ERC FUNDED PROJECTS

Blood and lymphatic vessels have been the subject of intense investigation due to their important role in cancer development and in cardiovascular diseases. The significant advance in the methods used to modify and analyse gene function have allowed us to obtain a much better understanding of the molecular mechanisms involved in the regulation of the biology of blood vessels. However, there are two key aspects that significantly diminish our capacity to understand the function of gene networks and their intersections in vivo. One is the long time that is usually required to generate a given double mutant vertebrate tissue, and the other is the lack of single-cell genetic and phenotypic resolution. We have recently performed an in vivo comparative transcriptome analysis of highly angiogenic endothelial cells experiencing different VEGF and Notch signalling levels. These are two of the most important molecular mechanisms required for the adequate differentiation, proliferation and sprouting of endothelial cells. Using the information generated from this analysis, the overall aim of the proposed project is to characterize the vascular function of some of the previously identified genes and determine how they functionally interact with these two signalling pathways. We propose to use novel inducible genetic tools that will allow us to generate a spatially and temporally regulated fluorescent cell mosaic matrix for quantitative analysis. This will enable us to analyse with unprecedented speed and resolution the function of several different genes simultaneously, during vascular development, homeostasis or associated diseases. Understanding the genetic epistatic interactions that control the differentiation and behaviour of endothelial cells, in different contexts, and with high cellular definition, has the potential to unveil new mechanisms with high biological and therapeutic relevance.

1 481 375 €

Start date: 2015-03-01, End date: 2020-02-29

Work conducted in my laboratory on the trypanosome killing factor of human serum led to the identification of the primate-specific Apolipoprotein L1 (APOL1) as a novel pore-forming protein with striking similarities with proteins of the apoptotic BCL2 family. APOL1 belongs to a family of proteins induced under inflammatory conditions in myeloid and endothelial cells. APOL1 is efficiently neutralized by the SRA protein of Trypanosoma rhodesiense, accounting for the ability of this trypanosome subspecies to infect humans and cause sleeping sickness. We found that natural APOL1 variants escaping SRA neutralization and therefore conferring human resistance to T. rhodesiense are associated with chronic kidney disease. Moreover, transgenic mice expressing these APOL1 variants exhibit an obese phenotype. Our unpublished results also indicate that APOLs control the lifespan of dendritic cells and podocytes activated by viral stimuli. Therefore, we propose that the pathology of APOL variants is due to their deregulated activity on the control of the cellular lifespan in myeloid/endothelial cells activated by pathogen detection. This project aims at characterizing (i) the molecular mechanism by which APOLs control the lifespan of activated dendritic cells and podocytes, which has direct impact on innate immunity and inflammation, and (ii) the mechanism by which APOL1 variants cause pathology. In addition, we plan to detail the physiological function of APOLs by studying the phenotype of transgenic mice either expressing human APOL1 (wild-type and variants) or devoid of APOL genes, which we have recently generated. Finally, we propose to exploit the extraordinary potential of trypanosomes for antigenic variation in order to produce SRA variants able to neutralize the pathogenic APOL1 variants. Preliminary experiments suggest that in podocytes SRA antagonizes APOL1 induction by viral stimulus and subsequent cell death, opening new perspectives to treat kidney disease.

2 250 000 €

Start date: 2015-09-01, End date: 2020-08-31

There is an intense interest in the function of human Myc proteins that stems from their pervasive role in the genesis of human tumors. A large body of evidence has established that expression levels of one of three closely related Myc proteins are enhanced in the majority of all human tumors and that multiple tumor entities depend on elevated Myc function, arguing that targeting Myc will have significant therapeutic efficacy. This hope awaits clinical confirmation, since the strategies that are currently under investigation to target Myc function or expression have yet to enter the clinic. Myc proteins are global regulators of transcription, but their mechanism of action is poorly understood. Myc proteins are highly unstable in normal cells and rapidly turned over by the ubiquitin/proteasome system. In contrast, they are stabilized in tumor cells. Work by us and by others has shown that stabilization of Myc is required for tumorigenesis and has identified strategies to destabilize Myc for tumor therapy. This work has also led to the surprising observation that the N-Myc protein, which drives neuroendocrine tumorigenesis, is stabilized by association with the Aurora-A kinase and that clinically available Aurora-A inhibitors can dissociate the complex and destabilize N-Myc. Aurora-A has not previously been implicated in transcription, prompting us to use protein crystallography, proteomics and shRNA screening to understand its interaction with N-Myc. We have now identified a novel protein complex of N-Myc and Aurora-A that provides an unexpected and potentially groundbreaking insight into Myc function. We have also solved the crystal structure of the N-Myc/Aurora-A complex. Collectively, both findings open new strategies to target Myc function for tumor therapy.

2 455 180 €

Start date: 2015-08-01, End date: 2020-07-31

The production of antibodies against pathogens is an effective mechanism of protection against a wide range of infections. However, some pathogens evade antibody responses by rapidly changing their composition. Designing vaccines that elicit antibody responses against invariant parts of the pathogen is a rational strategy to combat existing and emerging pathogens. Production of antibodies is initiated by binding of B cell receptors (BCRs) to foreign antigens presented on the surfaces of antigen presenting cells. This binding induces B cell signalling and internalisation of the antigens for presentation to helper T cells. Although it is known that T cell help controls B cell expansion and differentiation into antibody-secreting and memory B cells, how the strength of antigen binding to the BCR regulates antigen internalisation remains poorly understood. As a result, the response and the affinity maturation of individual B cell clones are difficult to predict, posing a problem for the design of next-generation vaccines. My aim is to develop an understanding of the cellular mechanisms that underlie critical B cell activation steps. My laboratory has recently described that B cells use mechanical forces to extract antigens from antigen presenting cells. We hypothesise that application of mechanical forces tests BCR binding strength and thereby regulates B cell clonal selection during antibody affinity maturation and responses to pathogen evasion. We propose to test this hypothesis by (1) determining the magnitude and timing of the forces generated by B cells, and (2) determining the role of the mechanical properties of BCR-antigen bonds in affinity maturation and (3) in the development of broadly neutralising antibodies. We expect that the results of these studies will contribute to our understanding of the mechanisms that regulate the antibody repertoire in response to infections and have practical implications for the development of vaccines.

1 999 386 €

Start date: 2015-09-01, End date: 2020-08-31

Introduction: Current anti-cancer treatments are often inefficient, while many patients initially benefit from anti-cancer drugs eventually experience relapse of resistant tumors throughout the body. Current clinical strategies mainly aim at inducing tumor cell death, but this induction may have unintentional and unwanted side effects on surviving tumor cells. Preliminary data: We show that after chemotherapy-induced initial regression, PyMT mammary tumors reappear. During regression, we observe an increased number of cells that have undergone epithelial-mesenchymal transition (EMT) and become migratory. We show that migration can be induced upon uptake of extracellular vesicles (e.g. apoptotic bodies). Our findings suggest that EMT is induced upon chemotherapy, through e.g. EV uptake, potentially leading to migration and growth of surviving cells. Hypothesis and main aim: Based on preliminary data, we hypothesize that tumor cell death induces migration and growth of the surviving tumor cells. We aim to identify the key cell types and mechanisms that mediate this effect, and establish whether interference with these cells and mechanisms can reduce recurrence of tumors after chemotherapy. Approach: We have developed unique intravital imaging tools and genetically engineered fluorescent mice to visualize and characterize if and how dying tumor cells can affect surrounding surviving tumor and stromal cells. We will test whether dying tumor cells can influence the growth, migration, dissemination and metastasis of surviving tumor cells directly or indirectly through stromal cells. We will identify potential targets to block the influence of the dying tumor cells, and test whether this blockade inhibits the unintended side-effects of tumor cell death. Conclusion: With the studies proposed in this grant, we will gain fundamental insights on how induction of tumor cell death, the universal aim of therapy, could play a role in growth and spread of surviving tumor cells.

