THE NETHERLANDS CANCER INSTITUTE SCIENTIFIC ANNUAL REPORT 2010

Transcription

1 THE NETHERLANDS CANCER INSTITUTE SCIENTIFIC ANNUAL REPORT 2010

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3 SCIENTIFIC ANNUAL REPORT 2010

4 4 Patron HM Queen Beatrix

5 5 SCIENTIFIC ANNUAL REPORT 2010 THE NETHERLANDS CANCER INSTITUTE CANCER RESEARCH LABORATORY AND CANCER HOSPITAL

6 6copyright COPYRIGHT Scientific Annual Report 2010 Illustrations and unpublished data in these reports may not be used without permission of the author. Copyright The Netherlands Cancer Institute Antoni van Leeuwenhoek Hospital Plesmanlaan CX Amsterdam The Netherlands Phone: Fax: ISSN

7 7 contents CONTENTS Board Members 9 Research Divisions 10 Introduction 12 Education in Oncology 16 Division of Biochemistry 18 Division of Cell Biology I 22 Division of Cell Biology II 29 Division of Experimental Therapy 36 Division of Gene Regulation 47 Division of Division of Immunology 53 Division of Molecular Biology 61 Division of Molecular Carcinogenesis 73 Division of Molecular Genetics 79 Division of Psychosocial Research and Epidemiology 88 Division of Diagnostic Oncology 99 Division of Medical Oncology 114 Division of Radiotherapy 124 Division of Surgical Oncology 147 Biometrics Department 162 Clinical Trials 167 Invited Speakers 182 Projects 184 Personnel Index 202

8 8colophon COLOPHON Coordinators Suzanne Corsetto Henri van Luenen Monique Duyndam Photograph HM The Queen Beatrix Enquiries/permission: Rijksvoorlichtingsdienst, afd Pers en Publiciteit/Foto Postbus EA Den Haag Photo Ruud Taal/Capital Press Copyright RVD Photograph AJM Berns: Loek Zuijderduin Other photographs and illustrations: Audiovisual services The Netherlands Cancer Institute/ Antoni van Leeuwenhoek Hospital Printed by Drukkerij Badoux, Houten

9 9 board members BOARD MEMBERS INTERNATIONAL SCIENTIFIC ADVISORY BOARD T De Lange, Leon Hess Professor, The Rockefeller University, New York, USA RA Flavell, Professor of Immunobiology, Yale University School of Medicine, New Haven, USA WGJ Hol, Professor of Biochemistry and Biological Structure, University of Washington, Seattle, USA J Mendelsohn, President MD Anderson Cancer Center, University of Texas, Houston, USA P Nurse, Professor of Microbiology, President of The Rockefeller University, New York, USA R Nusse, Professor of Developmental Biology, Stanford University, Stanford, USA HL Ploegh, Professor of Biology, Whitehead Institute for Biomedical Research, Cambridge, USA S Powell, Chairman, Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, USA K Vousden, Director, Beatson Institute for Cancer Research, Glasgow, UK RA Weinberg, Professor of Biology, Massachusetts Institute of Technology, Whitehead Institute for Biomedical Research, Cambridge, USA NATIONAL SCIENTIFIC ADVISORY BOARD DD Breimer, Professor of Pharmacology, Leiden University JC Clevers, Professor of Clinical Immunology, Hubrecht Institute, Utrecht EGE De Vries, Professor of Medical Oncology, University of Groningen JHF Falkenburg, Professor of Experimental Hematology, Leiden University CG Figdor, Professor of Experimental Immunology, Radboud University Nijmegen JHJ Hoeijmakers, Professor of Molecular Genetics, Erasmus University Rotterdam P Lambin, Professor of Radiation Oncology, Maastricht University RH Medema, Professor of Experimental Oncology, Utrecht University CJH Van de Velde, Professor of Surgical Oncology, Leiden University President of Board of Governors W Kok BOARD OF DIRECTORS AJM Berns, chairman and director of research S Rodenhuis, director clinical research and development WH Van Harten, director organization and management BOARD OF GOVERNORS W Kok, president HCJ Van der Wielen, vice president T De Swaan, treasurer GH Blijham FH Schröder MC Smeets EH Swaab PC Van der Vliet MJ Van Mourik SCIENTIFIC ADVISORY COUNCIL AJM Berns, chairman JJ Neefjes, secretary S Rodenhuis B Van Steensel M Verheij (until September 2010) T Ruers (from September 2010) J Borst (until December 2010) J Jonkers (from December 2010)

