Browsing by Subject "TOR Serine-Threonine Kinases"
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Item Characterization of mTOR Inhibition and Autophagy Inhibition in Non-Small Cell Lung Cancer(2014-04-14) Britt, Rebecca; DeBerardinis, Ralph J.; Minna, John D.; Levine, Beth; Mendelson, Carole R.Lung cancer continues to be the leading cause of cancer related death in both men and women. Pre-clinical studies of targeted therapies are needed in order to improve upon the chemotherapeutics that are currently in use. The ability to identify subsets of patient tumors which will respond to a particular targeted agent using biomarkers to indicate an acquired vulnerability will improve selection of effective therapeutics and minimize time and money wasted on ineffective drugs. The goal of this dissertation has been to characterize NSCLC response to mTOR inhibition and determine whether there are any molecular biomarkers that can predict response. mTOR is a central regulator of several pro-oncogenic signaling pathways and plays a role in cell growth, proliferation, metabolism, and inhibition of autophagy. Early studies examining mTOR inhibitors were limited by a lack of proper patient selection and the inability of first generation drugs to completely inhibit mTOR signaling. In the present study, we screened a panel of well-characterized NSCLC cell lines with three mTOR inhibitors, classical mTORC1 inhibitor rapamycin, and two novel dual mTORC1/2 inhibitors, Torin1 and AZD8055 in order to identify potential biomarkers that may be used to predict response to these agents. Additionally, in order to further characterize vulnerabilities to mTOR related genes within lung cancer subsets, we performed a genetic knockdown screen individually targeting 55 genes in this important pathway. Because inhibition of mTOR frequently leads to a cytostatic rather than cytotoxic effect, mTOR targeting agents may have greater utility when used in combination with other chemo- and targeted-agents. Therefore, we screened the three mTOR inhibitors in combination with the chemotherapy doublet paclitaxel/carboplatin or the targeted agent erlotinib. Finally, mTOR inhibition and other drug treatments have been shown to lead to autophagy activation. This process of cellular "self-eating" is thought to protect cancer cells from low nutrient availability and therapy induced stress. We screened NSCLC cells for their response to autophagy inhibitors alone and in combination with chemo- and targeted-therapy agents. The studies described in this thesis led us to the following conclusions. A subset of NSCLCs are more responsive to rapamycin than to mTORC1/2 inhibition by Torin1, or AZD8055, and sensitivity to mTOR inhibition is associated with RTK activation such as ERBB2 amplification or EGFR mutation or amplification, while KRAS and/or LKB1 mutations were associated with resistance. RNAi knockdown of various components related to mTOR signaling and autophagy produce a heterogeneous growth effect response in NSCLCs cells, and potentially define subset-specific vulnerabilities. mTOR inhibitors sensitize NSCLC cells to standard targeted- and chemotherapy agents erlotinib and paclitaxel/carboplatin doublet in an additive or synergistic manner, with the greatest level of synergy occurring in cell lines which are resistant to single agent therapies, including those with KRAS mutations. Finally, inhibition of autophagy using chloroquine is not likely to be a successful therapeutic approach in lung cancer as no significant growth effect was seen at physiologically relevant concentrations and no sensitization to standard chemo- or targeted-therapies were observed.Item Context-Selective Support of the AKT/mTOR Regulatory Axis by Tank-Binding Kinase 1 (TBK1)(2016-11-28) Cooper, Jonathan Mark; Brugarolas, James B.; Cobb, Melanie H.; Brekken, Rolf A.; White, Michael A.Oncogenic mutation of Ras or Ras effector signaling characterizes roughly thirty percent of all cancers. Persistent obstacles to the treatment of these diseases by direct Ras inhibition prompt alternative strategies aimed at leveraging signaling networks downstream of Ras. Tank-Binding Kinase 1 (TBK1) is downstream of the RalGEF/RalB arm of Ras effector signaling and supports Ras-driven oncogenic transformation via direct regulation of AKT. While TBK1 has been nominated as a therapeutic