novo pyrimidine synthesis is particularly dependent on cyclin D1. Cyclin D1/Cdk4 phosphorylates the initial enzyme of this pathway, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and metabolomic analysis indicates that cyclin D1 depletion markedly reduces the activity of this enzyme. Pharmacologic inhibition of Cdk4 along with the downstream pyrimidine synthesis enzyme dihydroorotate dehydrogenase synergistically inhibits proliferation and survival of hepatocellular carcinoma cells. These studies demonstrate that cyclin D1 promotes a broad network of biosynthetic pathways in hepatocytes, and this model may provide insights into potential metabolic vulnerabilities in cancer cells.

Portable optical spectroscopic assay for non-destructive measurement of key metabolic parameters on in vitro cancer cells and organotypic fresh tumor slices.

To enable non-destructive metabolic characterizations on in vitro cancer cells and organotypic tumor models for therapeutic studies in an easy-to-access way, we report a highly portable optical spectroscopic assay for simultaneous measurement of glucose uptake and mitochondrial function on various cancer models with high sensitivity. Well-established breast cancer cell lines (MCF-7 and MDA-MB-231) were used to validate the optical spectroscopic assay for metabolic characterizations, while fresh tumor samples harvested from both animals and human cancer patients were used to test the feasibility of our optical metabolic assay for non-destructive measurement of key metabolic parameters on organotypic tumor slices. Our optical metabolic assay captured that MCF-7 cells had higher mitochondrial metabolism, but lower glucose uptake compared to the MDA-MB-231 cells, which is consistent with our microscopy imaging and flow cytometry data, as well as the published Seahorse Assay data. Moreover, we demonstrated that our optical assay could non-destructively measure both glucose uptake and mitochondrial metabolism on the same cancer cell samples at one time, which remains challenging by existing metabolic tools. Our pilot tests on thin fresh tumor slices showed that our optical assay captured increased metabolic activities in tumors compared to normal tissues. Our non-destructive optical metabolic assay provides a cost-effective way for future longitudinal therapeutic studies using patient-derived organotypic fresh tumor slices through the lens of tumor energetics, which will significantly advance translational cancer research.

Asparagine restriction enhances CD8+ T cell metabolic fitness and antitumoral functionality through an NRF2-dependent stress response.

Robust and effective T cell immune surveillance and cancer immunotherapy require proper allocation of metabolic resources to sustain energetically costly processes, including growth and cytokine production. Here, we show that asparagine (Asn) restriction on CD8+ T cells exerted opposing effects during activation (early phase) and differentiation (late phase) following T cell activation. Asn restriction suppressed activation and cell cycle entry in the early phase while rapidly engaging the nuclear factor erythroid 2-related factor 2 (NRF2)-dependent stress response, conferring robust proliferation and effector function on CD8+ T cells during differentiation. Mechanistically, NRF2 activation in CD8+ T cells conferred by Asn restriction rewired the metabolic program by reducing the overall glucose and glutamine consumption but increasing intracellular nucleotides to promote proliferation. Accordingly, Asn restriction or NRF2 activation potentiated the T cell-mediated antitumoral response in preclinical animal models, suggesting that Asn restriction is a promising and clinically relevant strategy to enhance cancer immunotherapy. Our study revealed Asn as a critical metabolic node in directing the stress signaling to shape T cell metabolic fitness and effector functions.

Differential Inhibition of Anaplerotic Pyruvate Carboxylation and Glutaminolysis-Fueled Anabolism Underlies Distinct Toxicity of Selenium Agents in Human Lung Cancer.

Past chemopreventive human trials on dietary selenium supplements produced controversial outcomes. They largely employed selenomethionine (SeM)-based diets. SeM was less toxic than selenite or methylseleninic acid (MSeA) to lung cancer cells. We thus investigated the toxic action of these Se agents in two non-small cell lung cancer (NSCLC) cell lines and ex vivo organotypic cultures (OTC) of NSCLC patient lung tissues. Stable isotope-resolved metabolomics (SIRM) using 13C6-glucose and 13C5,15N2-glutamine tracers with gene knockdowns were employed to examine metabolic dysregulations associated with cell type- and treatment-dependent phenotypic changes. Inhibition of key anaplerotic processes, pyruvate carboxylation (PyC) and glutaminolysis were elicited by exposure to MSeA and selenite but not by SeM. They were accompanied by distinct anabolic dysregulation and reflected cell type-dependent changes in proliferation/death/cell cycle arrest. NSCLC OTC showed similar responses of PyC and/or glutaminolysis to the three agents, which correlated with tissue damages. Altogether, we found differential perturbations in anaplerosis-fueled anabolic pathways to underlie the distinct anti-cancer actions of the three Se agents, which could also explain the failure of SeM-based chemoprevention trials.

Bayesian kinetic modeling for tracer-based metabolomic data.