10 10 research divisions RESEARCH DIVISIONS Biochemistry Titia Sixma (head) Anastassis Perrakis Caroline Kapper (office manager) Cell Biology I Arnoud Sonnenberg (head) Wim van Blitterswijk John Collard Metello Innocenti Kees Jalink Wouter Moolenaar Ed Roos Patty Lagerweij (office manager) Cell Biology II Jacques Neefjes (head) Rob Michalides Huib Ovaa Peter Peters Marieke van der Velde (office manager) Experimental Therapy Adrian Begg (head) Jan Schellens Fiona Stewart Marcel Verheij Jelle Wesseling Thea Eggenhuizen (office manager) Gene Regulation Reuven Agami (head) Jan-Hermen Dannenberg Maarten Fornerod Fred van Leeuwen Bas van Steensel Suzanne Corsetto (office manager) Immunology Jannie Borst (head) Christian Blank John Haanen Heinz Jacobs Ton Schumacher Florry Vyth-Dreese José Overwater (office manager) Molecular Biology Hein Te Riele (head) Piet Borst (honorary staff member) Jos Jonkers Sabine Linn Alfred Schinkel Lodewyk Wessels Tom de Knegt (office manager) Linda Römer (secretary) Molecular Carcinogenesis René Bernards (head) Roderick Beijersbergen Rob Wolthuis Molecular Genetics Maarten van Lohuizen (head) Anton Berns Jacqueline Jacobs Anna-Pavlina Haramis John Hilkens Paul Krimpenfort Daniel Peeper Margriet Snoek Erica Delwel (office manager) Marie Anne van Halem (secretary) Psychosocial Research and Epidemiology Flora van Leeuwen (head) Neil Aaronson Eveline Bleiker Wim van Harten Matti Rookus Sanne Schagen Marjanka Schmidt Yvonne Driessen-Ruwaard (office manager) Diagnostic Oncology Laura van t Veer (head) Tanja Alderliesten Priscilla Axwijk Philippe Baars Olga Balague Ponz Peter Besnard Hans Bonfrer Daphne de Jong Kenneth Gilhuijs Cees Hoefnagel Frans Hogervorst Irma Kluijt Wim Koops Robert Kröger Charlotte Lange Claudette Loo Saar Muller Petra Nederlof Willem Nooijen Frank Pameijer Renée van Pel Warner Prevoo Marc Roef Efraim Rosenberg Marielle Ruijs Michiel Sinaasappel Ferida Sivro Marcel Stokkel Jelle Teertstra Renato Valdes Olmos Hester van Boven Fijs van Leeuwen Olaf van Tellingen Loes van Velthuysen Mark van de Vijver Lizet van der Kolk Senno Verhoef Wouter Vogel Jelle Wesseling Bart van de Wiel Christine Arkes (secretary) Carla van Tiggelen (secretary) Medical Oncology John Haanen (head) Joke Baars Paul Baas Jos Beijnen André Bergman Christian Blank Jan Paul de Boer Willem Boogerd Henk Boot Dieta Brandsma Wieneke Buikhuisen Sjaak Burgers Annemieke Cats Leo Gualtherie van Weezel Michel van den Heuvel Alwin Huitema Martijn Kerst Sabine Linn Anne Lukas Serena Marchetti Sjoerd Rodenhuis Jan Schellens Gabe Sonke Neeltje Steeghs Babs Taal Margot Tesselaar Marchien van der Weide Mariëlle de Kwant (secretary) Karin van Leuveren (secretary) Radiotherapy Marcel Verheij (head) Berthe Aleman Harry Bartelink José Belderbos Monique Bloemers Eugène Damen Roel de Boer Luc Dewit Paula Elkhuizen Rick Haas Olga Hamming-Vrieze Wilma Heemsbergen

11 11 research divisions Frank Hoebers Edwin Jansen Joost Knegjens Han Krewinkel Joos Lebesque Ben Mijnheer Luc Moonen Arash Navran Heike Peulen Floris Pos Coen Rasch Babs Reichgelt Peter Remeijer Nicola Russell Govert Salverda Christoph Schneider Jan-Jakob Sonke Joep Stroom Marcel van Herk Baukelien van Triest Karijn Verschueren Corine van Vliet-Vroegindeweij Marieke van Zwienen Thelma Witteveen Frits Wittkämper Patricia Haye-Fewer (section coordinator) Surgical oncology Theo Ruers (head) Marc van Beurden Michiel van den Brekel Alfons Balm Annemieke Ackerstaff Arend Aalbers Axel Bex Biljana Zupan-Kajcovski Charlotte Zuur Dirk Buitelaar Emiel Rutgers Frans Hilgers Frits van Coevorden Gemma Kenter Henk van de Poel Hester Oldenburg Houke Klomp Bing Tan Ingeborg Vergouwe Inka Nieuweboer-Krobotova Joris Hage Johanna van Sandick Jos van der Hage Julia ten Cate Katina Efthymiou Leonie Woerdeman Lotti Lubsen-Brandsma Ludi Smeele Marianne Piek-den Hartog Marie-Jeanne Baas-Vrancken Peeters Marieke van der Berg Martine van Huizum May Ronday Michael Srámek Michel Wouters Omgo Nieweg Peter Schutte Peter Lohuis Petra Biewenga Simon Horenblas Vic Verwaal Wietze van der Veen Willemien van Driel Wim Meinhardt Annemieke Hoogland (office manager) Biometrics department Otilia Dalesio Financial Administration Frieda Boekweit General Facilities, ICT and Personnel Department Eric de Wilde General Research Coordination Jacques Neefjes, deputy scientific director Henri van Luenen, director of operations

