More information on: https://www.rockefeller.edu/our-scientists/heads-of-laboratories/899-eric-d-siggia/

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Human embryonic stem cells when confined within two dimensional micropatterns with diameters in the 0.5-1mm range and subject to uniform Wnt or BMP stimulation, will spontaneously form radially symmetric spatial patterns that recapitulate the proximal-distal axis of the mammalian embryo at the time of gastrulation. This assay, when combined with inducible cell lines that express the canonical morphogens or inhibitors, becomes a very powerful technique to understand how an epithelium with ~2000 cells can self organize. To eliminate the boundaries that are inherent in 2D culture we have extended the assay to cells in defined hydrogels. They form closed apical-basal polarized shells that form a localized primitive streak in response to suitable morphogens. This quantitative assay shows in a context very different from the embryo how the canonical signaling pathways, generate spatial patterns over large scales, but it also reveals cell biological aspects of signaling that are difficult to study in mammalian embryos.

Maintenance of genome integrity requires the functional interplay between Fanconi anemia (FA) and homologous recombination (HR) repair pathways. Endogenous acetaldehyde, a product of cellular metabolism, is a potent source of DNA damage, particularly toxic to cells and mice lacking the FA protein FANCD2. We recently investigated whether HR-compromised cells are sensitive to acetaldehyde, similarly to FANCD2-deficient cells. We demonstrate that inactivation of HR factors BRCA1, BRCA2 or RAD51 hypersensitizes cells to acetaldehyde treatment, in spite of the FA pathway being functional. Aldehyde dehydrogenases (ALDHs) play key roles in endogenous acetaldehyde detoxification and their chemical inhibition leads to cellular acetaldehyde accumulation. We find that disulfiram (Antabuse), an ALDH2 inhibitor in widespread clinical use for the treatment of alcoholism, selectively eliminates BRCA1/2-deficient cells. Consistently, Aldh2 gene inactivation suppresses proliferation of HR-deficient mouse embryonic fibroblasts (MEFs) and human fibroblasts. Hypersensitivity of cells lacking BRCA2 to acetaldehyde stems from accumulation of toxic replication-associated DNA damage, leading to checkpoint activation, G2/M arrest and cell death. Acetaldehyde-arrested replication forks require BRCA2 and FANCD2 for protection against MRE11-dependent degradation. Importantly, acetaldehyde specifically inhibits in vivo the growth of BRCA1/2-deficient tumors and ex vivo in patient-derived tumor xenograft cells (PDTCs), including those that are resistant to poly (ADP-ribose) polymerase (PARP) inhibitors. This work therefore identifies acetaldehyde metabolism as a potential therapeutic target for the selective elimination of BRCA1/2-deficient cells and tumors.
 

Professor Will Wood received a Ph.D. in Developmental Biology from University College London before moving to Instituto Gulbenkian de Ciencia, Portugal, to work as a postdoctoral fellow. In 2006 he started his own research laboratory in the Department of Biology and Biochemistry at the University of Bath (UK) as a Wellcome Trust Career Development Fellow and later as a Wellcome Trust Senior Research Fellow. In April 2014 he moved to the School of Cellular and Molecular Medicine at the University of Bristol, UK where his lab investigates the molecular mechanisms that underlie Drosophila macrophage migration in vivo. He is particularly interested in how these immune cells prioritise competing cues such as damage signals released from wounds and 'eat me' signals arising from apoptotic corpses as well as the guidance cues that direct their developmental dispersal during embryogenesis.

The study of rare cancers and clinical phenotypes can reveal therapeutic opportunities as well as novel fundamental biological mechanisms. We will discuss multi-platform genomic profiling and functional studies in high risk hairy cell leukemia, pediatric gastrointestinal stromal tumor, and osteosarcoma, revealing genotype/phenotype associations in mitogenic signaling networks, metabolic effects on chromatin regulation, and mechanisms of telomere maintenance.

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Les signatures mutationnelles de sous-types moléculaires de tumeurs du sein dans l’étude PRECAMA (Premenopausal Breast Cancer in Latin American Women).

Degradation of essential amino acids is an immunosuppressive tumour escape strategy exploited by many tumours to hamper tumour-specific immune responses. However, the mechanisms by which cancer cells compensate for the shortage of essential amino acids in the local microenvironment remain unclear. We performed RNA-seq analysis of tumour cells expressing the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which revealed extensive remodelling of tumour cell metabolism, amino acid biosynthesis and amino acid transport gene expression. In particular, our findings demonstrated that expression of IDO or tryptophan 2,3-dioxygenase (TDO) induces up-regulation of several amino acid transporter genes, which allows improved glutamine and tryptophan import into tumour cells. We extended these results by demonstrating that re-programming of the amino transporter profile is caused by shortage of tryptophan, rather than accumulation of the tryptophan degradation by-product kynurenine, and is dependent on ATF4 expression, as ATF4 knockdown cells failed to up-regulate appropriate amino acid transporters under low tryptophan conditions. Our studies shed light on the compensatory mechanisms employed by tumours to allow survival under conditions of nutritional stress and have relevance for the identification of novel targets for pharmacological inhibition of tumours expressing amino acid-degrading enzymes IDO and TDO.

