by the Data Management and analysis center of IPC-PACA...

CANCER RESEARCH CENTER OF MARSEILLE (CRCM)CONNECTING TALENTSFUELING THE NEXT WAVE OF CANCER SCIENCE

TYPES OF CANCER TREATED AT IPC (2016)

“Today, almost 800 molecules are being evaluated in clinical

oncology, a win-win partnership for oncologists and patients

“

1612

1365

1257

960

550

368227111 Breast

Digestive tract

Hematology

Urology

Gynecology

Lung

Onco-endocrinology

ENT

Dermatology

Soft tissue

Bone, cartilage

Other

Strong basic scienceCRCM hosts outstanding basic science teams in cell biology, in the field of DNA repair and genome instability. The CRCM research teams focus on the fundamental aspects of oncogenesis, progression and tumor dissemination, with the aim of identifying the molecular alterations at the origin of cancer and their functional consequences by experimental in vitro and in vivo approaches at the molecular and cellular levels or in model organisms such as bacteria, yeast or mice. The main topics of investigation are the mechanisms that maintain genome stability, the basic concepts in cell polarity and cell signalling, the mechanisms involved in cell fate and epigenetics, and the mechanisms of host-tumour interactions and immunity.

Translational, preclinical and clinical research teamsAt IPC and CRCM, translational research is based on project teams with experts in our therapeutic areas of excellence: immunology, onco-hematology, breast tumors, digestive cancers, urological cancers and new endocrine tumours. Oncologists, surgeons, radiotherapists, bio-pathologists, basic researchers and specialists in applied research (biomarker experts in clinical trials) work together to identify high-potential molecules which are then evaluated in animals and humans by IPC (alone or in partnership).

These teams benefit from the complementary know-how and technologies available at IPC: › an integrated biological laboratory to identify genetic abnormalities or establish the identity card of the

tumors (genomic, expression profiling, proteomics); › animal models of human diseases and 80 000 samples from our biobank challenger for new therapeutic

targets; › an immunomonitoring platform to quantify and qualify the immune response of patients to these new

molecules and imaging and advanced medical science techniques such as endoscopy to evaluate their functional effect in situ.

In addition, three platforms are dedicated to clinical research in order to conduct clinical trials in compliance with regulatory and ethical requirements: the Department of Clinical Research and Innovation, the Data Management and Analysis Center, and a unit dedicated to early clinical trials called ETOH (Evaluation of Therapeutic Onco-Hematology).

RESEARCH CONTINUUM : FROM SCIENCE TO HEALTH

THE CANCER RESEARCH CENTER OF MARSEILLE (CRCM)

Affiliated to Inserm (UMR1068), CNRS (UMR7258) and Aix-Marseille University (UM105), CRCM is located at the heart of Institut Paoli-Calmettes and has a unique mission:

to increase the biology and medicine knowledge of cancer through translational programs from bench science to bedside clinical practice.

This continuum between basic, translational and clinical research is a hallmark of CRCM. We explore communication networks (signals, surface receptors, communication nodes...) that regulate the action of cancer and immune cells and seek to identify genetic alterations (from point mutations of a gene to

complex chromosomal abnormalities) leading to the transformation of a healthy cell into a tumor cell.

Our aim is to ensure better and faster understanding, diagnosis and treatment of cancer diseases.

At CRCM, comprehensive research programs have already improved our knowledge on cancer, by showing the important role of immune cells fighting cancer and the strategies used by tumor cells in an attempt to escape them.

These programs also led to new molecular target discoveries and their translation into diagnostic and therapeutic applications in oncology.

346 CRCM STAFFscientists, professors, clinicians, engineers, technicians, administrative staff and PhD students

17 TECHNOLOGICAL PLATFORMSwhere clinicians and scientists work together on a daily basis at IPC and CRCM

8.000 SQ. METERSof lab space

19 TEAMSdedicated to research working in close collaboration with IPC's clinical teams

KEY FIGURES

OUR FOUR RESEARCH PRIORITIES

Public / private partnerships are a prerequisite for innovation.IPC provides patients with the latest innovative treatments, and pharmaceutical partners with experience in running clinical trials according to industrial, ethical and regulatory quality standards. Today, almost 800 molecules are being evaluated in clinical oncology, a win-win partnership for oncologists and patients. Clinical trials are conducted within the IPC expertise or in partnership with other institutions: our researchers and clinicians work closely with drug professionals to accelerate the development of more active therapies and ensure that our patients get quick access to the best of innovation in cancer therapy.

› NEW TARGET IDENTIFICATION Identification of new targets specifically expressed by tumor cells and validation of their therapeutic potential.

› TUMOR/HOST RELATIONSHIP Understanding of the complex relationship between the tumor and its host and the implementation of new strategies in order to limit the formation of metastases.

› NEW BIOMARKER CHARACTERIZATION Identification of new biomarkers to improve cancer classification, facilitate its diagnosis, prognosis and monitoring, and predict responses to treatments.

› INNOVATIVE CLINICAL TRIALS Launch of innovative clinical trials within our fields of expertise and scientific treatment (breast, blood and digestive tract).

Created in 1945, the Institute originated from the outcome of a major French government ordinance. It is a private, independent, non-profit center involved in the management of more than 8.600 new cases of cancer each year. Its main areas of activity are breast cancers, digestive tract cancers and hematological cancers.

KEY FIGURES

246 ONGOING CLINICAL TRIALS(26 promoted by IPC)OPERATING BUDGET 209 M€100.250 CONSULTATIONS IN 201710.083 NEW PATIENTS/YEAR247 CONVENTIONAL BEDS91 OUTPATIENT BEDS1.554 STAFF 210 PHYSICIANS300 SCIENTISTS

Driving innovation,

personalized for patients

› technological innovation,› patient-centered care strategies,

› innovative multidisciplinary cancer care strategy.

The strengths of IPC are a world-class scientific network and medical excellence

combining research programs to the most advanced technology. At IPC, biologist experts and clinicians are

developing a collaborative culture to translate fundamental and applied science discoveries into remarkable

health gains for patients, every day.

Our mission› to prevent, diagnose and treat adult cancer patients, › to educate and train young professionals to the most advanced translational cancer research and cancer

patient care,› to develop world-class basic translational and clinical research programs.

Our assets› scientists, and translational research experts who lead and convert breakthrough discoveries into

successful applications,› oncologists and medical staff who evaluate new treatment efficiency and safety through clinical

development, › patients who benefit from all these programs.

The Institute also provides researchers with› a comprehensive biobank including biological collections of more than 80,000 samples

available for medical care and research purposes, › state-of-the-art medical and technological platforms and equipment.

INSTITUT PAOLI-CALMETTES is one of the French largest university-affiliated cancer center,exclusively devoted to research and patient care

246 ONGOING CLINICAL TRIALS(26 promoted by IPC)

INSTITUT PAOLI-CALMETTES

Driving innovative programs in immunology and oncology

EMPOWERS RESEARCHERS AND CLINICIANS

To provide the best patient care

INSTITUTPAOLI-CALMETTES232 boulevard Sainte Marguerite13009 MarseilleFrance

www.institutpaolicalmettes.fr/en tel + 33 (0)4 91 22 37 00

Director General: Prof. Patrice [email protected]

CANCER RESEARCH CENTER OF MARSEILLE27 boulevard Leï Roure13009 MarseilleFrance

www.crcm.marseille.inserm.fr/en tel + 33 (0)4 86 97 72 01

Director: Prof. Jean-Paul [email protected]

Design Angélique GERRARD & Production KOM Agency www.kom-fr.com

TEAM ADHESION MOLECULES IN TUMOR/HOST INTERACTIONS

MICHEL AURRAND-LIONS

The laboratory has a long lasting experience in the study of vascular and perivascular adhesion molecules involved in leukocyte adhesion and migration. We have signifi cantly contributed to this fi eld by cloning the endothelial adhesion molecules JAM-B (Jam2) and JAM-C (Jam3) in the early 2000s.

The JAM protein family belongs to the immunoglobulin superfamily (Ig Sf) and is composed of three classical members, JAM-A, JAM-C, JAM-B sharing up to 35% amino-acid identity. Their cytoplasmic tails are relatively conserved in length and composition with C-terminal sequences containing a type II PDZ binding motif.

JAM-C interacts with JAM-B through its two extracellular Ig domains and inhibition of the interaction with antibodies against JAM-C inhibits tumoral angiogenesis and infl ammation. This has been attributed to targeting of inter-endothelial junctions since neither JAM-B nor JAM-C is expressed by mature leukocyte in mouse. This contrasts with the human situation in which JAM-C is expressed by platelets, NK cells, dendritic cells, activated T cells, B cells or lymphoma cells and supports leukocyte adhesion to JAM-B expressing cells.

More recently, we and others have found that JAM-C is expressed by HSC mice and interacts with JAM-B expressed by bone marrow endothelial

cells and Mesenchymal Stem Cells (MSCs). JAM-B/JAM-C interaction contributes to HSC homeostasis as demonstrated by HSC exhaustion in Jam-b defi cient mice and by the downregulation of JAM-B/JAM-C interaction during HSC mobilization.

In follow-up studies, we have found that JAM-C is expressed by human HSC and interacts with Jam-B mice

across species.

Expression of JAM-C on human stem and progenitor cells (HSPC) is functional since it mediates HSPC adhesion to MSCs but not to other JAM-B-expressing stromal cells such as osteoblastic-diff erentiated MSCs. These results prompt us to further study adhesion mechanisms in normal and pathological hematopoiesis.

The major aim of our projects is to demonstrate that targeting leuko-stromal adhesion mechanisms is a valuable therapeutic approach to reduce drug-resistance and relapse in hematological malignancies. Inhibition of JAM-B/JAM-C interaction is used as a paradigm.

Selected recent publications1 ) Arcangeli et al. Function of Jam-B/Jam-C interaction in homing and mobilization of human and mouse hematopoietic stem and progenitor cells. Stem Cells. 2014 Apr;32(4):1043-54.

2 ) Stalin et al. Soluble melanoma cell adhesion molecule (sMCAM/sCD146) promotes angiogenic eff ects on endothelial progenitor cells through angiomotin. J Biol Chem. 2013;288:8991-9000.

3 ) Chai et a.. KIT-D816V oncogenic activity is controlled by the juxtamembrane docking site Y568-Y570. Oncogene. 2013.

4 ) Belotti et al. The human PDZome: a gateway to PSD95-Disc large-zonula occludens (PDZ)-mediated functions. Mol Cell Proteomics. 2013;12:2587-2603.

5 ) Wyss et al. Junctional adhesion molecule (JAM)-C defi cient C57BL/6 mice develop a severe hydrocephalus. PLoS One. 2012;7:e45619.

6 ) Arcangeli et al. The Junctional Adhesion Molecule-B regulates JAM-C-dependent melanoma cell metastasis. FEBS Lett. 2012;586:4046-4051.

7 ) Frontera et al. Cutting edge: JAM-C controls homeostatic chemokine secretion in lymph node fi broblastic reticular cells expressing thrombomodulin. J Immunol. 2011;187:603-607.

8 ) Arcangeli et al. JAM-B regulates maintenance of hematopoietic stem cells in the bone marrow. Blood. 2011;118:4609-4619.

9 ) Reynolds et al. Tumour angiogenesis is reduced in the Tc1 mouse model of Down’s syndrome. Nature. 2010;465:813-817.

Michel Aurrand-LionsCRCM

27 Boulevard Leï Roure13009 Marseille, France

[email protected]+33 (0)4 86 97 72 91

CONTACT

TEAM MOLECULAR MECHANISMS OF TUMOR CELL MOTILITY

ALI BADACHE

Cell motility and invasion underlie the dissemination of cancer cells away from the primary tumor site to colonize secondary sites. This process known as metastasis is the main cause of cancer patient lethality. The development of therapies to fi ght metastasis requires a better understanding of the molecular mechanisms of tumor cell motility. The role of the actin cytoskeleton in cell migration has been extensively studied. In contrast, the contribution of microtubules to cell motility and the mechanisms of action at the cellular and molecular levels clearly deserve further investigation. Our present projects focus on the identifi cation and the structural and functional characterization of protein complexes regulating microtubule dynamics and capture. In particular, our original fi ndings underscore the central role of proteins that specifi cally associates with microtubule + end: the + end tracking proteins (or +TIPs), in microtubule capture and directed cell migration.

Overexpression of the ErbB2 receptor tyrosine kinase in breast cancer is associated with an aggressive form of the disease. We have been working towards deciphering how ErbB2-dependent signaling pathways drive cell migration and metastasis and at determining the contribution of the microtubule cytoskeleton to these processes. We have identifi ed Memo (Mediator of erbB2-driven motility) as a novel eff ector of ErbB2 (Zaoui et al., 2010). Using live cell imaging, we showed that Memo controls microtubule capture at the leading edge of migrating cells. We further demonstrated that capture of microtubules at the cell front is necessary for directional migration towards growth factors. We delineated a Memo-dependent signaling pathway involving the small GTPase RhoA and its eff ector the formin mDia1, whose function is to target a microtubule capture complex that includes the tumor suppressor APC and the spectraplakin ACF7, to the cell cortex (Zaoui et al., 2010, Daou et al., 2014). APC and ACF7 belong to a particular category of microtubule-associated proteins, the +TIPs, localizing at the + end of microtubules via their interaction with the protein EB1. Spatial and temporal coordination of microtubule capture by the Memo/ACF7 pathway, stabilization via PI3K/Akt signaling and polarization is required for effi cient directional migration (Benseddik et al, 2013). Our recent

data shows that expression of Memo in breast tumors correlates with poor prognostic factors and that Memo contributes to experimental lung metastasis (MacDonald et al, 2014). Therefore, it is important to further explore the contribution of microtubules to metastasis and the underlying molecular mechanisms.Our research strategy relies on a combination of targeted proteomics, cell imaging and structural approaches. We have initiated a program of systematic analysis of +TIP protein-protein interactions in cellular models of ErbB2-induced breast cancer cell migration. Using state of the art mass-spectrometry analysis, we have identifi ed + end associated complexes, but also centrosomal complexes. Functional approaches using down-regulation of proteins by siRNA, rescues by wild-type or interaction-defective proteins, selected those altering both directed migration

Research program

EB1 (red) which specifi cally associateswith the plus-end of microtubules (green)

is a critical player in microtubule nucleation, microtubule capture and directed cell motility.

Selected recent publications1 ) MacDonald G, Nalvarte I, Smirnova T, Vecchi M, Aceto N, Dolemeyer A, Frei A, Lienhard S, Wyckoff J, Hess D, Seebacher J, Keusch JJ, Gut H, Salaun D, Mazzarol G, Disalvatore D, Bentires-Alj M, Di Fiore PP, Badache A, Hynes NE. Memo is acopper-dependent redox protein with an essential role in migration and metastasis. Sci Signal. 2014 Jun 10;7(329):ra56.

2 ) Daou P, Hasan S, Breitsprecher D, Baudelet E, Camoin L, Audebert S, Goode BL, Badache A. Essential and nonredundant roles for Diaphanous formins in cortical microtubule capture and directed cell migration. Mol Biol Cell. 2014 Mar;25(5):658-68.

3 ) Benseddik K, Sen Nkwe N, Daou P, Verdier-Pinard P, Badache A. ErbB2-dependent chemotaxis requires microtubule capture and stabilization coordinated by distinct signaling pathways. PLoS One. 2013.

4 ) Gillibert-Duplantier J, Duthey B, Sisirak V, Salaün D, Gargi T, Trédan O,Finetti P, Bertucci F, Birnbaum D, Bendriss-Vermare N, Badache A. Gene expression profi ling identifi es sST2 as an eff ector of ErbB2-driven breast carcinoma cell motility, associated with metastasis. Oncogene. 2012.

5 ) Calligaris D, Manatschal C, Marcellin M, Villard C, Monsarrat B, Burlet-Schiltz O, Steinmetz MO, Braguer D, Lafi tte D, Verdier-Pinard P. Tyrosine-dependent capture of CAP-Gly domain-containing proteins in complex mixture by EB1 C-terminal peptidic probes. J Proteomics. 2012 Jun 27;75(12):3605-16.

6 ) Zaoui K, Benseddik K, Daou P, Salaün D, Badache A. ErbB2 receptor controls microtubule capture by recruiting ACF7 to the plasma membrane of migrating cells. Proc Natl Acad Sci U S A. 2010 Oct 26;107(43):18517-22.

Ali BadacheCRCM


[email protected] +33(0)4 86 97 73 21

CONTACT

(measured in various motility assays: Transwell, cell tracking, wound healing) and microtubule capture at the leading edge (evaluated by time-lapse microscopy). Our goal is to isolate functional protein complexes and perform mapping of protein-protein interactions by molecular biology and cross-linking coupled to mass spectrometry analysis; and to produce recombinant proteins for in vitro reconstitution experiments and structural characterization (by NMR, SAXS or ion-mobility MS). We also investigate the role of complexes harboring

kinase and phosphatase activity, in the fi ne-tuning of microtubule dynamics and cancer cell migration and how it may be connected to ErbB2 signaling pathways. To this end, we perform phospho-proteomic analyses on the protein complexes (via 1D/2D gel electrophoresis and mass spectrometry) and diff erential targeted proteomics using mutants for protein-protein interactions. Finally, we use small molecules (kinase or phosphatase inhibitors, chemotherapeutic microtubule targeting agents) or molecular tools to pinpoint signaling nodes involved in microtubule capture and cell migration.We plan to extend our observations on 3D invasion models to explore the role of microtubules in cancer cell invasion. We will also test the relevance of our microtubule-dependent migration models on patient-derived material (Institut Paoli-Calmettes breast cancer xenografts collection) using invasion assays and correlate this data to expression profi ling (cDNA microarrays, TMA) and targeted gene sequencing. This research program will provide further insights into the mechanisms of tumor cell motility, invasion and metastasis and may lead to the discovery of biomarkers and the expansion of the toolbox for targeting metastatic cancer.

Multiple approaches to tackle macromolecular complexes that control tumor cell motility.

TEAM COMPUTATIONAL BIOLOGY & DRUG DESIGN

PEDRO BALLESTER

Research in the CBDD group focuses on the development and application of computational methods to predict and analyse the modulation of proteins and cell functions by small organic molecules. Problems of interest include oncology biomarker discovery, modelling cancer pharmacogenomics, polypharmacology prediction and drug design (phenotypic, structure-based and ligand-based).

Biomarker Discovery

Computational Drug Design

Targeted drugs, which inactivate specifi c molecular targets upon which cancer tumours rely to drive cell growth, have delivered therapies that are more specifi c and thus with generally less side-eff ects than traditional cytotoxic chemotherapy. Unfortunately, these therapies are only eff ective in some patients and our current ability to identify these responsive patients before administering the drug is still very limited. This diff erential drug response is not only due to heterogeneities between patients but also those across cancer tumour types.The advent of Next Generation Sequencing (NGS) constitutes an unprecedented opportunity to study the molecular basis of this aspect of human variation.

However, new computational methods are needed to predict the effi cacy of a drug from the genomic and epigenomic status of patients and their tumours. Furthermore, these molecular profi les can also be used to predict prognosis given the clinical history of the patients from whom samples were taken.

Therefore, our main goals in this area are: a) to investigate optimal methodologies to build predictive biomarkers of drug effi cacy and prognosis and b) to apply these methodologies to leukemia, pancreatic and breast cancers in collaboration with preclinical and clinical scientists at the CRCM.

In addition to research intended to optimise the application of known drugs, there is a constant need to discover new drugs to treat cancer patients who are non-responsive, relapsed and/or have poor-prognosis. However, drug discovery is not possible without a way to identify molecules that modulate the biological function of a validated therapeutic target. There are now a range of computational methods that predict the biological activities of a molecule from ever-increasing volumes of relevant experimental data. When applied to Virtual

Screening (VS), these computational methods can be used to search vast databases of candidate molecules for those likely to be active against the considered target. In practice, these tools have been able to discover drug leads and/or chemical probes in a wide range of targets and are particularly useful in those targets where High-Throughput Screening (HTS) performs poorly or is not an option (e.g. technically not possible, too expensive or too slow).

Depending on the type of data available for the target, three classes of methods can be applied. First, cell-based methods, such as those based on pharmacogenomics data, aim at identifying molecules inhibiting cancer cell growth. A subsequent requirement is to fi nd the protein targets of such phenotypic hits in order to understand their whole-cell activity (beyond drug leads, this is also important to explain the effi cacy and side-eff ects of development or even approved drugs). Second, ligand-based methods relate the chemical structure of drug molecules with their bioactivities against a protein or cell

target. Third, structure-based methods often operate by exploiting the X-ray crystal structure of a protein target in order to predict whether and how the molecule binds the target.Our research in this broad area includes investigating various methods for molecular target prediction exploiting recently-available chemogenomics databases as well as machine learning methods to predict how cellular or protein activity is aff ected by small molecules regardless of the type data available (interaction, structural, chemical, etc.).

