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    Paterson Institute for Cancer Research Scientific Report 2011

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    Scientific Report 2011 Cover images Top Paterson Institute A histological section of a colorectal tumour, showing both the tumour itself on the right and the more organised normal tissue in the centre and on the left. for Cancer Research The section has been stained with haematoxylin and eosin to show the various structures within the tissue. The sample is approximately 5mm in length. Image provided by Darren Roberts from the Immunology Group. Bottom This is a cytospin preparation of bone marrow harvested following treatment of mice with 10mg/kg oxaliplatin & stained with May-Grunwald-Giemsa stain. Evidence of dying cells can be seen in this sample, as well as the usual bone marrow cells - macrophages, red blood cell precursors, megakaryocytes & neutrophils. This experiment forms part of a project that is looking to correlate the expression of a potential toxicity biomarker in the blood with toxicity in bone marrow. Image provided by Cassandra Hodgkinson from the Clinical and Experimental Pharmacology Group.

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    Contents Senior Management Team’s Introduction 5 John Brognard 38 Stuart Pepper 61 Signalling Networks in Cancer Group Cancer Research UK GeneChip Microarray Service Research Highlights 2011 8 Georges Lacaud 40 Morgan Blaylock 62 Stem Cell Biology Group Flow Cytometry Facility Research Groups - Valerie Kouskoff 42 Garry Ashton 63 Paterson Institute for Cancer Research Stem Cell and Haematopoiesis Group Histology Akira Orimo 44 Mark Craven 64 Crispin Miller 14 Stromal-Tumour Interaction Group Laboratory Services Applied Computational Biology and Bioinformatics Group Maurice Cowell 64 Logistics Geoff Margison 16 Research Groups – Carcinogenesis Group The University of Manchester School of Cancer and Enabling Sciences Stuart Pepper 63 Molecular Biology Core Facility Karim Labib 18 Cell Cycle Group Vaskar Saha 48 Children’s Cancer Group Research Publications 66 Iain Hagan 20 Cell Division Group Robert Hawkins 50 Seminar Series 2011 76 Medical Oncology: Clinical and Experimental Nic Jones 22 Immunotherapy Group Cell Regulation Group Postgraduate Education 78 Gordon Jayson 52 Angeliki Malliri 24 Medical Oncology: Translational Cell Signalling Group Anti-Angiogenesis Group Operations 80 Caroline Dive and Malcolm Ranson 26 Tim Illidge 54 Clinical and Experimental Pharmacology Group Targeted Therapy Group Cancer Research UK’s 84 Local Engagement and Development Ivan Ahel 28 Catharine M. L. West 56 DNA Damage Response Group Translational Radiobiology Group Acknowledgement for Funding 86 of the Paterson Institute Donald Ogilvie 30 Drug Discovery Group Research Services Career Opportunities at the 87 Steve Bagley 58 Paterson Institute Peter L Stern 32 Advanced Imaging Facility Immunology Group Duncan Smith 60 Contact Details 88 Nullin Divecha 34 Biological Mass Spectrometry Facility Inositide Laboratory Group Tim Somervaille 36 Biological Resources Unit 60 Leukaemia Biology Group

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    Senior Management Team’s Introduction Welcome to the 2011 Paterson Institute Annual Scientific Report. There have been significant changes at the Institute over the past year, most notably with Nic Jones stepping down as Director to take on the prestigious and challenging role of Chief Scientist at Cancer Research UK. We take this Professor Caroline Dive opportunity to reflect on Nic’s achievements during his tenure (Senior Group Leader) as Director, to celebrate our success stories of 2011 and to look forward to an exciting new era in the development of the Institute. Nic Jones joined the Institute in 1999 and, 2008 Research Assessment Exercise. following a scientific review, instigated a major Fortunately, Nic will retain strong links with re-organisation of its research programmes and Manchester, by continuing to run his research operations. He spent twelve years leading the group here and remaining as Director of the Professor Iain Hagan Paterson during which time it has undergone Manchester Cancer Research Centre. (Senior Group Leader) enormous development. The number of research groups has expanded, a CR-UK funded drug At the start of 2011, we formed a Senior discovery programme has been introduced, and Management Team to oversee the Paterson a research services strategy established to Institute in the interim period between Nic’s support the increasingly complex technology departure and the appointment of a new that underpins world class cancer research. Director. We have been helped enormously in These achievements have led to higher quality this task by the example set by Nic’s strong research and greater international recognition of leadership and the Institute infrastructure put in the Institute. place during his tenure as Director. Some new appointments also eased the operational burden One of Nic’s strongest legacies has been the significantly, Stuart Pepper as Head of Research Pippa McNichol development of strategic partnerships which Services and Caroline Wilkinson as Scientific (Director of Operations) serve to facilitate a fully integrated approach to Operations Manager. These positions were cancer research in Manchester. Most significantly, created upon the departure of Jenny Varley, the in 2006, the Paterson became an Institute of the Assistant Director of Research, who retired in University which was accompanied by the March after ten years in this post, preceded by creation of the Manchester Cancer Research nine years as a Paterson Institute Group Leader. Centre. This federation brings together the basic One of Jenny’s key responsibilities was to and translational cancer research carried out at develop and oversee the research services, the the University of Manchester with the clinical excellent standard of which serves as one of her research of the Medical School and The Christie strongest legacies. Pippa McNichol also moved NHS Foundation Trust. This helps to foster on from the Institute at the end of the year, after collaborations between basic scientists and nine years as Director of Operations. Her clinicians that are essential if we are to develop professionalism, energy and enthusiasm made a more effective ways of diagnosing and treating big impact upon the Institute, and along with cancer. The development of the MCRC, along Jenny, she will be greatly missed. with the activities of the Paterson Institute during Nic’s tenure, played a large part in the University The search for a new Director began in early of Manchester being judged the leading 2011 and was completed in September with the university in the UK for cancer research in the recruitment of Professor Richard Marais who 4 | Paterson Institute for Cancer Research Scientific Report 2011 Senior Management Team’s Introduction | 5

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    antisense regulatory transcripts, was made In an increasingly challenging economic climate, it A Cancer Research UK fundraiser possible by the addition of next generation is more vital than ever that we engage with our takes a closer look at our work during the Institute’s open day sequencing capability to our research services. fundraisers at every opportunity, not only to We look forward to migrating the expertise and communicate our progress, but also to convey knowledge gained from these model organism our gratitude for their support. Events which studies to human biology and ultimately to have enabled us to do this in 2011 included our clinical samples. These new and exciting Institute open day which saw ninety local technological platforms will keep us in a strong supporters attend lab demonstrations. Forty position to contribute to CR-UK’s Stratified local sixth form students got a taste of life in the Medicine Programme. The international profile of lab when they took part in practical scientific the Institute was boosted this year when Iain sessions as part of the Institute's annual schools’ Hagan teamed up with Jon Pines from the day. Our scientists also volunteered at local race Gurdon Institute to organise the UK-Japan Cell for life and relay for life events as well as Cycle workshop in a surprisingly sunny Lake providing support by manning an official pit stop District in April. Further enhancement came at the Institute during the Manchester Shine walk from the hosting of the European Bioconductor which aims to raise £2m for CR-UK. The Institute Developers’ Workshop by the ACBB Group also welcomed a variety of visitors onto our which attracted participants from all over the monthly lab tours including CR-UK shop world. volunteers, local fundraising groups, two local MPs, a MEP and a premiership footballer! It is takes up the Directorship in early 2012. We are panel highlighted the outstanding contributions Our Drug Discovery Group has expanded after always humbling to meet our supporters and we delighted by his appointment as it will initiate an made by the team to biomarker-enhanced early a successful period of recruitment. This includes a are immensely grateful to all who give their time exciting new phase in the development of the clinical trials as well as the significant impact new Head of Biology, Dr Ian Waddell, who and money so generously to help fund our Paterson Institute. Richard’s research focuses on made by their work on circulating tumour cells joined us after eighteen years as Director of research. signalling through the RAS/RAF pathway and the and circulating micrometastases. The award, Discovery Medicine at AstraZeneca. He will role that this plays in the development and consisting of a research grant of £25,000, was oversee a team of ten bio-scientists working The coming year promises to be particularly progression of melanoma, the most deadly form presented at the NCRI conference in Liverpool with a portfolio of early phase drug targets. eventful, not only due to the arrival of our new of skin cancer. His work was pivotal in where there was also success for Ahmet Acar Some of these have arisen as a result of Director and his research group, but also as a determining how mutated BRAF drives the from the Stromal-Tumour Interaction Group, led collaborations with Paterson Institute Group result of further Group Leader recruitment, formation of this disease and he is now by Dr Akira Orimo, who was runner up in the Leaders illustrating the benefit of having drug which had been put on hold pending the new translating his findings via the development of best student poster competition. His work discovery capacity embedded within the Director’s arrival. Five Senior Group Leaders will therapeutic agents that are undergoing early describes how notch signalling mediates Institute. undergo Quinquennial Reviews which will allow clinical trials. His wide span of interests from myofibroblast differentiation of carcinoma- us to take stock of past achievements and basic science to transgenic models of disease, associated fibroblasts. Caroline Dive and Malcolm Ged Brady was appointed as Deputy Group prioritise our future research activities. There will coupled with his active engagement in both drug Ranson led a successful application to renew the Leader of the Clinical and Experimental also be reviews of all our Research Services discovery and clinical trials, perfectly mirrors the funding of the Manchester Experimental Cancer Pharmacology (CEP) group where he has which will direct us towards the necessary range of research activities being undertaken at Medicine Centre which was one of three that expanded the CEP Biomarker Portfolio to changes that will enable us to meet the evolving the Institute. will receive the maximum level of funding include a Nucleic Acids Biomarkers team. needs of the Institute. It promises to be an (£500,000 per annum for five years). The Stephen Walker has also joined CEP and is exciting time and we look forward to the Despite changes to its leadership, the Institute renewal will allow the centre to develop its playing a critical role in the management of the continuing strengthening and development of the has continued to thrive, with our scientists experimental cancer medicine portfolio in areas increasing number of clinical studies underway in Institute under Richard Marais’ leadership enjoying a great deal of success in 2011. First of such as radiotherapy, proteomics, metabolomics this group and will also assist in the development allowing the Paterson to play its part in realising all, the DNA Damage Response Group, led by and imaging and continue its early phase clinical of circulating tumour cell and blood based the goals of Cancer Research UK. Ivan Ahel, in collaboration with Professor David trial programme. biomarker assays. Leys’ group at the University of Manchester, published their ground-breaking work describing Waleed Alduaij, who recently completed his PhD the crystal structure of a poly (ADP-ribose) thesis in the Targeted Therapy Group, was Senior Management Team: Professor glycohydrolase in the highly prestigious journal, awarded the Royal Society of Medicine Caroline Dive (Senior Group Leader), Nature. This valuable structural insight will help Oncology Section - Sylvia Lawler Prize. Matthew Pippa McNichol (Director of Operations), Professor Iain Hagan direct mechanistic studies which should further Krebs, from the Clinical and Experimental (Senior Group Leader) the ultimate goal of developing PARG inhibitors Pharmacology Group, won the Institute’s for use in future cancer therapies. Ivan’s ”Dexter Young Investigator” award. His PhD continuing success was rewarded with his studies have culminated in three first author acceptance onto the EMBO Young Investigator publications, including a report describing the Programme. presence and prognostic significance of circulating tumour cells in non-small-cell lung The Cancer Research UK Translational Cancer cancer which was published in the Journal of Research Prize for 2011 was awarded to the Clinical Oncology. The previous winner of this leaders of the Clinical Experimental award, Danny Bitton (ACBB Group), published Pharmacology team, Caroline Dive and Malcolm two first author papers this year in collaboration Ranson along with Fiona Blackhall, the lung with the Cell Division Group. One of these cancer translational research lead within CEP and studies, which showed that fission yeast meiosis Andrew Hughes from AstraZeneca. The judging is regulated by a novel mechanism using 6 | Paterson Institute for Cancer Research Scientific Report 2011 Senior Management Team’s Introduction | 7

