Structural and Functional Characterization of the mammalian Target of Rapamycin Complex 2

Proteins are the fundamental units of life. They take part in any process within the cells and their regulation is essential to adapt to different environmental and intracellular conditions. Cells integrate a large variety of inputs and in turn need to rapidly respond and generate outputs to control key mechanisms such as metabolism, growth and proliferation. Multisubunit protein complexes have evolved to sense and integrate these stimuli. The atypical protein kinase mTOR (mammalian target of rapamycin) is the master regulator of cell growth and proliferation. The association with other proteins enables mTOR to sense intracellular inputs, integrate these signals and respond by phosphorylation of downstream proteins that control cell physiology. Dysregulated mTOR signaling is linked to cancer, and to metabolic and neurodegenerative diseases. mTOR functions in two structurally and functionally distinct signaling complexes, mTORC1 and mTORC2. This thesis provides high-quality structural information on human mTORC2, containing the protein subunits mTOR, mLST8, Rictor and SIN1 determined by cryo-electron microscopy at 3.2 Å resolution. The work resolves the enigmatic structure and interplay of the core mTORC2 subunits Rictor and SIN1. Contrary to previous hypotheses, it is the Rictor C-terminal domain that blocks the rapamycin binding site and causes rapamycin insensitivity of mTORC2. We demonstrate how intrinsically disordered parts of SIN1 integrate into Rictor and wrap around mLST8 to position mTORC2 substrates. We rationalize modes of mTORC2 regulation via control of complex stability and visualize novel ligand binding sites for nucleotides in Rictor and for inositol hexakisphosphate in mTOR. In summary, the results presented in this thesis provide a completely new framework to analyze mTORC2 regulation and its function. These studies open the route for further analyzing interactions with signaling proteins and membranes and pave the way for the development of specific mTORC2 inhibitors

Investigating the effect of RICTOR-deficiency in mTORC2 assembly/activity and its impact on human embryonic stem cell properties

The mechanistic target of rapamycin (mTOR) signalling plays crucial roles in controlling mammalian cell behaviour and function, and thereby maintains tissue homeostasis and organism health. This signalling functions through two distinct multi-protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Both complexes are essential in embryo development as deficiency of either one in mouse leads to embryonic lethality. Considerable knowledge has been obtained regarding to mTORC1 while much less is known about mTORC2, including its function in human embryonic development. In the current study, I utilise CRISPR/Cas9 technology to generate mTORC2-deficient HEK 293T cells and human embryonic stem cells (hESCs) by disrupting one of the mTORC2 key components, RICTOR gene. With these RICTOR-knockout cell model systems, I explored the relationship between the two key subunits (RICTOR and mSIN1) of mTORC2 and their functions in mTORC2 assembly/activity. In particularly, I investigated the effect of mTORC2-deficiency on human pluripotent stem cell properties. Using RICTOR-KO 293T cells, I have demonstrated that the protein expression of mSIN1 is substantially diminished upon RICTOR-deficiency and so does the mTORC2 activity. Further analyses demonstrate that RICTOR/mTORC2 is crucial for mSIN1 protein stability, which may be partly attributed to the mTORC2-associated phosphorylation on mSIN1-Ser260/270 residues. More importantly, this phosphorylation may also affect substrate recognition of mTORC2, suggesting that mTORC2 may have an auto-regulatory function which affects its substrate selection. In addition to the 293T cells, my data in hESCs reveal that mTORC2 signalling may be dispensable for the hESC self-renewal but may have a function in their cell fate determination, particularly in the differentiation of the mesendoderm lineages. Collectively, my findings advance our understanding in both regulation and function of RICTOR/mTORC2 and knockout cells generated from this work are valuable cell models for the further studies on this important signalling pathway, which will provide potential insights for both new therapies development and regenerative applications.Open Acces