Pedro BallesterCRCM


[email protected]

CONTACT

Drug RepositioningAny of these computational methods can be applied to drug repositioning, which has become an area of intense interest in recent years. In drug repositioning, a drug is associated in some way to an indication other than that for which it was initially approved. This strategy has the advantage that all the toxicity-related requirements in the preclinical and clinical stages have already been met by the drug molecule. Therefore, if the predicted association is experimentally confi rmed, the repositioned drug should follow a faster and cheaper

route to Phase II clinical trials for the new indication. In a pilot study that used one of our methods to screen the set of all FDA-approved drugs, only the best hit was experimentally tested for anti-cancer activity and this was enough to discover that the anti-hypertension drug Telmisartan inhibits the growth of colon cancer cell lines at the low micromolar level. Ongoing research includes the application of cell-based methods to unveil further drug repositioning opportunities as well as positioning a targeted drug candidate in responsive cancer subtypes.

Selected recent publications1 ) Li, H., Leung, K.-S., Wong, M.-H., Ballester, P.J. (2016) “USR-VS: a web server for large-scale prospective virtual screening using ultrafast shape recognition techniques”. Nucleic Acid Research (doi: 10.1093/nar/gkw320)

2 ) Peon, A., Dang, C., Ballester, P.J. (2016) “How reliable are ligand-centric methods for target fi shing?” Frontiers in Chemistry 4:15.

3 ) Ain, Q.U., Aleksandrova, A., Roessler, F.D., Ballester, P.J. (2015) “Machine-learning scoring functions to improve structure-based binding affi nity prediction and virtual screening”. WIREs Computational Molecular Science 5, 405–424.

4 ) Hoeger, B., Diether, M., Ballester, P.J., Köhn, M. (2014) “Biochemical evaluation of new virtual screening methods reveals cell-active inhibitors of the cancer-promoting phosphatases of regenerating liver”. European Journal of Medicinal Chemistry 88, 89-100.

5 ) Ballester, P.J., Schreyer, A., Blundell, T.L. (2014) “Does a more precise chemical description of protein-ligand complexes lead to more accurate prediction of binding affi nity?”. Journal of Chemical Information and Modeling 54, 944–955.

6 ) Menden, M., Iorio, F., Garnett, M., McDermott, U., Benes, C., Ballester, P.J., Saez-Rodriguez, J. (2013) “Machine learning prediction of cancer cell sensitivity to drugs based on genomic and chemical properties”. PLOS ONE 8, e61318.

7 ) Ballester, P.J. and Mitchell, J.B.O. (2010) “A machine learning approach to predicting protein-ligand binding affi nity with applications to molecular docking”. Bioinformatics 26:9, 1169-1175.

TEAM PREDICTIVE ONCOLOGY

D. BIRNBAUM AND F. BERTUCCI

The objectives of the laboratory are to identify and characterize the metabolic pathways and protein complexes altered during oncogenesis, in breast cancers, visceral cancers, prostate cancer, lung cancer, and myeloid leukemia, and to transfer the results to the clinic, as means to improve classification, prognosis and therapy.

Building the next generation of Personalized MedicineGenomics of breast cancers MAIN INVESTIGATORS: D. BIRNBAUM, F.BERTUCCI, M. CHAFFANETBreast cancer (BC) is a heterogeneous disease that can be classified according to histoclinical features (i.e. inflammatory breast cancer - IBC) and to molecular biology. Five major molecular subtypes are distinguished: normal-like, luminal A, luminal B, ERBB2 and basal. The last three have a poor prognosis. Our goal is to progress in the molecular and cellular definition of breast cancers and help improve their treatment. For this, we work on taxonomy, molecular targets and cancer stem cells. Of note that the same approach was since successfully applied to other types of cancers. Our goals are to understand breast cancers heterogeneity, how normal and cancer cells function, how they dysfunction, and how they can be targeted. We thus conducted a whole-genome screen to identify genes that regulate cancer cells. By doing so, we improved BC classification, defined gene expression signatures (GES) and markers of prognosis. We contributed to show that a major parameter of progno-sis in Basal BC is the quality of the immune reaction that takes place in each tumor. IBC, a highly aggressive and metastatic form of breast cancers, remains a mystery. We characterized its GES and genome alterations. We established the genome, gene expression and methylation profiles of a series of luminal B BCs and identified candidate oncogenes and tumor suppressor. We characterized the ZNF703 oncogene, and two tumors suppressors, L3MBTL4 and Afadin. We showed that ZNF703 is amplified and overexpressed in 8p12-amplified breast cancers, and when overexpressed in cell line, it increases its stem cell population, represses estrogen receptor (ER)-associated pathway and it expands E2F1 transcription program. Because of the inter- and intra-heterogeneity of breast cancers, appropriate selection of treatment is necessary for each patient. In collaboration with clinicians, we initiated a personalized medicine program enrolling metastatic patients. The program comprises: metastasis biopsy, banking, extrac-tion of nucleic acids, NGS, grafting to obtain Patient-derived xenograft (PDX), establishment of the molecular identity of a metastasis and its drug response, as well as the study of circula-ting tumor cells. Therapies are selected upon two types of results: genomics studies and model-based. We established a bank of well characterized primary and metastatic tumors (mostly from breast metastases so far). These xenografts conserve the genome and gene expression of their cognate tumors and are therefore excellent models for functional studies and drug testing. A bank of on-label drugs and new compounds is tested in in vitro and in vivo studies using PDX on short-term cultures of bulk tumor cells. Repositioning already-approved drugs is of growing interest. We recently repositioned a drug that synergizes with chemotherapeutic drugs and targets cancer stem cells. We are now also involved in the next generation ex vivo modelling of tumors, using patient-derived tumoroids, including stromal cells (see below).

TEAM PREDICTIVE ONCOLOGY

Providing new Biomarkers and Models to anticipate tumor evolution Circulating tumor cells (CTCs) and metastases in visceral tumorsMAIN INVESTIGATORS: E. MAMESSIER & C. ACQUAVIVA

Malignant myeloid diseasesMAIN INVESTIGATORS: V. GELSI-BOYER, A. MURATIWe are interested in defining mutated genes and homogeneous entities in malignant myeloid diseases including myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). In the past we showed that ASXL1 mutations can be found in all types of malignant myeloid diseases and have a major prognostic impact. We identified new altered genes: cohesins, SUZ12, DOK2, BCOR and SETBP1 in myeloid diseases. We now study how combinations of mutated genes collaborate to transform MPN to AML and how they induce specific phenotype such as FAB M6.

Lung cancerMAIN INVESTIGATORS: F. BARLESI AND P. TOMASINILung cancer is the leading disease in the field of predictive oncology with 10 predictive biomarkers assessed routinely in advanced stages. However, some of the most frequent mutations (ie KRAS) are still undruggable, and our team wants to understand the heterogeneity of this protein and its related pathways in order to develop new therapeutic options. In addition, our works focus on known oncogenic addictions (ie EGFR, ALK, etc) and the prediction of mechanisms of resistance to current targeted agents and also occurrence of specific metastic sites (ie, brain).

Our current project is to develop tools able to predict drug efficiency in real-time for each patient. This will be achieved by addressing 2 challenges:∠ Establishing the role of CTCs as a biomarker (not very invasive) for therapeutic efficiency and more specifi-cally to detect early signs of drug resistance mecha-nisms development,

∠ Developing “complex tumor units”, the next genera-tion of patient-derived tumoroids. They are 3D tumor units composed of malignant cells in interactions with their microenvironment (particularly with fibroblasts which represent the major cell population within the stroma, endothelial and/or immune cells).

For this, we have access to a cohort of colorectal cancer patients, sampled at different stage of their disease (Stage II/III or stage IV), i.e. before surgery or neoadjuvant therapy, after surgery, after adjuvant

therapy and in case of relapse. CTCs will be characte-rized (morphological, phenotypic & molecular charac-terization) at all time points. Tumor samples will be used to engineered “complex tumor units” for drug screening purposes. All the scientific information extracted from this study, combined with microfluidic technology will contribute to lay the basis of our long-term ambitious project consisting in building a pertinent fully humanized and personalized tumor model, called “metastases-on-a-chip”. With this model, our aim is to understand the different steps involved in metastases occurrence and how they can be targeted at an early stage. These projects are feasible thanks to interactions with various scientific experts, such as oncologists, surgeons, computational analysts, mathematicians but also specialists in tissue engineering and microfluidic.

D. BIRNBAUM AND F. BERTUCCI

The “Grail”: providing the right Therapeutic innovation for the right patientAntibody-drug conjugate (ADC)MAIN INVESTIGATOR: M LOPEZ ADCs are a rapidly evolving therapeutic class and many efforts are done to improve efficacy and safety. Currently, 15 ADCs are being evaluated clinically in breast cancer. Design of an effective ADC for cancer therapy requires the identification of an appropriate target, a monoclonal antibody against the target, potent cytotoxic agents, and the conjugation of the monoclonal antibody to one of these agents.We identified nectin-4 as a potential therapeutic target in breast cancer. Nectin-4 (encoded by the PVRL4 gene) is a cell adhesion molecule involved in the formation and maintenance of adherens junctions. It is also a receptor for the measles virus, mediating its internalization via endocytosis. Nectin-4 is expressed during foetal development, but is re-expressed as a tumor-associated antigen with pro-oncogenic properties in various carcinomas. In breast cancer, we have shown the correlation of nectin-4 expression with basal biomarkers, Triple-negative (TN) status and HER2 expression. Recently, we showed that nectin-4 is both a new prognostic biomarker and a therapeutic target for ADC in patients with TN breast cancer. We thus developed an ADC (N41mab-vcMMAE) comprising a human anti-nectin-4 monoclonal antibody conjugated to the toxin monomethyl auristatin-E (MMAE). In vitro, this ADC bound to nectin-4 with high affinity and specificity and induced its internalization as well as dose-dependent cytotoxicity on nectin-4-expressing breast cancer cell lines. In vivo, it induced rapid, complete and long-lasting responses of xenografted nectin-4-positive TN BC samples including primary tumors, local relapses, and metastatic lesions; efficiency was dependent on both the dose and the nectin-4 expression level. Clinical development is currently under process.We plan to evaluate the targeting of nectin-4 in treatment-resistant HER2-positive breast cancer and in other carcinomas like lung cancer.

Prostate Cancer Mechanisms identification and Oligonucleotide-based NanotherapiesMAIN INVESTIGATOR: P. ROCCHI We identified Hsp27 as a highly overexpressed gene in castration resistant prostate cancer (CRPC). Hsp27 knockdown using antisense oligonucleotides (ASO) and siRNA increases apoptosis and enhances hormone- and chemo-therapy in PC. We developed a 2nd generation ASO targeting Hsp27 that has been licensed (OGX-427) and a clinical trial phase II is ongoing. We identified 226 Hsp27 interacting partners using two-hybrid screens and found new potential Hsp27 functions such as telomere maintenance, RNA splicing and DNA repair by NHEJ. We hope to understand the mechanisms leading to Hsp27 over-expression and identify the molecular switches underlying PC. We showed the role of Hsp27 partner TCTP during PC evolution: ASO-induced TCTP silencing restores hormone and docetaxel sensitivity by enhancing apoptosis and delaying tumor progression. We developed and patented a first generation TCTP phosphorothioate backbone ASO that downregulates mRNA and protein expression level. We recently developed and patented a second generation lipid-modified antisense oligonucleotides (LASO) with the ability to downregulate TCTP levels without any transfecting agent (TCTP-LASO) in prostate cancer cells, leading to restoration of the normal function of p53, a tumor suppressor protein. We also demonstrated that TCTP-LASO forms nanomicelles that we can use to include drugs for combined therapy. We will now develop functional versatile and biocompatible nanocarriers for PSMA-targeted delivery of radiopharmaceutics and anticancer drugs as innovative theranostics in prostate cancer therapy. This program is now labeled by Aix-Marseille Université Initiative d’excellence (Amidex Emergence et Innovation 2018 “Nucleolipid-based nanocarriers for theranostics in prostate cancer”).

CRCM27 Boulevard Leï Roure

13009 Marseille, France

Daniel [email protected]

Tel. +33 (0)4 91 22 33 54François Bertucci

[email protected] Tel. +33 (0)4 91 22 59 21

CONTACTS

Selected recent publications

1 ) Birnbaum DJ, et al. Molecular classification as prognostic factor and guide for treatment decision of pancreatic cancer. Biochim Biophys Acta. 2018 - 1869, 248-255.2 ) Bertucci F, et al. The Genomic Grade Index predicts post-operative clinical outcome in patients with soft tissue sarcoma. Annals of Oncology. 2017 - in press. 3 ) Birnbaum DJ, et al. A 25-gene classifier predicts overall survival in resectable pancreatic cancer. BMC Med. 2017 - 15, 170. 4 ) Braendlein M, et al. Lactate Detection in Tumor Cell Cultures Using Organic Transistor Circuits. Adv Mater. 2017 - 29. 5 ) Lopresti A, et al. Circulating tumor cells: a real time dive into malignant plasticity. Med Sci. 2017 - 33, 491-493. 6 ) Karaki S, et al. Lipid-oligonucleotide conjugates improve cellular uptake and efficiency of TCTP-antisense in castration-resistant prostate cancer. J Control Release. 2017 - 258, 1-9.7 ) Ziouziou H, et al. Targeting Hsp27/eIF4E interaction with phenazine compound: a promising alternative for castration-resistant prostate cancer treatment. Oncotarget. 2017.8 ) M-Rabet M, et al. Triple-negative breast cancer therapy via nectin-4 targeting. Med Sci. 2017 - 33, 936-939. 9 ) M-Rabet M, et al. Nectin-4: A new prognostic biomarker for efficient therapeutical targeting of primary and metastatic triple-negative breast cancer. Ann Oncol. 2017 - 28, 769-776.10 ) Bertucci F, et al. PDL1 expression is a poor-prognosis factor in soft-tissue sarcomas. Oncoimmunology. 2017 - 6(3),e1278100. 11 ) Ferrari A, et al. A whole-genome sequence and transcriptome perspective on HER2-positive breast cancers. Nat Commun. 2016 - 7,12222. 12 ) Bertucci F, et al. Bevacizumab plus neoadjuvant chemotherapy in patients with HER2-negative inflammatory breast cancer (BEVERLY-1): a multicentre, single-arm, phase 2 study. Lancet Oncol. 2016 - 17, 600-11.13 ) Lopez M, et al. Identification of a naturally processed HLA-A*02:01-restricted CTL epitope from the human tumor-associated antigen Nectin-4. Cancer Immunol Immunother. 2016 - 65(10), 1177-1188.14 ) Wei, T. et al. Anticancer drug nanomicelles formed by self-assembling amphiphilic dendrimer to combat cancer drug resistance. Proc Natl Acad Sci U S A. 2015 - 112, 2978-83.

Recent Patents

1 ) Rocchi, P. Encapsulation of Phenazine and derivatives thereof. Patent EB16349, 2018.2 ) Rocchi, P. Nanoparticles comprising a core with a phenazine derivative and a shell with a nucleolipid and uses thereof. European Patent BET 16P0495, 2018.3 ) Rocchi, P. Antisense Oligonucléotides Effective to reduce the expression of Menin in cancer cells of a subject, EP/2016/16305135.4 ) Lopez, M. Antibodies having specificity to nectin-4 and uses thereof. PCT/EP2016/071076.

TEAM CELL POLARITY, CELL SIGNALING & CANCER

JEAN-PAUL BORG

More than 90% of adult cancers result from the transformation of normal epithelial tissues to carcinoma. Some of the most prominent features of these tumors are a higher resistance to apoptosis, increased cell proliferation, and alteration of cell polarity. In the most aggressive cancers, cancer cells acquired a motile and invasive phenotype involved in the development of metastasis.

Cell polarity is a fundamental process required for the organization and maintenance of tissues, in particular of epithelial origin. Epithelial cells make physical contact with neighboring counterparts and the extracellular matrix to acquire an apico-basal polarity, and form a barrier between two chemically distinct compartments. This polarity is best characterized by the formation of an apical domain segregated from the basolateral domain by specialized junctions (tight and adherens junctions). Orthogonal to the apico-basal polarity axis, planar cell polarity (PCP) refers to an uniform cellular organization within the plan. PCP provides directional cues that control and coordinate the integration of cells in a tissue, and is essential for the multicellularization of a living organism.A large body of work has already identifi ed important components of epithelial cell polarity and, in some cases, has shed light on their role in cancer. This is particularly the case for molecules involved in apico-basal polarity such as the tumor suppressors E-cadherin and LKB1, and more recently, the Scribble scaff old protein. Deregulation of these polarity proteins is per se suffi cient to induce tumor formation and/or dissemination in animal models, and loss of expression has been shown in human cancer of various origins. A defaulted cell polarity is most systematically accompanied by deregulation of signaling pathways, suggesting a control of cell signaling by cell polarity components. Accordingly, these molecules belong to protein network containing protein kinases, small GTPases and regulators of these activities. One has thus to envision that cell polarity not only organizes the shape of a tissue but also regulates intracellular signaling pathways. Compared to apico-basal polarity, much less is known about the role of PCP molecules in cancer although recent data have started to highlight their contribution. Mammalian PCP is governed by Frizzled (Fz) receptors that are stimulated by Wnt ligands. Wnt signaling is split into two branches defi ned as the non-canonical and the canonical pathways -,

the latter leading to ß-catenin stabilization, nuclear accumulation and transcriptional activation in conjunction with TCF/LEF factors. Studies in invertebrates and vertebrates have led to consider PCP as a non-canonical Wnt process, often referred to as the Wnt/PCP pathway. How the PCP pathway is molecularly organized and how deregulation of this pathway contributes to tumorigenesis remain to be discovered. The lab has been pioneered in the defi nition of important signaling cascades associated to cell polarity proteins such as Erbin, Scribble, PTK7 and more recently, Vangl2. We are currently pursuing the study of these proteins at the organism, cellular and molecular levels, combining fundamental and translational research in strong collaboration with clinicians, and a stimulating network of national and international collaborations. When we started the lab ten years ago, we were the fi rst to identify Erbin, a basolateral scaff old protein containing a PDZ domain, for its interaction with the ErbB2 tyrosine kinase receptor. Since then, we have extended our interest to Erbin homologues, - Scribble and Lano -, and contributed to the characterization of these basolateral proteins in epithelial cells. For example, we fi rst depicted biochemically and functionally

VANGL2- and PTK7-associated protein complexes implicated in cell polarity (here in migratory breast

cancer cells) were identifi ed by a combinatory purifi cation/mass spectrometry identifi cation strategy.

the Scribble complex that comprises GEFs (Guanine Exchange Factors) and GAPs (GTPase Activating Proteins) proteins involved in cell migration. We are currently pursuing our investigations on Erbin and Scribble at the physiological and physiopathological levels using in vitro studies,- in particular state-of-the-art proteomics and functional cellular assays -, and mouse models. We also recently became interested in the tumor suppressor LKB1, in particular its mode of regulation at the plasma membrane, as well as in PTK7 and Vangl2, two cell polarity receptors genetically linked to Scribble.Our main achievements are the following:› First cloning of Erbin (ErbB2 interacting), a gene

encoding a protein containing LRR and PDZ domains (Nat. Cell Biol. 2000),

› First identifi cation of a signaling complex ßPIX-GIT1

associated to the Scribble tumor suppressor (Curr Biol 2004),

› In collaboration with Martin Schwartz’s team, fi rst demonstration that LKB1 is an adherens junction-associated protein regulated by E-cadherin engagement (Curr Biol 2009),

› First characterization of PTK7 overexpression in acute myeloid leukemia and correlation of this event to poor prognosis (Blood 2010),

› First characterization of PTK7 implication in Wnt/ß-catenin canonical signaling (EMBO Reports 2011),

› First purifi cation of the endogenous Vangl1/Vangl2 heteromeric complex (PLoS One 2012),

› Characterization of the human PDZome (Mol. Cell. Proteomics 2013).

Selected recent publications1 ) Puvirajesinghe T.M., Bertucci F., Jain A., Scerbo P., Belotti E., Audebert S., Sebbagh M., Lopez M., Brech A., Finetti P., Charafe-Jauff ret E., Chaff anet M., Castellano R., Restouin A., Marchetto S., Collette Y., Gonçalvès A., Macara I., Birnbaum D., Kodjabachian L., Johansen T. and Borg J.-P. (2015) Identifi cation of p62/SQSTM1 as a component of non-canonical Wnt VANGL2-JNK signaling in breast cancer. (2016) Nature Communications, 7:10318.

2 ) Martinez S., Scerbo P., Giordano M., Daulat A.M., Lhoumeau A.-C., Thomé V., Kodjabachian L.* and Borg J.-P*. (2015) The PTK7 and ROR2 receptors interact in the vertebrate WNT/PCP pathway. (2015) J. Biol. Chem., 290: 30562-72. *co-last authors.

