Existing treatments for mantle cell lymphoma (MCL) are non-curative, demonstrating a need for a refined treatment approach. Recent clinical trials have shown promising results with the use of mammalian target of rapamycin inhibitors. I hypothesize that the anti-tumor effect of mTOR inhibitors in mantle cell lymphoma is mediated by an increase in manganese superoxide dismutase (MnSOD) protein expression and accumulation of hydrogen peroxide (H₂O₂). Findings indicate that the rapamycin-induced cytostatic effect is characterized by increased levels of MnSOD and H₂O₂, and is necessary for the full growth inhibitory effect of rapamycin. Furthermore, over-expression of MnSOD elevated the level of H₂O₂ and increased sensitivity to MnSOD. Treatment with rapamycin resulted in a loss of serine 473 phosphorylation of AKT and increased levels of MnSOD were found to be due to inhibition of the mTORC2 complex. These results are the first to suggest that long term treatment of MCL cells with rapamycin inhibits the mTORC2 complex. By understanding the key signaling molecules and affected pathways in the anti-tumor effects of mTOR inhibitors, we may be able to identify additional predictive markers to improve the therapeutic value, or study drug combinations that will enhance the effect of ROSinduced cytotoxicity. A retrospective study utilizing samples from lymphoma patients receiving standard anthracycline-based therapies, identified single nucleotide polymorphisms in oxidative stressrelated genes associated with survival. Individuals carrying minor allele SNPs in myeloperoxidase (MPO) and an aldo-keto reductase (AKR1C3) were found to be associated with shorter time to disease progression and death. This data suggest that some patients may benefit from a different therapy than the current standard of care and that regulation of the redox environment plays a role in aggressive lymphoma treatment response.

Plusieurs études ont suggéré une implication des glycogène synthase kinases 3 (GSK3) dans la carcinogenèse, notamment du pancréas. Des études ont rapporté des résultats contradictoires quant à l’impact des GSK3 sur la survie cellulaire. Au niveau du pancréas, il a été observé que l’inhibition des GSK3 inhibe la croissance entre autres via la régulation de la voie JNK ou NFkB. Les inhibiteurs des GSK3 sont présentement à l’étude comme traitement de différentes pathologies, notamment pour le cancer pancréatique. Une meilleure compréhension des voies de signalisation régulées par les GSK3 sera donc nécessaire. Nous avons entrepris ces travaux afin de mieux comprendre les mécanismes impliqués dans la régulation de la survie des cellules pancréatiques tumorales par les GSK3. Nous avons démontré que l’inhibition des GSK3 induit l’apoptose et l’autophagie dans les cellules pancréatiques tumorales humaines. L’inhibition des GSK3 stimule l’autophagie autant dans les cellules pancréatiques tumorales que non tumorales, alors que l’apoptose est induite spécifiquement dans les cellules tumorales. Contrairement à l’apoptose, l’autophagie est induite indépendamment de la voie JNK-cJUN suite à l’inhibition des GSK3. Nos résultats démontrent que l’inhibition des GSK3 mène à l’inactivation de la voie mTORC1 qui pourrait contribuer à l’induction de l’autophagie. D’autre part, nos travaux ont démontré pour la première fois que les GSK3 régulent le facteur de transcription EB (TFEB) dans les cellules pancréatiques tumorales. En effet, l’inhibition des GSK3 entraîne la déphosphorylation de TFEB, notamment sur la Ser211, la dissociation des 14-3- 3 et sa translocation nucléaire. Nos résultats suggèrent que la régulation de TFEB par les GSK3 impliquerait des Ser/Thr phosphatases et pourrait être indépendante de l’activité mTORC1. L’inhibition de l’autophagie ou la déplétion de l’expression de TFEB sensibilise les cellules pancréatiques tumorales à l’apoptose induite suite à l’inhibition des GSK3 suggérant un rôle pro-survie de l’autophagie et de TFEB dans ces cellules. Enfin, l’inhibition des GSK3 semble mener à l’inhibition de la glycolyse qui contribuerait à l’induction de l’apoptose. En résumé, nos résultats démontrent que l’inhibition des GSK3 induit à la fois des signaux pro-apoptotiques et pro-survie suggérant que l’équilibre entre ces signaux dicterait l’impact des GSK3 sur la survie des cellules pancréatiques tumorales humaines.

