MENTOR is a multi-disciplinary training network which focuses on the mTOR pathway, a central mechanistic hub that integrates nutritional cues and controls cell growth and metabolism. Aberrant activation of mTOR underlies monogenic diseases with broad spectrum of pathological manifestations, including tissue overgrowth, polycystic kidneys and neuropsychiatric disorders affecting around two million of people worldwide. To avoid the medical errancy of patients in different clinical departments and surgical mutilations, approaches of personalized medicine depending on the genetic landscape need to be implemented, taking advantage of suitable pharmacological products.
The overall goal of the MENTOR research program is to: delineate molecular mechanisms of fundamental and aberrant mTOR functions, preclinically decipher the pathophysiology of mTOR, develop a novel arsenal of drugs to selectively target the pathway and provide proof-of-concept support for such interventions. This ambition can only be achieved by the multidisciplinary research program that MENTOR has conceived, through the training of a new generation of scientists at Graduate Schools of biology, pharmacy, chemistry or medicine that will become the leaders of mTOR research in both public and private sectors. MENTOR will establish a collaborative and efficient network from academia to industry, involving patient associations and clinicians with impact on scientific community and society.
Work package 1 will provide detailed cartography of new gene products acting in the mTOR pathway, as outlined by the following three complementary tasks:
Discover and characterize new mechanisms involved in nutrient sensing by mTORC1 which may drive the pathophysiological processes in mTORopathies. As a future outcome, preclinical models of these new genes will be developed following the path defined by Work Package 2, while potential new drug targets will be modeled by Work Package 3. The results will likely also apply to age-related diseases with high incidence
DC 1: Novel regulators of RAG GTPase mediated Non-Canonical (NC) mTORC1 signaling
Nidhi Bhasin individual web page
Project description:
Non-Canonical mTORC1 (NC-mTORC1) signaling controls the function of MiT-TFE transcription factors through substrate recruitment, mediated by RagC/D and Folliculin (FLCN), independently of the TSC-Rheb axis. Our discovery represents a paradigm shift in the field. However, we still lack knowledge of components, regulators and mechanisms by which this pathway contributes to mTORopathies such as Birt-Hogg-Dubé (BHD) syndrome and TSC. The doctoral candidate will perform interactome analysis of FLCN, FNIP1 and RagC by IPs or bimolecular complementation affinity purification (BiCAP) and LC-MS/MS at FTELE MS Facility. Candidate interactors will be validated by co-IP and a validated multiparametric high-content assay at TIGEM High Content Screening Facility (HCSF), to test TFEB nuclear localization and canonical mTORC1 activity (pS6). Hits inducing TFEB nuclear translocation without affecting pS6 (i.e. NC-mTORC1) will be characterized based on the biochemical structure and function, and regarding their tumorigenic potential (proliferation, clonogenic assays, tumor sphere formation) in cell and animal models of mTORopathies.
DC 2: Non-canonical Rag GTPase-TORC1 signalling in yeast
Alisa Kirkin individual web page
Supervisor: Claudio de Virgilio
Project description:
My project will address the still elusive mechanism of how the Rag GTPases control TORC1 in yeast. Unlike in mammalian cells where the Rag GTPases serve to tether mTORC1 to lysosomes, yeast Rag GTPases control vacuolar TORC1 primarily via an elusive mechanism that neither involves membrane recruitment nor the regulation via the Rheb-orthologous Rhb1 (own unpublished data). An appealing explanation for this mystery relates to the recently discovered non-canonical Rag GTPase-mTORC1 (NC-mTORC1) pathway.
We speculate that also the RagC/D orthologue Gtr2 in yeast locally couples TORC1 to specific substrates. To address this, the doctoral candidate will probe both the interactome and proxisome by MS-coupled Turbo-BioID of wild-type and GDP- or GTP-locked alleles of Rag GTPases. Our preliminary data with Gtr2 identify all known TORC1 effectors, and hitherto unknown candidates. Doctoral candidate will validate them by co-IP and in vitro TORC1 kinase assays followed by MS-based identification of the TORC1 target residues. Using a combination of two-hybrid, co-IP, and Alpha-Fold-assisted modeling, DC2 will map the interaction surfaces on both the Rag GTPase and the novel effectors to denominate a common Rag GTPase interaction motif in the effector proteins. Emerging models will be tested by CRISPR/Cas9-mediated introduction of point mutations in the interaction domains that should affect binding and the biological function of the respective TORC1 effectors.
