ISREC - Annual Report 2014 by EPFL School of Life Sciences

EPFL School of Life Sciences ISREC - Swiss Institute for Experimental Cancer Research Report 2014

EPFL School of Life Sciences - 2014 Annual Report

ISREC

The Swiss Institute for Experimental Cancer Research (ISREC) has continued its contributions to the School of Life Sciences at EPFL, via cutting-edge research, mentoring young scientists, and classroom teaching. In addition, ISREC is playing a key role in the new Swiss Cancer Center Lausanne (SCCL), a joint venture with the University of Lausanne and its Hospital and Medical Center (CHUV). This new cancer center, announced in January 2013, has a mission statement to become the first comprehensive cancer center in Switzerland, as defined by depth and breadth in basic and translational cancer research, in clinical research and clinical trials of new therapies, and excellent care of cancer patients. ISREC, with 15 faculty research groups focused on cancer research or fundamental cell and developmental biology, brings exceptional strength and talent to this new cancer center. ISREC also has been centrally involved in community-building initiatives for the SCCL, including a series of annual faculty-only and faculty + staff retreats, held in the Spring and Fall, respectively. Both retreats - initiated in 2013 - have been successful at building bridges across the multiple sites in Lausanne that house cancer-related faculty.

Douglas Hanahan - Director

In addition to the retreats, The Lola and John Grace Distinguished Lectures in Cancer Research – sponsored by the Grace family – bring in eminent cancer scientists for a once-monthly lecture at EPFL that is televised to the CHUV and Biopole/Epalinges sites of the SCCL. Furthermore, ISREC sponsors a monthly faculty-only research presentation, and a weekly informal seminar series for ISREC students and postdocs. Finally, ISREC had a very successful bi-annual symposium in January 2014, held in Crans Montana, on Metastatic Colonization. There were two notable faculty transitions in 2013-2014: Lukas Kühn retired after 25 years studying molecular biology as a member of ISREC, and Elisa Oricchio joined ISREC as an assistant professor; Elisa is developing a research program investigating mechanisms of and innovative therapeutic strategies for follicular lymphoma, an understudied cancer type in need of new insights. http://sv.epfl.ch/ISREC

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ISREC - Swiss Institute for Experimental Cancer Research

Swiss Institute for Experimental Cancer Research

EPFL School of Life Sciences - 2014 Annual Report

Aguet Lab Michel Aguet

Full Professor

http://aguet-lab.epfl.ch/

Introduction

Michel Aguet, MD, held positions in academia and industry (associate professor, Institute of Molecular Biology, University of Zürich; director Molecular Oncology, Genentech, San Francisco) before he was appointed director of ISREC (1996-2009). He joined EPFL when ISREC became integrated into the school and led the National Center of Competence in Research (NCCR) in Molecular Oncology from 2001-2013. In the past, his research focused on interferon signaling, more recently on the role of the Wnt pathway in cancer cell differentiation. His laboratory will close in 2015 due to retirement.

Mutational activation of Wnt signaling is a key oncogenic event in most colorectal cancers (CRCs), but approaches targeting the Wnt pathway downstream of driving mutations prove challenging. Bcl9/9l proteins are components of the Wnt- β-catenin transcriptional activation complex, which is situated downstream of driver mutations in CRC. Our work focuses on exploring the role of the β-catenin-Bcl9/9l complex in mouse models of CRC. We have previously shown that in adult mice Bcl9/9l proteins are dispensable for normal homeostasis of the gastrointestinal epithelium. These results contrasted with the severe phenotypes observed upon ablation of other Wnt pathway components, which elicited strong proliferative defects resulting in crypt loss. Thus, in adult mammals, Bcl9/9l proteins seem to play a less critical role in Wnt signaling. Our previous work indicated that despite the only partial contribution of β-catenin-Bcl9/9l to Wnt transcriptional output, this complex might play a critical regulatory role in the maintenance of intestinal stem cells (ISC). While there were no overt anomalies in intestinal homeostasis, mice lacking Bcl9/9l proteins were deficient in epithelial regeneration as shown in an ulcerative colitis model, pointing to a possible deficiency in ISC expansion and/or maintenance. The role of Bcl9/9l in regulating stemness properties became overtly apparent in a mouse chemical carcinogenesis model of CRC, in which ablation of Bcl9/9l proteins resulted in virtual loss of ISC markers concomitantly with vastly down-regulated expression of Wnt targets and genes related to epithelial-mesenchymal transition (EMT).

Results Obtained in 2014

We made use of chemically or genetically induced mouse CRC models and RNA sequencing to establish a robust Bcl9/9l dependent gene expression signature, which showed a positive correlation of Bcl9/9l induced effects across the models. Importantly, this overlapping gene expression signature, which corroborated the striking Bcl9/9l dependence of stemness and EMT traits, correlated strongly with outcome in human CRC patients. The prognostic capacity of this Bcl9/9l core signature was independent of known clinical and molecular parameters as revealed by multivariate analyses. Therefore, the involvement of Bcl9/9l-β-catenin signaling in human CRC outcome is likely to depend on other prognostic tumor properties, such as tumor dissemination and/or chemoresistance. Even though underlying mechanisms remain elusive, our findings corroborate that the Bcl9/9l-β-catenin arm of Wnt-β-catenin signaling is critical for stemness maintenance, possibly through maximally enhancing the Wnt-β-catenin transcriptional output. This is in accordance with the notion that a gradient of Wnt-β-catenin signaling along the crypt villus axis regulates cellular hierarchy and continuous renewal, whereby strongest Wnt signaling is required for stem cell maintenance at the crypt bottom. Collectively, the rather subtle role of Bcl9/9l-β-catenin signaling that is critical for wound healing but not homeostasis in the normal intestine, yet strongly affects tumor traits associated with patient outcome, renders it attractive as a potential therapeutic target.

Keywords

WNT pathway, Bcl9/9l, cancer stem cells, epithelial-mesenchymal transition, cancer cell differentiation, drug target validation.

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EPFL School of Life Sciences - 2014 Annual Report

Team Members MD/PhD Student Andreas Moor

Bioinformatician Pascale Anderle

Administrative Assistant NCCR Christine Skaletzka

ISREC - Swiss Institute for Experimental Cancer Research

Postdoctoral Fellows Frédérique Baruthio Patrick Rodriguez

Immunoﬂuorescence staining of chemically induced mouse colon adenocarcinomas. Wildtype tumors, which strongly express stemness and EMT-related genes, stain positively for the mesenchymal intermediate filament vimentin (green) and show a discontinuous basement membrane (laminin; red). Bcl9/Bcl9lko tumors virtually lost stemness and EMT gene expression, which is paralleled by the loss of vimentin staining and the restoration of a continuous basement membrane, indicative of a more differentiated tumor phenotype.

Selected Publications » Cantu, C., Valenta, T., Hausmann, G., Vilain, N., Aguet, M., and Basler, K. (2013). The Pygo2-H3K4me2/3 interaction is dispensable for mouse development and Wnt signaling-dependent transcription. Development 140, 2377-2386. » Cantu, C., Zimmerli, D., Hausmann, G., Valenta, T., Moor, A., Aguet, M., and Basler, K. (2014). Pax6-dependent, but beta-catenin-independent, function of Bcl9 proteins in mouse lens development. Genes & Dev. 28, 1879-1884. » Christensen, J., Bentz, S., Sengstag, T., Shastri, V. P., and Anderle, P. (2013). FOXQ1, a novel target of the Wnt pathway and a new marker for activation of Wnt signaling in solid tumors. PloS One 8, e60051. » Pisco, A. O., Brock, A., Zhou, J., Moor, A., Mojtahedi, M., Jackson, D., and Huang, S. (2013). Non-Darwinian dynamics in therapy-induced cancer drug resistance. Nat. Commun. 4, 2467.

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Brisken Lab Cathrin Brisken

Associate Professor

http://brisken-lab.epfl.ch/

Introduction

Cathrin Brisken received an MD and a Doctorate in Biophysics from the University of Göttingen in 1993. She worked as a postdoc and research scientist at the Whitehead Institute, MIT, Cambridge, USA. She was assistant professor at the MGH Cancer Center, Harvard University before joining the NCCR Molecular Oncology at ISREC in 2002. In 2012 she was appointed Associate Professor and Dean of EPFL Doctoral School. Cathrin Brisken is a member of various scientific advisory boards and the International Breast Cancer Study Group (IBCSG) and of the “Hinterzartener Kreis”, the cancer think tank of the German Science Foundation (DFG).

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Breast cancer is a complex disease that is under hormonal control. We are interested in understanding why breast cancer incidence continues to increase and how we can prevent it. Our research focusses on the cellular and molecular mechanisms by which reproductive hormones control cell proliferation, cell-cell communication and morphogenesis in the breast in vivo and how deregulation of the response of various breast cell populations to hormones contributes to human breast carcinogenesis. We characterized the role of the reproductive hormones estrogens, progesterone, and prolactin in mammary gland development using the mouse as a model and identified key downstream mediators. Contrary to the widely held view that estrogens are tightly related to breast carcinogenesis, we have provided evidence that in adulthood, a key to breast cancer development is repeated exposure to progesterone. We are using side branching in the mouse mammary gland as a model of progesterone-induced changes in the human breast and determine the cellular and molecular events that are elicited by progesterone in vivo in the mouse mammary gland. The work in mice is complemented by studies with fresh human breast tissue specimens, that we obtain in collaboration with groups at the CHUV and that we process in the laboratory to assess the relevance of the findings in mice to the human breast.

Keywords

Hormones, mammary gland development, breast carcinogenesis, paracrine signaling, estrogen, progesterone, RANKL, Wnt-4, stem cells, preclinical xenograft models.

Results Obtained in 2014

The ovarian hormones, estrogens and progesterone, increase breast cancer risk by poorly understood mechanisms. We have assessed the role of progesterone in mammary epithelial stem cell function. We have done so by serially transplanting mouse mammary epithelia that are either WT or lack specific genes. We found that in the absence of progesterone receptor signaling, the regeneration capacity of the mammary epithelium was severely impaired. Receptor activator of NF-kB ligand (RANKL), a progesterone receptor target gene, previously linked to stem cell function, was not required for the regenerative potential of the mammary epithelium. However, deletion of Wnt4, another factor we had identified as a downstream mediator of progesterone receptor signaling reduced the mammary regeneration capacity even more than ablation of progesterone receptor signaling. We used a novel sensitive reporter to reveal so far undetected perinatal expression of Wnt4 and showed that, at this developmental stage, Wnt4 expression is independent of hormone signaling. This early, previously unappreciated, Wnt4 expression was found to be functionally important. Later, during puberty and adulthood the central stem cell control factor, Wnt4 is exquisitely controlled by PR signaling. We provided genetic evidence that canonical Wnt signaling in the myoepithelium requires progesterone receptor signaling and Wnt4 expression, whereas the canonical Wnt signaling activities observed in the embryonic mammary bud and in the stroma around terminal end buds were independent of Wnt4. Thus, progesterone and Wnt4 control stem cell function through a luminal–myoepithelial crosstalk with Wnt4 acting independent of progesterone receptor signaling perinatally.

EPFL School of Life Sciences - 2014 Annual Report

Team Members Postdoctoral Fellows Valerian Dormoy Georgios Sflomos Stéphanie Cagnet

PhD Student Dalya Ataca Duje Buric Rachel Jeitziner Valentina Scabia Marie Shamseddin

Master’s Student Caroline Contat Lab Manager Ayyakkannu Ayyanan

Technicians Mélanie Wirth Laura Battista Administrative Assistant Valérie Pahud

ISREC - Swiss Institute for Experimental Cancer Research

Senior Scientist Cécile Lebrand

Model of Wnt4 action in the mammary epithelium. Progesterone stimulation results in Wnt4 induction in the progesterone receptor+ luminal cells (LC), the ‘sensor cells.’ The secreted Wnt4 acts on adjacent basal/myoepithelial cells (MC). In the myoepithelial cells, Wnt4 activates canonical Wnt signaling which induces changes in gene expression. This results in the secretion of factors and changes in the ECM (light blue arrows) that in turn impinge on stem cells (SC), luminal-restricted stem cells (L-RSC), and basal-restricted stem cells (B-RSC). Wnt4 may also act directly on stem cells that are found within the basal layer. Repeated activation of this intercellular signaling cascade downstream of progesterone receptor signaling may promote tumorigenesis by expanding luminal progenitor cells with oncogenic mutations and by expanding the stem/progenitor cell compartment.

Selected Publications » Tanos, T.,Sflomos, G., Echeverria, P. C., Ayyanan, A., Gutierrez, M., Delaloye, J.-F., Raffoul, W., Fiche, M., Dougall, W., Schneider, P., Yalcin-Ozuysal,O., Brisken C (2013) Progesterone/ RANKL is a major regulatory axis in the human breastSci Transl Med 5 (182) » Cimino, D., C. De Pittà, F. Orso, S. Casara, M. Zampini, C. Romualdi, E. Penna, E. Quaglino, M. Forni, C. Damasco, E. Pinatel, P. Provero, R. Ponzone, C. Brisken, M. De Bortoli, N. Biglia, G. Lanfranchi and D. Taverna.(2013) miR148b is a major coordinator of breast cancer progression in a relapse-associated microRNA signature by targeting ITGA5, ROCK1, PIK3CA, NRAS and CSF1FASEB J; 27(3):1233-35. » Hickman JA, Graeser R, de Hoogt R, Vidic S, Brito C, Gutekunst M, van der Kuip H; IMI PREDECT Consortium. (2014) Three-dimensional models of cancer for pharmacology and cancer cell biology; capturing tumor complexity in vitro/ex vivo Biotechnol J. (9):1115-28. » Brisken C., Sflomos, G., Tanos, T.(2013) Reply to Is progesterone a neutral or protective factor for breast cancer? Nat Rev Cancer.14(6):146. » Sflomos, G., Brisken C. (2013) A new Achilles Heel in breast cancer? Oncotarget Aug;4(8):1126-7. » Brisken C. (2013) Progesterone signalling in breast cancer: a neglected hormone coming into the limelight. Nat Rev Cancer.13(6):385-96. » Soto AM, Brisken C, Schaeberle C, Sonnenschein C. (2013) Does Cancer Start in the Womb? Altered Mammary Gland Development and Predisposition to Breast Cancer due to in Utero Exposure to Endocrine Disruptors.J Mammary Gland Biol Neoplasia.18(2):199-208. doi: 10.1007/s10911-013-9293-5.

