Two parallel pathways connect glutamine metabolism and mTORC1 activity to regulate glutamoptosis

Glutamoptosis is the induction of apoptotic cell death as a consequence of the aberrant activation of glutaminolysis and mTORC1 signaling during nutritional imbalance in proliferating cells. The role of the bioenergetic sensor AMPK during glutamoptosis is not defined yet. Here, we show that AMPK reactivation blocks both the glutamine-dependent activation of mTORC1 and glutamoptosis in vitro and in vivo. We also show that glutamine is used for asparagine synthesis and the GABA shunt to produce ATP and to inhibit AMPK, independently of glutaminolysis. Overall, our results indicate that glutamine metabolism is connected with mTORC1 activation through two parallel pathways: an acute alpha-ketoglutarate-dependent pathway; and a secondary ATP/AMPK-dependent pathway. This dual metabolic connection between glutamine and mTORC1 must be considered for the future design of therapeutic strategies to prevent cell growth in diseases such as cancer.Unión Europea(PGC2018-096244- B-I00, SAF2016-75442-R

A Method for Unsupervised Semi-Quantification of Inmunohistochemical Staining with Beta Divergences

In many research laboratories, it is essential to determine the relative expression levels of some proteins of interest in tissue samples. The semi-quantitative scoring of a set of images consists of establishing a scale of scores ranging from zero or one to a maximum number set by the researcher and assigning a score to each image that should represent some predefined characteristic of the IHC staining, such as its intensity. However, manual scoring depends on the judgment of an observer and therefore exposes the assessment to a certain level of bias. In this work, we present a fully automatic and unsupervised method for comparative biomarker quantification in histopathological brightfield images. The method relies on a color separation method that discriminates between two chromogens expressed as brown and blue colors robustly, independent of color variation or biomarker expression level. For this purpose, we have adopted a two-stage stain separation approach in the optical density space. First, a preliminary separation is performed using a deconvolution method in which the color vectors of the stains are determined after an eigendecomposition of the data. Then, we adjust the separation using the non-negative matrix factorization method with beta divergences, initializing the algorithm with the matrices resulting from the previous step. After that, a feature vector of each image based on the intensity of the two chromogens is determined. Finally, the images are annotated using a systematically initialized k-means clustering algorithm with beta divergences. The method clearly defines the initial boundaries of the categories, although some flexibility is added. Experiments for the semi-quantitative scoring of images in five categories have been carried out by comparing the results with the scores of four expert researchers yielding accuracies that range between 76.60% and 94.58%. These results show that the proposed automatic scoring system, which is definable and reproducible, produces consistent results.FEDER / Junta de Andalucía-Consejería de Economía y Conocimiento US-1264994Fondo de Desarrollo (FEDER). Unión Europea PGC2018-096244-B-I00, SAF2016-75442-RMinisterio de Economía, Industria y Competitividad (MINECO). España TEC2017- 82807-

Autophagy induced by Helicobacter pylori infection is necessary for gastric cancer stem cell emergence

Background: The main cause of gastric cancer is the infection by the bacterium Helicobacter pylori which induces a chronic inflammation and an epithelial-to-mesenchymal transition (EMT) leading to the emergence of cells with cancer stem cell (CSC) properties. However, the underlying mechanisms have not been fully characterized. Moreover, H. pylori modulates the host cell autophagic process, but a few studies have investigated the role of this process in tumoral transformation. The aim of this study was to determine whether H. pylori-induced autophagy has a role in CSC emergence. Methods: Autophagic flux in response to H. pylori infection was characterized in AGS cell line expressing the tandem-tagged mCherry-GFP-LC3 protein and using a ratiometric flow cytometry analysis. Then, AGS and MKN45 cell lines were treated with bafilomycin or chloroquine, two pharmaceutical well-known inhibitors of autophagy, and different EMT and CSC characteristics were analyzed. Results: First, a co-expression of the gastric CSC marker CD44 and the autophagic marker LC3 in mice and human stomach tissues infected with H. pylori was observed. Then, we demonstrated in vitro that H. pylori was able to activate the autophagy process with a reduced autophagic flux. Finally, infected cells were treated with autophagy inhibitors, which reduced (i) appearance of mesenchymal phenotypes and migration ability related to EMT and (ii) CD44 expression as well as tumorsphere formation capacities reflecting CSC properties. Conclusion: In conclusion, all these data show that H. pylori-induced autophagy is implicated in gastric CSC emergence and could represent an interesting therapeutic target.This work was supported by the French foundation Ligue contre le Cancer (Pyrénées Atlantiques)

Two parallel pathways connect glutamine metabolism and mTORC1 activity to regulate glutamoptosis.