Quelque 30 % de la population neuronale du cortex mammalien est composée d’une population très hétérogène d’interneurones GABAergiques. Ces interneurones diffèrent quant à leur morphologie, leur expression génique, leurs propriétés électrophysiologiques et leurs cibles subcellulaires, formant une riche diversité. Après leur naissance dans les éminences ganglioniques, ces cellules migrent vers les différentes couches corticales. Les interneurones GABAergiques corticaux exprimant la parvalbumin (PV), lesquels constituent le sous-type majeur des interneurones GABAergiques, ciblent spécifiquement le soma et les dendrites proximales des neurones principaux et des neurones PV+. Ces interneurones sont nommés cellules à panier (Basket Cells –BCs) en raison de la complexité morphologique de leur axone. La maturation de la connectivité distincte des BCs PV+, caractérisée par une augmentation de la complexité de l’axone et de la densité synaptique, se déroule graduellement chez la souris juvénile. Des travaux précédents ont commencé à élucider les mécanismes contrôlant ce processus de maturation, identifiant des facteurs génétiques, l’activité neuronale ainsi que l’expérience sensorielle. Cette augmentation marquante de la complexité axonale et de la synaptogénèse durant cette phase de maturation suggère la nécessité d’une synthèse de protéines élevée. La voie de signalisation de la cible mécanistique de la rapamycine (Mechanistic Target Of Rapamycin -mTOR) a été impliquée dans le contrôle de plusieurs aspects neurodéveloppementaux en régulant la synthèse de protéines. Des mutations des régulateurs Tsc1 et Tsc2 du complexe mTOR1 causent la sclérose tubéreuse (TSC) chez l’humain. La majorité des patients TSC développent des problèmes neurologiques incluant des crises épileptiques, des retards mentaux et l’autisme. D’études récentes ont investigué le rôle de la dérégulation de la voie de signalisation de mTOR dans les neurones corticaux excitateurs. Toutefois, son rôle dans le développement des interneurones GABAergiques corticaux et la contribution spécifique de ces interneurones GABAergiques altérés dans les manifestations de la maladie demeurent largement inconnus. Ici, nous avons investigué si et comment l’ablation du gène Tsc1 perturbe le développement de la connectivité GABAergique, autant in vitro que in vivo. Pour investiguer le rôle de l’activation de mTORC1 dans le développement d’une BC unique, nous avons délété le gène Tsc1 en transfectant CRE-GFP dirigé par un promoteur spécifique aux BCs dans des cultures organotypiques provenant de souris Tsc1lox. Le knockdown in vitro de Tsc1 a causé une augmentation précoce de la densité des boutons et des embranchements terminaux formés par les BCs mutantes, augmentation renversée par le traitement à la rapamycine. Ces données suggèrent que l’hyperactivation de la voie de signalisation de mTOR affecte le rythme de la maturation des synapses des BCs. Pour investiguer le rôle de mTORC1 dans les interneurones GABAergiques in vivo, nous avons croisé les souris Tsc1lox avec les souris Nkx2.1-Cre et PV-Cre. À P18, les souris Tg(Nkx2.1-Cre);Tsc1flox/flox ont montré une hyperactivation de mTORC1 et une hypertrophie somatique des BCs de même qu’une augmentation de l’expression de PV dans la région périsomatique des neurones pyramidaux. Au contraire, à P45 nous avons découvert une réduction de la densité des punctas périsomatiques PV-gephyrin (un marqueur post-synaptique GABAergique). L’étude de la morphologie des BCs en cultures organotypiques provenant du knock-out conditionnel Nkx2.1-Cre a confirmé l’augmentation initiale du rythme de maturation, lequel s’effondre ensuite aux étapes développementales tardives. De plus, les souris Tg(Nkx2.1Cre);Tsc1flox/flox montrent