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    Research Highlights Slade, D., Dunstan, M.S., Barkauskaite, E., Weston, R., Lafite, P., Dixon, N., Ahel, M., Leys, expressed, we selected AI467606 for further studies. AI467606 encodes a protein with no D. and Ahel, I. known function and with no homology to other The structure and catalytic mechanism of a proteins. We validated the difference in poly(ADP-ribose) glycohydrolase. AI467606 expression and demonstrated by Nature, 2011, 477: 616-620. chromatin immunoprecipitation and promoter assay the direct regulation of AI467606 Poly(ADP-ribosyl)ation is a post-translational expression by AML1/RUNX1. We further protein modification that serves to regulate established that AI467606 is specifically many cellular pathways important for genome expressed in the haematopoietic system from its stability including DNA repair, mitosis and establishment early in ontogeny to its apoptosis. The Poly(ADP-ribose) chemical signal maintenance throughout adult life. Taken In this section we are highlighting some research publications is synthesised by the PARP family of enzymes together our findings indicate that AI467606 is a using cofactor NAD as a substrate. PARP novel transcriptional target of RUNX1/AML1 from 2011 which report significant advances in specific areas. inhibitors have been showing great promise in widely expressed within the haematopoietic The selected papers demonstrate the breadth and the quality clinical trials for patients with breast, ovarian and system. We are currently evaluating to what prostate cancers caused by mutations in genes extent AI467606 expression is altered in of the research being undertaken by the groups at the called BRCA1 and BRCA2. The main protein leukaemia cells and are further investigating the Institute. that removes poly(ADP-ribose) signals after the function of this intriguing novel protein in specific pathway has been completed is called haematopoiesis. poly(ADP-ribose) glycohydrolase (PARG). Bitton, D.A., Grallert, A., Scutt, P.J., Yates, T., Li, Y., Mehrotra, P.V., Ahel, D., Ryan, D.P., Weston, R., Scientists believe that, similar to PARP inhibitors, Harrison, L. R., Micha, D, Brandenburg, M., Bradford, J.R., Hey, Y., Pepper, S.D., Hagan, I.M. Wiechens, N., Kraehenbuehl, R., Owen-Hughes, drugs designed to block the action of PARG Simpson, K. L., Morrow, C. J., Denneny, O., and Miller, C.J. T., and Ahel, I. could be effective in treating cancer, but the Hodgkinson, C., Yunus, Z., Dempsey, C. Roberts, Programmed fluctuations in sense/antisense DNA repair factor APLF is a histone chaperone. progress in developing permeable, small- D., Blackhall, F., Makin, G. and Dive, C. transcript ratios drive sexual differentiation in Mol Cell, 2011, 41: 46-55. molecule PARG inhibitors has been limited due Hypoxic human cancer cells are sensitized to S. pombe. to the lack of mechanistic and structural data for BH-3 mimetic-induced apoptosis via Mol Syst Biol, 2011, 7: 559. There are many pathways and signalling PARG. In this work we present the first insight downregulation of the Bcl-2 protein Mcl-1. strategies that control genome stability in into the three-dimensional structure of a PARG J Clin Invest, 2011, 121(3): 1075-87. It has recently been shown that the majority of humans. Many of these pathways are regulated enzyme. This structure, in combination with the human genome is transcribed into non- by poly(ADP-ribosyl)ation. Poly(ADP- functional analysis, provides a vital understanding The majority of solid tumours contain regions of coding RNAs that are never translated into ribosyl)ation is a post-translational protein of how PARG works and will facilitate the hypoxia, due to insufficient tumour vasculature. functional proteins. This revelation has led to modification synthesised by the poly(ADP- development of small cell-permeable PARG Tumour cells which reside in the hypoxic region considerable interest in the potential role of ribose) polymerase (PARP) family of enzymes, inhibitors. are resistant to the majority of conventional these novel transcripts. The fission yeast using the vital cellular cofactor NAD as a chemotherapeutic agents and radiotherapy. They Schizosaccharomyces pombe is an important substrate. The recent development of potent, Ferreras, C., Lancrin, C., Lie, A. L. M., Kouskoff, V. also have an increased invasive capacity and are model organism for studying these mechanisms, cell-permeable PARP inhibitors has provided and Lacaud, G. enriched for stem cell markers. Therefore, novel since many of the key pathways involved in RNA powerful tools to study the pathways regulated Identification and characterization of a novel agents which preferentially kill hypoxic cells are interference (RNAi) are conserved in humans. In by poly(ADP-ribose), as well as providing a transcriptional target of RUNX1/AML1 at the of great therapeutic interest. The BH3 mimetic collaboration with the Cell Division group, the promising class of novel drugs in the treatment onset of haematopoietic development. ABT-737 induces apoptotic cell death by Applied Computational Biology and of cancer. Poly(ADP-ribosyl)ation plays a major Blood, 2011, 118: 594-7. interrupting the protein-protein interactions Bioinformatics (ACBB) group has been using S. role in DNA repair, where it regulates chromatin between pro- and anti-apoptotic Bcl-2 family pombe to study these basic processes. Bitton, relaxation as one of the critical events in the The gene AML1/RUNX1 (Acute Myeloid proteins. Harrison et al investigated the effect of Grallert et al., performed strand specific deep repair process. However, the molecular Leukaemia 1) encodes a transcription factor that ABT-737 on normoxic and hypoxic small cell sequencing of total RNA over a timecourse of mechanism by which poly(ADP-ribose) regulates the expression of haematopoietic lung cancer and colorectal cancer cells. This sexual differentiation. These data revealed a set modulates chromatin remains poorly genes and is critical for haematopoietic work demonstrated that hypoxic cells were of ‘Antisense Regulatory Transcripts’, or ARTs, understood. Here we identified the poly(ADP- development. Alteration in the activity of more sensitive to ABT-737 induced apoptosis that on further manipulation were found to be ribose)-regulated protein, APLF (Aprataxin and AML1/RUNX1 by either mutation or than their normoxic counterparts and that this responsible for regulating the protein levels of PNK-Like Factor), as a protein with the translocation is the most frequent initiating event was due to down-regulation in hypoxia of the some of the key coordinators of sexual remarkable ability to bind histones – major leading to leukaemia. To better understand the ABT-737 resistance biomarker Mcl-1, a Bcl-2 differentiation. A significant number of ARTs arise structural components of chromatin. We consequences of alteration of the activity of the family protein for which ABT-737 has only poor from adjacent and overlapping pairs of protein demonstrate that APLF possesses histone protein AML1/RUNX1, it is important to define affinity. ABT-737 sensitisation was independent coding genes transcribing towards each other chaperone activity in vitro, which we suggest is the downstream transcriptional targets of of Hif-1α (the main regulator of hypoxic from opposite strands of the genome. As well as important in the regulation of chromatin AML1/RUNX1. response) and Mcl-1 down-regulation was demonstrating a critical role for these transcripts structure in response to DNA damage and also caused by reduced translation of the Mcl-1 gene. in controlling meiosis, a key finding of the paper, for efficient DNA repair. In this study, we compared gene expression These data suggest that combining ABT-737 with therefore, is that neighbouring genes can regulate between AML1/RUNX1+/- and AML1/RUNX1-/- other therapeutic agents such as radiotherapy each other; a critical factor when considering the cells at a stage where blood development is that preferentially target normoxic regions of effects of knockout or knockdown studies, since halted in the absence of AML1/RUNX1 activity. tumours may prove clinically beneficial and this is perturbations to one locus may lead to Among several other genes differentially being investigated in further studies. unintended disruptions to another. 8 | Paterson Institute for Cancer Research Scientific Report 2011 Research Highlights | 9