CONCOMITANT TARGETING OF THE MTOR/MAPK PATHWAYS: NOVEL THERAPEUTIC STRATEGY IN SUBSETS OF NON-SMALL CELL LUNG CANCER

Over the last decade, a paradigm-shift in lung cancer therapy has evolved into targeted-driven medicinal approaches. However, patients frequently relapse and develop resistance to available therapies. Herein, we utilized genomic mutation data from advanced chemorefractory non-small cell lung cancer (NSCLC) patients enrolled in the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE-2) clinical trial to characterize novel actionable genomic alterations potentially of clinical relevance. We identified RICTOR alterations (mutations, amplifications) in 17% of lung adenocarcinomas and found RICTOR expression correlates to worse overall survival. There was enrichment of MAPK pathway genetic aberrations in key oncogenes (e.g. KRAS, BRAF, NF1) associated with RICTOR altered cases, underscoring that RICTOR could serve as an important co-oncogenic driver in specific molecular settings. Moreover, we utilized a panel of RICTOR amplified NSCLC cell lines and found that RICTOR genetic blockade impaired malignant properties seen by reduced effects on cell survival and tumorigenicity potential. We uncovered a compensatory activation of the MAPK signaling pathway following RICTOR knockdown specifically in KRAS co-mutational settings, exposing a unique therapeutic vulnerability. Our in vitro and in vivo data testing concomitant pharmacologic inhibition of both pathways (PI3K/AKT/mTOR and MAPK) via AZD2014 (mTORC1/2 inhibitor) and selumetinib (MEK1/2) resulted in synergistic responses of antitumor effects. Given the large population of patients affected by NSCLC, our study provides a treatment rationale for a specific subset of patients who may benefit from genomic stratification based on RICTOR/KRAS alterations, further underscoring the need for proper patient selection to gain optimal therapeutic response

Deciphering the function and evolution of the TOR signaling pathway in microalgae

Microalgae constitute a highly diverse group of photosynthetic microorganisms that are widely distributed on Earth. The rich diversity of microalgae arose from endosymbiotic events that took place early in the evolution of eukaryotes and gave rise to multiple lineages including green algae, the ancestors of land plants. In addition to their fundamental role as the primary source of marine and freshwater food chains, microalgae are essential producers of oxygen in the planet and a major biotechnological target for sustainable biofuel production and CO2 mitigation. Microalgae integrate light and nutrient signals to regulate cell growth. Recent studies identified the target of rapamycin (TOR) kinase as central regulator of cell growth and nutrient sensor in microalgae. TOR promotes protein synthesis and regulates processes that are induced under nutrient stress such as autophagy and the accumulation of triacylglycerol and starch. A detailed analysis of representative genomes from the entire microalgal lineage revealed the high conservation of central components of the TOR pathway likely present in the last eukaryotic common ancestor and the loss of specific TOR signaling elements at an early stage in the evolution of microalgae. Here we examine the evolutionary conservation of TOR signaling components in diverse microalgae and discuss recent progress on the study of this signaling pathway in these organisms.Ministerio de Ciencia y Tecnología PGC2018-099048-B-I00, PID2019-110080GB-I0

Insights into the regulation of mTOR signaling and the consequences of pharmacological inhibition

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008.Includes bibliographical references.Cells have evolved a highly tuned system for driving growth in response to the right cues. Permissive signals initiate a cascade of events that send nutrient transporters to the membrane, suppress apoptosis, boost protein synthesis, and adjust metabolic processes to fuel the cell's energy demands. Increases in cell growth are often coordinated with cell division, though the two programs can be decoupled. The TOR complexes, TORCI and TORC2, are central regulators of cell growth and share the serine/threonine TOR kinase as their catalytic domain. In mammals, the TORC2 homolog mTORC2 is activated by growth factors through the lipid kinase PI3K, and is a primary effector for many of its functions, including regulation of the proliferation and survival kinase Akt/PKB. Activation of PI3K also leads to activation of mTORC1. Unlike mTORC2, mTORC1 is equally dependent on nutrient availability, and connects to the protein translation machinery through its substrates S6K and 4E-BP1. Additionally, S6K can suppress insulin signaling, establishing a negative feedback loop to PI3K. Consistent with its role in cell growth, derangements in mTOR signaling are increasingly associated with cancer and, more surprisingly, metabolic diseases. In the work described here, we have investigated the mechanism through which insulin activates mTORC1 and identified the protein PRAS40 as a growth factor-regulated inhibitor and mTORC1 component. PRAS40 cooperates with rheb, an mTORC1 activator, to regulate growth factor signaling through the pathway. We have also developed a potent and selective mTORC1/2 small molecule inhibitor and used this to probe the role of mTOR signaling in tumor cell growth and proliferation. Through this, we have identified common genetic mutations that determine sensitivity to mTOR inhibition and suggest a novel therapeutic anticancer strategy.by Carson Cornell Thoreen.Ph.D