BACKGROUND: Stable Isotope Resolved Metabolomics (SIRM) is a new biological approach that uses stable isotope tracers such as uniformly [Formula: see text]-enriched glucose ([Formula: see text]-Glc) to trace metabolic pathways or networks at the atomic level in complex biological systems. Non-steady-state kinetic modeling based on SIRM data uses sets of simultaneous ordinary differential equations (ODEs) to quantitatively characterize the dynamic behavior of metabolic networks. It has been increasingly used to understand the regulation of normal metabolism and dysregulation in the development of diseases. However, fitting a kinetic model is challenging because there are usually multiple sets of parameter values that fit the data equally well, especially for large-scale kinetic models. In addition, there is a lack of statistically rigorous methods to compare kinetic model parameters between different experimental groups. RESULTS: We propose a new Bayesian statistical framework to enhance parameter estimation and hypothesis testing for non-steady-state kinetic modeling of SIRM data. For estimating kinetic model parameters, we leverage the prior distribution not only to allow incorporation of experts' knowledge but also to provide robust parameter estimation. We also introduce a shrinkage approach for borrowing information across the ensemble of metabolites to stably estimate the variance of an individual isotopomer. In addition, we use a component-wise adaptive Metropolis algorithm with delayed rejection to perform efficient Monte Carlo sampling of the posterior distribution over high-dimensional parameter space. For comparing kinetic model parameters between experimental groups, we propose a new reparameterization method that converts the complex hypothesis testing problem into a more tractable parameter estimation problem. We also propose an inference procedure based on credible interval and credible value. Our method is freely available for academic use at https://github.com/xuzhang0131/MCMCFlux . CONCLUSIONS: Our new Bayesian framework provides robust estimation of kinetic model parameters and enables rigorous comparison of model parameters between experimental groups. Simulation studies and application to a lung cancer study demonstrate that our framework performs well for non-steady-state kinetic modeling of SIRM data.

Ultrahigh resolution MS1/MS2-based reconstruction of metabolic networks in mammalian cells reveals changes for selenite and arsenite action.

Metabolic networks are complex, intersecting, and composed of numerous enzyme-catalyzed biochemical reactions that transfer various molecular moieties among metabolites. Thus, robust reconstruction of metabolic networks requires metabolite moieties to be tracked, which cannot be readily achieved with mass spectrometry (MS) alone. We previously developed an Ion Chromatography-ultrahigh resolution-MS1/data independent-MS2 method to track the simultaneous incorporation of the heavy isotopes 13C and 15N into the moieties of purine/pyrimidine nucleotides in mammalian cells. Ultrahigh resolution-MS1 resolves and counts multiple tracer atoms in intact metabolites, while data independent-tandem MS (MS2) determines isotopic enrichment in their moieties without concern for the numerous mass isotopologue source ions to be fragmented. Together, they enabled rigorous MS-based reconstruction of metabolic networks at specific enzyme levels. We have expanded this approach to trace the labeled atom fate of [13C6]-glucose in 3D A549 spheroids in response to the anticancer agent selenite and that of [13C5,15N2]-glutamine in 2D BEAS-2B cells in response to arsenite transformation. We deduced altered activities of specific enzymes in the Krebs cycle, pentose phosphate pathway, gluconeogenesis, and UDP-GlcNAc synthesis pathways elicited by the stressors. These metabolic details help elucidate the resistance mechanism of 3D versus 2D A549 cultures to selenite and metabolic reprogramming that can mediate the transformation of BEAS-2B cells by arsenite.

GAB functions as a bioenergetic and signalling gatekeeper to control T cell inflammation.

γ-Aminobutyrate (GAB), the biochemical form of (GABA) γ-aminobutyric acid, participates in shaping physiological processes, including the immune response. How GAB metabolism is controlled to mediate such functions remains elusive. Here we show that GAB is one of the most abundant metabolites in CD4+ T helper 17 (TH17) and induced T regulatory (iTreg) cells. GAB functions as a bioenergetic and signalling gatekeeper by reciprocally controlling pro-inflammatory TH17 cell and anti-inflammatory iTreg cell differentiation through distinct mechanisms. 4-Aminobutyrate aminotransferase (ABAT) funnels GAB into the tricarboxylic acid (TCA) cycle to maximize carbon allocation in promoting TH17 cell differentiation. By contrast, the absence of ABAT activity in iTreg cells enables GAB to be exported to the extracellular environment where it acts as an autocrine signalling metabolite that promotes iTreg cell differentiation. Accordingly, ablation of ABAT activity in T cells protects against experimental autoimmune encephalomyelitis (EAE) progression. Conversely, ablation of GABAA receptor in T cells worsens EAE. Our results suggest that the cell-autonomous control of GAB on CD4+ T cells is bimodal and consists of the sequential action of two processes, ABAT-dependent mitochondrial anaplerosis and the receptor-dependent signalling response, both of which are required for T cell-mediated inflammation.