DC 3: New Elements of Nutrient Signal Integration by the TSC complex
Kexing Gao individual web page
Supervisor: Kathrin Thedieck
Project description:
Using high resolution mass spectrometry (HR-MS) combined with CRISPR, knockdown, small compound inhibitors and/or PROTAC degrader techniques, the PhD candidate will discover and characterize new elements mediating the integration of nutrient signals by the TSC complex. Mathematical model-based computational simulations will enable the systematic generation and ranking of hypotheses on the mechanisms via which the candidates act on TSC-mTORC1 signaling. Metabolic effectors will be identified by MS-based metabolomics. In collaboration with the MENTOR partners, we will delineate the role of novel TSC complex regulators in pathophysiological processes linked with mTORopathies.
DC 4: New approaches to screen for novel direct mTORC1 targets
Joséphine Cherrière individual web page
Project description:
Several mTORC1 substrates with functions in anabolic and catabolic processes have been uncovered but their contribution and interplay in mediating mTORC1’s pathophysiological outputs is poorly understood, and we likely still miss a plethora of targets involved in these processes. State of the art shotgun phosphoproteomic approaches do not provide the full picture as they do not allow to distinguish whether phosphorylations are mediated by the kinase-of-interest itself or by an unknown downstream effector kinase. We have developed an on-column in vitro kinase assay (OBIKA) that allows the unbiased screen of complex, proteome-scale samples for direct kinase targets. The PhD candidate will combine OBIKA with in vivo phosphoproteomics to comprehensively identify mTORC1 substrates. Kinases directly interact with their substrates to perform phosphorylation reactions. Proximity labelling based on the miniTurbo-approach will allow to investigate stimulus-dependent protein neighbourhoods of mTORC1. To study the relevance of newly identified proteins and phosphosites in metabolic regulation, the doctoral candidate will CRISPR or overexpress up to 10 candidates and analyse their effects on mTORC1-driven cell growth, proliferation and metabolism. Inactive Ala and phospho-mimicking Asp/Glu variants will allow to study the relevance of single phosphosites. The potential of new molecular targets to serve as drug targets will be evaluated jointly with medical chemistry labs in the MENTOR.
DC 5: Functional genomics of S6K1/2 specific substrates regulating cell size
Cas Koeman individual web page
Project description:
Among the mTORC1 targets, S6 kinases 1 and 2 (S6K1 and S6K2) are exquisitely sensitive to nutrient availability and rapamycin inhibition. Of note, in vivo analysis of mutant mice reveals that S6K1 regulates cell size in a predominant way as compared to S6K2. Mutant animals also mimic a caloric restriction phenotype with decreased adiposity and increased lifespan. S6K1 may be therefore be key to the interconnection between cell size, and regenerative and aging responses. The PhD candidate will perform structure-function studies and phospho-proteomics to reveal:
Molecular biology and genome editing tools will be used to produce mutant kinases and swap relevant domains in S6K1 and S6K2. Phosphoproteomics will broaden the list of S6K1 specific substrates that may be involved in cell size control. Protein modeling will clarify the kinase-substrate as well as inhibitor-kinase interactions in collaboration with the University of Tübingen (Prof. M. Gehringer). The molecular understanding of cell size control by mTOR/S6K1 should open new therapeutic perspectives for mTORopathy in which giant cells are a prominent pathological feature.
