Inhibiting phosphoglycerate dehydrogenase counteracts chemotherapeutic efficacy against MYCN‐amplified neuroblastoma

Here we sought metabolic alterations specifically associated with MYCN amplification as nodes to indirectly target the MYCN oncogene. Liquid chromatography-mass spectrometry-based proteomics identified seven proteins consistently correlated with MYCN in proteomes from 49 neuroblastoma biopsies and 13 cell lines. Among these was phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in de novo serine synthesis. MYCN associated with two regions in the PHGDH promoter, supporting transcriptional PHGDH regulation by MYCN. Pulsed stable isotope-resolved metabolomics utilizing C-13-glucose labeling demonstrated higher de novo serine synthesis in MYCN-amplified cells compared to cells with diploid MYCN. An independence of MYCN-amplified cells from exogenous serine and glycine was demonstrated by serine and glycine starvation, which attenuated nucleotide pools and proliferation only in cells with diploid MYCN but did not diminish these endpoints in MYCN-amplified cells. Proliferation was attenuated in MYCN-amplified cells by CRISPR/Cas9-mediated PHGDH knockout or treatment with PHGDH small molecule inhibitors without affecting cell viability. PHGDH inhibitors administered as single-agent therapy to NOG mice harboring patient-derived MYCN-amplified neuroblastoma xenografts slowed tumor growth. However, combining a PHGDH inhibitor with the standard-of-care chemotherapy drug, cisplatin, revealed antagonism of chemotherapy efficacy in vivo. Emergence of chemotherapy resistance was confirmed in the genetic PHGDH knockout model in vitro. Altogether, PHGDH knockout or inhibition by small molecules consistently slows proliferation, but stops short of killing the cells, which then establish resistance to classical chemotherapy. Although PHGDH inhibition with small molecules has produced encouraging results in other preclinical cancer models, this approach has limited attractiveness for patients with neuroblastoma

Fructose-driven glycolysis supports anoxia resistance in the naked mole-rat

The African naked mole-rat’s ($\textit{Heterocephalus glaber}$) social and subterranean lifestyle generates a hypoxic niche. Under experimental conditions, naked mole-rats tolerate hours of extreme hypoxia and survive 18 minutes of total oxygen deprivation (anoxia) without apparent injury. During anoxia, the naked mole-rat switches to anaerobic metabolism fueled by fructose, which is actively accumulated and metabolized to lactate in the brain. Global expression of the GLUT5 fructose transporter and high levels of ketohexokinase were identified as molecular signatures of fructose metabolism. Fructose-driven glycolytic respiration in naked mole-rat tissues avoids feedback inhibition of glycolysis via phosphofructokinase, supporting viability. The metabolic rewiring of glycolysis can circumvent the normally lethal effects of oxygen deprivation, a mechanism that could be harnessed to minimize hypoxic damage in human disease.Work was supported aEuropean Research Council (294678), the Deutsche Forschungsgemeinschaft SFB 665 and Go865/9-1, NSF (grant #0744979 ), NIH (grants HL71626 and HL606

Experimental and mathematical analysis of the central carbon metabolism in cancer and stem cells

