THE NETHERLANDS CANCER INSTITUTE SCIENTIFIC ANNUAL REPORT 2008

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1 THE NETHERLANDS CANCER INSTITUTE SCIENTIFIC ANNUAL REPORT 2008 p26.1 p25.3 p24.3 p24.2 p24.1 p22.3 p22.1 p21.31 p14.3 p14.2 p14.1 p13 p12.3 p12.2 p12.1 q11.2 q13.11 q13.13 q13.31 q21.3 q22.1 q23 q24 q26.1 q26.2 q26.31 q26.32 p26.33 p28 p29 p15.33 p15.31 p15.2 p15.1 p14.3 p14.1 p13.3 p13.2 p13.1 p12 q11.2 q12.1 q12.3 q13.2 q13.3 q14.1 q14.3 q15 q21.1 q21.3 q23.1 q23.2 q23.3 q31.1 q31.3 q32 q33.1 q33.2 q33.3 q34 q35.1 q35.2 q35.3

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3 SCIENTIFIC ANNUAL REPORT

4 4 Patron HM The Queen Beatrix

5 5 SCIENTIFIC ANNUAL REPORT 2008 THE NETHERLANDS CANCER INSTITUTE CANCER RESEARCH LABORATORY AND CANCER HOSPITAL

6 6 COPYRIGHT Scientific Annual Report 2008 Illustrations and unpublished data in these reports may not be used without permission of the author.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 COLOFON Coordinators Suzanne Corsetto Henri Van Luenen Photograph HM The Queen Beatrix Enquiry s/permission: Rijksvoorlichtingdienst, 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 Plesmanlaan CX Amsterdam The Netherlands Printed by Thieme Amsterdam

7 7 CONTENTS Board Members 8 Research Divisions 9 Introduction 11 Education in Oncology 16 Division of Biochemistry 18 Division of Cell Biology I 22 Division of Cell Biology II 31 Division of Experimental Therapy 38 Division of Gene Regulation 46 Division of Division of Immunology 53 Division of Molecular Biology 62 Division of Molecular Carcinogenesis 75 Division of Molecular Genetics 79 Division of Psychosocial Research and Epidemiology 89 Division of Diagnostic Oncology 100 Division of Medical Oncology 116 Division of Radiotherapy 126 Division of Surgical Oncology 143 Biometrics Department 156 Clinical Trials 161 Invited Speakers 177 Projects 180 Personnel Index 193

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9 9 BOARD MEMBERS President of Board of Governors W Kok 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 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 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 FH Schröder D Sinninghe Damsté MC Smeets PC Van der Vliet GP Vooijs SCIENTIFIC ADVISORY COUNCIL AJM Berns, chairman JJ Neefjes, secretary S Rodenhuis T Sixma M Verheij J Borst

10 10 RESEARCH DIVISIONS RESEARCH DIVISIONS Biochemistry Titia Sixma (head) Anastassis Perrakis Franciska Manuputty (office manager) Cell Biology I Arnoud Sonnenberg (head) Wim van Blitterswijk John Collard Kees Jalink Wouter Moolenaar Ed Roos Cell Biology II Jacques Neefjes (head) Rob Michalides Huib Ovaa Peter Peters Marieke van der Velde (office manager) Experimental Therapy Laura van t Veer Adrian Begg Jan Schellens Fiona Stewart Thea Eggenhuizen (office manager) Gene Regulation Reuven Agami 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 Rob Wolthuis Tom de Knegt (office manager) Linda Römer (secretary) Molecular Carcinogenesis René Bernards (head) Roderick Beijersbergen Franciska Manuputty (office manager) Molecular Genetics Maarten van Lohuizen (head) Anton Berns Jan-Hermen Dannenberg Jacqueline Jacobs Anna Haramis John Hilkens Paul Krimpenfort Daniel Peeper Margriet Snoek Erica Delwel (office manager) Marie Anne van Halem (secretary) Psychosocial Research and Epidemiology Neil Aaronson (head) Eveline Bleiker Wim van Harten Flora van Leeuwen Sanne Schagen 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 Efraim Rosenberg Michiel Sinaasappel Ferida Sivro Jelle Teertstra Renato Valdes Olmos Hester van Boven Fijs van Leeuwen Olaf van Tellingen Loes van Velthuysen Mark van de Vijver Senno Verhoef Wouter Vogel Jelle Wesseling Christine Arkes (secretary) Carla van Tiggelen (secretary)