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    Krebs, M. G., Sloane, R., Priest, L., Lancashire, L., tumorigenic phenotypes of lung cancer cells. We The anti-CD20 monoclonal antibody (mAb) functional assays, we showed that transgressing Hou, J. M., Greystoke, A., Ward, T.H., demonstrated the importance of DAPK3 rituximab has substantially improved the clinical cells are motile, invasive, express active RAC2 Ferraldeschi, R., Hughes, A., Clack, G., Ranson, mutations by reconstituting lung cancer cells that outcome of patients with a wide range of B-cell and have an organised actin cytoskeleton. CNS M., Dive, C. and Blackhall. F.H. harboured a loss-of-function mutation in DAPK3, malignancies. However, many patients relapse or infiltrating cells from both cell lines and primary Evaluation and Prognostic Significance of with the wild type functional kinase, which fail to respond to rituximab, and thus there is material expressed CD70, ICAM1 and LFA-1. Circulating Tumor Cells in Patients With resulted in the lung cancer cells being more intense investigation into the development of This suggests that subclones expressing key Non-Small-Cell Lung Cancer. responsive to apoptotic stimuli and novel anti-CD20 mAbs with improved adhesion/integrin molecules activate cytoskeletal J Clin Oncol, 2011, 29(12): 1556-63. chemotherapeutics. As we progress into the age therapeutic efficacy. The Targeted Therapy reorganisation promoting diapedesis on contact of personalized medicine it will be essential to Group, led by Tim Illidge, has previously shown with stromal cells. In the future, it may be Lung cancer is the leading cause of cancer- determine causal mutations in cancer patients that certain antibodies including anti-HLA-DR possible to target these interactions as an related death worldwide with Non-Small Cell and to use this knowledge to administer drug and type II anti-CD20 mAbs efficiently induce adjunctive to chemotherapy and prevent Lung Cancer (NSCLC) accounting for 85% of cocktails tailored to an individual tumour that programmed cell death (PCD) through a extramedullary recurrences. cases. There are few predictive biomarkers to target aberrantly activated or inactivated process involving lysosomsal death (Ivanov et al., help guide therapeutic choices. Circulating pathways ultimately leading to the specific killing J Clin Investigation, 2009). Now, for this first time, Di, Y., Holmes, E. J., Butt, A., Dawson, K., tumour cells (CTCs) are an appealing biomarker of cancer cells. they have demonstrated that the humanized, Mironov, A., Kotiadis, V. N. - as the cells responsible for metastasis, their glycol-engineered anti-CD20 mAb GA101, which Gourlay, C. W., Jones, N. and Wilkinson, C. R. molecular characterisation will lead to increased Tamm, T*., Grallert, A*., Grossman, E. P., Alvarez- is currently undergoing extensive clinical testing, H2O2 stress-specific regulation of S. pombe understanding of the biology of metastasis and Tabares, I., Stevens, F. E. and Hagan, I. M. can trigger non-apoptotic PCD in a range of B- MAPK Sty1 by mitochondrial protein will help develop novel treatment strategies. This Brr6 drives the Schizosaccharomyces pombe lymphoma cell lines and primary B-cell phosphatase Ptc4. study explored the presence and prognostic spindle pole body nuclear envelope malignancies. This research demonstrates that EMBO J, 2011, 31(3): 563-75. value of CTCs in patients with advanced stage insertion/extrusion cycle. GA101-induced cell death is dependent on actin NSCLC using the FDA approved CellSearch™ J Cell Biol, 2011, 195: 467-84. reorganization, can be abrogated by inhibitors of Cells need to respond in an appropriate and system. CTCs (≥2 CTCs/7.5ml blood) were actin polymerization and is independent of BCL- timely manner to a broad spectrum of stress detected in 32% of patients with advanced Yeast biology has contributed much to our 2 over-expression and caspase activation. conditions. A key feature of stress responses is NSCLC and the presence of 5 or more CTCs in understanding of the basic biology of a cell. GA101-induced PCD is executed by lysosomes the mobilisation of appropriate defence and 7.5ml blood was strongly associated with poor While the function of the majority of the genes which disperse their contents into the cytoplasm repair mechanisms. In fission yeast, many of these prognosis, compared to those patients with less encoded within the genome is understood or and surrounding environment. Taken together, responses are orchestrated through the Sty1 than 5 CTCs. In multivariate analysis, CTC can be derived from comparisons between these findings reveal that GA101 is able to MAP kinase. Like its mammalian counterpart, number was the strongest independent homologues in different systems, functional potently elicit actin-dependent, lysosomal cell p38, Sty1 is phosphorylated and activated by a prognostic factor amongst all known standard interrogation via genetic screens can still prove death, which may potentially lead to improved variety of stress stimuli. Similar to other MAPK prognostic variables. Furthermore, a informative. We identified the fission yeast Brr6 clearance of B-cell malignancies in vivo. pathways, Sty1 signalling is highly regulated to pharmacodynamic effect was demonstrated molecule in a screen for mutants that were control the magnitude and duration of signalling. where a reduction in CTC number after a single unable to form a mitotic spindle. Significantly Holland, M., Castro, F. V., Alexander, S., Smith, This is achieved through co-ordination of cycle of standard-of-care chemotherapy Brr6 is required for an aspect of mitosis that is D., Liu, J., Walker, M., Bitton, D., Mulryan, K., positive and negative acting signals. Positive predicted for better outcomes than in those unique to organisms that undergo a closed Ashton, G., Blaylock, M., Bagley, S., Connolly, Y., signals include stress conditions that result in the patients whose CTC number increased. This mitosis in which the nuclear envelope does not Bridgeman, J., Miller, C., Krishnan, S., Dempsey, phosphorylation of Sty1 whereas negative signals study has formed the basis for ongoing studies in break down: polar fenestration. Polar C., Masurekar, A., Stern, P., Whetton, A. and involve phosphatases that inactivate Sty1 through lung cancer focused on CTC characterisation fenestration is the localised disruption of Saha, V. dephosphorylation. We found that Ptc4, a PP2C- with a view to identification of new treatment envelope integrity to enable the spindle pole to RAC2, AEP, and ICAM1 expression are family phosphatase, regulates both the magnitude targets and to developing CTCs as predictive nucleate microtubules that contact the cell associated with CNS disease in a mouse and duration of Sty1 activation in response to biomarkers. cortex at the same time as nucleating the model of pre-B childhood acute lymphoblastic H2O2 but not other stresses. microtubules of the mitotic spindle. As the leukemia. Brognard, J., Zhang, Y. W., Puto, L. A. and nuclear envelope breaks down during every Blood, 2011, 118: 638-49. Surprisingly, Ptc4 localizes to the mitochondria Hunter., T. division in human cells they do not require a and is targeted there by an N-terminal Cancer-associated loss-of-function mutations member of this highly conserved protein family. Though <2% of children with acute mitochondrial targeting sequence (MTS) which is implicate DAPK3 as a tumor-suppressing kinase. The highly structured nature of the conserved lymphoblastic leukaemia have central nervous cleaved upon import. A fraction of Sty1 also Cancer Res, 2011, 71:3152-61. domain in Brr6 and its apparent role in system (CNS) disease at diagnosis, this rises to localizes to the mitochondria suggesting that modifying the lipid composition of the nuclear over 40% at recurrence. The pathogenesis of this Ptc4 attenuates the activity of a mitochondrial There has been a recent flood of cancer kinome envelope makes it an ideal target for the process has remained an enigma, with the pool of this MAPK and moreover, that Sty1 may sequence data, but the functional consequences development of novel anti-eukaryotic microbial dogma being that leukaemic cells enter the CNS phosphorylate and thereby regulate targets in of the reported protein kinase mutations have therapies. via burst capillaries. As conventional therapy this organelle. Cleavage of the Ptc4 MTS is been inferred largely through statistical does not cross the blood-brain barrier, these greatly reduced specifically upon H2O2, resulting approaches. Our study, published in Cancer Alduaij, W., Ivanov, A. Honeychurch, J., Cheadle, cells survive giving rise to recurrence. We in the full length form of the phosphatase; this Research, represents a critical first step in E., Potluri, S., Lim, S. H., Shimada, K., Chan, C. H., developed a murine model of CNS disease, displays a stronger interaction with Sty1 thus assessing the functional relevance of putative Tutt, A., Beers, S. A., Glennie, M. J., Cragg, M. S. which demonstrates that CNS disease occurs via suggesting a novel mechanism by which the driver mutations experimentally. The study and Illidge, T. M. haematogenous dissemination and cellular negative regulation of MAPK signalling is illustrates how bioinformatic approaches can be Novel type II anti-CD20 monoclonal antibody diapedesis of lymphoblasts across blood-brain controlled and providing an explanation for the utilized to mine large cancer genomic datasets to (GA101) evokes homotypic adhesion and actin- and blood- cerebrospinal fluid barriers. These oxidative stress-specific nature of the regulation accurately identify important functional dependent, lysosome-mediated cell death in B- findings replicate autopsy findings in children of Sty1 by Ptc4. mutations. We were able to demonstrate that cell malignancies. dying due to CNS leukaemia. Using a semi- mutations in DAPK3 are loss-of-function Blood, 2011, 117: 4519-29. quantitative proteomic discovery approach and mutations that are essential for maintaining the 10 | Paterson Institute for Cancer Research Scientific Report 2011 Research Highlights | 11

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    Research groups Paterson Institute for Cancer Research 12 | Paterson Institute for Cancer Research Scientific Report 2011 Research Groups - Paterson Institute for Cancer Research | 13