2 000 000 €

Start date: 2015-09-01, End date: 2020-08-31

"Our research group has worked over the years at the interface between cancer and ageing, with a strong emphasis on mouse models. More recently, we became interested in cellular reprogramming because we hypothesized that understanding cellular plasticity could yield new insights into cancer and ageing. Indeed, during the previous ERC Advanced Grant, we made relevant contributions to the fields of cellular reprogramming (Nature 2013), cellular senescence (Cell 2013), cancer (Cancer Cell 2012), and ageing (Cell Metabolism 2012). Now, we take advantage of our diverse background and integrate the above processes. Our unifying hypothesis is that cellular plasticity lies at the basis of tissue regeneration (“adaptive cellular plasticity”), as well as at the origin of cancer (“maladaptive gain of cellular plasticity”) and ageing (“maladaptive loss of cellular plasticity”). A key experimental system will be our “reprogrammable mice” (with inducible expression of the four Yamanaka factors), which we regard as a tool to induce cellular plasticity in vivo. The project is divided as follows: Objective #1 – Cellular plasticity and cancer: role of tumour suppressors in in vivo de-differentiation and reprogramming / impact of transient de-differentiation on tumour initiation / lineage tracing of Oct4 to determine whether a transient pluripotent-state occurs during cancer. Objective #2 – Cellular plasticity in tissue regeneration and ageing: impact of transient de-differentiation on tissue regeneration / contribution of the damage-induced microenvironment to tissue regeneration / impact of transient de-differentiation on ageing. Objective #3: New frontiers in cellular plasticity: chemical manipulation of cellular plasticity in vivo / new states of pluripotency / characterization of in vivo induced pluripotency and its unique properties. We anticipate that the completion of this project will yield new fundamental insights into cancer, regeneration and ageing."

2 488 850 €

Start date: 2015-10-01, End date: 2020-09-30

Leukocytes are the key components of the immune system that fight infections and provide tissue repair, yet their migration patterns throughout the body over the course of a day are completely unknown. Circadian, ~24 hour rhythms are emerging as important novel regulators of immune cell migration and function, which impacts inflammatory diseases such as myocardial infarction and sepsis. Altering leukocyte tissue infiltration and activation at the proper times provides an option for therapy that would maximize the clinical impact of drugs and vaccinations and minimize side effects. We aim to create a four-dimensional map of leukocyte migration to organs in time and space and investigate with epigenetics techniques the molecular mechanisms that regulate cell-type specific rhythms. We will functionally define the daily oscillating molecular signature(s) of leukocytes and endothelial cells with novel proteomics approaches and thus identify a circadian traffic code that dictates the rhythmic migration of leukocyte subsets to specific organs under steady-state and inflammatory conditions with pharmacological and genetic tools. We will assess the impact of lineage-specific arrhythmicities on immune homeostasis and leukocyte trafficking using an innovative combination of novel genetic tools. Based on these data we will create a model predicting circadian leukocyte migration to tissues. The project combines the disciplines of immunology and chronobiology by obtaining unprecedented information in time and space of circadian leukocyte trafficking and investigating how immune-cell specific oscillations are generated at the molecular level, which is of broad impact for both fields. Our extensive experience in the rhythmic control of the immune system makes us well poised to characterize the molecular components that orchestrate circadian leukocyte distribution across the body.

1 497 688 €

Start date: 2015-09-01, End date: 2020-08-31

Annually 1.2 million new cases of colorectal cancer (CRC) are seen worldwide and over 50% of patients die of the disease making it a leading cause of cancer-related mortality. A crucial contributing factor to these disappointing figures is that CRC is a heterogeneous disease and tumours differ extensively in the clinical presentation and response to therapy. Recent unsupervised classification studies highlight that only a proportion of this heterogeneity can be explained by the variation in commonly found (epi-)genetic aberrations. Hence the origins of CRC heterogeneity remain poorly understood. The central hypothesis of this research project is that the cell of origin contributes to the phenotype and functional properties of the pre-malignant clone and the resulting malignancy. To study this concept I will generate cell of origin- and mutation-specific molecular profiles of oncogenic clones and relate those to human CRC samples. Furthermore, I will quantitatively investigate how mutations and the cell of origin act in concert to determine the functional characteristics of the pre-malignant clone that ultimately develops into an invasive intestinal tumour. These studies are paralleled by the investigation of stem cell dynamics within established human CRCs by means of a novel marker independent lineage tracing strategy in combination with mathematical analysis techniques. This will provide critical and quantitative information on the relevance of the cancer stem cell concept in CRC and on the degree of inter-tumour variation with respect to the frequency and functional features of stem-like cells within individual CRCs and molecular subtypes of the disease. I am convinced that a better and quantitative understanding of the dynamical properties of stem cells during tumour development and within established CRCs will be pivotal for an improved classification, prevention and treatment of CRC.

1 499 875 €

Start date: 2015-04-01, End date: 2021-03-31

Genome maintenance, chromatin remodelling and transcription are tightly linked biological processes that are currently poorly understood and vastly unexplored. Nucleotide excision repair (NER) is a major DNA repair pathway that mammalian cells employ to maintain their genome intact and faithfully transmit it into their progeny. Besides cancer and aging, however, defects in NER give rise to developmental disorders whose clinical heterogeneity and varying severity can only insufficiently be explained by the DNA repair defect. Recent work reveals that NER factors play a role, in addition to DNA repair, in transcription and the three-dimensional organization of our genome. Indeed, NER factors are now known to function in the regulation of gene expression, the transcriptional reprogramming of pluripotent stem cells and the fine-tuning of growth hormones during mammalian development. In this regard, the non-random organization of our genome, chromatin and the process of transcription itself are expected to play paramount roles in how NER factors coordinate, prioritize and execute their distinct tasks during development and disease progression. At present, however, no solid evidence exists as to how NER is functionally involved in such complex processes, what are the NER-associated protein complexes and underlying gene networks or how NER factors operate within the complex chromatin architecture. This is primarily due to our difficulties in dissecting the diverse functional contributions of NER proteins in an intact organism. Here, we propose to use a unique series of knock-in, transgenic and NER progeroid mice to decode the functional role of NER in mammals, thus paving the way for understanding how genome maintenance pathways are connected to developmental defects and disease mechanisms in vivo.

1 995 000 €

Start date: 2016-01-01, End date: 2020-12-31

T lymphocytes display potent pro- or anti-inflammatory properties, which typically associate with distinct effector (Teff) versus regulatory (Treg) cell subsets. Based on published and our preliminary data showing a major impact of microRNAs on T cell differentiation and (auto)immune pathology, my proposal aims to dissect the miRNA networks that control the balance between Teff and Treg subsets in vivo, in various experimental models of infection and autoimmunity. We will focus on three critical mediators of T cell functions: interferon-gamma (IFN-g) and interleukin-17A (IL-17), highly pro-inflammatory Teff cytokines; and Foxp3, the transcription factor that confers Treg suppressive properties. To track the activity of these key genes, we will generate a new Ifng/ Il17/ Foxp3 triple reporter mouse, from which we will isolate Teff and Treg subsets to determine their genome-wide miRNA profiles and specific signatures in vivo. We will investigate both natural (thymic-derived and present in naïve mice) and induced (in the periphery upon challenge) Teff and Treg subsets, as they make distinct contributions to the immune response. We will identify miRNAs selectively expressed in Teff (Ifng+ or Il17+) versus Treg (Foxp3+) subsets of various lineages (CD4+, CD8+, gamma-delta or NKT) in each in vivo model; assess whether they are induced during thymic development or upon peripheral activation; and determine the robustness of subset-specific miRNA profiles across various in vivo challenges. We will then use loss- and gain-of-function strategies to define the individual miRNAs that impact Teff or Treg differentiation and disease pathogenesis; dissect the external cues and intracellular mechanisms that regulate miRNA expression; and identify the mRNA networks controlled by key miRNAs in Teff and Treg differentiation. I expect this project to provide major conceptual and experimental advances towards manipulating miRNAs either to boost immunity or to treat autoimmunity.