DC 6: Brain mosaicism affecting the mTOR pathway
Lucas van Endert individual web page
Project description:
Focal Cortical Dysplasia type II (FCDII) causes severe drug-resistant pediatric epilepsy through mTOR pathway hyperactivation. FCDII-associated epilepsy is typically resistant to anti-seizure medications, and patients ultimately require neurosurgical resection of the epileptogenic zone for seizure control, allowing direct analysis of diseased tissue. The Baulac lab is one of the pioneers in the identification of brain somatic mutations in FCDII affecting various genes belonging to the mTOR pathway and which cause mTOR hyperactivation. However, there are still ~40% of FCDII cases that remain unsolved genetically. Moreover, the cell-type and developmental origin of FCDII abnormal cells remain poorly understood. DC6 will:
PhD candidate will analyze surgical epileptic brain tissues using cutting-edge genomics (targeted panels, whole exome/genome sequencing, longread sequencing), single-nucleus RNA sequencing, laser-capture microdissection, and integrate functional electrophysiology with spatial transcriptomics.
Work Package 2 will address why aberrant mTOR activity often impacts both neuronal excitability and the oriented cell division underlying epilepsy and polycystic kidney:
Discover and characterize new mechanisms involved in nutrient sensing by mTORC1 which may drive the pathophysiological processes in mTORopathies. As a future outcome, preclinical models of these new genes will be developed following the path defined by Work Package 2 while potential new drug targets will be modeled by Work Package 3. The results will likely also apply to age-related diseases with high incidence.
DC 7: How does the mTOR pathway regulate neuronal excitability: towards new therapeutic targets
Lukas Vaišvilas individual web page
Project description:
Genetic diseases, such as Tuberous Sclerosis Complex (TSC), leading to the up-regulation of mTOR activity predispose to altered cortical development, now classified as mTORopathies, which are associated with epilepsy as well as intellectual disability and autism. However, why aberrant mTOR activity alters neuronal excitability remains unknown. By genetic epistasis experiments in mouse models of the disease, our preliminary data identified one mTOR substrate, S6 kinases 1 (S6K1), as an essential factor for cortical malformations and seizures in a mouse model of TSC. By phospho-proteomics and electrophysiological studies, we observed that S6K1 phosphorylates key determinants of neuronal excitability. We hypothesized that their regulation by S6K1-mediated phosphorylation may contribute to epileptic seizures in Tuberous Sclerosis. The PhD candidate will test this model by original pharmacological, nutritional, biophysical and genetic approaches.
DC 8: Mechanisms in neuropsychiatric-like behavioral phenotypes and brain connectivity in TSC
Vishrutha Prakash individual web page
Supervisor: Justyna Zmorzyńska
Project description:
Using zebrafish as an animal model, the PhD candidate will test various compounds and potentially epistatic genes for modification of neuropsychiatric-like behavioral phenotypes in the Tsc2 mutant zebrafish (automated behavioral analysis using Zebrabox with automated data analysis). The compounds will be selected based on our previously obtained RNAseq data from brain tissue. In addition, compounds acting as new pharmacological inhibitors (University of Tübingen- M. Gehringer and the University of Basel – M. Wymann) or interfering with movel molecular targets (Université Paris-Cité – M.Pende, University Clinic of Essen – K. Thedieck) will also be tested. For compounds that modify behaviors, the brain morphology will be assessed by whole- mount immunofluorescence of brain markers in the intact brains (single-cell-resolution whole brain imaging using light-sheet microscopy) and then the activity of specific regions and the connectivity development will be examined using transgenic lines with Ca-sensitive fluorescent probe GcaMP or fluorescently-labelled specific neuronal populations, respectively (live time-lapse imaging of connectivity development using light-sheet microscopy).
DC 9: General and mRNA-specific translation downstream of mTOR in human neurodevelopment
Kartik Jatwani individual web page
Supervisor: Christos G. Gkogkas
Project description:
One of the best ascribed functions of mTOR is regulation of protein synthesis, also termed translational control. mTOR exerts tight control on gene expression at the level of general translation, but also mRNA-specific translation by preferentially stimulating or inhibiting the synthesis of specific subsets of proteins. While mTOR-sensitive mRNAs have been identified in mature brain using mouse models, this analysis has not been performed during early development. Moreover, little is known about the role of mTOR in regulating protein synthesis in early human brain development. Human induced pluripotent stem cell-derived brain organoids have emerged as a powerful model to study early development, as they recapitulate >96% of human embryonic brain gene expression and cytoarchitecture to a large extent. To elucidate the role of mTOR in translational control during early human brain development, the doctoral candidate will use translational profiling to assess general (metabolic labelling) and mRNA-specific (ribosome profiling) in human brain organoids where mTOR is upregulated and downregulated genetically or pharmacologically. We will further use structural and sequence analysis for mTOR-sensitive mRNAs to identify common molecular features. Understanding mTOR-regulated mechanisms of translational control in early human brain development will also contribute to elucidate the pathophysiology of neurodevelopmental mTORopathies, such as autism spectrum disorder.