Die Entstehung von Tumoren und damit einhergehenden Veränderungen wurden insbesondere im letzten Jahrzehnt kontrovers diskutiert. Bisher standen nur wenige Datensätze mit ausreichender Datendichte zur Verfügung um eine umfassende Untersuchung der Regulation des Stoffwechsels durchzuführen. Die in dieser Arbeit zusammengefassten Projekte adressieren verschiedene Aspekte der Stoffwechselregulation und beschreiben die Verknüpfung von Zellkulturexperimenten mit innovativen Hochdurchsatz-Technologien, komplexer Datenanalyse und Computer-basierter Modellierung zur Bestimmung der Stoffwechselflüsse in eukaryotischen Zellen. Die Kombination von GC-MS und LC-MS basierten Technologien ermöglicht die quantitative Analyse des zentralen Kohlenstoffwechsels. Markierungsexperimente mit stabilen Isotopen (pSIRM) erlauben die dynamische Analyse der Stoffwechselaktivität. In verschiedenen Projekten wurden das Proteom und Metabolom von Krebszellen, humanen Stammzellen (hESCs), induzierten pluripotenten Stammzellen (iPS) und deren dazugehörigen differenzierten Vorläufer- oder Nachfolgerzellen bestimmt. Die multivariate, statistische Analyse der Daten ermöglichte die Differenzierung verschiedener Zelltypen basierend auf der Kombination aller quantitativ bestimmten Daten. Quantitative Bestimmungen der Poolgrössen, Isotopeninkorporationen, sowie der extrazellulären Raten in neuronalen, pluripotenten Vorläuferzellen (Luhmes d0) und Neuronen (Luhmes d6) ermöglichte die Bestimmung der Stoffwechselflusskarte beider Zelltypen unter Verwendung der instationären metabolischenen Flussanalyse (INST-MFA). Die Etablierung einer Qualitätskontrolle für GC-MS basierte Daten (MTXQC), sowie die Zuordnung der GC-MS Fragmente zur Molekülstruktur, ermöglichten den Ausbau des Netzwerkes des zentralen Kohlenstoffwechsels und die Implementierung der Daten für die metabolische Flussanalyse.Metabolic reprogramming of the central carbon metabolism (CCM) is highly debated during the last decade. It describes the rearrangement of nutrient consumption for providing energy and building blocks for cellular proliferation and maintenance. So far, only sparse data are available for an in-depth analysis of metabolic reprogramming events. The herein summarised projects address metabolic programming from different perspectives and show the implementation of cell culture experiments, cutting-edge high-throughput technologies, bioinformatics, and computational modelling into one workflow providing the determination of metabolic flux maps of mammalian cells. The combination of GC-MS and LC-MS-based methodologies enable the quantitative analysis of proteins and metabolites of the CCM. Pulsed stable isotope-resolved metabolomics (pSIRM) experiments allow monitoring the fate of nutrients within the network of the CCM. The time-dependent and position-specific incorporation of carbon-13 leads to an indirect measurement of the metabolic flux, the only one functional readout of a cell. High-throughput technologies were applied in four projects to gain insights in metabolic reprogramming in cancer cell lines, human embryonic stem cells (hESCs), induced pluripotent stem (iPS) cells and their derived fibroblasts. A global principal component analysis demonstrated the discrimination of phenotypes by different classes of quantitative data. The comparison of metabolic and protein levels confirms the presence of the Warburg effect in both cell types. Though, the executing enzymes vary regarding their isoenzyme identity and expression levels. Methodological improvements provided the implementation of GC-MS derived data for INST-MFA. The mapping of GC-MS derived fragments to the molecule structure enables an extension of the CCM network. Robustness of the input data has been improved by the development of a R-scripting based quality control tool (MTXQC)

Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2-deoxyglucose using pulsed stable isotope-resolved metabolomics

Background Cellular metabolism is highly dynamic and continuously adjusts to the physiological program of the cell. The regulation of metabolism appears at all biological levels: (post-) transcriptional, (post-) translational, and allosteric. This regulatory information is expressed in the metabolome, but in a complex manner. To decode such complex information, new methods are needed in order to facilitate dynamic metabolic characterization at high resolution. Results Here, we describe pulsed stable isotope-resolved metabolomics (pSIRM) as a tool for the dynamic metabolic characterization of cellular metabolism. We have adapted gas chromatography-coupled mass spectrometric methods for metabolomic profiling and stable isotope-resolved metabolomics. In addition, we have improved robustness and reproducibility and implemented a strategy for the absolute quantification of metabolites. Conclusions By way of examples, we have applied this methodology to characterize central carbon metabolism of a panel of cancer cell lines and to determine the mode of metabolic inhibition of glycolytic inhibitors in times ranging from minutes to hours. Using pSIRM, we observed that 2-deoxyglucose is a metabolic inhibitor, but does not directly act on the glycolytic cascade

Stage-specific metabolic features of differentiating neurons: Implications for toxicant sensitivity