12 12 introduction INTRODUCTION I am pleased to present our Scientific Annual Report It provides an overview of the scientific activities at The Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital (NKI-AVL). Additional information can be found at A lucid general overview of our activities with illustrations can be found in our brochure (available on our website). The NKI-AVL is a Comprehensive Cancer Centre, combining hospital and research laboratories under one roof in a single independent organization. The hospital comprises 180 beds, an outpatient clinic and a large radiotherapy department. Facilities for clinical research include a large patient database, clinical data management, extensive diagnostic facilities, a pharmacy with a production unit for experimental drugs, and active research groups in medical, surgical and diagnostic oncology, radiotherapy, pharmacy, epidemiology and psychosocial oncology. The laboratory covers all major areas of cancer research, with special emphasis on cell-based screens, mouse tumour models, cell biology, structural biology, and immunology. Translational research is an integral activity of many research groups and is fostered by collaborations between clinical and basic scientists. Clinical activities showed again substantial growth in 2010 facilitated by a new temporary outpatient clinic that became operational in January. In the course of 2011 we will decide on the further expansion of the hospital so that a previously planned 50% growth can be accommodated. We also try to accommodate a national children oncology hospital on the premises of the NKI-AVL. Earlier in the year an independent committee installed by the Dutch organisation of paediatric oncologists and parent organisation of children with cancer selected the NKI-AVL as the preferred location for such a specialised hospital. It will become clear in the course of 2011 whether the building of this hospital close to the NKI-AVL can be realised. Execution of the plan to establish satellite radiotherapy treatment centres near two other hospitals in the area will start with constructions in the course of 2011 at one of the locations. The activities to establish a proton therapy centre together with the Erasmus University, the University Medical Centre Leiden, and the Delft Technical University, have been slowed down temporarily due to uncertainties with regard to reimbursement guarantees and new developments with regard to instrumentation. We secured together with a number of other European cancer centres a 12 million EU grant to implement robust high-throughput diagnostic platforms for genomics/proteomics assays. We need such platforms to identify suitable biomarkers that predict response to therapy. We are also in the process to establish with UMC-Utrecht, Hubrecht Laboratory, and ErasmusMC a foundation that will provide deep sequencing services of tumours and normal tissue of individual patients. This is an integral part of our plans to advance personalised medicine. Furthermore, we have succeeded in filling most of the vacancies for nursing staff and pathologists, but are still struggling to attract personnel in some other clinical Director of Research Ton Berns disciplines. Since the shortage of medical experts and skilled nursing staff is expected to further increase in the years to come we have to make the NKI-AVL stand out as the place to be for employees. This is an important task for the Institute. We concluded 2010 with a healthy profit for the hospital. As a result no harsh measures with regard to personnel and services will be needed to accommodate the substantially reduced reimbursement that we expect for clinical services in Our research expenditure also remained within budget in However, this could only be achieved by not filling the positions of staff members that left the Institute. The renovation of the laboratories in the old research building is in full swing and we expect to move into the renovated labs in the course of Bids for building the new animal facility will be evaluated in the beginning of We expect to select the contractors shortly after. Discussions with the Ministry of Health, aimed to increase our research budget as was strongly advised by the site visit team that evaluated the Institute in 2009, have not been successful. While it is evident that the government has to cut costs, it is disappointing that in the current political climate investment in research has a relatively low priority. Many other countries clearly choose to increase the support for research as they all share the view that high quality research is critical for securing the future prosperity of a country. Fortunately, the Dutch Cancer Society is more receptive to our needs and we are negotiating a new 5-year agreement for core funding which we hope to finalise early We have installed an introductory program for basic researchers in the clinic. In this way they can get a feel for the daily work of their clinical colleagues. We expect that this will also foster discussions and collaboration between basic researchers and clinicians in the Institute. It also can serve as a step-up to the twinning of clinicians with investigators in the laboratory to become effective teams. In 2010 two new spin-off companies were founded. UbiQ focuses on reagents, both for research and diagnostic purposes as well as to identify lead compounds for targeting regulatory pathways controlled by ubiquitination and deubiquitination reactions. This is an important new field with promising perspectives. The second company, MODRA, has as general goal the commercialisation of

13 13 introduction Table 1 Core research funding NKI-AVL from the Dutch Cancer Society and the Ministry of Health in the period in million euro s. Year Cancer Society Ministry Health Total * In addition to the core funding the NKI acquired support through external grants, donations, and research agreements. The contribution of these sources to the total budget has steadily increased over the years and represents roughly 65% of the total budget in inventions jointly made by Slotervaart Hospital and NKI- AVL. MODRA initially focuses on making existing drugs orally available through co-administration of inhibitors of drug transporters that prevent intestinal uptake under normal conditions. The Technology Transfer Office of the NKI has played an important role in defining the conditions and licenses for these new start-ups. HIGHLIGHTS The institute continues to have a strong scientific output; 2010 was again a productive year. It is always difficult to estimate the impact of research when the results are still fresh. The research highlights summarised here serve primarily as a sampling of work currently on-going in the Institute. A more complete and detailed account of specific projects can be found in the reports of individual group leaders in this SAR and on the website ( The Neefjes lab finished a data-based systems description of an essential process in the immune system: antigen presentation by MHC class II molecules. This has yielded a series of novel pathways controlling transcription and transport of these molecules. Huib Ovaa and colleagues have established a novel synthesis scheme allowing the production of unique ubiquitin pairs. His group also succeeded in reversing the digestion of peptides by the proteasome. In a collaborative effort of the P Borst, Jonkers, Linn and Wessels groups, led by Sven Rottenberg, the mouse model for Brca1-associated breast cancer was used to identify a new marker for chemotherapy response: low expression of the Xist gene correlated with good response to cisplatin and this marker was also found to predict long recurrence-free survival of a group of breast cancer patients treated with platinum-based chemotherapy. The Piet Borst group found a function for base J, a DNA base modification they discovered in 1993 in the nuclear DNA of some pathogenic protozoa. Deep sequencing revealed base J to be present at transcription termination sites. In the absence of J, RNA Polymerase II fails to halt and reads on. The resulting RNA chaos in the cell is probably responsible for the death of the parasite. The biosynthetic pathway of base J remains therefore an interesting pharmacological target to eradicate these parasites. In the Jonkers group, Peter Bouwman and Mark Pieterse found that inactivation of 53BP1 suppressed the proliferation arrest induced by BRCA1 loss and partially restored homologous recombination. Aberrant 53BP1 expression was also frequently found in human BRCAmutated and triple-negative breast cancers and associated with decreased metastasis-free survival. Interestingly, Janneke Jaspers and Sven Rottenberg found that Brca1- mutated mouse mammary tumours acquired resistance to PARP inhibitors through inactivation of 53BP1. Tanja van Harn and Floris Foijer (Te Riele group) found that loss of the retinoblastoma (Rb) proteins can cause genomic instability. Upon mitogen deprivation, Rbdefective cells progressed through S phase, but then arrested with high levels of DNA breaks and defective sister-chromatid cohesion. When arrested cells were restimulated to proliferate, the daughter cells were often aneuploid. Thus, genomic instability can ensue from defective cell cycle control in combination with (transiently) inappropriate culture conditions. Using a mouse model for small cell lung cancer, Joaquim Calbo, Erwin van Montfort and Natalie Proost of my own group found that the tumours are often composed of phenotypically different cells with either a neuroendocrine or a mesenchymal marker profile, a feature also noted in human SCLC cell lines. Crosstalk between these mesenchymal and neuroendocrine endowed the neuroendocrine cells with metastatic capacity, illustrating the potential relevance of tumor cell heterogeneity in dictating tumour properties. Chromatin composition is still poorly characterized. The Van Steensel group has generated genome-wide highresolution binding maps of 53 chromatin components in Drosophila cells. They demonstrated that the genome consists of five discernable chromatin types. One represents a new repressive chromatin type that covers about half of