3 ) Daulat A.M., Bertucci F., Audebert S., Sergé A., Finetti P., Josselin E., Castellano R., Birnbaum D., Angers S. and Borg J.-P. PRICKLE1 contributes to cancer cell dissemination through its interaction with mTORC2. (2016) Developmental Cell, 37:311-25.

4 ) Lhoumeau A.-C., Martinez S., Monges G., Castellano R., Poizat F., Saillard C., Viens P., Raoul J.-L., Prebet T., Aurrand-Lions M., Borg J.-P* and Gonçalves* A. Overexpression of the promigratory and prometastatic PTK7 receptor has an adverse clinical outcome in colorectal cancer. (2015) PLoS ONE, 10: e0123768. *co-last authors.

5 ) Sebbagh M. and Borg J.-P. Insight into Planar Cell Polarity. (2014) Invited review in Experimental Cell Research, 328:284-295.

6 ) Tao Y., Shen C.*, Luo S., Traoré W., Marchetto M., Santoni M.J., Xu L., Wu B., Shi C., Mei J., Bates R., Liu X., Zhao K., Xiong W.-C., Borg J.-P.*, and Mei L.* A role of Erbin in ErbB2-dependent breast tumor growth. (2014) PNAS, 111: E4429-38. *co-last authors.

7 ) Belotti E., Polanowska J., Daulat A.M., Lembo F., Audebert S., Thomé V., Lissitzky J.-C., Lembo F., Blibek K., Omi S., Lenfant N., Gangar A., Montcouquiol M., Santoni M.-J., Sebbagh M., Aurrand-Lions M., Angers S., Kodjabachian L., Reboul J., and Borg J.-P. The human PDZome: a gateway to PDZ mediated functions. (2013) Mol Cell Proteomics, 12: 2587-603.

8 ) Belotti E. , Puvirajesinghe T.M., Audebert S., Baudelet E., Camoin L. , Pierres M., Lembo F., Montcouquiol M., and Borg J.-P. Evidence for Vangl2/Vangl1 heteromeric complexes. (2012) PLoS One, 7(9):e46213.

9 ) Puppo F., Thomé V., Lhoumeau A.-C., Gangar A., Lembo F., Belotti E., Marchetto S., Lécine P., Prébet T., Sebbagh M., Shin W.-S., Lee S.-T., Kodjabachian L.*, and Borg J.-P.* PTK7 plays a conserved role in Wnt/ ß-catenin canonical signaling, (2011) EMBO Reports, 12:43-9 (*co-last authors).

10 ) Prébet T., Lhoumeau A.-C., Arnoulet C., Aulas A., Marchetto S., Audebert S., Puppo F., Chabannon C., Sainty D., Santoni M.-J., Sebbagh M., Summerour V., Huon Y., Lee S.-T., Esterni B., Vey N., and Borg J.-P. The cell polarity PTK7 receptor acts as a modulator of the chemotherapeutic response in acute myeloid leukaemia and impairs clinical outcome. (2010) Blood, 116: 2315-2323.

Jean-Paul BorgCRCM


[email protected]+33 (0)4 86 97 72 51

CONTACT

TEAM ANTIBODY THERAPEUTICS & IMMUNOTARGETING

PATRICK CHAMES & DANIEL BATY

Recent exciting progress in cancer immunotherapy has ushered in a new era of cancer treatment. It is now fi rmly established that tumor cells harbor numerous mutations leading to the expression of fully tumor-specifi c antigens. These so-called neoantigens are targeted by eff ector cells of the immune system such as NK and T cells. To become a clinically apparent tumor, cancer cells must induce an microenvironment able to dampen this immune response. New antibody-based therapies aiming at blocking inhibitory receptors or activating stimulatory receptors expressed by infi ltrated eff ector cells can revert the induced tolerance leading to impressive and long lasting clinical responses.

We are using nanobodies, i.e. small single domain antibodies derived from llamas to generate small multispecifi c constructs aiming at manipulating the immunological synapse for cancer immunotherapy.A second research axis relies on the inherent propensity of nanobodies to target clefts and cavities to generate conformational sensors. These innovative tools able to trace the conformational changes of receptors or signaling proteins upon activation on living cells are used to answer unsolved biological questions.

Manipulation of the immunological synapse using bispecifi c antibodiesSeveral actors of an anti-tumoral immune response express the activating receptor CD16, including Natural Killer cells. We have generated a new generation of nanobody-based bispecifi c antibodies called bsFabs. By simultaneously binding to the well characterized tumor marker HER2 expressed on breast cancer cells and CD16, they recruit and activate CD16+ cells in the vicinity of breast cancer cells. We could demonstrate that bsFabs lead to similar results than clinically validated antibody trastuzumab on cells expressing very high amount of HER2 and spare normal cells barely expressing HER2. However, bsFabs still lead to effi cient tumor lysis on cells expressing much lower amounts of the tumor marker that are clinically resistant to trastuzumab, such as MDA-MB-231, both in vitro and in animal models. Moreover, this bsFab is insensitive to CD16 polymorphism issues that

limit the immunological mode of action of conventional mAbs such as trastuzumab to 20% of patients. All together, this approach has the potential to expand the antibody-based treatments of breast cancers to a much larger proportion of patients. Recent clinical trials have demonstrated the impressive potential of combination treatments using immunomodulating antibodies such as anti-CTLA-4 and anti-PD-1 mAbs. We are taking benefi t of the modular nature of our nanobody-based molecules to create multispecifi c antibodies able to agonize activating receptors or block inhibitory receptors on the same cell populations. The possibility to modulate multiple inhibitory and/or activating pathways simultaneously is a unique and powerful attribute not provided by conventional mAbs.

Nanobodies as conformational sensors Unlike conventional mAbs, nanobodies have a natural preference for clefts and cavities. As a consequence, they are a rich source of conformational antibodies. Such antibodies can be used to discriminate the diff erential conformational states of a receptor or a signaling molecule on living cells, thereby behaving as innovative conformational sensors.We are exploring this potential to solve unanswered biological questions. The EGF receptor, overexpressed in a number of cancer types, contributes to tumor cell

Selected recent publications1 ) Nevoltris D, Lombard B, Dupuis E, Mathis G, Chames P, Baty D. Conformational nanobodies reveal tethered epidermal growth factor receptor involved in EGFR/ErbB2 predimers. ACS Nano (2015) 9(2): 1388-1399.

2 ) Del Bano J, Chames P, Baty D, Kerfelec B. Taking up Cancer Immunotherapy Challenges: Bispecifi c Antibodies, the Path Forward? Antibodies (2015) 5(1): 1.

3 ) Even-Desrumeaux K, Nevoltris D, Lavaut MN, Alim K, Borg JP, Audebert S, Kerfelec B, Baty D, Chames P. Masked selection: a straightforward and fl exible approach for the selection of binders against specifi c epitopes and diff erentially expressed proteins by phage display. Mol Cell Proteomics (2014) 13(2): 653-665.

4 ) Turini M, Chames P, Bruhns P, Baty D, Kerfelec B. A FcgammaRIII-engaging bispecifi c antibody expands the range of HER2-expressing breast tumors eligible to antibody therapy. Oncotarget (2014) 5(14): 5304-5319.

5 ) Hafi an H, Sukhanova A, Turini M, Chames P, Baty D, Pluot M, Cohen JH, Nabiev I, Millot JM. Multiphoton imaging of tumor biomarkers with conjugates of single-domain antibodies and quantum dots. Nanomedicine (2014) 10(8): 1701-1709.

6 ) Matz J, Herate C, Bouchet J, Dusetti N, Gayet O, Baty D, Benichou S, Chames P. Selection of Intracellular Single-Domain Antibodies Targeting the HIV-1 Vpr Protein by Cytoplasmic Yeast Two-Hybrid System. PLoS One (2014) 9(12): e113729.

7 ) Rakovich TY, Mahfoud OK, Mohamed BM, Prina-Mello A, Crosbie-Staunton K, Van Den Broeck T, De Kimpe L, Sukhanova A, Baty D, Rakovich A, Maier SA, Alves F, Nauwelaers F, Nabiev I, Chames P, Volkov Y. Highly Sensitive Single Domain Antibody-Quantum Dot Conjugates for Detection of HER2 Biomarker in Lung and Breast Cancer Cells. ACS Nano (2014) 8(6): 5682-5695.

8 ) Matz J, Kessler P, Bouchet J, Combes O, Ramos OH, Barin F, Baty D, Martin L, Benichou S, Chames P. Straightforward Selection of Broadly Neutralizing Single-Domain Antibodies Targeting the Conserved CD4 and Coreceptor Binding Sites of HIV-1 gp120. J Virol (2013) 87(2): 1137-1149.

9 ) Sukhanova A, Even-Desrumeaux K, Kisserli A, Tabary T, Reveil B, Millot JM, Chames P, Baty D, Artemyev M, Oleinikov V, Pluot M, Cohen JH, Nabiev I. Oriented conjugates of single-domain antibodies and quantum dots: toward a new generation of ultrasmall diagnostic nanoprobes. Nanomedicine (2012) 8(4): 516-525.

10 ) Bouchet J, Basmaciogullari SE, Chrobak P, Stolp B, Bouchard N, Fackler OT, Chames P, Jolicoeur P, Benichou S, Baty D. Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody. Blood (2011) 117(13): 3559-3568.

Daniel Baty & Patrick ChamesCRCM

Parc Scientifi que de LuminyINSERM Unité 1068

163 Avenue de Luminy13288 MARSEILLE cedex 9, France

[email protected]+33 (0)4 91 82 88 23

[email protected]+33 (0)4 91 82 88 33

CONTACTS

proliferation, evasion of apoptosis, angiogenesis, and metastatis. This receptor is targeted by several inhibitors such as small molecules and mAbs. Its mode of action is well described and involves a ligand-induced conformational change leading to homo or heterodimerization and activation. However, the existence of inactive dimers (called “predimers”) is documented in several studies. The conformation of EGFR in such predimers in living tumor cells remains elusive. We have generated nanobody-based EGFR conformational sensors able to selectively bind to the active or inactive conformation of EGFR. Using these tools, we have demonstrated that within EGFR/HER2 predimers, EGFR adopts its inactive conformation, thus

questioning the established mode of activation.We have extended this approach to the study of receptors of the metabotropic glutamate receptors. These receptors normally expressed on neurons are overexpressed in a number of cancers. Because the glutamate concentration found in the periphery is much higher than the concentration found in brain, these receptors can lead to strong signaling via PI3K/AKT/mTOR and MAPK pathways, leading to growth, migration, invasion and resistance to apoptosis. To better study the implication of mGluR in cancer, we are generating conformational sensors of these receptors and of their downstream signaling G proteins.

Identify sources of heterogeneity

within cancer

Conventional therapies

Anti-CSC therapies

Understanding CSC’s biology to cure cancer

How essential is the niche in regulating CSC metastatic spreading?

TEAM EPITHELIAL STEM CELLS AND CANCER

EMMANUELLE CHARAFE-JAUFFRET CHRISTOPHE GINESTIER

Our team aims at deciphering the role of CSCs at diff erent steps of carcinogenesis from tumor initiation to metastatic spreading in order to prevent and/or override therapeutic resistance.

Epithelial cancers are known to present a major intratumoral heterogeneity that contributes to therapy failure and disease progression. The origin of this cellular heterogeneity is mainly explained by a hierarchical organization of tumor tissues where several subpopulations of self-renewing cancer stem cells (CSCs) sustain the long-term oligoclonal maintenance of the neoplasm. CSCs drive tumor growth, resist to conventional therapies and initiate metastasis development. Thus, developing CSC-targeting therapies is becoming a major challenge requiring the understanding of the unique molecular circuitry of CSCs as compared to non-CSCs.

Our research programs focus on three main questions:

∠What is the contribution of lineage-restricted mechanisms that normally maintain epithelium homeostasis during tumor progression? ∠Can we modulate CSC’s intrinsic programs to develop new therapeutic strategies to cure cancer? ∠How essential is the niche in regulating CSC metastatic spreading?

To answer these questions we use two tissue models: the human mammary gland and the epithelial transition zones.

Breast Cancer ProgramOur team is a pioneer in the identifi cation and characterization of breast cancer stem cells (bCSCs) (Ginestier et al., Cell Stem Cell, 2007; Charafe-Jauff ret et al., Cancer Res., 2009). We are pursuing our eff ort by developing research programs to decipher intrinsic molecular pathways regulating bCSC-fate. We aim at identifying new therapeutic targets to develop anti-CSC therapies. All these translational research programs are based on the development of innovative tools and models (functionnal screens, patient-derived xenografts).

‘Epithelial Transition Zones’ ProgramTransition zones represent an abrupt transition between two types of epitheliums, and are ubiquitously found in our body such as in the eye, cervix, between the esophagus and stomach and between the anal canal and rectum. These

zones can develop cancer with poor prognosis in human and mouse associated with metastasis. Despite their clinical signifi cance, the underlying molecular and cellular reasons for this tumor susceptibility remain elusive. Our group is using various molecular and biochemistry techniques, fl ow cytometry, 3D cellular cultures and genetically modifi ed mouse models to characterize these transition zones in normal and pre-lesional states. We are also investigating the role of stem cells and the microenvironment in squamous cell carcinoma development.

Program leader: Géraldine GUASCH, for more information visit http://guaschresearch.info

Selected recent publications1) McCauley HA, Chevrier V, Birnbaum D, Guasch G. De-repression of the RAC activator ELMO1 in cancer stem cells drives progression of TGFβ-defi cient squamous cell carcinoma from transition zones. Elife. 2017 Feb 21;6. pii: e22914. doi: 10.7554/eLife.22914.

2) Mai T.T., Hamai A., Hienzsch A., Caneque T., Muller S., Wicinski J., Cabaud O., Leroy C., David A., Acevedo V., Ryo A., Ginestier C., Birnbaum D., Charafe-Jauff ret E., Codogno P., Mehrpour M., Rodriguez R. Salinomycin kills cancer stem cells by sequestering iron in lysosomes. Nat Chem. 2017/10 - volume: 9 - pages: 1025-1033.

3) Morel A.P., Ginestier C., Pommier R.M., Cabaud O., Ruiz E., Wicinski J., Devouassoux-Shisheboran M., Combaret V., Finetti P., Chassot C., Pinatel C., Fauvet F., Saintigny P., Thomas E., Moyret-Lalle C., Lachuer J., Despras E., Jauff ret J.L., Bertucci F., Guitton J., Wierinckx A., Wang Q., Radosevic-Robin N., Penault-Llorca F., Cox D.G., Hollande F., Ansieau S., Caramel J., Birnbaum D., Vigneron A.M., Tissier A., Charafe-Jauff ret E., Puisieux A. A stemness-related ZEB1-MSRB3 axis governs cellular pliancy and breast cancer genome stability. Nat Med. 2017/05 - volume: 23 - pages: 568-578.

4) El Helou R., Pinna G., Cabaud O., Wicinski J., Bhajun R., Guyon L., Rioualen C., Finetti P., Gros A., Mari B., Barbry P., Bertucci F., Bidaut G., Harel-Bellan A., Birnbaum D., Charafe-Jauff ret E., Ginestier C. miR-600 Acts as a Bimodal Switch that Regulates Breast Cancer Stem Cell Fate through WNT Signaling. Cell Rep. 2017/02 - volume: 18 - pages: 2256-2268.

5) Ginestier C., Birnbaum D., Charafe-Jauff ret E. Flick the cancer stem cells’ switch to turn cancer off . Mol Cell Oncol. 2017/01 - volume: 4 - pages: e1319896-.

6) Ke J., Zhao Z., Hong S.H., Bai S., He Z., Malik F., Xu J., Zhou L., Chen W., Martin-Trevino R., Wu X., Lan P., Yi Y., Ginestier C., Ibarra I., Shang L., McDermott S., Luther T., Clouthier S.G., Wicha M.S., Liu S. Role of microRNA221 in regulating normal mammary epithelial hierarchy and breast cancer stem-like cells. Oncotarget. 2015/01 - volume: 6 - pages: 3709-3721.

7) Deng L., Shang L., Bai S., Chen J., He X., Trevino R.M., Chen S., Li X., Meng X., Yu B., Wang X., Liu Y., McDermott S.P., Ariazi A.E., Ginestier C., Ibarra I., Ke J., Luther T.K., Clouthier S.G., Xu L., Shan G., Song E., Yao H., Hannon G.J., Weiss S.J., Wicha M.S., Liu S. MicroRNA100 inhibits self-renewal of breast cancer stem-like cells and breast tumor development. Cancer Res. 2014/09 - volume: 74 - pages: 6648-6660.

8) El Helou R., Wicinski J., Guille A., Adelaide J., Finetti P., Bertucci F., Chaff anet M., Birnbaum D., Charafe-Jauff ret E., Ginestier C. A distinct DNA methylation signature defi nes breast cancer stem cells and predicts cancer outcome. Stem Cells. 2014/07 - volume: 32 - pages: 3031-3036.

9) Lombardi S., Honeth G., Ginestier C., Shinomiya I., Marlow R., Buchupalli B., Gazinska P., Brown J., Catchpole S., Liu S., Barkan A., Wicha M., Purushotham A., Burchell J., Pinder S., Dontu G. Growth Hormone Is Secreted by Normal Breast Epithelium upon Progesterone Stimulation and Increases Proliferation of Stem/Progenitor Cells. Stem Cell Rep. 2014/06 - volume: 2 - pages: 780-793.

10) Honeth G., Lombardi S., Ginestier C., Hur M., Marlow R., Buchupalli B., Shinomiya I., Gazinska P., Bombelli S., Ramalingam V., Purushotham A.D., Pinder S.E., Dontu G. Aldehyde dehydrogenase and estrogen receptor defi ne a hierarchy of cellular diff erentiation in the normal human mammary epithelium. Breast Cancer Res. 2014/05 - volume: 16 - pages: R52-.

11) Liu S., Cong Y., Wang D., Sun Y., Deng L., Liu Y., Martin-Trevino R., Shang L., McDermott S.P., Landis M.D., Hong S., Adams A., D’Angelo R., Ginestier C., Charafe-Jauff ret E., Clouthier S.G., Birnbaum D., Wong S.T., Zhan M., Chang J.C., Wicha M.S. Breast Cancer Stem Cells Transition between Epithelial and Mesenchymal States Refl ective of their Normal Counterparts. Stem Cell Rep. 2014/01 - volume: 2 - pages: 78-91.

12) Salvador M.A., Wicinski J., Cabaud O., Toiron Y., Finetti P., Josselin E., Lelievre H., Kraus-Berthier L., Depil S., Bertucci F., Collette Y., Birnbaum D., Charafe-Jauff ret E., Ginestier C. The histone deacetylase inhibitor abexinostat induces cancer stem cells diff erentiation in breast cancer with low Xist expression. Clin Cancer Res. 2013/10 - volume: 19 - pages: 6520-6531.

Emmanuelle Charafe-Jauff ret& Christophe Ginestier

CRCM27 Boulevard Leï Roure

13009 Marseille, France

jauff [email protected]@inserm.fr

+33 (0)4 91 22 35 09

CONTACT

TEAM INTEGRATED STRUCTURAL & CHEMICAL BIOLOGY

YVES COLLETTE & XAVIER MORELLI

The team develops a fundamental research to identify, validate, understand and target the PPI involved in signaling and epigenetics processes, especially in oncology. The range of applicability of this fundamental research covers the identifi cation, optimization and validation of novel targets and compounds with high therapeutic potential.

The drug discovery process is inherently ineffi cient, especially in oncology. The challenge of matching the vastness and complexity of the chemical world to a physiological eff ect in daily clinical study is unfortunately illustrated by limitations such as harmful side eff ects and drug resistance that challenge the most powerful available chemotherapeutics. It is therefore an urgent need to identify new therapeutic targets and develop approaches to propose and characterize a new generation of anticancer drugs. Most drug discovery eff orts by

pharmaceutical companies concerning the development and / or expanding of their pre-clinical and clinical pipeline primarily target G protein-coupled receptors, nuclear receptors, ion channels and active sites of enzymes (i.e kinases). Although this strategy is perfectly understandable for historical reasons and risk management, inhibitors of protein-protein interactions (PPI) represent an alternative and powerful reservoir, almost unfi lled in oncology, in which we can tap new sources to meet this challenge in the 21st century.

Our activities include:› Developments in bioinformatics, chemoinformatics

and molecular modeling to provide dedicated virtual libraries for PPI and to predict parameters of «druggability» for identifi cation (in silico) of original new targets in oncology. (P. Roche & X. Morelli)

› Developments and Screening of a materialized PPI-focused library of drug compounds to search for original compounds targeting in house therapeutic targets, or in collaboration with academics or pharmaceutical companies. (X. Morelli).

› Developments in structural biochemistry and Drug Design to characterize the structural interfaces and develop innovative methodologies to accelerate the discovery of bioactive compounds targeting PPI in cancer. (S. Betzi, V. Receveur-Bréchot & X. Morelli).