Pathogenesis and treatment of Kaposi's sarcoma-associated herpesvirus related diseases

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and KSHV-associated multicentric Castleman disease (KSHV-MCD) and KSHV-associated inflammatory cytokine syndrome (KICS). KICS is a newly described clinical entity associated with lytic reactivation and high-level, systemic replication of KSHV. We characterized the clinical and laboratory features of a KICS patient over-time. Additionally, we compare the KICS patient to Kaposi’s sarcoma (KS) (n=11) and non-KS (n=6) cases. The KICS patient presented elevated levels of KSHV and IL-10, as expected. Treatment with tocilizumab effectively reduce his IL-10 level to undetectable. Surprisingly, this patient did not present high levels of IL-6. We successfully sequenced the whole KSHV genome, which showed no differential mutation before KICS was officially diagnosed and after diagnosis. Phylogenetic analysis of the KICS consensus sequences showed that both sequences aligned to the K1 clade and were closely related to BAC16, JSC1 (cell line) and GK1B sequences. Additionally, KSHV-related malignancies have a highly active mTOR pathway, which makes mTOR a potential therapeutic target. Therefore, we sought to test MLN0128, an ATP-competitive inhibitor of mTOR, as treatment for PEL. Our results demonstrated that MLN0128 has a greater effect on inhibiting proliferation than allosteric mTOR inhibitor rapamycin. MLN0128 has ~30 nM IC50 values across several PEL cell lines, including PEL that is resistant to conventional chemotherapy. MLN0128 induced apoptosis in PEL, whereas rapamycin only induced G1 arrest. MLN0128 inhibited phosphorylation of mTOR complex 1 and 2 targets, while rapamycin only partially inhibited mTOR complex 1 targets. PEL xenograft mouse models treated with MLN0128 showed reduced effusion volumes in comparison to the vehicle group. Rapamycin resistant (RR) clones with an IC50 for rapamycin 10 times higher than the parental clones emerged consistently after rapamycin exposure as a result of transcriptional adaptation. MLN0128 was nevertheless capable of inducing apoptosis in these RR clones. Our results suggest that MLN0128 might offer a new approach to the treatment of chemotherapy resistant PEL.Doctor of Philosoph

Dual abrogation of Mnk and mTOR; a novel therapeutic approach for the treatment of aggressive cancers

Targeting the translational machinery has emerged as a promising therapeutic option for cancer treatment. Cancer cells require elevated protein synthesis for cell growth and exhibit augmented activity to meet the increased metabolic demand. Eukaryotic translation initiation factor 4E (eIF4E) is necessary for mRNA translation, its availability and phosphorylation are regulated by the PI3K/AKT/mTOR and Mnk1/2 pathways, respectively. The phosphorylated form of eIF4E drives the expression of oncogenic proteins including those involved in metastasis. This article will review the role of eIF4E in cancer, its regulation, and discuss the benefit of dual-inhibition of upstream pathways. The discernible interplay between the Mnk1/2 and mTOR signaling pathways provides a novel therapeutic opportunity to target aggressive migratory cancers through the development of hybrid molecules

Host Cell Responses to Zika Virus Infection

The re-emergence of Zika virus (ZIKV) in 2015 as a significant human pathogen causing neurological diseases in infants as well as adults is a serious global health concern. A clear understanding of the mechanisms involved in ZIKV replication in infected host cells as well as the host responses to virus infection are keys to the development of therapeutic strategies against ZIKV. Studies conducted in this dissertation demonstrate that ZIKV infection induces the activation of mTOR signaling cascade that promotes viral protein accumulation and infectious progeny production. While both mTORC1 and mTORC2 are essential for ZIKV replication, the observation that depletion of Raptor, the unique component of mTORC1, imposes a robust negative effect on ZIKV protein expression and progeny production also suggests a mTOR- independent role played by Raptor. Additionally, the activation of autophagy at early times of infection indicates an antiviral role for autophagy in ZIKV infection. The observation that pharmacological inhibition of autophagy led to increased accumulation of ZIKV proteins further strengthens this contention. Since infection-induced oxidative stress contributes to ZIKV pathogenesis, studies reported in this dissertation also show that ZIKV infection alters the redox homeostasis in infected cells and triggers Nrf2- mediated antioxidant response. Depletion of Nrf2 downregulates the cellular pool of GSH and NADPH leading to enhanced ZIKV replication thereby underscores a role for cellular antioxidants in the suppression of ZIKV replication. The dependency of ZIKV replication on host cell glycolysis is highlighted by significant reduction in viral protein expression and virus yield due to pharmacologic inhibition. When glycolysis is inhibited, gluconeogenesis was found to facilitate ZIKV replication by compensating carbon input via oxidative mitochondrial metabolism. Further experimentation comparing the metabolic profiles of mock- and ZIKV-infected cells may provide important information in understanding the role of cellular metabolism in virus replication. Advisors: Asit K. Pattnaik and Rodrigo Franco Cru