des déficits dans la mémoire de travail et le comportement social et ce d’une façon dose-dépendante. En somme, ces résultats suggèrent que l’activation contrôlée de mTOR régule le déroulement de la maturation et la maintenance des synapses des BCs. Des dysfonctions de la neurotransmission GABAergique ont été impliquées dans des maladies telles que l’épilepsie et chez certains patients, elles sont associées avec des mutations du récepteur GABAA. De quelle façon ces mutations affectent le processus de maturation des BCs demeuret toutefois inconnu. Pour adresser cette question, nous avons utilisé la stratégie Cre-lox pour déléter le gène GABRA1, codant pour la sous-unité alpha-1 du récepteur GABAA dans une unique BC en culture organotypique. La perte de GABRA1 réduit l’étendue du champ d’innervation des BCs, suggérant que des variations dans les entrées inhibitrices en raison de l’absence de la sous-unité GABAAR α1 peuvent affecter le développement des BCs. La surexpression des sous-unités GABAAR α1 contenant des mutations identifiées chez des patients épileptiques ont montré des effets similaires en termes d’étendue du champ d’innervation des BCs. Pour approfondir, nous avons investigué les effets de ces mutations identifiées chez l’humain dans le développement des épines des neurones pyramidaux, lesquelles sont l’endroit privilégié pour la formation des synapses excitatrices. Somme toute, ces données montrent pour la première fois que différentes mutations de GABRA1 associées à des syndromes épileptiques peuvent affecter les épines dendritiques et la formation des boutons GABAergiques d’une façon mutation-spécifique. / About 30% of the total neuronal population in the mammalian cortex is composed by a very heterogeneous population of GABAergic interneurons. These interneurons differ in their morphology, gene expression, electrophysiological properties and subcellular targets, thus establishing a rich diversity. After birth in the ganglionic eminences these cells migrate to distinct cortical layers. Parvalbumin (PV) expressing cortical GABAergic cells which constitute the major GABAergic subtype specifically targets the soma and proximal dendrites of principal neurons and PV+ cells. These cells are often referred as Basket cells (BCs) because of the intricate morphological complexity of their axons. The maturation of the distinct connectivity of PV+ BCs, characterized by an increase of axon complexity and synapse density, occurs gradually in juvenile mice. Previous studies started to elucidate the mechanisms controlling this maturation process, including genetic factors, neuronal activity and sensory experiences. The striking increase in axonal complexity and synaptogenesis occurring during the maturation phase suggests the requirement for elevated proteins synthesis in order to sustain the developmental process. The Mechanistic Target Of Rapamycin (mTOR) pathway has been implicated in controlling several aspects of neurodevelopment by regulating protein synthesis. Mutations in the regulatory components Tsc1 and Tsc2 of mTOR-Complex1 (mTORC1) cause the disease Tuberous Sclerosis (TSC) in humans. The majority of TSC patients develop neurological problems including seizures, mental retardation and autism. Recent studies investigated the role of mTOR pathway dys-regulation in excitatory cortical cells, however its role in the development of cortical GABAergic interneurons and the specific contribution of altered GABAergic cells in disease manifestation remain largely unknown. Here, we investigated whether and how Tsc1 knockout perturbs GABAergic circuit development, both in vitro and in vivo. To investigate the role of mTORC1 activation in BC development, we knocked out Tsc1 expression, by