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    Paterson Institute for Cancer Research SCIENTIFIC REPORT 2006

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    Leukapheresis blood product is depleted of CD25+ lymphocytes prior to infusion back into the patient. CD4+CD25+ regulatory T-cells are thought to be instrumental in allowing a growing cancer to evade immunological attack and the rationale behind this clinical trial is that their removal may unmask an anti-tumour immune response in patients with cancer (See Biological Immune and Gene Therapy Group report on page 36). Illustration Credits Many illustrations in this report were taken by Jenny Varley and Paul Cliff

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    Canc e r R es e arch UK Paterson Institute for Cancer Research SCIENTIFIC REPORT 2006

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    Cancer Research UK Paterson Institute for Cancer Research Scientific Report 2006 Edited by Professor Jenny Varley, Assistant Director (Research) Design & Layout by Mark Wadsworth (Web & Graphic Designer) Paterson Institute for Cancer Research Wilmslow Road, Manchester M20 4BX Tel: 0161 446 3156 http://www.paterson.man.ac.uk ISSN 1740-4525 Copyright © 2006 Cancer Research UK Printed by Paramount Print Cancer Research UK Registered Charity No. 1089464 Registered as a company limited by guarantee in England and Wales No. 4325234 Registered address 61 Lincoln’s Inn Fields, London WC2A 3PX Tel +44 (0) 20 7242 0200 http://www.cancerresearchuk.org

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    Contents Director’s Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 The Manchester Cancer Research Centre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 The Paterson Institute for Cancer Research Bioinformatics Group........................................................................ Crispin Miller . . . . . . . . . . . . . . . . . . . . . . . . 10 Carcinogenesis Group .................................................................... Geoff Margison . . . . . . . . . . . . . . . . . . . . . . 12 Cell Cycle Group................................................................................. Karim Labib . . . . . . . . . . . . . . . . . . . . . . . . . 14 Cell Division Group ........................................................................... Iain Hagan. . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Cell Regulation Group...................................................................... Nic Jones . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Cell Signalling Group ......................................................................... Angeliki Malliri . . . . . . . . . . . . . . . . . . . . . . . 20 Clinical and Experimental Pharmacology Group.............. Caroline Dive and Malcolm Ranson . . . . . 22 Immunology Group............................................................................ Peter L Stern . . . . . . . . . . . . . . . . . . . . . . . . 24 Radiochemical Targeting and Imaging Group ...................... Jamal Zweit . . . . . . . . . . . . . . . . . . . . . . . . . 26 Stem Cell Biology Group................................................................ Georges Lacaud. . . . . . . . . . . . . . . . . . . . . . 28 Stem Cell and Haematopoiesis Group .................................. Valerie Kouskoff . . . . . . . . . . . . . . . . . . . . . . 30 Structural Cell Biology Group...................................................... Terence D Allen. . . . . . . . . . . . . . . . . . . . . . 32 The University of Manchester Division of Cancer Studies Academic Radiation Oncology Translational Radiobiology Group.............................................. Catharine West . . . . . . . . . . . . . . . . . . . . . 34 Biological, Immune and Gene Therapy Group ................... Robert Hawkins and Peter L Stern. . . . . . 36 Children’s Cancer Group................................................................ Vaskar Saha . . . . . . . . . . . . . . . . . . . . . . . . . 38 Medical Oncology: Breast Biology Group ............................. Rob Clarke . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Medical Oncology: Cell and Gene Therapy Group......... Robert Hawkins. . . . . . . . . . . . . . . . . . . . . . 42 Medical Oncology: Translational Angiogenesis Group.... Gordon Jayson . . . . . . . . . . . . . . . . . . . . . . . 44 Medical Oncology: Proteoglycan Group................................ John T Gallagher . . . . . . . . . . . . . . . . . . . . . 46 Targeted Therapy Group................................................................. Tim Illidge . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Research Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Publications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Seminars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Postgraduate Education . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Operations Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Acknowledgement for funding of the Paterson Institute . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Career Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 How to Find us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . inside back cover P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 5

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    Director’s Introduction 2006 has been a very important year in the history of the In 1991, the Paterson Institute was incorporated into the Christie NHS Trust, a relationship that Paterson Institute. It became an Institute of The University of existed until the beginning of 2006. Over these Manchester and at the heart of the newly developed years the Institute was the major driver of the Manchester Cancer Research Centre. Trust’s overall research efforts and close working relationships developed between the scientists in the Institute and the clinical researchers within the Hospital. The Trust provided significant financial support to the Institute as well as lab- oratory facilities to house our research efforts. However, on the 1st January of this year the Institute transferred to become an Institute of The University of Manchester retain- ing its current level of operational autonomy. The major reason for such a move was to initiate the development of The Manchester Cancer Research Centre (MCRC) which inte- grates all the cancer research efforts of the University and to provide a comprehensive approach to cancer research in very close partnership with the Christie NHS Trust and Cancer Research UK. The goals and aspirations of the Centre are described in the next section and are focused on ensuring that advances in the understanding of the molecular Nic Jones, Director of and cellular basis of cancer is translated into new approaches to therapy and meaningful the Paterson Institute diagnostics. Through the development of this Centre the Paterson can not only continue to support basic research but also facilitate translational and clinical research and thereby achieve its full potential as a Cancer Research UK core funded Institute. In addition, not only are our interactions with the rest of the University research activities enhanced, but our interaction within the Christie NHS Trust is also strength- ened through the MCRC-Christie partnership and the increased investment in research that this partner- ship will bring. A major ambition of the MCRC is to significantly increase the volume of cancer research on the Paterson/Christie site which will necessitate major investment in new laboratory facilities. A new research building is being planned but in the meantime it is important that existing facilities are renovated and devel- oped to maximise their use for supporting our research efforts. This year saw the completion of a signif- icant phase in such development with the opening of the TRF1 (Translational Research Facilities 1) labo- ratories. These laboratories provide first class facilities for many of our translational research activities – some that exist already and some for new activities such as developments in molecular pathology. One floor of the new facility is devoted to activities in pharmacolo- gy supporting the major phase 1 trial activities in the Christie NHS Trust. This includes the Clinical and Experimental Pharmacology Group headed by Caroline Dive and Malcolm Ranson (see pages 22-23) and the Translational Angiogenesis Group headed by Gordon Jayson (pages 44-45). The refurbishment of this space has allowed us to optimise the use of these facilities, such as the provision of a Good Clinical Laboratory Practice (GCLP) laboratory that facilitates our ability to assess clinical samples from early phase The newly refurbished basement labs within TRF1 trial patients under the very strict regulations that are now required. The provision of the GCLP laboratory was aided by a very generous donation from the PACCAR Foundation. We are very grateful for this sup- port and the support we receive from numerous other private donations (see pages 78-79). In addition to the academic interactions discussed above, it is vital that we have strong interaction with industry especially in the translational and clinical research areas. This year has seen the development of fruitful interactions with AstraZeneca one of the biggest pharmaceutical companies in the world. An P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 6

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    alliance between the MCRC and AstraZeneca has resulted in joint initiatives in biomarker research and in training which is of considerable benefit to the translational research of the Institute. A novel training pro- gramme for clinical pharmacologists has been initiated which we hope will help to alleviate the shortage of qualified and experienced researchers in this important area. This year has also seen the completion and implementation of a new Pay and Grading system for all Institute staff. This has been developed with Cancer Research UK and will operate across all their core funded Institutes. Although it involved considerable effort, overhauling our previous sys- tem was much needed and has resulted in a fair and trans- parent system that allows us to reward achievements and success and hopefully will prove motivating to all Institute staff. Our career structure for Group Leaders has two main categories: Junior Group Leaders who are at an early stage of their independent career development and Senior Group Leaders who have well established, inter- nationally competitive research programmes. It is vitally important that the balance between Junior and Senior Group Leaders reflects the opportunities the Institute has in developing scientific careers but equal- ly important that we ensure that only Junior Group Leaders with outstanding records and a high reputation in their respective fields are promoted to a senior position. This was certainly the conclusion that an inter- national appointments panel reached when they considered the promotion of Karim Labib who was made a Senior Group Leader in August. Karim has made outstanding contributions to our understanding of the process of DNA replication, a process that is fundamentally important for the stable inheritance of the genome and is known to be defective in cancer. It is a very conserved process and Karim’s studies using budding yeast as a convenient model system have provided direct insights into this process in mammalian cells. Not surprisingly given the importance of replication and its correct timing in the cell cycle, it is a process that is very highly regulated. Karim’s research has led to the identification of a new and essential complex called GINS which interacts with additional proteins to form a ‘Replication Progression Complex’ (see pages 14-15). This work is at the forefront of his field and his promotion and consequent expansion of his research group is richly deserved. The coming year will provide many new and exciting challenges and opportunities. Up to three additional Group Leaders will be recruited to enhance our research activities and strengthen our research focus in tumour cell biology, tumour microenvironment and stem cell biology. We will initiate new programmes in the development of advanced mouse models of cancer for pre-clinical research and therapeutic assessment and in the development of drug discovery. This latter activity is a major new initiative undertaken with Cancer Research UK aimed at the development of small molecule drugs against new potential targets some of which will emerge from our basic science programmes. This initiative will strongly complement the research that we currently do and plan for the future. The first step in this initiative will be to recruit a Senior Drug Discovery Scientist to lead the development. We will invest further in our research services by enhancing our capabilities in mass spectrometry based proteomics and advanced imaging. Finally we will continue to play a central role in the development of the MCRC to ensure that the promise and potential of the Centre will be realised. P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 7

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    Manchester Cancer Research Centre The Manchester Cancer Research Centre (MCRC) has been established to integrate the cancer research efforts within the University of Manchester in partnership with the Christie Hospital NHS Trust and Cancer Research UK. Its goal is to become a world-leading centre for basic, translational and clini- cal cancer research by 2015 with the major aims of: ● Supporting fundamental scientific research to better understand the processes that drive the initiation, progression and maintenance of cancer in different tissues and through which new targets for therapeutic intervention are identified and validated. ● Support translational and clinical research to develop and test new promising therapies as well as advances in diagnosis and prognosis. ● Support extensive clinical and non-clinical cancer research training and development. In order to achieve these aims the Centre has developed a strategic plan for future development and for the focus of its cancer research efforts, is coordinating the recruitment of world-class basic and clinical researchers, is developing laboratory-based and clinically-based research infrastructure and developing a matrix of basic and clinical research programmes which will provide a framework for matching strengths in basic research with interests and strengths in translational and clinical research. The Joint Research Strategy Group of the MCRC and the Christie Trust is where the main objectives are developed and steps for delivery identified. The opportunities and strengths of the MCRC come from the expertise and ambitions of the partners involved. The University of Manchester is the biggest single-site University in the country with the stated ambition to be among the top 25 Universities in the world by 2015. It has significant existing activities in cancer research within the Faculties of Medical and Human Sciences and Life Sciences augmented by the recent incorporation of the Paterson Institute. The Paterson Institute is a leading cancer research institute core-funded by Cancer Research UK, supporting programmes in basic and translational research. The Christie Hospital NHS Trust is a specialist tertiary centre for cancer treatment and one of the major cen- tres in the UK for clinical trials and radiotherapy research involv- ing cancer patients. It is the largest single-site cancer treatment centre in Europe treating approximately 12,000 new patients per year and supporting a network population of 3.2 million across Greater Manchester and Cheshire. Cancer Research UK is the world’s leading cancer charity supporting the research activities of over 3000 scientists, doctors and nurses working across the UK. Over the next 5-10 years significant investment in people, technol- ogy and infrastructure across the whole basic to clinical research spectrum will take place. Further investment in basic research will be made to understand in more depth the events critical for the ini- tiation, progression and maintenance of tumours, the interactions between the tumour cells and their immediate cellular environment and the mechanistic basis of drug resistance. This will be enhanced by continued provision of state-of-the-art technologies and new laboratory facilities. At the heart of translational research in the MCRC is experimental therapeu- tics and biomarker discovery. Early phase clinical trial activity is already a significant strength at the Christie P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 8

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    MANCHESTER CANCER RESEARCH CENTRE Hospital site with dedicated patient facilities and expertise associated with clinical and experimental phar- macology. The MCRC will significantly increase first-into-man trials of mechanism based therapies partic- ularly focussing on targets that match its biological strengths and interests. In addition it will expand cell- based therapies that aim to harness the immune system to recognise and kill tumour cells. This drug devel- opment and biomarker discovery focus is being augmented by further development of molecular imaging and clinical proteomics as well as the development of a new programme in drug discovery. To fully capi- talise on the large patient population associated with the Christie and the associated cancer network, the Centre will increase and strengthen its activities in clinical and population-based research by facilitating increased recruitment into clinical trials, developing pharmacogenomic research to identify and validate bio- markers with diagnostic and prognostic potential using major patient cohort studies and sample collections and by developing tumour-specific research programmes. Fundamental Translational & Clinical Tumour Specific Research Research Programmes Abnormal AbnormalGrowth: growth:proliferation, proliferation, apoptosis, genome genomestability, stability,signalling signalling TumourMicroenvironment: Tumour microenvironment:angiogenesis, hypoxia, angiogenesis, hypoxia,adhesion, adhesion, immunology immunology Stem Cell Stem cell biology Biology Tumour-Specific Programmes: breast, haematology (plus two others) Basic Diagnostics: Experimental Clinical Trials Discovery Imaging, Molecular Therapeutics Pharmacogenomics Labs Pathology, Proteomics, Genomics, Bioinformatics The MCRC is still in its infancy and many challenges lie ahead. However, the opportunities to develop a world-class comprehensive centre are great. In its first year significant progress has already been made. Most notable is its success in attracting over £3M to expand and develop the phase I clinical trial unit which will double its current capacity and in so doing, will become the biggest phase 1 unit in the world. It is anticipated that this expansion will take place over the next year. Recruitment of basic and clinical scien- tists and development of the plans for a new laboratory build will be the focus of the coming year. P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 9

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    GROUP LEADER POSTDOCTORAL SYSTEM Crispin Miller FELLOWS ADMINISTRATOR/ SCIENTIFIC Michal Okoniewski PROGRAMMER Carla Möller Levet Zhi Cheng Wang (with Catharine West) Páll Jónsson GRADUATE STUDENTS Graeme Smethurst (with Peter Stern) RESEARCH Laura Edwards (with Georges APPLICATIONS Lacaud) PROGRAMMER Danny Bitton Tim Yates Bioinformatics Group http://www.paterson.man.ac.uk/groups/bioinf.jsp The majority of the work in the Bioinformatics Group groups in the Institute, most notably through Graeme Smethurst and Laura Edwards, who have involves interpreting high-throughput gene expression data, joint PhD projects with Peter Stern (page 24) and most of it arising from the Institute’s Affymetrix system. The Georges Lacaud (page 28). group is strongly interdisciplinary by nature (the team’s back- Detailed annotation of microarray data grounds bring together a mix of computer science, mathe- The more computational side of our research is matics, and biology), and the aim is to maintain a friendly and also yielding exciting data. We have continued our collaborative environment that brings the informatics and work with BioConductor and R, and have con- tributed the package plier to the BioConductor biology together as much as possible. project, which provides access to Affymetrix’ plier algorithm for expression summarization. Affymetrix microarrays use 25-mer oligonucleotide Microarray gene expression data probes to target individual transcripts of interest. Over the last year, a number of biological collabo- Typically, 11 probes are used to target each tran- rations have begun to come to fruition; Claire script, and these are grouped in software to form a Wilson (a previous postdoc in the group) spent a probeset, and their combined data summarized to significant amount of time working on a microarray generate an estimated concentration for each tran- project investigating the effects of oestrogen on script. Michal Okoniewski has been interested in gene expression in epithelium and stroma of nor- the specificity of the probeset to transcript relation- mal human breast tissue in collaboration with Andy ship, and, along with Tim Yates, built a database of Sims and the Breast Biology group, headed by Rob probe-probeset-transcript mappings by searching, Clarke. Claire has also worked with Tony Whetton’s in silico, the target probe sequences against a data- Stem Cell and Leukemia Proteomics Laboratory base of well annotated mRNA sequences. Michal (SCALPL) at the University of Manchester, on was able to use this database to identify ‘multiply- work that revealed post-translational control as a targeted’ probes that were able to bind to multiple regulatory factor in primary hematopoietic stem transcripts, possibly from different genes, and then cells. Her contribution was towards the underlying to show that this was a significant effect that could bioinformatics necessary to bring the quantitative lead to apparent, but spurious, relationships proteomics data alongside Affymetrix gene expres- between genes. This work is also interesting sion data. Other projects, including one looking at because these events are most likely to happen Formalin Fixed Paraffin Embedded Tissue when sequences are similar. Since sequence similar- (FFPET) in collaboration with Stuart Pepper and ity is often used to infer functional relationships, we the Molecular Biology Core Facility (MBCF; page might expect the effect to be most frequently 54), Kim Linton, Tony Freemont at the University observed between functionally related genes. This of Manchester and John Radford at the Christie is important to consider, when, for example, using Hospital are also generating promising results, and correlation based techniques to predict functional we have continued to work extensively with other associations by clustering gene expression data. P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 10

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    B I O I N F O R M AT I C S Analysis of Exon arrays this has given us enough confidence to conduct Recently, Affymetrix released a new generation of some new projects to these arrays. microarrays that aim to interrogate every known and predicted exon in the human genome. The Development of novel statistics for gene expres- ability to investigate transcription at such fine levels sion analysis of detail is clearly exciting, but makes some signifi- Meanwhile, Carla Möller Levet has been working cant demands on data analysis, not least, because with Catharine West from the University of their interpretation requires access to a comprehen- Manchester’s Academic Department of Radiation sive description of the fine-grained structure of Oncology (ADRO), looking at clinical microarray every gene of interest. Michal and Tim have been data. Carla has also been developing novel analysis applying their expertise in databases and their techniques, and this has led to the development of understanding of probeset-transcript-gene relation- a promising new method for finding correlations ships to these arrays for some time now, and have within gene expression, important because correla- developed a set of software tools to support their tion based techniques are a fundamental set of tools analysis. Tim’s database, X:MAP, can be found for gene expression analysis. online at http://xmap.picr.man.ac.uk. We are now starting to use their software to explore microarrays Finally, in the last few months, Danny Bitton has at the exon level, and have a number of collabora- joined us as a PhD student investigating novel tran- tive exon array projects underway. scriptional and translational events in microarray and proteomics data, and Páll Jónsson has just Underpinning this work was a validation study done arrived as a postdoc to bring knowledge of protein in collaboration with the MBCF and Cancer interaction networks. Over the next year we aim to Research UK Affymetrix service (page 51), to con- begin to bring all of these different strands togeth- sider how well these new arrays perform in compar- er, and to continue to apply them to our datasets. ison to the previous generation of expression arrays from Affymetrix. We found that with the right data analysis high-correspondence could be found, and X:MAP, an annotation database representing the relationship between Affymetrix Exon 1.0ST array probesets and gene structure. Publications listed on page 56 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 11