Developmental neurotoxicity (DNT) may be induced when chemicals disturb a key neurodevelopmental process, and many tests focus on this type of toxicity. Alternatively, DNT may occur when chemicals are cytotoxic only during a specific neurodevelopmental stage. The toxicant sensitivity is affected by the expression of toxicant targets and by resilience factors. Although cellular metabolism plays an important role, little is known how it changes during human neurogenesis, and how potential alterations affect toxicant sensitivity of mature vs. immature neurons. We used immature (d0) and mature (d6) LUHMES cells (dopaminergic human neurons) to provide initial answers to these questions. Transcriptome profiling and characterization of energy metabolism suggested a switch from predominantly glycolytic energy generation to a more pronounced contribution of the tricarboxylic acid cycle (TCA) during neuronal maturation. Therefore, we used pulsed stable isotope-resolved metabolomics (pSIRM) to determine intracellular metabolite pool sizes (concentrations), and isotopically non-stationary 13C-metabolic flux analysis (INST 13C-MFA) to calculate metabolic fluxes. We found that d0 cells mainly use glutamine to fuel the TCA. Furthermore, they rely on extracellular pyruvate to allow continuous growth. This metabolic situation does not allow for mitochondrial or glycolytic spare capacity, i.e. the ability to adapt energy generation to altered needs. Accordingly, neuronal precursor cells displayed a higher sensitivity to several mitochondrial toxicants than mature neurons differentiated from them. In summary, this study shows that precursor cells lose their glutamine dependency during differentiation while they gain flexibility of energy generation and thereby increase their resistance to low concentrations of mitochondrial toxicants

Stage-specific metabolic features of differentiating neurons: Implications for toxicant sensitivity

Developmental neurotoxicity (DNT) may be induced when chemicals disturb a key neurodevelopmental process, and many tests focus on this type of toxicity. Alternatively, DNT may occur when chemicals are cytotoxic only during a specific neurodevelopmental stage. The toxicant sensitivity is affected by the expression of toxicant targets and by resilience factors. Although cellular metabolism plays an important role, little is known how it changes during human neurogenesis, and how potential alterations affect toxicant sensitivity of mature vs. immature neurons. We used immature (d0) and mature (d6) LUHMES cells (dopaminergic human neurons) to provide initial answers to these questions. Transcriptome profiling and characterization of energy metabolism suggested a switch from predominantly glycolytic energy generation to a more pronounced contribution of the tricarboxylic acid cycle (TCA) during neuronal maturation. Therefore, we used pulsed stable isotope-resolved metabolomics (pSIRM) to determine intracellular metabolite pool sizes (concentrations), and isotopically non-stationary 13C-metabolic flux analysis (INST 13C-MFA) to calculate metabolic fluxes. We found that d0 cells mainly use glutamine to fuel the TCA. Furthermore, they rely on extracellular pyruvate to allow continuous growth. This metabolic situation does not allow for mitochondrial or glycolytic spare capacity, i.e. the ability to adapt energy generation to altered needs. Accordingly, neuronal precursor cells displayed a higher sensitivity to several mitochondrial toxicants than mature neurons differentiated from them. In summary, this study shows that precursor cells lose their glutamine dependency during differentiation while they gain flexibility of energy generation and thereby increase their resistance to low concentrations of mitochondrial toxicants

The B-cell receptor controls fitness of MYC-driven lymphoma cells via GSK38\u9f inhibition

Similar to resting mature B cells, where the B-cell antigen receptor (BCR) controls cellular survival, surface BCR expression is conserved in most mature B-cell lymphomas. The identification of activating BCR mutations and the growth disadvantage upon BCR knockdown of cells of certain lymphoma entities has led to the view that BCR signalling is required for tumour cell survival. Consequently, the BCR signalling machinery has become an established target in the therapy of B-cell malignancies. Here we study the effects of BCR ablation on MYC-driven mouse B-cell lymphomas and compare them with observations in human Burkitt lymphoma. Whereas BCR ablation does not, per se, significantly affect lymphoma growth, BCR-negative (BCR(-)) tumour cells rapidly disappear in the presence of their BCR-expressing (BCR(+)) counterparts in vitro and in vivo. This requires neither cellular contact nor factors released by BCR(+) tumour cells. Instead, BCR loss induces the rewiring of central carbon metabolism, increasing the sensitivity of receptor-less lymphoma cells to nutrient restriction. The BCR attenuates glycogen synthase kinase 3 beta (GSK3β) activity to support MYC-controlled gene expression. BCR(-) tumour cells exhibit increased GSK3β activity and are rescued from their competitive growth disadvantage by GSK3β inhibition. BCR(-) lymphoma variants that restore competitive fitness normalize GSK3β activity after constitutive activation of the MAPK pathway, commonly through Ras mutations. Similarly, in Burkitt lymphoma, activating RAS mutations may propagate immunoglobulin-crippled tumour cells, which usually represent a minority of the tumour bulk. Thus, while BCR expression enhances lymphoma cell fitness, BCR-targeted therapies may profit from combinations with drugs targeting BCR(-) tumour cells