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    Applied Computational Biology and meiosis. Strand-specific deep sequencing of total RNA taken from S. pombe cells over a time from overlapping protein coding genes transcribing towards each other on opposite Bioinformatics Group course of meiosis was used to investigate how strands of the genome. This led to the the transcript profile of the cells changed as they conclusion that adjacent genes can regulate each http://www.paterson.man.ac.uk/bioinformatics underwent sexual differentiation (Bitton et al. other, and has profound implications for gene 2011, Mol Syst Biol, 7:559). Analysis of these deletion experiments, since care must be taken data identified hundreds of novel transcripts, and to ensure that the phenotype observed when revealed that a significant number of these were knocking out one gene is not the result of an differentially expressed, suggesting that they are unexpected interaction with one of its under regulatory control and may therefore be neighbours. functional. A subset of these molecules originated from the opposite strand to a known Software engineering The advent of high density tiling microarrays and deep protein coding gene, resulting in an antisense Bioconductor is an international collaboration to transcript. We hypothesised that these transcripts write open source software for the analysis of sequencing has revealed that the majority of the human may serve to regulate the amount of protein biological data. With the advent of high genome is transcribed, even though less than 2% of it encodes expressed by their corresponding protein coding throughput technologies including deep gene. We were able to test this hypothesis for sequencing, proteomics and microarrays, protein sequences. A major focus of the Applied four key regulators of meiosis (Spk1,Dis1,Spo4, computing is becoming increasingly ubiquitous Computational Biology and Bioinformatics (ACBB) Group is Spo6), each of which was found to have an within the biosciences, and the software tools associated antisense transcript. In every case, generated by projects such as Bioconductor are to develop a better understanding of the roles played by these over-expression of the antisense transcript from fundamental to much of the research conducted Group Leader novel non-coding RNA molecules, and to determine how an integrated ectopic locus resulted in the same in an institute such as the Paterson. Some of the phenotype as a deletion of the corresponding software packages developed by the ACBB Crispin Miller their behaviour is altered in tumours. The group is highly protein coding gene. For one locus, dis1+, the group are downloaded thousands of times each Postdoctoral Fellows inter-disciplinary and features a mixture of computer scientists, availability of an antibody made it possible to month. In December, we hosted the European measure protein expression. As predicted, over- Bioconductor Developers’ Workshop, a meeting Danny Bitton mathematicians and biologists. It collaborates widely with other expression of the dis1 antisense transcript designed to help foster the exchange of technical John Hall (joint with Translational Radiobiology Group) groups in the Paterson Institute and the Manchester Cancer resulted in a reduction in Dis1 protein levels. We expertise, to keep contributors up to speed with Hui Sun Leong refer to these transcripts as Antisense Regulatory the latest developments in Bioconductor, and to Yaoyong Li Research Centre. Transcripts, or ‘ARTs’. Further analysis coordinate related efforts. This year, the meeting demonstrated that for each of the four loci featured speakers from the Paterson Institute, Software Architect Identification of novel protein coding genes Schizosaccharomyces pombe, and to revise the tested, the action of these ARTs was dependent the Fred Hutchinson Cancer Research Center, Tim Yates One possibility raised by the widespread structure of many other protein coding genes. S. Figure 1 on core components of the RNAi machinery. Harvard University, Johns Hopkins University, A small region of the S. pombe Cambridge University, the CR-UK Cambridge Bioinformatics Programmer transcription of non-coding RNAs is that some pombe is an important model organism that genome showing sites of ART Chris Wirth of them encode for previously undiscovered shares many key pathways with human cells (see Antisense expression can arise from a number of Research Institute, IPB Halle and The University (Antisense Regulatory Transcript) proteins. Using the fission yeast below). In collaboration with the Cell Division activity (blue) as identified in Bitton sources including the expression of independent of Zurich. Bioinformatician Schizosaccharomyces pombe as a model organism, Group, we were then able to demonstrate that et al., (2011, Mol Syst Biol, 7:559). non-coding RNA transcripts, the transcription of Jan Taylor (joint with Translational Radiobiology Group) Danny Bitton (at the time, a graduate student these predictions did indeed result in the Each grey line represents a fragment protein coding genes that overlap at their 3’ Building gene expression classifiers from of chromosome, with known genes ends, and a phenomenon known as ‘bi- clinical data within the group), was able to use protein mass expression of novel proteins, that some of these represented as grey boxes Scientific Officer spectrometry to test this hypothesis (Bitton et al. proteins were essential for cell viability, and that directional transcription’ in which the polymerase Archival Formalin Fixed Paraffin Embedded (highlighted by black lines) appearing Paul Scutt appears to transcribe away from its promoter in (FFPE) tissue is an immensely valuable source of 2011, Genetics). He did this by searching the the deletion of others resulted in strong above or below, according to whether output of a mass spectrometer against a phenotypes including slow-growth and delayed- they are expressed on the forward or the ‘wrong’ direction. All these phenomena were information pertaining to cancer. Unfortunately, Graduate Students Danish Memon database derived from the set of all the known division. These novel genes (new1-new25 and reverse strand. observed in our data, including antisense arising the preservation process damages RNA, making Sharmin Naaz (joint with Stem and predicted protein sequences in the genome, tam1-tam14; transcripts altered in meiosis) have it hard to extract meaningful transcription data Cell and Haematopoiesis Group) generated by translating it in all six possible been incorporated in PomBase, the fission yeast from FFPE material. In collaboration with the Andrzej Rutkowski (joint with Translational Radiobiology Group we have been Immunology Group) forward and reverse reading frames. This differs reference genome database from a typical mass spectrometry experiment, (http://www.pombase.org). CENPW/Cug2, the developing bioinformatics approaches to support María José Villalobos Quesada (joint with Cell Division Group) which searches only against a database of known human homologue of one of these genes, new1, the analysis of microarray data generated from proteins, and allows it to be used for protein is a putative oncogene found upregulated in FFPE tissue. Recently, we were able to show not Systems Administrator discovery as well as identification. One of the many human cancers (McAinsh & Meraldi 2011 only that it is possible to use FFPE RNA to build Zhi Cheng Wang (joint with IT a meaningful gene expression based classifier department) challenges of such an approach is that the Semin Cell Dev Biol; Chun et al. 2011 J Biol Chem). candidate database contains a large number of capable of separating squamous cell carcinoma potential sequences that will never be expressed, ARTs – Antisense Regulatory Transcripts (SCC) and adenocarcinoma (AC), but that substantially increasing the possibility of spurious RNA interference (RNAi) is the process by transcripts from this classifier could be used to false positive matches. This requires the careful which double strand RNA molecules are used to correctly segregate an independent cohort of application of statistics and the incorporation of regulate gene expression. Many of the key samples using a different technology, QuantiGene annotation data from other sources, in order to components of the RNAi pathways found in (Hall, Leong et al. 2011 BJC). ensure that the error rate is minimised whilst human cells are also present in fission yeast, allowing the maximum scope for real matches to making it an important model system for be identified. Using these approaches, we were studying non-coding RNAs. In collaboration with Publications listed on page 66 able to add an additional 0.8% to the protein the Cell Division group, we have been studying coding complement of the fission yeast the role of non-coding RNAs in regulating 14 | Paterson Institute for Cancer Research Scientific Report 2011 Applied Computational Biology and Bioinformatics Group | 15

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    Carcinogenesis Group The subsequent steps in Atl1-mediated processing of the lesion have not been http://www.paterson.man.ac.uk/carcinogenesis completely elucidated but current evidence indicates that the bound Atl1 is a substrate for and/or recruits components of the nucleotide excision repair system which ultimately results in the elimination of the lesion from DNA. Figure 1 shows our current thinking of the factors involved. It is clear that in some cases, repair is probably mediated by the downstream components of the global genome repair (GGR) pathway involving the interaction of the S.pombe The group’s work focuses on the mechanism of action of a equivalents (Rhp7 and Rhp14 respectively) of the human DNA damage response (DDR) group of chemical compounds called alkylating agents. Agents factors DDB1 and DDB2. This is probably of this type display a wide range of biological effects in living followed by the binding of the S.pombe equivalents (Rhp41 and Rhp23 respectively) of organisms all of which are attributed to the introduction of the human proteins Xeroderma Pigmentosum various types of DNA damage. The ability of these agents to complementation factor C (XPC) and homologous recombination factor 23b kill cells is exploited in their use as antitumour agents in the (hHR23b). After this, transcription factor TFIIH is Group Leader treatment of certain types of cancer. Although a number of recruited and two helicase proteins therein unwind the DNA, allowing the region containing Geoff Margison different lesions can be generated in DNA, one of these, the lesion to be removed by dual endonuclease Postdoctoral Fellow O6-alkylguanine, is considered to be the most important. Figure 1 One of the original observations we made was activities. This is followed by copying of the that both eATL and Atl1 were able to rapidly complementary strand to fill in the gap and Vitaly Latypov We are trying to establish precisely how cells respond to this Current model for the processing of bind to methylated DNA and inhibit the action rejoining of the break by DNA ligase. In other O6-alkylguanines in S.pombe DNA Scientific Officers damage and the impact that this has on the biological effects by Atl1. GGR; global genome repair, of MGMT on its normal substrate, O6- cases, however, the Atl1-Rhp7/14/23/41 complex Mandy Watson (Until June) NER; nucleotide excision repair, TCT; methylguanine. One of the many questions this might be more stable. If such a complex is Gail McGown of these agents. transcription-coupled repair. See text raised was to what extent these proteins would present in the transcribed regions of transcribed Mary Thorncroft for other abbreviations and genes, transcription-coupled repair (TCR) factors explanation. bind to other O6-alkylguanines in DNA. We have Background haematological toxicity was constructed. Nadir been able to pursue this through a collaboration seem likely to be involved in processing the Graduate Students Pat Senthong (Jointly with Dr The Cancer Research UK drug Temozolomide, white-cell and platelet counts were related to, with Dr David Williams in the Chemistry lesions (see Figure 1). In non-transcribed genes, Andy Povey, Health Sciences which is used in the treatment of glioma and and hence could be predicted from, Department of Sheffield University who has and in cases where TCR is impaired, the stable Group at the University of melanoma, kills cells by chemical modification of pretreatment MGMT. Leucopenia and synthesized a large number of short complex probably stalls DNA replication forks. Manchester) Our current concept is that such structures may DNA, in particular at the O6-position of guanine. thrombocytopenia were more prevalent oligonucleotides containing a wide range of O6- Such lesions can be repaired by the damage amongst patients with low pretreatment PBMC alkylguanines. We have been examining the be extremely difficult to resolve and may Undergraduate Students Amy Hatch (From July) reversal protein O6-methylguanine-DNA MGMT, according to the highest grades of interaction of Atl1 and eATL with these oligos by account for the lethality of the agents that Matthew Humphreys methyltransferase (MGMT), which simply toxicity experienced and/or the dose intensity a variety of methods. More recently, in generate such lesions. Further studies will (until February) removes the methyl group from O6- patients could sustain. It is reasonable to collaboration with Andy Povey at the University hopefully confirm or refute our hypotheses. Jo Kelly (Until September) Sarah Pinder (From July) methylguanine and restores the DNA to its pre- conclude that the determination of MGMT in of Manchester, we have also investigated them as damaged state and hence protects against PBMC may be used to identify patients at substrates for MGMT. John Tainer and his group CHEMORES Vitaly Sukhinin (Until August) Emma Williams (From July) Temozolomide toxicity. To combat this, in greatest risk of toxicity or who are suitable for (Scripp’s research Institute, La Jolla), have We are a member of a European Union collaboration with Prof Brian McMurry and the dose intensification. obtained additional crystal structures with Atl1 Framework 6 programme-supported late Dr Stanley McElhinney (and their group at and these are telling us more about the Consortium that is investigating alternative the Chemistry Department, Trinity College, Alkyltransferase-like proteins molecular interactions that take place. The crystal mechanisms of chemotherapy resistance. Our Dublin) we developed the potent MGMT We have previously described some of our structures add further support to our previous focus is on aspects of DNA repair in a number inactivating drug, Lomeguatrib. However, in studies of a novel family of proteins, the suggestion that Atl1 binds to DNA, “flips” out of human melanoma cell lines, some of which we clinical trials, Lomeguatrib did not enhance the Alkyltransferase-like (ATL) proteins that we so the O6-alkylguanine from the base stack using an are generating by repair gene transfection. We therapeutic effect of Temozolomide and we are named because they resemble the arginine “finger”, rotates the phosphodiester are using biochemical assays of functional activity now investigating the possible basis of this by alkyltransferase family, but with the major bond by means of a tyrosine residue and of DNA repair proteins along with highly using fission yeast to characterise alternative difference that the cysteine residue which accommodates the base in the binding pocket, sensitive methods of quantifying DNA lesions repair pathways. accepts the alkyl group is replaced, usually by stabilized by various ionic interactions with generated by treatment with Temozolomide in tryptophan. Genes encoding these proteins are amino acid residues in the vicinity. In the case of attempts to identify pathways, perhaps Clinical studies present in prokaryotes, archaea and eukaryotes, MGMT, this allows alkyl group transfer to the resembling the Atl1 pathway in S.pombe, that The dose-limiting toxicity of Temozolomide but it is intriguing that some organisms seem to cysteine residue and rapid dissociation of the may provide resistance. occurs in bone marrow cells. To assess if this need both functions. Thus budding yeast MGMT, but ATL, unable to transfer the alkyl could be predicted using cells in the circulation expresses only an alkyltransferase and S.pombe group, remains bound. However, it is worth (peripheral blood mononuclear cells: PBMCs) we only has an ATL (Atl1), whereas E.coli expresses noting that this juxtapositioning is not the only Publications listed on page 66 quantified MGMT levels in ninety-three both alkyltransferase genes (in fact two: ada and prerequisite for alkyl transfer. We previously melanoma patients treated with temozolomide ogt) and an ATL gene (eATL). The evolutionary showed that mutation of the tryptophan in Atl1 or dacarbazine in four clinical trials. A model of basis of these differences are open to to cysteine, as in MGMT did not confer the interaction between MGMT expression and speculation. alkyltransferase activity. 16 | Paterson Institute for Cancer Research Scientific Report 2011 Carcinogenesis Group | 17