› Developments in organic chemistry targeting asymmetric synthesis and/or heterocyclic to characterize the chemical space of class-specifi c PPI complexes and to provide new libraries dedicated to these interfaces, but also to optimize compounds with high therapeutic potential by structure-activity relationship (SAR) studies. (C. Barral, S. Combes & JM Brunel).

› Developments in chemistry and biology to manipulate biological systems, to identify therapeutic targets by chemical proteomics, and to elucidate the mechanisms of action of drugs, with the prospect of optimizing therapeutic strategies. (Yves Collette).

› Preclinical evaluation in vitro and in vivo of optimized drug inhibitors (TrGET preclinical platform at CRCM) to provide proof of concept studies of drug effi cacy. (Yves Collette).

Research programWe aim to study and to target the interactions and pathological regulations involving Src Homology (SH)-3 domain-mediated PPI in cancer, using the oncogenic Bcr-Abl and Src family kinases involved in chronic myeloid leukemia (CML) as experimental model for proof of concept of our strategy.

The program is divided into two areas:› A « biased» approach that includes, on the one hand, the

development of structural biology-oriented molecules targeting PPI selected for their therapeutic value, and, on the other hand, the development of «druggable» PPI targets as defi ned by original algorithms developed by the team. Moreover, a diverse PPI-focused chemical library (which provides a particular chemical space) has been designed, assembled and is undergoing experimental validation in collaboration with the IbiSA screening platform ‘AD2P’, using both validated (e.g. with known PPI inhibitors available, such as p53/MDM2, and BRD4/ acetylated histones) and original PPI targets (including Bcr-Abl and Src family SH3 domains, as well as undisclosed PPI targets). The resulting selected compounds are biologically evaluated in the team and collaboration with the TrGET platform, for their anti-leukemic activity in vitro, on established cell lines, ex vivo, on CML patient samples,

and in vivo, in xenograft models.› An “unbiased” or reverse approach, based on chemo-

proteomic profi ling of drug inhibitors immobilized as affi nity columns and used to identify by mass spectrometry (in collaboration with the MaP proteomic platform) the actual proteome targeted by the studied PPI inhibitors. This approach was successfully applied to enzymatic kinase and histone deacetylase inhibitors, but to our knowledge it has not hitherto been achieved in view of inhibiting PPI. We expect a two-fold benefi t from our strategy: fi rst, to functionnaly orientate the selectivity of the studied compound, by synthesis of derivatives displaying optimized binding to the desired target, as opposed to “off ” targets, while preserving or optimizing their biological activity; second, by comparing the targeted proteome of cancer cells as compared to normal cells, we aim to reposition drug inhibitors by identifying original PPI targets relevant in cancer processes.

Selected recent publications1 ) Loosveld et al. Therapeutic Targeting of c-Myc in T-Cell Acute Lymphoblastic Leukemia, T-ALL. Oncotarget. 2014 May 30;5(10):3168-72.

2 ) Hamon et al. 2P2IHUNTER: a tool for fi ltering orthosteric protein-protein interaction modulators via a dedicated support vector machine. J. R. Soc. Interface. 2013 Nov 6;11(90):20130860.

3 ) Basse et al. 2P2Idb: A Structural Database Dedicated to Orthosteric Modulation of Protein-Protein Interactions. Nucleic Acid Research, 2013 Jan; 41: 41, D824-827.

4 ) Morelli & Hupp. Searching for the holy grail; protein-protein interaction analysis and modulation. EMBO Rep. 2012 Oct;13(10):877-9. doi: 10.1038/embor.2012.137.

5 ) Djouhri-Bouktab et al. Synthesis of new 3,20-bispolyaminosteroid squalamine analogues and evaluation of their antimicrobial activities. J. Med. Chem. (2011) Oct 27 ; 54(20) :7417-21.

6 ) Combes et al. Synthesis and biological evaluation of 4-arylcoumarin analogues of combretastatins. Part 2. J. Med. Chem. (2011) 54, 3153–3162.

7 ) Morelli et al. Chemical and structural lessons from recent successes in protein-protein interaction inhibition (2P2I). Curr Opin Chem Biol. 2011 Jun 17.

8 ) Chaib et al. Anti-leukemia activity of chaetocin via death receptor-dependent apoptosis and dual modulation of the histone methyl-transferase SUV39H1. Leukemia (2011) 26 (4): 662-674.

9 ) Lugari et al. Molecular mapping of the RNA Cap 2’-Omethyltransferase activation interface between SARS coronavirus nsp10 and nsp16. J. Biol. Chem. (2010) Oct 22;285(43):33230-41.

10 ) Salmi-Smail et al. Structure-activity relationships of a series of SAHA analogues histone deacetylase inhibitors. J. Med. Chem. (2010) 53(8):3038-47.

Most recent Patents1 ) Preparation of sulfonylthiomorpholine-3-carboxamides as apoptosis inducing compounds. Lopez, M.; Collette, Y.; Mezil, L.; Brunel, J.M.; Combes, S. PCT int. Appl. 2013, WO 2013050476 A1 20130411.

2 ) «Use of squalamine or analogue as a disinfecting agent» Brunel, J. M.; Raoult, D.; Rolain, J. M. PCT Int. Appl. WO 2013104849 (23/01/2013).

3 ) Arylcoumarine derivatives and based oncology drug. Fedorov, A.Y.; Sitnikov, N.S.; Vodovozova, E.L.V, Moiseeva, E.V.; Boldyrev, I.A.; Kuznetsova, N.Y.R.; Beletskaya I.P.; Combes, S. Brevet n° RU2440998 (C1), 2012.

4 ) Dérivés de 4-arylcoumarine et de 4-arylquinoléine, leurs utilisations thérapeutiques et leur procédé de synthèse. Combes, S.; Boutonnat, J.; McLeer-Florin, A.; Daras, E.; Peyrot, V.; Fedorov, A. Fr.Demande 2012, FR 2973703 A1 20121012.

5 ) «Utilisation de squalamine ou analogues comme agent désinfectant». Brunel, J. M.; Rolain, J. M.; Raoult, D. European patent N°1250312 (12/01/2012).

Yves Collette & Xavier MorelliCRCM


[email protected]@inserm.fr

+33 (0)4 86 97 73 31

CONTACTS

TEAM SIGNALING, HEMATOPOIESIS & MECHANISM OF ONCOGENESIS

PATRICE DUBREUIL

Hematopoiesis is a perfect model of cellular diff erentiation. It allows to study molecular mechanisms involved in the transition between a highly regulated system and deregulated situations , either diff erentiation programs and homeostasis or proliferative syndromes and leukaemia, respectively.

Our group contributes to the evaluation of mechanisms, transduction pathways and protein eff ectors initiated by genetic alterations aff ecting protein kinases. After identifi cation of such alterations, mainly in c-Kit receptor kinase in blood disorders and mastocytosis, our goal was along these years to generalize our strategy to other kinases important in onco-hematology as well as in solid cancers.

Our project is based on three main approaches in which we have a recognized expertise:

This expertise acquired in the c-Kit receptor kinase and mastocytosis models (with the AFIRMM network and on behalf of the CEREMAST label – MASTocytosis REference CEntre) is the basis of collaborations with clinicians and other research groups interested in the identifi cation of mutations in kinases. Mastocytosis is characterized by pathologic accumulation of mast cells in bone marrow and other tissues, often resulting in severe and multiple symptoms due to increased release of mast cell mediators. Most patients with systemic mastocytosis bear mutations in the receptor tyrosine kinase gene c-Kit. We used mastocytosis as a model of cancer progression by searching for new genetic alterations in addition to c-Kit mutations which could lead to aggressiveness and could play a role in leukemic progression through a mechanism of oncogenic cooperation. Recent sequencing in our large patient cohort of a panel of genes for mutations previously

identifi ed in other hemopathies has provided us with a list of recurrent somatic mutations in mastocytosis patients, including novel KIT mutations, mutations in epigenetic factors, splicing factors and other signaling molecules. The most frequent mutations, we identifi ed to date are KIT, TET2 and SRSF2 followed by more rare mutations (IDH2, U2AF1, SF3B1, CBL...). Other mutations frequently found in MDS/MPN/AML are less frequent or absent in SM patients (e. g. IDH1, DNMT3a, EZH2…). By the use of diff erent animal models (transgenic, KO, KI…), bone marrow derived mast cells and genetic modifi cation by lentiviral infection, we are investigating the role of each of these alterations in normal and pathologic mast cell diff erentiation as well as the mechanisms of their oncogenic cooperativity. Mechanisms and genes targeted by this cooperation will be identifi ed since they could represent new therapeutic approaches.

Our studies on receptor tyrosine kinase signaling in the context of hematological neoplasms, has led to the identifi cation of essential eff ectors of c-Kit and FLT3 oncogenic signals. We are currently studying the function of three protein kinases, CDK6, FES and FER, in the context of physiological and pathological conditions.CDK6 is a serine threonine kinase with a known function in G1 phase of the cell cycle. Using a siRNA library screen, we have shown that CDK6 is required for FLT3 mutant dependent signaling in AML cell lines. This is unique to FLT3 as oncogenic forms of the closely related receptor c-Kit are not dependent on CDK6. We have delineated the pathway linking FLT3 and CDK6 and we are now using

CDK6 defi cient mice to confi rm the results in the context of primary cells.FES and FER are non-receptor tyrosine kinases distinct from other family of kinases, which share a similar structure: an NH2 F-BAR domain, a central SH2 domain and the catalytic region. We have previously shown that FES is essential for KIT signaling while FER is required downstream of both for KIT and FLT3. We have now started a structure/function study of mutations (of FES and FER) found in various tumors. Using animal models, cell models and primary human samples we are investigating the role of these kinases in cell transformation.

Characterization of kinases involved in oncogenic signaling

The identifi cation and functional characterization of activating mutations in protein kinases.

Selected recent publications1 ) Soucie et al. In aggressive forms of mastocytosis, TET2 loss cooperates with c-KITD816V to transform mast Cells. Blood, 2012, 120(24):4846-9.

2 ) Georgin-Lavialle et al. Mast cell sarcoma: a rare and aggressive entity-report of two cases and review of the literature. Journal of Clinical Oncology, 2013;31(6):e90-7.

3 ) Georgin-Lavialle et al. Mast cell leukemia. Blood, 2013, 121(8):1285-95.

4 ) Chaix et al. KIT-D816V oncogenic activity is controlled by the juxtamembrane docking site Y568-Y570. Oncogene. 2014;33(7):872-81.

5 ) Rusakiewicz et al. Immune infi ltrates are prognostic factors in localized gastrointestinal stromal tumors. Cancer research 2013;73(12):3499-510.

6 ) Sokol et al. Gastrointestinal manifestations in mastocytosis: a study of 83 patients. J Allergy Clin Immunol. 2013, 132(4):866-73.

7 ) Hanssens et al. SRSF2-p95 hotspot mutation is highly associated with advanced forms of mastocytosis and mutations in epigenetic regulator genes. Haematologica. 2014 Jan 3.

8 ) Damaj et al. ASXL1 but not TET2 mutations adversely impact overall survival of patients suff ering systemic mastocytosis with associated clonal hematologic non-mast-cell diseases. PLoS One. 2014; 9(1): e85362.

9 ) Saleh et al. A new human mast cell line expressing a functional IgE receptor converts to tumorigenic growth by KIT D816V transfection. Blood, 2014 Jul 3;124(1):111-20.

10 ) Soucie et al. Molecular basis of mast cell disease. Mol Immunol., 2015 Jan;63(1):55-60.

11 ) PCT Patent Dubreuil P “Use of small molecule inhibitors/activators in combination with (deoxy)nucleoside or (deoxy)nucleotide analogs for treatment of cancer and hematological malignancies or viral infections”.

Patrice DubreuilCRCM


[email protected]+33 (0)4 86 97 72 81

CONTACT

Identifi cation, optimization and evaluation of functional activitiesof tyrosine kinase inhibitors In collaboration with AB Science company we identifi ed in 2010 a tyrosine kinase inhibitor, masitinib showing a high selectivity for c-Kit kinases. To date masitinib has been approved by health agency in veterinary medicine (mastocytoma) and is presently in multiple clinical trials in human medicine. In addition to its potent activity against its primary targets, we have previously shown that masitinib enhances the antiproliferative eff ects of gemcitabine in human pancreatic cancer, demonstrating potential as a chemosensitizer. This property could not be explained by the kinase selectivity profi le of the drug since masitinib exhibits a remarkably restricted protein kinase selectivity profi le. In order to associate a target to this chemosensitization eff ect, we performed a powerful direct chemical proteomic approach. This study has led to the identifi cation of several non-protein kinases targets of masitinib including the human deoxycytidine

kinase (hdCK). hdCK is an essential nucleoside kinase implied in the biosynthesis of nucleotides precursors used for cellular DNA synthesis. hdCK is also responsible for the activation by phosphorylation of a number of nucleoside-like prodrugs widely used in anti-cancer and/or antiviral chemotherapy. Using purifi ed hdCK proteins in the presence of masitinib and enzymatic kinetics assays, we found that masitinib is capable of positively modulating dCK activity and thus modulating phosphorylation of diff erent nucleotide analog drugs. This counterintuitive observation suggests that the pharmacological modulation of other nucleotide kinases activity could constitute a good strategy to identify new compounds for the potentiation of nucleoside-derived-based chemotherapy. Biophysical studies are ongoing to analyze the structural mechanism of such interaction.

TEAM EPIGENETIC CONTROL OF NORMAL & PATHOLOGICAL HEMATOPOIESIS

ESTELLE DUPREZ

Our group aims to understand the mechanisms involved in the regulation of proliferation versus diff erentiation of normal or pathological hematopoietic cells. Our hypothesis is that transcription and epigenetic factors act in combination to regulate the fate of the hematopoietic stem cell (HSC).

Role of the transcription factor PLZF in hematopoietisThe tumor suppressor gene PLZF (Promyelocytic Leukemia Zinc Finger) functions either as a transcriptional repressor or activator depending on the cell context and achieves its transcriptional regulation by binding to many secondary molecules to form large multi-protein complexes that bind to the regulatory elements in the promoter region of the target genes. Our team has contributed to the understanding of the function of PLZF as a repressor by identifying a new functional interaction between PLZF and the epigenetic Polycomb group protein (PcG) complexes (Boukarabila et al, Gene & Dev; 2009; Spicuglia et al, PloS ONE; 2011). Using a natural PLZF mutant mouse model: the Zbtb16lu model, in which a non-sense mutation leads to the production of a dominant negative mutant PLZF protein. We assess the roles of PLZF in the maintenance and diff erentiation of HSCs in relation to PcG function.

The role of PLZF on HSC cell cycle and agingBy combining cell cycle and micro-array analysis to multi-colour cell sorting strategies, we showed that:

› PLZF is highly enriched in HSCs and suppressed with age,› PLZF controls young HSC cell cycle and regulates HSC lineage priming,› Inactivation of PLZF accelerates HSC aging (Vincent etal. Blood 2016).PLZF and stress response in HSCAs our work points to an important role of PLZF in HSC aging and as recent studies have identifi ed intracellular levels of Reactive Oxygen Species (ROS) as an important regulator of the size of the HSC pool and function during the course of aging, we are investigating the role of PLZF in redox sensing. This part of our works aims to uncover the molecular mechanisms that hematopoietic stem cells have developed to avoid or endure physiological stress.PLZF and HSC mobilizationHSC motility (migration, homing, and release) is essential for BM repopulation and for the development of the hematopoietic system, In addition, various therapeutic agents function by recruiting hematopoietic stem and progenitor cells to the blood. We will study the eff ect of PLZF suppression on stem cell homing/mobilization.

In mammalian cells, the PcG proteins form two multiprotein repressive complexes called Polycomb repressive complexes (PRCs), which repress transcription through chromatin modifi cations. EZH2, the catalytic core protein in the PRC2, which catalyses the trimethylation of histone3 lysine27 (H3K27me3) and mediates gene silencing, is deregulated in a wide range of cancer types and can act both as a tumor suppressor or an oncogene. We aim to defi ne the specifi city of PcG (EZH2) protein recruitment, to further understand how PcG deregulation impacts on leukemogenesis.Defi ning PLZF and EZH2 interactionCombining co-immunoprecipitation and ChIp-seq analyses on hematopoietic cells, we are characterizing PLZF et EZH2 interplay (Koubi et al., unpublished data).

De-silencing the TAL1 locus in leukemiaIn collaboration with Bertrand Nadel lab (CIML), we have revealed a new adverse mechanism of mono-allelic oncogenic activation through site-specifi c loss of PcG-mediated epigenetic silencing (Navarro et al., 2015 Nat Comm). Dynamics of EZH2 recruitment during hematopoietic diff erentiationTo better assess the dynamics and specifi c recruitment of EZH2 during hematopoietic development, and provide an experimental in vivo tool for further identifi cation of PcG recruitment factors and mechanisms in leukemia, we are analysing epigenetic deregulation through hematopoiesis development using mouse models.

Understanding EZH2 recruitment to chromatin in hematopoietic cells

Selected recent publications1 ) Vincent-Fabert C, Platet N., Vandevelde, A., Poplineau M., Koubi, M., Finetti., P, Tiberi, G., Imbert, AM., Bertucci, F., Duprez, E. PLZF mutation alters mouse hematopoietic stem cell function and cell cycle progression , Blood (2016).127(15):1881-5

2 ) Kühnl, A et al., Down-regulation of the Wnt inhibitor CXXC5 predicts a better prognosis in acute myeloid leukemia. Blood, 7;125(19):2985-94 (2015).

3 ) Navarro et al. Site-& allele-specifi c de-silencing of polycomb repressive activity by insertional oncogenesis: a new recurrent mechanism of TAL1 activation in T-ALL. Nat Commun. 2015 Jan 23;6:6094.

4 ) Tiberi et al. PcG methylation of the HIST1 cluster defi nes an epigenetic marker of Acute Myeloid Leukemia. Leukemia. 2014 Dec 8. doi: 10.1038/leu.2014.339.

5 ) Santini et al. Epigenetics in focus: recent insights into the pathogenesis of MDS and the role of hypomethylating agents in the reprogramming of the epigenome. Critical Reviews in Oncology/Hematology; 2013 Nov;88 (2):231-45.

6 ) Ordoñez-Rueda et al. A novel form of mouse neutropenia resulting from a point mutation in the zinc fi nger protein Gfi 1. Eur J Immunol. (2012) 42:2395-408.

Estelle DuprezCRCM


[email protected]+33 (0)4 86 97 73 11

CONTACT

Identifi cation and characterization of new epigenetic markers in AMLAMLs are highly heterogeneous at the molecular level and a better characterization of the genetic and epigenetic modifi cations of each tumor is essential to refi ne diagnosis. As Polycomb complexes are altered in a wide range of hematologic malignancies, we used genome-wide mapping of the Polycomb Repressive Complex 2 (PRC2)-signature histone mark, H3K27me3 to study PcG activity in AML samples. Our work has enabled the discovery of a new epigenetic alteration that aff ects Cytogenetically normal (CN)-AML and impacts

on prognosis. Our data provide a proof of concept that epigenetic profi ling could be used to discover new biomarker with prognosis value (Patent # EP14305672.9and Tiberi et al., Leukemia, 2015).We are currently:› Investigating the mechanism underlying the abnormal

H3K27me3 patterns discovered in CN-AML patients.› Extending the potential of our signature on other

hematologic disorders.

Epigenetic profi ling reveals striking H3K27me3 dysregulation at the HIST1 cluster region in CN-AML.

Heatmap representing H3K27me3 promoter enrichment of the HIST1 cluster (55 histone genes) located on chromosome 6 (p22.2-p22.1) for 35 CN-AML samples. Chromosome 6 is represented at the top of the fi gure and the HIST1 cluster is indicated by a red square. Only HIST1 promoter regions are analysed and gaps between HIST1 promoter regions greater than 40 kbp are indicated. Each line represents a patient sample and each column represents a HIST1 promoter region ordered accordingly to the HG18 version of the human genome.

TEAM CONTROL OF STRUCTURE-SPECIFIC ENDONUCLEASES & GENOME STABILITY

PIERRE-HENRI GAILLARD

The main goal of our research is to strengthen our understanding of the regulation of structure-specifi c endonucleases, key players required for the resolution of potentially toxic DNA secondary structures in DNA repair and recombination mechanisms.

Controlling the action of Structure-Specifi c Endonuclease for Genome Stability

Despite their fundamental nature, the mechanisms that control structure-specifi c endonucleases remain poorly understood. To gain insight into these mechanisms we are carrying out in parallel two main projects. A fi rst project (A-) is aimed at unraveling some of the mechanisms that control the Mus81-Eme1 endonuclease, which is required for the resolution of DNA structures, in particular Holliday junctions, generated during repair of broken replication forks or during meiotic recombination. For this project we are exploiting the tremendous investigational power

of the fi ssion yeast Schizosaccharomyces pombe to combine genetic, cell biology, biochemical and proteomic approaches. The second project (B-) has initially consisted in the identifi cation and characterization of the human SLX4 protein. The rational to launch into this project stemmed from the fact that in budding yeast, Slx4 was found to interact with two diff erent structure-specifi c endonucleases: the Slx1 nuclease required for maintenance of the rDNA and the Rad1-Rad10 nuclease involved in various DNA repair and recombinations pathways.