Glutamoptosis is the induction of apoptotic cell death as a consequence of the aberrant activation of glutaminolysis and mTORC1 signaling during nutritional imbalance in proliferating cells. The role of the bioenergetic sensor AMPK during glutamoptosis is not defined yet. Here, we show that AMPK reactivation blocks both the glutamine-dependent activation of mTORC1 and glutamoptosis in vitro and in vivo. We also show that glutamine is used for asparagine synthesis and the GABA shunt to produce ATP and to inhibit AMPK, independently of glutaminolysis. Overall, our results indicate that glutamine metabolism is connected with mTORC1 activation through two parallel pathways: an acute alpha-ketoglutarate-dependent pathway; and a secondary ATP/AMPK-dependent pathway. This dual metabolic connection between glutamine and mTORC1 must be considered for the future design of therapeutic strategies to prevent cell growth in diseases such as cancer.This work was supported by funds from the following institutions: Agencia Estatal de Investigación/European Regional Development Fund, European Union (PGC2018-096244- B-I00, SAF2016-75442-R), Ministry of Science, Innovation and Universities of Spain, Spanish National Research Council—CSIC, Institut National de la Santé et de la Recherche Médicale —INSERM, Université de Bordeaux, Fondation pour la Recherche Médicale, the Conseil Régional d’Aquitaine, SIRIC-BRIO, Fondation ARC, and Institut Européen de Chimie et Biologie. C.B. was recipient of fellowships from the Minister of Higher Education, Research and Innovation (France) and the Fondation ARC (France). We thank Prof. Patricia Boya (Centro de Investigaciones Biologicas, Madrid, Spain) for kindly providing with the ATG5+/+ and ATG5−/− MEFs. We thank Prof. Benoit Viollet (Institute Cochin, Paris, France) for kindly providing with the AMPK+/+ and AMPK−/− MEFs, and the CA-AMPK plasmid

Downregulation of Glutamine Synthetase, not glutaminolysis, is responsible for glutamine addiction in Notch1-driven acute lymphoblastic leukemia

The cellular receptor Notch1 is a central regulator of T-cell development, and as a consequence, Notch1 pathway appears upregulated in > 65% of the cases of T-cell acute lymphoblastic leukemia (T-ALL). However, strategies targeting Notch1 signaling render only modest results in the clinic due to treatment resistance and severe side effects. While many investigations reported the different aspects of tumor cell growth and leukemia progression controlled by Notch1, less is known regarding the modifications of cellular metabolism induced by Notch1 upregulation in T-ALL. Previously, glutaminolysis inhibition has been proposed to synergize with anti-Notch therapies in T-ALL models. In this work, we report that Notch1 upregulation in T-ALL induced a change in the metabolism of the important amino acid glutamine, preventing glutamine synthesis through the downregulation of glutamine synthetase (GS). Downregulation of GS was responsible for glutamine addiction in Notch1-driven T-ALL both in vitro and in vivo. Our results also confirmed an increase in glutaminolysis mediated by Notch1. Increased glutaminolysis resulted in the activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway, a central controller of cell growth. However, glutaminolysis did not play any role in Notch1-induced glutamine addiction. Finally, the combined treatment targeting mTORC1 and limiting glutamine availability had a synergistic effect to induce apoptosis and to prevent Notch1-driven leukemia progression. Our results placed glutamine limitation and mTORC1 inhibition as a potential therapy against Notch1-driven leukemia.This work was supported by funds from the followinginstitutions: Agencia Estatal de Investigacion/Euro-pean Regional Development Fund, European Union(PGC2018-096244-B-I00, SAF2016-75442-R), Ministryof Science, Innovation and Universities of Spain,Spanish National Research Council—CSIC, InstitutNational de la Sante et de la Recherche Medicale—INSERM, Ligue Contre le Cancer—Gironde, Univer-site de Bordeaux, Fondation pour la Recherche Medi-cale, the Conseil Regional d’Aquitaine, SIRIC-BRIO,Fondation ARC and Institut Europeen de Chimie etBiologie. MJN was supported by a bourse d’excellencede la Federation Wallonie-Bruxelles (WBI) and a post-doctoral fellowship from Fondation ARC. We thankVincent Pitard (Flow Cytometry Platform, Universitede Bordeaux, France) for technical assistance in flowcytometry experiments. We thank Diana Cabrera(Metabolomics Platform, CIC bioGUNE, Spain) fortechnical assistance in metabolomics analysi