2 000 000 €

Start date: 2015-07-01, End date: 2020-06-30

Despite the clear importance and multiple functions of the bacterial cell wall, many bacteria appear to be able to switch into a cell wall deficient or “L-form” state. L-forms are very heterogeneous in size and shape and generally require osmotic stabilisers, such as 0.5 M sucrose, for viability. However, by lacking the requirement for a cell wall, L-forms are completely resistant to common cell wall antibiotics, such as β-lactams, and they are probably protected from some elements of innate immune recognition. L-forms are therefore of potential interest in relation to their possible involvement in human disease. They have often been reported in clinical specimens obtained from patients with recurrent or persistent infections or on long term prophylaxis with β-lactam antibiotics. Unfortunately, until recently, most of the work on L-forms had been done in the pre-molecular era, when it was difficult to characterise the L-forms and particularly to identify their origins and relationship with other resident pathogenic bacteria. Recently, several labs have revisited the L-form issue and started to apply modern molecular and cell biological methods. The proposal is divided into three Themes: • Improve our understanding of key features of the L-forms of our best characterised model system, B. subtilis, including both basic science and possible biotechnological applications. • Extend our analysis of basic L-form biology into several diverse bacterial systems, of relevance to both biotechnology and infectious disease. • Explore in detail the possible clinical relevance of L-forms, aiming to identify specific clinical situations in which they are relevant or, at least, to establish model systems in which the interactions between L-form and mammalian systems can be studied.

2 428 621 €

Start date: 2015-10-01, End date: 2020-09-30

Dynamic interplay between histone modifications and DNA methylation defines the chromatin structure of the humane genome and serves as a conceptual framework to understand transcriptional regulation in normal development and human disease. The ultimate goal of this research proposal is to study the chromatin architecture during normal and malignant T cell differentiation in order to define how DNA methylation drives oncogenic gene expression as a novel concept in cancer research. T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of pediatric and 25% of adult ALL cases and was originally identified as a highly aggressive tumor entity. T-ALL therapy has been intensified leading to gradual improvements in survival. However, 20% of pediatric and 50% of adult T-ALL cases still relapse and ultimately die because of refractory disease. Research efforts have unravelled the complex genetic basis of T-ALL but failed to identify new promising targets for precision therapy. Recent studies have identified a subset of T-ALLs whose transcriptional programs resemble those of early T-cell progenitors (ETPs), myeloid precursors and hematopoietic stem cells. Importantly, these so-called ETP-ALLs are characterized by early treatment failure and an extremely poor prognosis. The unique ETP-ALL gene expression signature suggests that the epigenomic landscape in ETP-ALL is markedly different as compared to other genetic subtypes of human T-ALL. My project aims to identify genome-wide patterns of DNA methylation and histone modifications in genetic subtypes of human T-ALL as a basis for elucidating how DNA methylation drives the expression of critical oncogenes in the context of poor prognostic ETP-ALL. Given that these ETP-ALL patients completely fail current chemotherapy treatment, tackling this completely novel aspect of ETP-ALL genetics will yield new targets for therapeutic intervention in this aggressive haematological malignancy.

958 750 €

Start date: 2015-07-01, End date: 2020-06-30

Identification of the full complement of genes and other functional elements in any virus is crucial to fully understand its molecular biology and guide the development of effective control strategies. Our recent discoveries of new 'hidden' genes in the potyviruses, alphaviruses, arteriviruses, flaviviruses and influenza A virus have demonstrated that, even in the most well-studied and economically-important viruses, small overlapping genes can remain undetected throughout decades of research. Comparative computational analyses can be used to efficiently identify hidden features and target experimental analyses, thus saving time and cost, and minimizing animal experiments. With the rapid increase in sequencing data, for the first time it is now possible to map out at high resolution functional elements genome-wide in hundreds of important virus species. Our research involves the development of powerful new tools for virus comparative genomics, and the application of these tools to uncover hidden genes and other functional elements in RNA virus and retrovirus genomes. Hidden genes are often translated via non-canonical mechanisms, such as programmed ribosomal frameshifting, and we are particularly interested in discovering and characterizing new types of non-canonical translation. Deciphering these 'exceptions-to-the-rule' enhances our understanding of the mechanics of protein synthesis. Further, these novel mechanisms may also be relevant to cellular gene expression. The goals of this project are: 1) To computationally identify all 'hidden' genes and major functional non-coding elements in the genomes of RNA viruses and retroviruses of medical, veterinary and agricultural importance. 2) To experimentally characterize the most interesting new features. 3) To characterize novel translation mechanisms utilized by RNA viruses. 4) To develop web interfaces to our software and an interactive RNA virus comparative genomics database.

1 780 000 €

Start date: 2015-09-01, End date: 2020-08-31

Originally described by cytogeneticists, common fragile sites (CFSs) are chromosomal regions known for their susceptibility to break and rearrange aberrantly, thus altering the expression of genes located therein. CFS instability is associated with tumor development and pathogenic copy number variations. Recent advances have significantly contributed to dissect the molecular bases of CFS instability, yet a unifying model for their unique breakage propensity has not been determined. Fanconi anemia (FA) is a chromosomal instability syndrome featuring congenital abnormalities, bone marrow failure and cancer predisposition, characterized by an increased CFS fragility. FA is thus an ideal model to understand the mechanisms underpinning CFS instability and the mechanistic link between CFS instability and the pathogenesis of disease phenotypes. I propose to use FA cellular models to examine the molecular events leading to CFS instability, and FA mouse models to investigate the consequences of deletions, amplifications or rearrangements involving CFSs on the expression of genes regulating critical signal transduction pathways involved in cell survival, proliferation, and differentiation. Exploring these mechanisms can lead to the development of chemopreventive or therapeutic strategies targeting aberrant gene expression or pathological pathways.

1 462 383 €

Start date: 2015-06-01, End date: 2020-05-31

In vertebrates, a receptor-based, innate sensing machinery is used to detect the presence of microbederived molecules or the perturbation microbial infection causes within the host. In the context of viral infection, non-self nucleic acids are sensed by a set of intracellular receptors that upon activation initiate broad antiviral effector responses to eliminate the imminent threat. Over the past years our understanding of these processes has considerably grown, mainly by employing murine knockout models. Recent advances in genome engineering now provide the opportunity to knockout genes or even to perform functional genetic screens in human cells, providing a powerful means to validate and generate hypotheses. We have developed a high-throughput genome targeting and validation platform that allows us to tackle large-scale loss-of-function studies both at a polyclonal as well as an arrayed format. In addition, we have invested considerable efforts to render this technology applicable to study innate immune sensing and signalling pathways in the human system. GENESIS will combine these efforts to tackle pertinent questions in this field that could not have been addressed before: We will systematically dissect known nucleic acid sensing pathways in the human system to explore their unique roles, cooperativity or redundancy in detecting non-self nucleic acids. We will perform polyclonal, genome-wide loss-of-function screens to elucidate signalling events downstream of intracellular DNA and RNA sensing pathways and their roles in orchestrating antiviral effector mechanisms. Moreover, in a large-scale perturbation study, we will specifically address the role of the kinome in antiviral innate immune signalling pathways, exploring the role of its individual members and their epistatic relationships in orchestrating gene expression. Altogether, these studies will allow us to obtain insight into innate immune signalling pathways at unprecedented precision, depth and breadth.