11 11 RESEARCH DIVISIONS Medical Oncology Sjoerd Rodenhuis (head) Joke Baars Paul Baas Jos Beijnen André Bergman Christian Blank Willem Boogerd Henk Boot Dieta Brandsma Sjaak Burgers Annemieke Cats Jan Paul de Boer Leo Gualtherie van Weezel John Haanen Alwin Huitema Martijn Kerst Sabine Linn Anne Lukas Jan Schellens Jan Schornagel Marianne Smits Gabe Sonke Babs Taal Wim ten Bokkel Huinink Margot Tesselaar Michiel van den Heuvel Marchien van der Weide Nico van Zandwijk Mariëlle de Kwant (secretary) Radiotherapy Marcel Verheij (head) Berthe Aleman Harry Bartelink José Belderbos Eugene Damen Roel de Boer Luc Dewit Paula Elkhuizen Rick Haas Olga Hamming-Vrieze Guus Hart Wilma Heemsbergen Frank Hoebers Edwin Jansen Han Krewinkel Joos Lebesque Corrie Marijnen Harry Masselink Ben Mijnheer Luc Moonen Floris Pos Coen Rasch Babs Reichgelt Peter Remeijer Nicola Russell Govert Salverda Christoph Schneider Joep Stroom Bart van Bunningen Marcel van Herk Baukelien van Triest Corine van Vliet Marieke van Zwienen Frits Wittkämper Patricia Haye-Fewer (management assistant) Surgical Oncology Theo Ruers (head) Marc van Beurden Michiel van den Brekel Alfons Balm Annemieke Ackerstaff Axel Bex Biljana Zupan-Kajcovski Christiaan Keijzer Dirk Buitelaar Emiel Rutgers Frans Hilgers Frits van Coevorden Henk van de Poel Hester Oldenburg Houke Klomp Bing Tan Ingeborg Vergouwe Inka Nieuweboer-Krobotova Joris Hage Johanna van Sandick Julia ten Cate Katina Efthymiou Leonie Woerdeman Lotti Lubsen Brandsma Ludi Smeele Marc van Beurden Marianne Piek-den Hartog Marie-Jeanne Baas Vrancken Peeters Marieke van der Berg Martine van Huizum May Ronday Michael Srámek, Michel Wouters Michiel van den Brekel Omgo Nieweg Peter Schutte Peter Lohuis Sanne Vos Simon Horenblas Tino Stoppa Vic Verwaal Wietze van der Veen Willemien van Driel Wim Meinhardt Annemieke Hoogland (office manager) Biometrics Department Otilia Dalesio Financial Administration Didier Dohmen 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 Director of Research Ton Berns 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 overview of our activities with illustrations can be found in our brochure (available on our website) The NKI-AVL is a Comprehensive Cancer Center, 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 pharmacy, epidemiology and psychosocial oncology. The laboratory covers all major areas of cancer research, with special emphasis on cellbased screens, mouse tumor models, cell biology, structural biology, and immunology. Translational research is an integral activity of many research groups and is fostered by collaboration between clinical and basic scientists. This scientific report deals mainly with the basic and clinical science in the NKI-AVL. Information on patient-care is described in our General Annual Report. In 2008 most of our research groups relocated to the renovated research labs that were constructed in the former hospital building. We have taken this opportunity to restructure the divisions in order to enhance interactions between the research groups. As a result, two new divisions were formed while two existing divisions were combined. The transition to the new labs went very smoothly thanks to the excellent preparation and organization of the whole process. Our clinical activities again exhibited growth, although not as much as expected due to the space limitations in the outpatient clinics and the operating rooms. In 2009, we will add an extra operating room specially equipped for minimally invasive surgery. We also foresee additional, temporary facilities to relieve the pressure on our outpatient facility. Filling current vacancies for nursing staff, however, is at present our most serious concern. Expansion of our radiotherapy department is planned to take place through the establishment of two off-site satellites at clinical centers elsewhere. In addition, we are planning, together with the Erasmus University, the University Medical Center Leiden and the Delft Technical University, a proton/heavy ion radiation center for treating specific groups of patients that will benefit from the more targeted delivery of radiation. The transition to a new reimbursement system in the health care system is progressing, and this enables us to further expand our clinical activities. However, we foresee harsher financial challenges due to the current economic crisis. This is a more general problem we are facing, although we do not expect major setbacks in the coming year. We concluded 2008 with a small profit for the hospital. Also our research expenditure remained within budget in We expect to balance the budget for both the hospital and the research in 2009 as well, despite steep increases in some expenses beyond our control. In 2008 our PhD students organized, together with Cancer Research UK, an international scientific meeting for PhD students only. The meeting was held at the NKI and was much appreciated by the participants. The International Scientific Advisory Board spent two full days at the Institute to critically evaluate our research portfolio. This was also meant as a rehearsal for the site visit evaluation that will take place in the Fall of 2009 in which an international site visit team appointed by the Royal Netherlands Academy of Arts and Sciences will evaluate the Cancer Institute on behalf of the Dutch Cancer Society and the Ministry of Health, the organizations that provide the core funding for The Netherlands Cancer Institute. Our International Scientific Advisory Board was very positive about the quality of our research program, but it also gave valuable advice with regard to policies for recruiting new staff members, coaching young group leaders, and fostering translational research. Many of their suggestions have already been implemented in the course of Table 1 Core research funding NKI-AVL from the Cancer Society and the Ministry of Health in 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 this latter income to the total budget has been steadily increasing over the years and represents roughly 60% of the total budget in 2008.