14 14 introduction the genome and lacks classic heterochromatin markers whereas two represent transcriptionally active euchromatin. Their results provide an overarching view of chromatin diversity and domain organisation in metazoan cells. Oncogene-induced senescence is a p53-dependent defence mechanism against uncontrolled proliferation. Consequently, about 50% of human tumours harbour p53 mutations while others show a dysfunctional p53 pathway, frequently by unknown mechanisms. The group of Agami used functional genetic screens with RNA-interference libraries and identified the gene BRD7 as a tumour suppressor that is part of the p53 pathway. In human breast tumours harbouring wild-type p53, the BRD7 gene locus is frequently deleted and low BRD7 expression caused by promoter silencing was found in a subgroup of tumours. This knowledge can be used to develop novel cancer drugs. Autotaxin (ATX) or ecto-nucleotide pyrophosphatase/ phosphodiesterase-2 (ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. The role of autotaxin is extensively studied by the group of Wouter Moolenaar. ATX-LPA signalling has roles in various pathologies including tumour progression and inflammation. However, the molecular basis of substrate recognition, catalysis, and the mechanism of interaction with target cells, has remained elusive. The group of Tassos Perrakis has determined the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. They identified a hydrophobic lipid-binding pocket and mapped key residues required for catalysis and selection between nucleotide and phospholipid substrates. They showed that ATX interacts with cell-surface integrins via its N-terminal somatomedin- B-like domains, using an atypical mechanism. Their results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localised LPA signalling, and enable new approaches to target ATX with small-molecule therapeutics. The Begg group and colleagues from the division of Radiotherapy found that expression of the putative stem cell marker CD44 was strongly associated with local recurrence after radiotherapy for laryngeal cancer. The finding was made from genome-wide expression profiling (microarrays) of pre-treatment biopsies, and then validated on a separate clinical series using immunohistochemical staining for CD44. The predictive potential of this marker was independent of known clinical predictors. This finding is likely to lead to a better understanding of treatment failure and more individualised treatments for this type of head and neck tumour. The release of the 4D-CBCT guided radiotherapy software application now permits high dose - high precision lung cancer irradiation in other radiotherapy centres in the Table 2 Short-term citations and impact of scientific articles published by the NKI research staff Publication year Publications Citations* Citations/publication Impact** As from 1997 publication year of articles is the criterion, instead of Scientific Report-year listing. From 2008 the online publication data are used as criteria for the year of publication. * Citations in the two years after publication, excluding self-citations. Starting 1989 citation analysis has been carried out on line. This allows detection (and elimination) of all self-citations. Before 1989 this pruning was limited to first authors. ** The impact factor is the average number of citations per year of an article in a given journal. The total impact is the sum of the impact of all articles published that year.