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    GROUP LEADER POSTDOCTORAL SCIENTIFIC OFFICERS Geoff Margison FELLOWS Gail McGown Amna Butt Mary Thorncroft Barbara Verbeek Mandy Watson Stephen Wharton GRADUATE STUDENT CLINICAL FELLOW Andrew Marriott Phil Crosbie UNDERGRADUATE STUDENTS Ghazala Begum Catia Caetano Jill Hunter Jeff Hollins Natalia Rukazenkova Carcinogenesis Group http://www.paterson.man.ac.uk/groups/carcino.jsp Many types of cancer treatment exploit the cell killing effects apeutic efficiency and also the toxic side effects of alkylating agents. Thus there is increasing interest of therapeutic agents that can generate a variety of types of in attenuating the expression of such pathways in damage in DNA. But the cell killing effects can be prevented tumours, and enhancing them in normal tissues, in by a variety of cellular DNA repair pathways that operate to order to increase the differential killing effects and thus to improve clinical outcome. These repair remove this potentially lethal damage from DNA. processes, especially that repairing damage at the Understanding how DNA damage leads to cell death, and O6-position of guanine, i.e. MGMT, and more how these repair systems process the damage, may provide recently alkylpurine-DNA glycosylase (APG) which instigates the base excision repair system that opportunities to improve the effectiveness of existing cancer processes 3-methyladenine, have therefore become therapies, and develop new agents. Our main focus is on targets for modulation. MGMT removes alkyl DNA damage and the ensuing DNA repair processes that groups from the O6-position of guanine by stoi- chiometric transfer to a cysteine residue in its active follow exposure to certain types of alkylating agents, one site, a process that results in its irreversible inactiva- example of which is the CR-UK drug, Temozolomide. Other tion. Because of this, inactivation of MGMT results projects include studies of the relationship between single in increased cellular sensitivity until more protein is synthesised. nucleotide polymorphisms in the O6-methylguanine-DNA methyltransferase (MGMT) gene and cancer risk, and the Development of the MGMT inactivating drug, characterisation, in fission yeast, of a close relative of MGMT, Lomeguatrib Over the last decade, in collaboration with Prof which appears to be part of a novel DNA alkylation damage Brian McMurry and the late Dr Stanley McElhinney repair pathway. (and their group at the Chemistry Department, Trinity College, Dublin), and with the support of Background CR-UK Drug Development and Formulation Units Chemotherapeutic alkylating agents generate vary- and also Cancer Research Technology, we have ing amounts of a dozen different types of lesions in developed a drug that is a “pseudosubstrate” for DNA and there is increased understanding of the MGMT. This drug is PaTrin-2, officially called mechanism/s by which some of these lesions result Lomeguatrib (Figure), and it effectively inactivates in cell killing. Thus, methylating agents such as MGMT and sensitises human tumour xenografts to Dacarbazine and Temozolomide produce O6- the killing effect of Temozolomide. The first-time- methylguanine in DNA and this kills cells via the into-man Phase I clinical trial of this drug started action of the post replication mismatch repair here at Christie Hospital in 2000, and established a (MMR) system. These agents also generate 3- dose combination of Lomeguatrib and methyladenine, which kills cells by blocking DNA Temozolomide for use in Phase II trials. These tri- replication. als, carried out under the auspices of KuDOS Pharamaceuticals (who were recently purchased by Repair pathways that probably evolved to deal with Astra-Zeneca), to whom Lomeguatrib is licensed, low levels of endogenously produced damage, and are ongoing in several centres in the UK, USA and which may be important in preventing the carcino- Australia. The dose and schedule of the two drugs genic effects of such damage, reduce both the ther- is currently being optimised. In addition, another P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 12

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    CARCINOGENESIS trial, sponsored by CR-UK is addressing the effec- retroviral vectors. tiveness of Lomeguatrib in inactivating MGMT in a number of tumour types in order to establish the It has been reported previously that over-expres- doses required for complete inactivation. These tri- sion of MGMT might be disadvantageous to als have required us to develop and validate to haematopoietic cells. To allow regulated functional- Good Clinical Laboratory Practice standards, quan- ity of MGMT, a 4-hydroxytamoxifen (4-OH-T) titative assays for both functionally active and total inducible MGMT(P140K) was constructed by fus- MGMT protein. Brain, prostate, breast and col- ing a mutated version of the mouse oestrogen orectal tumours are being examined in dose escala- receptor (ERT) to the C-terminus of tion studies. Once the effective dose is established, MGMT(P140K). Following transduction with a Phase II trials will be undertaken. We are also inves- retrovirus containing the MGMT(P140K)-ERT tigating the role of MGMT promoter methylation fusion, the human cell line K562, which does not in regulating MGMT expression and influencing express endogenous MGMT, expressed high levels response. of MGMT activity. Growth analysis showed that the negative effect of MGMT(P140K) overexpres- Aspects of these clinical trials and also of our can- sion in the absence of drug selection was reduced cer chemotherapy molecular epidemiology studies by the ERT-fusion. Further evaluation is in hand. have involved the determination of the major DNA methylation product, 7-methylguanine in DNA by Alkyltransferase-like (ATL) proteins: a novel means of a sensitive immunoslot-blot assay. The DNA repair pathway levels of this product in lymphocyte DNA of The alkyltransferase-like (ATL) proteins contain temozolomide-treated patients varies very consider- primary sequence motifs resembling those found in ably and recent results suggest that this may be a MGMT but in the putative active site of some ATL consequence of the activity of APG (see above). proteins a tryptophan residue replaces the cysteine found in the alkyltransferases. In collaboration with MGMT polymorphism studies David Williams (Sheffield) we have shown using gel Our previous single nucleotide polymorphisms shift assays that the ATL from E.coli binds to short (SNP) studies that demonstrated allelic expression single- or double-stranded oligonucleotides con- imbalance (AEI) of the two MGMT alleles in sub- taining a number of O6-alkyl-substituted guanines jects that are heterozygous for an expressed poly- including Benzyl, hydroxyethyl and 4-bromothenyl morphism have been confirmed and extended. A (i.e. Lomeguatrib embedded into an oligonu- human cell line that contains an expressed polymor- cleotide, which is the most potent MGMT inactivat- phism has been identified and this may allow us to ing agent so far described). Inactivation of the establish a possible mechanism of MGMT AEI ATL gene (that we have christened Atl1) in S. pombe that could then be explored in human samples. sensitises them to the toxic effects of a wide range of O6-alkylating agents, demonstrating that, in wild- Preclinical studies with MGMT inactivating agents type cells, the Atl1 protein confers resistance to We are also involved in preclinical studies aimed at such agents. The mechanism of this repair pathway establishing clinical strategies that will reduce the is currently being investigated. dose-limiting bone marrow toxicity of agents like Temozolomide, work that we had been undertaking in collaboration with the late, and greatly missed, Br Lez Fairbairn and his group. We have shown that the P(140)K mutant of MGMT is highly resistant to inactivation by Lomeguatrib and that a human S haemopoietic cell line (K562) transduced with a retroviral vector encoding MGMT(P140K) is high- ly resistant to the cytotoxic effects of O Temozolomide in combination with Lomeguatrib. This is therefore a suitable combination for chemo- N protective gene therapy. We have also shown in col- N laborations with Prof Chris Baum (Hamburg and Cincinnati) and Dr Samy Chinnasamy (Milwaukee) H 2N N N that self-inactivating gammaretroviral and lentiviral H vectors with comparable internal expression cas- Chemical structure of the MGMT inactivating drug, settes had similar MGMT expression properties. Lomeguatrib (O6-(4-bromothenyl)guanine; previously called These vectors are of interest because of the safety PaTrin-2, and having the trade mark, Patrin™) Publications listed concerns associated with the use of the standard on page 56 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 13

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    GROUP LEADER POSTDOCTORAL GRADUATE STUDENTS Karim Labib FELLOWS Agnieszka Gambus Ben Hodgson Vanessa Marchesi Masato Kanemaki Hiroko Nishikawa Alberto Sanchez-Diaz Magdalena Przywara SCIENTIFIC OFFICER Frederick van Deursen Cell Cycle Group http://www.paterson.man.ac.uk/groups/cellcycle.jsp Our group studies chromosome replication and cytokinesis important reason for this, namely that GINS main- tains the interaction of MCM with Cdc45 that is in eukaryotic cells. During 2006 we identified a key role for also essential for fork progression. the four-protein GINS complex at DNA replication forks. We showed that GINS allows the MCM helicase to interact Hiro Araki’s lab previously identified Sld3 as a DNA replication protein that appeared to behave in with the Cdc45 protein that is essential for the progression a very similar manner to Cdc45. Both proteins are of DNA replication forks. In contrast, we found that the recruited to early origins during G1 phase in bud- Sld3 protein is required to establish but not maintain MCM- ding yeast, in a mutually dependent manner, but Sld3 and Cdc45 are only recruited to late origins Cdc45-GINS complexes. Sld3 is recruited to origins before during S phase around the time that these origins GINS but is subsequently displaced during the initiation are activated. Sld3 and Cdc45 can apparently inter- process, and does not normally travel with DNA replication act with each other, and Sld3 is required to establish the interaction between Cdc45 and MCM during forks. In parallel with this work, we identified a novel factor initiation. More recently, Araki’s lab showed that that we call “Ingressin” or Inn1, which is required for cytoki- Sld3 could also interact with GINS in 2-hybrid nesis. We found that Inn1 associates with the actomyosin screens, and Sld3 is required for loading of GINS at origins during the establishment of DNA replica- ring at the end of mitosis and is required for contraction of tion forks. the ring to be coupled to ingression of the plasma mem- brane. We were struck, therefore, by the fact that Sld3 is not a component of Replisome Progression Complexes. This observation led us to suspect that Regulation of the establishment and progression Sld3 may only be required during initiation to medi- of DNA replication forks ate events such as the recruitment of Cdc45 and In a systematic study of the essential budding yeast GINS, but might then be excluded from the nas- proteins of previously unknown function we iden- cent replisome, in contrast to Cdc45 and GINS that tified four factors required for chromosome replica- are required together with MCM for progression of tion (Kanemaki, Sanchez-Diaz et al, (2003), Nature forks. To address this possibility, Masato Kanemaki 423, 720-724). Together with Hiro Araki’s group in used a sensitive and quantitative chromatin Japan we showed that these proteins interact to immunoprecipitation (ChIP) assay based on real- form a complex called GINS. By purifying GINS time PCR to follow the association of replication from yeast cell extracts, Aga Gambus found that proteins with DNA replication forks (Kanemaki GINS associates with the MCM helicase at nascent and Labib, 2006). He found that Sld3 is indeed dis- DNA replication forks and allows large “Replisome placed from origins during initiation without Progression Complexes” (RPCs) to form around becoming incorporated into the replisome, in con- MCM (Gambus et al, 2006). It appears that RPCs trast to Cdc45 and GINS that move away with exist uniquely at DNA replication forks as they are DNA replications forks, presumably as part of only found during S phase, on chromatin, require RPCs. He then used degron technology to gener- prior recruitment of the MCM helicase to origins ate a novel and extremely tight new allele of SLD3. during G1 phase, and are preserved if the progres- This required the addition of the degron cassette to sion of replication forks is blocked artificially. a version of SLD3 that also contained a mutation GINS is required for the normal progression of that contributed to the resultant temperature-sensi- DNA replication forks and Aga identified one tive phenotype. Masato used this new allele, called P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 14

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    CELL CYCLE sld3-7td, to show that Sld3 is not required for the quent abscission of the plasma membrane, thus completion of chromosome replication after the producing two cytoplasms from one. Alberto has establishment of DNA replication forks, in agree- shown that the localisation of Inn1 requires other ment with our finding that Sld3 is not part of RPCs essential components of the ring, and Inn1 remains and does not move with DNA replication forks associated with the ring during subsequent contrac- (Kanemaki and Labib, 2006). tion. Ingressin links contraction of the actomyosin He then used the degron strain to show that Inn1 is ring to ingression of the plasma membrane specifically required for ingression of the plasma during cytokinesis membrane. The Inn1 protein contains a predicted In our systematic screen for new cell cycle proteins lipid-binding domain, and a purified recombinant Alberto Sanchez-Diaz identified a protein that we form of Inn1 is able to bind to inositol phospho- call “Ingressin” or Inn1, which is essential for cell lipids in vitro. These data suggest that Inn1 may link division but dispensable for the continuation of the the actomyosin ring to the plasma membrane dur- nuclear cycle of replication and mitosis. In collab- ing cytokinesis. oration with Terry Allen’s group we have used elec- tron microscopy to show that cells lacking Inn1 are truly defective in cytokinesis. Alberto fused Inn1 to Green Fluorescent Protein (GFP) and found that it associates at the end of mitosis with the actomyosin ring that marks the site where cell division will subsequently occur. Contraction of the actomyosin ring is coupled by an unknown mechanism to ingression and subse- Figure 2 Inn1-GFP associates with the contractile actomyosin ring at the end of mitosis. The picture shows cells that express Inn1 fused to Green Fluorescent Protein, as well as a fusion of Red Fluorescent Protein to a Spindle Pole Body component. Cells were fixed and stained with a DNA binding dye. Figure 1 A model for the role of Sld3 during the initiation of chromosome repli- cation. See text for details. Publications listed on page 58 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 15

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    GROUP LEADER POSTDOCTORAL SCIENTIFIC OFFICERS Iain Hagan FELLOWS Deepti Wilks Agnes Grallert Nimesh Joseph GRADUATE STUDENTS Tamara Krsmanovic Dorota Feret Najma Rachidi Daphne Garcin Alasdair Robertson Kate Sloan Victor Tallada Cell Division Group http://www.paterson.man.ac.uk/groups/celldiv.jsp Microtubule behaviour underpins many aspects of cancer induced by GTP hydrolysis within the lattice induces the subunits to peel away from the micro- biology. Alteration in the function of these filaments within tubule lattice leading to the rapid depolymerization the mitotic spindle can affect chromosome transmission to of the microtubule. Thus, if at any point in the life alter the balance of tumour suppressor and tumour promot- of a microtubule, the hydrolysis rate exceeds the polymerisation rate, the microtubule will loose its er genes and so precipitate carcinogenic changes in genome protective cap of GTP tubulin and depolymerise. composition. Microtubules also play an integral part in the As the loss of a GTP cap is a random event, micro- migration of these cancer cells during their invasion of sec- tubules are constantly switching from growth to shrinkage independently of one another. The result ondary sites to form metastases and again in the generation is a dynamic microtubule cytoskeleton that is con- of the vasculature to provide the blood supply that is essen- stantly probing the 3-dimensional space. This tial for the survival of these secondary tumours. Thus, drugs enables it to rapidly establish contacts that are crit- ical in cell migration or chromosome segregation. that disrupt microtubule function, such as taxol, have proved highly effective chemotherapeutic agents. Further, under- The +TIP class of microtubule associated pro- standing microtubule biology will extend therapeutic options teins While the inherent GTPase activity of tubulin gen- by identifying novel ways to target the microtubule erates a dynamic cytoskeleton, it is the activity of a cytoskeleton and define better ways to exploit existing number of molecules that associate with micro- drugs. As the structure, composition and function of the tubules that modulate microtubule behaviour and it is the control of these microtubule associated pro- microtubule cytoskeleton is highly conserved, we study the teins (MAPs) that underpins the activity of the simple and highly malleable, unicellular yeasts to learn lessons microtubule cytoskeleton. There are several types that can then be applied to tumour biology. of MAP: motor proteins that migrate along micro- tubule lattice, structural MAPs that associate along the length of the microtubule and the +TIPs that Microtubule growth associate specifically with the +ends of micro- Microtubules form by the polymerisation of tubu- tubules. Because it is the dynamic +end of the lin heterodimers. They are polar structures. microtubule that mediates many microtubule func- Subunits add onto one end much faster than the tions, there is considerable interest in understand- other. Consequently this end is known as the plus ing how these +TIPs function. The +TIP CLASP end and the opposing end the minus end. Free was identified by virtue of its association with the tubulin subunits only associate with microtubules founder +TIP, CLIP170, leading to the proposal when it has bound GTP. The incorporation of that the association with CLIP170 enables CLASP GTP tubulin into the polymeric microtubule lattice to modulate microtubule dynamics. Subsequent then stimulates the hydrolysis of the GTP to GDP. analyses demonstrated that a number of other This hydrolysis induces a conformational switch in +TIPs also associate with one another, and the pro- the tubulin molecule. Thus, the microtubule poly- posal that CLASP influences microtubule function merises as a flat sheet of GTP tubulin that curls up through its association with a second +TIP, EB1. upon GTP hydrolysis to form a tube of GDP tubu- lin. If there are no GTP associated molecules at the end of the microtubule, the structural change P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 16

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    CELL DIVISION Fission yeast CLASP goes to minus ends or pulls the plus ends towards a We have been studying fission yeast CLASP. Like structure to which the dynein is anchored. We its higher eukaryotic counterparts, fission yeast found striking similarities in the consequences of CLASP regulates microtubule behaviour at the cell losing the function of CLASP and dynein. cortex. When microtubules arrive at the ends of these highly polarised cells they establish an end-on Lessons from yeast association with the cortex, continue to polymerise The ability to manipulate genes at will in a simple for a while at this attached end before finally organism whose primary purpose is to divide is depolymerising so that their end ultimately shrinks enabling us to explore the finer points of micro- away from the cell tip. The polymerisation rate of tubule biology. This information informs studies in microtubules that have established end-on associa- higher systems that, in turn, raise models that can tions at cell tips is slower than before contact is be most readily tested in yeast. This re-iterative made. We found that this reduction in polymerisa- cycle of comparative studies ensures that great tion rate depended upon CLASP function. Other strides are being made in understanding micro- aspects of cortical function were also altered by tubule biology. ablating CLASP function and many microtubules failed to make an end-on attachment but continued elongating to curl around cell tips. Unexpectedly we failed to find strong evidence for any interplay between fission yeast CLASP and its CLIP170 or EB1 homologues. Instead, we found a strong link between CLASP and the multi-subunit motor pro- tein dynein. Because dynein moves towards the minus end of microtubules it either transports car- Left: Consecutive images of a wild type and a clasp mutant cell. Arrows indicate the ends of dark microtubules that elongate to cell tips where they pause before de-polymerising. Right: Placing mutations in a back- ground that produces highly elongated cells shows that clasp plays no role in morphogenesis. Publications listed on page 58 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 17