The Mus81-Eme1 endonuclease is an heterodimeric endonuclease related to the evolutionarily conserved XPF family of 3’-fl ap endonucleases. In both budding and fi ssion yeast, Mus81-Eme1 is essential for cell viability in absence of the Sgs1 and Rqh1 RecQ helicases, respectively. Importantly, these are orthologs of the human BLM helicase that is defective in Bloom syndrome patients, a hereditable syndrome associated with remarkable chromosomal instability and cancer predisposition. BLM-related helicases are required to “dissolve” double Holliday junctions in a non-endonucleolytic process that prevents sister-chromatid exchange.Our research has led to the discovery of a novel DNA

damage-induced activation of Mus81-Eme1 in fi ssion yeast. This regulation requires both Cdc2CDK1 and Rad3ATR-dependent phosphorylations of Eme1. Mus81-Eme1 activation prevents gross chromosomal rearrangements in cells lacking the BLM-related DNA helicase Rqh1. We propose that linking Mus81-Eme1 DNA damaged-induced activation to cell cycle progression ensures effi cient resolution of Holliday junctions that escape dissolution by Rqh1-TopIII while preventing unnecessary DNA cleavages.We are pursuing our investigations on the control of the Mus81-Eme1 endonuclease in fi ssion yeast exploiting its tremendous investigational power allowing

Regulatory Mechanisms of Mus81-Eme1

Selected recent publications1) Guervilly J.H. and Gaillard P.H.L. (2015) SLX4 gains weight with SUMO in genome maintenance Mol. Cell. Oncology 10.1080/23723556.2015.1008297

2) Guervilly J.H.#*, Takedachi A.#, Naim V., Scaglione S., Chawhan C., Lovera Y., Despras E., Kuraoka I., Kannouche P., Rosselli F., Gaillard P.H.L.#* (2015) The SLX4 complex is a SUMO E3 ligase that impacts on replication stress outcome and genome stability Mol. Cell 57(1) :123–37 (Note: Article previewed in Molecular Cell) #co-fi rst authors *co-corresponding

3) Dehé, P.-M.#, Coulon, S.#, Scaglione, S., Shanahan, P., Takedachi, A., Wohlschlegel, J. A., Yates J.R., Llorente B., Russell P. and Gaillard P.-H.L. (2013). Regulation of Mus81-Eme1 Holliday junction resolvase in response to DNA damage. Nat Struct Mol Biol, 20(5), 598–603. #co-fi rst authors

4) Crossan G.P., van der Weyden L., Rosado I.V., Langevin F., Gaillard P.H.L, McIntyre R.E., Sanger Mouse Genetics Programme, Gallagher F., Kettunen M.I., Lewis D.Y., Brindle K., Arends M.J., Adams D.J. and Patel K.J. (2011) Disruption of mouse Slx4, a regulator of structure-specifi c nucleases, phenocopies Fanconi Anemia. Nat. Genet. 43(2) :147-52

5) Fekairi S.#, Scaglione S.#, Chahwan C., Tayor E., Tissier A., Coulon S., Dong M.Q., Ruse C., Yates J.R., Russell P., Fuchs R., McGowan C., Gaillard P.H.L. (2009) Human SLX4 is a Holliday Junction Resolvase Subunit that Binds Multiple DNA Repair/Recombination Endonucleases. Cell 138(1) : 78-89 (Note: Article previewed in Cell) #co-fi rst authors

Pierre-Henri GaillardCRCM


[email protected]+33 (0)4 86 97 73 71

CONTACT

SLX4, Structure-Specifi c Endonucleases and Genome StabilityThe identifi cation of the human SLX4 protein has become particularly relevant to the scientifi c interests of our laboratory with the fi nding that SLX4 in human cells appears as a docking platform involved in the control and coordination of not only SLX1 and XPF-ERCC1Rad1-Rad10 endonucleases but also of the human MUS81-EME1 complex. Furthermore, mutations in SLX4 have been found as causative of Fanconi Anemia (FA), a rare genetic disease associated with bone marrow failure, cancer predisposition and cellular sensitivity to DNA inter-strand crosslinks. Moreover, SLX4 hypomorphic mice display a phenotype that recapitulates many key features of Fanconi anemia and develop multiple cancers underlining the fundamental importance of SLX4 as a tumour suppressor. Our main focus today is to understand how the interactions between SLX4 and its partners, especially

its endonuclease partners, are modulated and how this contributes to the key functions of SLX4 in the maintenance of genome integrity. We have recently unraveled new SUMO-related functions of human SLX4. The SLX4 complex has SUMO E3 ligase activity that SUMOylates SLX4 itself and the XPF subunit of the DNA repair/recombination XPF-ERCC1 endonuclease. This is mediated by a unique interaction between SLX4 and the activated SUMO E2 conjugating enzyme UBC9. Unexpectedly, this not only relies on newly identifi ed SUMO interacting motifs (SIMs) in SLX4 but also on its BTB domain. In contrast to its ubiquitin-binding UBZ4 motifs, SLX4 SIMs are dispensable for its DNA interstrand crosslink repair functions. Instead, while detrimental in response to global replication stress, the SUMO E3 ligase activity of SLX4 is critical to prevent mitotic catastrophe following common fragile sites expression.

a combination of genetic, cell biology, biochemical and proteomic approaches. We are also extending our analysis to human cells. We have identifi ed cross-talks between a variety of post-translational modifi cations of Eme1 and its partners and we are investigating their functional signifi cance. Our results suggest a remarkable

complexity of what appears to be elaborate regulation mechanisms that control the catalytic activity of the complex, channel its activity to the appropriate structures and regulate its intra-nuclear distribution throughout the cell-cycle and in response to DNA damage.

TEAM TELOMERES & CHROMATIN

VINCENT GÉLI

Regulation of chromatin structure through covalent histone modifi cations is a central paradigm to modulate DNA-directed biological processes. In the past years, methylation of lysine 4 on histone H3 (H3K4) arouse considerable interest. The family of histone H3 lysine 4 (H3K4) methylases is highly conserved from yeast to human. They share a canonical organization in which the catalytic subunit acts as a docking platform for multiple subunits that regulate the enzymatic activity. Set1 complex (Set1C)-mediated H3K4 methylation is one of the most prominent histone modifi cations that mark active transcription.In budding yeast, Set1C and H3K4 methylation have not only been involved in transcription but also in multiple processes such as chromosome segregation, DNA replication, and meiotic recombination. Recent reports led to the concept that Set1C subunits, in addition to

regulating H3K4 methylation, may be directly involved in some of these biological functions by recruiting specifi c protein partners. However, the mechanisms by which such factors modulate enzymatic activity and relay regulatory signals remain to be determined. Our project aims to uncover how the Set1C protein interaction network regulates yet poorly understood functions of Set1C.

In view of our current data, our team investigates : › How Set1C regulates diff erent aspects of mRNA

biogenesis, including transcription termination and mRNA processing.

› The role played by Set1C during replication stress.› The mechanism by which the Spp1 subunit of the Set1C

controls meiotic double strand breaks performed by the Spo11 nuclease.

Telomeres are nucleoprotein structures that protect chromosome ends against degradation, fusion, recombination and recognition by the DNA damage machinery. The maintenance of telomere length depends on a specifi c reverse transcriptase called telomerase. The telomerase is expressed in most eukaryotic cells but in metazoan its expression is limited to germinal cells and some stem cells. When cells expressing low levels of telomerase divide, telomeres shorten until they reach a critical size that activates checkpoint signaling and associated cellular responses, such as senescence and/or apoptosis. Telomere shortening therefore acts as a cell-intrinsic mechanism limiting cellular proliferation and contributing to aging. Upon mutational inactivation of checkpoint proteins, continuous proliferation and cell survival provide a pro-carcinogenic mutational mechanism that favors genome instability and cell viability. These results raise the crucial question of the

long-term maintenance of telomeres when telomerase activity is limited. The focus of our group is to understand the mechanisms of telomere maintenance in dividing and quiescent cells and the cellular responses to telomere erosion.

Our fi rst project aims to understand how telomeres are repaired in telomerase negative budding yeast cells. Notably, we address how spatial relocation of eroded telomeres from the nuclear envelope to the Nuclear Pore Complex ensure unconventional recombination. In the second project, we investigate the Telomere Length Maintenance mechanisms in senescent and in quiescent S. pombe cells. Indeed fi ssion yeast is an excellent model system to study telomere biology with the discovery of a shelterin-like complex highlighting evolutionarily conserved elements of telomere

Our main topics of interest relate to the understanding, at the molecular level, of two major aspects of eukaryotic chromosome physiology, i.e. the way chromatin structure infl uences various DNA-dependent processes and the mechanisms that maintain telomeres integrity as well as the cellular responses to their dysfunction. Indeed, various defects in chromatin structure or telomere maintenance have been linked to human cancer. We are studying these processes in budding and fi ssion yeasts as model organisms.

Chromatin

Telomeres

Selected recent publications1 ) Churikov D, Charifi F, et al. Sumoylation of telomeres and tethering to the nuclear pore complex controls telomere recombination. Cell Reports, (2016) in press.

2 ) Niño CA et al. Posttranslational marks control architectural and functional plasticity of the nuclear pore complex basket. J Cell Biol, (2016) 212(2):167-80.

3 ) Audry J. et al. RPA prevents G-rich structure formation at lagging strand telomeres to allow maintenance of chromosomes ends. EMBO J. (2015) 34, 1942-1958. * corresponding authors.

4 ) Luciano P. Replisome function during replicative stress is modulated by histone H3 lysine 56 acetylation through Ctf4. Genetics (2015) 199(4):1047-63.

5 ) Churikov D et al. Rad59-Facilitated Acquisition of Y’ Elements by Short Telomeres Delays the Onset of Senescence. PLoS Genet. (2014) 10(11): e1004736.

6 ) Hardy J, Churikov D et al. Sgs1 and Sae2 promote telomere replication by limiting accumulation of ssDNA. Nat. Commun. 2014. 5:5004 doi: 10.1038/ncomms6004.

7 ) Acquaviva L et al. The COMPASS Subunit Spp1 Links Histone Methylation to Initiation of Meiotic Recombination. Science. 2013. 339(6116) :215-8. * corresponding authors.

8 ) Luciano P, Coulon S et al. RPA facilitates telomerase activity at chromosome ends in budding and fi ssion yeasts. EMBO J. (2012). 31(8):2034-46.

9 ) Faure V, Coulon S, et al. (2010) The telomeric single-stranded DNA-binding protein Cdc13 and telomerase bind through diff erent mechanisms at the lagging and leading telomeres. Mol Cell 38:842-52.

10 ) Khadaroo B et al. The DNA damage response at eroded telomeres and tethering to the nuclear pore complex. (2009). Nature Cell Biology, 11(8): 980-7.

Vincent GéliCRCM


[email protected]+33 (0)4 86 97 74 01

CONTACT

length regulation with mammalian cells. We expect to discover new key drivers of senescence. Besides, we also investigate how telomeres are maintained in quiescent S. pombe cells that can be experimentally maintained for weeks in quiescence. Finally, we have started a new study that aims to understand how cancer driver mutations regulate the balance between telomerase re-activation

(or upregulation) and induction of ALT. More specifi cally, we investigate how the oncogenic mutation IDH1-R132H that aff ects the global epigenetic landscape by interfering with DNA and histone demethylation, impacts the ALT/telomerase re-activation balance in specifi c types of brain tumours.

Relocation of eroded telomeres to the NPC promotes unconventional recombination.

TEAM CELL STRESS

JUAN IOVANNA

Our team is interested in the molecular aspects of the pancreatic cancer development and progression and is aimed to improve the current available therapeutic approaches. We focus on several complementary aspects such as: the role of the stress proteins (Nupr1 and TP53INP1) in the carcinogenesis and on the resistance to the accessible treatments;

the molecular dialogue established between the pancreatic transformed cells with the environmental stroma; the molecular mechanisms involved in endothelial-to-mesechymal transition since it could be a major source of non-inflammatory stromal cells;

the metabolic changes occurring in the transformed pancreatic cancer cells; the post translational modifications associated to resistance to conventional treatments; and finally, we are developing a translational study, named PaCaOmics, named PaCaomics aims to charaterize to characterize by means of Omics approaches, the primary pancreatic cancer cells obtained from patients and to correlate it with their clinical behavior.

Nupr1 is involved in cell resistance and in the control of tumor progression: The gene encoding the stress protein Nupr1, which is also known as Com1, has been identified in our laboratory because its expression is strongly induced in the pancreas during the acute phase of experimental pancreatitis. We have demonstrated that the expression of this gene is necessary for tumor development. We then focused on its mechanism of action. Recently, we were able to assign a major role for Nupr1 in the regulation of Epithelial-to-Mesenchymal Transition (EMT). In particular, we demonstrated that expression of Nupr1 is necessary for the classical TFGb-dependent EMT since when Nupr1 expression is knocked-down upon TGFb stimulation, pancreas cancer cells may undergo a novel EMT subtype yielding a phagocytic phenotype. Upon this Phagocyte-EMT, pancreas cancer cells become capable of cell cannibalism and undergo concomitant cell death, suggesting that Phagocyte-EMT is detrimental to tumor progression. We propose that upon TGFb exposure, pancreas cancer cells can undergo different EMT subtypes directed to distinct differentiation fates, which can either promote or suppress tumor progression involved in a new mechanism of cell death called cannibalism or entosis cell. We have partially dissected the molecular mechanism of this new function of Nupr1 and explored the pathways involved. On the other hand, our more recent results reveal a novel stress-related pathway that

requires the functional interaction of Nupr1 RelB IER3 in KrasG12D-dependent transformation of the pancreas. We have also established the pivotal role of Nupr1 in the formation of PanINs (precancerous ductal lesions known as Pancreatic Intraepithelial Neoplasia) and in PDAC progression beyond PanIN and in PDAC EMT in vivo, with a potential impact in PDAC cell stemness. Finally, we have shown that Nupr1 is an epigenetic regulator that counteracts senescence induced by the oncogenic Kras. Altogether, Nupr1 expression seems indispensable for Pancreatic cancer transformation and progression by regulating at least three independent major cancer process named EMT, Kras transformation and senescence. Therefore Nupr1 could be an important target to treat pancreatic cancer patients.Future developmentsNupr1 is a small, highly basic and unstructured protein that interacts with some partners which we are in the process of identifying and modulates their activities, and furthermore, as a co-transcriptional factor, regulates the expression of several genes. Therefore, Nupr1 may act at several steps in pancreatic cancer progression. Our project aims to characterize these functions and try to use the inhibition of Nupr1 as a new therapeutic target. In this way, we have recently developed an original screening, which is specific for unfolded proteins including Nupr1, andfind some promising inhibitors.

The Nupr1 protein

in vivo

PaCaOmicsA major impediment to the effective treatment of patients with PDAC (Pancreatic Ductal Adenocarcinoma) is the molecular heterogeneity of this disease, which is reflected in an equally diverse pattern of clinical outcome and in responses to therapies. We developed an efficient strategy in which PDAC samples from patients from three specialized centers of Marseille were collected by Endoscopic Ultrasound-Guided Fine-Needle Aspiration (EUS-FNA) or surgery and preserved as breathing tumors by xenografting and as a primary culture of epithelial cells. Transcriptomic analysis was performed from breathing tumors by an Affymetrix approach. On one hand, we observed a significant heterogeneity in the RNA expression profile of tumors. However, the bioinformatic analysis of this data was able to discriminate between patients with long- and short-term survival corresponding to patients carrying moderately- or poorly-differentiated PDAC tumors respectively. On the other hand, primary culture of cells allowed us to analyze their relative sensitivity to anticancer drugs in vitro by a chemogram, by similarity with the antibiogram for microorganisms, establishing an individual profile of drug sensitivity. As expected, the response was patient-dependent. Interestingly, we also found that the transcriptome analysis predict the sensitivity of cells to the five anticancer drugs the most frequently used to treating patients with PDAC. In conclusion, using this approach, we found that the transcriptomic analysis could predict the sensitivity to anticancer drugs and the clinical outcome of patients with PDAC.

Juan IovannaCRCM

Parc Scientifique de LuminyINSERM Unité 1068

163 Avenue de Luminy13288 MARSEILLE cedex 9, France

[email protected]. +33 (0)4 91 82 88 03

CONTACT

1 ) Hamidi et al. Nupr1 regulates RelB-dependent events necessary for pancreatic cancer development in mice. J. Clin. Invest. 122:2092-2103, 2012.2 ) Garcia et al. Tie1 deficiency induces Endothelial-Mesenchymal-Transition. EMBO Rep. 13:431-439, 2012.3 ) Seillier et al. TP53INP1, a tumor suppressor, interacts with LC3 and ATG8-family proteins through the LC3-interacting region (LIR) and promotes autophagic cell death. Cell Death Diff. 19:1525-1535, 2012.4 ) Cano et al. Cell cannibalism, a cell-death process driven by the Transforming Growth Factor-β and the Nuclear Protein 1, opposes to metastasis in pancreatic cancer. EMBO Mol. Med. 4:964-979, 2012. 5 ) Hamidi et al. A novel Nupr1-Aurora kinase A pathway provides protection against metabolic stress-mediated autophagic-associated cell death. Clin. Cancer Res. 18:5234-5246, 2012.6 ) Tomasini et al. TAp73 is required for macrophage-mediated innate immunity and the resolution of inflammatory responses. Cell Death Diff. 20:293-301, 2013.7 ) -Iovanna et al. Mechanistic insights into self-reinforcing processes driving abnormal histogenesis during the development of pancreatic cancer. Am J Pathol. 182(4):1078-86, 2013. 8 ) Guillaumond et al. Strengthened glycolysis under hypoxia supports tumor symbiosis and hexosamine biosynthesis in pancreatic adenocarcinoma. Proc. Natl. Acad. Sci. USA. 110:3919-3924, 2013.9 ) Gilabert et al. Pancreatic cancer-induced cachexia is Jak2-dependent in mice. J Cell Physiol. 2014 Oct;229(10):1437-43.10 ) Peuget et al. Oxidative stress-induced p53 activity is enhanced by a redox-sensitive TP53INP1 SUMOylation. Cell Death Differ. 2014 Mar 7. doi: 10.1038/cdd.2014.28. 11 ) Cano et al. Genetic inactivation of Nupr1 acts as a dominant suppressor event in a two-hit model of pancreatic carcinogenesis. Gut. 63(6):984-95, 2014.12 ) Garcia et al. IER3 supports KRASG12D-dependent pancreatic cancer development by sustaining ERK1/2 phosphorylation. J Clin Invest. 2014 Nov;124(11):4709-22. 13 ) Brisson et al. The Thymus-Specific Serine Protease TSSP/PRSS16 Is Crucial for the Antitumoral Role of CD4(+) T Cells. Cell Rep. 2015 Jan 6;10(1):39-46.

TEAM DNA INTERSTRAND CROSSLINK LESIONS AND BLOOD DISORDER

CHRISTOPHE LACHAUD

Our studies aim to understand the various mechanisms of interstrand crosslinks (ICLs) repair in higher eukaryotes using of new in vivo assays to dissect the molecular mechanisms underlying the repair of ICLs. The implications for both cancer development and treatment are extremely important both for patients with ICL repair-related diseases, and also for those undergoing ICL-based chemotherapeutics. A better knowledge of the ICL repair mechanisms will allow us to move towards rational therapeutic targeting the ICL repair-response in tumors.

Unlike lesions that aff ect only one DNA strand, which can simply be excised, interstrand crosslinks (ICLs) involve both strands of DNA, blocking the essential processes that require translocation along the DNA, namely DNA replication and transcription. In addition to this physical constraint on DNA, ICLs require repair of damage on both strands of the DNA. Comparative studies ranking in vitro and in vivogenotoxicity of large sets of compounds have ranked crosslinking agents among the most toxic (Lohman, 1999).

In actively dividing cells, the replicative machinery will encounter an ICL during S-phase, which will act as a physical barrier to replisome progression. This is thought to be a prevalent mechanism for sensing ICL lesions. In this setting, ICL repair involves the collision of a replication fork with the lesion as a trigger to initiate repair (Haynes et al., 2015; Raschle et al., 2008).

It has been assumed that this replication-dependent mode of repair should be suffi cient to cope with and repair ICLs in a timely fashion. However, in non-proliferating cells such as post-mitotic diff erentiated cells or quiescent stem cells, endogenously generated ICLs need to be repaired in the absence of DNA replication.

In this situation, if the ICL is positioned in an actively transcribed gene, a similar sensing process could take place following stalling of the RNA polymerase, with transcriptional blockage acting as the initiating event. Finally, it is thought that distorting ICLs can be recognized in the absence of collision with the replication or transcription machinery moving along DNA (Vogel et al., 1996).