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EPFL School of Life Sciences - 2014 Annual Report

Constam Lab Daniel B. Constam

Associate Professor

http://constam-lab.epfl.ch/

Introduction

Daniel Constam obtained his doctoral degree from ETH Zürich for studies on TGFβ isoforms in the central nervous system. He conducted postdoctoral studies as an EMBO fellow at Harvard University to investigate the regulation of TGFβ signaling by proprotein convertases. He joined ISREC as a group leader in 2000 and EPFL in 2005. He has been associate professor at the School of Life Sciences since 2007. He is interested in the molecular interactions between stem cells and their microenvironments that govern morphogenesis and tissue homeostasis.

We investigate novel molecular mechanisms that govern the differentiation and self-renewal of pluripotent stem cells in the mammalian embryo. Genetic and biochemical approaches are used to decipher specific interactions with the microenvironment, and how certain cancers may co-opt such developmental programs to promote tumor progression. Identifying cues that guide the maturation of progenitor cells into functional tissues during development is important because impaired differentiation increases the aggressiveness of tumor cells and limits the use of stem cell-derived transplants in regenerative medicine. A current focus is to image proprotein convertases in cells and transgenic mice using new reporter substrates as biosensors, and on elucidating the role of these secreted enzymes in the processing of specific growth factors and cell-cell adhesion molecules. Of particular interest to us are TGFβrelated factors and how dimerization and cleavage of their prodomains regulates ligand stability and signaling range. Proprotein convertases also activate other oncogenic factors as well as neurotrophic signals, but the regulation of these proteases at the level of subcellular compartmentalization and their potential as drug targets remain to be investigated. We also study gene silencing by the RNA-binding protein Bicc1 and its role in regulating cAMP/PKA and Wnt signaling. We have shown that mutations in Bicc1 randomize visceral left-right asymmetry and instigate cystic growth in the kidney and pancreas. Identifying relevant interacting proteins and target RNAs should shed important new light on the etiology of polycystic kidney diseases and pave a way for improved therapy of renal cysts.

Keywords

Imaging, proprotein convertases, TGFβ signaling, stem cells and cancer, cell adhesion, mRNA silencing, polycystic kidney diseases, ciliopathies.

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Results Obtained in 2014

Development of placental mammals until the blastocyst stage segregates a pluripotent inner cell mass (ICM) from trophectoderm that enables survival in the uterus. Proliferating stem cells in the ICM give rise to pluripotent epiblast cells that form endoderm, mesoderm or ectoderm during gastrulation. Epiblast stem cells and their germ layer allocation are specified by distinct signaling thresholds of the TGFβ family member Nodal. Potentiation of Nodal signaling in the epiblast requires cleavage of the Nodal precursor by cell non-autonomous forms of the proprotein convertases (PC) Pace4 and Furin that are secreted by the extra-embryonic microenvironment. However, cleavage mutant Nodal precursor can still induce mesoderm, raising the question “how”. To address this, we compared mature and uncleaved forms of Nodal for their potential to synergize with the co-ligands Gdf1 or Gdf3. Biochemical analysis revealed that Nodal heterodimerizes with Gdf1 and remains associated with cleaved prodomains, and that cleavage of the Gdf1 subunit was sufficient to significantly synergize even with uncleaved Nodal. To our surprise, soluble mature Nodal was unable to signal in the absence of serum except as a heterodimer with full-length Gdf1. Our results suggest that paracrine Nodal signaling requires heterodimerization with Gdf1 or Gdf3 and binding to their cleaved prodomains. Recent live imaging revealed Furin and Pace4 activities already in the blastocyst, together with an unidentified convertase. Analysis of compound mutant embryos showed that this activity involves PC7 and mediates morula compaction (manuscript submitted). Analysis of PC7 single mutants in addition led to the identification of BDNF as the first validated in vivo substrate. Impaired BDNF processing may accelerate the onset of Alzheimer’s disease. However, this hypothesis awaits further investigation since a mouse model that we used to test this prediction failed to develop AD-like disease under pathogen-free conditions.

EPFL School of Life Sciences - 2014 Annual Report

Team Members PhD Students Florian Bernet Teresa Didonna Lucia Leal-Esteban Pierpaolo Ginefra

Master’s Student Mariela Castelblanco

Laboratory Assistants Stéphane Baflast Séverine Urfer

Administrative Assistant Virginie Kokocinski

ISREC - Swiss Institute for Experimental Cancer Research

Postdoctoral Fellows Sylvain Bessonnard Prudence Donovan Christophe Fuerer Benjamin Rothé

Characterization of Nodal.Gdf1. A) Nodal expression and B) signal transduction. C) Purification of Nodal.Gdf1 heterodimer. D) Activity of Nodal.Gdf1 in HepG2 reporter cells. Gdf1 alone is inactive (not shown). E) Nodal.Gdf1 is serum (FBS)-independent. F) Active Nodal.Gdf1 is bound to its prodomains.

Selected Publications » Constam, D. B. (2014). Regulation of TGFβ and related signals by precursor processing. Semin. Cell Dev. Biol. 32, 85-97. » Fuerer, C., Nostro, M. C. and Constam, D. B. (2014). Nodal·Gdf1 heterodimers with bound prodomains enable serum-independent Nodal signaling and endoderm differentiation. J. Biol. Chem. 289, 17854-17871. » Tzanoulinou, S., Brandi, R., Arisi, I., D’Onofrio, M., Urfer, S. M., Sandi, C., Constam, D. and Capsoni, S. (2014). Pathogen-Free Husbandry Conditions Alleviate Behavioral Deficits and Neurodegeneration in AD10 Anti-NGF Mice. Journal of Alzheimer’s Disease 38, 951-964. » Wetsel, W. C., Rodriguiz, R. M., Guillemot, J., Rousselet, E., Essalmani, R., Kim, I. H., Bryant, J. C., Marcinkiewicz, J., Desjardins, R., Day, R., et al. (2013). Disruption of the expression of the proprotein convertase PC7 reduces BDNF production and affects learning and memory in mice. Proc. Natl. Acad. Sci. U. S. A. 110, 17362-17367. » Essalmani, R., Susan-Resiga, D., Chamberland, A., Asselin, M. C., Canuel, M., Constam, D., Creemers, J. W., Day, R., Gauthier, D., Prat, A., et al. (2013). Furin is the primary in vivo convertase of angiopoietin-like 3 and endothelial lipase in hepatocytes. J. Biol. Chem. 288, 26410-26418.

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De Palma Lab Michele De Palma

Tenure-track Assistant Professor

http://depalma-lab.epfl.ch

Introduction

Miki De Palma graduated in Biology (1999) and obtained a PhD degree in cell biotechnologies (2004) from the University of Torino Medical School, Italy, with a thesis on the regulation of tumor angiogenesis by bone-marrowderived cells. He performed post-doctoral training (20052008) at the Telethon Institute for Gene Therapy in Milan, where he developed strategies for engineering monocytes and reprogramming them into antitumoral immune cells. He was appointed group leader at the San Raffaele Institute, Milan, in 2008 (tenured in 2011), and joined ISREC/EPFL in 2012. He serves on the advisory boards of several international journals, including Science Translational Medicine and Cell Reports.

Early works of De Palma and colleagues have illustrated the important contribution of monocytes/macrophages to tumor angiogenesis and progression, and their functional heterogeneity in mouse and human cancer. By employing genetically engineered mouse models (GEMMs) of cancer, our laboratory investigates the interplay among macrophages, blood vessels and T-cells in tumors. By pharmacologically or genetically targeting these tumor-associated cells and their reciprocal cross talk, we aim to reprogram the immunosuppressive tumor microenvironment and rationally design combination treatments that may enhance the efficacy of conventional anticancer drugs and achieve more effective and enduring responses in cancer patients. Current research topics include: • The analysis of the molecular and functional heterogeneity of macrophages in mouse and human tumors; • The engineering and reprogramming of monocytes for adoptive cell therapy of cancer; • The role of macrophages in tumor responses to antiangiogenic (anti-VEGFA/angiopoietin-2) and immunostimulatory (immune check-point blockade) therapies; • The influence of macrophages on tumor evolution and response to therapies targeting pro-proliferative signaling (RTK inhibitors); • The mechanisms of tumor resistance to antiangiogenic therapy, including compensatory proangiogenic pathways (angiopoietin-2/TIE2) and the role of the extra-cellular matrix; • microRNA regulation of the macrophage phenotype in tumors; • The molecular determinants of microRNA sorting to exosomes and their transfer to acceptor cells (macrophage-endothelial cross talk).

Keywords

Macrophages, tumor angiogenesis, antiangiogenic therapy, immunotherapy, angiopoietin-2, microRNAs, exosomes, GEMMs of cancer, gene modification of hematopoietic cells, lentiviral vectors.

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Results Obtained in 2014

During the past two years we have mainly focused on the following projects: 1) Tumor resistance to anti-VEGFA therapy. Angiopoietin-2 (ANG2/ANGPT2) is a pro-angiogenic factor that promotes tumor angiogenesis in concert with vascular-endothelial growth factor-A (VEGFA). We previously showed that blocking ANG2 in mouse models of cancer inhibits tumor angiogenesis and metastasis. However, it was unclear whether blocking ANG2 could induce long-lasting antitumor responses, and/or overcome tumor resistance to VEGFA-targeted drugs. We have now shown that ANG2 plays an important role in driving tumor resistance to anti-VEGFA therapy, but only in selected cancer types. Specifically in these tumors, targeting VEGFA induced a compensatory response involving increased ANG2 levels, which promoted tumor resistance to anti-VEGFA therapy. Co-targeting VEGFA and ANG2 signaling in these tumors effectively blocked angiogenesis, halted cancer progression, and reversed tumor resistance to anti-VEGFA therapy (Rigamonti, Kadioglu, et al., 2014). 2) Role of microRNAs in exosome-mediated macrophage-to-endothelial communication. Our unpublished data show that microRNAs operate to sustain the protumoral functions of macrophages while limiting their immunostimulatory and antitumoral potential. We also found that macrophages secrete microRNAs via small microvesicles, called exosomes, which can fuse with and deliver their RNA cargo to neighboring cells. Interestingly, we noted that the microRNA composition of exosomes differed from that of the producer macrophages, suggesting an active mechanism of exosomal sorting. By employing cellular assays, RNA sequencing and bioinformatics tools, we determined that the sorting of microRNAs to macrophage-derived exosomes is partly regulated by the cellular levels of their targeted gene transcripts, which fluctuate in response to cell activation (see Figure). These findings have revealed a general (motif-independent) mechanism of microRNA sorting to exosomes, which may have implications for macrophage-to-endothelial cell communication in the tumor microenvironment (Squadrito, Baer, et al., 2014).

EPFL School of Life Sciences - 2014 Annual Report

Team Members PhD Students Caroline Baer Ece Kadioglu Shodai Takahashi (visiting)

Master’s Students Daniela Pais Ferreira Lucie Giesbrecht Luisa Spisak Fabien Jammes

Lab Manager Celine Rmili Wyser Bachelor’s Students Ka Ho Nicholas Cheung Yannick Francioli

Technicians Claudio Maderna; Axel Bellotti Administrative Assistant Soledad Andany

ISREC - Swiss Institute for Experimental Cancer Research

Postdoctoral Fellows Mario Leonardo Squadrito Nicolo’ Rigamonti Daniela Biziato Ioanna Keklikoglou

Mechanism of microRNA sorting to macrophage-derived exosomes. RNA profiling of macrophages and their exosomes shows that microRNA sorting to exosomes is modulated by cell-activationdependent changes of microRNA target levels in the producer cells. Genetically perturbing the expression of individual microRNAs or their targeted transcripts promotes bidirectional microRNA relocation from the cell cytoplasm/P bodies (sites of microRNA activity) to multivesicular bodies (sites of exosome biogenesis) and controls microRNA sorting to exosomes. For details, see Squadrito et al. (2014).

Selected Publications » Squadrito, M.L.*, Baer, C.*, Burdet, F., Maderna, C., Gilfillan, G.D. Lyle, R., Ibberson, M., & De Palma, M. (2014). Endogenous RNAs modulate microRNA sorting to exosomes and transfer to acceptor cells. Cell Rep. 11;8(5):1432-1446. » Keklikoglou I. & De Palma, M. (2014). Vascular modulatory functions of macrophages. In Macrophages: Biology and Role in the Pathology of Diseases. Edited by S. Biswas and A. Mantovani. 2014 Springer Science, New York, NY USA, pp 131-168. » Rigamonti, N.*, Kadioglu, E.*, Keklikoglou, I., Wyser Rmili, C., Leow, C.C., & De Palma, M. (2014). Role of angiopoietin-2 in adaptive tumor resistance to VEGF signalling blockade. Cell Rep. 7;8(3):696-706. » Escobar, G., Moi, D., Ranghetti, A., Ozkal-Baydin, P., Squadrito, M.L., Kajaste-Rudnitski, A., Bondanza, A., Gentner, B., De Palma, M., Mazzieri, R., & Naldini, L. (2014). Genetic engineering of hematopoiesis for targeted IFN-α delivery inhibits breast cancer progression. Sci Transl Med. 6(217):217ra3. » Squadrito, M.L., Etzodt, M., De Palma, M.*, & Pittet, J.M.* (2013). MicroRNA-mediated control of macrophages and its implications for cancer. Trends Immunol. 34(7):350-9. » Patel, A.S., Smith, A., Nucera, S., Biziato, D., Saha P, Attia RQ, Humphries J, Mattock K, Grover SP, Lyons OT, Guidotti LG, Siow R, Ivetic A, Egginton S, Waltham M, Naldini L, De Palma, M.*, & Modarai, B.* (2013). TIE2-expressing monocytes/macrophages regulate revascularization of the ischemic limb. EMBO Mol Med. 5(6):858-69. » De Palma, M. & Lewis, C.E. (2013). Macrophage regulation of tumor responses to anticancer therapies. Cancer Cell 18; 23(3):277-286.