1 970 000 €

Start date: 2015-10-01, End date: 2020-09-30

Chronic hepatotropic infections including hepatitis B (HBV) and C (HCV) are a major public health concern. Even though both viruses belong to completely distinct families the pathogenesis they elicit is strikingly similar, leading to liver fibrosis and cirrhosis. Treatment for HBV and HCV consists of either direct-acting antivirals or pegylated interferon (IFN)α. In contrast to HCV, these treatment regimen are noncurative for HBV. Little is known to date about the host/pathogen interactions determining viral persistence. Both viruses are sensitive to IFN, activating the JAK/STAT signalling pathway to activate interferonstimulated gene expression (ISG), which are ultimately acting as antiviral immune effectors. Nevertheless, neither type I or III IFN are very effective in their treatment. Here, we suggest investigating the mechanistic details of type I and type III IFN action on HCV and HBV in vitro and vivo with the goal of uncovering not only the differential ISG induction but furthermore characterise viral immune evasion strategies. Building on our previous success in dissecting the host response to HCV and creating the first immunocompetent mouse model for HCV we aim at using both, novel microfluidic culture systems based on 3D hepatocyte cultures susceptible to both HCV and HBV as well as human liver-chimeric mice in combination with single-cell analysis of the antiviral response against HBV and HCV elicited by type I and III IFN. Additionally, we will utilize lentiviral high throughput screening used previously for HCV to identify interferon effector molecules active against HBV. This project will not only provide new insights into the innate immune response to chronic hepatotropic virus infections but furthermore holds the potential of uncovering novel drug targets, aiding in the curative therapy for both, HCV and HBV and offer novel insights into vaccine design. This project has the aim of identifying novel host factors and drug targets enabling the development of immunomodulatory antiviral drugs. This ranks the scope of the proposal between LS6 Immunity and Infection and LS9 Applied Life Sciences and Non-Medical Biotechnology. Evaluating novel bioengineered human liver culture systems and building on human liver-chimeric mice clearly places this proposal at the forefront of identifying novel drug targets and assisting in the development of novel biotechnology and preclinical projects.

1 498 312 €

Start date: 2015-06-01, End date: 2020-05-31

Antibodies are destined to neutralize pathogens and can prevent and fight infectious diseases. Over the last years, advances in single B cell cloning resulted in the isolation of highly potent and broad HIV-1 neutralizing antibodies (bNAbs) that have been shown to prevent SHIV infection in non-human primates (NHPs). Recently, we have demonstrated that a combination of bNAbs can suppress HIV-1 replication in humanized mice, reducing viremia to undetectable levels. Moreover, bNAb therapy of SHIV-infected NHPs induced a rapid decline in viremia, followed by a prolonged control due to the long half-life of the antibodies. While these results strongly encourage the clinical evaluation of bNAbs in HIV-1 therapy, it is of critical importance to understand how the therapeutic potential of antibodies can be harnessed in the most effective way. Therefore, we aim to: I.) Identify exceptionally potent HIV-1 neutralizing antibodies that will be a crucial component of immunotherapy. By establishing novel methods for single-cell sorting and high-throughput sequencing we want to identify bNAbs targeting novel epitopes. II.) Prevent HIV-1 escape applying rationally designed treatment strategies targeting conserved functional sites for HIV-1 entry. III.) Evaluate immune markers and function in relation to bNAb administration in humans. Being at the forefront of one of the first clinical trials studying an HIV-1-directed bNAb, we will have the unique opportunity to investigate the interplay of antibody therapy and the host immune system. This proposal aims to strongly advance the field of HIV-1 antibody therapy and therefore enable the introduction of a new therapeutic modality for HIV-1, and will gain insights for antibody-mediated therapy in other infectious diseases.

1 500 000 €

Start date: 2016-01-01, End date: 2020-12-31

Infant cancer is very distinct to adult cancer and it is progressively seen as a developmental disease. An intriguing infant cancer is the t(4;11) acute lymphoblastic leukemia (ALL) characterized by the hallmark rearrangement MLL-AF4 (MA4), and associated with dismal prognosis. The 100% concordance in twins and its prenatal onset suggest an extremely rapid disease progression. Many key issues remain elusive: Is MA4 leukemogenic? Which are other relevant oncogenic drivers? Which is the nature of the cell transformed by MA4? Which is the leukemia-initiating cell (LIC)? Does this ALL follow a hierarchical or stochastic cancer model? How to explain therapy resistance and CNS involvement? To what extent do genetics vs epigenetics contribute this ALL? These questions remain a challenge due to: 1) the absence of prospective studies on diagnostic/remission-matched samples, 2) the lack of models which faithfully reproduce the disease and 3) a surprising genomic stability of this ALL. I hypothesize that a Multilayer-Omics to function approach in patient blasts and early human hematopoietic stem/progenitor cells (HSPC) is required to fully scrutinize the biology underlying this life-threatening leukemia. I will perform genome-wide studies on the mutational landscape, DNA and H3K79 methylation profiles, and transcriptome on a uniquely available, large cohort of diagnostic/remission-matched samples. Omics data integration will provide unprecedented information about oncogenic drivers which must be analyzed in ground-breaking functional assays using patient blasts and early HSPCs carrying a CRISPR/Cas9-mediated locus/allele-specific t(4;11). Serial xenografts combined with exome-seq in paired diagnostic samples and xenografts will identify the LIC and determine whether variegated genetics may underlie clonal functional heterogeneity. This project will provide a precise understanding and a disease model for MA4+ ALL, offering a platform for new treatment strategies.

2 000 000 €

Start date: 2016-01-01, End date: 2020-12-31

Severe sepsis remains a poorly understood systemic inflammatory condition with high mortality rates and limited therapeutic options outside of infection control and organ support measures. Based on our recent discovery that anthracycline drugs prevent organ failure without affecting the bacterial burden in a model of severe sepsis, we propose that strategies aimed at target organ protection have extraordinary potential for the treatment of sepsis and possibly for other inflammation-driven conditions. However, the mechanisms of organ protection and disease tolerance are either unknown or poorly characterized. The central goal of the current proposal is to identify and characterize novel cytoprotective mechanisms, with a focus on DNA damage response dependent protection activated by anthracyclines as a window into stress-induced genetic programs conferring disease tolerance. To that end, we will carry out a combination of candidate and unbiased approaches for the in vivo identification of ATM-dependent and independent mechanisms of tissue protection. We will validate the leading candidates through adenovirus-mediated delivery of constructs for overexpression (gain-of-function) or shRNA for gene silencing (loss-of-function) to the lung, based on our recent finding that rescuing this organ is essential and perhaps sufficient in anthracycline-induced protection against severe sepsis. The candidates showing the most promise will be characterized using a combination of in vitro and in vivo genetic, biochemical, cell biological and physiological methods. The results arising from the current proposal are likely not only to inspire the design of transformative therapies for sepsis but also to open a completely new field of opportunity to molecularly understand core surveillance mechanisms of basic cellular processes with a critical role in the homeostasis of organ function and whose activation can ultimately promote quality of life during aging and increase lifespan.