13 13 INTRODUCTION The staff retreat that will be held in January 2009 will focus on how to further enhance the synergy between the preclinical and clinical research within the NKI-AVL. In 2008, we continued to expand collaborative efforts and signed an agreement with the Technical University Twente to work together in developing minimally invasive surgical techniques. We also concluded an agreement with Astra Zeneca to serve as one of their preferred academic partners both with regard to preclinical and clinical research. We signed an agreement with the Karolinska Institutet in Stockholm and the Institut Gustave Roussy in Paris to collaborate more closely on innovative clinical trials and exchanging expertise in specific diagnostic areas. This collaborative effort between three European cancer centers will give access to powerful technological platforms of genomics/ proteomics assays. These are important to refine diagnosis and measure responses to therapy, thereby assisting in defining more effective combination therapies. These initiatives should help us to advance personalized medicine. We are actively involved in establishing a broader alliance of European Comprehensive Cancer Centers of Excellence, which can also serve as a more influential companion of the pharmaceutical industry. Highlights Our institute continues to have a strong scientific output; 2008 was again a productive year. It is always difficult to estimate the impact of research when the results are still fresh. The research highlights summarized 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 ( Nuno Rocha, Rik van der Kant and Coenraad Kuijl from the Neefjes lab (Division of Cell Biology) performed an in-depth cell biological study explaining the contribution of cholesterol to lysosomal behavior as detected in various genetic diseases like Gaucher and Niemann Pick disease. They showed that a unique cholesterol sensor on lysosomes attracts a protein from the ER to remove a motor protein on late endosomes. This is the first study showing how different intracellular compartments communicate to control each other s behavior. Eva Wielders in the group of Hein te Riele (Division of Molecular Biology) has used a novel technique, oligonucleotide-directed gene modification, to recreate allelic variants of the central mismatch repair gene MSH2 in mouse embryonic stem cells. By studying the phenotype of mutant ES cells she could unambiguously classify two MSH2 variants of uncertain significance as innocent polymorphisms and a third one as a deleterious mutation causative of hereditary non-polyposis colorectal cancer. This work will help medical geneticists in the counseling of (suspected) HNPCC families. Breast cancers that are deficient in homologous recombination are exquisitely sensitive to bifunctional alkylating agents, such as were given in the high-dose chemotherapy programs in the late 20 th century. Marieke Vollebergh in the group of Sabine Linn (Divisons of Medical Oncology and Molecular Biology), in collaboration with Petra Nederlof and Jelle Wesseling (Division of Diagnostic Oncology), has shown that sporadic tumors more likely respond to such regimens and can be recognized by either establishing a BRCA1 signature in an array-cgh test or by finding an expanding growth pattern at microscopic examination of a resection specimen. The group of Bas van Steensel (Division of Gene Regulation) mapped some 1,300 large domains in the genome that associate with Lamin (LADs). These LADs represent a repressive chromatin environment, demonstrating that the human genome is divided into large, discrete domains that are units of chromosome organization within the nucleus. The group of Fred van Leeuwen (Division of Gene Regulation) developed a novel assay in yeast to determine protein turnover in vivo and applied it to histones. They found that histones throughout the genome are subject to extensive replication-independent exchange, demonstrating that histones and their posttranslational marks are not permanent residents in chromatin. Daniel Peeper s group (Division of Molecular Genetics) has previously found that cells can respond to the activation of oncogenes by entering a long-term proliferative arrest, known as Oncogene-Induced Senescence (OIS). As such, OIS serves as a potent mechanism protecting us against cancer. Recently, they uncovered an unanticipated role for secreted proteins, including interleukins, in this process. Interleukins are known for their important role in the immune system, but Peeper s group found that they are also indispensable for OIS. These secreted factors, collectively called the Senescence-Messaging Secretome (SMS), allow for communication ( SMS-ing ) between cells under stress (like senescent cells that carry an activated oncogene) and their environment. Alexandra Pietersen in the lab of Van Lohuizen (Division of Molecular Genetics) described an important role for the Polycomb gene Bmi1 in the regulation of differentiation and proliferation of mammary epithelial stem cells and progenitors Anthony Uren and Jaap Kool (Berns and Van Lohuizen labs, Division of Molecular Genetics) in collaboration with the groups of Lodewyk Wessels (Division of Molecular Biology) and David Adams (Sanger Centre, UK), highlighted the power of high throughput insertional mutagenesis screens as a cancer gene network discovery platform in cancer-predisposed p19arf and p53 deficient mice. The group of Laura van t Veer (Division of Experimental Therapy) found that primary colorectal tumors that develop liver metastasis have a unique chromosomal signature that might be suitable to identify patients at risk to develop liver metastasis. The observed genomic aberrations also provide an inroad to understand the underlying mechanism and may lead to more individualized disease management.