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    GROUP LEADER ASSOCIATE GRADUATE STUDENTS Nic Jones SCIENTIST Gemma Thornton Caroline Wilkinson Orestis Mavroudis-Chocholis POSTDOCTORAL SCIENTIFIC OFFICERS FELLOWS Keren Dawson Julien Ackermann Steve Lyons Wolfgang Breitwieser Yujun Di Dominic James Clare Lawrence Wolfgang Reiter Cell Regulation Group http://www.paterson.man.ac.uk/groups/cellreg.jsp Cells commonly respond to extracellular signals by modulat- stress responses is highly conserved. In fission yeast the major transcriptional responses to stress ing the activity of specific transcription factors and subse- conditions are coordinated by the transcription fac- quently the expression of many target genes. We are partic- tors Atf1 and Pap1, which are related to mammalian ularly interested in the response to cytotoxic and genotoxic ATF and Jun proteins respectively. In addition the activity of Atf1 is regulated by the Sty1 kinase, a stress which results in the mobilisation of a battery of pro- homologue of the mammalian p38 kinase. Thus tective and repair mechanisms or the induction of apoptosis. fission yeast serves as a useful model for under- Failure to respond appropriately can result in cellular damage standing the role and regulation of AP-1 proteins in mediating stress responses. and thereby drive tumourigenesis. Functional Characterisation of ATF2 The AP-1 transcription factor plays a key role in the ATF2 is a member of the AP-1 family and can bind response of cells to extracellular signals. In mam- to DNA either as a homodimer or as a heterodimer malian cells it is regulated by a plethora of physio- with other AP-1 family members, most prominent- logical and pathological stimuli including mitogens, ly c-Jun. ATF2 is activated by the p38 or JNK hormones, genotoxic agents, stress signals, viral kinases through phosphorylation of two N-termi- infections and cytokines. Not surprisingly there- nal threonine residues T69 and T71. Many reports, fore, it has been linked to many cellular events mostly using in vitro systems, have implicated ATF2 including cell proliferation, differentiation as well as in numerous growth and developmental programs apoptosis. AP-1 plays important roles in tissue and in response pathways after stimulation with stress responses such as inflammation and ischemia geno- and cytotoxic stresses. and is implicated in the onset and progression of tumours. The factor and its regulation is complex To better understand the biological importance of since it is not a single entity but rather a mixture of ATF2 we have generated a number of genetically dimeric complexes composed of members of the modified mice where ATF2 activity is compro- Jun, Fos, ATF and MAF protein families. Different mised. The germline deletion of the DNA binding dimeric combinations can recognise slightly differ- domain in ATF2 leads to postnatal lethality due to ent sequence elements and be regulated by distinct deficiencies in the control of breathing. A similar signalling pathways. A well characterised signalling defect is uncovered by specifically deleting ATF2 in cascade involves the activation of the mitogen-acti- neuronal cells by Cre/loxP mediated conditional vated protein (MAP) kinases ERK and stress- gene inactivation. Therefore, we could show that induced MAP kinases JNK and p38 which directly ATF2 has essential roles in the central nervous sys- phosphorylate and modulate the activity of various tem, and at least one essential function is the estab- members of the AP-1 complex. Over the last few lishment or correct placement of respiratory cen- years considerable progress has been made in eluci- tres in the hindbrain. dating the function of individual AP-1 proteins through the characterisation of genetically modified The essential role of ATF2 in embryonic develop- mice and cells that derive from them. ment is uncovered by simultaneously deleting ATF2 and its closest homologue ATF7 in the germline. Homologues of AP-1 family proteins are found in The resulting double homozygous embryos display all eukaryotic organisms and their involvement in P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 18

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    C E L L R E G U L AT I O N severe deficiencies in the developing liver due to isms, in particular, must contend with fluctuations severely enhanced apoptosis. During large periods in nutrients, pH, temperature and external osmolar- of embryogenesis, the embryonic liver consists of ity, as well as exposure to UV irradiation and a range developing hepatocytes as well as haematopoietic of potentially toxic environmental compounds. precursor cells and in the absence of functional Appropriate responses to these environmental ATF2 and ATF7, both cell types showed increased stresses must be induced for cell survival and pro- apoptosis. This increase is driven by high levels of liferation. activated p38 kinase and is reversed by the addition of a specific p38 inhibitor. The accumulation of Both the Sty1 kinase and the Atf1 transcription fac- active p38 is due to a decrease in a number of dual tor are crucial for fission yeast to respond normally specificity phosphatases, including DUSPI/MKPI, to a range of different stress conditions by orches- which serve to negatively regulate the activity of trating the expression of a common set of environ- MAP kinases. ATF2 binds directly to the promot- mental stress response genes encoding numerous er regions of these phosphatases and regulates their defence and repair proteins. We are investigating transcription, thereby establishing a negative feed- how Sty1 modulates Atf1 activity. Atf1 is directly back loop. We also generated mice containing an phosphorylated by Sty1 and we have shown that allele of ATF2 where the phospho-acceptor threo- this regulates Aft1 stability. Our results suggest that nine residues (T69 and T71) are altered to alanines Atf1 phosphorylation by Sty1 is not required for (ATF2-AA) and thus rendering ATF2 inactive for activation of gene expression per se. However, Atf1- response to upstream MAP kinases. On an ATF7 dependent transcription absolutely requires Sty1 mutant background, these mice demonstrate the which suggests that the MAPK plays some other same embryonic phenotypes as the deletion in the role in the activation of gene expression. We are DNA binding domain. Furthermore, since the currently investigating the mechanism whereby Sty1 ATF2/7 mutations phenocopy the germline muta- activates expression and find that the kinase gets tion of JNK and p38 activating kinases MKK4, and recruited to Atf1 dependent promoters upon stress. MKK7, it is likely that during liver development ATF2/7 are essential effectors of MAP kinase sig- We have gone on to identify an E3 ubiquitin ligase naling. involved in Atf1 turnover and are currently charac- terising the mechanisms by which phosphorylation Cells derived from ATF2/7 mutant embryos have interferes with this turnover. Furthermore, this lig- been established in culture. Compared to wild-type ase itself seems to be subject to stress dependent control cells ATF2/7 mutant cells are characterised regulation. In addition, we have used mass-spec- by saturation at a higher cell density suggesting a trometry approaches to identify a number of novel defect in contact inhibition of growth. The cells protein interactions with Atf1. The functional are also sensitive to specific pro-apoptopic signals importance of these interactions is being charac- such as TNFα. When immortalised and expressing terised and should lead to a more detailed under- oncogenic Ras, ATF2 deficient cells generate signif- standing of how this factor is regulated. icantly larger tumours following injection into immunodeficient mice suggesting a negative role for ATF2 in tumour growth. In this context, the importance of regulation of MAP kinase activity through the transcriptional activity on MAP kinase phosphatase genes is currently being established. ATF2/7 knockout cells, as well as other tissue spe- cific knockout models, are being used to further address the role of ATF2 in tumourigenesis or the response of tumour cells to therapeutic approach- es. Stress Response in Fission Yeast Chemical inhibition of p38 kinase blocks growth arrest in ATF2/7 mutant We use fission yeast as a model system for studying embryonic hepatocytes. stress responses since there appears to be remark- able conservation involving similar signalling path- ways and the mobilisation of closely related tran- scription factors. All cells sense and react to changes in their environment. Single-celled organ- Publications listed on page 58 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 19

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    GROUP LEADER POSTDOCTORAL SCIENTIFIC OFFICER Angeliki Malliri FELLOWS Gavin White Eduardo Castaneda Saucedo GRADUATE STUDENTS Sonia Castillo-Lluva Lucy Dalton (funded by an Natalie Reeves EMBO Long-Term Claire Rooney fellowship) Simon Woodcock Cell Signalling Group http://www.paterson.man.ac.uk/groups/cellsig.jsp Tumour initiation and progression result from inappropriate transformation (Malliri et al., Nature 2002; 417: 867). activation of intracellular signalling cascades. Rho-like GTPases are molecular switches in signalling pathways that Tiam1/Rac signalling and tumorigenesis in vivo regulate cytoskeletal and junctional organisation, as well as Mice deficient for Tiam1 are resistant to the forma- tion of skin tumours induced by application of a gene transcription. In this way, Rho proteins influence cell two-stage chemical carcinogenesis protocol (Malliri morphology, adhesion, motility, as well as cell cycle progres- et al., Nature 2002; 417: 867). This protocol entails sion and cell survival. Rho proteins are essential for Ras- tumour initiation in epidermal keratinocytes by treatment with the carcinogen 7,12-dimethyl-ben- mediated in vitro transformation. Recently, data has emerged zanthracene, which induces oncogenic activation of to directly implicate Rho proteins in tumour initiation and the c-Ha-Ras gene. Subsequent repeated treatments progression in vivo. Our group’s focus is on identifying sig- with the tumour promoter 12-O-tetradecanoylphor- bol 13-acetate (TPA) result in the outgrowth and nalling events downstream of Rho proteins that modulate progression of initiated cells. Tiam1-deficient tumour susceptibility and disease progression. tumours were not only fewer but also smaller than wild-type tumours and this correlated with Similarly to Ras, Rho proteins such as Rac1, RhoA increased apoptosis and reduced proliferation in and Cdc42 are guanine nucleotide binding proteins carcinogen-exposed skin of Tiam1-deficient mice. that cycle between an inactive GDP-bound state and an active GTP-bound state. In the active state, Tiam1 is also a potent modifier of intestinal Rho proteins bind and stimulate effector molecules tumourigenesis (Malliri et al., J Biol Chem 2006; that in turn govern cell morphology, adhesion, 281: 543). The majority of intestinal tumours are motility, as well as cell cycle progression and cell caused by mutations in the canonical Wnt signaling survival. Of relevance to cancer, Rho proteins are pathway, leading to its activation. However, few transforming in in vitro assays, particularly when genes targeted by this pathway have been demon- expressed in combination with Ras effectors, and strated to affect tumour development in vivo. Tiam1 they are required for Ras-induced transformation. is a Wnt-responsive gene. It is expressed in the pro- The activity of Rho proteins is controlled by gua- liferative compartments (crypts) of the adult mam- nine nucleotide exchange factors (GEFs) and malian intestine where the Wnt pathway is normal- GTPase-activating proteins (GAPs). GEFs activate ly active. It is also up-regulated in adenomas from small GTPases by promoting the exchange of GDP patients with either sporadic colorectal polyps or for GTP, whereas GAPs enhance the intrinsic rate familial adenomatous polyposis (FAP), as well as in of hydrolysis of bound GTP for GDP, leading to adenomatous polyps in Min (multiple intestinal inactivation. Tiam1 (for T-lymphoma invasion and neoplasia) mice. In each instance, the Wnt pathway metastasis protein) belongs to the GEF family of is hyperactivated due to a mutation in the apc proteins and selectively activates Rac in response to tumour suppressor gene. Further, by comparing growth factors and cell-substrate interactions. tumour development in Min mice expressing or Interestingly, Tiam1 preferentially associates with lacking Tiam1, it was found that Tiam1 deficiency activated GTP-bound Ras through a Ras-binding significantly reduces the formation as well as domain (RBD). Activated Ras and Tiam1 synergize growth of polyps in vivo (Malliri et al., J Biol Chem to induce formation of Rac-GTP (Lambert et al., 2006; 281: 543). Nature Cell Biol 2002; 4: 621). Further, Tiam1- deficient cells are resistant to Ras-induced cellular These two studies on tumourigenesis in vivo demon- P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 20

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    CELL SIGNALLING strate that two independent oncogenic signalling do more than simply activate Rho molecules, and pathways of major clinical significance (Ras and several studies now point to their role in influencing Wnt) recruit the Tiam1-Rac signalling pathway by the choice of biological response elicited by a given specific, albeit distinct mechanisms. In the context Rho protein. GEFs have been shown to bind to of oncogenesis, activation of this signaling module effectors directly or to scaffold proteins that com- promotes tumour initiation and growth. Moreover, plex with components of effector pathways. Tiam1 this role is specific to Tiam1 since its loss cannot be contributes to the signalling specificity downstream compensated for by other Rho GEFs. of Rac via associating with IB2/JIP2, a scaffold that promotes Rac activation of p38 kinase cascade Tiam1/Rac signalling and tumour invasion over JNK MAP kinase cascade (Buchsbaum et al., The skin carcinogenesis model revealed an addi- Mol Cell Biol 2002; 22: 4073). Tiam1 can also influ- tional role for Tiam1 in tumourigenesis. The few ence Rac signalling specificity through its interac- skin tumours arising in Tiam1-deficient mice pro- tion with spinophilin, a scaffold that binds to p70 gressed more frequently to malignancy than those S6K, another kinase regulated by Rac. Spinophilin in wild-type mice, suggesting that Tiam1 deficiency binding supresses the ability of Tiam1 to activate promotes malignant conversion (Malliri et al., Pak1, a different Rac effector (Buchsbaum et al., J Nature 2002; 417: 867). Analysis of Tiam1 expres- Biol Chem 2003; 278: 18833). In our lab, we are sion in skin tumours of wild-type mice revealed that using biochemical approaches to identify Rac and benign papillomas maintained high levels of Tiam1 Rac GEF interacting proteins involved in different expression, whereas expression was reduced in aspects of transformation including malignant pro- squamous cell carcinomas and was completely lost gression (acquisition of invasiveness). We are also in highly invasive spindle cell carcinomas. investigating the potential role of the Tiam1 homo- Paradoxically, the increased Ras signalling associat- logue, Stef, in tumourigenesis and the impact of ed with advanced skin malignancies (resulting from down-regulating more than one GEF simultaneous- amplification of the mutated Ras allele) seems to be ly in different aspects of the transformed pheno- responsible for the reduction or loss of Tiam1 type. expression in the later stages of tumour progres- sion, as demonstrated in vitro for Ras-transformed MDCK cells (Zondag et al., J Cell Biol 2000; 149: 775). Thus, while Tiam1/Rac co-operate with Ras in establishing tumours, they antagonize Ras during tumour invasion. Similarly in intestinal tumours, lack of Tiam1 increased the invasiveness of malig- nant cells (Malliri et al., J Biol Chem 2006; 281: 543). One probable mechanism by which Tiam1/Rac antagonizes malignant progression is through their ability to stimulate cell-cell adhesion. In vitro studies have shown that over-expression of activated Rac or Tiam1 can promote the formation of adherens junctions and the accompanying induction of an epithelioid phenotype in a number of cell lines (Malliri & Collard, Curr Opin Cell Biol 2003; 15: 583). Moreover, using both RNA interference and cells derived from Tiam1-deficient mice, it has been shown that endogenous Tiam1 is required for both The Rho GTPase cycle. Rho-like GTPases cycle the formation as well as the maintenance of cad- between an active GTP-bound and an inactive GDP- herin-based adhesions (Malliri et al., J Biol Chem bound form. This is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating pro- 2004; 279: 30092). Intriguingly, Asef, another Rac teins (GAPs). Guanine nucleotide dissociation specific exchange factor, promotes intestinal inhibitors (GDIs) inhibit nucleotide dissociation and tumour cell migration and invasiveness in in vitro control cycling of Rho GTPases between membrane models through down-regulating cadherin-mediat- and cytosol. Signals like growth factors, extracelullar matrix (ECM) or lysophosphatidic acid (LPA) are able ed adhesion (Kawasaki et al., Nature Cell Biol 2003; to activate Rho-like GTPases. Active GTPases interact 5: 211). with effector molecules to elicit various cellular responses. Additionally GEFs could work as scaffold The diverse and even opposing roles of Rho GEFs proteins by either binding directly to Rac effectors or other scaffold proteins that bind to effectors. in certain processes clearly indicate that Rho GEFs Publications listed on page 58 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 21

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    GROUP LEADERS CLINICAL FELLOWS UNDERGRADUATE Caroline Dive Ruth Board STUDENT Malcolm Ranson Emma Dean Emma Judson (Director of the Derek Alastair Greystoke Crowther Unit) Gavin Wilson KEY COLLABORATORS Gordon Jayson SENIOR LECTURER / SCIENTIFIC OFFICERS (Medical Oncologist, PAEDIATRIC ONCOLOGIST Juliana Bales gynaecological tumours) Guy Makin Karen Brookes Fiona Blackhall Fouziah Butt (Medical Oncologist, lung STAFF SCIENTISTS Martin Dawson cancer) Tim Ward Olive Denenny Mark Saunders Jeff Cummings Martin Greaves (GI Clinical Oncologist) Cassandra Hodgkinson Noel Clarke (GU Surgeon) POSTDOCTORAL FELLOWS Alison Hogg Richard Byers (Pathologist) Clare Dempsey Amanda Lomas Tony Whetton (Clinical Sarah Holt Lourdes Ponce Perez Proteomics) Jian Mei Hou Nigel Smith Crispin Miller (Bioinformatics) Deema Hussein Elizabeth Sweeney Andrew Hughes David Moore (Translational Oncology) Flavia Moreira Leite GRADUATE STUDENTS Christopher Morrow Jane Barraclough Darren Roberts Martin Brandenburg Stephen St George Smith Christina Fernandez Kathryn Taylor Dimitra Micha Arek Welman Isabel Pires Clinical and Experimental Pharmacology Group http://www.paterson.man.ac.uk/groups/cep.jsp CEP incorporates teams in pre-clinical drug target validation Clinical Trial Facilities at the Christie Hospital’s DCU and biomarker discovery. Within the recently refurbished, Our translational research is associated directly with specialised PACCAR Good Clinical Laboratory Practice clinical trials in DCU. During 2005/2006, DCU had 118 trials involving 635 patients. The Christie (GCLP) laboratory CEP also develop, validate and imple- Hospital NHS Trust, became an Experimental ment pharmacokinetic (PK) and pharmacodynamic (PD) Cancer Medicines Centre (ECMC) led by Malcolm assays for Phase I trials at the Christie Hospital’s Derek Ranson in 2006 and recent award of a Clinical Research Infrastructure bid will fund a doubling of Crowther Unit (DCU). Our research focuses on novel DCU capacity making it one of the largest units of agents targeted to apoptosis pathway components (e.g. IAP its kind worldwide. The CR-UK Phase I trial of and Bcl-2 inhibitors) and, in collaboration with Gordon Aegera Therapeutics’ AEG35156 (anti-sense XIAP) showed that it is well tolerated up to Jayson, we are validating a panel of biomarkers for trials of 125mg/m2 x 7 days every 3 weeks with dose limit- anti-angiogenic drugs (e.g. GSAO). Technology development ing toxicity of asymptomatic, reversible increase in initiatives in CEP include generation of preclinical models liver enzymes. There was preliminary evidence of single agent anti-tumour activity in refractory breast with inducible expression of wild type or mutant drug tar- cancer and in refractory non-Hodgkin’s lymphoma. gets, optimisation of serum proteomics in a new clinical pro- The Phase I Trial of Allos Therapeutics’ bio-reduc- teomics facility, the isolation of circulating tumour cells, the tive alkylating agent RH-1 concluded this year. Clinical PK/PD data are now being integrated and use of Q Dots to enhance immuno-histochemical (IHC) modelled in collaboration with Prof Leon Aarons, analyses and implementation of multiplexed bead arrays. UoM. P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 22