1 985 375 €

Start date: 2015-10-01, End date: 2020-09-30

Lymphatic vasculature maintains tissue fluid homeostasis and has important emerging roles in inflammation, immunity, lipid metabolism, blood pressure regulation and cancer metastasis. Lymphatic vessels are specialised to fulfil the functional needs of different organs while diseases associated with lymphatic dysfunction frequently affect vessels of specific tissues. How functional specialisation of vessels is achieved and what underlies tissue-specific vessel failure is not understood. I hypothesise that organ-specific manifestation of lymphatic dysfunction in disease is due to vascular bed-specific differences in vessel formation. In this project my aim is to identify genes and mechanisms required for organ-specific lymphatic development. Building on our recent discovery of a previously unknown progenitor cell type that is required for lymphatic development in an organ-specific manner I set out to identify the origin and function of lymphatic endothelial progenitor cells (LEPC) during development and assess their potential for therapeutic lymphatic regeneration. Towards this aim, we will identify organ-specific origins of lymphatic vasculature using lineage tracing and determine genetic signatures of lymphatic endothelial progenitors by mRNA sequencing. Cells and tissues from normal and mutant mice that show organ-specific lymphatic defects will be analysed. To identify molecular and cellular mechanisms of LEPC derived vessel formation, we will functionally characterise LEPC signature genes using mouse models and visualise vessel development by in vivo two-photon microscopy. The function and therapeutic potential of LEPCs and LEPC derived vessels will be assessed using mouse models of tolerance, inflammation, obesity and lymphoedema. This work will provide novel insights into organ-specific mechanisms of vascular morphogenesis and identify a progenitor cell that may be expoited to restore lymphatic function in disorders associated with lymphatic vessel failure.

2 368 439 €

Start date: 2015-05-01, End date: 2020-04-30

In the vascular system, cell phenotype and fate are driven by the mechanical environment. Whereas physiological mechanical stress defines and stabilizes normal cell phenotype, aberrant mechanical signals trigger phenotypic alteration, leading to inflammation and vascular remodelling. Despite recent advances, how mechanical cues impact gene expression to specify cell phenotype remains poorly understood. Our hypothesis is that mechanical stresses are transmitted to the nucleus where they activate signaling pathways, which in turn regulate gene expression, but what are these mechanotransduction mechanisms occurring within the nucleus? Besides, while most vascular cells respond to mechanical force, Resident Stem Cells (RSCs) are virtually insensitive and remain undifferentiated despite constant cyclic stretch. What are the molecular mechanisms which protect RSCs from stretch-induced differentiation? To answer these questions, we designed an interdisciplinary proposal which gathers biophysical, biochemical and genetic assays, with the following objectives: I) To determine how nuclear mechanotransduction pathways regulate vascular cell phenotype in response to mechanical cues. By combining proteomic and biophysical assays, we will identify nuclear proteins that are post-translationally modified in response to mechanical stress, then we will determine their contribution to gene expression regulation and vascular cell differentiation. II) To identify the molecular mechanisms which protect RSCs from stretch-induced differentiation. We will identify differentially expressed force-bearing structural elements in RSCs compared to more differentiated vascular cells and we will evaluate their impact on gene expression, stress transmission, RSC differentiation and blood vessel formation. The proposed project will yield new insights in different areas of life science from cell biology to potential identification of new therapeutic targets in cardiovascular and regenerative medicine.

1 498 413 €

Start date: 2016-01-01, End date: 2020-12-31

In most western countries, life expectancy is increasing by 3 months a year. As the average age of the population increases, so too does the prevalence of age-related diseases such as cancer, cardiovascular and metabolic diseases, and neurodegenerative disorders. An essential component of ageing research is to understand the biological mechanisms that contribute to the ageing process at the cellular and molecular levels. This has major implications not only for the treatment of age-associated diseases but also for the promotion of healthy ageing. Studies of experimental animals and observations in humans have identified an array of genes and nutritional conditions that increase lifespan. However, these manipulations (genetic or nutritional) often have detrimental effects on other biological processes; for example, reproduction, metabolism, immunity or growth. This is an area of ageing research that has largely been ignored but that is critical to the success of strategies intended to slow ageing and thus the onset of disease. The primary goal of this research proposal is to understand how lifespan extension is linked to reproduction. We will use the nematode Caenorhabditis elegans as a model organism to identify novel conserved genes, molecules, and metabolic networks that link reproduction and longevity through nutrition. The proposed study is based on a unique set of preliminary data that identifies the first clear molecular links between these traits: a steroid hormone receptor and a reproduction-responsive lipase, both of which modulate lifespan extension achieved through changes in nutrition. Understanding the regulation and function of these genes and pathways will clarify at the molecular level how reproduction is linked to longevity. This may ultimately lead to interventions that optimise metabolic activity to promote healthy ageing.

1 941 499 €

Start date: 2015-10-01, End date: 2020-09-30

Ageing is a complex physiological process that affects almost all species, including humans. Despite its importance for all of us, the biology of ageing is insufficiently understood. To uncover the molecular underpinnings of ageing, I propose an interdisciplinary research program that will identify and investigate metabolic and genetic regulators of ageing. Progressive loss of cellular homeostasis causes ageing and an age-associated decline in protein quality control has been implicated in numerous diseases, including neurodegeneration. Seeking for ways to improve protein quality, I have identified a novel longevity pathway in Caenorhabditis elegans. In a forward genetic screen, I found a link between metabolites in the hexosamine pathway and cellular protein quality control. Hexosamine pathway activation extends C. elegans lifespan, suggesting modulation of ageing by endogenous molecules. In a first step, I will explore the mechanism by which hexosamine metabolites improve protein quality control in mammals, using cultured mammalian cells and a mouse model for neurodegeneration. Preliminary data show that hexosamine pathway metabolites enhance proteolytic capacity in cells and reduce protein aggregation, suggesting conservation. Second, I will investigate molecular mechanisms that activate the hexosamine pathway to promote protein homeostasis and counter ageing. Third, I will perform a direct forward genetic screen for modulators of ageing in C. elegans. For the first time, mutagenesis and next generation sequencing can be paired in forward genetic screens to interrogate the whole genome for lifespan-extending mutations in a truly unbiased manner. This innovative approach has the potential to reveal novel modulators of the ageing process. Taken together, this work aims to understand molecular mechanisms that maintain cellular homeostasis to slow the ageing process, and to develop a new technology to identify yet unknown genetic modulators of ageing.

1 500 000 €

Start date: 2015-08-01, End date: 2020-07-31

MicroRNAs (miRNAs) can be transferred between cells, representing an exciting new dimension to intercellular communication, referred to as non-cell-autonomous gene regulation. We recently identified that distinct miRNAs are packaged and exported from TREG cells and delivered directly to TH1 cells, suppressing T cell-mediated disease. Different T cell populations express different miRNAs and release a distinctive set of extracellular miRNAs. In this proposal we will identify whether the transfer of miRNAs between cells contributes to T cell development, T cell differentiation and TH2-mediated allergy and anti-helminth immunity. miRNA-mediated gene silencing requires one of four catalytically active Argonaut (Ago) proteins to regulate gene expression. To investigate miRNA transport between cells, we have generated novel mice with miRNA-deficient T cells that can (Dicer–/–) or cannot (Dicer–/–Ago-1,-3,-4–/– Ago-2fl/fl) respond to exogenous miRNAs. Using these novel mice we will identify which Ago protein(s) specific miRNAs associate with and which Ago proteins are required for miRNA-mediated gene regulation in T cells. TH2 cells express unique miRNAs, which can be found within TH2 cells and in extracellular vesicles released from TH2 cells. We have generated several new TH2-associated miRNA-deficient mice to investigate the cell intrinsic (cell-autonomous) and extrinsic (non-cell-autonomous) role of these miRNAs in TH2-mediated allergy and anti-helminth immunity. Studies in plants and worms have identified various mechanisms of RNA transfer between cells, involving cell-contact dependent and independent mechanisms. We will translate these observations into mammalian systems and identify the mechanisms of miRNA transfer. Results from this work will identify novel miRNA-mediated pathways and incentivise state-of-the-art approaches for novel therapeutic intervention to treat inflammatory diseases.

