La biogénèse des ribosomes consiste à assembler les ARN ribosomiques (ARNr) et les protéines ribosomiques de la petite sous unité (RPSs) ou de la grande sous unité (RPLs) afin de former les sous unités 40S et 60S du ribosome. Ce processus est l’un des plus complexes des cellules dont il utilise une grande quantité des ressources. Un taux élevé de biogénèse des ribosomes est une caractéristique de la prolifération cellulaire dans les conditions physiologiques ou pathologiques. L’inhibition de la biogénèse des ribosomes active un checkpoint du cycle cellulaire qui induit un arrêt du cycle cellulaire, et selon le contexte, l’apoptose. L’activation de ce checkpoint est due au facteur suppresseur de tumeur p53 qui s’accumule lorsque la biogénèse des ribosomes est inhibée grâce à l’inhibition de son facteur de dégradation, l’ubiquitine ligase E3 MDM2. Cette inhibition de MDM2 se fait par la fixation d’un complexe formé par les protéines ribosomiques RPL11 et RPL5 et l’ARNr 5S. Des études ont montré le potentiel thérapeutique de l’activation de ce checkpoint dans des cancers caractérisés par une biogenèse ribosomique élevée. Par contre l’activation de p53 semble avoir un rôle pathologique dans les ribosomopathies, un ensemble de pathologies causées par un défaut dans la biogénèse des ribosomes comme l’anémie macrocytaire de Diamond-Blackfan (ABD). p53 est clairement impliqué dans les effets anti-prolifératifs de l’inhibition de la biogénèse des ribosomes, cependant de nombreuses évidences montrent l’existence de mécanismes indépendants de p53 qui affectent l’homéostasie cellulaire. On observe par exemple dans l’ABD, des mutations de RPL11/RPL5 dont la déplétion in-vitro n’induit pas p53. Mon travail de thèse a consisté à élucider les mécanismes mis en place par les cellules pour répondre à l’inhibition de la biogénèse des ribosomes, dans un modèle in-vitro de lignées cellulaires. Dans ces lignées, nous avons inhibé la biogénèse des ribosomes par déplétion des RPs de la grande ou de la petite sous unité, indépendamment de l’induction ou pas de p53, à savoir, RPs6, RPL7a et RPL11. Nous avons mis en évidence des liens entre l’inhibition de la biogénèse des ribosomes et l’homéostasie du réticulum endoplasmique, ou la régulation de l’expression de gènes du métabolisme tels que l’enzyme oncogénique PHGDH. / Ribosome biogenesis is the process that leads to the assembly of ribosomal RNA (rRNA) and ribosomal proteins of the small (RPS) or the large (RPL) subunit into ribosomal 40S and 60S subunits. This is a highly complex process in the cells which uses a large amount of energy and resources. High rate of ribosome biogenesis is a trait of cell proliferation in physiological or pathogenic conditions. Inhibition of ribosome biogenesis activates a cell cycle checkpoint which induces a cell cycle arrest, and apoptosis. Activation of this checkpoint is due to the inhibition of ubiquitin ligase E3 MDM2, which does not anymore address the tumor suppressor factor p53 to proteasome. The p53 tumor suppressor factor then accumulates in cells and blocks the cell cycle progression. The inhibition of MDM2 is caused by the binding of a complex formed by RPL11, RPL5 and rRNA 5S. Few studies reveal that activation of this checkpoint has a therapeutic effect on cancer cells characterized by high rate of ribosome biogenesis. However, p53 activation seems to have pathogenic effects in ribosomopathies, a set of disorders characterized by ribosome biogenesis impairment, like Diamond-Balckfan macrocytic anemia (DBA). It is clear that p53 has anti-proliferative effects when ribosome biogenesis is inhibited, but evidences show that p53independants mechanisms also exist. In DBA for example, mutations in RPL5 and RPL11 that do not lead to p53 activation are observed. The goal of this study was to investigate the cellular mechanisms induced in response to inhibition of ribosome biogenesis. These investigations have been performed in an in-vitro system of cell lines. In those cell lines, ribosome biogenesis has been inhibited by depletion of RPs of the 40S or 60S ribosomal independently of p53 status. We brought out links between inhibition of ribosome biogenesis and endoplasmic reticulum homeostasis, or metabolic genes expression regulation like oncogene PHGDH.