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    Cell Cycle Group Figure 1 A A -4 -4 Stability of the replisome at defective f4 f4 http://www.paterson.man.ac.uk/cellcycle DNA replication forks is independent 0 0 b b 10 10 of S-phase checkpoint kinases. Cells d d Æ Æ 1Æ 1Æ l l l l A A 1- 1- ro ro ro ro were synchronised in G1-phase and 53 53 3- 3- nt nt nt nt ec ec ec ec then released for 90 minutes into S- d d d d co co co co phase in the presence of 0.2M m ra m m ra m sl sl hydroxyurea (HU), to induce G1 HU HU HU HU HU G1 HU HU HU HU HU replication stress. Replisome material was isolated by Pol2 immunoprecipitation of the Sld5 Pol1 subunit of the GINS complex. Mcm6 Our group studies the mechanisms and regulation of Mcm5 chromosome replication and cytokinesis. Eukaryotic cells have Mcm3 evolved complex regulatory pathways to survive replication Mcm2 stress and preserve genome integrity, and this regulation is Cdc45 likely to be of particular importance in many human tumours Pob3 that have inherent defects in chromosome replication. In Ctf4 Group Leader response to replication stress, cells activate a signalling pathway Psf3 Karim Labib called the S-phase checkpoint, which involves activation of a Sld5 Postdoctoral Fellows series of checkpoint kinases. Previous work indicated that a Cell extract IPs of Sld5 Magdalena Foltman key role of the checkpoint kinases was the preservation of Giacomo de Piccoli Luis Garcia-Rodriguez replisome stability at defective DNA replication forks. To test subsequently when the source of replication stability is independent of the checkpoint Alberto Sanchez-Diaz stress is removed or repaired. This conclusion response, it now seems very likely that the Sugopa Sengupta this model directly, we isolated replisome material from came from a variety of chromatin checkpoint kinases will regulate replisome Scientific Officers extracts of budding yeast cells suffering replication stress. Our immunoprecipitation experiments that function, as part of a highly complex and multi- Frederick van Deursen data indicate that replisome stability is actually independent of monitored the association of replisome faceted response that preserves genome Pedro Junior Nkosi components with DNA around origins of integrity in response to replication stress. S-phase checkpoint kinases, which instead might regulate replication following replication stress. Whereas Previous large-scale screens for targets of Graduate Students replisome factors were clearly still associated checkpoint kinases identified several replication Asli Devrekanli replisome function in ways that help preserve genome integrity with DNA at replication forks in the presence of factors, and we have identified a series of new Tim Maculins Marija Maric at defective replication forks. checkpoint kinases, this association appeared to targets by analyzing replisome material isolated be lost in cells lacking Rad53 or Mec1. from cells suffering replication stress, and looking for gel-shifts that represent phosphorylations For most of the cell cycle, eukaryotic In human cells, the ATR kinase (ATR = Ataxia To study how the checkpoint response might (Giacomo de Piccoli and Karim Labib, chromosomes exist in a highly stable form as and rad related) is recruited to defective DNA regulate the replisome following replication unpublished data). The nature of this regulation double strand DNA that is compacted into replication forks and leads to activation of the stress, we developed a new approach based on will be explored in the future, but one attractive chromatin. Nevertheless, duplication of the downstream kinase Chk1, which is thought to the direct isolation of replisome material from idea could be that checkpoint kinases reduce the genome during the S-phase of the cell cycle mediate many of the roles of the checkpoint extracts of budding yeast cells. Surprisingly, we rate of fork progression in response to involves unwinding of the DNA duplex by a response. ATR also seems to have additional found that replisome stability is actually replication stress, as this might reduce the risk of DNA helicase, to produce the single strand functions not shared with Chk1, such as independent of checkpoint kinases, and in fact mutations that could result from DNA synthesis DNA template upon which DNA polymerases phosphorylation of Histone H2AX that helps more replisome material can be isolated from on a damaged template, and might also reduce can act. At each DNA replication fork the recruit other checkpoint proteins and repair cells lacking Mec1 or Rad53, due to the the exposure of unprotected ssDNA at replicating DNA has a unique structure that factors. The budding yeast orthologue of ATR is activation of both early and late origins when defective forks. The underlying mechanisms exposes single strand DNA as well as known as Mec1 and leads to activation of the cells lacking checkpoint kinases suffer replication remain to be determined, but our unpublished unprotected DNA ends on the leading and Rad53 kinase in response to replication defects. stress (Figure 1). The amount of replisome data indicate that the replicative DNA helicase is lagging strands, greatly increasing the possibility Rad53 protects cells from replication stress in material recovered from mec1∆ or rad53∆ cells a direct target of multiple checkpoint kinases. that nucleases or recombination factors might many ways, such as by inhibiting mitosis, is very similar to the amount that can isolated Future work will focus on mapping novel gain access and generate DNA damage or preventing the activation of late origins of from other mutants such as mec1-100 or sld3-A phosphorylation sites amongst replisome chromosomal rearrangements. This risk is much replication, and stimulating the activity of dbf4-4A, which cannot restrain the activation of components, and exploring whether such higher when problems in DNA synthesis ribonucleotide reductase (Labib, K. and De late origins following replication stress but do modifications do indeed regulate fork increase the amount of unprotected ssDNA at Piccoli, G., 2011, Phil. Trans. R. Soc. B, 366, 3554- preserve the functional integrity of defective progression, or else act in other ways, for replication forks, and eukaryotic cells have 3561). Previous studies indicated that both replication forks (De Piccoli et al, submitted). example by recruiting other factors to defective evolved sophisticated regulatory pathways to Mec1 and Rad53 were required for an additional DNA replication forks. detect problems in DNA replication and and apparently critical aspect of the checkpoint The resumption of DNA synthesis at defective preserve the functional integrity of defective response, namely the stabilization of the DNA replication forks is highly defective in the DNA replication forks. replisome at defective DNA replication forks, so absence of Mec1 or Rad53, and the same is true Publications listed on page 67 that DNA synthesis is able to resume in human cells lacking ATR. Although replisome 18 | Paterson Institute for Cancer Research Scientific Report 2011 Cell Cycle Group | 19

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    Cell Division Group we drew upon the observation that removal of Wee1 function enables cells to survive without Brr6 – a new target for the fight against eukaryotic microbial infections? http://www.paterson.man.ac.uk/celldivision Cdc25 because there can be no requirement for A major challenge in the fight against eukaryotic a phosphatase to remove a phosphate from microbial infections is to identify “druggable” Cdc2 if the kinase that puts this phosphate there targets or pathways within the microbe that are is absent. Thus, MPF activation and mitotic sufficiently distinct from those of the host that commitment will occur in the absence of Cdc25 they can be targeted to kill the microbe while when Wee1 is inhibited. A cue as to how this leaving the host unscathed. A screen for essential inhibition may arise in cut12.s11 mutants came molecules that are required for division of fission from the key role played by Polo kinase in the yeast conducted in the lab may well have MPF positive feedback loop in higher eukaryotes. identified a suitable target, Brr6. The basis for We therefore considered the possibility that this optimism lies in one fundamental difference Errors in chromosome transmission alter the balance Cut12 suppresses ablation of Cdc25 because it between the nuclear divisions of many microbes inappropriately prompts Polo to shut down and humans. between tumour suppressor and tumour promoting genes. Wee1. This line of reasoning uncovered a direct This imbalance favours changes in genome composition in the relationship between Polo activity and Cut12 When our cells divide we break down the status; Polo activity is elevated when Cut12 nuclear envelope that separates the nucleoplasm ensuing cell divisions that can lead to cancer. Chromosome function is enhanced and severely reduced when from the cytoplasm. This dispersal enables the segregation during mitosis is initiated by the attachment of Cut12 function is compromised. Furthermore, centrosome to nucleate two sets of while polo normally associates with the SPB for microtubules; those that form the bipolar spindle the microtubules of the mitotic spindle to the chromosomes. 30 minutes prior to mitosis, this association and the astral microtubules that anchor the Group Leader Once all chromosomes have become attached to both spindle occurs 30 minutes earlier in cut12.s11 cells. We spindle at the cortex to ensure that genome are now exploiting a range of approaches partitioning is perpendicular to the plane of Iain Hagan poles the chromosomes split into two identical chromatids that including the mass spectrometric mapping of division. For a variety of reasons many microbes Associate Scientist then move to opposite poles. Because the regulatory networks phosphorylation sites in both targeted and global maintain an intact nuclear envelope throughout approaches to identify the means by which a division in a “closed mitosis”. Because Agnes Grallert that regulate mitotic commitment and progression are highly structural component of the spindle pole can microtubules cannot penetrate the nuclear Postdoctoral Fellows conserved, studying the complexities of cell division in the exert such a strong influence upon the mitotic envelope, this preservation of envelope integrity Kuan Yoow Chan commitment switch. throughout division presents a major challenge if Marisa Alonso-Nuñez relatively simple unicellular yeasts greatly accelerates the astral microtubules are required to anchor the Ye Dee Tay analysis of the more complex issue of cell division control Figure 1 G2 M Legend spindle to the cortex. To meet this challenge many microbes undergo “polar fenestration” in Graduate Students Elvan Boke in man. SPB nuclear envelope which a localised breakdown of the nuclear P phosphate Avinash Patel ?+ envelope at the poles can enable microtubules Maria-Jose Villalobos Quesda We use the fission yeast Schizosaccharomyces critical threshold level of MPF is reached a P P to span the nuclear envelope. In some microbes, Cdc25 (joint with ACBB Group) Cdc2 Cdc2 including yeasts, the polar fenestra is used as a pombe to study cell division because it is a positive feedback loop is promoted that boosts simple, unicellular organism with excellent Cdc25 activity and suppress Wee1 activity, CyclinB Wee1 CyclinB site into which the SPB is inserted to become an genetics that is cheap to grow and divides thereby driving full-scale commitment to mitosis. ? integral part of the nuclear envelope. rapidly. While the major focus of the laboratory Fully activated MPF then activates a number of Nucleation from the inner surface of this asks how cells take the decision to divide, we highly conserved kinases that are named after Plo1 integrated SPB then generates the microtubules also use genetic screens to identify proteins that the founder members of each group Polo, that form the spindle, while the outer face Cut12 are required for cell division and have recently Aurora and NIMA (Figure 1). nucleates the astral microtubules that anchor the developed collaborations with the Applied spindle to the cortex (Figure 2). Computational Biology and Bioinformatics Group Cut12, the spindle pole and mitotic to use fission yeast as a model organism in which commitment Figure 2 We found that Brr6 is an essential molecule that to develop approaches for the interrogation of Our studies of the spindle pole body (SPB) Human cells: nuclear envelope breakdown is recruited to the SPB in order to promote SPB genome function on a global scale (see account component Cut12 have uncovered a critical role insertion into the envelope during mitotic by Crispin Miller in the ACBB Group section of for events on the spindle pole in mitotic control. commitment. A domain within the Brr6 amino this report). Specifically, they suggest that the MPF amplifying acid sequence that is bordered by two positive feedback loop is primed from the SPB. membrane spanning domains is highly structured Mitotic Commitment The foundations for this view lie in the reciprocal and shows striking conservation in a large Commitment to mitosis is regulated by the genetic interactions between cut12 and cdc25. number of microbes (including malaria and activity of a protein kinase called MPF. MPF is The cut12.s11 gain of function mutation pathogenic fungi). Importantly it is only composed of a catalytic sub-unit encoded by the suppresses loss of function mutations in cdc25. S. pombe and many microbial pathogens: polar fenestra on microbes that undergo polar fenestration that cdc2+ gene and a regulatory sub-unit called Conversely, mutational enhancement of Cdc25 have a molecule that harbours this domain. Cyclin B. Prior to mitosis MPF is inhibited via activity suppresses loss of Cut12 function. Given the prevalence of opportunistic fungal phosphorylation by the protein kinase Wee1 on Consistently, combining conditional loss of infections, the Brr6 pathway offers an attractive a residue (tyrosine 15) that lies within the ATP function mutations within cdc25 and cut12 in the set of targets for the development of novel binding pocket of p34cdc2. This phosphate is same strain generates synthetic lethality. therapeutics. removed by the protein phosphatase encoded Cdc25. The balance of activity between Cdc25 Cut12 and polo in mitotic commitment and Wee1 determines when MPF will be In seeking ways to understand how an SPB spindle pole Publications listed on page 67 chromosome activated to drive mitotic commitment. Once a component could compensate for loss of Cdc25, microtubule kinetochore microtubule kinetochore 20 | Paterson Institute for Cancer Research Scientific Report 2011 Cell Division Group | 21