14 14 INTRODUCTION The group of Jan Schellens (Division of Medical Oncology) has documented a promising anticancer activity of the combination of the PARP inhibitor AZD2281 alone and in combination with carboplatin in early clinical studies. PARP inhibitors selectively target tumors that are defective in DNA repair by homologous recombination, such as breast cancer in BRCA1 or BRCA2 carriers. Adrian Begg and coworkers (Division of Experimental Therapy) tested the predictive potential of signatures known or suspected to affect response to radiotherapy. The putative stem cell marker CD44 was the most significant predictor of outcome (local control), with an acute hypoxia signature showing a strong trend. CD44 also emerged as the most significant gene in a datadriven approach searching for any set of genes distinguishing cures from recurrences. 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 since the beginning of the 1980 s (Table 2). It is satisfying to note that the Institute is retaining an internationally prominent position in cancer research, despite its limited budget that is relatively modest in comparison with many comparable institutes abroad. 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 prestigious scientific meetings, awards obtained by our staff. We score high on all these accounts. We are also successful in securing grant support, e.g. Sjoerd Rodenhuis and colleagues successfully competed for a large grant together with industry from the Center for Translational Molecular Medicine (CTMM) aimed at identifying new diagnostic markers for therapy response. Several groups in the Institute also participate with university groups in a CTMM program on lung cancer. Bas van Steensel received a VICI grant from NWO, and VIDI awards were given to Karin de Visser and Elisabetta Citterio. I myself Table 2 Short-term citations and impact of scientific articles published by the NKI research staff Publication year Publications Citations* Citations/publication Impact** *** **** # * Citations in the two years after publication, excluding self-citations. Starting 1989 citation analysis has been carried out online. 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. *** As from 1997 publication year of articles is the criterion, instead of Scientific Report-year listing. **** Self-citations not excluded for publications 2001 and later. # From 2008 the online publication date is used as criteria for the year of publication.