transfecting Cre-GFP driven by a promoter specific for BCs in cortical organotypic cultures prepared from Tsc1lox mice. Tsc1 knockdown in vitro caused a precocious increase in bouton density and terminal branching formed by mutant BCs, which was reversed by Rapamycin treatment. These data suggest that mTOR pathway hyperactivation affects the timing of BC synapse maturation. To investigate the role of mTORC1 in GABAergic cells in vivo, we bred Tsc1lox mice with Nkx2.1-Cre and PV-Cre mice. At P18, Tg(Nkx2.1Cre),Tsc1flox/flox mice showed both mTORC1 hyperactivation and somatic hypertrophy in BCs along with increased expression of PV in the perisomatic region of pyramidal neurons. In contrast, by P45 we found a reduction of PV-gephyrin (post-synaptic GABAergic marker) perisomatic puncta density. Study of BC morphology in organotypic cultures from the Nkx2.1-Cre conditional knockout confirmed the occurrence of a faster maturation rate initially which however collapsed at later stages. Additionally Tg(Nkx2.1Cre),Tsc1flox/flox mice exhibit Tsc1 dose-dependent deficits in working memory and social behaviour. All together, these results suggest that controlled mTOR activation regulates both the time course and the maintenance of BC synapses. Dysfunction of GABAergic neurotransmission has been implicated in several disease states like epilepsy and in some patients it is associated with mutations in the GABAA receptor. How these mutations affect the BC cell maturation process remains largely unknown. To address this question, we used the Cre-lox strategy to knockout the endogenous GABRA1 gene coding for the GABAA-receptor alpha-1 subunit in single PV-expressing basket cells (BCs) in organotypic cultures. Cell-autonomous loss of GABRA1 reduced the extent of BC innervation field suggesting changes in inhibitory inputs caused by the absence of GABAAR α1 subunit may alter BC development. Over-expression of mutant GABAAR α1 subunits (found in patients diagnosed with epilepsy) show similar effects in terms of BC target coverage. Further studies involved the effect of these human mutations in the development of Pyramidal cell dendritic spines, which are the preferential site for excitatory synapse formation. Altogether, this data show for the first time that different GABRA1 mutations associated with genetic epilepsy syndromes can affect dendritic spine and GABAergic bouton formation in a mutation-specific manner.

iv Abstract Crosstalk of integrin and mTOR signalling is an essential process that monitors cellular interaction with extracellular matrix and transmits these inputs to cell growth signalling. Although adhesion status of the cell monitored by integrin signalling is clearly important for regulation of cellular growth, a little is known about the crosstalk of integrin and mTOR signalling. In this study, we employed two different approaches to describe and elucidate character of this crosstalk. p130Cas is an adaptor protein phosphorylated by Src kinase and focal adhesion kinase upon integrin ligand binding and implicated in cell adhesion, motility and survival in both Src-transformed and untransformed cells. Recently, p130Cas was also described in cellular pathology, mainly by its ability to stimulate cell invasion and metastasis. In this study, we described that p130Cas affects mTOR signalling in Src-transformed cells. Substrate domain of p130Cas was found to be indispensable for this effect and is also responsible for serum-induced activation of mTOR signalling. In addition, we prepared cell lines overexpressing various Rheb protein versions and characterized them in context of mTOR signalling, integrin signalling and cell cycle progression. Interestingly, a cell line overexpressing constitutively active...