Polarization and ß-Glucan Reprogram Immunomodulatory Metabolism in Human Macrophages and Ex Vivo in Human Lung Cancer Tissues.

Immunomodulatory (IM) metabolic reprogramming in macrophages (Mϕs) is fundamental to immune function. However, limited information is available for human Mϕs, particularly in response plasticity, which is critical to understanding the variable efficacy of immunotherapies in cancer patients. We carried out an in-depth analysis by combining multiplex stable isotope-resolved metabolomics with reversed phase protein array to map the dynamic changes of the IM metabolic network and key protein regulators in four human donors' Mϕs in response to differential polarization and M1 repolarizer ß-glucan (whole glucan particles [WGPs]). These responses were compared with those of WGP-treated ex vivo organotypic tissue cultures (OTCs) of human non-small cell lung cancer. We found consistently enhanced tryptophan catabolism with blocked NAD+ and UTP synthesis in M1-type Mϕs (M1-Mϕs), which was associated with immune activation evidenced by increased release of IL-1ß/CXCL10/IFN-γ/TNF-α and reduced phagocytosis. In M2a-Mϕs, WGP treatment of M2a-Mϕs robustly increased glucose utilization via the glycolysis/oxidative branch of the pentose phosphate pathway while enhancing UDP-N-acetyl-glucosamine turnover and glutamine-fueled gluconeogenesis, which was accompanied by the release of proinflammatory IL-1ß/TNF-α to above M1-Mϕ's levels, anti-inflammatory IL-10 to above M2a-Mϕ's levels, and attenuated phagocytosis. These IM metabolic responses could underlie the opposing effects of WGP, i.e., reverting M2- to M1-type immune functions but also boosting anti-inflammation. Variable reprogrammed Krebs cycle and glutamine-fueled synthesis of UTP in WGP-treated OTCs of human non-small cell lung cancer were observed, reflecting variable M1 repolarization of tumor-associated Mϕs. This was supported by correlation with IL-1ß/TNF-α release and compromised tumor status, making patient-derived OTCs unique models for studying variable immunotherapeutic efficacy in cancer patients.

Resolving Enantiomers of 2-Hydroxy Acids by Nuclear Magnetic Resonance.

Biologically important 2-hydroxy carboxylates such as lactate, malate, and 2-hydroxyglutarate exist in two enantiomeric forms that cannot be distinguished under achiral conditions. The D and L (or R, S) enantiomers have different biological origins and functions, and therefore, there is a need for a simple method for resolving, identifying, and quantifying these enantiomers. We have adapted and improved a chiral derivatization technique for nuclear magnetic resonance (NMR), which needs no chromatography for enantiomer resolution, with greater than 90% overall recovery. This method was developed for 2-hydroxyglutarate (2HG) to produce diastereomers resolvable by column chromatography. We have applied the method to lactate, malate, and 2HG. The limit of quantification was determined to be about 1 nmol for 2HG with coefficients of variation of less than 5%. We also demonstrated the method on an extract of a renal carcinoma bearing an isocitrate dehydrogenase-2 (IDH2) variant that produces copious quantities of 2HG and showed that it is the D enantiomer that was exclusively produced. We also demonstrated in the same experiment that the lactate produced in the same sample was the L enantiomer.

A Micro-Scale Analytical Method for Determining Glycogen Turnover by NMR and FTMS.

Glycogen is a readily deployed intracellular energy storage macromolecule composed of branched chains of glucose anchored to the protein glycogenin. Although glycogen primarily occurs in the liver and muscle, it is found in most tissues, and its metabolism has been shown to be important in cancers and immune cells. Robust analysis of glycogen turnover requires stable isotope tracing plus a reliable means of quantifying total and labeled glycogen derived from precursors such as 13C6-glucose. Current methods for analyzing glycogen are time- and sample-consuming, at best semi-quantitative, and unable to measure stable isotope enrichment. Here we describe a microscale method for quantifying both intact and acid-hydrolyzed glycogen by ultra-high-resolution Fourier transform mass spectrometric (UHR-FTMS) and/or NMR analysis in stable isotope resolved metabolomics (SIRM) studies. Polar metabolites, including intact glycogen and their 13C positional isotopomer distributions, are first measured in crude biological extracts by high resolution NMR, followed by rapid and efficient acid hydrolysis to glucose under N2 in a focused beam microwave reactor, with subsequent analysis by UHR-FTMS and/or NMR. We optimized the microwave digestion time, temperature, and oxygen purging in terms of recovery versus degradation and found 10 min at 110−115 °C to give >90% recovery. The method was applied to track the fate of 13C6-glucose in primary human lung BEAS-2B cells, human macrophages, murine liver and patient-derived tumor xenograft (PDTX) in vivo, and the fate of 2H7-glucose in ex vivo lung organotypic tissue cultures of a lung cancer patient. We measured the incorporation of 13C6-glucose into glycogen and its metabolic intermediates, UDP-Glucose and glucose-1-phosphate, to demonstrate the utility of the method in tracing glycogen turnover in cells and tissues. The method offers a quantitative, sensitive, and convenient means to analyze glycogen turnover in mg amounts of complex biological materials.