15 15 INTRODUCTION received a program award from the Dutch Cancer Society. The quality of research of the groups in each division is evaluated approximately every 5 years by an external site visit team. Because of the reshuffling in 2008 of research groups as a result of the relocation to the new research building and the fact that our International Scientific Advisory Board conducted an overall evaluation of the research in 2008, no site visit of individual research groups was held in Honors 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 2008, 27 staff members had professorships at one of the Dutch Universities. John Haanen was appointed as Professor of Translational Immunotherapy of Cancer at the Leiden University, and Daniel Peeper as professor of Functional Oncogenomics at the Free University in Amsterdam. Harry Bartelink received a doctorate honoris causa from the Academia of Medicine in Gdansk, Poland. Titia Sixma became member of the Royal Netherlands (Dutch) Academy of Arts and Sciences. Bas van Steensel and Daniel Peeper were elected as EMBO members. The AvL-prize 2008 for young, promising NKI-scientists was awarded to Wilbert Zwart for his work on tamoxifen resistance mechanisms. There were substantial changes in our clinical staff mostly due by retirements. Harry Masselink, Joos Lebesque and Bart van Bunningen retired as radiation oncologists after serving the Institute for periods ranging from 27 to 37 years. Corrie Marijnen left the Radiotherapy Department to become the new chair of the subsection Radiation Oncology at the Leiden University Medical Center. Jan Schornagel, Wim ten Bokkel Huinink, Nico van Zandwijk retired as medical oncologists and Jaap van der Sande as a neurooncologist. Fortunately, we have been able to recruit excellent successors. Sanne Schagen and Eveline Bleiker were appointed as tenured staff members in the Division of Psychosocial and Epidemiological Research, and Maarten Fornerod and Jos Jonkers became tenured staff members in the Division of Gene Regulation and Molecular Biology, respectively. Staff of the NKI-AVL fulfilled numerous functions in national and international organizations, on boards of scientific journals, as members of study sections, and as organizers or co-organizers of scientific meetings, workshops and congresses. Outlook and acknowledgements We are confident that the NKI-AVL will continue to flourish, even in the face of a worldwide economic crisis which may force us to be even more frugal than we already are. Collectively, we have the scientific skills and expertise, as well as the drive and motivation to effectively compete for resources to support our activities. In the institute-wide evaluation that will take place later this year, we aim to convince the site visit team that the NKI is a unique institution that should be given full support by the Dutch Cancer Society and the Ministry of Health that provide the core funding for the Institute. Thus far, we have been very successful in securing support through a variety of funding mechanisms, and with the on-going initiatives to combine forces with other institutions and pharmaceutical industries within and outside the Netherlands we are in a good position to raise additional funds for our research. But we also need to access new sources to fund our research. The Antoni van Leeuwenhoek Research Fund that we founded together with the Dutch Cancer Society is one such endeavour. This fund can also help us to further increase our visibility to industry and private sponsors as an organization worth investing in. Real breakthroughs are more likely to come from of the combination of basic understanding of the underlying mechanisms and stateof-the-art implementation, and offer the best perspectives to substantially improve cure rates and long-term survival rates of cancer patients. This is an area in which the NKI has a strong record. I am convinced that breakthroughs in cancer diagnosis and treatment can be realized with approaches that are now broadly applied in the NKI. Our scientific output has been very robust over the years, indicating that the organization is stable and capable of making substantial contributions to reduce significantly the burden of cancer. This, together with the drive and enthusiasm that is so characteristic of the individuals that shape our institute, should convince donors that the NKI-AVL is an institution at the international forefront of cancer research and therefore worth supporting. In closing, I want to end by thanking 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 a new hospital and 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 are inspired by this report. Ton Berns Director of Research

16 16 EDUCATION IN ONCOLOGY EDUCATION IN ONCOLOGY In the Netherlands Cancer Institute, student training takes place at various levels, from Master, PhD to postdoctoral level and involves medical workers, technical personnel and scientists. Research and clinical staff as well as their group members are involved in theoretical and practical training. A number of staff members have joint appointments as professors at Dutch universities and also contribute to the regular curriculum at these 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 outside the education committee as dean for the post-docs. MASTER STUDENTS The program in Experimental Oncology attracts Master students of all national universities. 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 four months. In 2008, 31 Dutch university students completed a placement of 5-12 months at the biomedical research divisions. As in previous years, the students came primarily from the Amsterdam Universities UvA (7) and VU (9), as well as Utrecht University (7), but also from Nijmegen (4), Wageningen (2), Leiden (1) and Delft (1). The new Life Science and Technology Masters is particularly suited to the biomedical research environment in the NKI. 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. The core element of theoretical training is the course in Experimental Oncology, given twice yearly (table 1). This course is compulsory for NKI Master students who do an internship, but in addition attracts many as 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. 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 2008, the institute had 144 PhD students registered with the OOA. Of these, 22 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 participate in 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. Also, students with no prior background in cancer research can participate in the Experimental Oncology course. 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 year, 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 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 Table 1 Courses in Experimental Oncology Epidemiology Surgery Radiodiagnostics Pathology* Molecular diagnostics** Drug therapy Radiotherapy* DNA damage response and apoptosis** Signal transduction** Cell cycle** Cell Division Cell senescence and genomic instability** Cell adhesion** Tumor microenvironment Mouse models of cancer** Immunology and immunotherapy** Analysis of protein structure Rational drug development and drug delivery** * including tour ** including tutorial Flora van Leeuwen Emiel Rutgers Renato Valdes Olmos, Michiel Sinaasappel Daphne de Jong Ron Kerkhoven, Sabine Linn Sjoerd Rodenhuis Marcel Verheij Adrian Begg, Jannie Borst Wouter Moolenaar René Bernards, Daniel Peeper Rob Wolthuis Roderick Beijersbergen, Hein te Riele Ed Roos Karin de Visser Jos Jonkers Ton Schumacher, John Haanen Titia Sixma Jos Beijnen, Alfred Schinkel