target, the field lacks knowledge of the mechanisms whereby TBK1 inhibitors mediate lethality and of the preferential context(s) for their application. We therefore leveraged toxicity profiles for TBK1 inhibitors in 100 NSCLC cell lines and identified robust correlation between TBK1 inhibitors and a cadre of mTOR direct and upstream regulatory network signaling inhibitors. This observation, along with orthogonal phosphoproteomics data, suggested an intersection exists between TBK1 and mTOR regulation and mechanistic target space. We identified that TBK1 is required for AKT/mTOR activation during the nutrient starved-to-fed state transition. Furthermore, we established that TBK1 physically intersects with the AKT/mTOR regulatory axis signaling at multiple nodes and can follow permissive and instructive mechanistic routes to regulate mTORC1 activation in response to nutrients. In parallel, we utilized a bioinformatics approach to identify that "Ras-mutant/mesenchymal" status serves as a molecular indicator of TBK1 inhibitor sensitivity in NSCLC. Concordantly, signaling through the AKT/mTOR regulatory axis was acutely attenuated by TBK1 inhibition in Ras-mutant/mesenchymal but remained unresponsive in Ras-mutant/epithelial NSCLC, indicating TBK1-resistant NSCLC may have uncoupled AKT/mTOR signaling from substantive TBK1 regulation. We furthermore demonstrated that TBK1 inhibition synergizes with Transforming Growth Factor-beta (TGF-beta)-mediated induction of the epithelial-to-mesenchymal transition (EMT) to reduce cancer cell viability. Together, these observations suggest that TBK1 supports pro-survival signaling downstream of Ras and EMT/TGF-beta signaling through the AKT/mTOR regulatory axis. Our findings, therefore, reveal novel mechanistic contributions of TBK1 in the regulation of AKT/mTOR signaling, and also nominate Ras-mutant/mesenchymal NSCLC as the preferential context for therapeutic interventions targeting TBK1.Item Dynamic Modulation of Exocyst Interaction Networks Integrates Hippo and mTOR Pathway Response to Pathogen Detection(2017-11-16) Zaman, Aubhishek; Brekken, Rolf A.; White, Michael A.; Schmid, Sandra; Mirzaei, HamidMonomeric RALGTPases, via direct binding to the exocyst (a.k.a Sec6/8 complex), help mount productive cell autonomous responses to trophic and immunogenic signals. However, RAL-exocyst downstream effectors register in a bewildering array of signaling events-- suggesting the presence of mechanisms that confer context dependent coordination amongst them. Here, we employed quantitative proteomics-based characterization of dynamic signal-dependent modulation of the exocyst interactome as an approach to detect such mechanisms. We identified sentinel innate immune kinases- PKR and TBK1 as host defense stimulus induced effectors of distinct exocyst subcomplexes specified by the presence of Exo84 versus Sec5 subunits. We find that, under virally compromised conditions, the Exo84 subcomplex accommodates Hippo signaling kinase MST1 together with PKR whereas a Sec5 subcomplex assists assembly of anabolic growth regulatory kinase mTOR together with TBK1. Detailed functional and biochemical analysis indicated that PKR directly phosphorylates MST1 for activation of Hippo signaling and consequent YAP1 inactivation. In parallel, TBK1 was found to be a positive regulator for mTOR and a negative regulator of YAP1 activity. Furthermore, RALB, which is activated by the host defense response, was found to be required and sufficient for induction of both Hippo and mTOR signaling through dual exocyst subcomplex engagement. RALB-dependent activation of these pathways can help cells deflect viral challenge and can be corrupted by oncogenes to help deflect apoptotic checkpoint activation. Thus, RAL-exocyst signaling complexes can be recognized as context-dependent mechanisms for integrated engagement of Hippo and mTOR signaling pathways.Item Studies on Cellular Nutrient Responses and Protein Degradation(2015-06-01) Ghosh, Anwesha; Goodman, Joel M.; Cobb, Melanie H.; Albanesi, Joseph P.; Sternweis, Paul C.I have worked on two projects. The first project investigates mechanisms involved in cellular responses to amino acids. Amino-acid abundance promotes protein synthesis and cell growth via activation of the protein kinase mTOR, while amino-acid deprivation promotes protein degradation by