15 15 introduction world. The in-house developed 3D-EPID dosimetric verification of radiation treatment software received a 3 rd ranking in the National Patient Safety Contest. QUALITY OF RESEARCH The quality of research can be monitored in several ways. Our scientific productivity as assessed by objective bibliometric parameters (citations and impact of scientific articles published by the NKI staff) has shown a steady increase over time (Table 2). It is satisfying to note that the Institute retains an internationally prominent position in cancer research. While a high citation score is gratifying, it is only one measure of scientific productivity. The quality of our work should also be gauged by the invitations of our staff to present at international scientific meetings, awards obtained by our staff and significant grants that were obtained. We score high on all these accounts. Sjaak Neefjes and Ton Schumacher both obtained a prestigious ERC grant. Flora van Leeuwen obtained a Queen Wilhelmina Research Award, Reuven Agami received a VICI grant, and VIDI grants were awarded to Sven Rottenberg and Fijs van Leeuwen. Lodewyk Wessels, Jos Jonkers and Bas van Steensel were successful in securing a 4.5 million grant to establish a Cancer System Biology Centre at the NKI. The quality of research of the groups in each division is evaluated approximately every 5 years by an external site visit team. In November 2010 the Division of Radiotherapy was evaluated. The reviewers were very positive about the Division, which they rated to the top in the world. HONOURS AND APPOINTMENTS The NKI-AVL cannot award university degrees. However, many of our staff members hold special part-time chairs at Dutch Universities. This allows them to award PhD degrees to graduate students receiving their training at The Netherlands Cancer Institute. In 2010, 32 staff members had professorships at one of the Dutch Universities. Theo Ruers was appointed professor of Oncology at the University of Twente, Bas van Steensel as Professor of Chromosome Biology at the Erasmus Medical Centre in Rotterdam, and Paul Baas as professor of Pulmonary Oncology at the Academic Medical Centre in Amsterdam. Jos Beijnen was elected as member of the Royal Netherlands Academy of Sciences and Ton Schumacher as member of the European Molecular Biology Organisation (EMBO). There were substantial changes in our clinical staff mostly due to retirements. Fortunately, we have been able to recruit a number of excellent successors. Fred van Leeuwen was appointed as tenured staff member in the Division of Gene Regulation. Wim van Blitterswijk and Wouter Moolenaar retired. However, Wouter Moolenaar will continue to run his group as an extraordinary staff member. Laura van t Veer left the Institute to take a prestigious position at the Cancer Center at UCSF. Maarten Fornerod left the institute to become staff member at the Erasmus MC, and Kenneth Gilhuijs moved to the UMC Utrecht. Staff of the NKI-AVL fulfilled numerous functions in national and international organisations, on boards of scientific journals, as members of study sections, and as organisers or co-organisers of scientific meetings, workshops and congresses. OUTLOOK AND ACKNOWLEDGEMENTS I am confident that the NKI-AVL will continue to flourish, even in the face of all the uncertainties that are associated with the current economic crisis. However, it is imperative that our core funding increases or, alternatively, that a full-costs reimbursement model is installed for external project grants. We are very successful in securing external grants but these grants provide almost no overhead thereby putting considerable strain on the core budget. The arrangements we hope to finalise with the Dutch Cancer Society will be critical for our capacity to stay in the forefront of international cancer research. More than ever breakthroughs will come from the close collaboration of basic and clinical investigators. A continuous flow of ideas in both directions is needed not only to bring new concepts and drugs to the clinic, but also to understand at the molecular level why new promising treatments often do not work or quickly result in resistance. In this regard, state of the art molecular typing of tumours using genomic and proteomic techniques combined with robotic screens to identify pathways responsible for drug resistance are critical for unravelling mechanisms of resistance. I firmly believe that breakthroughs in cancer diagnosis and treatment can be realised with approaches that are now broadly applied in the NKI. However, to make an impact in this area, we do need to conduct these studies at a sufficiently large scale. This requires additional investments in molecular pathology and therefore has high priority. With the drive and enthusiasm that is so characteristic of the individuals that shape our Institute, we should be able to convince funding agencies as well as individuals to support the NKI- AVL, an institution that is at the international forefront of cancer research. I want to end by thanking all our employees and those who continue to support us: The Dutch Cancer Society, the most reliable and significant sponsor of our research; the Ministry of Health, which provides a substantial core grant to the Institute and has provided the funds for the renovation of our research facilities; and, last but not least, the many individuals who provide us with financial, moral, and practical support. Only with their help can we continue to develop new ideas that can improve the perspectives of cancer patients. I hope you will be inspired by this report. Ton Berns Director of Research

16 16 education in oncology EDUCATION IN ONCOLOGY The Netherlands Cancer Institute offers a variety of opportunities for practical and theoretical training to (trainee) technicians, University Master students, PhD students and post-doctoral fellows. Research and clinical staff and their group members are involved in theoretical and practical training. Many staff members have joint appointments as professors at Dutch universities and an even larger number contribute to the regular curriculum at various universities. The research divisions attract students from universities throughout the country. The NKI has a formal affiliation with the Science faculty of the University of Amsterdam (UvA) and is committed to make a contribution to Master student teaching. The institute participates in the Oncology Graduate School Amsterdam, together with the medical faculties of the UvA and the Free University (VU), referred to as Academic Medical Center (AMC) and VU medical center (VUmc), respectively. All educational activities are supervised by the Teaching Committee, which consists of Jannie Borst (chair and dean Master students), Hein te Riele (general affairs), Roderick Beijersbergen (Master course), John Hilkens, John Collard (HLO students and publicity), Titia Sixma (dean PhD students) and Fons Balm (clinical teaching). Peter Peters functions as dean for the post-docs. MASTER STUDENTS The program in Experimental Oncology attracts Master students of all national universities, see Research/Career/Masters+students. Students generally have a background in (Medical) Biology, Health Sciences, Chemistry, Pharmacology, Medicine, or Psychology. The program offers combined practical and theoretical training in various aspects of experimental oncology. Practical training includes participation in ongoing research projects for a minimum of 4 months. In 2010, 41 Dutch university Master students and 2 students from abroad completed a placement of 5-9 months at the biomedical research divisions. The students came primarily from the Free University Amsterdam (VU) (19) and the University of Amsterdam (UvA) (10), but also from Utrecht University (5), Wageningen (4), Leiden (1), Nijmegen (1), and Groningen (1). The institute also provides practical training opportunities to trainee technicians, who stay for similar periods of time as the university students and like these, often make significant contributions to research progress of the PhD students and post-docs who supervise them. There is an increasing demand from Universities for placing Bachelor students for the three month internship that concludes their program and we have accommodated 3 of them this year. The core element of theoretical training is the course in Experimental Oncology, given twice yearly (Table 1). This course is compulsory for Master students who do an internship at the NKI, but in addition attracts many students from throughout the country. We routinely host about 40 students per course. It includes lectures and tutorials given by our highest level clinical and research professionals and is rated very highly in University evaluations. Table 1 - Course in Experimental Oncology Epidemiology F van Leeuwen, M Rookus Surgery E Rutgers Radiodiagnostics W Vogel Pathology D de Jong Molecular diagnostics* R Kerkhoven, S Linn Conventional pharmacotherapy S Rodenhuis Radiotherapy** M Verheij DNA damage response and apoptosis* A Begg, J Borst Tumor development* R Beijersbergen Genomic instability* H te Riele Signal transduction* W Moolenaar Cell cycle R Bernards Oncogene-tumor suppressor gene interactions D Peeper Cell division R Wolthuis Cell adhesion* E Roos Tumor microenvironment K de Visser Mouse models of cancer* J Jonkers Immunology and immunotherapy* T Schumacher, J Haanen Analysis of protein structure T Sixma Rational drug development A Huitema Drug delivery* A Schinkel * including tutorial ** including tour PHD STUDENTS The PhD students at the NKI-AVL participate in the Oncology graduate school Amsterdam (OOA) together with the oncology departments of the VUmc and the AMC. In 2010, the institute had ~170 PhD students registered with the OOA and 24 students defended a PhD thesis at a Dutch university. Students participate in research of their group and in interdepartmental work discussions. In addition, the students follow the OOA training program, which consists of courses (Table 2) and an annual retreat on the island Texel. Apart from courses on different topics in cancer research, the OOA offers a course on scientific English. Students with no prior background in cancer research can participate in the Experimental Oncology course. Part of the training of the NKI students are discussions with experts in the field of oncology. The Friday morning seminar speakers take their lunch with a delegation of the students. Twice a year a list of speakers is sent round and each graduate student participates several times per year in these opportunities to exchange views with experts in the field. The PhD student retreat focuses entirely on the research of the graduate students themselves. At this retreat, students are not only presenting their work in the form of a poster in the first year and presentations in subsequent years, but they are also in charge of chairing sessions and discussions. In recent years, they also participate in peer review, giving a prize for the best poster and best presentation. In this