17 17 EDUCATION IN ONCOLOGY 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 Ann Bless April Epigenetics R Steenbergen, T vd Elsen, J Kooter May Signalling Wouter Moolenaar and Arnoud Sonnenberg Spring Stem cells and Cancer Jan Paul Medema October Annual Graduate Student Retreat October November Titia Sixma Inflammation and Cancer Ed Roos en Karin de Visser English Writing and Presenting Ann Bless 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 2008, this event was hosted by the NKI and the excellent organization was performed by four NKI graduate students (Floor Frederiks, Bernike Kalverda, Jasper Mullenders and Daan Peric Hupkes). Participants from British CrUK institutes, the Italian IFOM and the Spanish CNIO contributed 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. Each student has a supervisory committee that meets once a year to monitor scientific progress. The committee discusses progress with the supervisor and student separately and participates in a joint discussion of the research. The students are represented in the OIOcouncil that meets with the Dean of graduate student affairs on a regular basis, as well as upon special request. about all the possibilities for the next step in your career. This year we were very happy to be able to receive a grant from the ministry of economic affairs to professionalize our ten year NKI-AVL history of postdoc activities in order to create an organization which we called PCDI for Postdoc Career Development Initiative with a nationwide activity. We have not only offered new perspectives, but also helped improving skills that will increase career choices, both within and outside academia. The activities we have offered can be visited at The PCDI organized two postdoc retreats at Kapellerput, which were held from 9-11th of April and from 5-7th of November Each time we reached hundred participants and were overbooked. Thanks to the organizing committees we had great inspirational speakers, among which Bruce Alberts, Hans Clevers, Peter Friedl, Bart Noordam but also young successful scientists, business managers and entrepreneurs. The workshops on transferable skills were well received, and we look back at stimulating networking days with enthusiastic ex-postdocs and other representatives working in academia, companies and non-profit organizations. The retreats scored in an anonymous evaluation between very good and excellent, which will hopefully inspire the participating postdocs and last year PhD students to make the right choices with regard to their career. POST-DOCS Post-docs in the Netherlands Cancer 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 often opportunities to receive training in supervision, manuscript writing and presentation. In many cases, postdocs in the biological sciences, medicine and technology have a preference for working in an institute or university and indeed, many of our post-docs go on to careers in science. Still, there are many other options available for an interesting career after a successful postdoc period. Many postdocs are unaware of these possibilities, or they may consider it too great a step to go into biotech, industry, journalism or a government position. Since only 20% of postdocs are likely to get a permanent position in academia, it can be of interest to have a good look around during your postdoc period and to find out

18 18 BIOCHEMISTRY DIVISION OF BIOCHEMISTRY STRUCTURAL BIOLOGY Division head, group leader Titia Sixma Titia Sixma PhD Group leader Alex Fish PhD Post-doc Rick Hibbert PhD Post-doc Prakash Rucktooa PhD Post-doc Andrea Alfieri PhD Post-doc Alex Faesen MSc PhD student Mark Vargas MSc PhD student Annet Reumer MSc PhD student Francesca Mattiroli MSc PhD student Judith Smit 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. Structural biology can help to understand these errors at the atomic level, by studying the molecules involved. We use X-ray crystallography as a tool to provide threedimensional 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 in eukaryotes. 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. Mismatch binding triggers the uptake of ATP in the MutS nucleotide-binding domain and it is this mismatchdependent ATP state that authorizes repair by recruitment of additional mismatch repair components. In collaboration with Robert van den Heuvel in Utrecht we have used native mass spectrometry to study MutS DNA complexes. In a collaboration with Serge Cohen and Tassos Perrakis a novel method was developed that will allow us to analyze the detailed nucleotide status of these large complexes. In collaboration with Peter Friedhoff and Martin Marinus we studied a dimeric mutant of the MutS protein. This mutant allows detailed kinetic studies and structure determination of the full-length protein, alone and in complexes. Analysis of these data is beginning to yield interesting insight into the mechanism of mismatch discrimination (figure 1). Figure 1: Surface plasmon resonance analysis of DNA mismatch repair protein MutS biniding to mismatched DNA. Use of a dimer mutant allows kinetic fitting of DNA binding. Interestingly, it is not the search process, but the off-rate that provide discrimination between mismatch and homoduplex DNA. 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 of so 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 contribute to the development of novel drugs wich are directed towards critical