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    C L I N I C A L A N D E X P E R I M E N TA L P H A R M AC O L O G Y The Critical Importance of Biomarkers in Early across our paediatric cell line panel. Repression of Clinical Trials XIAP by shRNAi or antisense oligonucleotide can In an era of mechanism-based therapies there is sensitise to cytotoxic agents. A novel XIAP small clear imperative to determine the right dose and molecule inhibitor is effective against these cell lines schedule for the right patient and thus increased and synergizes with conventional cytotoxic agents requirement for pharmacodynamic (PD) biomark- in a number of them. ers. These biomarkers allow proof of concept demonstrations of target modulation in a patient; Biomarker Discovery they can facilitate rational choice of correct dose In a joint venture with Tony Whetton’s group and schedule, and, if implemented to the appropri- (UoM) and in collaboration with Beyond Genomics ate quality standard they can contribute to clinical Medicine Inc, we are optimising a workflow for decision making. Ultimately biomarkers can help large-scale proteomic screening of human plasma. explain or predict clinical outcomes and decrease With a pilot clinical study and process validation costs of drug development. CEP has therefore put underway, and recruitment of a bioinformatician increased emphasis on development and validation (in collaboration with Crispin Miller, PICR), we of PD biomarkers and on biomarker discovery anticipate our first clinical proteomics data sets strategies. early in 2007. Pharmacodynamic Assay Development Biomarkers Club and the AstraZeneca CEP has focussed on IHC, flow cytometric and Serological Biomarker Alliance ELISA-based PD biomarkers. With a Bcl-2 small AZMU Biomarkers Club, a forum to discuss bio- molecule inhibitor phase I trial due to be initiated in markers of efficacy and toxicity with colleagues at DCU in 2007, we worked up a series of IHC meth- AstraZeneca met on the topics of apoptosis, inva- ods to detect Bcl-2 family proteins and apoptosis. sion and hypoxia. The recruitment of three staff We have progressed and validated markers of and purchase of an ELISA Robotic station in 2006 tumour vasculature and vascular density assessment activated the AZ/CEP Serological Biomarker within biopsies. In collaboration with AstraZeneca Alliance to investigate surrogate markers of tumour and Richard Byers (UoM), CEP is also evaluating cell death in AstraZeneca-driven clinical trials. the utility of Q-dot technology for multi-analyte detection in IHC. Multiple serological ELISA-based biomarkers have been evaluated for clinical use including three surrogate markers of cell death and a panel of biomarkers of angiogenesis. CEP is cur- rently developing methods to utilise cytometric bead arrays to measure multiple analytes in small volume clinical samples. Protocol development is ongoing to implement 4 colour flow cytometric analysis of apoptosis in serial samples from lym- phoma patients (see Figure). Development of a GCLP Quality System To use biomarkers for clinical decision making, studies must be performed to GCLP standard. With expansion of the QA team and relocation to the TRF, the QA system now covers facilities com- pliance, equipment qualification, resource logs and archiving. CEP continues to innovate in QA/lab management with introduction of pro forma lab books and fast track validation, establishing the lab/clinic (CEP/DCU) QA interface and develop- ment of custom designed electronic resources. Four-colour Flow Cytometric Apoptosis Assay Pre-clinical validation of novel agents against paediatric tumours. Cell death in lymphoma cells from patients treated with RH-1 and a novel HDAC inhibitor demonstrated AEG 35156 XIAP antisense was assessed using CD20 pos- effectiveness against paediatric tumour cell lines in itive staining to discriminate lymphoma cells, Annexin V for vitro; RH-1 also showed efficacy in vivo. We validat- early apoptosis and cleaved caspase 3,7 for late apoptosis with membrane permeable (necrotic) cells staining positive- ed two agents that target XIAP which is expressed ly for 7-AAD. Publications listed on page 58 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 23

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    GROUP LEADER POSTDOCTORAL CLINICAL RESEARCH Peter L Stern FELLOWS FELLOWS Eyad Elkord Likhith Alakandy Hui-Rong Jiang Sai Daayana David Shaw (with HC Kitchener) Tom Southgate Christy Ralph (with Medical Oncology) SCIENTIFIC OFFICERS Ursula Winters Debbie Burt (with HC Kitchener) Kate Mulryan GRADUATE STUDENTS Graeme Smethurst (with Crispin Miller) Helen Spencer (with Chris Ward) ROTATION STUDENTS Ester Martin Immunology Group Rebecca Roberts http://www.paterson.man.ac.uk/groups/immuno.jsp A consistent theme of the Immunology Group’s research has tion of N-cadherin proteins. The E- to N-cadherin switch during ES cell differentiation correlates with been the investigation of shared properties of developmental up-regulation of Snail and Slug proteins, both of tissues and tumours or cancer cells with a view to identifying which show nuclear localisation in OCT-4 negative new targets/markers of value in diagnosis, prognosis or ther- cells. ES cell differentiation is also associated with up-regulation of transcripts encoding matrix metal- apy. One particular success has been the 5T4 oncotro- loproteinases (MMP)-2 and -9 and increased gelati- phoblast molecule where several different types of nase activity. Although E-cadherin is down-regulat- immunotherapies have progressed through preclinical and ed during ES cell differentiation it is not required for maintenance of undifferentiated ES cells. early clinical studies and are now entering phase III studies. Furthermore, E-cadherin, N-cadherin and 5T4 pro- Our clinical studies of 5T4 and HPV related therapies are teins are independently regulated during ES cell dif- now reported in the Biological Immune and Gene Therapy ferentiation and are not required for induction of epithelial-mesenchymal transition (EMT)-associat- section. In the past year we have investigated the early dif- ed transcript expression. Abrogation of E-cadherin ferentiation of embryonic stem cells of both mouse and mediated cell-cell contact in undifferentiated ES human to further study the cellular role of 5T4 molecules. cells resulted in a reversible mesenchymal pheno- type in the absence of EMT-associated transcript In addition we have exploited the early upregulation of sur- expression. The loss of cell surface E-cadherin in face 5T4 expression of embryonic stem (ES) cells committed undifferentiated ES cells results in translocation of to differentiation rather than self renewal, to identify addi- the 5T4 antigen from the cytoplasm to the cell sur- face in an energy-dependent manner. We conclude tional gene expression changes which may be important in that E-cadherin protein is likely to function in ES both development and cancer. cells to stabilise cortical actin cyoskeletal arrange- ment and this can prevent cell surface localisation 5T4 expression in ES cells of the 5T4 antigen, a newly identified component The 5T4 oncofoetal antigen is a cell surface glyco- of the EMT process. Interestingly, a study (with protein that is transiently expressed during mouse Renehan, O’Dwyer et al. Dept. Surgery) of ES cell differentiation and correlates with decreased pseudomyxoma peritonei (PMP), a rare neoplasm pluripotency of such cells. We have now shown of mainly appendiceal origin, demonstrated a spe- that the 5T4 antigen is a transient marker of human cific pattern of adhesion-related protein expres- ES cell differentiation and that 5T4 phenotype, sions of increased N-cadherin, reduced E-cadherin, colony seeding density and culture conditions sig- and increased vimentin (P=0.004), a phenotype nificantly influence the maintenance of pluripotent suggesting a possible epithelial–mesenchymal tran- hES cells and their differentiation to neural lineag- sition state. The similarities between PMP and col- es (Exp Cell Res. 2006; 312: 1713-26). In collabo- orectal adenocarcinoma, also reveal a specific cad- ration with Chris Ward, we have shown that mouse herin phenotype that may characterise the distinct and human ES cell differentiation is also associated non-metastasising behaviour of PMP (Brit. J. with loss of cell surface E-cadherin and up-regula- Cancer 2006; 95: 1258–1264) P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 24

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    IMMUNOLOGY 5T4 has been used as a marker to define and sepa- numbers of CD4+CD25high regulatory cells which rate populations of pluripotent mouse ES cells express the FOXP3 marker. We are examining the (5T4 negative) and early differentiating cells (5T4 modulation of repertoire of recognition of 5T4 in positive) for comparison by microarray analysis. patients in trials aimed at reducing T regulatory Many established transcriptional changes that occur activity in CD25+ depletion in renal cell carcinoma during differentiation were identified, including the and in upper gastrointestinal cancer patients receiv- down-regulation of the transcription factors ing anti-CTLA-4 therapy. Krüppel like factor 4 and oestrogen receptor relat- ed ß, very recently suggested to have roles in ES cell HPV studies self-renewal. In addition, a number of previously High risk HPV infection is a necessary cause of cer- unreported transcriptional changes were identified. vical cancer. An ideal vaccine would have both pro- One of these was the significant down-regulation phylactic and therapeutic effects. Such a vaccine of CD26 during differentiation. CD26 is a multi- would have immediate benefit in contrast to the functional cell surface molecule which inhibits inevitable delay before the clinical impact of pro- SDF-1 chemokine induced cell migration. In vari- phylactic vaccines. A fusion protein comprising ous cancers loss of CD26 expression is associated HPV16 L2, E6 and E7 is a candidate combination with increased metastatic potential and SDF-1 preventive and therapeutic HPV vaccine. In collab- responsiveness (Figure). The inverse correlation oration with Richard Roden at Johns Hopkins between 5T4 and CD26 expression during mouse University we have analysed sera from previous ES cell differentiation, and the known roles of clinical trials of the TA-CIN vaccine for L1 and L2- these molecules in cell migration/motility, suggests specific and neutralising serum antibody titers. In that molecular events are common to both ES cell particular vaccination three times at monthly inter- differentiation and tumour metastasis. vals in a phase I randomised double-blind placebo Understanding the co-ordination of these events controlled dose escalation trial in 40 healthy volun- may provide new strategies for cancer treatment. teers, and a phase II trial of TA-CIN at the maxi- mum dose in 29 women with high grade anogenital In our ongoing studies into the 5T4 oncofoetal intraepithelial neoplasia (AGIN). Vaccination of antigen we have backcrossed 5T4 KO heterozygote healthy volunteers induced L2-specific serum anti- mice against the 129 and C57BL/6 backgrounds bodies that were detected one month after the final allowing us to assess the consequences of the KO vaccination and neutralized both HPV16 and phenotype. The null 5T4 C57Bl/6 animals are HPV18 in vitro. There was also an antigen-specific viable but adult animals show some structural disor- proliferative response of the vaccinated healthy vol- ganisation within the brain and exhibit a high fre- unteers that showed a significant trend with increas- quency of hydrocephaly. Importantly, the 5T4 null ing vaccine dose and correlated with the L2-specif- mice have been used to generate useful monoclon- ic antibody response. However, the AGIN patients al antibodies and cytotoxic T cell responses against responded less effectively to vaccination at the the mouse 5T4 target. This will allow the detailed highest dose than the healthy patients with respect analysis of the consequences of generation of to the induction of HPV16 L2-specific and neutral- autologous antigen specific immunity in animals as ising antibodies and proliferative responses. We are a result of vaccination (Cancer Immunol exploring the means to increase such cross neutral- Immunother. 2006; 56 : 165-180), adoptive transfer ising antibody titres with different adjuvants. of natural or targeted T cells or their combination use (e.g. J Immunology 2006; 177: 4288-98), all of which are potential goals for clinical trials in man. 5T4 immunotherapy We have shown that human CD8-T cell repertoire versus 5T4 antigen exists and can be detected in the absence of CD4 T cells (either helper or regulatory) and HLA-A2 epitopes have been identified (Int J Cancer. 2006; 119: 1638-47). Significant 5T4 responses of CD4 T cell enriched populations are most apparent with depletion of the CD25+ T cells and some MHC Class II restricted epitopes have been identified. These observations imply that the availability of the 5T4 repertoire of both CD4 and CD8 T cells may be optimally accessed in the absence of T regulatory activity. In many different types of cancer there are significantly increased Publications listed on page 60 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 25

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    GROUP LEADER POSTDOCTORAL SCIENTIFIC OFFICERS Jamal Zweit FELLOWS John Bailey James Gillies Lynn Disley Christian Prenant Nigel Smith David Shaw (with Immunology) GRADUATE STUDENT Sadia Ashraf Radiochemical Targeting and Imaging Group http://www.paterson.man.ac.uk/groups/rti.jsp Research activity over the last year focussed on i) developing radio-quantum dot approach to multi-modality imaging. We have synthesised radiotracer quantum multi-modality imaging with radio-quantum dots, allowing dots which emit both gamma-ray and fluorescence both radionuclide and fluorescent signals to be detected signal. Such probes were found to be non-toxic in using the same probe and ii) investigation of 18F- various cell lines and were significantly (40-60% in 30 minutes) incorporated in lung and colorectal Fluorothymidine ([18F]-FLT) as a PET probe for cell prolifera- tumour cells. Whole body autoradiography of tion. In the quantum dot project, a 109CdSe(ZnS) probe 109 CdSe/ZnS quantum dots (630nm) showed accu- capable of detecting a combined fluorescence and radionu- mulation mainly in the RES system followed by metabolism and clearance through the liver and kid- clide signal was developed and evaluated in in vitro and in neys (figure 1). In tumour-bearing mice, the uptake vivo studies. Radio-quantum probes were synthesized at a of 109CdSe/ZnS quantum dots at 24 hours was 10- tracer level, hence overcoming a major toxicity issue associ- fold higher than at 1 hour. Uptake is mainly driven by endocytosis with greater retention in tumour ated with conventional fluorescence only quantum dots. The cells than in normal organs including non-RES tis- bio-distribution and autoradiography profile of intravenously sues. To demonstrate non-toxicity, stability and administered probe showed accumulation in reticulo- biological compatibility, the radio-quantum dots were conjugated to the anti-CD20 monoclonal anti- endothelial system (RES) tissues, particularly the liver and body rituximab and the binding of the probe was spleen, followed by metabolism and significant clearance over demonstrated in lymphoma xenografts expressing 24 hours. To demonstrate biological targeting, the radio- the CD20 protein (figure 2). quantum dot was conjugated to the monoclonal antibody Nucleoside-based PET probes retuximab and found to bind at the surface of lymphoma The development of [18F]-FLT as a probe to meas- cells expressing the anti-CD20 protein target. In the second ure cell proliferation has continued over the last year. In terms of thymidine kinase activity, in both project we have investigated the uptake kinetics of [18F]-FLT normal and tumour bearing animals, FLT showed and enzyme activity in tumour cell lines and related the higher level of the kinase in proliferative tissues nucleoside kinase activity to the concentration of radiotracer (bone marrow, spleen, intestine) as well as in NSCLC xenografts. Despite high thymidine phos- in tumour samples. Nucleoside kinase activity (pmoles per phorylase activity of [124I]-IUdR, measured as the µg protein) of tissues from H460 tumour-bearing mice were number of cleaved iodouracil molecules, the higher higher for 3H-thymidine and [18F]-FLT than for radioiodinated stability of [18F]-FLT resulted in higher and more specific uptake of the PET signal in both tumours IUdR and FIAU. The higher in vivo stability of [18F]-FLT com- and the proliferative cells of the bone marrow, pared to the iodinated analogues enabled better cell incor- spleen and intestine. These studies, along with our poration and its improved overall kinetics showed better previous observation, of the [124I]-IUdR kinetic profiles in animal and human studies indicate that reflection of S-phase enzyme activity than in the case of the despite the higher DNA incorporation of [124I]- iodinated nucleosides. IUdR, it has lower specific signal sensitivity com- pared to [18F]-FLT due to the extensive metabolism Radiotracer quantum dots of the radioiodinated pyrimidine analogue. This We have established a model system, based on observation was substantiated in our pilot clinical 109 CdSe, for the development and validation of the study which showed a biodistribution pattern main- P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 26

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    R A D I O C H E M I C A L TA R G E T I N G A N D I M A G I N G ly of radiolabelled metabolites in the case of the study the pharmacokinetics of such therapy, we [124I]-IUdR compared to [18F]-FLT. have, in collaboration with Immunology and Medical Oncology, designed, optimised and validat- Imaging pharmacokinetics of superantigen tar- ed a clinical labelling strategy to label the drug geted therapy ANYARA with the positron emitting isotope 124I. Clinical trials of superantigen targeted therapy Proof of principle of drug targeting was demon- directed against the tumour associated antigen 5T4 strated in renal cancer patients and the radiolabelled have shown clinical efficacy in lung cancer (unpub- drug uptake was found to be antigen-specific as val- lished) and in renal cancer (Shaw et al in press). In idated by immuno-histochemistry on biopsy sam- the latter study, a sub-set of patients was assessed ples. for clinical effect using FDG-PET, which gave a reliable, early indication of disease control which correlated with long term survival. In order to Figure 1. Whole body autoradiographs of 109 CdSe/ZnS radio-quantum dots following intravenous administration of the probes into mice. Figure 2. Conjugation of 109CdSe/ZnS radio-quantum dots to the anti-CD20 monoclonal antibody rituximab (top) and binding of the conjugated antibody to the surface of lymphoma cells expressing the CD20 protein (bottom). Publications listed on page 61 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 27