17 17 education in oncology manner, the retreat provides training in presentation and interaction skills, but it also provides an overview of the research in the OOA at an early stage of the student s career. This provides a good opportunity for translational interaction and bottom-up research, allowing the graduate students themselves to contribute significantly to interaction between different research groups. In addition, senior graduate students can participate in a joint retreat with other cancer institutes in Europe. In 2010, this event was hosted by IFOM in Milan, with participants from among others British CrUK institutes, the Italian IFOM and the Spanish CNIO contributing to a program of scientific lectures and posters as well as an enthusiastic social session. This retreat gives students the opportunity to compare notes among excellent cancer institutes throughout Europe. The progress and work is monitored annually by a supervisory committee. This committee has independent members within and outside the division. The committee discusses progress with the supervisor and student separately and participates in a joint discussion of the research. After two years of PhD research the student, supervisor and committee evaluate the state of the project in a midterm review. At this more elaborate meeting the likelihood of achieving a PhD within a reasonable time frame is discussed. This meeting can be used to redefine goals if necessary. The students are represented in the OIO-council that meets with the Dean of graduate student affairs on a regular basis, as well as upon special request. They also mediate communication between the graduate students and research manager or board of directors. Table 2 - OOA graduate student courses March The Macroscopic and Pathologic Anatomy of the mouse WH Lamers, CJF van Noorden April English Writing and Presenting November Annual Graduate Student Retreat, Texel T Sixma In the Footsteps of Antoni van Leeuwenhoek, basic course P Peters, C van Noorden, K Jalink, E Reits et al. November English Writing and Presenting RETREAT parallel workshops, 9 keynote lectures of senior peers, 3 days away from the lab. These were the main ingredients for the retreat which took place from April in Kapellerput, Heeze, where postdocs and final year PhD students came together to reflect on their current position and their career perspectives. There are more determinants to success than a publication list alone. Each year, about 100 young scientists gather to reflect on their current position and to learn what opportunities there are. Love what you are doing, choose a position that suits your interests and skills best and to know how to sell yourself, were among the things they heard at the retreat. At the retreat, successful academics gave career advice: know how to get and use feedback from colleagues, how to establish collaborations, how to present yourself and to gain independence. Being aware of your transferable skills is part of the masterplan to success. Participants were also put to work during three parallel sessions of workshops, to start drafting their individual masterplan. The sessions focused on what the postdocs actually enjoy doing and on the way to achieve that goal: it is important that they know how to present themselves and convince potential employers, collaborators and peers of their qualities. In the workshops they learned the do s and don ts when writing an application letter, and to improve public speaking and presentations. Finally, participants improved a transferable skill of choice: non-verbal behaviour, grant writing, negotiation, project management or how to give/ask/receive feedback. On the final day, PCDI invited a few dozen ex-researchers to talk about their career choices and their current positions. Networking is another part of the masterplan to success, because the world of Life Sciences is not limited to academic research, it includes technology transfer, biotech companies, government and media, with a common goal: improved health. Under the leadership of Peter Peters the retreats have been attended by more then 1100 postdocs over the last 11 years. Our institute has landed again in the Scientist s list 2011 of top 10 best international places to work for postdocs. POST-DOCS Post-docs in our Institute perform challenging research in an internationally competitive environment. They participate in work discussions both within the group and among different divisions. Within the research group there are opportunities to receive training in supervision, manuscript writing and presentation. In many cases, our postdocs have a preference for working in science and indeed, many of our post-docs go on in academia. There are however many alternative options available for an interesting career after a successful post-doc period. The Postdoc Career Development Initiative (PDCI) was initiated based on the post-doc platform at the NKI-AVL. The activities can be seen at