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Duboule Lab Denis Duboule

Full Professor - EPFL & University of Geneva

http://duboule-lab.epfl.ch/

Introduction

Denis Duboule earned his PhD in Biology in 1984. He is currently Professor of Developmental genetics and genomics at the EPFL and at the department of Genetics and Evolution of the University of Geneva. Duboule has a longstanding interest in the function and regulation of Hox genes, a family of genes responsible for the organization and evolution of animal body plans. More generally, he is interested in transcriptional regulation during development and evolution. He is an elected member of several academies and societies and has received many national and international awards.

Our laboratory opened at EPFL in 2007. Its major aim is to study some of the principles of mammalian embryological developmental genetics by using the recent tools of functional genomics. A special focus is given to those similarities and differences that exist between the embryological development of vertebrates (to whom mammals belong) and those of other animals (invertebrates), from whom vertebrates derive. To achieve this task, we use the developing mouse embryo in vivo as an experimental system, and try and apply the methodology developed following the sequencing of complex genomes. Our major aim is the understanding of the regulation of a critical family of transcription factors during the construction of the animal body plan, referred to as architect genes (the Hox gene family). These genes have a special interest in the study of both our ontogeny (our development as individuals) and our phylogeny (our origin as a group of individuals) and the detailed understanding of their regulations and functions will be an important step in the deciphering of our evolutionary and developmental trajectories.

Keywords

Embryos, development, evolution, transcription, epigenetic regulation, Hox gene clusters, enhancers.

Results Obtained in 2014

Over the past two years, progress has been made along several lines of research. By using biochemical, genetic and epigenetic approaches, we have finally managed to obtained a fair understanding of the collinear mechanism at work during limb development, a project that started in 1989 with the discovery of this intriguing phenomenon, whereby the order of Hox genes along the chromosomal locus does reflect the place and the time where these genes will be transcribed during development. To understand the molecular mechanisms underlying this process, we first had to localize and characterize the enhancer sequences driving Hox gene expression during limb development. We observed that such sequences are found within large gene deserts flanking the HoxD cluster. We next deciphered the chromatin structure around the HoxA and HoxD clusters, precisely in those places where such numerous long-range acting enhancers are located. We observed that the two gene deserts flanking the HoxD cluster perfectly match the extent of Topologically Associating Domains (TADs), as defined by the HiC technology (data from the Bing Ren laboratory). These large structural and regulatory domains are specifically acting during the development of different structures, with Hox genes contacting alternatively either the telomeric, or the centromeric TAD. We also observed that both the HoxA and HoxD clusters evolved similar regulatory structures and hence we speculate that such a large integrated regulatory landscapes were already present in an ancestor animal, which only had one gene cluster, before the emergence of present days vertebrate animals. We also speculate that these TADs may have been instrumental in the emergence of long-range acting enhancer sequences, by providing chromatin niches where to evolve regulatory sequences. Likewise, these structures may have helped develop pleiotropic regulation, i.e. to recruit an entire regulatory machinery from one tissue to the other, in the course of evolution. We provided a possible example of such a process by showing how similar Hox gene regulations are between the development of digits and the external genital organs. In parallel, we imported and developed in the laboratory optical tools allowing for the direct visualization of these regulatory structures (figure 1). In this context, we have used either classical DNA FISH methodologies or super-resolution (STORM) approaches in collaboration with the laboratory of Sulyana Manley (School Basic Science - EPFL).

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Team Members PhD Students Fabrice Darbellay Nicolas Lonfat Rita Lhopitallier

Master’s Students Cécile Fruchard Olivier Selmoni Alexandra Iouranova

SNF Ambizione Fellow Anouk Necsulea

Technician Elisabeth Joye

Visiting Fulbright Student Ben Mormann

Administrative Assistant Doris Sapin

ISREC - Swiss Institute for Experimental Cancer Research

Postdoctoral Fellows Pierre Fabre Daan Noordermeer Nayuta Yakushiji-Kaminatsui

Super-resolution (STORM) microscopy is used to resolve the various levels of decompaction of the HoxD cluster, using DNA probes (A) scanning the locus (B,C,D : coll. with the laborartory of Sulyana Manley (EPFL). E, F. 3D DNA-FISH to assess the relative conformations of Topologically Associating Domains (TADs) in the developing forelimb.

Selected Publications » » » » »

N. Lonfat, T. Montavon, F. Darbellay, S. Gitto and D. Duboule (2014). Convergent evolution of complex regulatory landscapes and pleiotropy at Hox loci. Science, 346, 1004-1006, 2014. J. Lopez-Rios, A. Duchesne, D. Speziale, G. Andrey and K. A. Peterson et al. (2014) Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs. Nature, 511, 548-553. G. Andrey and D. Duboule. SnapShot: Hox Gene Regulation (2014). Cell, 4. De Laat, W. and Duboule, D. (2013) Topology of mammalian developmental enhancers and their regulatory landscapes. Nature, 502, 499-506. Andrey, G., Montavon, T., Mascrez, B., Gonzalez, F., Noordermeer, D., Leleu, M., Trono, D., Spitz, F. and Duboule, D. A switch between topological domains underlies collinearity in mouse limbs. (2013) Science, 340(6137):1234167

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Gönczy Lab Pierre Gönczy

Full Professor

http://gonczy-lab.epfl.ch/

Introduction

Pierre Göncyz obtained his PhD from The Rockefeller University (New York City, USA) in 1995. Thereafter, he conducted postdoctoral work at the EMBL (Heidelberg, Germany), before starting his laboratory in Lausanne in 2000 at ISREC, joining EPFL in 2005.

We are interested in understanding fundamental cell division processes and focus on two that are crucial for genome integrity: centriole formation and asymmetric cell division. To uncover the underlying mechanisms, we use a combination of genetic, functional genomic, biochemical, proteomic and cell biological approaches, primarily in the nematode C. elegans and human cells in culture. Centriole formation - Centrioles are evolutionarily conserved organelles essential for the assembly of cilia, flagella, and centrosomes, and which are characterized by a striking 9-fold radial symmetry of microtubules. Despite their importance, the mechanisms governing centriole formation have remained elusive until recently. We and others identified five proteins required for centriole formation in C. elegans, the relatives of which are likewise crucial in other organisms. In collaboration with the Steinmetz laboratory, we discovered that one of these protein families (SAS-6 proteins) can form 9-fold symmetric rings, leading us to propose that their self-assembly dictates the 9-fold symmetry of centrioles. Asymmetric cell division - Asymmetric division is crucial for generating diversity during development and stem cell lineages. For successful asymmetric division, the mitotic spindle must be positioned in a manner that ensures proper segregation of cytoplasmic constituents to daughter cells. Work performed in many laboratories, including ours, indicates that spindle positioning requires an evolutionary conserved ternary complex, which anchors the minus end directed motor protein complex dynein at the cell cortex. Dynein is thought to generate pulling forces on astral microtubules that emanate from the spindle poles, thus positioning the mitotic spindle.

Keywords

Results Obtained in 2014

Centriole formation - We have pursued an innovative multidisciplinary research program to gain novel insight into the mechanisms governing centriole formation. Thus, we elucidated the complete architecture of the centriole proximal part using cryo-tomography (Guichard et al., 2013; Figure 1). Moreover, we conducted an siRNA-based functional genomic screen that enabled us to identify novel genes that regulate centriole number in human cells (Balestra et al., 2013). In C. elegans, we reported that SAS-1 encodes a C2 domain protein that is critical for centriole integrity and whose homologue is mutated in a human ciliopathy syndrome (von Tobel et al., 2014). Overall, these and other findings have led to a better understanding of the mechanisms governing centriole formation. Asymmetric cell division - We have continued our work on spindle positioning in both C. elegans and human cells to better understand the mechanisms governing asymmetric cell division. In human cells, we uncovered that levels of cortical dynein increase during anaphase in a manner that depends on dephosphorylation of the ternary component NuMA at a specific threonine residue (Kotak et al., 2013). Further work established that NuMA directly associates with PtdInsP (PIP) and PtdInsP2 (PIP2) phosphoinositides in vitro, and that depletion of PIP/PIP2 prevents NuMA cortical localization in vivo. These findings uncover a novel function for plasma membrane phospholipids in governing cortical dynein distribution (Kotak et al., 2014). In C. elegans, we have shown notably that clathrin negatively regulates pulling forces acting on centrosomes during interphase and on spindle poles during asymmetric division. Our work, which includes computational modeling, reveals that clathrin plays a critical role in this instance by promoting acto-myosin cortical tension (Spiro et al., 2014). The above experiments have contributed to enhance understanding of the mechanisms governing metazoan spindle positioning.

Cell biology, developmental biology, cell division, centriole formation, spindle positioning, C. elegans, human cells.

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EPFL School of Life Sciences - 2014 Annual Report

Team Members PhD Students Alessandro De Simone Melina Scholze Veronika Villimova

Technicians Coralie Busso Isabelle Fluckiger

Administrative Assistant Nicole De Montmollin

ISREC - Swiss Institute for Experimental Cancer Research

Postdoctoral Fellows Nicola Brown Paul Guichard Virginie Hamel Sachin Kotak Jian Quiu Lukas von Tobel Benita Wolf

Schematic (left), super-resolution image (SIM, center) and interpretation thereof (right) of centriole pair isolated from the green alga Chlamydomonas reinhardtii stained with antibodies against acetylated-tubulin (green) and the SAS-6 protein Bld12p (purple). See Hamel et al. 2014 for details.

Selected Publications » von Tobel L, Mikeladze-Dvali T, Delattre M, Balestra FR, Blanchoud S, Finger S, Knott G, Müller-Reichert T, Gönczy P. (2014) SAS-1 is a C2 Domain Protein Critical for Centriole Integrity in C. elegans. PLoS Genet. Nov 20;10(11):e1004777. doi: 10.1371/journal.pgen.1004777. » Kotak S., Busso C. and Gönczy P. (2014) NuMA interacts with phosphoinositides and links the mitotic spindle with the plasma membrane EMBO J. 33:1815-1830. » Spiró Z., Thyagarajan K., De Simone A., Träger S., Afshar, K. and Gönczy P. (2014) Clathrin regulates centrosome positioning by promoting acto-myosin cortical tension in C. elegans embryos Development 141:27122123. » Keller D., Orpinell M., Olivier N., Wachsmuth M., Mahen R., Wyss R., Hachet V., Ellenberg J., Manley S. and Gönczy P. (2014) Mechanisms of HsSAS-6 assembly during centriole formation in human cells. J. Cell Biol. 204:697-712. » Guichard P., Hachet V., Majubu N., Neves A. , Demurtas D., Olieric N., Fluckiger I., Yamada A., Kihara K., Nishida Y., Moriya S., Steinmetz M.O., Hongoh Y. and Gönczy, P. (2013) Native architecture of the centriole proximal region reveals novel features underlying their 9-fold radial symmetry Curr. Biol. 23:1620-1628. » Balestra F.R., Strnad P., Flückiger I. and Gönczy P. (2013) Discovering Regulators of Centriole Biogenesis through siRNA-Based Functional Genomics in Human Cells. Dev Cell. 25:555-571.

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EPFL School of Life Sciences - 2014 Annual Report

Hanahan Lab Douglas Hanahan

Full Professor - Director of Swiss Institute of Experimental Cancer Research - ISREC - Merck-Serono Professor of Molecular Oncology

http://hanahan-lab.epfl.ch/

Introduction

Douglas Hanahan, born in Seattle, Washington, USA, received a bachelorâ&#x20AC;&#x2122;s degree in Physics from MIT (1976), and a Ph.D. in Biophysics from Harvard (1983). He worked at Cold Spring Harbor Laboratory in New York (1978-88) initially as a graduate student and then as a group leader. From 1988-2010 he was on the faculty of the Department of Biochemistry & Biophysics at UCSF in San Francisco. He has been elected to the American Academy of Arts & Sciences (2007), the Institute of Medicine (USA) (2008), the US National Academy of Science (2009), and EMBO (2010). In 2011, Hanahan received an honorary degree from the University of Dundee (UK).

The Hanahan group investigates tumor development and progression using genetically engineered mouse models of cancer that recapitulate important characteristics of human cancers, with strategic goals to elucidate pathogenic mechanisms underlying multi-step tumorigenesis and malignant progression, and to develop new therapeutic strategies based on knowledge of mechanism for translation to clinical trials aiming to improve the treatment of human cancers. Currently the lab focuses on melanoma, glioblastoma, pancreatic cancer and squamous carcinomas elicited by human papillomaviruses. Topics include mechanistic studies on acquired capabilities â&#x20AC;&#x201C; hallmarks of cancer - including resistance to programmed cell death, tumor angiogenesis, and invasion and metastasis. A crosscutting theme is the role of the heterotypic tumor microenvironment and the accessory cells that collaborate with cancer cells to manifest malignant disease. In addition, the lab is studying mechanisms of adaptive resistance to therapies targeting these and other hallmark capabilities, which present fascinating perturbations into the regulatory systems, and offer potential avenues to circumvent such drug resistance with combinatorial therapies.

Keywords

Cancer, translational oncology, genetically engineered mouse models of human cancer, transgenic mice, tumor microenvironment, angiogenesis, invasion, metastasis, metabolism, pre-clinical trials.