UVA

Click reaction

ICL inducing drug

A clickable drug is activated by UVA to induce interstrand crosslink lesions (ICL). Click reaction is then used to labbel the drug and allow detection by fl uorescence.

After incubation with clickable drugs, ICL is induced using a UVA laser assisted by microscope. DAPI is used to monitor DNA and a click reaction is performed to label the lesions.

DAPI clicked-ICL

Detection of ICLs

Selected recent publications1) Feeney L, Muñoz IM, Lachaud C, Toth R, Appleton PL, Schindler D, Rouse J. RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for Interstrand Crosslink Repair and Human Health. Mol Cell. 2017 Jun 1;66(5):610-621.e4.

2) Lachaud C, Rouse J. A route to new cancer therapies: the FA pathway is essential in BRCA1- or BRCA2-defi cient cells. Nat Struct Mol Biol. 2016 Aug 3;23(8):701-3.

3) Lachaud C, Slean M, Marchesi F, Lock C, Odell E, Castor D, Toth R, Rouse J. Karyomegalic interstitial nephritis and DNA damage-induced polyploidy in Fan1 nuclease-defective knock-in mice. Genes Dev. 2016 Mar 15;30(6):639-44.

4) Lachaud C, Moreno A, Marchesi F, Toth R, Blow JJ, Rouse J. Ubiquitinated Fancd2 recruits Fan1 to stalled replication forks to prevent genome instability. Science. 2016 Feb 19;351(6275):846-9.

5) Rojas-Fernandez A, Herhaus L, Macartney T, Lachaud C, Hay RT, Sapkota GP. Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9. Sci Rep. 2015 Apr 29;5:9811.

6) Perez-Oliva AB, Lachaud C, Szyniarowski P, Muñoz I, Macartney T, Hickson I, Rouse J, Alessi DR. USP45 deubiquitylase controls ERCC1-XPF endonuclease-mediated DNA damage responses. EMBO J. 2015 Feb 3;34(3):326-43.

Christophe LachaudCRCM


[email protected]+33 (0)4 86 97 73 82

CONTACT

Because ICLs are profoundly cytotoxic, especially for replicating cells, ICL-inducing agents are widely used for cancer treatment, in particular mitomycin C, nitrogen mustard, and platinum compounds (Deans and West, 2011). These are front-line chemotherapeutic agents used to treat a wide range of solid tumors and blood cancers. ICL-based chemotherapy is eff ective in treating leukaemia or breast and ovarian cancers, and especially testicular cancer with a high cure rate. However, the eff ectiveness is limited by side eff ects such as renal toxicity, neuropathy, and development of further tumors. Additionally, tumors often develop resistance to ICL-inducing agents. This chemoresistance is partially due to the restoration of the ICL repair mechanism in which the cell was initially defi cient. Overcoming these limitations would greatly enhance the eff ectiveness of treatment, but the underlying mechanisms are unclear.

Improving our understanding of the mechanisms of ICL repair, and the implications for both cancer development and treatment is extremely important for patients with ICL repair-related diseases and also for those undergoing ICL-based chemotherapeutics. A better knowledge of the ICL repair mechanisms will allow us to move towards rational therapeutic targeting the ICL repair-response in tumors. Our studies, carried out in both human cells and mouse model, constitute a complementary approach for the analysis and understanding of the various mechanisms of ICL repair in higher eukaryotes. Particular attention is placed on the development of new in vivo assays to dissect the molecular mechanisms underlying the repair of ICLs. Results from our investigations in cells and mouse provide a solid base to extend our analyses to patient tumors in close collaboration with Institut Paoli-Calmettes.

TEAM GENOME DYNAMICS & RECOMBINATION

BERTRAND LLORENTE

Through original molecular genetics and genomics studies in Saccharomyces cerevisiae and in other hemiascomycetous yeasts, we wish to help deciphering the regulation of HR from the initiation to the resolution step, and to unveil the infl uence of critical cellular parameters such as chromatin structure, chromosome environment and sequence polymorphisms.

Specifi cally the goals of my team are to:› Decipher the control mechanism of meiotic DSB interference and its impact on genome stability.› Dissect the genetic and epigenetic control of DNA end resection and one-ended recombination.› Determine the fi ne structure of meiotic recombination intermediates to deeply revisit meiotic recombination pathways

mostly based on a 30 years old model.› Establish meiotic DSB and recombination maps in non-conventional yeast species to shed light the genetic control

of meiotic recombination and its evolution, this latter approach being expected to pave the way for genome biology studies.

› We anticipate that this work will bring signifi cant breakthroughs in our understanding of how genomes are maintained and evolve, a matter at the intersection of fundamental genetics, medicine and biotechnology.

Homologous recombination (HR) is a ubiquitous DNA repair process that fi xes deleterious lesions, including double strand breaks (DSBs). DSBs are inherent to the meiotic program but mostly result from replication accidents in mitosis. The essence of HR is to promote the interaction of a broken DNA molecule with an intact homologous donor to copy its genetic information and ultimately fi x the initial lesion.This unilateral transfer of information, in addition to the crossovers that can occur when recombining DNA strands are disentangled, promotes vital genetic diversity but also potentially detrimental mutations playing key roles in cancer formation and development.

Control mechanism of meiotic DSB interference & impact on genome stabilityValérie Garcia, a senior CNRS researcher that recently joined the lab, showed that meiotic DSBs display inter-hotspots interference in cis, in which the occurrence of a DSB within a hotspot suppresses DSB formation within adjacent hotspots (Garcia et al. Nature 2015). This new process, likely to be conserved across evolution, is mediated by the Tel1 yeast orthologue of the checkpoint protein Ataxia-telangiectasia mutated (ATM). While the role of ATM/Tel1 in DNA damage response in mitotic cells is well described, in meiosis the mechanism behind Tel1-mediated DSB interference and its impact on genome stability are yet unexplored and form our objectives.

Dissect the genetic and epigenetic control of mitotic recombinationThe aim of this project is to focus on the control of mitotic DNA end resection and one-ended recombination.This is done by:› Pursuing the characterization of the chromatin remodeler Fun30 that we showed to promote DNA end resection in

yeast, a function that we also showed to be conserved by its human counterpart SMARCAD1 (Costelloe et al, 2012).› Performing genetic screens to identify mutants defi cient for second end capture.› Measuring HR effi ciency throughout the genome to identify factors that impact on HR effi ciency.

Dissect the genetic control of meiotic recombination pathwaysThe aim of this project is to revisit current models of meiotic recombination. This is done by analyzing genome wide the heteroduplex DNA patterns associated with recombination events in critical mutants. This study is the follow up of our seminal work performed in collaboration with Emmanuelle Martini (Martini et al, 2011).

Selected recent publications1 ) Vakirlis N, Sarilar V, Drillon G, Fleiss A, Agier N, Meyniel JP, Blanpain L, Carbone A, Devillers H, Dubois K, Gillet-Markowska A, Graziani S, Huu-Vang N, Poirel M, Reisser C, Schott J, Schacherer J, Lafontaine I, Llorente B*, Neuvéglise C* and Fischer F*. Reconstruction of ancestral chromosome architecture and gene repertoire reveals principles of genome evolution in a model yeast genus. Genome Research, in press, *Co-corresponding authors.

2 ) Tomimatsu N, Mukherjee B, Catherine Hardebeck M, Ilcheva M, Vanessa Camacho C, Louise Harris J, Porteus M, Llorente B, Khanna KK, Burma S (2014) Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice. Nat Commun. Apr 7;5:3561.

3 ) Dehé PM, Coulon S, Scaglione S, Shanahan P, Takedachi A, Wohlschlegel JA, Yates 3rd JR, Llorente B, Russell P and Gaillard PHL (2013) Regulation of Mus81-Eme1 Holliday Junction Resolvase in Response to DNA Damage. Nature Structural and Molecular Biology, May;20(5):598-603.

4 ) Costelloe T*, Louge R*, Tomimatsu N*, Mukherjee B, Martini E, Khadaroo B, Dubois K, Wiegant W, Thierry A, Burma S, van Attikum H, Llorente B (2012) The yeast Fun30 and human SMARCAD1 chromatin remodellers promote DNA end resection. Nature Sep 27;489(7417): 581-4, *Co-fi rst authors.

5 ) Martini E, Borde V, Legendre M, Audic S, Regnault B, Soubigou G, Dujon B, Llorente B (2011) Genome-wide analysis of heteroduplex DNA in mismatch repair-defi cient yeast cells reveals novel properties of meiotic recombination pathways. PLoS Genet. Sep;7(9):e1002305.

Bertrand LlorenteCRCM


[email protected]+33 (0)4 86 97 74 11

CONTACT

Top: yeast chromosome after meiotic recombinationBottom: representative heteroduplex DNA

associated with crossover (left) and non-crossover (right) recombination

50000 100000 150000 200000 250000

ABCD

12121212

A 12

CD

1212

2kb

Use of biodiversity to understand meiotic recombinationBiodiversity within species has been underappreciated so far while it harbors many resources of potential fundamental and biotechnological interests.Here, we use non-conventional yeast species to bypass classical model organisms in order to shed light on the genetic control of meiotic recombination and its evolution. We plan to generate meiotic DSBs maps and maps of meiotic recombination events in several non Saccharomyces yeast species.

TEAM HOMOLOGOUS RECOMBINATION, NON-HOMOLOGOUS END JOINING & MAINTENANCE OF GENOME INTEGRITY

MAURO MODESTI

DNA damage in the genome of our cells is inevitable. Errors in DNA transactions, ultraviolet or ionizing radiations, genotoxic chemicals all may generate lesions in DNA and in turn lead to mutations that can change the original genetic program. While potentially detrimental, these genetic changes are important to fuel evolution and are purposely exploited by specialized cell types such as germ line and immune system cells to create genetic diversity. However, robust DNA damage monitoring and repair systems operate in our cells to maintain genome integrity. Perturbations of these systems cause a state of genome instability that predispose cells to escape their normal genetic program, elevating the risk of cancer.

DNA double-strand breaks are lesions that are under strict surveillance in our cells. When the detection or repair of DNA double-strand breaks fails the rate of incidence of chromosomal aberrations strongly increases. These gross chromosomal alterations may activate oncogenes after chromosomal translocations or inactivate tumor suppressor genes after chromosomal deletions. To repair DNA double-strand break, our cells can chose between Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ), two mechanistically distinct and mutually exclusive pathways (Figure 1). Many of the players involved in these two pathways are genome caretakers. The proteins encoded by Breast and Ovarian Cancer Susceptibility genes including BRCA1, BRCA2, PALB2 and the RAD51 paralogs are tumor suppressors crucial for HR. They regulate and guide the central RAD51 strand exchange catalyst. Likewise, products of

the LIG4, XRCC4 and XLF genes act together to seal DNA double-strand breaks by NHEJ. Defects in these NHEJ genes lead to Severe Combined ImmunoDefi ciency. Recent work revealed that NHEJ proteins are implicated in the promotion of translocations. Overall our work aims at providing a better understanding of these DNA double-strand break repair pathways in order to fi nd ways to improve existing cancer therapies or discover new ones. In particular, our focus is to characterize at the molecular level the protein-DNA transactions involved in these pathways. To investigate these mechanisms we use multidisciplinary approaches. We combine biochemical and biophysical methods in bulk phase or at the single-molecule level to reconstitute key mechanistic steps in vitro (Figure 2). The precise deciphering of these mechanisms tremendously helps the design of screens for inhibitors of HR and NHEJ that

Figure 1 Figure 2

Selected recent publications1 ) Menchon G, Bombarde O, Trivedi M, Négrel A, Inard C, Giudetti B, Baltas M, Milon A, Modesti M, Georges Czaplicki1 G, Calsou P. Structure-Based Virtual Ligand Screening on the XRCC4/DNA Ligase IV Interface. Sci Rep. 2016, In press.

2 ) Silva-Portela RC, Carvalho FM, Pereira CP, de Souza-Pinto NC, Modesti M, Fuchs RP, Agnez-Lima LF. ExoMeg1: a new exonuclease from metagenomic library. Sci Rep. 2016 Jan 27;6:19712.

3 ) Roy S, de Melo AJ, Xu Y, Tadi SK, Négrel A, Hendrickson E, Modesti M, Meek K. XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation. Mol Cell Biol. 2015 Sep 1;35(17):3017-3028.

4 ) Tomaszowski KH, Aasland D, Margison GP, Williams E, Pinder SI, Modesti M, Fuchs RP, Kaina B. The bacterial alkyltransferase-like (eATL) protein protects mammalian cells against methylating agent-induced toxicity. DNA Repair (Amst). 2015 Apr;28:14-20.

5 ) Vlijm R, Mashaghi A, Bernard S, Modesti M, Dekker C. Experimental phase diagram of negatively supercoiled DNA measured by magnetic tweezers and fl uorescence. Nanoscale. 2015 Feb 21;7(7):3205-3216.

6 ) Candelli A, Holthausen JT, Depken M, Brouwer I, Franker MA, Marchetti M, Heller I, Bernard S, Garcin EB, Modesti M, Wyman C, Wuite GJ, Peterman EJ. Visualization and quantifi cation of nascent RAD51 fi lament formation at single-monomer resolution. Proc Natl Acad Sci U S A. 2014 Oct 21;111(42):15090-15095.

7 ) Frykholm K, Alizadehheidari M, Fritzsche J, Wigenius J, Modesti M, Persson F, Westerlund F. Probing physical properties of a DNA-protein complex using nanofl uidic channels. Small. 2014 Mar 12;10(5):884-887.

8 ) Candelli A, Modesti M, Peterman EJ, Wuite GJ. Single-molecule views on homologous recombination. Q Rev Biophys. 2013 Nov;46(4):323-348.

9 ) King GA, Gross P, Bockelmann U, Modesti M, Wuite GJ, Peterman EJ. Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fl uorescence microscopy. Proc Natl Acad Sci U S A. 2013 Mar 5;110(10):3859-3864.

10 ) Cottarel J, Frit P, Bombarde O, Salles B, Négrel A, Bernard S, Jeggo PA, Lieber MR, Modesti M, Calsou P. A noncatalytic function of the ligation complex during nonhomologous end joining. J Cell Biol. 2013 Jan 21;200(2):173-186.

11 ) Persson F, Fritzsche J, Mir KU, Modesti M, Westerlund F, Tegenfeldt JO. Lipid-based passivation in nanofl uidics. Nano Lett. 2012 May 9;12(5):2260-2265.

12) Andres SN, Vergnes A, Ristic D, Wyman C, Modesti M, Junop M. A human XRCC4-XLF complex bridges DNA. Nucleic Acids Res. 2012 Feb;40(4):1868-1878.

13 ) Roy S, Andres SN, Vergnes A, Neal JA, Xu Y, Yu Y, Lees-Miller SP, Junop M, Modesti M, Meek K. XRCC4’s interaction with XLF is required for coding (but not signal) end joining. Nucleic Acids Res. 2012 Feb;40(4):1684-1694.

14) Meek K, Lees-Miller SP, Modesti M. N-terminal constraint activates the catalytic subunit of the DNA-dependent protein kinase in the absence of DNA or Ku. Nucleic Acids Res. 2012 Apr;40(7):2964-2973.

Mauro ModestiCRCM


[email protected]+33 (0)4 86 97 73 91

CONTACT

Figure 3

could one day be used as drugs to improve cancer therapies. We also use CRISPR/Cas9 mediated genome engineering to inactivate or modify our

favorite DNA repair genes and study repercussions in cells. To this end, we combine state-of-the-art cell biology and advanced microscopy

methods to image individual proteins involved in DNA repair and study their organization, dynamical interactions and

recruitment in response to diff erent types of DNA damage in situ in cells (Figure 3).

Our scientific activity is focused on immunity against cancer and chronic viral infections with the goal of developing a continuum leading from basic research to diagnostic and therapeutic applications. We investigate the mechanisms used by cancer cells to escape immune surveillance systems with a major emphasis on innate immunity and cosignaling pathways.

in vitro

in vitro in vivo.

in vitro

Tregs -

Escape mechanisms in NHL: role for BTLA and HVEM -

Viral or tumoral immune escape

Blood

Blood

Blood

J Allergy Clin Immunol

Blood

BMC Cancer

EMBO J

Leukemia

Int J Obes

Cell Mol Life Sci

Oncotarget

Front Immunol

Cancer Research

Cytometry A

J Immunol

Cell Rep

Oncoimmunology

J Allergy Clin Immunol

Antioxid Redox Signal

Cancer Research

TEAM DNA DAMAGE & GENOME INSTABILITY

VINCENT PAGÈS

Our research projects focus on understanding the biological consequences that result from replication of damaged genomes. The presence of damage in template DNA during replication is the major source of point mutations, the initiating cause of Cancer. Our team investigates the mechanisms of DNA damage tolerance and their impact on genome instability from prokaryotic to eukaryotic cells.

Despite effi cient repair mechanisms, the presence of unrepaired lesions at the replication fork is a frequent event in all dividing cells. Cells possess two major strategies to tolerate lesion in their DNA:› Translesion Synthesis (TLS) where specialized DNA

polymerases insert a few nucleotides opposite the lesion with the possibility of introducing a mutation;

› Damage Avoidance (DA) strategies that are error-free as they rely on mechanisms related to homologous recombination with the sister chromatid. The balance between these two strategies is very important since it defi nes the level of mutagenesis during lesion bypass.

We have developed a novel technology that allows us to insert a single lesion at a specifi c site of the bacterial chromosome. We can thus monitor in vivo and quantitatively the partition between Translesion Synthesis (TLS) and Damage Avoidance (DA) in diff erent genetic backgrounds, in order to defi ne the genes that regulate the choice among these pathways. For several replication-blocking lesions, we demonstrate that DA events massively

outweigh TLS events and that DA events are highly dependent upon the presence of the RecA protein. We also showed that tolerance events are executed in chronological order with TLS coming fi rst, followed by DA. We are currently further exploring the genetics of error-free damage tolerance pathways.

We are also analyzing the early replication intermediates that form near the lesion site, at the molecular level (qPCR, 2D-gels, Electron Microscopy), to defi ne the structure of the replication fork that encounters a lesion, and the molecular mechanisms involved in lesion tolerance.

Using a similar approach based on the Cre/lox system, we are exploring DNA damage tolerance pathways in the yeast Saccharomyces cerevisiae. In the near future, we plan to apply the same approach to human cells in culture.

Selected recent publications1) A defect in homologous recombination leads to increased translesion synthesis in E. coli Karel Naiman, Vincent Pagès, Robert P., Fuchs. Nucleic Acids Research, 2016 in press.

2 ) Pagès, V. (2016) Single strand gap repair involves both RecF and RecBCD pathways. Current Genetics DOI 10.1007/s00294-016-0575-5.

3 ) Laureti, L., Demol, J., Fuchs, R. P., & Pagès, V. (2015) Bacterial Proliferation: Keep Dividing and Don’t Mind the Gap. PLoS Genet 11: e1005757.

4 ) Naiman, K., Philippin, G., Fuchs, R. P., & Pagès, V. (2014) Chronology in lesion tolerance gives priority to genetic variability. Proc Natl Acad Sci USA 111: 5526–5531.

5) Fuchs, R. P. & Fujii, S. (2013) Translesion DNA synthesis and mutagenesis in prokaryotes. Cold Spring Harb Perspect Biol 5: a012682.

6 ) Pagès, V., Mazon, G., Naiman, K., Philippin, G., & Fuchs, R. P. (2012) Monitoring bypass of single replication-blocking lesions by damage avoidance in the Escherichia coli chromosome. Nucleic Acids Res 40: 9036–9043.

7 ) Pagès, V. & Fuchs, R. P. (2003) Uncoupling of leading- and lagging-strand DNA replication during lesion bypass in vivo. Science 300: 1300–1303.

Vincent PagèsCRCM


[email protected]+33 (0)4 86 97 73 84

CONTACT

Integration of a single lesion into the chromosome. The recipient strain contains a single attR integration site in fusion with the 3’ end of the lacZ gene at minute 17 in the Escherichia coli chromosome. Following ectopic expression of phage lambda’s integrase and excisionase, the lesion-carrying construct is introduced by electroporation. Its attL site will recombine with the chromosomal attR, leading to integration of the entire lesion-containing construct. Integration events are selected on the basis of their resistance to ampicillin. Integration at nucleotide level resolution restores a functional lacZ gene on the damaged strand, allowing TLS events to be monitored as blue sectors on X-gal indicator plates whereas DA events are represented by the remaining white colonies on the plate.

3’-lacZOriC

AttR

chromosome

electroporation

DNA lesion

1. Recipient strain

2. Plasmid vectorcontaining the

single lesion

integration

pVP135

kan®

ori

xisint

pVP135

kan®

ori

xisint

5’-lacZ

AttL

Amp®

R6K

chromosome

LacZ

AttBAttP

OriC Amp®

sequence heterology (loop) used as molecular/genetic marker

The syntenin-syndecan traffi cking pathway allows heparan sulfate-dependent cell signaling receptor systems to escape degradation. It boosts signaling in cis (receptor recycling) and in trans (cell-cell communication via exosomes).