DC 10: Kidney on a chip for kidney mTORopathies research
Project description:
DC10 will develop a modular, 3D human kidney model in the microfluidic platform OrganoPlate to mimic a kidney architecture and function. In this plate, two channels are separated by a hydrogel. One channel is populated with RPTECs or tubuloid-derived epithelium, the adjacent channels will be used for co-culture with kidney fibroblasts, endothelial and immune cells. This platform will be then used to model disease-kidney model-based readouts mimicking diseased kidney phenotypes suitable for high-throughput screening. We will then test and validate the effect of mediators and compounds on correcting the modelled kidney alterations present in mTORopathies.
DC 11: Shifted metabolism in TSC patients: a new avenue for interventions
Seema Ouhadi individual web page
Project description:
Metabolic differences are promising non-genetic candidate modifiers of the disease course in mTORopathies. Disturbed energy metabolism has been reported in mouse and rat models of TSC, but it is unknown whether this observation translates to human patients. DC11 will be assess this in TSC cell models by fluxomics and quantitation of intra- and extracellular metabolites in response to changes in nutrient supply. Recruited scientist will test the impact of bioenergetics by modulating gene expression and by pharmacological targeting in vitro and in vivo. In patient cohorts, the PhD candidate will evaluate the correlation between nutrient levels in TSC individuals with disease burden related to brain and kidney function.
DC 12: Cell-autonomous effects of activating RagGTPase mutants in heart and kidney
Sofia Garcua Vazquez individual web page
Project description:
Outgrowths, tumors, cysts, renal, cardiac neurological defects and a compromised immune system are present in mTORopathies. Somatic mutations in RagC have been linked to fatal dilated cardiomyopathy, and various members of the MENTOR (Efeyan, Pende, Ballabio) have described life threatening pathologies in the kidney, in agreement with those observed in familial mTORopathies (Birt Hogg Dube and TSC). We have previously generated knock-in mice expressing activating mutations in RagA that mimics several features of mTORopathies. We have also KI activating mutations in RagC originally found in B-cell lymphomas, and such systemic expression of RagC mutations result in alterations in the heart that are consistent with dilated cardiomyopathy. In addition, these mice also exhibit multi-organ features of accelerated aging, and importantly for this proposal, progressive inflammation and other phenotypic alterations in RagC-mutant kidneys. Understanding the cellular and molecular underpinnings of these pathologies is somehow obscured by the pleiotropic effects that RagC mutations have on immune cells and on inflammation. We want to dissect the effects that are cell-autonomous in the kidney and heart parenchyma, thus mirroring somatic mutations in cells from these two organs, and in contrast to those systemic effects secondarily driven by deregulation of inflammatory cells. This will be followed by the dissection of the molecular mechanisms underlying such alterations by means of primary cultures and organoids derived from these models under hypothesis-driven and unbiased – omics interrogation. We will then test genetic epistasis and selected compounds in vitro and in vivo.