19 19 BIOCHEMISTRY pathways in ubiquitin and SUMO conjugation. The process of conjugation by ubiquitin-(like) proteins involves covalent linking of one or more of the 76-aminoacids within ubiquitin 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, many questions on selectivity and specificity remain. We study distinct E2/E3 complexes involved in conjugation of ubiquitin and the related modifier SUMO as well as proteins involved in de-ubiquitination. One interesting aspect of SUMO compared to ubiquitin is its specificity for certain sequences. In collaboration with Klaus Schwamborn at Pepscan a chip was developed that allows studying this specificity in an efficient manner (Schwamborn et al, 2008). The E2 ubiquitin conjugating enzyme for sumoylation, Ubc9, can itself be modified by SUMO (figure 2). Publications Bertaccini EJ, Lindahl E, Sixma T, Trudell JR. Effect of cobratoxin binding on the normal mode vibration within acetylcholine binding protein. J Chem Inf Model. 2008;48: Cohen SX, Ben Jelloul M, Long F, Vagin A, Knipscheer P, Lebbink J, et al. ARP/wARP and molecular replacement: the next generation. Acta Crystallogr D Biol Crystallogr. 2008;64:49-60 Crosetto N, Bienko M, Hibbert RG, Perica T, Ambrogio C, Kensche T, et al. Human WRNIP1 is localized in replication factories in a UBZ-dependent manner. J Biol Chem Figure 2: The activity for the E2 for sumoylation, Ubc9, can be modulated by SUMO itself in two different manners: Right: Binding of SUMO to Ubc9 in a non-covalent interaction, the so-called back-side binding, affects SUMO chain formation (Knipscheer et al, EMBO J. 2007). Left: The covalent SUMO modification of mammalian Ubc9 on K14 affects the target choice, increasing the rate of modification of the SIM containing target Sp100, but limits its activity against RanGAP (Knipscheer et al, Mol Cell 2008) In collaboration with Andrea Pichlers group in Vienna, we showed that this modification is important for target choice of the E2 enzyme (Knipscheer et al, 2008). It can enhance modification of the Sp100 transcription factor and we showed that this enhanced modification is due to binding of the covalently bound SUMO to the target via a SUMO Interaction Motif (SIM). Surprisingly the site of SUMO modification on Ubc9 is equivalent to the site of SUMO modification of E2-25K, an E2 enzyme for ubiquitin, but the consequence of the latter modification is radically different (Pichler et al, 2005). Our crystal structure suggested that this difference could be due to intramolecular interaction of the attached SUMO with a Ubc9- specific beta-hairipin and indeed, specific removal of the hairpin, by replacing it with a di-glycine sequence causes a shift in the effect of E2 modification to resemble that in E2-25K (Knipscheer et al, 2008). DNA translesion synthesis (TLS) allows for tolerance to DNA damage and synthesis beyond the mismatch during replication and avoids replication stallimg. This process is initiated by ubiquitin modification of PCNA by the ubiquitin conjugating E2/E3 enzyme complex Rad6/Rad18. In our studies of the Rad6/Rad18 activity we used thermodynamic analysis of the DNA binding properties. Studying the ZnF domain we were able to compare the binding properties of ubiquitin and short chains of ubiquitin (Crosetto et al, 2008). Knipscheer P, Flotho A, Klug H, Olsen JV, van Dijk WJ, Fish A, et al. Ubc9 sumoylation regulates SUMO target discrimination. Mol Cell. 2008;31: Schwamborn K, Knipscheer P, van Dijk E, van Dijk WJ, Sixma TK, Meloen RH, et al. SUMO assay with peptide arrays on solid support: insights into SUMO target sites. J Biochem. 2008;144:39-49 van Leuken RJ, Luna-Vargas MP, Sixma TK, Wolthuis RM, Medema RH. Usp39 is essential for mitotic spindle checkpoint integrity and controls mrna-levels of aurora B. Cell Cycle. 2008;7: Nicotinic Acetylcholine receptor homolog AchBP In collaboration with the groups of Guus Smit and Iwan de Esch at the Free University in Amsterdam we study the Acetylcholine Binding Proteins (AChBP) as tools for understanding the ligand binding in pentameric ligand-gated ion-channels. AChBP has strong sequence similarity to the a-subunits of the nicotinic acetylcholine receptor ligand-binding domain and our AChBP crystal structures are the established high-resolution model for the ligand-binding domains in this class of ion channels. Based on in silico screening we found a number of novel molecules that bind AChBP. Structural analysis showed that binding was indeed present, but with considerable variability in the binding. The analysis of these compounds on nachrs indicate that these compounds compete with bungarotoxin, implying binding to the ligand binding site. In addition, however, they seem to bind to the transmembrane pore, precluding further development as lead compounds. Further studies focus on additional hits from the in silico screen.