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    Cell Regulation Group motor neurons and to profile gene expression from recovered RNA. We found that, as mammalian counterpart, p38, Sty1 is phosphorylated and activated by a variety of http://www.paterson.man.ac.uk/cellregulation observed in the developing liver, specific DUSP stress stimuli and inactivation of the kinase gene expression was impaired in these neurons, results in pleiotropic stress sensitivity. For any which could explain the increased levels of MAP kinase, both the amplitude and duration of activated JNK and p38, and concomitant signalling is carefully controlled. These parameters apoptosis. Thus, in development ATF2 is critical can greatly influence the cellular response to the in limiting apoptosis-inducing activities of MAP input signal and their control is orchestrated kinases. through positive- and negative-acting signals, as well as through the spatial distribution of the In tumorigenesis, ATF2 can exert pro- pathway’s components. The negative signals are tumorigenic but also anti-tumorigenic activities mediated through phosphatases which remove MAP kinase pathways are central in the cellular response to depending on the tumour type. Therefore one the activating phosphate groups. Two classes of focus of our current research has been to phosphatase act upon Sty1: the Pyp and the Ptc exogenous and endogenous stimuli, and are involved in a decipher these context-dependent activities. In phosphatases which dephosphorylate P-tyr and multitude of biological activities ranging from cell proliferation, an ongoing project exploiting a mouse model of P-thr residues respectively. Ptc4 is a PP2C family hepatocellular carcinoma, we found that ATF2 phosphatase and we have found that it regulates differentiation, and cell death. However, distinct MAP kinase and ATF7 exert novel tumour suppressing both the magnitude and duration of Sty1 activities regulate different and diverse cellular programmes. activities. Here, double mutant hepatoblasts activation in response to hydroperoxides, but not transformed with oncogenic H-Ras show a to other stress conditions. Ptc4 localises Generally, growth factor mediated activation of MAP kinases significantly stronger tendency to develop into exclusively to the mitochondria and is targeted Group Leader of the ERK family is commonly associated with cell survival and HCC after orthotopic transplantation into there by an N-terminal mitochondrial targeting recipient livers compared to ATF2 active sequence (MTS) which is cleaved upon import. Nic Jones growth promotion, while stress stimulus activated MAP kinase controls. We also find that active ATF2 induces As well as controlling MAPK activation, our data p38 can induce cell cycle arrest and apoptosis and is therefore cell death in transformed hepatoblasts in culture indicates that Ptc4 is also a critical regulator of and reduces colony formation in soft agar. These various mitochondrial functions including Associate Scientists Wolfgang Breitwieser generally regarded as growth suppressive. In contrast, the activities appear to be dependent on the oxidative phosphorylation. Using immuno- Caroline Wilkinson JNK family of MAP kinases has been associated with both activation of the upstream kinase JNK and could electron microscopy as well as sub-cellular and explain some of the tumour suppressive sub-mitochondrial fractionation techniques, we Postdoctoral Fellows growth-promoting as well as suppressing activities. Not functions that have been reported for JNK. found that a fraction of Sty1 also localises within Yujun Di Malgorzata Gozdecka surprisingly, deregulated MAP kinase signalling is a critical the mitochondria, and to the same membrane Stress Responses in Fission Yeast fraction as Ptc4, suggesting that Ptc4 attenuates Saki Kondo feature in tumour development. The Cell Regulation Group also uses fission yeast the activity of a mitochondrial pool of this Hayley Thirkettle as a convenient model to gain insights into the MAPK. Cleavage of the Ptc4 MTS is abrogated MAP kinases exert their activities, to a great shown to be in limiting the activities of upstream nature and regulation of stress responses. As specifically upon H2O2, resulting in the precursor Scientific Officers extent, through changes in transcriptional acting p38 kinase in a negative acting feedback these processes are conserved in eukaryotes, it form of the phosphatase; this displays a stronger Keren Dawson Steve Lyons programmes. Critical targets of their activities loop. This is achieved by the ATF2 dependent is anticipated that these insights will be relevant interaction with Sty1 thus suggesting a novel include members of the AP-1 complex, a dimeric activation of specific MAP kinase phosphatase to mammalian cells. In Schizosaccharomyces mechanism by which the negative regulation of transcription factor consisting of Jun, Fos, and genes of the DUSP (dual specificity pombe, the Sty1 MAP kinase plays a key role in MAPK signalling is controlled and explaining the Graduate Students Emily Holmes ATF family DNA binding proteins. Depending on phosphatases) family. Hence in the absence of mediating a general stress response. Like its oxidative stress-specific nature of the regulation Jacek Walczynski the cellular context the composition of the functional ATF2/7 in embryonic liver cells, p38 of Sty1 by Ptc4. We have identified the Lu Zhang dimeric complexes determines the regulation of MAP kinase was abnormally upregulated, at mitochondrial protease responsible for cleaving Figure 1 growth, survival, or apoptosis. For example, JNK levels which were shown to be apoptosis A. Western blot of fission yeast whole Ptc4 and we are currently investigating the phosphorylates c-Jun, leading to the dimerisation inducing. cell extract illustrating that Ptc4 mechanism by which oxidative stress inhibits this with c-Fos and activation of AP-1 target genes. exists as an extra isoform upon process. We hypothesize that the fraction of Sty1 oxidative stress induced by hydrogen localised to the mitochondria contributes to the JNK, and p38, also phosphorylate ATF2 which We also discovered a critical role for ATF2 in peroxide but not upon other forms of leads to the activation of ATF2 homodimers, or the developing central nervous system regulation of this organelle through stress. ATF2-c-Jun heterodimers and specific (Ackermann et al., 2011, PLoS One, 2011, phosphorylation of targets therein. Indeed, transcription activation. Thus, depending on the 6(4):e19090). B. Fluorescence microscopy indicates localising a stress-signalling MAPK module to a external stimulus, the AP-1 dependent that Ptc4 localizes to the compartment responsible for the majority of mitochondria as indicated by co- intracellular ROS production could promote a transcriptional programme contributes to cell Deletion of ATF2 caused the loss of specific localization with Cox4, a known proliferation (e.g. through cyclin gene expression) motor neurons of the hindbrain, which resulted timely response to oxidative damage. One protein of the inner mitochondrial or cell death (e.g. through BH3 domain gene in failure to coordinate breathing activities in membrane. possible function is that Sty1 regulates expression). postnatal stages and led to invariable death. respiration as deletion of sty1 results in reduced Analysis in the embryonic brain showed that in C. Model to explain how oxygen consumption although further work will mitochondrial Sty1 is regulated by be required to determine if this is a result of ATF2 in development and tumorigenesis the absence of ATF2, specific motor neurons of Ptc4 specifically upon oxidative To explain ATF2-dependent activities in the hindbrain developed normally but gradually directly regulating mitochondrial components stress. mammalian systems we undertook mouse underwent processes reminiscent of through phosphorylation. We are currently trying knockout studies. We showed that ATF2 (in neurodegeneration. This observation was to identify mitochondrial targets of Sty1 as well overlapping functions with its closest homologue correlated with increased levels of as determining the mechanism by which ATF7) is essential in the development of phosphorylated (activated) JNK and p38 in these oxidative stress affects the cleavage of Ptc4. embryonic liver and heart (Breitwieser et al. neurons. Laser capture dissection of embryonic 2007). Here, one critical function for ATF2/7 was motor neurons allowed us to isolate knockout Publications listed on page 67 22 | Paterson Institute for Cancer Research Scientific Report 2011 Cell Regulation Group | 23