1 762 510 €

Start date: 2015-08-01, End date: 2020-07-31

Melanoma incidence continues to increase across Europe and compared to other cancers, it disproportionately affects young people, causing a significant loss in life-years in those affected. Ultraviolet radiation (UVR) is the only environmental risk factor in melanoma, but the underlying genetic constitution of the individual also plays an important role. However, our knowledge of the gene-gene and gene-environment interactions in melanomagenesis is still very limited and here we will use various cutting-edge technologies to investigate the role of UVR in melanoma initiation and progression. We have developed mouse models of UVR-driven melanoma that closely mimic UVR-driven melanoma in humans and these provide an unprecedented opportunity to dissect how different wavelengths and patterns of UVR exposure affect melanomagenesis. We propose a multidisciplinary programme of work to examine how host genetic susceptibility factors and responses such as DNA damage repair and inflammation affect melanoma development and progression following UVR exposure. We will integrate knowledge from our animal experiments with epidemiological, histopathological, clinical, and genetic features of human tumours to improve stratification of human melanoma and thereby assist clinical management of this deadly disease. Our overarching aim is to develop a validated stratification approach to melanoma patients that will assist in the development of effective public health campaigns for individuals at risk across Europe.

2 171 623 €

Start date: 2016-03-01, End date: 2021-02-28

A major role of metabolic alterations in the development of several human diseases, as diabetes, cancer and in the onset of ageing is becoming increasingly evident. This has a deep impact for human health due to the alarming increase in nutrient intake and obesity in the last decades. Fundamental aspects of how aberrant nutrient fluctuations trigger deregulated hormone levels and endocrine signals have been elucidated, being a prime example the phenomenon of insulin resistance. In contrast, how changes in nutrient levels elicit direct cell-autonomous signal transduction cascades and the consequences of these responses in physiology are less clear. The signalling circuitry of direct nutrient sensing converges with that of hormones in the regulation of the mechanistic target of rapamycin (mTOR) kinase, a driver of anabolism, cell growth and proliferation. Deregulation of mTORC1 activity underlies the pathogenesis of cancer and diabetes, and its inhibitor rapamycin is approved as an anti-cancer agent and delays ageing from yeast to mammals. In spite of its importance for human disease, our understanding of the nutrient sensing signalling pathway and its impact in physiology is largely incomplete, as only a few years ago the direct molecular link between nutrients and mTORC1 activation, the Rag GTPases, were identified. The present proposal aims to determine how the nutrient sensing signalling pathway affects mammalian physiology and metabolism, and whether its deregulation contributes to cancer, insulin resistance and aging. In particular, the objectives are: 1) To identify novel regulators of the Rag GTPases with unbiased and candidate-based approaches. 2) To establish the consequences of deregulated nutrient-dependent activation of mTORC1 in physiology, by means of genetically engineered mice. 3) To determine the impact of the nutrient sensing pathway in the effects of dietary restriction and nutrient limitation in glucose homeostasis and cancer.

1 846 494 €

Start date: 2016-01-01, End date: 2020-12-31

Maintenance and drug resistance of pancreatic ductal adenocarcioma (PDAC) depends on cancer cell intrinsic mechanisms and a stroma that supports tumor growth. Mouse models of human PDAC have provided important insights into the evolution of this highly lethal tumor, but there are no models that allow secondary genetic manipulation of autochthonous tumors, the tumor microenvironment or the metastatic host niche once the tumor has formed. We generated an inducible dual-recombinase system by combining Flp/frt and Cre/loxP. This novel PDAC model permits spatial and temporal control of gene expression enabling unbiased genetic approaches to study the role of tumor cell-autonomous and non-autonomous functions in endogenous cancers. This tool provides unparalleled access to the native biology of cancer cells and their hosting stroma, and rigorous genetic validation of candidate therapeutic targets. We performed tumor cell-autonomous and non-autonomous targeting, uncovered hallmarks of human multistep carcinogenesis, validated genetic tumor therapy, and showed that mast cells in the tumor microenvironment, which had been thought to be key oncogenic players, are in fact dispensable for tumor formation. In the proposed research program, we will 1) develop and further improve next-generation PDAC models, 2) deploy these systems to identify and target key features of PDAC maintenance in tumor cells and their microenvironment, and 3) discover mechanisms of treatment resistance. The application of cutting edge genetic engineering and screening technologies will allow us to address biological questions that could not be addressed before. The PanCaT project will open new horizons for the functional understanding of pancreatic cancer biology with a strong impact on clinical management and prognosis of PDAC patients. It will also produce a unique set of highly versatile and widely applicable genetic tools that will facilitate the study of PDAC at an organismal level.

2 440 275 €

Start date: 2016-02-01, End date: 2021-01-31

Defining the molecular mechanisms governing memory T cell differentiation and homeostasis is of pivotal importance to generate durable and protective T cell responses against infections and cancers. Considerable knowledge in this regard has been acquired in mouse models but is still limited about human T cells. In particular, some mechanisms are assumed to occur in humans but were never formally demonstrated. We showed that memory T cells adoptively-transferred with bone marrow transplantation failed to persist in recipient hosts in the absence of antigen. By contrast, self/tumor-specific naïve T cells rapidly acquired T memory stem cell (TSCM) attributes and subsequently reconstituted the memory T cell pool by homeostatic differentiation. Current models indicate human TSCM cells as superior to conventional memory T cells in regards to effector potential and persistence capacity. Genome-wide expression analysis identified candidate TSCM cell-specific transcriptional regulators that were shown to inhibit senescence, promote self-renewal and regulate somatic differentiation. In this project, by using single cell technologies, primary human samples and in vivo humanized models, we will define the molecular mechanisms at the basis of memory T cell formation and maintenance in humans. We will initially define the antigenic requirement for the long-term persistence of memory T cells by following the fate of adoptively-transferred T cells. As the field remains unexplored, we will investigate the acquisition of memory attributes by self/tumor-specific T cells on multiple functional levels. The gene products specifically expressed by self-renewing TSCM cells will be finally tested for their capability to arrest T cell differentiation and generate long-lived memory T cells with enhanced stem cell-like properties. Our results will impact multiple physiological and pathological situations involving T cell-mediated immune responses.