Master of Science in Biomedical Sciences / Department of Anatomy and Physiology / Yongming Sang / Animal metabolic and immune systems integrate and inter-regulate to exert effective immune responses to distinct pathogens. The signaling pathway mediated by mechanistic target of rapamycin (mTOR) is critical in cellular metabolism and implicated in host antiviral responses. Recent studies highlight the significance of the mTOR signaling pathway in the interferon (IFN) response. Type I IFNs mediate host defense, particularly, against viral infections, and have myriad roles in antiviral innate and adaptive immunity. In addition to their well-known antiviral properties, type I IFNs also affect host metabolism. However, little is known about how animal type I IFN signaling coordinates immunometabolic reactions during antiviral defense. Therefore, understanding the interaction of mTOR signaling and the type I IFN system becomes increasingly important in potentiating antiviral immunity. Tissue macrophages (MФs) are a primary IFN producer during viral infection, and their polarization to different activation statuses is critical for regulation of immune and metabolic homeostasis. Using porcine reproductive and respiratory syndrome virus (PRRSV) as a model, we found that genes in the mTOR signaling pathway were regulated differently in PRRSV-infected porcine alveolar MФs at different activation statuses. Therefore we hypothesize that: 1) the mTOR signaling pathway involves host anti-PRRSV regulation; 2) mTOR signaling interacts with IFN signaling to modulate the antiviral response; and 3) different type I IFN subtypes (such as IFN-α1 and IFN-β) regulate mTOR signaling differently. We show that modulation of mTOR signaling regulated PRRSV infection in MARC-145 cells and porcine primary cells, in part, through regulating production and signaling of type I IFNs. In addition, expression and phosphorylation of two key components in the mTOR signaling pathway, AKT and p70 S6 kinase, were regulated by type I IFNs and PRRSV infection. Taken together, we determined that the mTOR signaling pathway, a key pathway in regulation of cell metabolism, also mediates the type I IFN response, a key immune response in PRRSV infection. Our findings reveal that the mTOR signaling pathway potentially has a bi-directional loop with the type I IFN system and implies that some components in the mTOR signaling pathway can serve as targets for augmentation of antiviral immunity and therapeutic designs.

The role of the immune system in periodontal disease has been well established. Individuals who smoke are more prone to developing periodontitis because of the excess plaque buildup and the immune system's attack of the bacteria on the gingiva. This study aims to examine the role of immunotherapy in the reversal of periodontal disease in individuals who smoke. Further research will not only assist with the reversal of periodontitis, but may also improve other debilitating co-morbid diseases including autoimmune diseases such as human immunodeficiency virus. To examine this question, a variety of research studies from various sources were examined. There are numerous factors, which contribute to the initiation and progression of periodontal disease. Use of nicotine products enhances the accumulation of plaque formation. Accumulation of large amounts of plaque and calculus assist in the progression of periodontal disease. Disease of the gingiva may lead numerous other debilitating diseases such as respiratory infections, Alzheimer's disease and unfavorable pregnancy outcomes. Two components of the immune system play a major role in gingival inflammation observed in smokers; these include cytokine production and inflammation. The concentration of pro-inflammatory cytokines released from the macrophages of smokers is significantly higher than that observed in non-smokers. Pro-inflammatory cytokines, such as TNF-alpha, are responsible for inflammation of the gingiva. The use of pre-existing immunotherapy treatments may be beneficial and assist in the reversal of periodontal disease. Further researcher on immune therapy, which has been shown to be successful in other disease treatments will not only be beneficial for the oral cavity, but for overall systemic health. Both immune and bacterial components should be taken into consideration when developing therapies for periodontal disease. Pro-inflammatory cytokines such as TNF-alpha are dramatically increased in smokers. Researchers should model therapies after existing therapies that have been successful for other diseases such as immunotherapy studies with lamvidine (Hepatitis B), lambrolixumab (melanoma) and infliximad (oral plaque). The mTOR signaling pathway controls the release of pro-inflammatory and anti-inflammatory cytokines. Deletion of the raptor protein causes more inflammation in the colon due to an increased release of pro-inflammatory cytokines. Therapies which directly act upon this signaling pathway should be further researched upon to reverse the effects of periodontal disease. Chromogranin A release showed a significant increase in smokers because of the body's ability to want to automatically attach the harsh environment created by tobacco. Modulating the immune response induced by Chromagranin A or possibly through the use of anti-TNF-alpha therapies which have shown success in patients with rheumatoid arthritis also is open to future research. The shift from gram-negative to gram-positive bacteria was noted in periodontal disease patients. The less diverse bacteria lead to the loss of the ability of ligaments to attach to one another in the oral cavity. The development and testing of bacteria specific antibiotics such as Fusobacterium, Prevotella, and Selenomonas may prove to be very beneficial. There is much optimism among the dental professionals that immunotherapy may lead to the development of future therapies for periodontitis in both smokers and non-smokers. However we must also encourage patients to stop smoking through awareness and education.