18 18 biochemistry DIVISION OF BIOCHEMISTRY STRUCTURAL BIOLOGY Division head, group leader Titia Sixma Titia Sixma PhD Group leader Marcello Clerici PhD Post-doc Alex Fish PhD Post-doc Rick Hibbert PhD Post-doc Prakash Rucktooa PhD Post-doc Mariano Stornaiuolo PhD Post-doc Azusa Seto PhD Post-doc Alex Faesen MSc PhD student Mark Vargas MSc PhD student Francesca Mattiroli MSc PhD student Judith Smit MSc PhD student Flora Groothuizen MSc PhD student Danny Sahtoe MSc PhD student Pim van Dijk Technical staff Herrie Winterwerp Technical staff Development of cancer is generally due to errors that occur in cellular pathways. Understanding mechanisms of the underlying processes will help to determine where the errors occur and how they can be treated. We use X-ray crystallography as a tool to provide three-dimensional structures and we interpret the structural data using a variety of biochemical and biophysical techniques. These studies provide more insight in the molecular processes and they can also provide targets for drug design studies. In our group we focus mainly on proteins involved in ubiquitin conjugation in chromatin regulation, on DNA mismatch repair and nicotinic receptor signaling. DNA mismatch repair DNA mismatch repair plays a crucial role in ensuring genomic stability. Defects in the mismatch repair cascade in humans predispose to hereditary non-polyposis colorectal cancer and are associated with a variety of sporadic cancers. DNA mismatch repair is initiated by the protein MutS (in Escherichia coli) or its MSH homologs. MutS recognizes and binds to mismatches or unpaired bases that have escaped the proofreading capacity of the DNA replication machinery or are present in the genome during recombinatorial events between non-fully complementary DNA strands. We make use of variants of MutS that specifically maintain the dimer or tetramer state of these proteins in order to be able to provide quantitative data on the mismatch recognition cycle. In collaboration with the groups of Terence Strick (Paris), Peter Friedhoff (Giessen) and Joyce Lebbink (Rotterdam) this allows a precise analysis of the steps involved in mismatch recognition and the initiation of repair. These mutants allowed novel crystallographic analysis of the proteins with and without DNA, leading to new insight into the state of the protein prior to DNA binding. Our detailed analyses of the steps following mismatch recognition n are building up to a model that reveals eals the details of the complex ATPase cycle of the mismatch repair proteins. Publications Lebbink JH, Fish A, Reumer A, Natrajan G, Winterwerp HH, Sixma TK. Magnesium coordination controls the molecular switch function of DNA mismatch repair protein MutS. J Biol Chem. 2010;285: Shanmugham A, Fish A, Luna-Vargas MP, Faesen AC, El Oualid F, Sixma TK, Ovaa H. Nonhydrolyzable ubiquitinisopeptide isosteres as deubiquitinating enzyme probes. J Am Chem Soc. 2010;132: El Oualid F, Merkx R, Ekkebus R, Hameed DS, Smit JJ, de Jong A, Hilkmann H, Sixma TK and Ovaa H. Chemical Synthesis of Ubiquitin, Ubiquitin-based Probes and Diubiquitin Angewandte Chemie 2010;49: Figure 1: Novel crystal form of E.coli MutS in complex with 16-mer DNA containing an AA mismatch revealed that crystal packing did not affect protein-dna interactions, since all contacts were conserved between the two crystal forms (Lebbink et al, 2010)

19 19 biochemistry Ubiquitin and SUMO conjugation Ubiquitin conjugation is critical for signaling in almost all cellular processes, including DNA repair, apoptosis, cell cycle, chromatin regulation and endocytosis. Since these processes are important for cellular stability, deregulation of ubiquitin-dependent processes often leads to cancer. Structure analysis of the proteins involved in ubiquitin and SUMO conjugation could help the development of novel drugs inhibiting critical pathways in ubiquitin conjugation. The process of conjugation by ubiquitin-(like) proteins involves covalent linking of one or more 76-amino-acid ubiquitins to a target protein by an E1/E2/E3 cascade of enzymes. Correct ubiquitination requires the complex spatial arrangement of ubiquitin, E2, E3 proteins and the target simultaneously in a precise but flexible manner. Although the overall mechanism has been defined, the atomic details have been lacking and the specificity determining factors are unclear. We study several E2/E3 complexes involved in conjugation of ubiquitin and the related modifier SUMO as well as proteins involved in de-ubiquitination. Deubiquitinating enzymes are found in five classes, and there are many less than there are E2/E3 combinations. This indicates that regulation may well be taking place at a different level. In order to understand this we have undertaken a comparison of a number of ubiquitin specific proteases. In collaboration with Huib Ovaa we are making use of novel tools to study these enzymes. Thus we can incorporate the analysis of all diubiquitin pairs for this type of analysis. In terms of regulation we are studying the role of the ubiquitin-like Ubl domains in a number of DUB enzymes. Although these domains have a similar fold to ubiquitin, their sequence properties are very different. Sofar our studies have included the Ubl domain that is internal to the Usp4 catalytic domain and we also analyzed the role of the C-terminal domain of Usp7 and its activating properties for catalytic activity. In collaboration with Rolf Boelens in Utrecht we studied the chain formation activity of the Rad6 E2 enzyme and how this is modulated by the presence of Rad18. We studied the PCNA modification by Rad6/Rad18 and showed that Rad18 binding inhibits the chain forming activity of Rad6 (Hibbert et al, submitted for publication. Figure 2: Crystals of catalytic domain of Usp4 Nicotinic Acetylcholine receptor homolog AChBP The acetylcholine binding protein (AChBP) is a homolog of the extracellular domain of the nicotinic acetylcholine receptor, and a representative of the cys-loop receptor family. It remains the best model for atomic resolution structures of ligand binding to this pharmaceutically important family of ion channels. In collaboration with the group of Iwan de Esch (VU) novel interactors for AChBP have been identified, using a fragment growing approach, into a specific binding pocket (Edink et al, submitted for publication). Using a combination of different thermodynamic analyses and structural studies we were able to explain the behavior of the different ligands that were studied as part of this procedure. Interestingly the comparison of surface plasmon resonance and isothermal calorimetry indicated very similar results and provided an explanation for the observed variations in binding mode. Structural studies of a series of reference compounds including toxins and anti-smoking compounds provide insight in the details of subunit specificity of the nicotinic receptor homologs.