1 500 000 €

Start date: 2015-09-01, End date: 2020-08-31

Background: The human protozoan malaria parasite P. falciparum causes approximately 200 million infections and 0.7 million deaths (mainly children) per year. In the well-studied asexual blood stages, cyclic monocistronic gene activation occurs at the transcriptional level; however, relatively few transcription factors have been identified, thus other types of regulatory processes that contribute to this coordinated gene expression are believed to exist. Through the study of molecular process of monoallelic expression of immune evasion genes in P. falciparum (project funded by a previous ERC grant to A. Scherf), we discovered an entirely new mechanism of gene silencing. We demonstrated that an exoribonuclease silences genes linked to severe malaria. A non-canonical 3’-5’exoribonuclease termed PfRNase II destroys nascent RNA made from promoter regions, leading to cryptic unstable mRNA. Parasites carrying a deficient PfRNase II produce full-length mRNA and long noncoding RNA. The molecular events and the number of genes directly controlled by this novel type of posttranscriptional gene silencing remain elusive. Aim: This proposal aims to investigate the molecular mechanisms controlling PfRNase II-dependent gene silencing using innovative strategies such as the new genome editing technique (Cas9/CRISPR) developed in my laboratory for use in P. falciparum. We will study i) the recruitment of PfRNase II to promoter regions of severe malaria related genes using protein pull-down assays and ii) the genome occupancy of PfRNase II and two other 3’-5’ exoribonucleases to determine the total number of genes controlled by this mechanism. Impact: This project represents a major change in mainstream malaria parasite gene regulation paradigms with repercussions for other organisms. The proposed research will both open new avenues in molecular process that control severe malaria and appeal to young researchers to join this rather ‘untouched’ topic.

2 499 761 €

Start date: 2015-11-01, End date: 2020-10-31

The herpesvirus human cytomegalovirus (HCMV) infects the majority of the world's population, leading to severe diseases in millions of newborns and immunocompromised adults annually. During infection, HCMV extensively manipulates cellular gene expression to maintain conditions favorable for efficient viral propagation. Identifying the pathways that the virus relies on or subverts is of great interest as they have the potential to provide new therapeutic windows and reveal novel principles in cell biology. Over the past years high-throughput analyses have profoundly broadened our understanding of the processes that occur during HCMV infection. However, much of this analysis is focused on transcriptional changes at the lytic phase of infection leaving posttranscriptional regulation and the latent phase of the virus relatively untouched. Novel emerging technologies have the potential to extend our knowledge in areas that were heretofore unattainable. My overall goal is to decipher the multiple mechanisms by which HCMV modulates the host cell. For this, I will use multiple cutting-edge deep-sequencing and imaging technologies that will allow the analysis of novel aspects of host gene regulation during infection. Accordingly, the primary objectives of this research proposal are: 1) Deciphering posttranscriptional mechanisms that control cellular gene expression during HCMV infection; 2) Identifying and characterizing cellular protein diversification during infection; and 3) Uncovering the changes that occur in infected cells during latent infection. The knowledge generated from these objectives will provide us with a clearer depiction of the changes that take place during HCMV infection, which in turn can facilitate the development of novel anti-viral strategies. More broadly, with its comprehensive and complementarity approaches, this work will provide a paradigm for understanding how gene expression is regulated during a complex biological process.

1 500 000 €

Start date: 2015-06-01, End date: 2020-05-31

Small and large non-coding RNAs are essential components at the heart of gene expression regulation. The past fifteen years have witnessed the emergence of a new field of research impacting diverse domains of biology. Among these, virology is no exception and discoveries such as the antiviral role of RNA silencing, virus-encoded microRNAs (miRNAs), or miRNA-based regulation of viruses have notably shifted our views of host-virus interactions. Although we know a lot about the mechanisms of action of ncRNAs, and their role in the context of viral infections, we know much less regarding the control of the regulatory RNAs themselves. In other words, how are the regulators regulated? To provide answers to this burning question, we propose to use different viruses as models to investigate the various levels where modulation of regulatory RNA can occur. Thus, we will study the importance of RNA secondary and tertiary structure as well as accessory proteins in the regulation of miRNA primary transcript processing. In a second axis, we propose to investigate how the functional, mature miRNAs can be controlled. To this end, we will focus on the mechanisms of target-mediated miRNA decay and the role of competing endogenous RNAs. We will finally turn to the regulation of antiviral RNA silencing. Although it seems that this kind of defence mechanism exist in mammalian cells, it is not yet clear how physiologically relevant it is and how it interfaces with other innate immune mechanisms. In this multidisciplinary project, we will use a combination of techniques ranging from bioinformatics to cellular biology to achieve our goal to get a comprehensive view of how RNA silencing processes are regulated during virus infection.

1 998 291 €

Start date: 2016-01-01, End date: 2020-12-31

Endothelial cells (ECs) exhibit a remarkable and unique plasticity in terms of redox biology and metabolism. They can quickly adapt to oxygen, nitric oxide and metabolic variations. Therefore, EC must be equipped with a selective and unique repertoire of redox and metabolic mechanisms, that play a crucial role to preserve redox balance, and adjust metabolic conditions in both normal and pathological angiogenesis. The identification of such redox signaling and metabolic pathways is crucial to the gaining of better insights in endothelial biology and dysfunction. More importantly, these insights could be used to establish innovative therapeutic approaches for the treatment of those conditions where aberrant or excessive angiogenesis is the underlying cause of the disease itself. However, the formation, actions, key molecular interactions, and physiological and pathological relevance of redox signals in ECs remain unclear. Here, by using cutting-edge real-time redox imaging platforms, and innovative molecular and genetic approaches in different in vivo animal models, we will (1) reveal the working of redox signaling in EC in health and disease, (2) shed light on the novel role for the mevalonate metabolic pathway in angiogenesis and (3) provide solid evidence, that manipulation of endothelial redox and metabolic state by genetic alteration of the redox rheostat UBIAD1, is a valuable strategy by which to block pathological angiogenesis in vivo. The ultimate objective is to open the way for the development of innovative (cancer) therapeutic strategies and complement the existing ones based on genetic or pharmacological manipulation of redox rheostats to balance oxidative or reductive stress in angiogenic processes. The success of this project is built upon our major expertise in the field of angiogenesis in small vertebrate animal models as well as on the collaborations with leading laboratories that are active in research on the pre-clinical stages for angiogenesis-rel

1 999 827 €

Start date: 2016-07-01, End date: 2021-06-30

"RNA editing is a type of programmed RNA sequence alteration that can result in a range of proteomic changes, from subtle fluctuations in output, to specific alterations in protein content. Editing is catalyzed by two classes of deaminases: those which convert adenosine to inosine (ADARs) and those which convert cytosine to uracil (APOBEC1). We have previously shown that APOBEC1-catalyzed editing in the transcriptome of macrophages leads to the generation of populations that are heterogeneous, and functionally diverse, enabling rapid population adaptation to different environmental settings. Our first aim for this proposal is to extend our studies to additional immune cell subsets, focusing on cells that are recently recognized as ""plastic"" to define the contribution of editing to this plasticity of fate and function. RNA editing of the type we study has also been demonstrated to be crucial for cancer progression. For instance, APOBEC1-deficiency significantly reduces tumour burden on cells of the intestine and colon that are prone to adenocarcinomas in the context of the APC-min mutation. This is also the case for testicular carcinomas in mouse models of such tumours. Thus, there is genetic evidence for a requirement for APOBEC1 and RNA editing to drive tumour progression, in two tumour contexts. Based on these data and on our recently deciphered role for APOBEC1 as a ""stealthy"" diversifier of cellular transcriptomes (and proteomic outcomes), we hypothesize that APOBEC1 drives tumour progression by editing select transcripts in tumour cells (or tumour stem cells), thus enabling the rapid adaptation of the tumour to the onslaught of the immune response. Our second aim is to characterize the subset of edited transcripts in these model tumours (either at the population or at the single cell level) and understand their role to tumour survival and progression, both in mouse models of disease, and in human tumour samples (in collaboration with QP Hammarstrom, KI)."