The PI3K/mTOR signalling pathway plays a major role in biology and disease. Therefore, effective inhibitors that target proteins of this pathway have been developed. However, acquired resistance of cancer cells is a prevalent phenomenon that limits the durable response of these compounds. It is becoming apparent that experimental approaches for comprehensive biochemical analysis contribute to understand the complex mechanisms that confer drug resistance, and advances in largescale technologies including genomic sequencing and proteomics allow unprecedented molecular coverage without being biased for specific genes/cellular pathways. Initially, the phenotypic response of sensitive and resistant cells to the absence or presence of a PI3K inhibitor (PI3Ki), as well as other kinases, was examined. This study revealed that PI3Ki-resistant cells experience extensive phenotypic changes upon withdrawal of the PI3Ki from the culture media. The regulation of the proteome and phosphoproteome of sensitive and PI3Ki-resistant cells grown with or without the PI3Ki was analysed by shotgun mass spectrometry-based label-free quantitative technology. This analysis demonstrated that the proteomes and phosphoproteomes of drug-resistant cells are remodelled conditional to the presence of PI3Ki, and that the levels of enzymes with metabolic roles are modulated in resistant cells. Functional analysis of the metabolism of cells capable to survive in absence of PI3K/mTOR activity demonstrated that the bioenergetic activity of these cells is contingent on the presence of the selection drug. The complete set of protein-coding regions of the genome (exome) of sensitive and PI3Ki-resistant cells was then sequenced. This study unveiled common alterations in exome regions across PI3Kiresistant cell lines, as well as a degree of genomic heterogeneity between them. Lastly, the impact of lactic acid, a metabolic product, on a defined signalling network of the MCF7 breast cancer cell line was analysed. This study described the capacity of this metabolite to change the activity of signalling network branches.

mTOR is the central kinase in biochemical pathways that regulate cellular growth, protein synthesis and cell survival. Deregulation of mTOR signaling results in uncontrolled cell proliferation and hence is implicated in various cancers and autoimmune diseases. mTOR functions through two distinct signaling complexes, called mTORC1 and mTORC2. CCT is a cytosolic chaperonin that assists in folding of several protein substrates. In these studies, we have identified two components of the mTOR complexes, mLST8 and Raptor, as substrates of CCT. We have performed biochemical and signaling studies which indicate that CCT is involved in assembly and signaling of mTOR complexes by folding β-propeller domains of mLST8 and Raptor. We have also obtained high resolution structural information of the mLST8-CCT complex by cryo-EM and mass spectrometric cross-linking. Moreover, we have explored the role of PhLP1 as a co-chaperone for CCT in the assembly of mTOR complexes. Interestingly, we found that PhLP1 plays very different roles in the case of mLST8 and Raptor. While PhLP1 participate in assembly of mLST8 into mTOR complexes, it facilitates degradation of Raptor. These biochemical data, combined with structural information, can be used to design small molecules that modulate mTOR signaling by affecting the formation of intact mTOR complexes.

