Browsing by Subject "mTOR"

Germinal center kinases (GCKs) belong to the mammalian Ste20-like family of serine/threonine kinases and participate in various signaling pathways needed to regulate a wide range of cellular activities. GCK-like kinase (GLK), also known as MAP4K3, belongs to the MAP kinase kinase kinase kinase (MAP4K) family of proteins and has recently been established as a key node in the amino acid response pathway and putative nutrient sensing regulator in cells, as it is required for the amino acid-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1)—a central regulator of cell growth and metabolism. The precise mechanism(s) by which MAP4K3 activates mTORC1 under conditions of amino acid satiety, however, are undefined. Recent studies in the La Spada lab suggest MAP4K3 activates mTORC1 by phosphorylating the NAD-dependent deacetylase sirtuin 1 (Sirt1) and subsequently, inhibiting the LKB1-AMPK pathway—a pathway that suppresses mTORC1 activation during starvation. MAP4K3 has additionally been linked to the regulation of cellular stress responses, autophagy, growth, survival, and organismal lifespan through largely unknown pathways. My working hypothesis is that MAP4K3 serves as an amino acid sensor and activates mTORC1 through phosphorylation of Sirt1 and subsequent inhibition of the mTORC1-suppressing Sirt1-LKB1-AMPK pathway under conditions of amino acid satiety and engages different biological pathways by virtue of its protein interacting partners to control critical cellular processes involved in cell growth, survival, and lifespan. In study 1, I used amino acid depletion/restimulation experiments and phospho mass spectrometry to establish a direct link between MAP4K3 and the Sirt1-LKB1-AMPK pathway and determines that Sirt1 is phosphorylated at Threonine 344 (T344) in a MAP4K3- and amino acid-dependent manner. Furthermore, I showed that phosphorylation of T344 inhibits Sirt1 and is sufficient to restore amino acid-dependent mTORC1 activation in cells lacking MAP4K3. To elucidate additional pathways regulated by MAP4K3, in study 2, I sought to discover novel MAP4K3 interacting partners by integrating proteomics interactome data and phosphoproteomics data followed by validation studies in cells. Experiments from these studies indicate a novel role for MAP4K3 in regulating DNA double-strand break (DSB) sensing and repair in the nucleus, mTOR localization to the lysosome through the GATOR2 complex, and endocytosis. Recent discoveries regarding the important role for MAP4K3 in nutrient sensing through mTORC1 activation and other cellular activities, including cell growth, autophagy, and survival are significant because deregulation of these cellular processes has been implicated in aging, as well as a wide array of human diseases including cancer, immunological disorders, and neurodegeneration. This dissertation, thus, sheds light on the molecular mechanisms by which MAP4K3 regulates these processes and provides significant insight into the modulation of these pathways in health and disease states.

During the immune response, helper T cells must proliferate and upregulate key metabolic programs including glucose and glutamine uptake. Metabolic reprogramming is imperative for appropriate T cell responses, as inhibition of glucose or glutamine uptake hinders T cell effector responses. Glutamine and glutaminolysis use in cancer cells has partially been explored. However, the role of glutamine and its downstream metabolites is incomplete and unclear in T cells. The first step of glutamine metabolism is conversion to glutamate via the hydrolase enzyme glutaminase (GLS). To target glutaminolysis, two different methods were employed: 1) genetic knockout of GLS using a CRE-recombinase system specific for CD4/CD8 T cells, and 2) pharmacological inhibition of GLS via the potent and specific small molecular CB839. These two models of glutaminase insufficiency were used as a tool to target glutamine metabolism during T cell activation and differentiation both in vitro and in vivo.

GLS-deficient T cells had decreased activation at early time points compared to control. Over several days, these GLS-deficient T cells differentiated preferentially to Th1-like effector cells. This was reliant on increased glucose carbons incorporating into Tri-Carboxylic Acid (TCA) metabolites. This increased effector response in vitro occurred in both CD4+ T helper cells and CD8+ cells (Cytotoxic lymphocytes, or CTLs). Differentiation of CD4+ T cells to Th1 or Th17 subsets showed decreased Th17 differentiation and cytokine production, while Th1 effector responses were increased. This increased Th1 function was dependent on IL-2 signaling and mTORC1, as reducing IL-2 or inhibiting mTORC1 with rapamycin prevented GLS inhibition-induced Th1 effector function. Th17 cells, meanwhile, were inhibited by changes in reactive oxygen species, and recovery of Th17 function was achieved with n-acetylcysteine treatment.