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    Cell Signalling Group SUMOylation-induced maintenance of active Rac separation during prophase, demonstrated its requirement in balancing Eg5-induced forces http://www.paterson.man.ac.uk/cellsignalling SUMO during bipolar spindle assembly in vitro and in Activation vivo, and showed that proper centrosome GEFs Rac GTP separation in prophase facilitates subsequent (Tiam1) chromosome congression (Woodcock et al. Curr Biol. 2010; 20:669). Subsequent to this study, we PIAS3 Inactive Active have found that Tiam is phosphorylated by Cyclin B/CDK1 in mitosis. This phosphorylation, RhoGDI Rac GDP Rac GTP while not required for Tiam1 localisation to Rac GDP centrosomes, appears essential for its role in Ub HACE1 Ub regulating centrosome separation. Currently we Tumour initiation and progression result from inappropriate Sequestration of GAPs Ub Ub are investigating the mechanism by which inactive Rac P phosphorylation of Tiam1 influences its role at activation of intracellular signalling cascades. Rho-like GTPases Inactivation Rac GTP centrosomes through attempting to identify are molecular switches in signalling pathways that regulate Ubiquitylation-induced interacting partners whose interaction with Tiam1 at centrosomes is dependent on cytoskeletal and junctional organisation, as well as gene degradation of active Rac phosphorylation. transcription. In this way, Rho proteins influence cell Figure 1 rescues the migration defect of Rac1-null cells to a greater extent than wild-type Rac1. These Tiam1 antagonizes malignant progression morphology, adhesion, motility, as well as cell cycle progression Multiple mechanisms exist to regulate Rac activity. The Rac GTPase findings identified HACE1 as an antagonist of cell Despite its role in promoting tumour survival Group Leader and cell survival. cycles between inactive GDP-bound and active GTP-bound states. Rac migration through its ability to degrade active and growth, Tiam1 appears to suppress Rac1 (manuscript submitted). malignant progression since the few skin activation is facilitated by the action Angeliki Malliri of GEFs (such as Tiam1), which tumours arising in Tiam1-deficient mice Rho proteins are transforming in vitro and are migration in response to Hepatocyte Growth Tiam1-Rac signalling regulates bipolar spindle progressed more frequently to malignancy than promote GDP dissociation from Rac Postdoctoral Fellows essential for Ras-mediated in vitro transformation. Factor (HGF) signalling. Subsequently we those in wild-type mice (Malliri et al., Nature and allow GTP to bind instead. assembly, chromosome congression and mitotic Sonia Castillo-Lluva (until Moreover, data has emerged to directly implicate demonstrated that Rac1 can be conjugated to Through the association with GAPs progression dependent on phosphorylation of 2002; 417: 867). One mechanism by which Tiam1 November 2011) Natalie Mack Rho proteins in tumour initiation and SUMO-1 in response to hepatocyte growth the intrinsic GTPase activity of Rac is Tiam1 by Cyclin B/CDK1 and Rac suppress malignant progression is progression in vivo. Our group investigates the factor treatment and that SUMOylation is accelerated thereby inactivating Rac. through promoting cell–cell adhesion. Over- Andrew Porter (since Through association with RhoGDIs Tiam1 (for T-lymphoma invasion and metastasis August 2011) mechanisms by which certain regulators of the enhanced by PIAS3. We also showed that the protein) is a GEF that selectively activates Rac. expression of activated Rac or Tiam1 promotes Rac can be sequestered in its Helen Whalley Rho protein Rac control cell cycle progression GTP-bound form of Rac is a better substrate for inactive state. Activated Rac can also Mice deficient for Tiam1 are resistant to the the formation of adherens junctions (AJs) and and cell adhesion and how their activities, as well SUMOylation. Furthermore, we identified non- be removed through ubiquitylation- formation of skin tumours induced by chemical the accompanying induction of an epithelial-like Scientific Officer Gavin White as activity of Rac itself, are controlled. consensus sites within the polybasic region of induced degradation (mediated by carcinogens and the few resulting tumours grow phenotype in a number of mesenchymal cell Rac1 as the main location for SUMO HACE1 following a migration lines (Malliri & Collard, Curr Opin Cell Biol 2003; stimulus) or it can be maintained very slowly (Malliri et al., Nature 2002; 417: 867). Graduate Students Rac1 cycles between a GDP- and a GTP-bound conjugation. We demonstrated that PIAS3- To better understand the role of Tiam1 in 15: 583). Moreover, Tiam1 is required for both following its modification by SUMO Chong Tan state. When GTP-bound, it interacts with various mediated SUMOylation of Rac1 controls Rac1- (mediated by PIAS3). promoting tumour growth we have examined its the formation as well as the maintenance of Hadir Marei effector molecules that elicit downstream GTP levels and the ability of Rac1 to stimulate role in the cell cycle. During this cycle, cadherin-based adhesions (Malliri et al., J Biol Erinn-Lee Ogg (since October 2011) responses including notably actin cytoskeletal lamellipodia, cell migration and invasion. The centrosomes separate and co-ordinate bipolar Chem 2004; 279: 30092). The oncoprotein Src, a reorganisation. Multiple mechanisms control Rac1 finding that a Ras superfamily member can be spindle formation required for subsequent non-receptor tyrosine kinase implicated in activity including control of nucleotide binding SUMOylated provides an insight into the chromosome segregation during mitosis. malignant progression, potently induces and hydrolysis by Guanine nucleotide Exchange regulation of these critical mediators of cell Centrosome separation occurs via distinct epithelial–mesenchymal transition (EMT) by Factors (GEFs) and GTPase Activating Proteins behaviour. Moreover, our data revealed a role for prophase and prometaphase pathways. Kinesin-5 targeting AJs for disassembly. We recently (GAPs) respectively, regulation of subcellular SUMO in the regulation of cell migration and (Eg5), a microtubule (MT) motor, pushes showed that direct phosphorylation of Tiam1 by localisation and modulation of Rac1 protein invasion (Castillo-Lluva et al. Nat Cell Biol. 2010; centrosomes apart during bipolar spindle Src is required for the initial stages of Src- levels. More recently, regulation by post- 12:1078). assembly and its suppression results in induced EMT. We showed that Src translational modification has emerged as a monopolar spindles and mitotic arrest. Forces phosphorylates Tiam1 on tyrosine 384 (Y384). significant means of regulating Rac activity. The Rac1 activity is also regulated through that antagonise Eg5 in prophase are unknown. This occurs predominantly at AJs during the first such modification identified was C-terminal ubiquitylation and subsequent degradation. We identified a new force generating mechanism initial stages of Src-induced EMT triggering the prenylation to facilitate membrane association However, the E3 ubiquitin ligase responsible for mediated by Tiam1, dependent on its ability to localised degradation of Tiam1 at AJs by calpain but more recently phosphorylation as well as Rac1 degradation following activation by a activate Rac. We revealed that Tiam1 and Rac proteases. Abrogating Tiam1 phosphorylation modification with ubiquitin and ubiquitin-like migration stimulus was unknown. Recently, we localize to centrosomes during prophase and and degradation suppressed Src-induced AJ proteins have been described. identified this to be the tumour suppressor prometaphase, and Tiam1, acting through Rac, disassembly and inhibited cell migration. HACE1. We showed that the HACE1 and Rac1 ordinarily retards centrosome separation. Significantly, we demonstrated that in human Post-translational modifications of the GTPase interaction is enhanced by HGF signalling. Importantly, both Tiam1-depleted cells in culture lung, colon, and head and neck cancers Rac1 during cell migration Furthermore we showed that HACE1 catalyses and Rac1-deficient epithelial cells in vivo escape phosphorylation of Y384 of Tiam1 positively To gain further insight into the regulation of Rac the poly-ubiquitylation of Rac1 at lysine 147 the mitotic arrest induced by Eg5 suppression. correlated with Src activity, while total levels of during cell migration, we performed a screen for following its activation by HGF, resulting in its Moreover, Tiam1-depleted cells transit more Tiam1 were inversely correlated with Src activity, proteins that interact with Rac under such proteasomal degradation. HACE1-depletion is slowly through prometaphase and display consistent with the above-mentioned post- conditions. This revealed the small ubiquitin-like accompanied by increased total Rac1 levels and increased chromosome congression errors. translational regulatory mechanism operating in modifier (SUMO) E3-ligase, PIAS3, as a novel Rac accumulation of Rac1 in membrane ruffles. Significantly, Eg5 suppression in Tiam1-depleted malignancies (Woodcock et al. Mol Cell. 2009; interacting protein. We found that PIAS3 Moreover, HACE1-depletion enhances cell cells rectifies not only their increased 33: 639). interacts better with the GTP-bound form of migration independently of growth factor centrosome separation but also their Rac. We then showed that PIAS3 is required for stimulation, which may have significance for chromosome congression errors and mitotic increased Rac activation and optimal cell malignant conversion. A non-ubiquitylatable Rac1 delay. These findings identified Tiam1-Rac Publications listed on page 67 signalling as the first antagonist of centrosome 24 | Paterson Institute for Cancer Research Scientific Report 2011 Cell Signalling Group | 25