autophagy. The heterodimeric G protein coupled receptor (GPCR) T1R1-T1R3 can act as an extracellular sensor for amino acids, promoting mTOR activity while repressing autophagy in cells. Quantitative PCR analysis revealed that T1R3 depletion increases mRNA expression of amino acid transporters as a compensatory mechanism induced by perceived starvation. The arrestin proteins can bind GPCRs to mediate their internalization or to facilitate downstream signaling. I tested the hypothesis that β-arrestin 2 might participate in regulation of mTOR activity and autophagy by amino acids. siRNA-mediated β-arrestin 2 depletion decreased T1R1-T1R3 protein expression, reduced mTOR activity and increased autophagy in different cell types. β-arrestin 2 loss increased phosphorylation of the MAP kinase ERK1/2, which may play a role in promoting autophagy. Taken together, these findings demonstrate a role for β-arrestin 2 in promoting mTOR activity and suppressing autophagy. The second project examined the role of different protein degradation pathways and an E3 ubiquitin ligase UBR5 in regulating the stability of the protein kinase WNK1, a key regulator of cellular ion homeostasis. Mutations that increase WNK1 protein expression cause familial hypertension, highlighting the importance of understanding the regulation of WNK1 protein expression. Cycloheximide chase experiments revealed that WNK1 degradation may be complex, as it does not follow simple exponential decay kinetics. Pharmacological inhibition of different protein degradation pathways showed that autophagy and the calpain system of non-lysosomal cysteine proteases, but not the proteasome, can promote WNK1 degradation. Inhibition of the protein chaperone Hsp90 increased WNK1 protein levels, possibly through stabilization of WNK1 by Hps70. Immunoprecipitation experiments demonstrated that UBR5 can associate with WNK1. siRNA-mediated silencing of UBR5 increased WNK1 stability, decreased the ubiquitination of an overexpressed N terminal fragment of WNK1, and reduced the levels of KLHL3, an adaptor protein that recruits WNK1 to the Cullin3-RBX1 E3 ligase complex for ubiquitination and degradation. Taken together, these findings identify degradation pathways and molecular players that regulate WNK1 stability.Item T Cell Intrinsic BCAP Links IL1R to the PI3K-mTOR Pathway and Regulates Pathogenic Th17 Differentiation(2018-07-24) Deason, Krystin Leigh; Hooper, Lora V.; Pasare, Chandrashekhar; van Oers, Nicolai S. C.; Satterthwaite, Anne B.Toll-IL-1R homology (TIR) domains are found within adaptor proteins involved in the signaling of Toll like receptors (TLRs) and Interleukin 1 receptor (IL1R) families. Previous work by our lab identified a TIR domain in the protein B cell adaptor for phosphoinositide 3-kinase (BCAP) and determined a role for BCAP in the TLR signaling pathway in myeloid cells. Due to the shared use of TIR domains by TLR and IL1R signaling pathways, I hypothesized that BCAP would also be involved in signaling downstream of the IL1 family of receptors. The IL1 cytokine family has been shown to play a major role in T cell activation, survival, and differentiation; IL1b, in particular, plays a critical role in the differentiation of Th17 lineage cells. Here, I discovered that BCAP functions downstream of IL1R in CD4 T cells and thereby regulates Th17 lineage differentiation and function. IL1b-induced PI3K-Akt-mTOR signaling is compromised in BCAP deficient T cells which leads to decreased mTOR activation, decreased glycolysis, and defective Th17 lineage commitment. Transcriptional analysis of BCAP deficient CD4 T cells revealed that BCAP is critical for the expression of genes associated with pathogenic Th17 lineage cells. Mice specifically lacking BCAP in T cells have normal development of steady state Th17 cells in vivo yet have decreased development of pathogenic Th17 diseases, such as experimental autoimmune encephalomyelitis (EAE) and T cell transfer colitis. Further, the use of a potent inhibitor of mTOR, which is downstream of BCAP activation, mimics BCAP deficiency by preventing IL1b induced differentiation of pathogenic Th17 cells. This study establishes BCAP as a critical link between IL1R and the metabolic status of activated Th17 cells and further demonstrates that BCAP is critical for the generation of pathogenic Th17 cells in vitro and in vivo.