Inaccurate repair of broken chromosomes generates structural variants that can fuel evolution and inflict pathology. Homologous recombination (HR) seals DNA double strand breaks by using an intact donor molecule as a template for repair. As such it is considered a high fidelity repair mechanism. Yet, multiple mutant contexts have revealed that the HR machinery is also prone to induce chromosomal rearrangements and toxic intermediates. We describe a novel genomic instability mechanism in which translocation between intact chromosomes is induced by a lesion on a third chromosome. We named it “multi-invasions-induced rearrangement” (MIR), as it stems from a byproduct of the multiplexed homology search process in which a single Rad51-ssDNA filament invades more than one molecule simultaneously. MIR does not required the translocating donors to share homology, and the intervening sequence from the invading molecule is inserted at the translocation site. MIR is stimulated by increasing homology length and spatial proximity of the donors, and depends on the overlapping activities of the structure-selective endonucleases Mus81-Mms4, Slx1-Slx4, and Yen1. Conversely, the 3’-flap nuclease Rad1-Rad10 and enzymes known to disrupt recombination intermediates (Sgs1-Top3-Rmi1, Srs2 and Mph1) inhibit MIR. Resolution of MIR intermediates propagates secondary chromosome breaks that frequently cause additional rearrangements. MIR features have implications for the formation of simple and complex rearrangements underlying human pathologies.

https://research.pasteur.fr/fr/team/spatial-regulation-of-genomes/

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Gisou van der Goot studied engineering at the Ecole Centrale de Paris, then did a PhD in Molecular Biophysics at the Nuclear Energy Research Center, Saclay, France, followed by a postdoc at the European Molecular Biology Laboratory (EMBL) in Heidelberg. She started her own group in 1994 in the department of Biochemistry, University of Geneva, became Associate professor at the Faculty of Medicine (Univ. Geneva) in 2001 and finally Full Professor at the EPFL in 2006, where she co-founded the Institute of Global Health. In 2014, she was appointed Dean of EPFL's School of Life Sciences.

The integration of distinct endocytic routes is critical to regulate receptor signaling. A non-clathrin endocytic pathway (NCE) of the epidermal growth factor receptor (EGFR) is activated at high ligand concentrations, in parallel to clathrin-mediated endocytosis (CME). EGFR-NCE requires receptor ubiquitination, which also occurs at high EGF doses, and targets EGFRs to degradation, attenuating signaling. Despite its critical role in restricting EGFR signaling, the molecular mechanism of EGFR-NCE was poorly defined. Through an unbiased proteomic approach coupled to siRNA screening, we identified NCE-specific regulators, including the endoplasmic reticulum (ER)-resident protein reticulon-3 (RTN3), and a specific cargo, CD147. RTN3 was critical for EGFR/CD147 internalization via NCE, promoting the creation of plasma membrane (PM)–ER contact sites that were required for the formation/elongation of NCE tubular invaginations. Local Ca2+ release at these sites, triggered by IP3-dependent activation of ER Ca2+ channels, was needed for the completion of EGFR internalization and vesicle fission, in concert with the action of the GTPase dynamin. Thus, we identified a novel mechanism of EGFR endocytosis that relies on ER-PM contact sites and local Ca2+ signaling. This modality of internalization integrates numerous cellular functions towards its execution - as NCE regulators are proteins belonging to different cellular compartments, including ER, mitochondria and nucleus - and it is critical to mediate EGF-dependent cellular responses.

Cell membranes have developed a tremendous complexity of lipids and proteins geared to perform the functions cells require. To facilitate these functions, the bilayer has evolved the propensity to segregate its constituents laterally. This capability is based on dynamic liquid-liquid immiscibility and underlies the raft concept of membrane sub-compartmentalization. Key to understanding the principles underlying liquid-liquid de-mixing in cell membranes is the mutual weak interactions between sterols, sphingolipids
and raft proteins. The potential for sphingolipid-cholesterol self-assembly combines with protein specificity to dynamically regulate protein segregation within the membrane plane. The regulation of the two dimensional separation of lipids and proteins in membranes into dynamic liquid membrane rafts, separating from the surrounding bilayer, is dependent on the propensity for liquid phase separation. Most importantly, cellular plasma membranes seem to be poised close to a phase transition, facilitating dynamic sub-compartmentalization with little energetic cost.
The capability of liquid-liquid phase transitions depends on a tight control of cellular lipid composition. Amazingly, this stringent regulation translates into a new measure for metabolic status. We have developed a shotgun mass spectrometry platform that can analyse hundreds of lipids in only a few minutes with absolute quantification. This effective routine of shotgun lipidomics became possible by introducing a unique workflow that combined improved extraction protocols with cutting edge mass spectrometry and novel software. We have used this technology to analyse blood lipidomes from thousands of human subjects. The data show that the blood lipidomics can be used to measure health and disease.