We are currently investigating the impact of the deregulation of this pathway in cancer signaling and systemic progression.

We are also trying to identify druggable compounds inhibiting this pathway.

TEAM SPATIO-TEMPORAL CONTROL OF CELL SIGNALING - SCAFFOLDS& PHOSPHOINOSITIDES

PASCALE ZIMMERMANN

Our aim is to identify novel mechanisms of cellular signaling important in cancer and to propose innovative diagnostic and therapeutic approaches. We concentrate on scaff old proteins (heparan sulfate proteoglycans and PDZ proteins) and signaling lipids (phosphoinositides and others) that are important molecules for the spatio-temporal organisation of cell signaling. In particular we study their eff ect on the traffi cking and the turnover of signaling receptors, their role in cell-cell communication and their impact on nuclear processes. We thereby hope to pave the way for innovative therapeutic approaches.

We investigated the impact of the PDZ protein syntenin-1 on the traffi cking of heparan sulfate proteoglycans (syndecans) and their associated signaling receptors. We showed that the syntenin-1-syndecan-ARF6 recycling pathway controls early polarized movements in zebrafi sh (Lambaerts et al., 2012). We established that by interacting with ALIX, syntenin-1 also controls the biogenesis of exosomes and

thereby trans-cellular communication and signaling (Baietti et al., 2012). We showed that exosome biogenesis relies on ARF6 and its eff ector phospholipase D2 (Ghossoub et al., 2014). So syntenin-1 appears to allow a plethora of signaling receptors to escape degradation with potential, cell autonomous and non-cell autonomous signaling eff ects (Figure).

Recent investigations

We assessed the prevalence of PDZ-PtdInsPs interactions by screening the human PDZ proteome (246 PDZ domains). We identifi ed a new subset of PtdInsPs-interacting PDZ domains, and characterized PtdInsPs-modifying enzymes controlling PDZ protein distribution. We established that PDZ-PtdInsPs interactions are context dependent and that the subcellular localization of PDZ domains/proteins can be driven by the combination of diff erent interactions (Ivarsson et al., 2013). Investigating more specifi cally the determinants of the membrane targeting of syntenin-1 we found that the subcellular distribution of syntenin-1 is not solely regulated by its PDZ domains but also by its N- and C-terminus. A phosphorylation switch may regulate the N-terminal autoinhibition of the PDZ domains and the C-terminus adds electrostatic interactions that contribute to syntenin-1 membrane targeting (Wawrzyniak et al., 2012).As our screen of the human PDZ proteome identifi ed an unexpected high number of potential PDZ-PtdInsPs

interactions taking place in the nucleus, we decided to address the functional relevance of such interactions taking the PDZ protein syntenin-2 as a model. We characterized the nuclear dynamics of syntenin-2 and the impact of its interaction with PtdInsPs in living cells (Geeraerts et al., 2013).

Selected recent publications1 ) Egea-Jimenez et al. Frizzled 7 and PIP2 binding by syntenin PDZ2 domain controls Frizzled 7 traffi cking and signaling. Nat Commun. 2016, in press.

2 ) Ghossoub et al. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2. Nat Commun. 2014 Mar 18;5:3477.

3 ) Wawrzyniak et al. Phosphoinositides and PDZ domain scaff olds. Adv Exp Med Biol. 2013;991:41-57. Review.

4 ) Ivarsson et al. Prevalence, specifi city and determinants of lipid-interacting PDZ domains from an in-cell screen and in vitro binding experiments. PLoS One. 2013;8(2):e54581.

5 ) Baietti et al. Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nat Cell Biol. 2012 Jun 3;14(7):677-85.

Pascale ZimmermannCRCM


[email protected]+33 (0)4 86 97 73 51

CONTACT

One of our major goals is to investigate the eff ect of syntenin-1 knock-in and knock-out on known oncogenic signaling loops driving specifi c tumor types, and in particular the role of syntenin-1 in the tumor microenvironment. In parallel, we are assessing whether we can develop specifi c PDZ inhibitors, in particular for syntenin-1. To support rational drug design, we recently revealed the molecular determinants of syntenin PDZ-lipid interaction (Egea-Jimenez et al., 2016). Finally, we plan to elucidate the biological role of syntenin-2 and other nuclear PDZ-PtdInsPs interactions. We combine structural, biophysical, cell biological and model organism’s approaches. We have a quite broad expertise in standard approaches of molecular biology, cell biology (in particular fl uorescence confocal and live imaging), biochemistry (in particular surface plasmon resonance/Biacore) and animal models. We have ongoing collaborations for NMR, crystallization, drug discovery and cancer studies.

Projects and technological approaches

Reconstituted liposome are immobilized on a lipophilic surface and used to measure protein-membrane interactions by surface plasmon resonance (BIAcore).

THE CRCM INTEGRATIVE BIOINFORMATICS PLATFORM

PLATFORM MANAGER: GHISLAIN BIDAUT

CIBI

Ghislain [email protected]

+33 (0)4 86 97 73 01

CONTACT

The CRCM Integrative Bioinformatics platform (CIBI)focuses on bioinformatics and systems biology. It is staff ed with two research engineers with complementary expertise, a requirement for the analysis of complex and large data sets. It off ers a wide range of expertise, from high throughput biological data management and analysis (e.g. proteomics and genomics), to multi-technology data integration and public repositories data mining (Gene Expression Omnibus, ArrayExpress). Its expertise in bioinformatics specifi cally covers DNA array analysis, ChIP-Seq and RNA-SEQ, variant search, Next-gen sequencing (NGS) gene interaction networks (human interactome) and proteomics.It has developed an advanced systems biology approach, the Interactome-Transcriptome Integration that allows integration of gene expression, protein-protein interactions, regulation data (vertical integration) and meta-analysis of multiple datasets (horizontal integration). This is based on a pipeline developed locally, available either for unsupervised (version 1.0) or supervised analysis (version 2.0) and published in multiple papers with application to cancer.It has also developed Infocyt, an automated analysis method for Flow Cytometry data, that allows the complete cell population analysis is a large dimensional space, as opposed to the classical approach, where investigators tend to study pre-defi ned populations in a restricted

space using 2D-projections and manual gating. InfoCyt includes both a cell population analysis part, based on the FlowClust package, and a cell population signature visualization part.For data management, the platform has developed the database Djeen, (Database for Joomla!’s extensible engine). Djeen allows for multi technological data management (CGH-SNPs-DNA microarray, ChIP-Chip arrays and Flow Cytometry data) and is specifi cally designed for clinical/experimental annotation ina high throughput manner.In this regard, Cibi off ers collaborative and fee-for-service opportunities to help laboratories set up their own Djeen database, or design integrated bioinformatics analysis.

The main tasks of the «Cibi» platform are to:› Provide tools and necessary data for bioinformatics

analysis to biologists and research projects.› Support the development strategy in bioinformatics

in connection with existing services and bioinformaticians working in research teams.

› Provide IT development (database, automation, pipeline ...).

› Organize training cycles in statistics and bioinformatics.

› Provide assistance, advice and expertise on its activities.

Equipment› The platform acquired an High Performance Computing infrastructure (See DISC platform)

DATACENTRE IT & SCIENTIFIC COMPUTING OF CRCM PLATFORM

PLATFORM MANAGER: BERNARD CHETRIT

DISC

Bernard Ché[email protected]

+33 (0)4 86 97 72 07

CONTACT

DISC is the computing and operating center for scientifi c data of the CRCM. It represents an exchange area and a vital gateway across users and national and international networks related to computing.This platform provides a computing environment with strong means and support necessary for the conception of new tools, applications and methods, for the development of computing-based scientifi c projects.The networks partners of the DISC platform are: MobyleNet (Bioinformatics and chemoinformatics tools), ReNaBi (Bioinformatics Computing Grid), ENMR (European Grid computing for computational tools related to NMR, Molecular Modelling, Molecular Docking, …). In addition to user support, DISC proposes the following services:› A high-performance computing infrastructure,

allowing running and testing applications and codes in

conditions of highly parallelized computation.› An off er of virtualization to quickly deploy specifi c

computing or development environments.› Space for the storage and preservation of scientifi c

data.› Visualization tools for advanced remote displays

from personal computers without high performance graphics card.

› Set of tools for management, development project, continuous integration, technical debt measurement.

› Assistance through its expertise in the development, adaptation and deployment of software.

› Training by example (GNU/LINUX, use computing cluster, HDF5, ...)

All provided services are available to all engineers and researchers from the CRCM and IPC teams and platforms, as well as their collaborators, at the national and international level, but also to industrial partners.

Equipment› Alambic cluster (236 CPU-CORES, 4 TFLOPS)› Agui cluster (260 CPU-CORES, 4,2 TFLOPS)› Virtualization servers (60 CPU-CORES 2 TFLOPS)› 2 x 3D vizualisation servers (20 CPU-CORES,

2xQuadro K5200, 256G RAM)

› High-performance storagefor backing up scientifi c data- redundant NAS 2x145 TO in active/active replication

› Online fi le distribution service HPFS - redundant NAS 2x20 TO in active/active replication

› Router, Network Data Commutator 10GBs point to point

“The evolution of research through in silico method and not just in vivo or in vitro, requires high-performance infrastructure for computing and storage.Based on computer simulation or modeling, in silico research allows the testing of chemicals in the place of animal testing or in vitro assays. The results of existing tests are used to model, predict and evaluate the properties of chemical substances on the human body and the environment. In this way, it is possible to predict and assess the nature of a particular chemical substance used in a specifi c context.The potential benefi ts of in silico methods are huge, because they can reduce the need for animal testing, reduce research and production costs and delays, allow a large number of chemicals to be tested, increase the quality and quantity of information.”

More informations at http://disc.marseille.inserm.fr

THE FLOW CYTOMETRY & CELL SORTING PLATFORM

PLATFORM MANAGERS: DANIEL OLIVE & MARIE-LAURE THIBULT

CYTOMETRY


+33 (0)4 86 97 72 71

Marie-Laure [email protected]

+33 (0)4 86 97 73 63

CONTACTS

Flow cytometry allows the quantitative analysis of microscopic particles, such as cells and chromosomes, by suspending them in a stream of fl uid and passing them by an electronic laser detection apparatus.

The facility allows the simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second.Flow cytometry is routinely used in the diagnosis of health disorders, especially blood cancers, but has many other applications in both research and clinical practice. This technology also allows the physical sorting of particles based on their properties, so as to purify populations of interest.

Equipment› 2 Flow cytometers for analysis and cell sorting:

› 1 Aria II (3 lasers 405 /488/633nm - 9 PMT) (Becton Dickinson)› 1 Aria III SORP (5 lasers 350/405/488/561/630 nm - 18 PMT) (Becton Dickinson)

› 4 Flow cytometers for analysis only:› 1 LSRII SORP (4 lasers 405/488/532/630- 17 PMT) (Becton Dickinson)› 1 Fortessa (3 lasers 405/488/630- 11 PMT) (Becton Dickinson)› 1 Canto (2 lasers 488/630 - 6 PMT) (Becton Dickinson)› 1 Gallios (3 lasers 405/488/630- 10 PMT) (Beckman Coulter)

› 2 Stations for data analysis (softwares: FlowJo, Kaluza, Diva)› 1 Bioplex 200 (BioRad)› 1 Automacs Pro (Miltenyi)

THE IMMUNOMONITORING PLATFORM

PLATFORM MANAGER: DANIEL OLIVE

IMMUNOMONITORING


+33 (0)4 86 97 72 71

CONTACT

This question is at the heart of the concerns of scientists, clinicians and industrialists who seek to demonstrate the effi cacy and safety of a new drug.Ultimately, the answer will largely determine their decision: the continuation or discontinuation of development of the compound, especially when the latter acts directly on the immune system as a cytokine, a monoclonal antibody or a vaccine.The immunomonitoring platform designs, performs and interprets the molecular and cellular tests that quantify and qualify the many facets of the immune response and in turn reduce the uncertainties of the development of these agents. Data from these tests also permit exploration of new directions that complement to the study of archived material from the tumor bank.The platform of Immunomonitoring in Cancerology is labelled IBiSA (2008) and certifi ed ISO 9001:2008 and NFX 50-900 which is specifi c to life science technology platforms, since January 2015. The platform is open to the medical, scientifi c community as well as industry within the framework of projects related to the cancerology and immunology. Our clients are biotechnology and biopharmaceutical companies.The platform has expertise in tumor immunology

(NK cells functions in hematological malignancies, breast cancer and congenital immunodefi ciency, cellsin AML and NHL, pDC), in signaling (co-signalingmolecules in hematological malignancies and solid tumors) and also in the analysis of tumor microenvironment.

Platform activities include many domains such as:› Biological follow-up of cancer biotherapies protocols:

therapeutic vaccination, monoclonal antibodies (immunotherapy); allogenic stem cells and cord blood graft (transplantation)

› Pre-clinical development of new biotherapies (dendritic cells, NK cells, adjuvant)

› Alterations of immune system identifi cation induced by neoplastic process (dendritic cells, NK and T cells abnormalities)

› Analysis of the immune response against pathogens associated with immunodefi ciency

The platform uses diff erent approaches to develop the research projects on the basis of fundamental immunology, cellular and molecular biology.

Equipment› Cell culture rooms (2 L1 and 1 L2) fi tted with 4 PSMs, microscopes and incubators › 1 FacsCanto II BD fl ow cytometer for analysis (3 lasers 405nm/488nm/633nm – 8 PMT) fi tted with

a tube conveying system › 3 Stations for data analysis (software: Diva, FlowJo, Infi nicyt)› 1 Cr51 manipulation room for cytotoxicity assays› 1 CTL Immunospot 5 UV core for fl uorescence ELISpot assays› 1 BioAnalyzer 2100 for qualitative and quantitative RNA analysis (Agilent)

How does the immune system of the animal or the patient respond to the treatment?

THE IPC & CRCM EXPERIMENTAL PATHOLOGY PLATFORM

PLATFORM MANAGERS: EMMANUELLE CHARAFE-JAUFFRET & MICHEL AURRAND-LIONS

ICEP

Emmanuelle Charafe-Jauff retjauff [email protected]

+33 (0)4 91 22 35 09

Michel [email protected]

+33 (0)4 86 97 72 91

CONTACTS

ICEP performs paraffi n-embedding of research samples and conventional staining (H&E) or immunohistochemistry (IHC) to investigate the expression and localization of new biomarkers which could go on to be developed as new therapeutic targets.In addition, thin sections can be prepared for immunohistochemistry on frozen material. The platform also performs tissue microarrays (TMA) from paraffi n-embedded samples to study the expression of a maker simultaneously on a wide range of cases, with clinical records. IHC can be done on TMA.We deliver scans of slides that can be further analyzed. We are also able to provide IHC analysis by a pathologist

dedicated to the platform.More than 5,000 samples with clinical data available are stored in TMA and can be provided for research purposes on request. Automated computer-assisted quantifi cation of white fi eld pictures is used to allow standardized and objective analysis of IHC results. After image cropping of the slide’s scans, automated densitometric measurements of immunoprecipitates in each TMA are scored individually. The resulting quantitative data are appropriate for statistical analysis and correlation with prognostic and therapeutic responses.

Equipment› Immunohistochemistry devices (Ventana (Roche) Discovery XT, DAKO Autostainer Plus, MicromFrance

Thermoscientifi c 480S)› Deparaffi nors (MicromFrance PT1057T0905 and DAKO PTLink)› Microtomes (MicromFrance HM340E and Leica RM2235)› Cryostats (Leica CM3050S and MicromFrance Cryostar NX70)› Cryojane (Leica) › Vibratome (LeicaCryoJane1373)› Cold plate (MicromFrance Thermoscientifi c HFU 300TV)› Unwringler (MicromFrance LP60)› Microscopes (Nikon DiaPath, Nikon double headed CIL LED)› Stainer (Leica Autostainer XL)› Dehydration device (Leica ASP300/ASP300S)› Paraffi n inclusion device (Leica EG 1150 C/H)› Chemical hood (MicromFrance EG 1150 C/H)› Ultra fast tissue specimen freezing system SnapFrost 2 (Excilone)› Tissue arrayers (Excilone Minicore, Alphelys)› Slide scan device (Hamamatsu, Nanozoomer 2.0 HT)

The IPC and CRCM Experimental Pathology (ICEP) platform is dedicated to paraffi n blocks or tissue slides delivering of research samples. The platform performs as well morphological/molecular analysis of patient tissue sections for research purposes.

THE MICROSCOPY & SCIENTIFIC IMAGING PLAFORM

PLATFORM MANAGERS: DANIEL ISNARDON & ALI BADACHE

MISC


+33 (0)4 86 97 73 64

Ali [email protected]

+33 (0)4 86 97 73 21

CONTACTS

The Microscopy and Scientifi c Imaging facility off ers a wide array of imaging equipment that allows investigating the organization and the dynamics of tissue, cellular and sub-cellular structures using the latest cellular imaging techniques for research purposes: structured light fl uorescent microscopy, time-lapse microscopy, improved resolution confocal microscopy and high speed confocal microscopy (spinning disk technology) that permit to acquire fl uorescent and phase contrast images in four dimensions. It also gives access to image processing and

analysis tools for multi-dimensional images (x,y,z, t and λ dimensions).This facility is shared by all groups of the CRCM and open to outside investigators. The facility is supervised by a research scientist and managed by a research engineer (with a PhD degree). It provides training and support to all users, maintains and upgrades materials and software, develops analytical tool and performs technological watch.

EquipmentMICROSCOPES› LSM 880 confocal microscope (Zeiss) with spectral and Airyscan modes,› LSM 510 META confocal microscope (Zeiss),› A fl uorescence structured light microscope - Apotome type (Zeiss) with a color camera for colored samples,› Two time-lapse epifl uorescence microscopes (Zeiss-Ropper Scientifi c),› A confocal multiple-point microscope (Zeiss-Ropper Scientifi c) with a « spinning disk », a FRAP, FLIP and photo-

activation system, able to operate in TIRF mode.

SOFTWARE› Metamorph,› Huygens (deconvolution 3, 4 and 5D) in a network hooked on a calculation cluster,› ImageJ, Icy (image analysis freeware),› MATLAB software in a network hooked to a cluster.

The platform is continuously adapted to best suit user needs and projects.

The Microscopy and Scientifi c Imaging facility mainly focuses on photonic microscopy techniques: widefi eld microscopy, confocal microscopy, structured light microscopy…

MASS SPECTROMETRY & PROTEOMICS FACILITY

PLATFORM MANAGERS: STÉPHANE AUDEBERT, JEAN-PAUL BORG, LUC CAMOIN, NELSON DUSETTI & ANTHONY GONCALVES

MAP

CONTACTS

MaP is a four-site proteomics facility, which off ers a wide panel of complementary techniques for protein identifi cation and characterization, biomolecule quantifi cation, biomarker discovery, binding studies and bioinformatics services. MaP was labelled by IBiSA since its creation in 2008 and off ers partnership, collaborative and fee-for-service opportunities for basic and clinical research but also for industry. This structuration led to strengthen expertise in the diverse proteomics fi eld and technologies needed to accompany local, national or international scientifi c projects.We have expertise in all the diff erent fi elds of proteomics required for the qualitative as well as quantitative description of complex mixtures of proteins. The technologies we provide our users allow the identifi cation, the characterization (molecular mass,

sequence, isoforms, post-translational modifi cations) of proteins, as well as their quantifi cation by chemical tagging (iTRAQ, 2D-DiGE), metabolic tagging (SILAC) or in the absence of tagging (Label Free). The quantitative methods we use are particularly well suited for the identifi cation of cancer biomarkers.

Our proteomics platform has three major axes:› Discovery and quantifi cation of new biomarkers in

clinics› Mass spectrometry identifi cation and deciphering of

new protein complexes › Discovery of protein-protein interaction by Y2HWe also perform two-dimensional electrophoresis using Diff erential in-Gel Electrophoresis (2DE-DiGE) and chemoproteomics.

Equipment› Mass Spectrometer Q-Exactive (ThermoScientifi c) coupled to a nanoLC RSLC Ultimate (Dionex) (2013)› Mass Spectrometer LTQ-Velos Orbitrap ETD (ThermoScientifi c) coupled to a nanoLC RSLC Ultimate

(Dionex) (2010)› Mass Spectrometer Maldi-ToF MS/MS UltraFlex III (Bruker) (2009)› Automate Freedom Evo 100, (Tecan), (2007)› Automate Freedom Evo 150, (Tecan), (2010)› Automate spot picker/digester Xcise (Shimadzu), (2008)

Proteomics technologies allow the simultaneous structural and functional analysis of large sets of protein samples both for medical (diagnosis or prognosis) and research purposes.