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    GROUP LEADER POSTDOCTORAL SCIENTIFIC OFFICER Georges Lacaud FELLOWS Catherine Gavin Mark Holland Christophe Lancrin GRADUATE STUDENTS Flor Perez-Campo Kelly Chiang (with V. Kouskoff) Laura Edwards (with C. Miller) Patrycja Sroczynska Malgorzarta Gozdecka Stem Cell Biology Group http://www.paterson.man.ac.uk/groups/scbio.jsp The AML1/Runx1 transcription factor is a frequent target of The existence of the haemangioblast has been demonstrated recently in vivo in early embryos. gene rearrangements and mutations in human acute myel- ogenous leukemia (AML) and acute lymphoblastic leukemia Development of blast colonies and characteriza- (ALL). Consistent with its initial implication in leukemias, tion of the onset of blood cell development. We have initiated a series of studies to determine Runx1 has been shown to be critical for normal the series of cellular events leading to the genera- haematopoietic development. The MOZ gene is involved in tion of a blast colony from a BL-CFC. For this, we three independent myeloid chromosomal translocations fus- have followed the maturation of the precursor in time lapse experiments and identified different ing MOZ to the partner genes CBP, P300 or TIF2. developmental stages. In collaboration with Terry Using the in vitro differentiation system based on Allen (Structural Cell Biology Group) we are cur- mouse embryonic stem (ES) cells and mouse mod- rently further investigating by electron microscopy els, our goals are to further define the role of Runx1 these early events of generation of blood cells. and MOZ in early haematopoietic development and how alterations of their function leads to leukemo- genesis. Early haematopoietic development and haemangioblast The earliest site of blood cells development in the mouse embryo is the yolk sac where blood islands, derived from mesodermal cells, develop at approxi- mately day 7.5 of gestation. The yolk sac blood islands consist of two lineages, a population of primitive erythroid cells surrounded by a layer of angioblasts that eventually form the developing vas- culature. The parallel development of these lineag- es in close association provided the basis for the hypothesis that they arise from a common precur- sor, a cell called the haemangioblast. Figure1: Electron microscopy of early haematopoietic cells generated from ES cells. The differentiation of embryonic stem (ES) cells in culture offers a powerful alternative approach to study the development of lineages that are estab- A critical function of Runx1 in Haemangioblast lished very early in embryonic life. Using this development. model system, a precursor was identified that gen- To investigate the role of Runx1 at the earliest stage erates blast colonies containing precursors of of haematopoietic commitment, we have analysed endothelial and haematopoietic lineages. The blast its expression pattern and function during ES/EB colony-forming cells (BL-CFC) that generate these differentiation and in early yolk sac development. colonies represent a transient population that Expression analyses indicated that Runx1 is appears in the embryoid bodies (EBs) prior to the expressed in yolk sac mesodermal cells prior to the emergence of any other haematopoietic lineage establishment of the blood islands and within the precursors. The characteristics of the BL-CFC sug- BL-CFC in EBs. Analysis of early EBs revealed a gest that it represents the in vitro equivalent of the profound defect in the potential of the Runx1-/- haemangioblast and as such the earliest stage of ES cells to generate blast colonies. Altogether these haematopoietic development described to date. results provide evidence that Runx1 does function P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 28

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    STEM CELL BIOLOGY at the haemangioblast stage of development and the specific expression of these isoforms. We are position Runx1 as a master regulator of the onset of currently investigating the respective expression of blood development. these isoforms, the biological potential of the cells expressing them and the respective function of Downstream transcriptional targets of Runx1 at each isoform. the onset of blood development Our results have indicated that Runx1 is required A critical function of MOZ in haematopoietic for definitive haematopoietic development at the development. level of the BL-CFC and is therefore likely to regu- MOZ is found translocated in cases of AML with late a specific set of genes at this time of develop- either CREB binding protein (CBP), the nuclear ment. To identify these genes, we have compared receptor TIF2 or the P300 transcriptional co-activa- the patterns of gene expression of haemangioblast- tor. All these genes encode enzymes containing a enriched cell populations or haemangioblast- histone acetyl transferase domain (HAT) suggesting derived cell populations from either Runx1 deficient that aberrant modification of histones or other fac- or Runx1 competent ES cells. We have then validat- tors could provide the first step in the route to ed the differential expression of candidates on sam- oncogenicity. To evaluate the role of the HAT ples generated from the ES/EB system. We have activity of MOZ, we have created a mouse model further documented the regulation by Runx1 of the with a point mutation inactivating the HAT activity transcription of several of these genes by promot- of MOZ. Analysis of these mice has revealed a er assays or chromatin immunoprecipitation. We profound defect in haematopoiesis. The numbers are currently evaluating the specific function of of haematopoietic foetal liver precursors is dramat- these genes at the onset of haematopoietic develop- ically affected by the mutation. Haematopoietic ment. stem cells carrying the mutation present a strong defect in competitive repopulation assays. These in Runx1 DNA binding partners vivo results were substantiated with ES cells mutat- We have created Runx1 variants containing a N- or ed for the HAT activity of MOZ. Much less primi- C- tag which can be biotinylated in vivo by a biotin tive and definitive haematopoietic precursors were ligase enzyme. We have validated that both con- generated with these mutated cells. These studies structs are still able to rescue the haematopoietic with embryonic stem cells allowed us to further pin- defects in Runx1 deficient cells. We have expressed point the defect in absence of the HAT activity of both constructs in human Jurkat T cells and puri- MOZ to the balance between normal proliferation fied proteins associated with Runx1 using strepta- and differentiation of haematopoietic precursors. vidin beads. Peptide sequencing and mass spec- We are currently trying to identify the critical genes trometry indicated the association of Runx1 with whose expression is epigenetically regulated by the several components of the nucleosome remodeling HAT activity of MOZ. and histone deacetylase (NuRD) complex, one of the major transcriptional co-repressor complexes in 2500 100 EryP HAT +/+ EryD mammalian cells. Future experiments will investi- 2000 HAT +/- 80 HAT -/- gate using this experimental approach the nature of 1500 60 the proteins associated with Runx1 at the onset of 1000 40 haematopoietic development. 500 20 0 0 Runx1 isoforms 5 6 7 8 9 10 11 5 6 7 8 9 10 11 Days Days Previous studies have shown that Runx1 is 500 1200 Mac Mast expressed as multiple naturally occurring spliced 1000 400 isoforms that generate proteins with distinct activi- 800 300 ties on target promoters. One intriguing hypothesis 600 200 is that the different isoforms of this transcription 400 100 factor could fulfill distinct functions at the different 200 0 0 stages of the establishment of the haematopoietic 5 6 7 8 9 10 11 5 6 7 8 9 10 11 Days system. We have determined that expressions of Days some of these isoforms are differentially regulated Figure 2: Analysis of primitive and definitive haematopoietic potential of during early haematopoietic development. We have HAT+/+, HAT+/- and HAT-/- MOZ ES cells. Primitive erythroid (EryP), generated ES cells containing reporter genes knock- definitive erythroid (EryD), macrophage (Mac) and mast cell (Mast) in in the different isoforms or knock-out altering colonies generated by HAT+/+, HAT+/- and HAT-/-. Publications listed on page 61 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 29

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    GROUP LEADER POSTDOCTORAL GRADUATE STUDENTS Valerie Kouskoff FELLOWS Rebecca Baldwin Arnaud Gandillet (50%, with Cathy Merry) Alicia Gonzalez- Katalin Boros Serrano Kelly Chiang Andrew Williamson (with Georges Lacaud) Sarah Lewis SCIENTIFIC OFFICER Stella Pearson Stem Cell and Haematopoiesis Group http://www.paterson.man.ac.uk/groups/sch.jsp The differentiation of embryonic stem (ES) cells offers a conditions, fetal calf serum been one of the key components of the culture conditions. Although powerful approach to study mechanisms implicated in cell the inherent characteristics of ES cell lines are like- fate decision. A major hurdle however is to promote the ly to be important for an efficient differentiation, directed and efficient differentiation of ES cells toward a spe- the batch of serum used appears critical in obtain- ing the most effective differentiation. This fact cific lineage. To overcome that challenge, we have estab- illustrates the complexity of serum composition lished a serum-free culture system allowing the selective and and the many parameters that may modulate posi- efficient differentiation of ES cells toward haematopoietic tively or negatively the differentiation process. To dissect and understand the molecular mechanisms progenitors. We have defined in serum-free media the mini- of cell fate specification, refined culture conditions mal growth factors that are required to control each step of are needed to allow the specific and efficient differ- the differentiation process. BMP4 (Bone morphogenetic entiate of ES cells toward haematopoiesis. protein 4) promotes the very efficient formation of meso- Selective and efficient differentiation of ES cells derm, FGF (Fibroblast growth factor) and Activin A induce toward the formation of blood progenitors: the differentiation of these mesodermal precursors to the To define the minimal requirement for the genera- tion of blood progenitors from mouse ES cells in haemangioblast fate, and VEGF (Vascular endothelial growth absence of serum, we assessed several serum-free factor) is required for the production of fully committed media and soluble factors previously shown to haematopoietic progenitors. This step-wise control of differ- induce some degree of mesoderm or haematopoiesis specification. To monitor mesoder- entiation is extremely efficient and provides a perfect system mal differentiation, we used an ES line (GFP-Bry) for understanding the molecular machineries involved in carrying the cDNA for green fluorescence protein blood progenitor commitment. targeted to the Brachyury locus. We have previous- ly shown using this ES cell line that GFP expression In vitro differentiation of ES cells as a model sys- recapitulated effectively Brachyury expression and tem to study lineage specification: could be used to track mesoderm formation in vitro Upon differentiation, mouse ES cells can give rise and in vivo. Using the in vitro differentiation of to primitive and definitive haematopoietic precur- GFP-Bry ES line, we have studied the requirement sors, an in vitro process that was shown to accurate- for each step of the differentiation (figure 1): ES ly recapitulate the in vivo development of yolk sac cells to epiblast-like cells (step 1), epiblast-like cells haematopoiesis. This progressive differentiation to mesodermal precursors (step 2), mesodermal can be monitored by measurement of gene expres- precursors to haemangioblast (step 3) and haeman- sion and quantitative analysis of biological poten- gioblast to committed blood precursors (step 4). tial. As they lose their self-renewal and pluripotent characteristics, ES cells form epiblast-like cells Altogether, our data demonstrate that commitment which differentiate further to give rise to mesoder- to the haematopoietic lineage can be driven effi- mal precursors. The first blood precursor, the hae- ciently by only four factors in the absence of serum mangioblast, derives from the mesoderm and gives and that each step of this differentiation can be rise to primitive and definitive haematopoiesis, controlled by only one or two factors (Figure 1). smooth muscle and endothelium. This carefully The removal of leukemia inhibitor factor (LIF) and orchestrated process can be reproduced in vitro feeder cells which together keep ES cell undifferen- upon the differentiation of ES cells in well-defined tiated, is sufficient to trigger the progression from P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 30

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    S T E M C E L L A N D H A E M ATO P O I E S I S ES cell to epiblast-like cell stage (step 1). These either without (T6) or with the addition of Activin data suggest that no added factors are necessary for A/FGF (T6+). Secondary replating assay was per- the transition from ES cells to epiblast-like cells. formed to assess the number of haemangioblast The transition from epiblast-like cells to mesoder- precursors in each population (Figure 2A). Analysis mal precursors (step 2) requires the addition of of the microarray data revealed that 33 genes were BMP4 in a dose dependent manner. The specifica- differentially regulated with a fold change greater tion of haemangioblast from the mesodermal pre- than 2 and with a p value lower than 0.001 (Figure cursors (step 3) is induced very efficiently and rap- 2B). We are now in the process of investigating the idly upon stimulation with FGF and Activin A. The role of these selected genes in the specification of last step of differentiation (step 4), allowing the for- haemangioblast precursors. mation of committed blood precursors from hae- mangioblast is triggered upon the addition of VEGF to the culture already stimulated by the ES cells sequential addition of BMP4, Activin A and FGF. The very high fraction of haematopoietic cells Step 1: LIF and feeders within the EBs underscores the efficiency with removal which this combination of factors specifically pro- Epiblast-like cells motes haematopoietic commitment at the expense of other lineages. Step 2: BMP4 Molecular analysis of haemangioblast precursor specification: Mesoderm A robust increase in the number of haemangioblast precursors can be detected within a few hours of Step 3: Activin A stimulation with Activin A and FGF (Figure 2). FGF This very rapid response suggest that Activin A/ Haemangioblast FGF might promote the specification of mesoder- mal precursors to the haematopoietic program Step 3: VEGF rather that inducing the proliferation of a few hae- mangioblast already present in the EBs. To dissect Committed the molecular mechanisms implicated in haeman- Haematopoietic gioblast commitment, we performed a screening for Precursors genes implicated in this process via Affymetrix microarray analysis. Three independent samples for Figure 1: Schematic representation of ES cell differentiation to the haematopoietic fate. each time point were harvested: (T0) prior to stim- ulation with Activin A and FGF and 6 hours later B A 4000 T6+ B MP 4 B M P 4 + Ac tiv in/F G F 3000 33 genes FC>2 2000 Comparison of gene expression by gene chip micro-array analysis P<0.001 1000 T0 Blast per 100,000 cells T6 0 0 3 6 12 Activin A h o u r s p o s t in d u c tio n T0 T6 T6+ FGF Figure 2: (A) Kinetic of blast colony formation upon stimulation with Activin A and FGF. (B) Heat map representation of transcripts found to be differentially expressed upon Activin A and FGF induction. Publications listed on page 62 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 31

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    GROUP LEADER POSTDOCTORAL VISITING FELLOWS Terence D Allen FELLOWS Elena Kiseleva Sheona Drummond Fiona Gardiner SCIENTIFIC OFFICERS Sandra Rutherford Stephen Murray Structural Cell Biology Group http://www.paterson.man.ac.uk/groups/scb.jsp Higher eukaryotes are characterised by the separation of The structural visualisation of cohesin and con- densin in complex with condensing chromatin. nucleus and cytoplasm with a nuclear membrane, which Cohesin is a multisubunit complex that mediates undergoes two major dynamic events during the cell cycle. sister-chromatid cohesion. Cohesin complexes link At mitosis the entire structure is dismantled and reformed, replicated DNA duplexes by forming huge ring structures which ‘embrace’ both duplexes until and during interphase the nuclear surface area and pore cleavage is brought about during anaphase by the complex populations increase by a factor of two. Our cysteine protease separase. We are studying cohesin experimental endpoint is direct, in situ, 3D macromolecular loading onto DNA duplexes in S-phase, investigat- ing both the possible de novo assembly of cohesin on visualisation of specific proteins and multiprotein complexes. chromatin and the transient opening of the cohesin These studies incorporate GFP labelling for live cell imaging ring to allow entry of DNA molecules. Cohesin coupled with immunogold localization at the EM level. We may also mediate the repair of double-strand DNA breaks. We are using immunogold and fluoro- are also investigating the roles of condensin and cohesin in nanogold labeling by light microscopy (LM) and the processes of chromatin condensation and chromosome Field Emission In Lens Scanning Electron separation. Collaborations within the Institute currently Microscopy (FEISEM) to investigate the predicted ring structure of cohesion. involve projects on apoptosis, yeast mitosis and haematopoi- etic stem cell interactions. Condensins are structurally related to cohesin, pos- sessing two SMC (Structural Maintenance of Chromosomes) core subunits, but differ in action to Nucleoporin interactions during nuclear pore produce condensation of replicated chromatin. formation. Condensins I and II consist of the hSMC2 and New nuclear pore complexes (NPC) are inserted hSMC4 core subunits in combination with three directly into the nuclear envelope (NE) throughout non-SMC regulatory subunits, forming distinct interphase. At mitotic NE reformation however, complexes which interact with chromatin at specif- the nucleoporin (nup)107/160 complex associates ic stages during condensation. Our data support a directly with the chromatin surface before NE model in which the condensins function as molecu- membrane reformation. This complex locates to lar hinges, contacting two points on a chromatin kinetochores during prophase, but also over the fibre in their open conformations and bringing chromosome arms from metaphase onwards. them into closer proximity upon its closure. Labelling for other structural nucleoporins by mAb 414 is absent from the chromosomal surface until Maintenance and regulation of structural anaphase, and appears to be concentrated around organization at the nuclear surface the spindle poles. Thus although NPC formation is Our studies into the function of the human ortho- initiated by binding of the nup107/160 complex to logue of the integral membrane nucleoporin gp210 chromosome surfaces at late metaphase, it may not have involved down-regulation of protein expres- be completed until the chromosomes have migrat- sion using the pSUPER vector system. FEISEM ed towards the spindle poles for recruitment of the studies have revealed that a reduction in gp210 remaining NPC structural elements. expression results in a loss of spatial organisation where individual NPCs are no longer maintained at P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 32

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    STRUCTURAL CELL BIOLOGY a uniform distance from each other. Ultra-structur- increase in c-SRC expression levels in a colorectal al visualisation indicates that the nuclear lamina cancer cell line (Welman et al Neoplasia 2006, 8, interacts with the “spoke-ring” component of 905), ultrastructural observations confirmed the NPCs. This is a large annular structure, thought to induction of a cell death pathway in detached cells be at least partly composed of gp210 oligomers, characterised by nuclear alteration and intense cyto- that encircles the channel through which molecules plasmic vacuolization, which was associated with are transported and is embedded within the lumen the accumulation of cells in the G2 phase (Figure of the NE. Therefore it is likely that this structure 1). contributes to the positional regulation of NPCs within the plane of the NE. FACS analysis of In collaboration with the Stem Cell Biology group, HeLa cells transfected with our pSUPER gp210 we have initiated investigations into the cellular knock-down constructs shows that a significant interactions during the early stages of blast colony proportion of these cells contain activated caspase- forming cell differentiation (see page 28). Figure 2 3. This strongly suggests that gp210 is an essential shows images visualizing cell interactions in protein without which cells initiate apoptosis. haematopoietic differentiation in vitro. Cultures have been observed by LM time lapse (Fig 2:2), then fixed in situ for both SEM and TEM with spe- cific areas relocated for direct comparison by verti- cal or ‘en face’ sectioning by TEM, (Fig 2:3) or whole cell interactions by SEM (Fig 2:1). Fig 1 Increased c-SRC expression in HCT 116 colorectal cancer cells results in intense cytoplasmic vacuolation and nuclear disorganization of detached cells, which undergo cell death in a characteristic manner, but without classic apoptot- ic morphology. (top micrographs from attached cells, lower micrographs from detached cells). Ultrastructural aspects of cell death Accidental (necrosis) versus Programmed (apopto- sis) cell death is now too limited an option to describe the spectrum of cellular demise. Necrosis is best used for features that appear after the cell has died, and Oncosis, (Greek –swelling), describes accidental death. Apoptosis is the mechanism for reducing cell numbers in tissue, characterized by nuclear alteration of chromatin condensation and fragmentation, blebbed membranes, loss of endo- plasmic reticulum and mitochondrial integrity, fol- lowed by phagocytosis by adjacent cells. Of the 3 various pathways leading to an apoptotic endpoint, ‘anoikis’ (Greek - state of homelessness) is trig- Fig 2 Haematopoietic cell association in culture, visualized by SEM (1), gered by ‘loss of cell anchorage’ The ability to LM (2), and sections cut parallel to the growing surface for TEM (3). overcome detachment-induced apoptosis is a cru- cial step in the oncogenic transformation pathway. In a recent collaborative study involving an induced Publications listed on page 62 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 33