Downregulation of Glutamine Synthetase, not glutaminolysis, is responsible for glutamine addiction in Notch1-driven acute lymphoblastic leukemia

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Caractérisation biochimique de la régulation de mTORC1 par le métabolisme de la glutamine

Glutamine is the most abundant amino acid in the blood of mammals and its metabolism is particularly important for tumour cell proliferation. Cancer cells metabolize glutamine mostly through glutaminolysis, a metabolic process catabolized by glutaminase (GLS) and glutamate dehydrogenase (GDH) that activates mTORC1 signalling. Together with AMPK, the mTORC1 pathway is a key regulator of cell growth and proliferation. The unbalanced activation of mTORC1 by glutaminolysis during amino-acid starvation leads to a non-canonical apoptotic cell death known as “glutamoptosis”. In this thesis project, we identified that the reactivation of AMPK prevented both mTORC1 activation and cell death during glutamoptosis both in vitro and in vivo; suggesting an active role of AMPK during this process. Surprisingly, the connection between glutamine and AMPK, mediated by ATP, did not involve the necessary participation of glutaminolysis. Rather, we demonstrated the crucial role of the asparagine synthetase (ASNS) and the GABA shunt for the production of ATP during glutamine sufficiency, necessary for the metabolic control of the AMPK/mTORC1 axis. Indeed, the complete inhibition of mTORC1 required both the inhibition of GLS and the ASNS. Hence, we propose a model by which glutamine metabolism regulates mTORC1 pathway through two independent branches involving glutaminolysis and ASNS/GABA shunt that should be considered for potential targeted therapies against cancer.La glutamine est l'acide aminé le plus abondant dans le sang des mammifères et son métabolisme est particulièrement important pour la prolifération des cellules tumorales. Les cellules cancéreuses métabolisent la glutamine principalement par la glutaminolyse, un processus métabolique catabolisé par la glutaminase (GLS) et la glutamate déshydrogénase (GDH) qui active la signalisation mTORC1. Avec l'AMPK, la voie mTORC1 est un régulateur clé de la croissance et de la prolifération cellulaire. L'activation déséquilibrée de mTORC1 par la glutaminolyse en absence d'acides aminés entraîne une mort cellulaire apoptotique non canonique appelée "glutamoptose". Dans ce projet de thèse, nous avons identifié que la réactivation de l'AMPK empêchait à la fois l'activation de mTORC1 et la mort cellulaire pendant la glutamoptose, in vitro et in vivo ; ce qui suggère un rôle actif de l'AMPK pendant ce processus. De façon surprenante, le lien entre la glutamine et l'AMPK, médié par l'ATP, n'a pas nécessité la participation de la glutaminolyse. Nous avons cependant démontré le rôle crucial de l'asparagine synthétase (ASNS) et du GABA shunt dans la production d'ATP en présence seulement de glutamine, qui s’est révélé nécessaire au contrôle métabolique de l'axe AMPK/mTORC1. En effet, l'inhibition complète de mTORC1 a nécessité à la fois l'inhibition de la GLS et de l'ASNS. Par conséquent, nous proposons un modèle par lequel le métabolisme de la glutamine régule la voie mTORC1 par deux branches indépendantes impliquant la glutaminolyse et l’ASNS/GABA shunt qui devrait être envisagé pour d'éventuelles thérapies ciblées contre le cancer

Caractérisation biochimique de la régulation de mTORC1 par le métabolisme de la glutamine