Results Obtained in 2014

The Hanahan laboratory continues to study the RIP-Tag mouse model of multi-step pancreatic neuroendocrine cancer (PanNET), which is still proving instructive about mechanisms of cancer 30 years after its inception (Hanahan, Nature, 1985). Recent results include the discovery that this ostensibly homogeneous oncogene-driven mouse tumor model is actually heterogeneous, being defined by two molecular subtypes identified by transcriptome profiling, that recapitulate similar subtypes in the cognate human pancreatic neuroendocrine cancer (Sadanandam et al, 2015). The tumors are fueled by both aerobic glycolysis and oxidative phosphorylation, the latter of which is restricted in productivity by limited bioavailable copper in the tumor microenvironment (Ishida et al, PNAS, 2014). New insights have also been gained into the invasive growth state, which is facilitated by activation of an autocrine signalling circuit involving glutamate ligand and NMDA receptor (Li and Hanahan, Cell, 2013). Additionally, impairment of tumor angiogenesis heightens invasiveness as an alternative route to vascularization via normal vessel co-option, most recently exemplified by genetic knockout of the gene encoding the proangiogenic protease MMP-9 (Shchors, et al Oncogene, 2013). Another study (Sadanandam, Nature Medicine, 2013) reported on distinctive molecular subtypes that differentiate human colorectal carcinomas, reflecting different physiological phenotypes and likely cell-of-origin. Another project area has focussed on a mouse model of the lethal brain cancer glioblastoma, assessing new therapeutic strategies targeting the p53 tumor suppressor or the autophagic cellular recycling system (Shchors, et al, PNAS, 2013; Shchors, et al Cancer Cell, 2015).

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EPFL School of Life Sciences - 2014 Annual Report

Team Members PhD Students Gabriele Galliverti Leanne Li Julie Scotton

Bachelor Students Agata Mlynska Sadegh Saghafina Ismael Wafy

Lab Manager Ehud Drori Sabbatical Professors Luisa Arispe (UCLA) David Helfman (KAIST)

Technical Staff Sylvie André Balfast Pasqualina Magliano Aristea Massaras Mei-Wen Peng Bruno Torchia Administrative Assistant Annissa Bayer

ISREC - Swiss Institute for Experimental Cancer Research

Postdoctoral Fellows Elizabeth Allen Krisztian Homiscko Iacovos Michael Ksenya Shchors Courtney Thomas Stephan Wullschleger Qian (Sophia) Zeng

A new mode of invasive growth regulation has been discovered, involving activation of glutamate-stimulated NMDA Receptor signaling (LI & Hanahan, 2013). Interstitial ﬂuid pressure/ ﬂow in solid tumors induces autocrine glutamate secretion and NMDAR activity, leading to heightened invasiveness.

Selected Publications » Sadanandam, A., Wullschleger, S., Lyssiotis, C., Grötzinger, C., Barbi, S., Bersani, S., Körner, J., Wafy, I., Mafficini, A., Lawlor, R.T., Asara, J., Bläker, H., Cantley, L.C., Wiedenmann, B., Scarpa, A., & Hanahan, D. (2014). Pancreatic neuroendocrine tumors: cross-species analysis reveals molecular subtypes with distinctive metastatic, developmental, and metabolic characteristics. Manuscript submitted. » Shchors, K. Massaras, A. & Hanahan, D. (2015). Dual targeting of the autophagic regulatory circuitry in gliomas with repurposed drugs elicits cell-lethal rates of autophagy and therapeutic benefit. Manuscript in revision, Cancer Cell. » Hanahan, D. (2014). Rethinking the war on cancer. Lancet 383: 558-563. » Ishida, S., Andreux, P., Poitry-Yamate, C., Auwerx, J., and Hanahan, D. (2013). Bioavailable copper modulates oxidative phosphorylation and growth of tumors. PNAS. 110: 19507-19512. » Shchors, K., Persson, A.I., Rostker, F., Tihan, T., Lyubynska, N., Li, N., Swigart, L.B., Berger, M.S., Hanahan ,D., Weiss, W.A., & Evan, G.I. (2013). Using a preclinical mouse model of high-grade astrocytoma to optimize p53 restoration therapy. Proc Natl Acad Sci U S A. 110: E1480-9. » Shchors, K., Nozawa, H., Xu, J., Rostker, F., Swigart-Brown, L., Evan, G., & Hanahan, D. (2013). Increased invasiveness of MMP-9-deficient tumors in two mouse models of neuroendocrine tumorigenesis. Oncogene 32: 502-513. » Sadanandam, A., Lyssiotis, C.A., Homicsko, K., Collisson, E.A., Gibb, W.J., Wullschleger, S., Ostos, L.C., Lannon, W.A., Grotzinger, C., Del Rio, M., Lhermitte, B., Olshen, A.B., Wiedenmann, B., Cantley, L.C., Gray, J.W., & Hanahan, D. (2013). A colorectal cancer classification system that associates cellular phenotype and responses to therapy. Nat Med. 19: 619-625. » Li, L., & Hanahan, D. (2013). Hijacking the neuronal NMDAR signaling circuit to promote tumor growth and invasion. Cell. 153: 86-100.

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EPFL School of Life Sciences - 2014 Annual Report

Hantschel Lab Oliver Hantschel

Tenure-track Assistant Professor - ISREC Foundation Chair for Translational Oncology

http://hantschel-lab.epfl.ch/

Introduction

Oliver Hantschel studied biochemistry at the University of Regensburg and at Rockefeller University in New York City. He received his PhD in 2004 from the European Molecular Biology Laboratory in Heidelberg and did postdoctoral work at the Research Center for Molecular Medicine of the Austrian Academy of Sciences in Vienna. In 2010, he obtained his Venia Docendi (Habilitation) in Experimental Haematology from the Medical University of Vienna and joined the EPFL School of Life Sciences as a Tenure Track Assistant Professor in 2011.

Protein kinases are involved in almost all aspects of oncogenesis. The inhibition of particular aberrantly activated kinases is considered to be beneficial for cancer treatment for numerous tumor types. Over the past ten years, 25 different inhibitors of a few oncogenic driver kinases in haematological and solid tumor types have received regulatory approval and entered clinical practice. Despite remarkable clinical responses that could be achieved in selected diseases, it is now well-established that most kinase inhibitors merely improve progression-free survival, but not overall survival. The main reasons is the development of drug resistance, often caused by point mutations in the targeted kinase that prevents or inhibits drug binding. Moreover, it is difficult to develop highly selective kinase inhibitors, as there are more than 500 kinases in humans with a conserved sequence and structure. Therefore, side effects caused by the inhibition of off-target kinases may also limit its clinical utility. The Hantschel lab studies oncogenic kinase signaling pathways by using interdisciplinary approaches at the interface of biochemistry, proteomics, chemical biology and protein engineering with the aim to identify innovative and novel ways for therapeutic intervention. Main research avenues include: • Structure-function analysis of protein kinases • Analysis of oncogenic signaling networks using interactionand phospho-proteomics. • Targeting of intracellular protein-protein interactions and posttranslational modifications with engineered high-affinity protein antagonists • Mechanism-of-action and specificity studies of kinase inhibitors.

Results Obtained in 2014

Chronic myelogenous leukemia (CML) is caused by BCR-ABL, which is a constitutively active form of the Abelson tyrosine kinase. While treatment with the different kinase inhibitors leads to durable remissions in the majority CML patients, drug resistance remains a clinical problem. Targeting additional sites in BCR-ABL may be an alternative strategy to restrict drug resistance. Our recent work has shown that an intramolecular interaction of the BCR-ABL SH2 domain with its kinase domain is critical for leukemogenesis and can be targeted with an engineered high-affinity binding protein. In the past two years, we have elucidated the molecular mechanisms responsible for the regulation of BCR-ABL by its SH2 domain: Using an efficient E. coli expression system for the BCR-ABL SH2-kinase domain unit allowed us to quantitatively study regulation of BCR-ABL with purified recombinant proteins in vitro. Unexpectedly, we found that the interaction of the SH2 domain with the kinase domain is the critical switch that shifts the BCR-ABL activation loop from an otherwise closed to a fully open conformation and enables its autophosphorylation. The activation loop is a central control element that regulates the activity of protein kinases. We show that the SH2-kinase interaction enables autophosphorylation of the activation loop in trans by rendering a key phosphorylation site (Tyr-412) highly accessible. In summary, our study demonstrates a novel mechanism by which a protein-protein interaction domain may allosterically mediate the transition of an inactive to an active kinase conformation in a key oncoprotein. This work may serve as an archetype to identify further allosteric regulatory mechanisms in other tyrosine kinases that are activated in haematological malignancies and facilitate the development of new allosteric inhibitors targeting oncogenic tyrosine kinases.

Keywords

Leukemia, oncoproteins, tyrosine kinases, kinase inhibitors, protein engineering, protein structures, protein phosphorylation, proteomics, proteinprotein interaction domains.

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EPFL School of Life Sciences - 2014 Annual Report

Team Members PhD Students Emel Gencer Barbara Gerig Allan Lamontanara Nadine Schmit

Master’s Students Delphine Harduin

Technician Sandrine Georgeon

Administrative Assistant Christine Skaletzka

ISREC - Swiss Institute for Experimental Cancer Research

Postdoctoral Fellows Tim Kükenshöner Sina Reckel

The Abl kinase domain has a predominantly inactive activation loop conformation that is similar to the conformation once imatinib or other type 2 kinase inhibitors are bound (upper panel). Upon formation of the SH2-kinase interface, the equilibrium is shifted to a predominantly open activation loop similar to the conformation once dasatinib or other type 1 inhibitors bind to the kinase domain (lower panel).

Selected Publications » Lamontanara, A.J., Georgeon, S., Tria, G., Svergun, D.I. and Hantschel, O. (2014). The SH2 domain of ABL kinases regulates kinase autophosphorylation by controlling activation loop accessibility, Nat. Commun. 5, 5470. » Mahul-Mellier, A.-L., Fauvet, B., Gysbers, A., Dikiy, I., Oueslati, A., Georgeon, S., Lamontanara, A.J., Bisquertt, A., Eliezer, D., Masliah, E., Halliday, G., Hantschel, O. and Lashuel, H.A. (2014). c-Abl phosphorylates α-syn and regulates its degradation, implication for α-syn clearance and contribution to the pathogenesis of Parkinson’s Disease. Hum. Mol. Genet. 23(11), 2858-2879. » Zhou, T., Georgeon, S., Moser, R., Moore, D.J., Caflisch, A., Hantschel, O. (2014). Specificity and mechanism-of-action of the JAK2 tyrosine kinase inhibitors ruxolitinib and SAR302503 (TG101348) Leukemia. 28, 404–407. » De Keersmaecker, K., Porcu, M., Cox, L., Girardi, T., Vandepoel, R., Girardi, T., Gielen, G., Mentens, N., Bennett, K.L. and Hantschel, O. (2014). NUP214-ABL1 mediated cell proliferation in T-cell acute lymphoblastic leukemia is dependent on the LCK kinase and various interacting proteins. Haematologica, 99(1), 85-93. » Sha, F., Gencer, E.B., Georgeon, S., Koide, A., Yasui, N., Koide, S. and Hantschel, O. (2013). Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains. Proc. Natl. Acad. Sci. USA, 110(37), 14924-14929.

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EPFL School of Life Sciences - 2014 Annual Report

Huelsken Lab Joerg Huelsken

Associate Professor - Debiopharm Chair in Signal Transduction in Oncogenesis

http://huelsken-lab.epfl.ch

Introduction

Joerg Huelsken received his PhD in 1998 at the Humboldt University, Berlin, and did postdoctoral research at the Max-DelbrĂźck Center for Molecular Medicine, Berlin. He joined ISREC as an associate scientist and an NCCR project leader in January 2003 and was nominated Associate Professor at the EPFL School of Life Sciences in 2011. He holds the Chair in Signal Transduction in Oncogenesis sponsored by Debiopharm, Lausanne.

The last years of cancer research have established the concept of cancer stem cells (CSC) as sub-population of cells within a tumor responsible for tumor initiation and long-term tumor growth. We are interested in understanding the biology of these cells, studying their interaction with other tumor cells and the tumor stroma and to revealing how targeting this population could be achieved and whether this would improve overall survival. We have explored several therapeutic options to interfere with these cells at the root of disease and have identified a number of successful strategies which can cure breast and colon cancer by eliminating cancer stem cells in pre-clinical models. We further provided evidence that these cancer stem cells are also essential for the initiation of metastatic disease and characterized the interaction between stem cells and their environment as an essential factor for metastatic growth. Understanding in detail the communication between cancer cells and surrounding stroma will help to define new therapeutic options to block spreading of cancer to secondary sites. The process of metastasis is rather inefficient and requires formation of local support structures, so called metastatic niches, which exploit wound repair mechanisms of the normal tissue to drive tumor progression. Since this support relies on activated, but otherwise normal, non-mutated cells, we are optimistic that it should be possible to interfere with these support signals and thereby prevent metastasis formation in the future.

Keywords

Cancer stem cells, metastatic colonization, differentiation therapy, immunotherapy.

136

Results Obtained in 2014

Using an experimental system to study the process of hepatic metastasis formation, we characterized in detail factors which are altered in response to metastatic seeding using RNA expression profiling. We identified key components which determine metastatic success, and in particular the family of Hedgehog (Hh) ligands was found to play a major role in enabling tumor cells to initiate metastasis. These results are supported by analysis of human pancreatic cancer samples which revealed increased expression of Hh ligands in pancreatic liver metastases. Liver stromal cell respond to this Hh signal and support tumor cell survival by blocking resident innate immune cells which would otherwise attack and eliminate invading tumor cells. Interestingly, tumor cells of human colon cancers appear to have adapted this mechanism in an alternative, more direct way enabling them to block innate immune reactions in the liver. Overcoming this novel, immune suppressive activity should help to prevent metastatic spread to the liver, one of the major complications of abdominal cancers. Most cancers, even in an advanced stage, resemble their tissue of origin indicating that tumor cells maintain parts of the normal differentiation program of their non-transformed ancestors. We now have identified the homeobox transcription factor HoxA5 as an important inducer of intestinal epithelial differentiation. In colon cancer, HoxA5 is down-regulated during cancer progression, but when re-activated can induce loss of the cancer stem cell phenotype and can strikingly overcome tumor growth and metastasis in vivo. HoxA5 is interconnected with the Wnt pathway in a negative feedback loop which ensures definitive bimodal fate decisions enforcing cells to halt cell cycling and exit the stem cell pool. Since HoxA5 expression can be triggered by certain drugs, this may allow to treat colon cancer patients by Hox-mediated elimination of cancer stem cells.