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    GROUP LEADER CLINICAL RESEARCH GRADUATE STUDENT Catharine M.L. West FELLOWS Stephanie Donaldson (joint Ewen Griffiths with Imaging Science and POSTDOCTORAL Ashirwad Merve Biomedical Engineering) FELLOWS Karim Sillah Sara Bhana UNDERGRADUATE Carla Möller Levet SCIENTIFIC OFFICERS STUDENTS (joint with Bioinformatics) Helen Valentine Gemma Foley Ruth Swann Joely Irlam-Jones (joint with Genitourinary SCIENTIFIC Group) ADMINISTRATORS Jo Cresswell (30%) Rebecca Elliott RESEARCH NURSE Teresa Bailey Cancer Studies Academic Radiation Oncology: Translational Radiobiology Group The Translational Radiobiology Group, part of Academic dataset, again independent of established variables and a trained intrinsic signature. During the past Radiation Oncology (ADRO) in the Christie Hospital (head year work was established to validate the signature Prof Pat Price) has laboratory space within the Paterson in terms of investigating the hypoxia induction of Institute. The group explores and develops methods for the novel genes identified in the work (Sara Bhana). individualisation of radiotherapy. With the advent of high- Development of a homogeneous database of throughput techniques, the group is interested in characteris- head and neck patients ing molecular profiles that reflect relevant biological pheno- Validation work for the hypoxia signature will include examining the prognostic significance of types and predict tumour and normal tissue response to novel genes. A homogeneous database of head and radiation. neck cancer cases was established, therefore, com- Development of a hypoxia transcriptome in head prising patients with oropharyngeal cancers (ton- and neck cancer sil/tongue base) who received radiotherapy for their The group has a long interest in evaluating methods primary tumour; 133 patients were identified and for assessing tumour hypoxia and a prospective tumour blocks obtained from 79 (Priy Silva, Helen study was established in patients with head and Valentine). Features associated with poor locore- neck cancer to investigate the potential of RNA gional control were low Hb level (p=0.035) and microarrays (Priy Silva). This work involves collab- advancing disease stage (p=0.007). HIF-1α expres- oration with Prof Adrian Harris at the Weatherall sion was a more significant adverse prognostic fac- Institute of Molecular Medicine in Oxford, Dr tor in tonsil (HR=23.1, 95% CI 3.04-176.7) than Francesca Buffa at the Gray Cancer Institute, Dr tongue base (HR=2.86, 95% CI 1.14-7.19) tumours Crispin Miller (Bioinformatics Group), Dr Nick (p=0.03, interaction test). High tumour HIF-1α Slevin (Clinical Oncology), Mr Jarrod Homer expression associated with low Hb levels (p=0.03). (Surgery) and Prof Phil Sloan (Pathology, In multivariate analysis, the only independent prog- Manchester Royal Infirmary). Tumour (n=59) and nostic factors for locoregional control were increas- normal (n=11) tissue samples were arrayed and a ing Hb level (HR=0.84, 95% CI 0.72-0.98, p=0.03), signature representing 99 up-regulated genes was tongue base cancer (HR=2.03, 95% CI 1.04-3.96, derived by clustering around the tumour expression p=0.04) and high tumour HIF-1α expression of 10 well-validated hypoxia-associated genes (eg (HR=6.60, 95% CI 2.92-14.86, p<0.001). The VEGF, CA9). The median RNA expression of all work showed differences in radiotherapy outcome 99 genes was used to investigate the prognostic abil- within a homogeneous subsite of head and neck ity of the signature (HS-up). In multivariate analy- cancers related to molecular marker expression. sis in an independent head and neck cancer dataset including an intrinsic classifier and established clin- Assessment of hypoxia-associated markers in ico-pathological prognostic variables, HS-up was a oesophagogastric cancer significant and independent prognostic factor out- Evidence for the presence and prognostic signifi- performing the intrinsic classifier. HS-up also had cance of hypoxia is lacking in oesophagogastric prognostic information in a published breast cancer cancer. The latter, along with the need to develop P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 34

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    CANCER STUDIES ACADEMIC RADIATION ONCOLOGY: TRANSLATIONAL RADIOBIOLOGY GROUP approaches for selecting patients likely to benefit VORTEX-BIOBANK from adjuvant radiotherapy, led to the initiation of Funding was obtained from the CR-UK for VOR- a project looking at the role of hypoxia in the dis- TEX-BIOBANK. VORTEX is a national ran- ease. This work (Ewen Griffiths, Helen Valentine) domised trial currently in set-up. Patients with soft involves collaboration with Mr Ian Welch and Dr tissue sarcoma of the extremity who undergo post- Sue Pritchard (Wythenshaw Hospital). Hypoxia- operative radiotherapy: 66 Gy in 33 fractions once associated markers were studied in oesophageal and daily for 5 days a week over 6.5 weeks will be ran- gastric carcinogenesis sequences, and 177 surgical- domised to one of two different radiation volumes ly-treated oesophagogastric cancers. Marker expres- and the primary outcome measures are limb func- sion increased with progression along the carcino- tionality and time to local recurrence. The target genesis sequences. HIF-2α was expressed late in accrual is 400 patients based on non-inferiority of the Barrett’s sequence, and was only seen in dyspla- local control but improved limb function in patients sia and cancer samples. HIF-1α and HIF-2α were with reduced irradiation volumes. Joely Irlam-Jones expressed in 53% (2% >30% staining) and 63% will manage the prospective sample collection for (44% >30% staining) of tumours, respectively. the trial. The translational hypothesis underlying HIF-1α expression at the invasive tumour edge was the work is that a pre-treatment tumour molecular associated with a median survival of 18 vs 33 profile will define patients with a high risk of treat- months for negative tumours (p=0.019). High ment failure following surgery and post-operative HIF-2α was an adverse prognostic factor radiotherapy, and that the profile could be used in a (p=0.015). Neither protein provided independent subsequent study to identify patients who might prognostic information in multivariate analysis. benefit from adjuvant systemic therapy. The sec- The late expression of HIF-2α in the carcinogene- ondary hypothesis is that there is an association sis models and its high expression in tumours between SNPs in relevant candidate genes and indi- (Figure) suggest it is worth further study as a mark- vidual patient variability in normal tissue radiation er of disease progression in patients with Barrett’s toxicity. It is envisaged that samples for microarray dysplasia and as a cancer therapeutic target. analysis will be collected at surgery from at least half of the patients. Paraffin blocks for tissue microar- Radiogenomics: assessment of polymorphisms rays and blood samples for future genotyping for predicting the effects of radiotherapy (RAP- should be collected from the majority of patients. PER) a Individual radiosensitivity is considered to be an inherited complex trait dependent on interactions between multiple genes/gene products. RAPPER is designed to explore associations between single Box and whisker plot nucleotide polymorphisms (SNPs) in candidate of HIF-2α expression in the Barrett’s (15 genes and radiotherapy toxicity. It is a multi-centre columnar-lined meta- study with a planned recruitment of 2200 patients plasia, 20 intestinal and is powered to detect common alleles conferring metaplasia, 17 dyspla- b sia, 20 adenocarcino- a moderate risk of late morbidity and rarer ones ma) carcinogenesis with larger effects. Breast, prostate or gynaecolog- sequence (a). Tumour ical cancer patients are being recruited to identify HIF-2α expression vs polymorphisms affecting radioresponse across overall survival in 177 tumour types. The project involves collaboration oesophagogastric can- with Drs Neil Burnet and Alison Dunning cer patients (b). Tumour section (Cambridge), Prof Soeren Bentzen (Wisconsin) and showing high HIF-2α numerous clinical oncologists locally and nationally. expression (c). The day-to-day administration of RAPPER is car- ried out by Rebecca Elliott. The work began in July 2005 and sample accrual is on target with blood col- lected from 1075 patients. Radiation toxicity is c being scored using LENT-SOMA. Rapid develop- ments in high-throughput genotyping may allow study of 120 genes using SNP-tags to cover all common variation in each gene. Selection of the candidate list will be finalised in ~2 years but will focus on cell cycle checkpoint control, DNA dam- age response and cytokine pathways. Publications listed on page 62 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 35

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    GROUP LEADERS SCEINTIFIC OFFICERS CLINICAL RESEARCH Robert E Hawkins Hayley Bartha FELLOWS Peter L Stern Debbie Burt Adam Dangoor Natallia Kirillova (at NBS) Richard Griffiths SENIOR CLINICAL Christy Ralph RESEARCH FELLOW RESEARCH NURSES Alaaedin Shablak Fiona Thistlethwaite Jackie Fenemore Ursula Winters (with HC Carmel Garner Kitchener) POSTDOCTORAL Caroline Hamer SCIENTISTS Jackie O’Dwyer Eyad Elkord (Immunology) ADMINISTRATORS David Gilham (Cell and Debbie Maskell Gene Therapy) Catriona Parker Ryan Guest (at NBS) Jane Rogan Dominic Rothwell Zihni Yazici Cancer Studies: Biological, Immune and Gene Therapy Group In order to translate laboratory discoveries into effective NCRN colorectal group combining TroVax with chemotherapy and Bevacizumab. clinical therapies, partnerships involving scientific and clinical researchers as well as the commercial sector are key. It is A mechanistic study sponsored by CR-UK investi- from this platform that the Biological, Immune and Gene gated the effect of TroVax in patients undergoing resection of colorectal cancer liver metastases. The Therapy Group has been established. The focus of the objectives in this trial included assessment of sys- group is on development of novel immunotherapies with a temic immune responses before and after surgery as particular interest in cellular based therapies, genetic vaccines well as local tumour immunity and clinical out- comes. Twenty patients undergoing resection of and antibodies. This report details the group’s current clinical liver metastases by David Sherlock at North trial activity and highlights the translational interface that Manchester General Hospital received 2 vaccina- exists from target discovery through to later stage Phase II tions pre-operatively, 2 post-operatively and 2 fur- ther boosting vaccinations if immune responses and III trials. In recent years the group has spearheaded the were detected. Immunological assays indicate that development of two novel agents from pre-clinical develop- 15 of the 20 patients have T-cell or T-cell plus anti- ment through Phase I and on to Phase III trials. The group is body responses to 5T4 or subunit peptide within 14 weeks. Of the 16 protocol-compliant patients 13 also involved with other trials and is the largest recruiter to have demonstrated responses. Currently we are trials in the Christie Clinical Trials Unit (Derek Crowther investigating the levels of regulatory T-cells (Tregs) in Unit). peripheral blood and local immune factors by Clinical trials of TroVax – 5T4 vaccine immunohistochemistry and isolated tumour infil- With Oxford Biomedica we have been involved in trating lymphocytes. the development of 5T4 vaccines from pre-clinical and through all phases of clinical trials. The main 5T4 superantigen therapy Phase I trial (Clin Can Res. 2006; 12: 3416-24) of Targeting of superantigen to a tumour-associated TroVax in colorectal cancer showed safety and antigen can direct T-cell mediated cytotoxicity. immunological efficacy as well as encouraging cor- Building on previous successful Phase II studies relates of survival with immune responses. with a first generation agent (Shaw et al in press) we Subsequent Phase II studies combining TroVax have now undertaken a Phase I trial (with Active with chemotherapy in colorectal cancer and with Biotech) with a re-engineered molecule. This interferon in renal cancer have been successfully shows improved tolerability and encouraging clini- completed and have lead to the development of cal results. A PET mechanistic study has also been Phase III studies in each of these diseases. An completed (with Peter Julyan / David Hastings / international Phase III study of TroVax in combi- Jamal Zweit) and this shows tumour localization of nation with standard therapy for renal cancer has the fusion protein in all patients tested. An been initiated by Oxford Biomedica with International Phase II/III study in renal cancer is Manchester as the lead UK site. For colorectal can- due to commence shortly with Manchester as the cer we are developing a national study with the lead centre. P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 36

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    CANCER STUDIES: BIOLOGICAL, IMMUNE AND GENE THERAPY GROUP Adoptive transfer of Treg depleted autologous that VIN lesions pre-treated with an immune T-cells response modifier would respond more favorably With the Urology Group (Noel Clarke/Vijay to PDT. Treatment consisted of Imiquimod and Ramani) we have demonstrated that PDT using methylated ALA as a photosensitiser. CD4+CD25high Tregs are present in increased num- Follow up at 12 months is available for 10 women bers in the peripheral blood of patients with and response rates are maintained at this time point advanced cancer compared to normal controls. Tregs with 3 complete responses and 4 partial responses. suppress proliferation and cytokine release from Pharmacodynamic analysis showed that Imiquimod effector cells and, in animal models, depletion of significantly increased CD8 T-cells within VIN CD25+ T-cells can induce tumour responses. We lesions and that non-response to Imiquimod was therefore initiated a study to examine the effects of associated with an increase in Tregs within VIN Treg depletion on renal cancer. Six patients with lesions. advanced renal cancer were recruited and under- went leukapheresis followed by conditioning Trials with engineered T-cells chemotherapy with cyclophosphamide and fludara- Trials of engineered T-cells (see Cell and Gene bine. The autologous leukapheresis product was Therapy Group) are awaiting final approval by the depleted of CD25+ cells by magnetic selection MHRA following recent changes in the regulations using the CliniMACS® system then reinfused into governing first into man trials of immunotherapy. the patient. Treg depletion of the leukapheresis For our CR-UK sponsored trial targeting CEA the product was efficient and there was no toxicity of GMP virus has been produced and production runs the infused cellular product. Transient reductions of gene modified T-cells carried out to GMP (with in Tregs in the peripheral blood of patients were Eric Austin at Manchester Blood Centre). Likewise noted with evidence of increased T-cell responses assay validation for immunological and molecular to the tumour-associated antigen 5T4 in at least one monitoring of the trial has been completed and eth- patient. This was associated with mixed/minor ical approvals are in place. The trial targeting CD19 responses in two patients. We are now looking to is at a similar point although delays in production of combine this treatment with vaccines or with engi- GMP virus mean the trial could not start before neered T-cells. March 2007. Anti-CTLA-4 monoclonal in advanced oesopha- Other large scale trials go-gastric cancer We are also involved in trials of agents targeting CTLA4 is a transmembrane protein which is a cru- other biological pathways in renal and oesphago- cial inhibitor of T-cell activation. Anti-CTLA4 gastric cancer. In renal cancer a number of major antibody blockade has been shown to have thera- trials have been completed and these are changing peutic effects in melanoma. We are therefore the way we treat renal cancer. Building on success- exploring the effects of an anti-CTLA4 monoclon- ful recruitment to trials of anti-angiogenic drugs we al antibody (Ticilimumab, Pfizer) in a Phase II study are investigating how these interact with the in advanced oesophago-gastric cancer. As well as immune system as a means of rationally developing clinical efficacy we are examining pharmacodynam- combination trials. Following our presentation at ic effects on lymphocyte phenotype (eg. CD25, ESMO of interesting results from a large Phase III CTLA4, and FoxP3), and for the development of trial with Lapatinib (Glaxo) in renal cancers over- active immune responses against 5T4. The trial expressing EGFR we are also planning to look at opened in September, and has recruited eleven combination studies. patients to date. Toxicity has been acceptable, and the first patients are due formal re-staging of their Summary disease by the New Year. The group, through interactions with laboratory groups in Manchester and elsewhere and with the Sequential therapy of imiquimod and photody- pharmaceutical industry, has a vibrant trials portfo- namic therapy for vulval intraepithelial neoplasia lio focusing on a variety of ways to manipulate the (VIN) immune system. The aim of this Phase II study (with Henry Kitchener) was to demonstrate the tolerability of Imiquimod and photodynamic therapy (PDT) used sequentially and to assess lesion and immunological response in women with VIN. The hypothesis was Publications listed on page 63 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 37

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    GROUP LEADER NON-CLINICAL LECTURER GRADUATE STUDENT Vaskar Saha Louise Jones Shai Senderovich POSTDOCTORAL FELLOW CLINICAL TRIALS MANAGER Olga Yiannikouris Carly Leighton SCIENTIFIC OFFICERS DEPARTMENT Seema Alexander ADMINISTRATOR Naina Patel Marita Marshall CLINICAL FELLOWS Shekhar Krishnan Doaa Khater Frederik van Delft Children’s Cancer Group The Children’s Cancer Group (CCG), formed in 2000, was allogeneic transplantation after completing 5 blocks of treatment. We have obtained funding from the previously located within the laboratories of the Institute of Leukaemia Research Fund to monitor the speed of Cancer, Queen Mary University of London Charterhouse response to therapy, identify key mutations and campus. In October of 2006, the group relocated to the unique gene expression patterns in these patients. Our preliminary studies suggest that mutations in Paterson Institute for Cancer Research at the University of the ABL kinase domain are present in some chil- Manchester. The focus of our research lies in improving the dren with Ph+ ALL, but this does not necessarily outcome of children with acute lymphoblastic leukaemia predict for a poor response to therapy. On the other hand mutations in cell cycle kinase genes (ALL). On the clinical front, this involves designing and run- appear to do so. This trial will continue to 2010. ning of novel international clinical trials for those with high risk disease. In the laboratory we are exploring biological BIOV-111 This is a phase II trial of the drug Clofarabine in explanations for the variations in response to therapy. resistant and relapsed ALL. It is currently recruit- ing in 10 countries throughout Europe and will Clinical Trials: close early next year. At the moment the response rate is 28% and some refractory patients have been ALL R3 transplanted in remission. This is highly promising This trial is for children with ALL who relapse after for a single agent in this population. Both pharma- initial therapy. The trial recruits patients from all 21 cokinetics and molecular pharmacology studies are paediatric centres in UK and Ireland as well as from incorporated into this trial. centres in Netherlands, Australia and New Zealand. An indigenously designed web-based remote entry Laboratory Investigations: database with decision support tools has been made to run the trial. Minimal residual disease estimation Targets of ETV6 after 5 weeks of therapy is used to risk stratify chil- The ETS-related transcription factor ETV6 is fre- dren. Those who have high levels of disease at this quently found disrupted in many human malignan- stage are offered allogeneic stem cell transplanta- cies, in particularly in childhood ALL, where 25% tion. The trial opened in the UK in 2003 and to of patients have ETV6-RUNX1, t(12;21) transloca- date has recruited 178 patients. The overall survival tion. ETV6 binds to a specific DNA sequence con- of patients is 70% at 3-years. A number of clinical sisting of GGAA/T core motif within the promot- and cytogenetic markers of poor prognosis have ers of its target gene and as a result, repress its tran- been identified. An interim analysis is being per- scription. To date, there are few known target formed to better inform us of the progress of the genes of ETV6, namely BCLXL, MMP3 and Fli1. trial and help us design the next trial when this one We have used a Gene-ACE technique to convert closes in 2010. ETV6 into an activator and used microarray analy- sis to identify putative direct targets of ETV6. EsPhALL Promoter and chromatin immunoprecipitation This trial is randomising the use of imatinib in chil- assays identify two new target genes of ETV6, dren with Philadelphia positive (Ph+) ALL. All namely the cytokines CCL3 and CSF2. children with this disease in the UK are eligible for P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 38