Tubulointerstitial fibrosis is the key histological predictor of the progression of declining renal function and the final common pathway of progressive kidney disease, regardless of aetiology. Despite its significance, there are currently no treatments available to abrogate this process and those that suffer with this burden eventually succumb to renal failure. Tubulointerstitial fibrosis is largely mediated by fibroblasts and myofibroblasts present in the interstitium. In response to injury, activated fibroblasts differentiate into myofibroblasts which serves as a histological hallmark of fibrosis. Myofibroblasts are characterised as the key contributors to interstitial volume and their presence ultimately leads to loss of renal function. The pathological entities leading to fibrosis inextricably depend on complex signalling pathways. Whilst many of the well-known growth factors that exert effects on renal fibroblasts (such as FGF, EGF and PDGF) involve the activation of receptor tyrosine kinases, the intracellular signalling events dictating the response of fibroblasts remain undefined. The kinase mTOR, responsible for integrating stress and amino acids and controlling cell growth, is increasingly recognised for its ability to integrate growth factor signals mediated through the upstream serine/threonine kinase PI3K. A number of recent studies have highlighted the role of PI3K and mTOR in the regulation of key events relevant to fibrosis, serving as a basis for Chapter 3: The role of PI3K and mTOR in the regulation of fibroblast proliferation and collagen synthesis, and the first part of Chapter 5: The role of PI3K and mTOR in the regulation of myofibroblast differentiation. These studies have identified a key role for PI3K and mTOR in the regulation of fibroblast proliferation, differentiation and collagen synthesis. The work described within has also attempted to examine the derivation of myofibroblasts via EMT. EMT is a process that is integral to embryogenesis and may act as an important source of myofibroblasts during fibrosis. This process is examined in Chapter 4: Development and validation of an ex vivo model of EMT. This model aims to better represent the in vivo environment and has also been used to identify novel regulators involved in EMT being utilised in the second part of Chapter 5: The role of PI3K and mTOR in EMT. Although cytokines and growth factors are thought to be chiefly responsible for tubulointerstitial fibrosis, we now know that serine proteases of the coagulation cascade may also play roles in renal disease. However, unlike their role in glomerular diseases, the role of coagulation in tubulointerstitial fibrosis is less well-known. The work described in Chapter 6: Constituents of the coagulation cascade are spatially and functionally related to experimental tubulointerstitial fibrosis has examined temporal and spatial in vivo relationships of coagulation factors and markers of fibrosis that aid our understanding of mechanisms of fibrosis. The aim of this thesis was to examine those facets of renal fibroblast function that are most devastating to renal function and culminate in an expansion of the renal interstitium during fibrosis. This work hopes to provide useful information to aid the understanding of the multifaceted mechanisms involved in renal tubulointerstitial fibrosis.

Hypoxic regions found in most solid tumors often contain cells which are resistant to various cancer therapies. However, hypoxia also forces cells to rely solely on the catabolism of glucose through glycolysis for ATP production and survival, thereby creating a therapeutic window that can be exploited by glycolytic inhibitors, such as 2-deoxy-D-glucose (2-DG). Previous studies in our lab demonstrated that activation of Hypoxia Inducible Factor (HIF-1) in hypoxic tumor cells confers resistance to glycolytic inhibition by 2-DG. In surveying a number of tumor types for differences in intrinsic levels of HIF-1 alpha under hypoxia, we found that pathways upstream of HIF -- i.e. AKT and mammalian target of rapamycin (mTOR) -- have significantly reduced activity in 2 human non-small lung cancer cell lines (NSCLC) as compared to 4 small cell lung cancer cell (SCLC) lines. This reduced activity of AKT and mTOR correlated with increased sensitivity to 2-DG under hypoxia. Since HIF-1 alpha translation is regulated by the mammalian target of rapamycin (mTOR), we examined the effects of blocking mTOR with an analog of rapamycin (CCI-779) in SCLC cells which express high levels of mTOR activity. Under hypoxia, treatment with CCI-779 resulted in HIF-1 alpha down-regulation. Furthermore, CCI-779 potentiated the cytotoxic effects of 2-DG in hypoxic SCLC cells. Conversely, CCI-779 did not increase 2-DG toxicity in NSCLC lines that do not express HIF, SCLC lines treated with siRNA against HIF-1 alpha, or HIF-deficient mutants. These latter results support the hypothesis that, although mTOR modulates numerous downstream pathways, mTOR inhibition by CCI-779 increases the toxicity of 2-DG in hypoxic cells through down-regulation of HIF-1 alpha. Overall, our findings show that CCI-779 hyper-sensitizes HIF-expressing hypoxic tumor cells to 2-DG. Additionally, our results suggest that the intrinsic expression of AKT, mTOR, and HIF in many tumor types may be important predictors of clinical responsiveness to 2-DG and could be used to guide future treatment decisions on whether to use 2-DG alone or in combination with an mTOR inhibitor.