Les patients atteints de la MA souffrent d'altérations métaboliques et synaptiques précoces. Via la glycolyse et le cycle de Krebs, le métabolisme du glucose permet la production d'ATP, essentielle à l'activité et la plasticité synaptique. Contrairement aux neurones, les astrocytes utilisent majoritairement la glycolyse pour métaboliser le glucose. En plus de la production d'énergie, la glycolyse fournit les précurseurs indispensables à la synthèse de biomolécules comme la L-sérine. Cet acide aminé est produit à partir du glucose par la déviation du 3-phosphoglycérate (3PG), un intermédiaire glycolytique, via l'enzyme 3-phosphoglycérate déshydrogénase (PHGDH), exprimée spécifiquement dans les astrocytes. La L-sérine est le précurseur de la D-sérine, le principal co-agoniste des NMDAR nécessaires à l'activité et la plasticité synaptique.Nous avons utilisé des souris 3xTg-AD, un modèle développant une MA progressive, afin d'étudier si une altération de la production de L-/D-sérine pouvait contribuer à des déficits synaptiques.A 6 mois, lorsque les souris 3xTg-AD ne possèdent pas encore de plaques amyloïdes dans l'hippocampe, nous avons observé in vivo une diminution du métabolisme du glucose, de la concentration de L-sérine et des déficits synaptiques (LTP). L'expression locale de la PHGDH est aussi altérée. L'application de D-sérine restaure complètement les déficits de LTP chez les souris 3xTg-AD.Ces données supportent l'hypothèse qu'un déficit de production de L-sérine par les astrocytes médié par une diminution du flux glycolytique serait responsable de l'altération synaptique observée dans l'hippocampe des souris 3xTg-AD. / An early alteration of both cerebral glucose metabolism and synaptic activity has been consistently described in Alzheimer's disease (AD) patients. Metabolism of glucose via glycolysis and the citric acid cycle produces ATP that is essential for synaptic activity and plasticity. In the brain, glucose is predominantly processed glycolytically into astrocytes and not by neurons. Beyond ATP production, a major function of aerobic glycolysis is to provide precursors to support macromolecular synthesis. L-serine, generated from glucose through diversion of the glycolytic intermediate 3-phosphoglycerate (3PG) into the phosphorylated pathway, is only produced in astrocytes by 3-phosphoglycerate dehydrogenase (PHGDH), selectively expressed in those glial cells. L-serine is the precursor of D-serine, the main co-agonist of synaptic NMDAR, required for synaptic activity and plasticity. We used 3xTg-AD mice, which develop a progressive pathology, to investigate whether a defective production of L-/D-serine contributes to early synaptic deficits in AD. We found that 3xTg-AD mice display early in vivo alterations of glucose metabolism, synaptic deficits (LTP) in the CA1 region and also lower concentration of L-serine. The local expression of PHGDH was significantly altered. Exogenous D-serine completely rescued LTP in 3xTg-AD mice. These data support the hypothesis that a deficit of L-serine synthesis by astrocytes likely mediated by a decreased glycolytic flux may be responsible for the synaptic alteration mediated by NMDAR in the hippocampus of 3xTg-AD mice.

Myc transcriptionally regulates genes involved in processes such as cell proliferation, metabolism, differentiation, and angiogenesis.  MYC expression is deregulated in many types of human cancer; therefore discovering the mechanisms behind MYCs role in tumorigenesis is essential.  In this dissertation, I have focused on several Myc target genes, Spermidine synthase (Srm); Lactate dehydrogenase (Ldh); 3-phosphoglycerate dehydrogenase (Phgdh); Serine hydroxymethyltransferase (SHMT) 1 and 2; and Pim-3 (a member of the Pim family of serine/threonine kinases).  These enzymes play a role in various functions: Spermidine synthase (polyamine synthesis); Lactate dehydrogenase (glycolysis); Phgdh and Shmt (serine metabolism); and Pim-3 (cell signaling).  In order to elucidate the impact Myc over-expression has on metabolism in tumorigenesis, we use human cell lines, and transgenic mice as well as cell lines and tissues derived from these mice.  The impact of inhibition of these target genes on Myc-driven tumorigenesis was done by genetically inhibiting the target gene (using RNAi or mouse models) or inhibiting the protein with a chemical inhibitor.  Investigating these Myc target genes will help determine if inhibition of Myc target genes is a viable approach for chemotherapeutics, and under what conditions this inhibition may be the most valuable.  In paper I, we examine SRM; a highly expressed enzyme in the polyamine synthesis pathway that converts putrescine to spermidine, and is important for actively growing cells.  Genetic inhibition via RNAi against Srm, or chemical inhibition of Srm, resulted in decreased proliferation of B-cell tumor lines from transgenic mice in vitro.  In vivo treatment of λ-Myc transgenic mice with a chemical SRM inhibitor exhibited a significant chemopreventative effect on tumor formation. These results support previous findings that inhibition of polyamine synthesis pathway enzymes has a place in cancer therapy.  Many Myc target genes have been suggested as attractive targets in battling Myc-driven tumorigenesis.  Surprisingly in paper II, when we analyzed the inhibition of other Myc target genes, such as Ldh, Shmt, and Phgdh, we found that inhibition of these genes did not inhibit Myc-driven tumorigenesis to any significant degree. However, inhibition of Ldh, Phgdh and Shmt2 had a notable effect on in vitro Ras-driven transformation.  These findings suggest that chemotherapeutic inhibition of metabolic genes such as Ldh, Phgdh and Shmt2 may be effective in genetically defined settings, keeping in mind the oncogenic lesion behind the tumor.  The Pim kinase family consists of three serine/threonine kinases, Pim1-3.  In paper III, we found that Pim-3 is a direct Myc target gene and that Pim-3 expression is high in Burkitt Lymphoma samples taken from human patients, as well as spontaneously arising lymphomas from Myc transgenic mice. We also found that inhibition of Pim-3 using a pan-Pim kinase inhibitor, Pimi, in these spontaneously arising Myc lymphomas resulted in caspase independent cell death.  These results indicate that Pim kinase inhibition may be a potential chemotherapeutic strategy in human lymphomas that rely on Pim-3 kinase expression.