EPFL School of Life Sciences - 2014 Annual Report

Team Members PhD Students Zuzana Tartarova Nicolas Desbaillets

Master’s Student Raphael Sommer

Technicians Fanny Cavat Pierre Dessen Nancy Thompson

Administrative Assistant Ursula Winter

ISREC - Swiss Institute for Experimental Cancer Research

Postdoctoral Fellows Jean-Paul Abbuehl Caroline Dafflon Anja Irmisch Stefania Mejetta Paloma Ordóñez Morán Albert Santamaria Martínez Patrick Schmidt

Intestinal organoids reﬂect the differentiation potential of intestinal stem cells (marked by Lgr5-GFP). Loss of the tumor suppressor gene APC expands the stem cell pool and blocks terminal differentiation. The homeobox gene HoxA5 overcomes this transformation by depleting Lgr5+ cancer stem cells.

Selected Publications » Dafflon, C., S. Mejetta, P. Ordonez-Moran, A. Santamaria-Martinez, T. Hussenet, F. Naef, J. Huelsken (2015). Stromal cross-talk prevents anti-tumor immunity and facilitates liver metastasis. in preparation » Ordóñez-Morán, P., C. Urech, J. Huelsken (2015). HoxA5 counteracts stem cell traits by inhibiting Wnt signalling in colorectal cancer. Cancer Cell, in revision » Ordóñez-Morán, P., A. Irmisch, A. Barbachano, I. Chicote,S. Tenbaum, S. Landolfi, J. Tabernero, J. Huelsken, A. Muñoz, H.G. Pálmer (2013). SPROUTY2 is a β-catenin and FOXO3a target gene indicative of poor prognosis in colon cancer. Oncogene, 33, 1975-85. » Ordóñez-Morán, P., J. Huelsken (2014). Complex metastatic niches: already a target for therapy? Curr. Opin. Cell Biol., 31, 29-38. » Irmisch, A. and J. Huelsken (2013). Metastasis: New insights into organ-specific extravasation and metastatic niches. Exp. Cell Res., 319,1604-10. » Petherick, K.J., A.C. Williams, J.D. Lane, P. Ordóñez-Morán, J. Huelsken, T.J. Collard, H.J.M. Smartt, J. Batson, K. Malik, C. Paraskeva and A. Greenhough (2013). Autolysosomal β-catenin degradation regulates Wntautophagy-p62 crosstalk. EMBO J. 32,1903-16.

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EPFL School of Life Sciences - 2014 Annual Report

Lingner Lab Joachim Lingner

Full Professor

http://lingner-lab.epfl.ch

Introduction

Joachim Lingner received his PhD in 1989 from the Biocenter, University of Basel under the supervision of Walter Keller. He then pursued a Postdoc working with Thomas Cech at the Howard Hughes Medical Institute in Boulder, Colorado. In 1997, he became a group leader at ISREC and then was promoted to Senior group leader in 2002. Prof. Lingner became an Associate Professor at EPFL in 2005 and then a Full Professor in 2009. He has received many honors including the STARTfellowship from the Swiss National Science Foundation in 1997; Friedrich Miescher Prize from the Swiss Society of Biochemistry in 2002; EMBO member in 2005; ERC advanced investigator grant in 2008.

The physical ends of chromosomes, known as telomeres, play critical roles in cancer development, other age-related disorders and short telomere syndromes. Telomeres protect chromosomes from degradation and rearrangements that are typically seen in cancer. Telomeres also serve as cellular clocks. They shorten in most normal, human, somatic cells with every round of DNA replication due to the DNA end replication problem and the absence of telomerase. Short telomeres elicit a DNA damage response triggering a permanent cell cycle arrest termed cellular senescence. Thus, the replicative potential of primary cells is limited, restraining the growth of pre-cancerous lesions that have lost normal growth control. During progression towards malignancy, senescence is overcome by mutations in cell cycle regulators such as p53 and pRB. Further telomere shortening, however, will lead to loss of telomere protection from the DNA end fusion machinery, which is repressed at intact telomeres. When telomeres are fused, cells enter a crisis state at which fused chromosomes that contain multiple centromeres become missegregated or become torn apart during mitosis due to pulling of centromeres towards opposite poles by the spindle apparatus. Cells can escape crisis by re-gaining telomerase expression. Through the expression of telomerase, human cancer cells acquire an immortal phenotype. Our laboratory combines telomeric chromatin analysis by mass spectrometry, biochemistry and molecular genetics to study the dynamics of telomere structure, function and replication in human cells under normal and pathological situations. Our work may allow manipulation of telomere functions in tumors and other diseased tissues in the future.

Keywords

Telomeres, TERRA, chromatin, long noncoding RNA, cellular senescence and immortality, genome stability, DNA replication, telomeropathies, cancer development.

138

Results Obtained in 2014

Our labâ&#x20AC;&#x2122;s work concentrated on the analysis of telomerase structure, the telomeric long noncoding (lnc) RNA TERRA and telomeric chromatin. In collaboration with Daniela Rhodes (Cambridge-UK), we determined the first three-dimensional structure of active human telomerase by single particle electron microscopy (Sauerwald et al., 2013). Human telomerase has a bilobal dimeric structure and it can bind two DNA substrates. We functionally characterized the single strand telomeric DNA binding complex CST. We identified the molecular defects in the CST complex that cause a telomere syndrome known as dyskeratosis congenita/Coats Plus. Our data indicate that telomere DNA replication defects in mutant CST are the primary cause of the disease (Chen et al., 2013). Our lab also discovered novel roles of the lnc RNA TERRA at telomeres when they become uncapped or critically short. TERRA is upregulated upon telomere uncapping (Porro et al., 2014a) promoting the recruitment of the LSD1 lysine demethylase to MRE11 at the damaged telomeres. LSD1 stimulates the nuclease activity of MRE11 and the nucleolytic processing of uncapped telomeres (Porro et al., 2014b). In addition, TERRA associates with SUV39H1 H3K9 histone methyltransferase, which promotes accumulation of H3K9me3 at damaged telomeres and sustains telomere endto-end fusions that occur upon loss of the telomeric shelterin component TRF2 during cell crisis. Finally, we established a quantitative telomeric chromatin isolation protocol (QTIP), to biochemically isolate the entity of proteins and nucleic acids present at telomeres and compare different telomeric states by mass spectrometry (Grolimund et al., 2013). We uncovered several new telomeric factors (Pfeiffer et al., 2013), which are under study. QTIP will provide the basis to tackle one of the biggest challenges in the telomere field, which is to identify the molecular changes at telomeres that occur during normal development, in cancer and in telomere syndromes.

EPFL School of Life Sciences - 2014 Annual Report

Team Members PhD Students Alix Christen Larissa Grolimund Jana Majerska Patricia Renck-Nunes Anna-Sophia Reis Aleksandra Vancevska

Technician Thomas Lunardi

Administrative Assistant Nicole de Montmollin

ISREC - Swiss Institute for Experimental Cancer Research

Postdoctoral Fellows Eric Aeby Reyes Babiano Marianna Feretzaki Verena Pfeiffer Sophie Redon Ivo Zemp

Quantitative telomeric chromatin isolation protocol (QTIP). (a) Workﬂow of QTIP. (b) Effects of TRF2depletion at telomeres. (c) Comparison of telomeric protein composition at long versus short telomeres. See Grolimund, Aeby et al., Nature Communications 4: 2848 (2013) for details.

Selected Publications » » » » » » »

Grolimund, L., Aeby, E., Hamelin, R., Armand, F., Chiappe, D., Moniatte, M., and Lingner, J. (2013). A quantitative telomeric chromatin isolation protocol identifies different telomeric states. Nat Commun 4:2848. Chen, L.Y., Majerska, J., and Lingner, J. (2013). Molecular basis of telomere syndrome caused by CTC1 mutations. Genes Dev 27:2099-2108. Pfeiffer, V., Crittin, J., Grolimund, L., and Lingner, J. (2013). The THO complex component Thp2 counteracts telomeric R-loops and telomere shortening. Embo J 32:2861-2871. **Sauerwald, A., **Sandin, S., Cristofari, G., Scheres, S.H., *Lingner, J., and *Rhodes, D. (2013). Structure of active dimeric human telomerase. Nat struct & mol biol 20:454-460. Porro A, Feuerhahn S, Delafontaine J, Riethman H, Rougemont J, Lingner J. (2014). Functional characterization of the TERRA transcriptome at damaged telomeres. Nat Commun 5:5379. Porro, A., Feuerhahn, S., and Lingner, J. (2014). TERRA-reinforced association of LSD1 with MRE11 promotes processing of uncapped telomeres. Cell Reports 6:765-776. Zemp I, Lingner J. (2014). The shelterin component TPP1 is a binding partner and substrate for the deubiquitinating enzyme USP7. J Biol Chem. 289:28595-28606.

» Redon S, Zemp I, Lingner J. (2013). A three-state model for the regulation of telomerase by TERRA and hnRNPA1. Nucleic Acids Res. 41(19):9117-9128.

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Meylan Lab Etienne Meylan

Tenure-track Assistant Professor - SNSF Professor

http://meylan-lab.epfl.ch/

Introduction

In our research laboratory, we are studying the mechanisms that govern the development of non-small cell lung cancer (NSCLC). To this end, we use a combination of bioinformatics analyses, cancer-derived mouse and human cell lines, tumor tissue material and genetically-engineered mouse models of human NSCLC. Our research focuses on two main areas: Etienne Meylan received a PhD in Life Sciences from the University of Lausanne in 2006, for his work on innate immunity performed in the laboratory of Jürg Tschopp. From 2007 to 2010, he worked as postdoctoral fellow in the laboratory of Tyler Jacks, at the Koch Institute for Integrative Cancer Research, MIT, Cambridge USA. In 2011, he established his research laboratory at ISREC, as a Swiss National Science Foundation Professor and later as tenure-track Assistant Professor. His laboratory focuses on the molecular mechanisms that contribute to the development of non-small cell lung cancer.

Alterations in glucose metabolism - Since the pioneering work of Otto Warburg almost a century ago, it is recognized that tumors are at increased need for glucose compared to normal cells. We aim to address the mechanisms of derailed glucose utilization during the development of NSCLC – and increasingly other tumor types –, and the consequences on tumor progression. Specifically, we focus on the first and rate-limiting step of glucose usage: its entry that is mediated by glucose transporters of the GLUT family. Hopefully, our findings will lead to possible developments of therapies aimed to inhibit tumor-specific characteristics of glucose utilization. Innate immune and inﬂammatory pathways - Building upon our previous studies where we identified the NF-kB signaling pathway being critical for lung tumor development, we are currently investigating the role of several proteins and pathways – known to regulate innate immunity and/ or inflammatory processes – in the development of lung cancer. Specifically, we use genetic approaches to perturb these pathways directly in the lung tumor epithelial cells in vivo, to obtain a clear picture of their role and impact on tumor growth control. Collectively, we hope our research will help to understand better how lung cancer grows and progresses, in order to develop knowledge-based strategies to combat this devastating disease.

Results Obtained in 2014

Because it is known that tumor cells consume increased quantities of glucose to build their biomass required for proliferation, it is important to understand the biological consequences of increased glucose utilization, and to identify the limiting factors for glucose entry into tumor cells. To this end, we began to analyze the regulation of various glucose transporters, and to explore their contribution to lung tumor progression. We initially focused on GLUT3, a glucose transporter best characterized as a neuronal transporter with high affinity for glucose. We observed that, although GLUT3 is not expressed in normal lungs, it is induced in tumors from genetically-engineered mouse models of human lung cancer. In human cell lines derived from non-small cell lung cancer, we made the intriguing observation that GLUT3 was expressed to high mRNA and protein levels, specifically in some cell lines but not others. We elucidated this dichotomy in GLUT3 expression, unraveling a strong induction during an epithelial-mesenchymal transition (EMT, an important developmental pathway reactivated in cancer to promote tumor progression). Specifically, we found that, during EMT, GLUT3 transcription is induced by ZEB1, a crucial transcription factor of the EMT program, which directly binds to the second intron of the GLUT3 gene to activate its transcription (Figure). Currently, we are focusing on the regulation and importance of this and other glucose transporters in tumor progression, in lung tumors and other cancer types. In other important projects of the laboratory, we are developing methods for genetic perturbation and monitoring of the activity of innate immune or inflammatory pathways directly in lung tumors in vivo, to better understand their impact on tumor growth, the tumor microenvironment and the response to chemotherapy.

Keywords

Lung cancer; glucose metabolism; glucose transporters; innate immunity; mouse models of cancer; NF-kappaB.

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Team Members PhD Students Caroline Contat Svenja Groeneveld Jawahar Kopparam Mark Masin

Master’s Student Bernard Moret

Technician Jessica Vazquez

Administrative Assistant Christine Skaletzka

ISREC - Swiss Institute for Experimental Cancer Research

Postdoctoral Fellows Stefania Crippa Julien Faget

GLUT3 regulation during EMT - During EMT, GLUT3 expression increases dramatically, due to ZEB1 binding to the intron 2 of GLUT3 gene. Cells with mesenchymal characteristics express GLUT3 to high levels, and need it for sustained proliferation. See also Masin et al., Cancer & Metabolism, 2014.

Selected Publications » Masin, M., Vazquez, J., Rossi, S., Groeneveld, S., Samson, N., Schwalie, P.C., Deplancke, B., Frawley, L.E., Gouttenoire, J., Moradpour, D., Oliver, T.G. and Meylan, E. (2014). GLUT3 is induced during epithelialmesenchymal transition and promotes tumor cell proliferation in non-small cell lung cancer. Cancer & Metabolism. 2:11. » Peters, S. and Meylan, E. (2013). Targeting receptor activator of nuclear factor-kappa B as a new therapy for bone metastasis in non-small cell lung cancer. Current Opinion in Oncology. 25(2):137-144.