La glutamine est l'acide aminé le plus abondant dans le sang des mammifères et son métabolisme est particulièrement important pour la prolifération des cellules tumorales. Les cellules cancéreuses métabolisent la glutamine principalement par la glutaminolyse, un processus métabolique catabolisé par la glutaminase (GLS) et la glutamate déshydrogénase (GDH) qui active la signalisation mTORC1. Avec l'AMPK, la voie mTORC1 est un régulateur clé de la croissance et de la prolifération cellulaire. L'activation déséquilibrée de mTORC1 par la glutaminolyse en absence d'acides aminés entraîne une mort cellulaire apoptotique non canonique appelée "glutamoptose". Dans ce projet de thèse, nous avons identifié que la réactivation de l'AMPK empêchait à la fois l'activation de mTORC1 et la mort cellulaire pendant la glutamoptose, in vitro et in vivo ; ce qui suggère un rôle actif de l'AMPK pendant ce processus. De façon surprenante, le lien entre la glutamine et l'AMPK, médié par l'ATP, n'a pas nécessité la participation de la glutaminolyse. Nous avons cependant démontré le rôle crucial de l'asparagine synthétase (ASNS) et du GABA shunt dans la production d'ATP en présence seulement de glutamine, qui s’est révélé nécessaire au contrôle métabolique de l'axe AMPK/mTORC1. En effet, l'inhibition complète de mTORC1 a nécessité à la fois l'inhibition de la GLS et de l'ASNS. Par conséquent, nous proposons un modèle par lequel le métabolisme de la glutamine régule la voie mTORC1 par deux branches indépendantes impliquant la glutaminolyse et l’ASNS/GABA shunt qui devrait être envisagé pour d'éventuelles thérapies ciblées contre le cancer.Glutamine is the most abundant amino acid in the blood of mammals and its metabolism is particularly important for tumour cell proliferation. Cancer cells metabolize glutamine mostly through glutaminolysis, a metabolic process catabolized by glutaminase (GLS) and glutamate dehydrogenase (GDH) that activates mTORC1 signalling. Together with AMPK, the mTORC1 pathway is a key regulator of cell growth and proliferation. The unbalanced activation of mTORC1 by glutaminolysis during amino-acid starvation leads to a non-canonical apoptotic cell death known as “glutamoptosis”. In this thesis project, we identified that the reactivation of AMPK prevented both mTORC1 activation and cell death during glutamoptosis both in vitro and in vivo; suggesting an active role of AMPK during this process. Surprisingly, the connection between glutamine and AMPK, mediated by ATP, did not involve the necessary participation of glutaminolysis. Rather, we demonstrated the crucial role of the asparagine synthetase (ASNS) and the GABA shunt for the production of ATP during glutamine sufficiency, necessary for the metabolic control of the AMPK/mTORC1 axis. Indeed, the complete inhibition of mTORC1 required both the inhibition of GLS and the ASNS. Hence, we propose a model by which glutamine metabolism regulates mTORC1 pathway through two independent branches involving glutaminolysis and ASNS/GABA shunt that should be considered for potential targeted therapies against cancer

Biochemical characterization of mTORC1 regulation by glutamine metabolism

La glutamine est l'acide aminé le plus abondant dans le sang des mammifères et son métabolisme est particulièrement important pour la prolifération des cellules tumorales. Les cellules cancéreuses métabolisent la glutamine principalement par la glutaminolyse, un processus métabolique catabolisé par la glutaminase (GLS) et la glutamate déshydrogénase (GDH) qui active la signalisation mTORC1. Avec l'AMPK, la voie mTORC1 est un régulateur clé de la croissance et de la prolifération cellulaire. L'activation déséquilibrée de mTORC1 par la glutaminolyse en absence d'acides aminés entraîne une mort cellulaire apoptotique non canonique appelée "glutamoptose". Dans ce projet de thèse, nous avons identifié que la réactivation de l'AMPK empêchait à la fois l'activation de mTORC1 et la mort cellulaire pendant la glutamoptose, in vitro et in vivo ; ce qui suggère un rôle actif de l'AMPK pendant ce processus. De façon surprenante, le lien entre la glutamine et l'AMPK, médié par l'ATP, n'a pas nécessité la participation de la glutaminolyse. Nous avons cependant démontré le rôle crucial de l'asparagine synthétase (ASNS) et du GABA shunt dans la production d'ATP en présence seulement de glutamine, qui s’est révélé nécessaire au contrôle métabolique de l'axe AMPK/mTORC1. En effet, l'inhibition complète de mTORC1 a nécessité à la fois l'inhibition de la GLS et de l'ASNS. Par conséquent, nous proposons un modèle par lequel le métabolisme de la glutamine régule la voie mTORC1 par deux branches indépendantes impliquant la glutaminolyse et l’ASNS/GABA shunt qui devrait être envisagé pour d'éventuelles thérapies ciblées contre le cancer.Glutamine is the most abundant amino acid in the blood of mammals and its metabolism is particularly important for tumour cell proliferation. Cancer cells metabolize glutamine mostly through glutaminolysis, a metabolic process catabolized by glutaminase (GLS) and glutamate dehydrogenase (GDH) that activates mTORC1 signalling. Together with AMPK, the mTORC1 pathway is a key regulator of cell growth and proliferation. The unbalanced activation of mTORC1 by glutaminolysis during amino-acid starvation leads to a non-canonical apoptotic cell death known as “glutamoptosis”. In this thesis project, we identified that the reactivation of AMPK prevented both mTORC1 activation and cell death during glutamoptosis both in vitro and in vivo; suggesting an active role of AMPK during this process. Surprisingly, the connection between glutamine and AMPK, mediated by ATP, did not involve the necessary participation of glutaminolysis. Rather, we demonstrated the crucial role of the asparagine synthetase (ASNS) and the GABA shunt for the production of ATP during glutamine sufficiency, necessary for the metabolic control of the AMPK/mTORC1 axis. Indeed, the complete inhibition of mTORC1 required both the inhibition of GLS and the ASNS. Hence, we propose a model by which glutamine metabolism regulates mTORC1 pathway through two independent branches involving glutaminolysis and ASNS/GABA shunt that should be considered for potential targeted therapies against cancer