Succinate dehydrogenase/complex II is critical for metabolic and epigenetic regulation of T cell proliferation and inflammation.

Effective T cell-mediated immune responses require the proper allocation of metabolic resources to sustain growth, proliferation, and cytokine production. Epigenetic control of the genome also governs T cell transcriptome and T cell lineage commitment and maintenance. Cellular metabolic programs interact with epigenetic regulation by providing substrates for covalent modifications of chromatin. By using complementary genetic, epigenetic, and metabolic approaches, we revealed that tricarboxylic acid (TCA) cycle flux fueled biosynthetic processes while controlling the ratio of succinate/α-ketoglutarate (α-KG) to modulate the activities of dioxygenases that are critical for driving T cell inflammation. In contrast to cancer cells, where succinate dehydrogenase (SDH)/complex II inactivation drives cell transformation and growth, SDH/complex II deficiency in T cells caused proliferation and survival defects when the TCA cycle was truncated, blocking carbon flux to support nucleoside biosynthesis. Replenishing the intracellular nucleoside pool partially relieved the dependence of T cells on SDH/complex II for proliferation and survival. SDH deficiency induced a proinflammatory gene signature in T cells and promoted T helper 1 and T helper 17 lineage differentiation. An increasing succinate/α-KG ratio in SDH-deficient T cells promoted inflammation by changing the pattern of the transcriptional and chromatin accessibility signatures and consequentially increasing the expression of the transcription factor, PR domain zinc finger protein 1. Collectively, our studies revealed a role of SDH/complex II in allocating carbon resources for anabolic processes and epigenetic regulation in T cell proliferation and inflammation.

Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile.

TP53 is the most commonly mutated gene in cancer, and gain-of-function mutations have wide-ranging effects. Efforts to reactivate wild-type p53 function and inhibit mutant functions have been complicated by the variety of TP53 mutations. Identified from a screen, the NSC59984 compound has been shown to restore activity to mutant p53 in colorectal cancer cells. Here, we investigated its effects on esophageal adenocarcinoma cells with specific p53 hot-spot mutations. NSC59984 treatment of cells reactivated p53 transcriptional regulation, inducing mitochondrial intrinsic apoptosis. Analysis of its effects on cellular metabolism demonstrated increased utilization of the pentose phosphate pathway and inhibition of glycolysis at the fructose-1,6-bisphosphate to fructose 6-phosphate junction. Furthermore, treatment of cells with NSC59984 increased reactive oxygen species production and decreased glutathione levels; these effects were enhanced by the addition of buthionine sulfoximine and inhibited by N-acetyl cysteine. We found that the effects of NSC59984 were substantially greater in cells harboring the p53 R248W mutation. Overall, these findings demonstrate p53-dependent effects of NSC59984 on cellular metabolism, with increased activity in cells harboring the p53 R248W mutation. This research highlights the importance of defining the mutational status of a particular cancer to create a patient-centric strategy for the treatment of p53-driven cancers.

In vivo deep network tracing reveals phosphofructokinase-mediated coordination of biosynthetic pathway activity in the myocardium.

Glucose metabolism comprises numerous amphibolic metabolites that provide precursors for not only the synthesis of cellular building blocks but also for ATP production. In this study, we tested how phosphofructokinase-1 (PFK1) activity controls the fate of glucose-derived carbon in murine hearts in vivo. PFK1 activity was regulated by cardiac-specific overexpression of kinase- or phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase transgenes in mice (termed GlycoLo or GlycoHi mice, respectively). Dietary delivery of 13C6-glucose to these mice, followed by deep network metabolic tracing, revealed that low rates of PFK1 activity promote selective routing of glucose-derived carbon to the purine synthesis pathway to form 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Consistent with a mechanism of physical channeling, we found multimeric protein complexes that contained phosphoribosylaminoimidazole carboxylase (PAICS)-an enzyme important for AICAR biosynthesis, as well as chaperone proteins such as Hsp90 and other metabolic enzymes. We also observed that PFK1 influenced glucose-derived carbon deposition in glycogen, but did not affect hexosamine biosynthetic pathway activity. These studies demonstrate the utility of deep network tracing to identify metabolic channeling and changes in biosynthetic pathway activity in the heart in vivo and present new potential mechanisms by which metabolic branchpoint reactions modulate biosynthetic pathways.