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Oricchio Lab Elisa Oricchio

Tenure-track Assistant Professor - ISREC Foundation Chair for Translational Oncology

http://oricchiolab.epfl.ch

Introduction and Research Interests

In November 2014, Elisa Oricchio was appointed as tenure track Assistant Professor at ISREC/EPFL. Prof. Oricchio graduated in Genetics with highest honor in 2004 and obtained a PhD in 2008 at the National Italian Institute of Health in Rome Italy. In Sept 2008, she joined the group of Dr. HG Wendel at Memorial Sloan Kettering (NY, USA) as a post-doc. Her research work focused on the genetics of Lymphoma exploring innovative therapeutic approaches.

Research in the Oricchio laboratory focuses on the genetics of lymphoma and its translation into new therapies. Lymphoma is a heterogeneous disease characterized by multiple genomic alterations. Our goal is to define the functional role of recurrent genetic lesions in the lymphomagenesis. We combine genomic analyses of human tumors with functional in vivo studies and we use mosaic models of lymphomas to functionally annotate genes of interest identified by genomic analyses and to perform preclinical treatment studies. Project Description Initially, we analyze lymphoma patient samples by RNA sequencing to quantitatively measure cancer associated variations in gene expression and specific isoform levels, as well as to identify potential novel gene fusions. We integrate these data with mutations and chromosomal aberrations to provide unprecedented insights into the genomics of lymphoma. We complement the genomic analyses with functional screenings to define the phenotypical contribution of these alterations to lymphomagenesis. This step is critical to the design of in vivo functional studies.

Our ultimate goal is to use our genetic and biological studies to design new therapeutic strategies. Frequent genomic alterations can influence therapeutic response, determine mechanisms of resistance and potentially targeted by using selective inhibitors. To test our hypotheses, we use highly controlled experimental systems that resemble the design of clinical trials in a physiological context. We test combination therapies in genetically defined tumors, we directly compare the impact of different genetic lesions on therapy and we measure the effect of tumor intrinsic heterogeneity to the treatment. The Oricchio laboratory opened at ISREC-EPFL in November 2014. A senior technician and a junior post-doc with experience in cancer biology have been recruited to start our research activity.

Next, we use genetically engineered mouse models of lymphoma to study the genetics and pathology of the disease. These murine models retain key human features and we will exploit these murine models to dissect the impact of recurrent genomic alterations on lymphoma initiation and transformation in vivo.

Keywords

Cancer genetics, mouse models, therapy.

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Team Members Technician Katanayeva Natalya

Administrative Assistant Demeester Dorothée

ISREC - Swiss Institute for Experimental Cancer Research

Postdoctoral Fellow Roli Misra

Functional in vivo studies using murine model of lymphoma. A) Schematic of adoptive transfer strategy that enable to quickly define the role of genetic lesions in lymphomagenesis and progression in vivo B) Representative data indicating how distinct genetic lesions affect tumor latency in vivo.

Selected Publications » Oricchio E. Papapetrou EP, Lafaille F, Ganat YM, Kriks S, Mark WH, Teruya-Feldstein J, Huse JT, Reuter V, Sadelain M, Studer L, Wendel HG (2014) “A cell engineering strategy to enhance the safety of stem cell therapies”. Cell Rep. Sep 25;8(6):1677-85. » Oricchio E., Ciriello G, Schatz JH, Jiang M, Heguy A, Viale A, de Stanchina E, Teruya-Feldstein J, Sander C, Wayne T, Seshan VE, Chaganti RSK Wendel HG. (2014) Frequent disruption of the RB pathway in indolent follicular lymphoma suggests a new combination therapy. J Exp. Med. Jun 30;211(7):1379-91.

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Radtke Lab Freddy Radtke

Full Professor

radtke-lab.epfl.ch

Introduction

Freddy Radtke graduated from the University of ZĂźrich in molecular biology 1994, a postdoctoral fellowship at Genentech Inc. USA 19951996 was followed by a postdoctoral position at ISREC Switzerland 1997-1999; Assistant Member of the Ludwig Institute for Cancer Research 1999-2004 promoted to Associate Member in 2004; joined ISREC as Senior Scientist in 2006, before joining EPFL in August 2006 as associate professor; promoted to full professor in 2012.

Our group is interested in the molecular mechanisms controlling stem cell maintenance, lineage commitment and differentiation in self-renewing systems (such as the hematopoietic system, the skin and the gut) and cancer. The basic principle of self-renewing tissues is that they continuously produce cells from a stem cell reservoir that gives rise to proliferating transient amplifying cells, which subsequently differentiate and migrate to the correct compartment. These processes have to be tightly regulated to ensure life-long homeostasis. In recent years a substantial body of evidence has accumulated to support the notion that signaling pathways known to be important during embryonic development (such as e.g. Wnt and Notch) play important roles in regulating self-renewing tissues. Moreover, these pathways are often deregulated during tumorigenesis due to mutations in key elements involved in these pathways. Using mouse genetics we study the role of evolutionarily conserved signaling pathways under physiological and pathological situations to gain a better understanding of their role in cancer. In addition, the lab optimizes and validates potential drug development candidates that target developmental signaling pathways to assess their mode of action and their efficacy in pre-clinical cancer models and in primary human tumor samples.The laboratory also studies how inflammation can either promote or inhibit tumor progression.

Keywords

Cancer, leukemia, stem cells, differentiation, immunity, notch, Wnt, preclinical drug development and trials.

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Results Obtained in 2014

Specific fibroblastic niches in secondary lymphoid organs orchestrate Notch-regulated immune responses : Primary and secondary lymphoid organs provide specialized microenvironments that support the development and maturation of immune cells as well as the induction and control of immune responses. Until recently the primary function of fibroblasts within secondary lymphoid organs (SLO) was thought to be mainly structural. However, fibroblasts of SLO are now known to consist of several subsets fulfilling distinct and important physiological functions through their cross talk with immune cells. These fibroblasts are distinct from endothelial cells, which control cell entry/exit from SLO. In addition, Notch signalling has been shown to play critical roles in multiple aspects of immune cell differentiation and function within SLO, although the cellular interactions that regulate these events have remained elusive. We identified and characterized specific fibroblast subsets within SLO that control and specify Notch mediated immune regulation for marginal zone B cells, and certain dendritic cell subsets in the spleen as well as T follicular helper cells in the lymph node. Notch signaling in the pigmented epithelium of the anterior eye segment promotes ciliary body development at the expense of iris formation: The iris and ciliary body are pigmented structures of the anterior eye segment and are each essential for normal ocular function. Perturbed development and/or function of either structure results in detrimental pathological conditions such as glaucoma and Phtisis bulbi. We showed that canonical Notch signaling promotes ciliary body development and opposes iris formation. In addition, we demonstrate that genetic manipulation of Notch signaling in ocular pigment cells facilitates robust modeling of human pathologies such as glaucoma and Phthisis bulbi.

EPFL School of Life Sciences - 2014 Annual Report

Team Members PhD Students Marzia Amaro Monique Coersmeyer Chhavi Jain Delphine Harduin Annaise Jauch Fabian Junker Viktoria Reinmüller Bhushan Sarrode Silvia Wirth

Technicians Laure Bardouillet Christelle Dubey Marianne Nkosi

Administrative Assistant Catherine Pache

ISREC - Swiss Institute for Experimental Cancer Research

Postdoctoral Fellows & Scientists Ute Koch Markus Germann Craig Nowell

Co-staining of EYFP and the Notch ligand DL4 (red) in activated LN of R26EYFP∆Ccl19Cre mice reveals overlapping expression in specific fibroblastic LN cells also known as follicular dendritic cells (FDCs, in blue).

Selected Publications » Fasnacht N., Huang HY., Koch U., Favre, S., Auderset F., Cai, Q., Onder L., Kallert S., Pinschewer D., MacDonald HR., Tacchini-Cottier F., Ludewig B., Luther SA. Radtke, F. Specififc fibroblastic niches in secondary lymphoid organs orchestrate distinct Notch-regulated immune responses. J Ex Med. (2014) Oct 20;211(11):2265-79. » Maes H., Kuchnio A., Peric A., Moens S., Nys K., De Bock K., Quaegebeur A., Schoors S., Georgiadou M., Wouters J., Vinckier S., Vankelecom H., Garmyn M., Vion AC., Radtke F., Boulanger C., Gerhardt H., Dejana E., Dewerchin M., Ghesquière B., Annaert W., Agostinis P and Carmeliet P. Tumor vessel normalization by chloroquine independent of autophagy Cancer Cell (2014) Aug 11;26(2):190-206. » Sarrode B., Nowell CS.,Ihm J., Kostic C., Arsenijevic Y., Moulin AP., Schorderet DF., Beermann F., Radtke F. Notch signaling in the pigmented epithelium of the anterior eye segment promotes ciliary body development at the expense of iris formation. Pigment Cell Melanoma Res. (2014) jul 27 (4):580-9. » Auderset F, Schuster S, Fasnacht N, Coutaz M, Charmoy M, Koch U, Favre S, Wilson A, Trottein F, Alexander J, Luther SA, MacDonald HR, Radtke F, Tacchini-Cottier F. Notch signaling regulates follicular helpoer T cell differentiation. J Immunol. (2013) Sep 1; 191 (5):2344-50. » Radtke F., MacDonald HR., and Tacchini-Cottier F. Regulation of Innate and Adaptive Immunity by Notch. (2013) Nature Reviews Immunology ,13(6):427-37. » Licciulli S. Aviala JL, Hanlon L, Troutman S, Cesaroni M, Kota S, Keith B, Simon MC, Pure E, Radtke F, Capobianco AJ, Kissil JL. Notch1 is required for Kras-induced lung adenocarcinoma and controls tumor cell survival via p53. Cancer Res. (2013) Oct 1;73(19):5974-84

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Simanis Lab Viesturs Simanis

Associate Professor

http://simanis-lab.epfl.ch

Introduction

Viesturs Simanis studied Biochemistry at Imperial College, graduating with a First Class Honours Degree in 1980. He did his Ph.D. studies with Professor Sir David Lane, at Imperial College London (awarded 1984). He did his postdoctoral studies with Professor Sir Paul Nurse, at ICRF in London, and the Department of Microbiology in Oxford (1984 to 1988). He then moved to the Swiss Institute for Experimental Cancer Research in Lausanne (Switzerland), as a junior, then senior group leader (1988 to 2006). He was appointed Associate Professor at EPFL in 2006.

Cell division is essential for the propagation of all organisms. If the fidelity of the processes involved in cell division is reduced, there is an increased risk that errors will occur in the transmission of genetic information from a cell to its daughters; this can result in cell death, or alter the cell’s behaviour, which can contribute to the development of diseases such as cancer. We study cytokinesis, the final event of the cell cycle, using the S. pombe model system. If a cell divides without segregating its DNA, the outcome is a dead cell devoid of chromosomal DNA and a cell with increased ploidy, which is genetically less stable than a normal cell. Alternatively, if cytokinesis is triggered before chromosome segregation has been completed, then the nucleus may be “cut”; resulting in cell death, or aneuploidy. Our goal is to understand how cytokinesis is regulated and coordinated with other events in the cell cycle. In S. pombe a GTPase-regulated NDR-kinase signalling network known as the SIN (septation initiation network) acts at multiple points during cytokinesis. Failure of SIN signalling results in the production of multinucleated cells that die, while inappropriate activation of the SIN promotes cytokinesis from any cell cycle stage. The SIN is considered to be the functional counterpart of the mammalian “Hippo” signalling pathway, which regulates growth and proliferation. The SIN also plays a role in meiosis, where it is essential for generating the spores/gametes following completion of the two meiotic divisions. Our primary tools are forward and reverse genetics using the S. pombe model, coupled to cell biology and biochemical analysis. Our goal is to identify regulators and targets of the SIN in mitosis and meiosis.

Keywords

Schizosaccharomyces pombe, cytokinesis, signal transduction, cell cycle, meiosis, protein kinase, protein phosphatase.

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Results Obtained in 2014

Semi-automated image analysis reveals new facets of SIN regulation Localisation of SIN proteins at the spindle pole bodies (SPBs) is important for SIN regulation. Quantitative analysis of SIN protein localisation was performed using a semi-automated image analysis plug-in developed for the Image J platform in collaboration with the Unser lab (EPFL); (Schmitter et al., 2013). We used this to analyse large numbers of mutant and wild-type cells, which revealed several novel facets of SIN regulation; (Wachowicz et al., 2014). First, the SIN exists in two states; in early mitosis the SPB localisation of SIN proteins is positionally unstable, and depends upon the conserved POLO-family kinase Plo1p. This corresponds to the time when the SIN plays its role in contractile ring assembly. In late mitosis, when the SIN signals the onset of cytokinesis, SIN protein localisation now becomes dependent upon the signalling GTPase Spg1p and its regulator Etd1p. The transition between these states is tightly coupled to other events in mitosis, and regulators that promote and delay it, were identified. This provides the basis for further studies of the nature of these two states and how the transition between them is regulated. Regulation of the SIN in meiosis We studied the regulation of the SIN in meiosis, and found that the regulatory hierarchy of the signalling is different from that seen in mitosis. We discovered that stage-specific protein degradation is important for the regulation of SIN signalling, with the conserved ubiquitin ligase Dma1p playing a central role (Krapp and Simanis, 2014). Future studies will examine the basis for these differences. Others We contributed to a study of mitotic and morphogenetic control in S. pombe (Grallert et al., 2013). We also collaborated to studies that optimised the analogue-sensitive cdc2 mutant (Aoi et al., 2014) and described new vectors for S. pombe (Fennessey et al., 2014).