Caractérisation biochimique de la régulation de mTORC1 par le métabolisme de la glutamine

Glutamine is the most abundant amino acid in the blood of mammals and its metabolism is particularly important for tumour cell proliferation. Cancer cells metabolize glutamine mostly through glutaminolysis, a metabolic process catabolized by glutaminase (GLS) and glutamate dehydrogenase (GDH) that activates mTORC1 signalling. Together with AMPK, the mTORC1 pathway is a key regulator of cell growth and proliferation. The unbalanced activation of mTORC1 by glutaminolysis during amino-acid starvation leads to a non-canonical apoptotic cell death known as “glutamoptosis”. In this thesis project, we identified that the reactivation of AMPK prevented both mTORC1 activation and cell death during glutamoptosis both in vitro and in vivo; suggesting an active role of AMPK during this process. Surprisingly, the connection between glutamine and AMPK, mediated by ATP, did not involve the necessary participation of glutaminolysis. Rather, we demonstrated the crucial role of the asparagine synthetase (ASNS) and the GABA shunt for the production of ATP during glutamine sufficiency, necessary for the metabolic control of the AMPK/mTORC1 axis. Indeed, the complete inhibition of mTORC1 required both the inhibition of GLS and the ASNS. Hence, we propose a model by which glutamine metabolism regulates mTORC1 pathway through two independent branches involving glutaminolysis and ASNS/GABA shunt that should be considered for potential targeted therapies against cancer.La glutamine est l'acide aminé le plus abondant dans le sang des mammifères et son métabolisme est particulièrement important pour la prolifération des cellules tumorales. Les cellules cancéreuses métabolisent la glutamine principalement par la glutaminolyse, un processus métabolique catabolisé par la glutaminase (GLS) et la glutamate déshydrogénase (GDH) qui active la signalisation mTORC1. Avec l'AMPK, la voie mTORC1 est un régulateur clé de la croissance et de la prolifération cellulaire. L'activation déséquilibrée de mTORC1 par la glutaminolyse en absence d'acides aminés entraîne une mort cellulaire apoptotique non canonique appelée "glutamoptose". Dans ce projet de thèse, nous avons identifié que la réactivation de l'AMPK empêchait à la fois l'activation de mTORC1 et la mort cellulaire pendant la glutamoptose, in vitro et in vivo ; ce qui suggère un rôle actif de l'AMPK pendant ce processus. De façon surprenante, le lien entre la glutamine et l'AMPK, médié par l'ATP, n'a pas nécessité la participation de la glutaminolyse. Nous avons cependant démontré le rôle crucial de l'asparagine synthétase (ASNS) et du GABA shunt dans la production d'ATP en présence seulement de glutamine, qui s’est révélé nécessaire au contrôle métabolique de l'axe AMPK/mTORC1. En effet, l'inhibition complète de mTORC1 a nécessité à la fois l'inhibition de la GLS et de l'ASNS. Par conséquent, nous proposons un modèle par lequel le métabolisme de la glutamine régule la voie mTORC1 par deux branches indépendantes impliquant la glutaminolyse et l’ASNS/GABA shunt qui devrait être envisagé pour d'éventuelles thérapies ciblées contre le cancer