Work Package 3 will translate the fundamental understanding of the mTOR pathway into therapeutic strategies for mTORopathy patients by:
To expand the pharmacological arsenal for precision medicine in mTORopathies. To optimize patient follow-up and biomarker discovery. The development of chemical compounds targeting different upstream and downstream elements of the mTOR pathway will be tested in Work Package 2 system-level pathway modeling will include new gene products from Work Package 3
DC 13: Establishment of novel covalent inhibitors targeting PI3K/mTOR while minimizing adverse effects
Christina Maria Kazakou individual web page
Project description:
PI3K and mTOR kinase inhibition are vital strategies for limiting cell growth in mTORopathies and cancer. However, it can lead to mechanism-based adverse effects especially drug-induced insulin resistance, causing hyperglycemia and elevated insulin levels. To minimize these adverse effects, we are developing inhibitors with a durable covalent and specific action on PI3Ka while sparing PI3Kb which has a redundant function in insulin signaling. The doctoral canddiate will circumvent a partial bypass of PI3Ka inhibition observed in previous studies by concomitantly targeting the mTOR kinase in a reversible manner. This will be achieved by adapting the reversible binding module of our inhibitors, which gives rise to different selectivities across the PI3K and PIKK family as we have previously shown by > 800 structural variations. Our covalent inhibitors will be fully characterized by measuring kchem, Ki (vs. PI3K and PIKK), kinact, cellular potencies for pathway (phosphorylation of Akt, S6K, S6, 4EBP etc), growth inhibition, and more. Resistance and escape mechanisms, time on target, and re-entry into protein synthesis and translation will be evaluated. Efficacy and adverse effects will be evaluated for key compounds in in vitro and in vivo mTORopathy models.
DC 14: Optimization of S6K2-Selective Inhibitors for In Vivo Application
Lucilla Paretti individual web page
Supervisor: Matthias Gehringer
Project description:
The p70 ribosomal protein S6 kinases S6K1 (RPS6KB1) and S6K2 (RPS6KB2) are major downstream effectors of the mTOR complex 1 (mTORC1). While the role of S6K1 is well studied, at least in cancer, knowledge on S6K2 function remains severely limited, especially in the context of mTORopathies. Investigation of S6K2 as a potential drug target is hampered by a lack of isoform-selective S6K2 inhibitors to dissect S6K1 and S6K2 functions. We recently reported the first selective S6K2 inhibitor which relies on a S6K2-specific covalent binding mechanism. DC14 aims at optimizing our S6K2 inhibitors for application in vivo, with a special emphasis in brain penetration. DC14 will improve the solubility and identify/mitigate metabolic hotspots, while maintaining potency and selectivity. In conjunction with molecular modelling, optimization will be guided by enzyme and reactivity assays, physicochemical property profiling and testing of in vitro absorption, distribution, metabolism, and excretion (ADME) properties (e.g. microsomal stability, permeability and efflux). In vivo PK/PD profiling of key compounds will be performed with collaborators from this consortium and the optimized inhibitors will be provided to partners for functional in vivo studies.
DC 15: S6K1- and S6K2-Selective PROTAC Degraders
Sveva Pidello individual web page
Supervisor: Matthias Gehringer
Project description:
The deconvolution of the individual roles of the mTORC1 downstream kinases S6K1 (RPS6KB1) and S6K2 (RPS6KB2) has mostly relied on genetic tools, and only in the case of S6K1, pharmacological inhibition. Proteolysis Targeting Chimera (PROTACs) are a new modality enabling the degradation of a target protein with high specificity and spatiotemporal resolution by hijacking the ubiquitin- proteasome system. So far, PROTACs have neither been developed for S6K1 nor for S6K2. DC15 will generate selective PROTACs for each kinase. In parallel to determining binding affinities, chimeric degraders will be characterized in cellular assays for their degradation potency (DC50) and maximal level of degradation (Dmax). Ternary complex formation and degradation kinetics will subsequently be determined for key compounds in commercial bioluminescence resonance energy transfer (BRET)-based assays. While S6K1-selective PROTACs will build on selective non-covalent S6K1 inhibitors concomitantly identified in our ongoing S6K2 program, S6K2 PROTACs will be based on new reversible-covalent S6K2 inhibitors to grant isoform-selectivity and a substoichiometric protein turnover.