T cells lacking GLS were unable to induce inflammation in a mouse model of Graft vs Host disease, an inflammatory bowel disease model, or in an airway inflammatory model. Importantly, Chimeric Antigen Receptor (CAR) T cells made from GLS knockout cells were unable to maintain B cell aplasia in recipient mice. Contrary to this, temporary inhibition of GLS via small-molecule inhibition increased B cell killing in vitro and enhanced T cell persistence in both the B cell aplasia and in a vaccinia virus recall response. These results indicate a balance, where permanent deficiency of GLS is detrimental to T cell responses, but acute inhibition can actually promote T effector responses and survival. Overall, this work aims to understand how perturbations in glutamine metabolism in T cells affects differentiation and function and the role of glutaminolysis and improve therapies for inflammatory disease and cancer.

Mast cells (MCs) are long-lived hematopoietic cells located within tissues. These cells are densely packed with granules containing preformed bioactive components that are released within seconds to minutes upon activation in a process called degranulation. MCs have beneficial roles in pathogen clearance and wound healing but are most widely associated with their deleterious effects in allergic diseases. Importantly, MCs have been shown to reform granules following degranulation in vitro. This capacity for multiple cycles of degranulation and regranulation is thought to contribute to chronic allergic diseases such as asthma and atopic dermatitis, however, MC regranulation has not been previously demonstrated in vivo. Additionally, how MCs regulate regranulation has not been previously shown. In this study, we demonstrate that following anaphylaxis, peritoneal MCs from mice can undergo regranulation. Additionally, using inducible Raptor knockout mice, we show that mTORC1, a well-known mediator of cellular metabolism, is necessary for MC regranulation in vitro and in vivo. Using a microarray approach, we determined that mTORC1 activity is regulated by Slc37a2. This glucose-6-phosphate transporter is necessary for increased glucose-6-phosphate and ATP concentrations during regranulation, two upstream signals of mTOR. Additionally, Scl37a2 was found localized to endosomes during regranulation, where it concentrated extracellular cargo which are trafficked into newly formed granules. Thus, MC regranulation is regulated by a metabolic reprogramming that requires the interaction of the glucose-6-phosphate transporter Slc37a2 and the nutrient sensor mTORC1.

Castration resistant prostate cancer (CRPC) causes significant morbidity and mortality around the world and improving treatment options for patients with CRPC is a major concern for biomedical research. Because of the importance of activating mutations in the PI3K/AKT/mTOR pathway in prostate cancer, several mTOR inhibitors have been tested for efficacy in CRPC but despite promising preclinical findings, the results of clinical trials have been disappointing. The findings of several groups, including a clinical trial of RAD001 conducted at Duke, suggest that feedback upregulation of PI3K and autophagy may be potential mechanisms for resistance of CRPC to mTOR inhibitor therapy.

The main goal of this dissertation was to explore these mechanisms in vitro and to determine if combinations of PI3K inhibitors and different classes of mTOR inhibitors can overcome resistance to mTOR inhibitor monotherapy. In particular, we used immunoblotting, reverse phase protein microarrays, polysome profile analysis, cell cycle analysis, and several techniques for determining cell survival and proliferation to explore the differences in survival, proliferation, autophagy, and activity of the AKT, translation initiation, and autophagy cell signaling networks between prostate cancer cell lines treated with different combinations of mTOR and PI3K inhibitors. Our findings revealed that the combination of PI3K and mTOR inhibition leads to a synergistic inhibition of prostate cancer cell survival and cytostasis that is correlated decreased translation rates, hypophosphorylation of 4E-BP1, autophagy, and an uncoupling of normal signaling between AKT and mTOR. We were able produce an effect on cell survival similar to treatment with high doses of mTOR/PI3K inhibitor combinations by inhibiting cap-dependent translation using a non-phosphorylatable mutant of 4E-BP1. In contrast, knocking down two major autophagy genes had little to no effect on the survival of prostate cancer cells treated with PI3K/mTOR inhibitors but did protect from cell death caused by the UPR activator tunicamycin.

We conclude that treatment strategies that target PI3K, mTORC1 and mTORC2 simultaneously have the potential to be clinically useful in CRPC, probably due to the increased inhibition of eIF4E activity and cap-dependent translation when compared to monotherapy with allosteric mTORC1 inhibitors. Although autophagic cell death can be induced in prostate cancer cells, the autophagy observed after inhibition of PI3K and mTOR does not appear to contribute to cell death and is not a major resistance mechanism under these conditions. Nevertheless, we did observe different roles for autophagy in the survival of cells exposed to different types of stressors, and further elucidation of autophagy signaling networks may yet provide useful clinical targets.