EPFL School of Life Sciences - 2014 Annual Report

Team Members PhD Students Manuela Moraru

Administrative Assistant Catherine Pache

ISREC - Swiss Institute for Experimental Cancer Research

Postdoctoral Fellows Andrea Krapp Paulina Wachowicz

The SPB association of Cdc7p early mitotic requires full Plo1p function. Cells were mixed, synchronised, incubated at 36°C and imaged. Note the presence of a Cdc7p-GFP signal on the SPBs of the plo1+ cell (blue nuclear signal) and its absence from the plo1ts4 cell (Wachowicz et al., 2014).

Selected Publications » Wachowicz, P., Chasapi, A., Krapp, A., Cano Del Rosario, E., Schmitter, D., Sage, D., Unser, M., Xenarios, I., Rougemont, J. and Simanis, V. (2014). Analysis of S. pombe SIN protein SPB-association reveals two genetically separable states of the SIN. J Cell Sci in press. » Krapp, A. and Simanis, V. (2014). Dma1-dependent degradation of SIN proteins during meiosis in Schizosaccharomyces pombe. J Cell Sci 127, 3149-61. » Aoi, Y., Kawashima, S. A., Simanis, V., Yamamoto, M. and Sato, M. (2014). Optimization of the analogue-sensitive Cdc2/Cdk1 mutant by in vivo selection eliminates physiological limitations to its use in cell cycle analysis. Open Biol 4. 140063 » Fennessy, D., Grallert, A., Krapp, A., Cokoja, A., Bridge, A. J., Petersen, J., Patel, A., Tallada, V. A., Boke, E., Hodgson, B. et al. (2014). Extending the Schizosaccharomyces pombe molecular genetic toolbox. PLoS One 9, e97683. » Schmitter, D., Wachowicz, P., Sage, D., Chasapi, A., Xenarios, I., Simanis, V. and Unser, M. (2013). A 2D/3D image analysis system to track fluorescently labeled structures in rod-shaped cells: application to measure spindle pole asymmetry during mitosis. Cell Div 8, 6. » Grallert, A., Patel, A., Tallada, V. A., Chan, K. Y., Bagley, S., Krapp, A., Simanis, V. and Hagan, I. M. (2013). Centrosomal MPF triggers the mitotic and morphogenetic switches of fission yeast. Nat Cell Biol 15, 88-95.

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Bucher Group Philipp Bucher

Group leader

http://bucher-lab.epfl.ch/

Introduction

New technologies allow for comprehensive characterization of the molecular changes that cause a healthy cell to become cancerous. These technologies produce vast amounts of data. We develop computational methods that will help to extract insights and knowledge from such data. Philipp Bucher was first trained as a molecular biologist at the University of Zürich, and subsequently received his PhD in computational biology at the Weizmann Institute of Science in Israel. He then worked as a postdoctoral fellow with Sam Karlin at Stanford University before he moved to ISREC where he was promoted senior scientist in 2001.

Our main focus is on gene regulation. Transcription factors are key elements of regulatory circuits that control the expression of genes. By binding to specific sites in the genome, they turn on and off the expression of neighboring genes. We are interested in the molecular processes that guide transcription factors to their target sites, in a developmental stage-, tissue- and environmental condition-specific manner, and we are studying these processes by analyzing high-throughput functional genomics data produced by technologies such as ChIP-Seq, DNase-seq or CAGE. We are also developing novel computer algorithms for this purpose. We are further interested in the use of molecular profiling data for medical diagnosis. To this end we develop and test machine learning methods in the framework of open prediction challenges organized by the DREAM and sbv IMPROVER consortia. Besides research, our group develops and maintains bioinformatics databases and web servers. Our best known resource is the Eukaryotic Promoter Database EPD, created in 1986 and regularly updated since then. The ChIP-seq server features web-based programs to access and analyze a large collection of public functional genomics data sets. The Signal Search Analysis (SSA) server offers DNA motif discovery and search tools. These three resources are tightly interlinked and together form a comprehensive web-based system for gene regulatory regions analysis.

Keywords

Gene regulation, epigenetics, ChIP-Seq data analysis, bioinformatics algorithms, computational molecular diagnostics.

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Results Obtained in 2014

Novel bioinformatics methods for epigenome analysis Together with Bernard Moret’s group from computer science, we are developing new computational methods to study epigenetic phenomena with Next-Generation Sequencing (NGS) data. We recently published a probabilistic partitioning algorithm which serves for the detection of subclasses and extraction of chromatin signatures from selected genomic regions. Moreover, we have applied phylogenetic inference methods to reconstruct cell differentiation trees from ChIP-seq data. Using molecular profiling data for clinical diagnosis We carried out a proof-of-concept study to evaluate the usefulness of ChIP-Seq data from surgically removed breast tumor tissues as prognostic and predictive signatures. In addition, we participated in the Acute Myeloid Leukemia Outcome challenge organized by the DREAM consortium (http://dreamchallenges.org/). The goal of this challenge was to predict drug-response, remission duration, and overall survival time from patient data composed of clinical correlates and phosphoprotein expression levels. EPDNew now covers 5 model organisms EPDnew is a recently introduced, automatically compiled section of EPD. Its goal is to provide comprehensive, single-base resolution annotation of transcription start sites (TSS) for important model organisms. The massive release of new TSS mapping data over the last two years has enabled us to substantially expand the contents of EPDnew. By the end of 2014, we reached nearly complete gene coverage for human, mouse, and Drosophila, and about 50% coverage for zebrafish and C. elegans. PWMscan: a fast tool for scanning genomes for DNA motifs. Responding to needs created by NGS data, we created a new web-server, PWMscan, for scanning whole genomes for transcription factor binding site motifs defined by a position weight matrix. High speed is achieved by the use of pre-computed indexed genomes together with fast string matching software. Searching the human genome for a PWM-defined motif usually takes less than 30 seconds.

EPFL School of Life Sciences - 2014 Annual Report

Team Members Administrative Assistant Sophie Barret

ISREC - Swiss Institute for Experimental Cancer Research

Postdoctoral Fellows Giovanna Ambrosini Rouayda Cavin Périer René Dreos Sunil Kumar

Input form and results page of the PWMScan server. Upper right: Input form. Center left: sequence logo of the position weight matrix entered. Bottom: results page with action buttons for saving the match list, for extracting surrounding DNA sequences or sending the results to another web application.

Selected Publications » Nair, N.U., Lin, Y., Manasovska, A., Antic, J., Grnarova, P., Sahu, A.D., Bucher, P. and Moret, B.M. (2014). Study of cell differentiation by phylogenetic analysis using histone modification data. BMC Bioinformatics 15:269. » Nair, N.U., Kumar, S., Moret, B.M. and Bucher, P. (2014). Probabilistic partitioning methods to find significant patterns in ChIP-Seq data. Bioinformatics 30(17), 2406-2413. » Aghaeepour, N., Finak, G., FlowCAP Consortium, DREAM Consortium, Hoos, H., Mosmann, T.R., Brinkman, R., Gottardo, R. and Scheuermann, R.H. (2013). Critical assessment of automated flow cytometry data analysis techniques. Nat. Method 10(3), 228-238. » Weirauch, M.T., Cote, A., Norel, R., Annala, M., Zhao, Y. et al. (2013). Evaluation of methods for modeling transcription factor sequence specificity. Nat. Biotechnol. 31(2), 126-134. » Dreos, R., Ambrosini, G., Cavin Périer, R. and Bucher. P. (2013). EPD and EPDnew, high-quality promoter resources in the next-generation sequencing era. Nucleic Acids Res. 41(Database issue), D157-164. » Dimitrieva, S. and Bucher, P. (2013). UCNEbase--a database of ultraconserved non-coding elements and genomic regulatory blocks. Nucleic Acids Res. 41(Database issue), D101-109.

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Frey Group Peter Frey

External Adjunct Professor EPFL, Honorary Professor UNIL

http://lmrp.epfl.ch

Introduction

The main research area of the lab is tissue engineering and regenerative medicine for urological applications in particular for the treatment of congenital malformations.

Peter Frey received his master’s in Medical Zoology from the University of Basel in 1973 and then his Swiss Medical Degree in 1980. He received a Fellowship in Pediatric Surgery for the Swiss Academy of Medicine in 1988 and later became a Senior Lecturer in Basel (1990) and in Lausanne (2000). Dr. Frey then became an Associate Professor and head of Pediatric Urology at the CHUV in 2003, Visiting Professor at the EPFL in 2004 and then an Adjunct Professor in 2008. Prof. Frey was also a Visiting Professor at the University of Malaya, Kuala Lumpur (2012). He is a Member of the Swiss Commission of Technology and Innovation (CTI) and of the European Research Council. Over the years, Prof. Frey has received several grants from the EC, SNF and from CTI.

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The lab is involved in investigating the biology of human urothelial and smooth muscle cells, and in molecular and stem cell biology with particular interest in urothelial precursor cells. Further it evaluates smart, morphogen/growth factor loaded collagen and collagen fibrin hybrid matrices for tissue engineering purposes and develops flow bioreactors, imitating physiological conditions. In addition the lab is involved in the development of smart injectable bulking biomaterials for the treatment of vesico-ureteral reflux and urinary incontinence.

Results Obtained in 2014

Mechanically compressed tubular collagen and IGF1 loaded smart collagen matrices as well as collagen-fibrin hybrid matrices were developed and further, “humanized” tubular cell-engineered matrices were created. These matrices were successfully investigated in a preclinical rabbit urethral model.

Keywords

Tissue engineering, urology, pediatrics, urothelial cells, smooth muscle cells, stem cells, collagen, fibrin, growth factors, bioreactors.

EPFL School of Life Sciences - 2014 Annual Report

Team Members PhD Students Elif Vardar

Master’s Students Xavier Mottart Ann-Lena Jahnsen

Administrative Assistant Carol Bonzon

External Adjunct Professors

Postdoctoral Fellows Eva Maria Balet Mattias Larsson Kalitha Pinnagoda

Cross section of urethral tubular collagen graft 3 months after replacement of excised urethra in the rabbit. Note urethral lining and smooth muscle regeneration.

Selected Publications » Arenas da Silva, LF., Micol, LA., Tiemessen, D., Kuppevelt, TH., Frey, P., Oosterwijk, E., Geutjes, PJ. and Feitz, WFJ. (2013) Is there a need for smooth muscle cell transplantation in urethral reconstruction? Tissue Engineering Part A 12/2013; DOI:10.1089/ten.TEA.2013.0185. » Larsson, HM., Gorostidi, F., Hubbell, JA. and Frey, P. (2014) Clonal, Self-Renewing and Differentiating Human and Porcine Urothelial Cells, a Novel Stem Cell Population PlosOne February 26, 2014 DOI:10.1371/ journal.pone.0090006.

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Molinari Group Maurizio Molinari

External Adjunct Professor - Institute for Reseasrch in Biomedicine - Bellinzona

http://www.irb.usi.ch/protein-folding-and-quality-control

Introduction

The aim of the research performed in our group is to understand how mammalian cells insure expression of the cellular proteome and how they respond to variations in ER load with folding-defective or foldingcompetent polypeptides. Maurizio Molinari earned a PhD in Biochemistry at the ETH-Zurich in 1995. After post-docs in the laboratory of Cesare Montecucco (Padua) and Ari Helenius (Zurich), in 2000 he accepted the position of group leader at the IRB, Bellinzona. Maurizio Molinari received the Science Award 2002 from the Foundation for the study of neurodegenerative diseases, the Kiwanis Club Award 2002, the Friedrich-Miescher Award 2006 and the Research Award Aetas 2007. Since 2008, he is Adjunct Professor at the EPFL. Since 2013 he is member of the Research Committee at the Università della Svizzera italiana.

The endoplasmic reticulum (ER) contains high concentrations of molecular chaperones and enzymes that assist maturation of newly synthesized polypeptides destined to the extracellular space, the plasma membrane and the organelles of the endocytic and secretory pathways. It also contains quality control factors that select folding-defective proteins for ER retention and/or ER-associated degradation (ERAD). Mutations, deletions and truncations in the polypeptide sequences may cause protein-misfolding diseases characterized by a “loss-of-function” upon degradation of the mutant protein or by a “gain-of-toxic-function” upon its aggregation/ deposition. Pathogens hijack the machineries regulating protein biogenesis, quality control and transport for host invasion, genome replication and progeny production. Our long-standing interest is to understand the molecular mechanisms regulating chaperone-assisted protein folding and the quality control processes determining whether a polypeptide can be secreted, should be retained in the ER, or should be transported across the ER membrane for degradation. A thorough knowledge of these processes will be instrumental to design therapies or to identify drug targets for diseases caused by inefficient functioning of the cellular protein factory, resulting from expression of defective gene products, or elicited by pathogens.

Results Obtained in 2014

We have pursued our studies to better characterize the mechanisms regulating protein folding and quality control, with particular emphasis on the role of the UDP-glucose:glycoprotein glucosyltransferase (UGGT1). In a collaboration with the group of Randy Kaufman we revealed the importance of UGGT1-mediated protein re-glucosylation in maintaining the solubility of folding-competent and folding-defective polypeptides expressed in the ER lumen. We are also interested in the characterization of novel and druggable quality control mechanisms operating in the secretory compartment. In a collaboration with the group of Paolo Paganetti, transgenic mice were prepared that express a single open reading frame encoding the heavy and light chain of the specific antibody to the N-terminus of the Aβ peptide linked by the mouth and foot virus peptide 2A (to promote equimolar expression of light and heavy chains that are efficiently processed and assembled in the ER of transgenic cells). The in situ production of the antibody substantially reduced Aβ formation and aggregation compared to age-matched, mock-treated APP23 micecombinant proteins to be employed in the clinics and in the industry.

Keywords

Endoplasmic reticulum (ER), proteostasis, molecular chaperones and folding enzymes, protein quality control, autophagy, ER-associated degradation (ERAD), conformational diseases, UPR.