Structural studies on the target of rapamycin complex 1

Proteins are the functional units executing the genetic program of living cells. Protein activity has to be modulated and adapted to environmental and intracellular conditions throughout the life cycle of every cell. In higher eukaryotes, complex multidomain proteins and multisubunit complexes have evolved to integrate large numbers of input signals to control key steps in metabolism, growth and proliferation. Structural studies of authentic eukaryotic multidomain proteins are required to understand their emergent properties resulting from interdomain and intersubunit crosstalk and conformational dynamics. However, due to problems in sample preparation and characterization, resolving the structures of large eukaryotic protein assemblies remains a considerable challenge. This thesis provides novel structural and mechanistic insights to three highly relevant eukaryotic protein systems, based on integrative multimethod approaches to tackle the inherent complexity of each case. Mammalian target of rapamycin (mTOR) is the master regulator of growth and proliferation; it senses nutrient and growth signals and in response mediates the switch between anabolism and catabolism. Dysregulation of mTOR signaling is implicated in metabolic diseases and cancer, and mTOR is an established drug target. mTOR is comprised in two structurally and functionally distinct signaling complexes, mTORC1 and mTORC2. In Chapter 2, we determine the structure of the human mTORC1, containing the protein subunits mTOR, Raptor and mLST8, bound to FKBP12, by combining cryo-electron microscopy of the assembled complex at 5.9 Å resolution and crystallographic studies of the 149 kDa Raptor from Chaetomium thermophilum at 4.3 Å resolution. The core scaffold of the complex is formed by mTOR; the Raptor N-terminal conserved (RNC) domain is bound in vicinity to the mTOR catalytic site, suggesting a key role of the RNC in substrate recognition and delivery. Polo-like kinase 4 (PLK4) is a central controller of centriole duplication. Chapter 3 identifies a mechanism for PLK4 regulation by the partner protein STIL by using biochemical mapping, kinase assays, super resolution microscopy, isothermal calorimetry in combination with structural studies of the interaction of the PLK4- polobox 3 (PB3) domain with a coiled-coil region of STIL (STIL-CC). NMR 5 spectroscopy provides a solution structure of the isolated PLK4-PB3 and crystallographic structure determination reveals the mode of complex formation of PLK4-PB3 and STIL-CC. Mutations in STIL-CC abrogate the interaction to PB3 and diminish centriole duplication in cells, demonstrating the relevance of the PLK4-STIL interaction for centriole duplication. Acetyl-CoA carboxylase (ACC) catalyzes the conversion of acetyl-CoA to malonyl- CoA, providing the building blocks for fatty acid synthesis. Eukaryotic ACCs are large multidomain proteins, that comprise a unique 120 kDa regulatory central domain (CD) besides the N- and C-terminal catalytic domains biotin carboxylase (BC) and carboxyl transferase (CT). In chapter 4 we determine the structure of the human and yeast CD and provide intermediate resolution crystal structures of up to nearly full-length ACC from Chaetomium thermophilum. In combination with functional assays, these data reveal the structural basis for phosphorylation-dependent control of yeast ACC activity. In summary, the results presented in this thesis provide new structural and mechanistic insights into crucial eukaryote-specific regulatory properties of large multidomain proteins and protein complexes. These studies open important routes for further dissecting functional mechanisms by targeted biochemical and biophysical approaches. In particular for mTORC1, the current results provide a basis for analyzing the interactions with signaling partner proteins. Interdomain crosstalk and regulated protein conformational dynamics in these systems are closely linked to disease. Targeting interdomain interactions may serve as a relevant strategy for therapeutic intervention, e. g. in cancer therapy. The detailed depiction of intact assemblies of ACC and mTORC1 provides the structural groundworks for such approaches