DC 16: Signaling network modeling of the mTOR and PK-PD modeling of the drug candidates
Muskan Madan individual web page
Supervisor: Jeroen Elassaiss-Schaap
Project description:
DC16 will predict the metabolic stability and blood brain barrier penetration of compounds from existing S6K2 and PI3Kα inhibitor libraries (EKUT-Gehringer and UNIBAS-Wymann). Since most of these compounds are targeted covalent inhibitors with a certain intrinsic reactivity, DC16 will place particular emphasis on the generation of suitable models for prediction of extrahepatic clearance. The simulations and models will support strategic decision making by prioritizing compounds for further evaluation. Moreover, DC16 will pursue predictive PK/PD modeling of promising virtual hit structures from structure-based design to select synthesis candidates. DC16 will also perform toxicity prediction and prediction of BBB penetration. Since PROTAC degraders (synthesized by EKUT-Gehringer) are particularly difficult to optimize in terms of ADMET properties due to their high molecular weight and (so far) limited training data, DC16 will use the specific PD Value expertise to establish tailor-made models enabling the simulation of the in vivo properties of such compounds. Jointly with Ukessen-Thedieck, PD value has linked their PK-PD models to ODE based dynamic models of the mTOR network to predict altered mTOR network dynamics upon physiological inhibitor concentrations. DC16 will link PK-PD outputs on S6K2 and PI3Kα inhibitors to ODE mTOR network models parameterized on dynamic data from TSC1 versus TSC2 deficient cell models and patients. Hence, DC16 will simulate the physiological drug response upon TSC1/2 deficiency.
DC 17: Association of metabolic markers and neurological manifestations in patients with mTOR pathway related epilepsy
Daoud Abu Husein individual web page
Project description:
The mTOR pathway regulates essential cellular functions, such as protein synthesis, cell growth, and synaptic plasticity, potentially impacting neuronal excitability and epileptogenesis. Genetic variants in pathway-related genes lead to neurological issues, including epilepsy and neurodevelopmental disorders. DC17 will focus on two genetic conditions: tuberous sclerosis complex (TSC) with TSC1 or TSC2 gene variants and GATOR 1 epilepsy with DEPDC5, NPRL2, and NPRL3 gene variants. The main objective is to improve our understanding of altered metabolism in TSC or GATOR1 epilepsy patients and the interplay between altered metabolism, mTORC1, and clinical manifestation. The ultimate goal is to identify novel targets and approaches for therapeutic intervention. Specifically, DC17 will assess blood amino acid levels and glucose after fasting in patients with TSC or GATOR1 epilepsy and associate alterations with neurological manifestations assisted by magnetic resonance spectroscopy. We will investigate amino acids and glucose levels in TSC patients under ketogenic diet or mTOR inhibitor treatment and study the relations with efficacy, tolerance and response to treatment. Finally, we will identify possible targets for nutritional interventions and conduct a proof-of-principle n of 1 trial.
DC 18: mTOR modulation in kidney mTORopathies: role and effects on epithelial and immunological level
Marta Zubizarreta Ruiz individual web page
Project description:
Despite the well-known contribution of the mammalian target of rapamycin (mTOR) in cyst growth and progression, clinical studies with mTOR inhibitors in the context of Autosomal Dominant Polycystic Kidney Disease (ADPKD) have been disappointing. DC18 aims at performing a deeper dissection of mTORs upstream regulators and downstream effects, identified in this consortium, involved in cyst progression. For this, we will modulate these players in the mTOR signaling pathway in human renal epithelial cell lines of human patients with ADPKD or contiguous gene syndrome (tuberous sclerosis complex associated with PKD) and analyze their effect on relevant disease-specific cellular phenotypes. In addition, DC18 will expand on the role of altered mTOR as an orchestrator of inflammation during cyst progression. DC18 will investigate the role of the upstream and downstream mTOR associates in human epithelial and immune cells (PBMCs and macrophages) on cytokine production, differentiation, and their interaction (co-cultures). We also hold an extensive longitudinal biobank of biological samples (urine, blood, tissue) of ADPKD and mTORopathy patients and healthy individuals available for the overall benefit of the project.
Université Paris-Cité (UPCité)
85 Boulevard Saint-Germain,
75006 Paris, France
Follow us on
Coordinator :
Project manager :
Funded by the European Union under the grant agreement No 101168624. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Executive Agency (REA). Neither the European Union nor the granting authority can be held responsible for them.