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    CHILDREN’S C ANCER GROUP Chromosome 21 and childhood ALL tumours in NOD-SCID mice. This behaviour is We have identified and characterised a new cytoge- inhibited by AEP specific inhibitors. AEP also netic sub-type of childhood ALL. This is charac- cleaves the drug Asparaginase and we believe this terised by a intrachromosomal amplification of not only activates the drug but may increase its chromosome 21 (iAMP21). A common region of MHC Class II processing resulting in the produc- amplification (CRA) has been identified between tion of inactivating antibodies. 33.192 and 39.796Mb and a common region of deletion (CRD) between 43.7 and 47Mb in 100% Plans for 2007 and 70% of iAMP21 patients, respectively. The group is in the process of reorganisation and Supervised gene expression analysis showed a dis- will add a new senior post doctoral fellow and clin- tinct signature for eight patients with iAMP21, with ical senior lecturer in 2007. The clinical trials will 10% of overexpressed genes located within the move towards further integration with the laborato- CRA. The mean expression of these genes was sig- ry with the development of a national cell bank in nificantly higher in iAMP21 when compared to Manchester for relapsed and refractory ALL. We other ALL samples. Although genomic copy num- plan to use the new chromosome 21 tiling arrays to ber correlated with overall gene expression levels further investigate the differences in the transcrip- within areas of loss or gain, there was considerable tome of chromosome 21 in childhood ALL. The individual variation. A unique subset of differen- ETV6 targets will be further examined using a ChIP tially expressed genes, outside the CRA and CRD, on chip approach and we will investigate the possi- were identified when gene expression signatures of bility of collaboration between ETV6 and RUNX1 iAMP21 were compared to ALL samples with on the CSF2 and CCL3 promoter. The role of ETV6-RUNX1 fusion or high hyperdiploidy with AEP in resistant disease is being investigated using additional chromosomes 21. From this analysis, a number of different tools. AEP was shown to be overexpressed in patients with iAMP21. Genomic and expression data has further characterized this ALL subtype, demon- strating high levels of 21q instability in these patients. AEP over expression and high risk ALL We have now further demonstrated that AEP is overexpressed in high risk ALL and those with extramedullary relapse. Western analysis identifies high levels of active AEP in blast cells obtained from these patients. Leukaemic cell lines over- expressing AEP show increased migration and inva- siveness in vitro assays and produce extramedullary The AEP expressing cells are tumour associated macrophages. Blue = DAPI; Red = antiCD68; Green = AEP HE section of glioblastoma showing cells highly positive for AEP expression (brown) at the edge of the tumour. Publications listed on page 64 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 39

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    GROUP LEADER POSTDOCTORAL SCIENTIFIC OFFICERS Rob Clarke FELLOWS Kath Spence Gillian Farnie Rognald Blance Helen Kalirai Pam Flint Rebecca Lamb Andy Sims GRADUATE STUDENTS Ngoc-Sa Nguyen-Huu CLINICAL FELLOWS Hannah Harrison Kai Ren Ong Rachael Johnson ROTATION STUDENTS Stephanie Jobling Clair Thomas Medical Oncology: Breast Biology Group Normal tissues are maintained by the self-renewal capacity Hoechst dye-efflux to obtain the mammary cell ‘side population’. More recently, we have exploited of a rare population of stem cells, which divide asymmetrical- the expression of ALDH1 in stem cells by using the ly both to replace themselves and to generate progenitors. commercial substrate Aldefluor to enrich for stem After limited cell division, progenitor cells produce the non- cell populations. Both these methods can be cou- pled with the use of mammary stem cell surface dividing differentiated cells specific for each tissue. An markers. To identify the factors that regulate breast emerging concept is that in leukemia as well as in neural and stem cell phenotype, we are collecting material from epithelial cancers, including breast cancer, only a minority of consenting patients undergoing surgery for either breast reduction, removal of a benign or primary cells, i.e. the “cancer stem cells”, have the capacity to initiate tumour, ductal carcinoma in situ or pleural effusions tumours; the others are committed to differentiation path- (from advanced breast cancer patients). ways and senescence. Thus, characterising the cancer stem One current focus is the signalling pathways regu- cell and understanding the molecular basis for dysregulated lating stem cell self-renewal of which Notch, Wnt, ErbB, Prl and ovarian hormones are of particular self-renewal will be crucial for identification of targets for interest. We have shown the aberrant activation of effective therapeutic intervention. the Notch receptor signaling pathway in breast can- cer (see Stylianou et al., 2006). We have extended Stem cell self-renewal pathways these studies of Notch signaling and have data Identification of stem cell self-renewal pathways is demonstrating its importance in regulation of emerging as important for cancer prevention, and breast cancer stem-like cells, using methods for for the future therapy of treatment-resistant cancer non-adherent mammosphere suspension culture stem cells that have the capacity to initiate tumours (see figure), analogous to neurosphere culture that and recurrence. Although some cell-specific mark- enriches for brain stem cells. Our recent results ers and signal transduction pathways are similar in indicate that in this culture system self-renewing normal and cancer stem cells, the tight regulation of stem cells can be enriched and passaged, and the self-renewal that is operative in the normal stem cell contribution of the above signalling pathways can may well be disrupted in cancer. Thus, understand- be assessed. Their importance in an in vivo model ing the regulation of self-renewal will be crucial for of tumourigenesis (the gold standard stem cell improving therapeutic intervention by targeting the assay) is also being actively investigated. cancer stem cell that is predicted to be inherently chemo- and endocrine resistant and to initiate Gene expression and functional genomics tumour recurrence. Gene expression arrays and functional genomics methods are being employed to identify novel path- Our current aim is to exploit culture and in vivo ways that participate in stem cell regulation. For the techniques to address the regulation of normal and functional genomics studies, we are collaborating cancer stem cells in the breast. We have already with Rene Bernards’ group at the NKI, Amsterdam established methods to isolate human mammary to use a retroviral short hairpin (sh) RNA library epithelial stem cells from normal tissue, using that targets approximately 8,000 genes (Nature, P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 40

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    MEDICAL ONCOLOGY: BREAST BIOLOGY GROUP 2004, 428: 431-7). We use the library to screen for women, then it may be possible to reduce the genes that function in stem cell self-renewal by increasing incidence of breast cancer. We therefore using several rounds of mammosphere culture to collaborate with Professors Howell and Evans at enrich for integrated shRNA sequences that the South Manchester Family History Clinic to increase production of non-adherent sphere examine the effects of preventative strategies to see colonies. DNA must be collected at each passage whether relevant gene expression is altered follow- and ‘bar-coded’ by amplification, labelling and ing a clinical intervention such as a calorie restrict- hybridisation to custom arrays of the shRNA ed diet. sequences. The enriched sequences light up on the It is hoped that the results of these investigations array and the most important ones will be increased should lead to an increased understanding of the by each passage of mammospheres. We are cur- biology of the normal human breast which, in turn, rently testing the candidate shRNA sequences in could lead to the development of new strategies or our normal and cancer stem cell assays. new targets for breast cancer prevention and therapy. Gene expression and risk of breast cancer Another area of focus is gene expression and risk of breast cancer where we compare tissues from women at high or normal risk of breast cancer, for example, parous versus nulliparous women or those with a family history of breast cancer versus popu- lation controls. To inform these investigations, we have developed an in vivo model where nulliparous human breast tissue is exposed to pregnancy levels of ovarian hormones and we can observe signifi- cant changes in genes previously identified in other model species. If these genes can be modulated in The photomicrograph shows a bright field image of mammospheres grown from dissociated normal human breast epithelial cells obtained from a reduction mammoplasty specimen. The mammospheres are colonies grown in sus- pension from single stem-like cells. They are analogous to neurospheres that have been demonstrated to enrich for brain stem cells. Magnification x400 Publications listed on page 64 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 41

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    GROUP LEADER POSTDOCTORAL SCIENTIFIC OFFICERS Robert Hawkins FELLOWS Hayley Batha David Gilham Alison O’Neill Eleanor Cheadle Amanda Russell Jennifer Loconto Simon Dovedi ADMINISTRATOR Vivien Watson Isabelle Wartelle Michael Lie-a-Ling GRADUATE STUDENTS CLINICAL RESEARCH John Bridgeman FELLOWS Alex Smith Wasat Mansoor Grazyna Lipowska-Bhalla Kallingal Riyad Medical Oncology: Cell and Gene Therapy Group The primary focus of the Cell and Gene Therapy group is CIR gene into T cells. T cells engrafted the CIR then target tumour cells through the scFv part of upon the development of engineered T cells to eradicate the CIR resulting in the specific destruction of the cancer. Pre-clinical studies generated over the past five years tumour cell. There are two principal targets used in have formed the basis of two clinical trials which are the group – carcino-embryonic antigen (CEA – gastro-intestinal cancers and others) and CD19 (B presently at the final regulatory hurdle (see Biological, cell lymphoma in collaboration with Prof. J. Immune and Gene Therapy Group report). Consequently, Radford, Medical Oncology) and these are the tar- the focus of our work has moved towards the use of model gets of the two clinical trials currently held up in regulation (see clinical trial section). Human T cells systems to investigate the mechanisms of activity of engi- engrafted with anti-CEA.CD3ζ or anti- neered T cell therapies with the aim of improving the overall CD19.CD3ζ receptors kill their respective antigen- therapeutic approach. A key recent development is the expressing tumour cell lines in vitro and also can effectively challenge the growth of these tumour ATTACK programme which is a Framework 6 EU funded lines in immuno-compromised animal models. project (Co-ordinator Professor Hawkins) which brings together 14 laboratories to work on the pre-clinical devel- While immuno-compromised animal models pro- vide a setting to investigate the functionality of opment of engineered T cell strategies. Importantly, local human T cells, these models lack the power to fully collaborative work with various groups within the PICR explore the implications of adoptive cell therapy in (including Immunology and Carcinogenesis) is exploiting the context of a normal, functioning host immune system. To this end, we have developed immuno- these model systems in order to discover how combinations competent models for both CEA and CD19 and of therapeutic approaches may synergise thereby producing mouse T cells harbouring the respective receptors more potent therapies. are indeed active in challenging the growth of tumours. However, in order to achieve high levels of T cell engraftment, lymphodepleting Engineered T cells chemotherapy is required which transiently reduces The core activity of the research group is the genet- the number of white cells in the circulation thereby ic engineering of T cells. In essence, this involves allowing the therapeutic T cells to survive long the stable introduction of genes encoding chimeric enough to carry out their anti-tumour function. immune receptors (CIR) into primary T cells. CIRs Optimising the combination of chemotherapy and consist of scFv (derived from antibodies) fused to engineered T cell infusions in these model systems the signalling domain of important T cell receptors is the key to the future translation of the therapy. (e.g. CD3ζ). The group has spent some consider- However, one safety concern is the possibility of able time optimising methods for the gene modifi- auto-immunity driven by the engineered T cells tar- cation of primary human and mouse T cells with geting tissues expressing normal or physiological the result that we have protocols which use retrovi- levels of target antigen. To investigate this, we are ral vectors to reliably and efficiently introduce the using mice transgenic for CEA (CEATg) which P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 42

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    MEDICAL ONCOLOGY: CELL & GENE THERAPY GROUP express CEA protein to levels similar to that in primary colorectal tumours (Cheadle et al., sub- observed in man. Initial experiments have proven mitted). Eotaxin-2 is more commonly found play- encouraging where CEATg animals given ing a role in allergy and at present it is not clear chemotherapy and CEA specific T cells remained what the biological significance of the presence of tumour-free with no evidence of auto-immunity this chemokine is in colorectal tumours. However, while cohorts treated with the same regime but with the presence of specific chemokines in tumours non-specific control T cells developed tumour. does provide a potential tag with which to attempt to target engineered T cells to specifically migrate The concept of combining therapies is being to tumour and our future work will involve explored in a broader context. Together with the attempts to manipulate T cells to respond to the Immunology group, we have recently demonstrated presence of these tumour derived chemokines. that mouse T cells engineered with specificity for the 5T4 tumour-associated antigen synergise with Summary 5T4 vaccination and dendritic cell therapy to signif- The translational research carried out by our group icantly improve therapy against established tumours has formed the basis for two clinical trials which are (Jiang et al., 2006). We are currently seeking to due to commence in 2007. Further development to improve the 5T4 specific chimeric receptor in order generate future trial proposals will be dependent to investigate the mechanisms underlying this upon model systems being currently used in the observation. In a similar vein, in a further collabo- laboratory to investigate the potential and also to ration with the Immunology and Carcinogenesis assess the potential risks of gene and immune ther- Groups, we are exploring whether DNA damaging apies. chemotherapy may potentiate immune therapies in our model systems. Furthermore, improving the power of the receptor itself is being undertaken with more powerful signalling domains being incor- porated which may further boost the activity of the engineered T cell. Chimeric immune receptors in other immune cell types. T cells are potent immune effector cells. However, many other immune cells can play an important role in eradicating tumour and exploiting these cells through chimeric receptor targeting has been one avenue of research. Our group was the first to suc- cessfully show that monocyte/macrophages can be transduced to express a chimeric receptor and to effectively combat the growth of tumour cells in vivo (Biglari et al. 2006). Current studies are work- ing upon the expression of chimeric receptors in Natural Killer cells (collaboration with Immunology) and also seeking to exploit gene- modified haematopoietic stem cells as a source to re-populate recipient animals with multiple lineages of immune cells harbouring tumour specific chimeric receptors. Homing of T cells to tumour. As an aside to our studies investigating engineered T cell function in patients with advanced CEA+ tumours, we have identified the presence of an Photo of ATTACK / CRUK sponsored Symposium in Manchester December 2006. The meeting featured the unusual chemokine within colorectal tumours global leaders in the field. which have metastasised to the liver. Chemokines play an important role in controlling the migration of cells around the body and we have identified that the chemokine Eotaxin-2 is present to very high levels in colorectal metatastases and is also present Publications listed on page 65 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 43

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    GROUP LEADER POSTDOCTORAL CLINICAL FELLOWS Gordon Jayson FELLOWS Andrew Clamp Bader Al Salameh Sin Lau Alison Backen Claire Mitchell Claire Cole Daniela Rosa Steen Hansen Will Stimpson Medical Oncology: Translational Angiogenesis Group We have established a Translational Angiogenesis Group in nous FGF2 we were able to probe for non-activat- ing sequences of HS but this was also reduced on collaboration with Professor Caroline Dive with the aim of the tumour cell surface suggesting that there is a defining biomarkers for anti-angiogenic agents. We will run global rather than sequence-specific reduction in the translational research programme for two MRC/NCRN cell surface HS. Both heparanase and H-sulf1 were expressed by the tumour cells but only heparanase international randomised trials of anti-angiogenic agents in could account for the findings. Thus these data ovarian cancer. The laboratory programme has identified suggest that heparanase is a key mediator of critical FGFs in ovarian cancer and developed heparin heparan sulphate depolymerisation at the tumour cell surface, augmenting its known role in invasion oligosaccharide synthesis. and angiogenesis. Ovarian Cancer Laboratory Programme In an extensive study of FGFs in ovarian cancer we The obligate dependency of the Fibroblast Growth have identified a switch in the receptor type upon Factors (FGF) on heparan sulphate (HS) for their transformation. This is associated with the biological activity led us to examine this axis in response of the tumour cells to the appropriate lig- ovarian cancer. In previous work we had shown and FGFs and enhanced chemosensitivity in inhibi- that stromal syndecan 1 was of prognostic signifi- tion studies. Thus our data (manuscript in prepara- cance in the disease and that syndecan 3, conven- tion) suggest that FGF2 is relevant to angiogenesis tionally found on neuronal tissue, was aberrantly while other FGFs play a role in transformation and expressed in the tumour endothelium. Using a the malignant phenotype, highlighting the potential unique molecular probe we showed that the of FGF inhibitors in the disease. heparan sulphate on the tumour vascular endotheli- um has the capacity to activate FGF2. While the Therapeutic Programme stroma bound this probe only moderately, the We had previously shown that heparin octasaccha- tumour cells were largely negative. rides inhibit angiogenesis in vivo and over the last year have developed the organic chemistry to make We investigated why the tumour cells do not synthetic heparin octasaccharides. This work is express growth factor activating heparan sulphate ongoing and will provide uniquely pure reagents to and showed, using in situ hybridisation, that the test structure-function relationships in the next tumour cells strongly express 6-O-sulphotrans- year. ferase, an enzyme implicated in the synthesis of biologically active heparan sulphate. In addition we Clinical Trial Programme showed that the core proteins for proteoglycans We have completed phase I trials of anti-angiogenic were present on the surface of ovarian cancer cells. agents that include a pure anti-αv integrin antibody These data implied that the tumour cells make and a pure anti-VEGFR2 antibody. These trials heparan sulphate but that active heparan sulphate is have yielded unique insights into the possible roles not present on the cell surface. By adding exoge- that the drugs might have. In the integrin trial we showed that the antibody was very well tolerated P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 44

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    MEDICAL ONCOLOGY: T R A N S L AT I O N A L A N G I O G E N E S I S G R O U P and that one patient with angiosarcoma underwent next 2 years and will test the acceptability of defer- rapid improvement of her disease with single agent ring surgery in ovarian cancer through the use of therapy. In the anti-VEGFR2 trial we demonstrat- pre-operative chemotherapy. ed growth retardation using serial imaging strategies (collaborators: Jackson, Parker, Univ. Manchester) but did not see changes in vascular permeability, thereby challenging the dogma that all active VEGF inhibitors reduce vascular permeability as assessed by dynamic contrast enhanced Magnetic Resonance Imaging. In collaboration with Prof. Sean Kehoe (Oxford) we have gained approval from CTAAC to expand the preliminary randomised study of neoadjuvant chemotherapy for the treatment of ovarian cancer. The CHORUS trial will now be completed over the A. HS6ST1 ISH shows RNA to be present in ovary tumour and endothelial cells, but absent in stroma and normal ovary (inset). B. HS6ST2 ISH shows RNA to be present in ovary tumour cells, but absent in endothelial cells, stroma and normal ovary (inset). Each scale bar represents 400mm. Publications listed on page 67 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 45