Stéphane Audebert - [email protected], +33 (0)4 91 22 33 07 Jean-Paul Borg - [email protected], +33 (0)4 86 97 72 51

Luc Camoin - [email protected], +33 (0)4 86 97 72 58Nelson Dusetti - [email protected], +33(0)4 91 82 88 28

Anthony Goncalves - [email protected], +33 (0)4 91 22 36 62

THE ONCOGENOMICS PLATFORM

PLATFORM MANAGERS: DANIEL BIRNBAUM, MAX CHAFFANET & FRANÇOIS BERTUCCI

ONCOGENOMICS


+33 (0)4 91 22 33 54

Max Chaff anetchaff [email protected]

+33 (0)4 91 22 34 77

CONTACTS

This platform contributes to better understand cancer diseases and currently participates to translational clinical research projects associated with the development of precision medicine in advanced cancers. The refi ned molecular characterization of the tumor and the identifi cation of potential actionable genes might contribute to help oncologists to better defi ne their therapeutic strategies i.e chose among available targeted therapies that present the best effi ciency with reduced secondary eff ects and less harmful to healthy tissues than standard therapies.Genomic analyses are then required for the development

of the precision medicine identifying the treatment which might be the most adapted to each cancer patient.

The aim is to › Predict the drug sensitivity profi le of each patient

based on the molecular profi le of their tumor, › Stratify patients according to their tumor’s

characteristics, › Identify the most suited available treatment (new drug

currently being tested within the frame of a clinical trial or FDA approved drug) for each group of patients.

Equipment› Illumina MiSeq and NextSeq500 sequencers (exome, targeted-sequencing, RNAseq)› Transcriptomics plat-form : Aff ymetrix GCS 3000 7G Genechip System analyses at the gene

and exon levels

The oncogenomics platform establishes genomic, DNA methylation and mRNA expression profi les of various cancers using whole human genome 180k array-CGH, custom promotor array (Agilent Technologies) and exon, miRNA, gene expression chips (Aff ymetrix), respectively. Mutations are identifi ed by Next Generation Sequencing on Illumina MiSeq and Next Seq500. Technological developments are ongoing to allow the profi ling of paraffi n-embedded patient tumor samples.All oncogenomic analyses are done by bioinformaticians associated with the platform.

François Bertucci [email protected]

+33 (0)4 91 22 34 77

THE TRGET PRECLINICAL ASSAY PLATFORM

PLATFORM MANAGERS: RÉMY CASTELLANO & YVES COLLETTE

TRGET

Rémy [email protected]

+33 (0)4 86 97 73 32

Yves [email protected]

+33 (0)4 86 97 73 31

CONTACTS

TrGET off ers a variety of fl exible and customizable services, with privileged development of bioluminescence-based technologies: In vitro (96 well-adapted):› Proliferation/cytotoxicity› Caspases/apoptosis› Soft agar/methylcellulose colony assays› Migration› ALDH activity

In vivo (murine - non invasive intravital imaging):› Subcutaneous, orthotopic (breast and prostate) and

hematopoietic xenograft models› Human primary leukemic (LAM, LLC, LAPDC) models› Tumour volume / time to progression or doubling› Tumoral dissemination (blood, bone marrow, spleen,…)

and metastasis› Survival studies› Tissue collection, preparation and preservation

Equipment› Small animal imaging platform:

› PhotonImager (BiospaceLab)› Gaz anesthesia station coupled to an O2 concentrator› Pseudo-3D reconstruction station

› Integrated information management system with infra-red bar code screening (ModulBio)› Luminometer: Centro XS3 LB960 with injectors (Bertdhold)› EOPS animal facility › P2 cell culture facility

TrGET is a preclinical platform, performing and developing in vitro and in vivo testing in cell culture and in xenografted mouse models of target genes involved in tumorogenesis or anti-tumoral therapeutics.

We develop a combinatorial screening strategy to assess the potential synegistic eff ect of candidate molecules with currently available chemotherapeutic drugs or targeted drugs to optimize therapeutic effi ciency (COMBO-SCREEN program).

We also perform chemo-proteomic profi ling and mass spectrometry to identify the proteomic target of candidate drugs and investigate their mode of action (in collaboration with the MaP IBiSA platform).

The data are statistically analyzed and additional investigations can be provided.

THE NANOBODY PLATFORM

PLATFORM MANAGER: DANIEL BATY & PATRICK CHAMES

NANOBODIES


+33 (0)4 91 82 88 23

Patrick [email protected]

+ 33 (0)4 91 82 88 33

CONTACTS

The specifi city of this platform is to yield single domain antibodies (Nanobodies). These small and compact fragments (13 kDa) corresponding to the variable domains of a subtype of llama antibodies are characterized by outstanding properties in terms of production, stability, and their propensity to bind buried epitopes inaccessible to conventional antibodies. Most nanobodies can be expressed as soluble and active fragments without disulfi de bond formation, in a reducing environment such as the cell cytoplasm or nucleus. This particularity allows their use as intracellular tool to precisely knock out a specifi c interaction of a target protein without interfering with other interactions of this target. Thanks to their high propensity to bind cavities, enzyme active sites or other binding sites, they represent a rich source of blocking antibodies. Their humanization is also straightforward.

The mission of the nanobody platform is to help research teams with the isolation of specifi c nanobodies. The selection and screening steps, based on phage

display, can be performed on recombinant proteins, transfected cells, intact cells or lysate of cell lines. Nanobodies are produced and purifi ed from E. coli, and can be fused to various tags (c-Myc, 6-His), modifi ed with an extra C-terminal free cystein allowing a site-directed conjugation to nanoparticles, or biotinylated in vivo.Genes coding for nanobodies can also be fused to the gene of a reporter molecule such as GFP, mRFP1. The resulting constructs can be directly used as probe in living cells or for protein localization.We have 26 nanobody libraries which are available for screening. They have been built after llama immunization with:› Various recombinant proteins› Human thymocytes› Biopsies of various cancer types› Transfected cells› Haptens, etc…The platform can also generate customized libraries: we will perform llama immunization and library construction.

Equipment› Gradient PCR cycler,› 3 laminar fl ow hoods for phage display,› 2 laminar fl ow hoods for cell culture,› Microincubators for 15 microtiter plates,› Tecan infi nite M1000,› Flow cytometer (3 lasers, 10 colors),› AKTA purifi cation system,› 15 L fermentor,› Robot Tecan Freedom EVO 150.

Monoclonal antibodies are major tools used in various fi elds including proteomic approaches, diagnosis or therapy.

Nanobody selection process

In this endeavor, the IPC Tumor Bank has joint forces with its AP-HM University Hospital counterpart- and with the joint University Hospital – Comprehensive Cancer Centre (CHUN-CAL) Tumor Bank in Nice. The two Marseille Tumor Banks established a common strategy to build signifi cant collections of appropriately and clinically annotated biological samples, jointly prepared the ISO 9001 certifi cation, and have worked together to favor access to molecular profi ling platforms, allowing innovative translational studies to be performed, including genomic, transcriptomic and proteomic tumor typing. The IPC Tumour Bank now maintains the ISO 9001 certifi cation, together with the DRCI (see below), providing to the scientifi c community a unique resource that associates latent information that can be revealed from sample analyses and existing clinical and biological information. The two biobanks are an important component and facility of the Marseille SIRIC. The two Biobanks (IPC and AP-HM) currently have more than 130,202 samples in store (tissue, blood cells, serum, DNA, RNA). Up to 1 228 samples from the biobank were used for research or medical care in 2015.

The two biobanks store and distribute samples of human origin that are representative of diff erent groups of cancers:› Breast cancer, gastro-intestinal and pancreatic carcinomas, haematological

malignancies at IPC › Brain tumours, sarcomas, prostate cancer and lung cancers at AP-HM.

These biological resources are available to research groups that conduct basic or translational programs in the fi eld of oncology.

The two Marseille Tumor Banks teamed up with the CHUN-CAL tumor Bank in Nice, to produce the fi rst virtual tumor bank in France: the Cancéropôle PACA Tumor Bank (www.biobank-paca.com)allowing for online multicriteria searches in a catalogue of anonymized biological resource. Together, the three PACA Tumor banks received support from the IBiSA organization in 2008, in order to improve the quality of samples and annotations.› Number of new samples stored in 2015: 25 096› Number of samples used in 2015: 1 228

THE TUMOR BANK / BIOLOGICAL RESOURCE CENTER IN ONCOLOGY

PLATFORM MANAGER: CHRISTIAN CHABANNON

BIOBANK

Christian [email protected]

+33 (0)4 91 22 34 79

CONTACT

The Institut Paoli-Calmettes (IPC) has long established the practice of collecting and storing human tissues and cells from cancer patients, and thus provides a resource for translational and clinical research to the onsite scientifi c community. The tumor bank was established as an independent and shared infrastructure in 2000, using the same cryopreservation equipment as the cell therapy facility. Since then, it has improved its operations through the implementation of SOPs, the installation of a digitalized inventory, the diversifi cation of tissue and cell processing procedures including a shared facility for the automated isolation of nucleic acids. With additional support from Inserm, Agence Nationale de la Recherche and Cancéropôle PACA, the hospital infrastructure worked towards the ISO 9001 certifi cation as well as IBISa recognition.

THE CELL THERAPY FACILITY & THE CENTER FOR CLINICAL INVESTIGATIONS IN BIOTHERAPY

PLATFORM MANAGER: CHRISTIAN CHABANNON

CBT-1409

Christian [email protected]

+33 (0)4 91 22 34 79

CONTACT

Biotherapies are therapies based on manufacturing of biological products, such as antibodies, cells, and blood-derived material.

The Cell Therapy Facility of IPC includes the infrastructures, equipment and human resources that are required for the collection and processing of hematopoietic cells obtained from patients or donors, and thus supports the autologous and allogeneic hematopoietic stem cell transplant programs respectively through the production and delivery of cell therapy products.

It is one of the largest European infrastructures active in this fi eld, and it benefi ts from its integration within the hospital, as well as of the presence of numerous research teams in its immediate vicinity.

Other innovative cellular therapies are also currently developed, such as cellular anti-tumoral vaccines and adoptively transfered immune eff ectors. The Cell Therapy facility works closely with the Center for Clinical Investigations in Biotherapy (CBT-1409).

The Center for Clinical Investigations in Biotherapy is affi liated with IPC, Inserm, Aix-Marseille University and Assistance Publique-Hôpitaux de Marseille.

It develops cellular and tissue engineering technologies, brings innovative technologies to clinical standards, develops biological assays to monitor the effi ciency and safety cell and tissue therapies (surrogate markers). It ensures that the latest regulation in biotherapy is enforced.

THE CLINICAL RESEARCH FACILITIES THE DEPARTMENT OF CLINICAL RESEARCH & INNOVATION

THE DATA MANAGEMENTAND ANALYSIS CENTER (DMAC)

PLATFORM MANAGER: DOMINIQUE GENRE

PLATFORM MANAGER: LILIAN LABORDE

CLINICAL RESEARCH

The Department of Clinical Research and Innovation (DCRI) provides regulatory, administrative and educational services to the Institute’s investigators or staff participating in multi-center, national and international clinical trials. This resource also serves to increase awareness and accrual to clinical trials as well as to improve the quality and effi ciency of conducting clinical trials in compliance with regulatory, documentation, and oversight requirements. The DRCI team brings together project leaders who work in close relation with the medical coordinator of the clinical trial, to write the trial protocol, record patients inclusions, control all stages of the trial, from its initiation to the analysis and publication of results, and supervise the administrative and fi nancial aspects of the study, a pharmacist who prepares the treatments prescribed in the trials, technicians and clinical research nurses who performs the day-to-day tests.

The DRCI supervises over 200 protocols each year, 18 of which were promoted by IPC itself in 2012. 831 people were included in clinical trials at IPC in 2012 (11,55% patients).

The DRCI platform received the ISO 9001:2008 certifi cation in 2013.

Lilian [email protected]

+33 (0)4 91 22 35 10

The Data Management and Analysis Center manages the clinical trial data and ensures the quality and accuracy of data collected and its security in strict

compliance with good clinical practice. It also allows randomization and statistical analysis and medical writing.

CONTACT

CONTACT

Dominique [email protected]+33 (0)4 91 22 37 43

THE EARLY PHASE TRIAL UNIT

PLATFORM MANAGERS: NORBERT VEY & ANTHONY GONCALVES

CLINICAL TRIALS

The aim of this department is to evaluate the benefi t of therapeutic innovations for hematological cancers as well as for solid tumors. This department is labeled by the Regional Health Agency (ARS) of Provence Alpes Côte d’Azur and by the National Institute of Cancer (INCa). It is the largest early phase trial unit in France for malignant hemopathies.

Early phase trials are critical for the development of new drugs. It is the fi rst time new molecules can be administered to patients, and their effi ciency and toxicity can be evaluated. This requires a specifi c procedures for patient care, follow up and security, as well as a strict monitoring protocol. Pharmacokinetic analysis are performed on all patients included in the trials at regular intervals.The early phase trial unit ensures the level of care and quality of the process imposed by this type of trial and accelerate the evaluation of innovative therapeutic approaches.It spreads over 250 sqm and is organized around 9 single rooms (5 beds are available on an outpatient basis), a treatment room, monitoring tools and a specifi c area allowing immediate treatment of blood and urine samples.

Norbert [email protected]

+33 (0)4 91 22 37 47

Anthony [email protected]

+33 (0)4 91 22 36 62

CONTACTS

The early phase trial unit provides eligible patients with access to the latest innovations in cancer therapy and allows them to benefi t from new, potentially more effi cient and less harmful therapeutic drugs, within the frame of phase I or phase II clinical trials.

The early phase trial hosts a multidisciplinary team which brings hematologists, oncologists, a pharmacist, a radiologist, biologists, intensive care anesthesists, and clinical research nurses. They work in tight collaboration with the intensive care unit and with the technological platforms of IPC. A hundred trials are currently underway, most of which within the frame o international collaborations. The early phase trial unit has 2 trials which were marked the fi rst administration in man of two drug candidates for malignant hemopathies. The main pathologies targeted are breast cancers, pancreatic cancer, acute myeloid leukemia and uro-gynecological cancers. The latest therapeutic innovations are in the areas of immunotherapy and targeted therapies based on the molecular profi ling of each patient’s tumor and on the identifi cation of specifi c biomarkers which allow the prediction of patient response to treatment.

5 TEAMS (CANCER & HEMATOLOGY)

MULTIDISCIPLINARY RESEARCH

Gynecological Cancer team - Manager: Eric Lambaudie, [email protected] Cancer team - Manager : Anthony Goncalves, [email protected]

Urological Cancer team - Manager : Gwenaëlle Gravis, [email protected] Cancer team - Manager: Jean-Luc Raoul, [email protected]

Onco-Hematology team - Manager: Norbert Vey, [email protected]

CONTACTS

Molecular characterization of the patients’ tumorRecent technological advances in sequencing now allow precise information about the genomic alterations of individual tumors to be obtained as part of the routine protocol within a time frame compatible with the implementation of a personalized care procedure, adapted to each tumor and to each patient. Information about the genomic alterations of a patient’s tumor provides insight into the mechanism of oncogenesis as well as allowing the physicians to predict the tumor’s sensititivity/resistance profi le to the various treatments available. Depending on the genomic alterations detected in their tumor, patients can be off ered to take part in clinical trials evaluating the effi ciency of targeting a specifi c genomic alteration. However, a specifi c treatment is not always available for all genomic alterations. Other alternatives can also be investigated.Targeting cancer stem cellsIt is becoming increasingly clear that cancer is a very heterogeneous disease; various sub-types of tumors exist and even within a given tumor, not all cells are identical. Resistance to treatment is often due to this intra-tumor heterogeneity: even when the bulk of the tumor is eradicated by treatment, a small number of cells can resist the treatment, and subsequently give rise to a new tumor. These cancer stem cells could account for relapse and metastasis formation.

Dr Daniel Birnbaum’s laboratory at the CRCM runs a specifi c program on mammary stem cells lead by Prof Emmanuelle Charafe-Jauff ret and Dr Christophe Ginestier. Biomarkers and functional assays are now available to monitor the evolution of stem cells in patients over the course of their treatment. Dr Birnbaum’s team is also investigating new potential stretegies to specifi cally target stem cells.Patient-derived xenografts (PDX)Animal models are also developed at IPC and at the CRCM in order to test a given tumor’s response to treatment and thus orient therapeutic decisions. Biopsies obtained from a patient’s tumor can be grafted into animal models: this allows the effi ciency of drugs to be evaluated in a physiological context for a given tumor. This approach is used at IPC and at the CRCM for patients with breast cancer, leukemia patients and pancreatic cancer.ImmunotherapyFinally, immunotherapy is another alternative. In this case, treatment is not directly targeting the tumor, but at the immune system, so as to boost its anti-tumoral activity. Prof Norbert Vey, head of the Early Phase Trial Unit of IPC has thus conducted the fi rst phase I clinical trial using a monoclonal antibody which modulates the activity of Natural Killer cells in order to boost the immune system’s response to cancer, in collaboration with the team of Daniel Olive at the CRCM: this was the fi rst example of NK-based immunotherapy in human.

Cancer care has made huge progress over the decade. New insight in tumor biology and technological innovations have now allowed biomarkers to be identifi ed, diagnosis to be refi ned and treatment to be better target to the tumor and healthy tissue better spared.A multidisciplinary approach to cancer care is thus necessary to orient therapeutic decisions and select the treatment most suited to a given patient at a given time. Oncologists, surgeons, radiotherapists, radiologists, and biopathologists at IPC, work together with biologists from the CRCM in translational research teams. Five such teams have been established for blood cancers (onco-hematology: leukemia, myelodystrophic disorders and lymphomas), breast tumors, digestive cancers (pancreatic but also colon and stomach), urological cancers (cancers of the kidney, prostate and bladder) and gynecological tumors (cancers of the uterus and ovary).

SIMULATION & MODELLING: ADAPTIVE RESPONSE FOR THERAPEUTICS IN CANCER

PLATFORM MANAGER: JOSEPH CICCOLINI

SMARTc

Joseph Ciccolini [email protected]

+33 (0)6 27 91 54 74

The SMARTc platform, located at the school of pharmacy of Marseille and La Timone University Hospital of Marseille, is a unique group dedicated to developing PK/PD models and decision algorithms to optimize combinational regimen in oncology.

The SMARTc platform is built around a group of mathematicians and modelers in PK, PK/PD and systems biology along with a scientifi c staff benefi ting from a fully equipped wet lab to perform experimental therapeutics in oncology with a strong focus on drug metabolism, pharmacogenetic and pharmacokinetic studies (in vitro, in vivo) and state-of-the-art ISO15189 clinical pharmacokinetic laboratory to assay drugs in biological matrix, including biologics and immune check point inhibitors.

SMARTc’s expertise focuses on, but not limited to, developing innovative tools using the latest advances in applied mathematics and phenomenological models to optimize the effi cacy/toxicity balance of anticancer drugs, especially when used as part of combination therapies in refractory tumors.

CONTACT

This covers ∠Developing biomarker-based dosing algorithms ∠ Identifying new covariates impacting on pharmacokinetics variability and PK/PD relationships of anticancer agents ∠Developing innovative drug regimen driven by PK/PD modeling support ∠Developing model-informed strategies to optimize metronomic regimen ∠Developing model-informed design of nanoparticles to optimize the PK/PD relationships of anticancer agents ∠Developing tools to optimize new combination regimen such as immunotherapy-based combos ∠Developing tools for model-informed design of clinical trial ∠Running phase I and phase I/II clinical trials in a FDA-approved, Iso15189 environment

THE PLATFORM FOR ORGANIC MOLECULES SYNTHESIS

PLATFORM MANAGER: SÉBASTIEN COMBES

DOSynth

Sébastien [email protected]

+33 (0)4 91 83 55 51

Paul Bré[email protected]

+33 (0)4 91 83 55 51

CONTACTS

DOSynth is a platform dedicated to the synthesis of organic molecules of medical interest, including biological tools and probes.

Researchers can access to DOSynth either through collaborations, especially for research projects in oncology, or services, for research projects in life sciences. DOSynth is intended to provide non-commercially available or hardly accessible organic compounds which require specifi c know-how. The platform is able to conduct automated synthesis for generating dedicated libraries of compounds and works in close collaboration with the molecular modeling platform ‘Int3D’.DOsynth benefi ts from the technologies available at the Aix-Marseille University ‘Faculty of Pharmacy’ and provides a fl exible access to chemical laboratories and specifi c equipment.

Services ∠Custom synthesis ∠Design and synthesis of dedicated library ∠Hit optimization ∠Functionalization and vectorization ∠Chemical analyses

Equipment ∠Chemspeed Synthesizer SLT II ∠Microwave Biotage Initiator + ∠Combi fl ash Rf Teledyne ISCO ∠Chaine HPLC préparative Agilent ∠Chaine LCMS 1260 infi nity Agilent

CONTACTS

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