2 270 000 €

Start date: 2016-01-01, End date: 2020-12-31

I propose to decipher the unresolved molecular paradox of positive versus negative gene regulation by the Glucocorticoid Receptor (GR). GR is one of the most potent anti-inflammatory drug targets in clinical use today, and one of the most powerful metabolic regulators. Unfortunately, its unique ability to efficiently shut off inflammatory gene expression is accompanied by serious side effects. These undesired effects are attributed to the transcriptional activation of its metabolic target genes and limit its therapeutic use. SILENCE uses cutting-edge genome-wide approaches to identify the molecular mechanisms underlying the transcriptional repression, or silencing, of inflammatory genes by GR. The general, open question I want to address is how one transcription factor can simultaneously both activate and repress transcription. GR is a member of the nuclear hormone receptor family of ligand-gated transcription factors. Upon hormone binding, GR can regulate gene expression both positively and negatively, but the mechanism governing this choice is unknown. I have previously shown that classical models and existing paradigms are insufficient to explain GR-mediated gene silencing. Therefore, I postulate the existence of unknown coregulator proteins, cis-regulatory DNA sequences, noncoding RNAs, or combinations thereof. To test these hypotheses, I plan 1. a large scale RNAi screen to identify those cofactors that specify repression versus activation, 2. ChIP-exo experiments to map genomic GR binding sites at an unprecedented resolution, and 3. GRO-Seq studies to define the role of noncoding RNAs during the silencing of inflammatory genes. Inflammation is known to contribute to the pathogenesis of numerous human illnesses, including cancer, autoimmune diseases, diabetes and cardiovascular disease. Understanding the specific mechanisms involved in the silencing of inflammatory gene expression carries transformative potential for novel therapies and safer drugs.

1 496 275 €

Start date: 2015-08-01, End date: 2020-07-31

Here we propose a strategy to control body weight and prevent/reverse obesity based on targeting brown adipose tissue (BAT) to facilitate negative energy balance and prevent adaptive responses to dietary restriction. However, BAT in humans is limited and poorly characterised. Thus, we propose to use stem cells as a tool to gain unique insights into the biology of human brown adipocytes. The General Objective is to identify pathways and factors of potential therapeutic relevance that promote BAT development and/or activation/recruitment using a stem cell based BAT differentiation approach, and then to functionally validate the role of these factors in vitro by genome engineering human stem cell derived adipocytes and in vivo by transplanting these cells into mice. Specific Aims are: 1.To identify molecular mechanisms involved in human brown adipose tissue development and activation. 2. To investigate the molecular mechanisms involved in human white adipose tissue browning/beige cells recruitment 3. To identify new agents/compounds of therapeutic value, able to activate or recruit human brown adipose tissue/brite cells. Experimental strategy: We will use a UCP1-reporter human pluripotent stem cell (PSC) line differentiated into brown and white adipocytes to identify genetic factors that may contribute to brown adipocyte differentiation/activation and white adipocyte browning. Following the identification of candidate genes, we will knock out, constitutively and/or inducibly, both alleles of these genes in human PSC cells, producing a total loss of function. Following the in vitro phenotyping of the cells we will proceed to the in vivo validation of the functional properties/phenotype of human PSC derived brown/brite adipocytes (wild-type and loss of function) by transplanting these cells into mice. Using these reporter tools we will also perform in vitro pharmacological screening and in vivo validation of new compounds that stimulate BAT activation and WAT browning

2 500 000 €

Start date: 2016-01-01, End date: 2020-12-31

The immunological synapse is a highly conserved scaffold for communication between immune cells built around cooperation of antigen and adhesion receptors. It often takes the form of a bull’s eye with a central cluster of antigen receptors surrounded by a ring of adhesion molecules. We have recently observed that antigen receptor coated extracellular microvesicles bud directly from the center of the immunological synapse- which we define as synaptic ectosomes. Synaptic ectosomes are transferred to the antigen- presenting cell and can generate signals after the T cell-APC synapse has dissolved. We aim to determine the composition of synaptic ectosomes, determine their fate in the antigen-presenting cell and identify approaches to manipulate their formation in vivo. The objectives will be to 1) isolate synaptic ectosomes from human T cells and determine their molecular composition; 2) determine the functional impact of synaptic ectosomes on the antigen presenting cell; and 3) use gene targeting to control the process in vivo to understand its role in T function of helper, cytotoxic and regulatory T cells. The technologies will include microscopy, proteomics, genomics, and in vivo models with constitutive and conditional gene targeting. This work will address fundamental gaps in our understanding of immune cell communication.

2 212 114 €

Start date: 2015-11-01, End date: 2020-10-31

The adult mammalian stomach can be divided into three distinct parts: From the proximal fore-stomach over the corpus to the distal pylorus. Due to constant exposure to mechanical stress and to hostile contents of the lumen, highly specialized cell types have to be constantly reproduced in order to maintain the function of the gastrointestinal tract. Recently, the applicant identified Troy+ chief cells as a novel stem cell population in the corpus epithelium. Troy+ chief cells displayed a very low proliferation rate indicating their quiescent nature compared to other known gastro-intestinal tract stem cells. Interestingly, these stem cells can actively divide upon tissue damage, suggesting distinctive statuses under conditions of homeostasis and injury. As Troy+ stomach stem cells exhibit interconvertible characteristics i.e. quiescent and proliferative, they represent a unique model of adult stem cells with which we can study 1) the dynamics of stem cell propagation in homeostasis and regeneration and the underlying mechanism of this switch by analysing molecular and epigenetic profiles. Subsequently, by analysing mRNA expression profiles and epigenetic changes in Troy+ stem cells between homeostasis and injury repair, we will generate a list of genes with potentially interesting functions in cell fate decisions. We will therefore investigate 2) the stomach stem cell programme in homeostasis and regeneration using in vitro and in vivo functional genetics. Lastly, we will characterise 3) human stomach stem cells in normal and pathological conditions. Here we pursue three main aims: - Investigating Troy+ stem cell dynamics during homeostasis and injury repair - Unmasking the stomach stem cell programme using in vitro and in vivo functional genetics - Characterising human stomach stem cells

1 570 399 €

Start date: 2015-08-01, End date: 2020-07-31

Vaccinia virus (VACV), the prototypic poxvirus, is a large, enveloped, DNA virus characterized by its cytoplasmic site of replication and large subset of genes. Due to the complexity of VACV, the majority of studies focus on the virus rather than the host cell. Thus the repertoire of cell factors and functions required for its replication remain largely unknown. Our previous work to define a subset of these, revealed the cellular degradation machinery as a key requirement of VACV replication. Our findings indicated that ubiquitin (Ub), Ub ligase activity, and proteasome-mediated degradation are required for multiple stages of the virus lifecycle. The aim of this proposal is to reveal how VACV differentially modulates or takes advantage of the Ub proteasome system during genome uncoating, the initiation of DNA replication, and the assembly of progeny virions. For genome uncoating we will characterize the spatial and temporal interactions between ubiquitinated viral proteins, proteasomes, the viral uncoating factor, and the viral genome that occur on cytoplasmic cores. To ascertain how Cullin-3 based ubiquitination and proteasome degradation facilitate the switch from uncoating to replication of the viral genome, we will identify the relevant Cullin-3 substrates in the context of a detailed characterization of viral replication initiation sites. Coming full circle, we will explore the mechanisms used by VACV to modulate cellular degradation such that ubiquitinated viral core proteins are packaged into newly forming virions without being degraded. Using systems biology, virology, cell biology, biochemistry, molecular biology and a wide range of microscopy approaches we will unravel the complex interactions between poxviruses and the host cell degradation machinery. In turn, as viruses often serve as valuable tools to study cell function, this work is likely to uncover new insights into how cells spatially and temporally regulate their own degradative capacities.

2 270 000 €

Start date: 2015-09-01, End date: 2020-08-31