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    GROUP LEADER SCIENTIFIC OFFICERS PLACEMENT STUDENT John Gallagher Nijole Gasiunas Nicole Yan Graham Rushton SENIOR FELLOW Jon Deakin RESEARCH Malcolm Lyon COLLABORATORS GRADUATE STUDENTS Valerie Kouskoff and Georges POSTDOCTORAL Rebecca Baldwin (joint Lacaud FELLOWS with Stem Cell Biology) Stem Cell Biology Group Chris Robinson Annie Wat Claire Johnson Eun –Ang Raber Professor Gordon Jayson and (joint with Salford Colleagues University) Translational Angiogenesis Group Dr Catherine Merry, Medical Oncology: Materials Science, University of Manchester Proteoglycan Group Heparan sulphate (HS) is a near-universal cell surface co- the highest affinity heparin- and dermatan sulphate- binding sites within NK1. Together, these data sug- receptor for many growth factors and cytokines. Its mode of gest that the N domain provides a single high affin- action is still unclear, although specific structural motifs in the ity site for both GAGs, but that long GAG chains, HS polysaccharide chain are known to be essential for bind- especially HS/heparin, may be able to engage in additional, weaker, cooperative interactions with ing growth factors and enabling their efficient engagement other HGF/SF modules. with tyrosine kinase receptors. We are investigating the molecular design of HS in different cell types, including Factor H Complement Regulatory Protein In collaboration with Dušan Uhrín, we have inves- embryonal stem (ES) cells, and examining how its unique tigated the GAG-binding properties of factor H, an domain structure and conformational flexibility drive the inhibitor of complement amplification on self-sur- assembly of ligand-receptor signalling complexes on the faces. Factor H is comprised of 20 tandem comple- ment control protein modules. Our gel mobility plasma membrane. shift assay demonstrated binding of a fully-sulphat- Glycosaminoglycan recognition sites in complex ed heparin tetrasaccharide to the C-terminal 19-20 modular proteins module pair. NMR chemical shift mapping allowed the GAG-binding site to be delineated within the Hepatocyte Growth Factor/Scatter Factor newly-derived 3D solution structure of this module Hepatocyte growth factor/scatter factor (HGF/SF) pair (Herbert et al. J. Biol. Chem. 2006; 281:16512). is comprised of an N-terminal domain (N), four Strikingly, missense mutations associated with atyp- kringle domains (K1 to K4) and a serine proteinase ical haemolytic uraemic syndrome congregate in homology domain (SP). It contains both Met this GAG-binding site and may thus disrupt the receptor and glycosaminoglycan (GAG) co-recep- GAG self-recognition properties of factor H. tor-binding sites. The alternatively transcribed Similarly, we have also shown that the Tyr-to-His NK1 fragment of HGF/SF retains the major mutation at residue 402 in module 7, that is known GAG-binding site that recognises two structurally- to predispose to acute macular degeneration, caus- distinct GAGs, HS/heparin and dermatan sulphate. es a reduction in affinity of the isolated module 7 We have recently investigated the independent for heparin oligosaccharides. This is consistent GAG-binding properties of the six individual N, K with the positioning of this residue on the edge of and SP domains (provided by Dr Ermanno a separate GAG-binding site. Gherardi, MRC Centre, Cambridge), using gel mobility shift and HPLC size exclusion chromato- Studies on mouse embryonic stem cells graphic assays. Interestingly, GAG-binding sites were revealed in three distinct domains, with rela- Characterisation of ES cells with mutations in tive affinities of N>K1>>SP. All three sites bound HS endosulphatases heparin but only the N domain bound dermatan The Sulfs are a family of extracellular enzymes sulphate with high affinity. NMR titration data (in which act to modify mature HS chains, removing 6- collaboration with Dušan Uhrín, University of O-sulphate groups from specific sites. The loss of Edinburgh), also indicate a substantial overlap of these groups significantly alters the biological func- P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 46

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    MEDICAL ONCOLOGY: P R OT E O G LY C A N G R O U P tion of HS by influencing the ability of the chains the Manchester Stem Cell Centre on a variety of to interact with a variety of growth factors and projects. morphogens, such as BMP and FGF. Although their activities are tightly controlled during develop- Observations on the interaction of HS with ment, Sulfs are mis-regulated in various tumours FGF1 using an enzyme protection-based including bladder and breast cancer. Working with approach collaborators in Germany (Prof. Dierks, Bielefeld) The fibroblast growth factors (in particular FGF1 we analysed the structure and function of HS and FGF2) are an important area of research extracted from mice mutants of one or both mem- because of the roles they play in tumour growth bers of the Sulf family. Surprisingly, we found that and angiogenesis. At the cell surface, 2:2 complex- there was an element of co-operativity in the func- es of FGF and FGFR (FGF-receptor) are respon- tions of these enzymes, a feature not observed in sible for signal transduction; however, these com- previous studies. In addition, we demonstrated that plexes cannot form in the absence of HS or heparin the pattern of HS sulphation observed in these in a chemical analogue of the sulphated domains (S- vivo models was different to that predicted from in domains) of HS. We have demonstrated that spe- vitro experiments, with considerably more 6-O-sul- cific heparin saccharides drive the assembly of 2:1 phate groups being targeted by the enzymes than FGF1: saccharide complexes by a co-operative previously thought. We have developed a non-inva- binding mechanism that correlates with the capaci- sive assay for detecting Sulf activity, and we hope to ty of these saccharides to induce FGF1-mediated use this method to define the extent of Sulf cell proliferation. We have now begun to analyse loss/gain in and around tumour sites in vivo. FGF1 interactions with HS using a protection assay in which preformed HS-FGF1 complexes are incu- HS and the regulation of ES cell biology bated with heparinase enzymes to degrade unpro- ES cells are a useful model system to study the tected HS. This showed that FGF1 conferred inter-relationships between HS structure and func- almost complete protection on the heparinase I tion. We can manipulate the levels of HS biosyn- cleavage sites in HS (i.e. the S-domains). This was thetic enzymes and core proteins using either surprising since only a minority of S-domains bind RNAi, or by deriving new ES cell lines from exist- FGF1 following their excision from HS. Another ing mutant mice. As pluripotent ES cells differen- surprising observation was that FGF1 also protect- tiate and commit to a defined lineage (e.g. neural, ed transition zones (T-zones) of HS from degrada- haematopoietic), they change the sulphation pat- tion. T-zones are positioned at the interface of the terns of their HS chains. The expression of line- S-domains and the non-sulphated sections of HS. age-specific HS may influence the ability of differ- They have an intermediate level of sulphation and entiating cells to respond to the range of HS- were not expected to interact with FGF1. Our dependent factors that direct ES cell differentiation. findings indicate that FGF1 binding to T-zones and ES cells deficient for the iduronate-2-O sulpho- S-domains may be facilitated by co-operative con- transferase enzyme (Hs2st) appear to be restricted formational effects resulting from their close appo- in their ability to differentiate to specific neural lin- sition in the HS chain. It is unclear whether FGF1 eages. Various members of the FGF family are crit- is dimerized at any of these binding regions in HS. ical in this pathway and we are investigating how In collaboration with Professor Tom Blundell and loss of 2-O-sulphation influences their activity. colleagues (University of Cambridge), we are now There is growing interest in the role of extracellular exploring the interaction of FGF-receptors with matrix components in directing ES cell behaviour, HS-FGF1 complexes with a view to identifying the Sulf enzymes “edit” and we have been able to conduct experiments active sites in HS for assembly of signalling com- the sul phation pat- tern of HS demonstrating that specific HS oligosaccharides plexes. can be used to influence neural and mesodermal Sulphation at C6 of differentiation pathways. We are also working with N-sulphated disac- charides in HS is dynamically regulated by an interplay of sulphotr ansfer ase and endosulphatase (Sulf) enzymes. The extracellular Sulfs specifically remove 6- O-sulphate groups and in so doing regu- late HS-mediated cell growth. Publications listed on page 67 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 47

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    GROUP LEADER GRADUATE STUDENT RADIOPHARMACIST Tim Illidge Andrei Ivanov Anna McNicholas CLINICAL SENIOR PHYSICIST PRECLINICAL GROUP RADIOIMMUNOTHERAPY Mathew Guy POSTDOCTORAL GROUP FELLOWS CLINICAL TRIALS James Hainsworth SENIOR CLINICAL ADMINISTRATOR Jamie Honeychurch SCIENTIST Emma Burke Lijun Mu Maureen Zivanovic RESEARCH NURSES Sue Neelson Tracy Gardener Cancer Studies: Targeted Therapy Group The research aims of the Targeted Therapy Group are to ation and mAb effector mechanisms. Our results confirm that RIT is much more than targeted radi- increase our understanding of the interactions between ation and provide a scientific rationale to support monoclonal antibodies (mAb) and irradiation in the treat- the use of selecting combinations of mAb in RIT ment of cancer and to develop novel clinical trial protocols rather than using a single mAb. We have shown that a radiation dose response exists for RIT in B- using radionuclide targeted therapies. There are two research cell lymphoma, when the targeted dose of radiation domains which inter-relate to form a cohesive programme of is augmented in the presence of signalling mAb. preclinical work under the title of radioimmunotherapy (RIT) We have demonstrated for the first time in vivo that this type of combination mAb approach is effective of cancer, which include optimisation of RIT and irradiation in RIT and are attempting to translate these preclin- and immunoregulation. In the first project, the roles of mAb ical findings to clinical studies. Our recent work has as both vectors to deliver radiotherapy to tumours and in focused on the importance of the micro-distribu- tion of radiolabelled mAb to the delivery of target- direct cancer cell killing are further explored. The specific ed radiotherapy to tumour. We have found impor- aims are to further understand the mechanisms of action of tant therapeutic differences between targeting dif- RIT and define the relative contributions of targeted radia- ferent tumour antigens and their ability to deliver tumouricidal doses of radiation at the microscopic tion and mAb effector mechanisms to the clearance of level. tumour. In the second project, mAb are used to augment T- cell responses to tumour by blocking or stimulating co- Another line of investigation has been the intracel- lular signalling pathways underlying the combined receptors in the immune system. This project aims to further treatment of anti-CD20 antibodies and irradiation define the factors which are important in combining irradia- in non-Hodgkin lymphoma cells. We have tion and immunoregulatory mAb. observed a synergistic cytotoxic effect when the anti-CD20 mAb B1 was combined with irradiation. Preclinical Group The additive effect seen with B1 mAb and radiation The last year has been very successful in creating a was not however observed with another anti-CD20 cohesive preclinical laboratory and clinical transla- mAb, Rituximab. This synergistic effect was tional groups. In February 2006 Dr Jamie accompanied by activation of ERK/MAPK path- Honeychurch relocated from Southampton and way and could be reversed with MEK inhibitors two new post-doctoral fellows Dr James and siRNA approaches. Hainsworth and Dr Lijun Mu were appointed later in 2006. Our other preclinical laboratory interests involve exploiting the therapeutic potential of combining Over the last years, we have substantially increased immuno-modulatory mAb such as anti-CD40 with our understanding of the relative contributions of irradiation or cytotoxic chemotherapy. We have antibody effector mechanisms and targeted radia- been able to demonstrate that irradiation and anti- tion to the eradication of tumour by using well CD40 mAb can act in concert to eradicate lym- defined syngeneic animal models of B-cell lym- phoma and induce long-term survival under condi- phoma. We have demonstrated for the first time in tions whereby either treatment alone is ineffective. a variety of different syngeneic models of lym- When anti-CD40 mAb is given in combination with phoma that successful RIT leading to long-term EBRT a clear radiation dose-dependent increase in tumour protection consists of both targeted irradi- survival is seen with long-term CD8 T-cell depend- ent protection. We are hopeful that this type of P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 48

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    CANCER STUDIES: TA R G E T E D T H E R A P Y G R O U P combination approach using tumour cytoreduction tan (Zevalin) in previously untreated follicular lym- with standard anti-cancer approaches such as cyto- phoma and will be led from the Christie and involve toxic chemotherapy and irradiation followed by collaborations with investigators in France and Italy. host immunostimulation may provide an excellent therapeutic opportunity for future clinical testing. The Phase I/II study in diffuse large B cell lym- Our current research is focused on improving our phoma is the first attempt to integrate RIT into a understanding of the fate of irradiated tumour cells combination chemotherapy schedule. It is being led in vivo following combination therapy, and deter- from the Christie along with 5 other UK sites. mining which type of cell death provides the great- More recently the CR-UK and an industrial partner- est stimuli to the host immune system and which ship have funded a further novel trial approach are more “immunologically silent”. To address using abbreviated chemotherapy followed by RIT in these issues we are currently developing a number relapsed follicular lymphoma. This study (SCRIFT) of systems for manipulating individual antigen pre- will again be led from the Christie but will run senting cell (APC) populations in vivo, including through the NCRI lymphoma group. transgenic models, specific depleting agents, and mechanisms for altering the recognition of dying Another novel clinical trial developed in tumour cells by phagocytes. Using fluorescently Manchester and launched this year is the GemBex labelled tumour cells, we have been able to demon- study in Cutaneous T cell lymphoma. This is first strate that following EBRT lymphoma cells are rap- multi-institutional national study ever to have been idly cleared in vivo by macrophages in a radiation attempted in this rare cancer and is again funded by dose-dependent manner. These studies are current- the CR-UK. An important element of this Phase II ly being extended to assess the role of macrophages clinical trial using Gemcitabine and Bexarotene in and other APC such as dendritic cells and B-cells in addition to the clinical response rates, will be the priming immune responses following combination skin assessment but a detailed quality of life assess- therapy. To increase our understanding of how the ment. Finally we plan to develop the anti-Idiotype mode, site and quantity of cell death can affect against Rituximab in clinical trials to measure serum immunogenicity we are also characterising and Rituximab levels in national clinical trials. This will comparing the efficacy of cells treated ex vivo with be done via a collaboration with the Clinical chemotherapy or radiotherapy in prophylactic and Experimental Group in the Paterson Institute, led therapeutic immunisation protocols. by Caroline Dive. Clinical Translational research Collaborators A major focus of the targeted therapy group is to Sue Owens Nuclear Medicine; John Radford work alongside and to interact with the clinical trial Medical Oncology; Richard Cowan Clinical programme. The clinical RIT group has made Oncology (all Christie Hospital) and members of tremendous progress this year in building sufficient Manchester Lymphoma Group; Richard Myers infrastructure to enable the delivery of high quality Pathology, MRI; Martin Glennie, Peter Johnson, novel clinical research over the coming years. We Mark Cragg; Cancer Sciences Division, were delighted to welcome Maureen Zivanovic, a Southampton University; Franck Morschausser, senior clinical scientist who relocated from Lille, France; Giovanni Martenelli, Milan, Italy Southampton, Anna McNicholas a radiopharmacist and to have secured the services of Mathew Guy to lead the clinical radionuclide dosimetry. A major project over the coming year and beyond will be the building of a new radiopharmacy following the recent Christie Trust approval. One of the major successes from the laboratory programme which we have translated from the clin- ic to the laboratory has been the use of fractionat- ed RIT as well as the development of a novel anti- Idiotype against Rituximab that can be used to measure serum Rituximab levels in patients. We have completed a Phase I/II dose escalation study of fractionated 131I Rituximab and are now embark- ing on a multicentre investigator led study using ERK protein activated in cells undergoing homotypical adhe- fractionated RIT. This phase II study (FIZZ) in fol- sion following treatment with the anti-CD20 antibody B1. licular lymphoma will be the first study to be per- Immunofluorescence of active ERK (Alexa fluor 488, green) and DNA (Propidium Iodide, red) Publications listed formed with fractionated 90Y Ibritumomab tiuxe- on page 69 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 49

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    HEAD OF RESEARCH SERVICES Jenny Varley Research Services http://www.paterson.man.ac.uk/facilities/scifacs.jsp There have been a number of significant developments over cell and the very act of imaging the cell has as low the past year in the Research Services. A review of the photo-toxic effect as possible. This microscope system enables the researcher to record not only in requirements of the Institute for Electron Microscopy has a manner that does not affect the biological activity resulted in the formation of a Transmission EM service unit of the cell but also allows large numbers of cells to which will roll out in January 2007, and which will be run by be examined thus producing statistically viable results. The requirement for specific processing Stephen Murray who currently works for Terry Allen. The tools to achieve numerical characterisation of parti- proteomics service component of the Molecular Biology cle and cell movement has increased which has led Core Facility has been boosted tremendously by the to the purchase of another 64-bit workstation and the building of software which utilises mathemati- appointment of Duncan Smith who is working closely with cal modelling software. Yvonne Connolly to provide an excellent proteomics serv- ice. We have continued to invest in equipment, including Currently usage of the microscopes is up to 80% which includes overnight and weekend usage, hence computers and software, to make sure that the services the amount of useful data being generated increas- remain at the forefront. This year major purchases have es exponentially year on year. As a result of this included a new Qiagen robot for the MBCF and a new time amount of data, together with that generated by other facilities and groups in the Institute, IT lapse microscopy system. The facilities work closely together department is looking at a storage and archival solu- to provide a range of integrated activities such as cell sorting tion due to the terabytes of data being generated. or laser capture microdissection for microarray work, and Challenges over the coming year include high genotyping transgenic pups. throughput imaging and three dimensional histo- logical imaging which would allow the automatical- ____________________________________ ly imaging and creation of whole tissue visualisa- tion. An important avenue of study would also be Advanced Imaging Facility the localisation of specific protein interactions with Steve Bagley tissue array data. The requirement for high resolu- http://www.paterson.man.ac.uk/facilities/advimg.jsp tion, low light, and low impact imaging also increas- es as it becomes a technique adopted by several One of the principal challenges in the study of bio- research groups within the institute consequently logical processes is the temporal localisation of there is a constant drive for the update of the cur- events hence the facility has concerned itself over rent equipment and software to allow the systems the last five years with the visualisation of some- to become more photon efficient and hence impact time fleeting relationships between proteins and a less upon biological activity. study of their function. Working in conjunction with other facilities within the institute, the analysis ____________________________________ of structure, function and relationship assists in building up a snapshot of biological activity. Biological Resources http://www.paterson.man.ac.uk/facilities/bru.jsp Over the previous year the institute has purchased another time lapse microscope which has been The Biological Resources unit is a modern trans- designed for the analysis of biological activity genic facility that continues to support the scientif- where the environment (temperature, nutrition, ic research programmes at the Paterson whilst vibration) around the microscope is neutral to the ensuring that the highest quality of welfare and P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6 50

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