20 20 BIOCHEMISTRY BIOCHEMISTRY STRUCTURAL BIOLOGY Group leader Anastassis Perrakis Anastassis Perrakis PhD group leader Patrick Celie PhD Post-doc Serge Cohen PhD Post-doc Valeria De Marco PhD Post-doc Dene Littler PhD Post-doc Krista Joosten PhD Post-doc Wijnand Mooij PhD Post-doc Eirini Mitsiki PhD Post-doc Jens Hausmann MSc PhD student Evangelos Christodoulou Technical staff Tatjana Heidebrecht Technical staff Diederick De Vries Technical staff Publications van de Weerdt BC, Littler DR, Klompmaker R, Huseinovic A, Fish A, Perrakis A, et al. Polo-box domains confer target specificity to the Polo-like kinase family. Biochim Biophys Acta Jun;1783(6): Repanas K, Fuentes G, Cohen SX, Bonvin AM, Perrakis A. Insights into the DNA cleavage mechanism of human LINE-1 retrotransposon endonuclease. Proteins Perrakis A, Romier C. Assembly of protein complexes by coexpression in prokaryotic and eukaryotic hosts: an overview. Methods Mol Biol. 2008;426: This year our biology focus remained around mechanisms for correct DNA inheritance: DNA replication and the subsequent chromatide separation between the daughter cells. This interest is demonstrated by our work in the DNA replication license and in spindle assembly checkpoint proteins. Our involvement on three in-house collaborations remained active, trying to determine structures that are of importance to in-house research: we work with Wouter Moolenaar on ATX, with Piet Borst on JBP1 and with Ton Schumacher on MHC class I structures. Work in computation methods went on, with many steps taken towards new algorithms for crystallographic model completion and consolidating the control structures of the ARP/wARP software. A new side product of our computation effort was a tool to facilitate the design of truncated proteins suitable for protein expression experiments. DNA replication licensing and the role of the Cdt1/Geminin complex This year we finalized our work on the function of the Geminin: Cdt1 complex, an inhibitor of the formation of the pre-replication complex, which is necessary to be assembled prior to DNA replication in eukaryotic cells. Based on our crystal structure of a human truncated Geminin/Cdt1 complex and X-ray solution scattering experiments (SAXS) we demonstrated that the complex can exist both as a heterotrimer and a heterohexamer under physiological conditions, possibly representing a conformation switch. In vivo experiments in mammalian cells and in Xenopus oocyte extracts conclusively support our conclusions for the function of this molecular switch. Structural studies of proteins involved in mitotic progression Our interest in this area was initiated by the work on Polo-like kinases, together with the group of Rene Medema. We have recently shown with biophysical measurements combined with cell-based assays that the polo binding domains (PBDs) of Plk1-3 show unexpected differences. We demonstrated a partial functional overlap but also distinct roles of the different PBDs. Last year we have initiated a high-throughput approach towards characterising different proteins involved in mitotic progression and obtained the first crystals. We have made good progress improving the crystals of a regulatory domain of BubR1 and we are about to determine the structure of a regulatory domain of TTK/Mps1. We have also determined the structure of the DNA-binding domain the FoxM1 transcription factor (figure 3). Langer G, Cohen SX, Lamzin VS, Perrakis A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat Protoc. 2008;3: Joosten K, Cohen SX, Emsley P, Mooij W, Lamzin VS, Perrakis A. A knowledgedriven approach for crystallographic protein model completion. Acta Crystallogr D Biol Crystallogr. 2008;64: Cohen SX, Ben Jelloul M, Long F, Vagin A, Knipscheer P, Lebbink J, et al. ARP/ warp and molecular replacement: the next generation. Acta Crystallogr D Biol Crystallogr. 2008;64:49-60 Figure 3: (A) A cartoon representation of the structure of the complex of the FoxM1 DNA binding domain bound to DNA and the assumed position of the N- and C-terminal domains. (B) Binding of FOXM1 to Random DNA, a singe recognition sequence and a double recognition sequence and (C) the occurrence of FOXM1 DNA recognition sequnces in the 3,000 bp flanking regions of human genes.