20 20 biochemistry STRUCTURAL BIOLOGY Group leader Anastassis Perrakis Anastassis Perrakis PhD Group leader Christophe Caillat PhD Post-doc Eleonora von Castelmur PhD Post-doc Krista Joosten PhD Post-doc Robbie Joosten PhD Post-doc Jens Haussman MSc PhD student Tatjana Heidebrecht Technical staff Figure 3: The HA155 inhibitor bound to ATX This year was marked by two breakthroughs on our major long-term in-house collaboration projects: determining the structure of Autotaxin (with Wouter Moolenaar, Division of Cell Biology I, and Huib Ovaa, Division of Cell Biology II) and of JBP1 (with Piet Borst, Division of Molecular Biology). Concurrently, our work on DNA replication licensing and on mitotic progression advanced and our adventures in X-ray crystallography model building and refinement are undermarked by a change of focus towards structural boinformatics. Structural studies of Autotaxin The role of the signaling phospholipid lysophosphatidic acid (LPA) and Autotaxin (ATX), the protein that produces LPA from lysophosphatidylcholine (LPC), was established over the last three decades, largely owing to the efforts of the group of Wouter Moolenaar at the NKI. LPA and ATX have been shown by numerous studies to be involved in cancer metastasis and other pathogenic situations, such as inflammation and neuropathic pain. ATX is the protein ecto-nucleotide phosphodiesterase 2 (ENPP2), a 100 kda glycoprotein, capable of both lysopld and nucleotide pyrophosphatase activities. Over the last few years we have engineered a human cell line (HEK293 derivative) that is stably expressing glycosylation-deficient mutants of rat autotaxin, which allowed us to crystallize ATX, and determine its structure this year. Autotaxin hydrolyzes different species of LPC (with different alkyl chain lengths) and some other lipids, as well as nucleotide substrates. Our structure shows that both nucleotides and lipids partially share the same binding pocket, but the alkylchain of lipid substrates form additional hydrophobic contacts with ATX. This model implies that the LPA product likely has higher affinity for ATX than any nucleotide substrate, a hypothesis consistent with the previous finding that LPA acts as an inhibitor of ATX activity against a variety of substrates, but not of LPC hydrolysis. Thus, LPA formed by the enzyme may act as a substrate specifying factor effectively inhibiting the hydrolysis of nucleotides because it can only be displaced by lysophospholipid substrates such as LPC. This mechanism could dictate ATX activity in vivo, because the spectrum of available LPA species could define ATX activity against specific lysophospholipid substrates. Our findings also suggest that further analysis of structural determinants of substrate discrimination could lead to the identification of molecules that inhibit the hydrolysis of specific substrates, e.g. long-chain rather than short-chain LPC species. The group of Huib Ovaa has developed a series of small-molecule boronic acid inhibitors of ATX-mediated LPC hydrolysis, that lower plasma LPA levels in mice following intravenous administration. We also determined a structure of ATX in complex with one of these inhibitors, HA155, which enables us to correlate the activity of this inhibitor with its binding mode. We show that the boron atom on one end of the inhibitor forms a reversible covalent bond with the nucleophile hydroxyl group of the active site threonine nucleophile. The other end of HA155, a hydrophobic fluoro-benzene, is pointing directly into the deep hydrophobic pocket, oriented perpendicular to the protein surface. Our results will allow us to provide feedback to inhibitor development studies, which are purused in-house in collaboration with CR-UK, but also by our collaborators in Pfizer and by Merck-Darmstadt. Importantly we have now formed the basis for understanding how autotaxin sequesters its lipid substrates and we formulated testable hypotheses for how ATX can specifically present the hydrolysis products to the cell-surface LPA receptors, to elicit a variety of important responses. This will allow us how Autotaxin is related to processes such as inflammation, neuropathic pain, obesity, and most importantly in cancer metastasis. Structural studies of JBP1 The JBP1 protein, discovered by Piet Borst and colleagues, binds to DNA that contains base J ( -D-glucosyl-hydroxymethyluracil). JBP1 has been shown to be essential for survival in many major protozoa human pathogens such as T. brucei (sleeping sickness), T. cruzi (Chagas disease) and Leishmania species (various types of Leishmaniasis). Last year, it has been shown that there exists a partial mammalian homologue of JBP1, which is localized in a region of the TET proteins that are involved in myeloid leukemia and