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Team Members PhD Students Giorgia Brambilla Pisoni Fiorenza Fumagalli Timothy Bergmann Ilaria Fregno

Visiting Scientists Tim Beltraminelli (UNIL) Ivan Hang (ETH-ZH) Ilaria Fegno (Erasmus)

External Adjunct Professors

Senior Scientists Carmela Galli Tatiana Soldà Elisa Fasana

Schematic representation of the proteostasis network

Selected Publications » » » »

Noack, J. and Molinari, M. (2014) RESETting proteostasis. Nature Chem. Biol. 10, 881-882. Noack, J., Brambilla-Pisoni, G. and Molinari, M. (2014) Proteostasis: Bad News and Good News From the Endoplasmic Reticulum. Swiss Medical Weekly 144:w14001, 1-13. Noack, J., Bernasconi, R. and Molinari, M. (2014) How Viruses Hijack the ERAD Tuning Machinery. J. Virology 88, 10272-10275. Paganetti, P., Reichwald, J., Bleckmann, D., Abramowski, D., Ammaturo, D., Barske, C., Danner, S., Molinari, M., Müller, M., Papin, S., Rabe, S., Schmid, P. and Staufenbiel, M (2013) , Transgenic Expression of β1 Antibody in Brain Neurons Impairs Age-Dependent Amyloid Deposition in APP23 Mice. Neurobiology of Aging 34, 2866-2874. » Ferris, S.P., Jaber, N.S., Molinari, M., Arvan, P. and Kaufman R.J. (2013) UDP-glucose: Glycoprotein Glucosyltransferase (UGGT1) Promotes Substrate Solubility in the Endoplasmic Reticulum. Mol. Biol. Cell 24, 25972608. » Merulla, J., Fasana, E., Soldà, T. and Molinari, M. (2013) Specificity and Regulation of the Endoplasmic Reticulum-Associated Degradation Machinery. Traffic 14, 767-777.

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EPFL School of Life Sciences - 2014 Annual Report

Rainer Group Gregor Rainer

External Adjunct Professor - University of Fribourg

http://www.unifr.ch/inph/vclab/

Introduction

Gregor Rainer is an ESF EURYI young investigator, Associate Professor at the University of Fribourg and adjunct Professor at EPFL. Previously he was a group leader at the Max-Planck Institute for biological cybernetics in T端bingen. He received his MSc in Physics from the Univ. of Vienna in 1994, a PhD in Systems Neuroscience from the Massachusetts Institute of Technology in 1999 and a Habilitation in Neurobiology from Univ. of T端bingen in 2003. He was awarded an APART scholarship, a DFG Heisenberg award, a teaching prize from the T端bingen graduate school and an Otto Hahn Medal.

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In the visual cognition laboratory, we perform research on various aspects of visual neurophysiology, including sensory representations of visual information, visual decision making and integration of sensory signals with reward-, motivation- and basal forebrain activation - related signals. We study both cortical and relevant subcortical components of the visual processing brain network. We complement this work with behavioural studies of visually guided behaviour, including exploratory behaviours as well as task performance after appropriate operant conditioning training. We are particularly interested in comparative aspects of visual cognition and the underlying neural mechanisms, and thus study these processes in several species including rodents, tree shrews and macaques. This approach allows us to distinguish species-specific adaptations from more general principles of neural processing related to visual cognition. Large scales electrophysiological recordings from multichannel arrays, iontophoretic drug application, deep brain stimulation, eye tracking and mass spectrometry based quantification of neuroactive biomolecules are among the techniques currently employed in the laboratory. .

Keywords

Primary visual cortex, basal forebrain, acetylcholine, psychopharmacology, pulvinar, lateral geniculate nucleus, eye tracking, mass spectrometry, liquid chromatography.

Results Obtained in 2014

In the period 2013/14, we have published several studies on neural activity in the tree shrew primary visual cortex (V1). For example, in one study we were able to show that tree shrew V1 contains virtually no simple cells, as estimated by the parameter of overlap between white and black visual receptive subfields. This is of interest because the visual cortex of the cat, the classical animal model for V1 function, contains many simple cells, and these simple cells are thought to represent and obligatory step in the cortical transformation of thalamic inputs. Our findings show that this is not the case in general, since the tree shrew V1 contains in fact almost exclusively complex cells. Although tree shrew V1 neurons are temporally modulated by drifting gratings, this is in fact not a result of separate subfields but rather due to a striking neural responses dominance for responses to black stimuli (i.e. luminance decrements) over white stimuli. Additional work in the lab has focused on how deep brain stimulation of the basal forebrain increases contrast sensitivity of V1 neurons, a finding that underscores the importance of ascending cholinergic projections in modulating cortical activation. These neurophysiological results are complemented by reports on behavioural findings during object exploration in tree shrews, and a detailed description of a hard- and software solution for eye tracking without the need for rigid fixation. In parallel, we have further developed mass spectrometry based neurochemical analysis methods for small molecule quantitation, and applied these methods to study neuropeptide regulation by chronic nicotine administration.

EPFL School of Life Sciences - 2014 Annual Report

Team Members PhD Students Paolo de Luna Mohammed Faiz Jordan Poirot Jayakrishnan Nair Wenxue Li

External Adjunct Professors

Postdoctoral Fellows Xiaozhe Zhang Abbas Khani

Visual receptive field of a single neuron recorded in the supragranular layer of Tree Shrew V1 for white (left) and black (right) sparse noise stimuli. The colorbar to the right of each panel denotes mean firing rate in Hertz. Oriented two dimensional Gaussian functions were fit to the activation maps to obtain its slant (θ), as well as the exact spread of the receptive field (σl and σs). Receptive fields tend to be elliptical and horizontally slanted.

Selected Publications » » » » »

Veit J., Bhattacharyya A., Kretz R., Rainer G. (2014) On the relation between receptive field structure and stimulus selectivity in the tree shrew primary visual cortex. Cerebral Cortex 24(10):2761-71. De Luna P., Faiz M., Rainer G. (2014) A MATLAB-based eye tracking control system using non-invasive helmet head restraint in the macaque J Neurosci Methods 235: 41–50. Falasca S., Ranc V., Petruzziello F., Khani A., Kretz R., Zhang X., Rainer G (2014) Altered neurochemical levels in the rat brain following chronic nicotine treatment J Chem Neu: 59-60:29-35. Nair J., Topka M., Khani A., Isenschmid M., Rainer G. (2014) Tree shrews (Tupaia belangeri) exhibit novelty preference in the novel location memory task with 24-h retention periods. Frontiers in Psychology 5:303. Bhattacharyya A., Veit J., Bondar I., Kretz R., Rainer G. (2013) Basal forebrain activation controls contrast sensitivity in primary visual cortex BMC Neuroscience 14:55.

» Petruzziello F., Falasca S., Andren P., Rainer G., Zhang X. (2013) Chronic nicotine treatment impacts the regulation of opioid and non-opioid peptides in the rat dorsal striatum. Mol Cell Prot 12(6):1553-62.

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EPFL School of Life Sciences - 2014 Annual Report

Schorderet Group Daniel Schorderet

External Adjunct Professor - Institute for Research in Ophthalmology (IRO) - Sion- Director

www.irovision.ch

Introduction

Born in Fribourg, Daniel Schorderet studied Pediatrics in Geneva and did a post-doctoral fellowship at the University of Washington in Seattle with Arno Motulsky (medical Genetics) and Stanley Gartler (genetics). He was appointed research assistant professor in 1988. On his return, he became associate professor of Molecular Genetics and head of the Unit of Molecular Genetics at the Division autonome de Génétique médicale of the CHUV. He took over the direction of that division in 1997. He is board certified in Pediatrics, Medical Genetics and in genetic analyses. In 2003, he became director of IRO in Sion, an institute founded among others by EPFL and UNIL. In 2005, he became adjunct professor of Genetics at EPFL and in 2013 full professor at UNIL.

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The Institute for Research in Ophthalmology (IRO) is dedicated to study vision. Since 2003, we run a translational program called “Genes & Vision”, that takes advantage of the patients seen at the genetics clinics of the Jules-Gonin Eye Hospital as well as at other places through national and international collaborations. Genes & Vision has 6 research avenues: gene identification, study of retinal degeneration induced by these genes, investigate ophthalmic malignancies, and develop new animal models specifically in the zebrafish. These animal models are then used to develop new molecular therapies based on small peptides and patent-free drugs. Currently, we are developing a new program called ZebEye that aims at generating zebrafish models for all retinal degeneration diseases in order to use them for new drug screening. The gene identification program is based on next-generation sequencing of patients mostly affected with recessive disorders. More than 20 genes have been identified recently. We also focused on the role of HMX1, a transcription factor responsible for the oculoauricular syndrome of Schorderet-Munier-Franceschetti.

Results Obtained in 2014

Our investigation of numerous families with anophthalmia/ microphthalmia (M/A) allowed us to identify a major role for ALDH1A3 in the development of the eye and to show that mutations in this gene represent an important factor in recessively inherited M/A. We also determined that MCT12 was implicated in cataract and glucosuria and acted as a creatine transporter and participated in the discovery of LRIT3, a new gene responsible for congenital stationary night blindness. A major part of our research is devoted to understand the role of HMX1, a gene that we discovered 5 years ago. We showed that it needs dimerization to be active, characterized the domain of dimerization, developed a predictive model to identify its targets and validated EPHA6 as its first target. Using a yeast two-hybrid approach, we characterized several proteins as member of the HMX1 inhibitory complex. We also evaluated several molecules and showed that a small peptide based on a BH3 domain is able to kill retinoblastoma cells.

Keywords

Oculogenetics, gene identification, HMX1, RPE65, NR2E3, retinal ischemia, hypoglycemia, age-related retinal degeneration, zebrafish.

EPFL School of Life Sciences - 2014 Annual Report

Team Members PhD Students Antille Mélanie Von Alpen Désirée Linda Wicht

Administrative Assistant Evéquoz Pascale

External Adjunct Professors

Postdoctoral Fellows Allaman-Pillet Nathalie Boulling Arnaud Marcelli Fabienne Renner Nicole

Family with anophthalmia. A. Pedigree showing 4 affected children in one generation. B. Partial electropherogram of ALDH1A3 exon 10 with homozygous and heterozygous (*) mutation. C. Picture of affected individuals including brain MRI of IV.1 showing absence of globe and hypoplastic optic nerves. (From Abouzeid et al. Human Mutation 2014)

Selected Publications » Marcelli F, Boisset G, Schorderet DF(2014). A Dimerized HMX1 Inhibits EPHA6/epha4b in Mouse and Zebrafish Retinas. PLoS One 9(6):e100096. » Abouzeid H, Favez T, Schmid A, Agosti C, Youssef M, Marzouk I, El Shakankiry N, Bayoumi N, Munier FL, Schorderet DF (2014). Mutations in ALDH1A3 represent a frequent cause of microphthalmia /anophthalmia in consanguineous families. Hum Mutation, 35(8):949-53. » Boulling A, Wicht L, Schorderet DF (2013). Identification of HMX1 target genes: a predictive promoter model approach. Molecular Vision 19:1779-1794. » Yahyavi M*, Abouzeid H*, Gawdat G, de Preux A-S, Xiao T, Bardakjian T, Schneider A, Choi A, Jorgenson E, Baier H, El Sada M, Schorderet DF*, Slavotinek AM* (2013). ALDH1A3 Loss of Function Causes Bilateral Anophthalmia/Microphthalmia and Hypoplasia of the Optic Nerve and Optic Chiasm. Hum Molec Genet, 22(16):3250-3258 (*contributed equally) » Abplanalp J, Laczko E, Philip NJ, Neidhardt J, Zuercher J, Braun P, Schorderet DF, Munier FL, Verrey F, Berger W, Camargo SMR, Kleockener-Gruissem B (2013). The cataract and glucosuria associated monocarboxylate transporter MCT12 is a new creatine transporter. Hum Molec Genet, 22(16):3218-3226. » Zeitz et al. (2013). Whole exome sequencing identifies mutations in LRIT3 as a cause for autosomal recessive complete congenital stationary night blindness. Am J Hum Genet 92:67-75.

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EPFL School of Life Sciences - 2014 Annual Report

Tanner - Swiss TPH Marcel Tanner

External Adjunct Professor - Swiss TPH Institute - Basel - Director

www.irovision.ch

Research Interests

Marcel Tanner holds a PhD in medical biology from the University of Basel and a MPH from the University of London. He is Director of the Swiss Tropical & Public Health Institute and Professor (chair) of Epidemiology and Medical Parasitology, University of Basel. The research ranges from basic research in cell biology and immunology on malaria, schistosomiasis, trypanosomiasis and filariasis to epidemiological and public health research. Research, teaching and health planning are based on long term work in Africa and Asia. He is a member of various national and international bodies and boards.

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Swiss TPH (Swiss Tropical and Public Health Institute) and the EPFL School of Life Sciences are collaborating with the goal to bring together complementary expertise of the two institutions in research on hostpathogen interaction in infectious and chronic diseases and the development of new diagnostics, drugs and vaccines. Besides the collaboration in research, exchanges of teaching faculty and students within the MSc courses is continuing. Furthermore, a collaboration with the EPFL School of Computer and Communication Sciences on the development of Massive Open Access Online Courses (MOOCs) is initiated.

and the preclinical profiling of vaccine candidates. These activities rely heavily on the BSL3 animal facilities at the GHI. A mouse model for Buruli ulcer has been successfully established and is used to evaluate the protective efficacy of candidate vaccines. The model is also used for studies towards the re-purposing of tuberculosis drug candidates.

The two mycobacterial pathogens, Mycobacterium tuberculosis and M. ulcerans, the causative agents of the human diseases tuberculosis and Buruli ulcer, are being investigated as part of the GHI-Swiss TPH collaboration. Collaborative activities towards the clinical testing of a tuberculosis drug candidate developed by GHI in Tanzania are one major focus. Work in vaccinology is concentrated on the emerging disease Buruli ulcer and involves studying the immune responses to infection with M. ulcerans

Keywords

The biannual report of Swiss TPH: http://www.swisstph.ch/resources/publications/biennial-reports/ br2013-14.html

Epidemiology, public health, vaccines, drugs and diagnostics.