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    Clinical and Experimental Pharmacology Group Clinical Fellows Kyaw Aung and mass spectroscopy facilities. With respect to target validation, CEP are investigating whether Geoff Parker, our CR-UK/AZ clinical pharmacology fellow Danielle Shaw has Jenny Adamski http://www.paterson.man.ac.uk/cep Matthew Krebs an enzyme involved in the repair of etoposide- undertaken two pilot studies using the death Leila Khoja induced damage is a potential drug target in switch model to investigate the effects of tumour Kalena Marti Marti Danielle Shaw combination with etoposide in SCLC. A joint cell death on the extracellular and extravascular Laura Cove Smith strategic plan has been constructed to begin space using Diffusion Weighted MRI. biomarker discovery/validation as drug discovery Graduate Students Elizabeth Hitchman proceeds from hit identification to lead Portfolio management of clinical biomarker Shaun Villa identification, making sure predictive and studies pharmacodynamic biomarkers aligned to DDG To ensure effective management of the significant Scientific Officers Jessica Booth leads are not only useful in preclinical number of biomarker studies in CEP, we have Damien Brown development but are also ‘fit for purpose’ for initiated the development of a portfolio CEP validates and implements pharmacodynamic, prognostic Debbie Burt Fouziah Butt deployment in early clinical trial scenarios. management system. A key first step has been the recruitment of Stephen Walker as portfolio and predictive biomarkers working in tandem with The Martin Dawson Olive Denneny Preclinical models of induced tumour cell leader who is responsible for 70 provisional or Christie Cancer Treatment Centre that incorporates one Maciej Dolniak Shital Dulabh death as tools for characterisation of ongiong clinical biomarker studies in CEP. imaging biomarkers Biomarkers are being deployed on a range of of the largest early clinical trials units worldwide. Suzanne Faulkner Grace Hampson Several collaborations on non invasive imaging of early clinical trials sponsored by the Manchester Cassandra Hodgkinson cell death have been carried out this year. We Cancer Research Centre (MCRC) and ECMC Matthew Lancashire developed and validated a colorectal cancer cell partner organisations (CR-UK, Christie Clinical Two senior appointments this year strengthened at the Dana Farber Cancer Institute in Boston in Daniel Morris Karen Morris line engineered to express a robust doxycycline Trials Unit (CTU), Manchester University, UCL, CEP; Ged Brady joined us to develop nucleic the regulation of cancer cell apoptosis. The Letai Andrew Price (dox) inducible constitutively active mutant of Cardiff, Glasgow and Birmingham CTUs) and acid based biomarkers and Stephen Walker will group have developed a technology, BH3 Lyndsey Priest effector caspase 3 (the death switch) that is used several pharmaceutical companies (e.g. oversee the CEP Biomarker portfolio. We also profiling, which allows the propensity of cancer Tony Price Amrita Shergil in tumour xenograft models. The model allows AstraZeneca, Roche, Novartis, GSK, Chugai, and welcomed back Alastair Greystoke and Emma cells to undergo apoptosis to be determined, Robert Sloane synchronous and regulated levels of apoptosis to this year via a new overarching Master Dean as NIHR Clinical lecturers developing their either in vitro or ex vivo. The BH3 profile is Nigel Smith be induced in vivo where changes in imaging Agreement with Abbott Laboratories). To independent translational research programmes predictive of response to various novel and Hannah Turpin tissue and blood biomarkers can be monitored facilitate management of the extensive within CEP. We continue our focus on circulating conventional anti-cancer agents however the Scientific Assistants and correlated, with the horizon of improved biomarker portfolio we have utilised a tumour cells (CTCs), designing and implementing protocol requires large numbers of cells and is Aileen Jardine interpretation of multi modality biomarker data Laboratory Information Management System novel proof of mechanism CTC assays and we currently limited to lymphomas or tumours Judith Thorp in the clinic. In collaboration with Dr Kaye (LIMS) to establish a single database for all initiated an exciting collaboration to develop a which can be readily biopsied. The aim of this Laboratory Manager Williams and Dr Chris Cawthorne at the biomarker studies. The LIMS database will new CTC technology. Joint working with the collaboration is to develop BH3 profiling for Martin Greaves Wolfson Molecular Imaging Centre (WMIC), contain key information for more than 250 MCRC Drug Discovery group is underway such single cell analysis by microscopy and to apply it pilot studies have been performed to study the biomarker projects enabling effective tracking of that drugs and biomarkers are developed in to CTCs isolated from patients with lung cancer. Group Leaders uptake of [18F]ML-10, a PET imaging probe all ongoing and future clinical biomarker studies. parallel. The highlight of 2011 was the award to Dr Radek Polanski received a CR-UK Travel Figure 1 Caroline Dive the CEP based team of the CR-UK Translational award to visit the Letai laboratory to progress The Figure presents an overview of designed to be specifically taken up by apoptotic cells to address the specificity and sensitivity of Overview of CEP/AstraZeneca Serological Malcolm Ranson Research Prize. these studies following the visit to PICR of CEP's blood based Nucleic Acids this novel tracer. An imaging collaboration with Alliance Jeremy Ryan from the Letai Lab. SCLC (small cell Biomarkers (NAB) strategy where Deputy Group Leader the aim is to couple a single GE Healthcare was initiated using this death Since 2006 an Alliance has been in place Ged Brady Expansion of the Biomarker Portfolio – lung cancer) is initially chemo-sensitive but it simplified clinical blood collection switch model investigating the biodistribution of between AstraZeneca (based at nearby Alderley The Nucleic Acids Biomarkers Team rapidly relapses as a chemo-resistant disease and protocol with DNA and RNA analysis Disease Focus Team Leaders three 99Tc-labelled compounds as specific Park) and CEP to analyse, report and advise on The CEP Biomarker Portfolio has expanded to the molecular mechanism(s) driving this are of both circulating tumour cells Fiona Blackhall (CTCs) as well as the examination of imaging probes targeted to apoptotic cells. In data obtained from blood borne serological Guy Makin include a Nucleic Acids Biomarkers (NAB) team unclear. By BH3 profiling SCLC patients’ CTCs at tumour derived nucleic acids present collaboration with Professor Tim Illidge, a murine biomarker assays validated for clinical use in our Andrew Renehan led by CEP Deputy Ged Brady. NAB projects presentation and relapse, in combination with in plasma and serum. The NAB cell line containing the death switch has been GCPL laboratories to support AZ’s oncology have been initiated to examine miRNA, NGS approaches in the NAB team, we hope to focus will be to carry out a Staff Scientists generated and grown in vivo and the impact of portfolio. The CEP/AZ Alliance now Jeff Cummings methylation patterns present in circulating free uncover new treatment strategies to overcome biomarker discovery phase through induction of tumour cell apoptosis on the encompasses a broad range of biomarkers (cell Dominic Rothwell DNA (cfDNA), RNA profiling and mutation drug resistance. exhaustive fractionation and profiling Stephen Walker of blood samples using CTC immune system is being examined. In death, invasion, angiogenesis, CTC enumeration detection in CTCs (Figure 1). NAB assay fractionation and next generation collaboration with the University of Manchester and characterisation) all established for high Clinical Lecturers development takes into consideration the Adding Value to the Drug Discovery Pipeline at sequencing followed by an evaluation Biomedical Imaging Department and Professor throughput clinical trial analysis where thousands Emma Dean requirement for simple sample processing PICR – joint working with the Drug Discovery of potential markers in the clinic Alastair Greystoke of samples are shipped to CEP from clinical trial amenable to upcoming multi-site clinical trials. Group (DDG) using simpler more streamlined sites across the globe. Two highlights in 2011 Associate Scientists Projects using next generation sequencing As the drug discovery programme initiated in methods. were a) the development of a bespoke assay to Kathryn Simpson (NGS) of lung cancer patients’ blood and 2009 by Donald Ogilvie in the PICR DDG takes Chris Morrow measure androgen receptor (AR) protein tumour samples are underway within CEP and in shape, CEP are working closely with DDG expression in CTCs as a Proof of Mechanism Service Manager collaboration with the Cancer Genome Project colleagues to develop biomarkers early during biomarker in Phase I clinical trials of the Selective David Moore group at the Wellcome Trust Sanger Institute. the drug discovery process, assist in the Androgen Receptor Down-regulator (SARD); New biomarker collaborations are being initiated validation of drug targets and facilitate in-house Postdoctoral Fellows and b) the development of a panel of highly Ivona Baricevic-Jones with companies developing novel CTC/blood in vivo drug metabolism and pharmacokinetic sensitive ELISAs to quantitate circulating levels of Alison Backen biomarker technology platforms. analyses. The two groups have also worked Jian Mei Hou the erbB ligands to trials of AZD8931 (pan-Erb collaboratively to develop analytical methods for (AZ secondment) inhibitor). Radek Polanski Determining the Apoptotic Potential of cell based assays that are helping to drive the Rajeeb Swain Circulating Tumour Cells DDG portfolio. DMPK (Drug Metabolism and Cong Zhou An exciting collaboration has begun combining Pharmacokinetics) studies on compounds Publications listed on page 67 CEP’s expertise in isolating and analysing CTCs produced by the DDG are ongoing utilising and the expertise of Professor Tony Letai’s group CEP’s in vivo preclinical pharmacology expertise 26 | Paterson Institute for Cancer Research Scientific Report 2011 Clinical and Experimental Pharmacology Group | 27

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    DNA Damage Response Group http://www.paterson.man.ac.uk/dnadamage Many cancer therapy procedures, such as radiotherapy and Recently, in screening for proteins with the ability to bind poly(ADP-ribose), we discovered a Another class of protein that remains a focus of our research is those containing a macrodomain. some types of chemotherapy, work by overwhelming the poly(ADP-ribose)-binding zinc finger motif The macrodomain is another evolutionary capacity of the cell to repair DNA damage, resulting in cell (PBZ). PBZ is a structurally distinctive, atypical type of zinc finger that is associated with several widespread module with the capacity to bind poly(ADP-ribose) and we recently identified death. Most rapidly dividing cells - cancer cells - are proteins involved in response to DNA damage several human macrodomain protein factors that preferentially affected by such treatments, providing the (Ahel at al, Nature, 2008). One of the human proteins containing a PBZ motif is a protein are recruited to broken DNA ends in a poly(ADP-ribose)-dependent manner. These opportunity to use DNA damaging agents to selectively kill called Checkpoint protein with FHA and RING include a histone H2A variant called MacroH2A Group Leader cancer cells. In addition, the genomic instability is the driving domains (CHFR). CHFR is an ubiquitin ligase frequently inactivated in human epithelial and several other uncharacterised macrodomain proteins. Ivan Ahel force of cancer development, which requires multiple DNA tumours, which acts as a key regulator of the mutations resulting in loss of cellular growth control. In order poorly understood early mitotic checkpoint that Structural and functional analysis of poly(ADP- Postdoctoral Fellows transiently delays chromosome condensation and ribose) glycohydrolase (PARG) and its Dragana Ahel to accelerate the accumulation of genetic changes, cancers nuclear envelope breakdown in response to a validation as a target for cell-permeable Rosa Morra often sacrifice specific DNA repair pathways. This can make variety of stresses. The elucidation of the function inhibitor design Scientific Officer of the PBZ motif gave us a vital clue to discover Available data indicates that inhibiting PARG Ria Weston cancer cells additionally susceptible to DNA damaging agents that the CHFR-dependent checkpoint is might offer a promising and beneficial approach Graduate Students and/or to inhibitors that block alternative repair pathways. For regulated by PARPs and that the PBZ motif in the CHFR protein is critical for checkpoint in the treatment of cancer and cardiovascular conditions. However, unlike the case of PARP Eva Barkauskaite Michael Tallis (from October) these reasons, studying the protein components involved in the activation. Another PBZ-regulated protein we are inhibitors, progress in developing permeable, repair of damaged DNA has proved to be a valuable strategy studying is a protein called Aprataxin-PNK-like factor (APLF). APLF uses tandem PBZ repeats small-molecule PARG inhibitors has been limited, partly due to the lack of mechanistic and in searching for novel approaches and targets in cancer for direct interaction with poly(ADP-ribosyl)ated structural data for the human PARG protein. therapy. PARP1, which allows APLF’s timely localisation to the sites of DNA damage. We recently Recently, we solved the structure of a bacterial PARG-like protein, which gave the first insight discovered that the role of APLF is to act as a into the basic principles of PARG structure and Poly(ADP-ribosyl)ation in regulation of arising at the sites of damaged DNA serves as a histone chaperone to modulate chromatin its mechanism of catalysis (Slade et al, Nature, DNA repair platform for specific recruitment and scaffolding structure and facilitate DNA repair reactions in 2011) (Figure 1). This structure revealed that the Poly(ADP-ribosyl)ation is a post-translational of DNA repair complexes. In addition, the response to poly(ADP-ribose) signalling PARG catalytic centre is a diverged type of protein modification that controls several nuclear damage-induced poly(ADP-ribosyl)ation has a (Mehrotra et al, Mol Cell, 2011). macrodomain. Despite this advance, structural processes known to be important for genome role in relaxation of the chromatin structure and information on the human PARG is still lacking. stability, including DNA repair, regulation of in apoptotic signalling. The recent development Figure 1 Our goal is to solve the structures of human chromatin structure, cell cycle checkpoint, of potent PARP inhibitors provided powerful Active site of the Thermomonospora PARG in complex with the substrate analogues transcription, apoptosis and mitosis. Poly(ADP- tools to study pathways regulated by poly(ADP- curvata PARG enzyme with bound and inhibitors by means of X-ray crystallography ADP-ribose. The two catalytic ribose) is a highly negatively charged polymer ribose), as well as providing a promising novel which in combination with solution and cell glutamate residues are shown in that is formed from repeating ADP-ribose units class of drugs for cancer treatment. Specifically, pink. biology studies should address the mechanism, linked via glycosidic ribose-ribose bonds, and is selective inhibition of the DNA break repair structure and regulation of human PARG, as well synthesised by the poly(ADP-ribose) polymerase pathway using permeable PARP inhibitors has as providing a foundation for the development (PARP) family of enzymes using a vital cellular proven highly effective against breast and ovarian of small, cell-permeable PARG inhibitors. cofactor NAD+ as a substrate. The reversion of cancers (Bryant at al, Nature 2005; 434: 913). poly(ADP-ribosyl)ation is performed by the Thus, understanding the molecular basis of hydrolytic action of an enzyme called poly(ADP- poly(ADP-ribose)-dependent DNA repair Publications listed on page 69 ribose) glycohydrolase (PARG), which specifically processes is likely of vital importance for targets ribose-ribose bonds and cleaves selecting and developing efficient therapies. poly(ADP-ribose) into ADP-ribose monomers. The role of poly(ADP-ribosyl)ation is best Identification and characterisation of novel understood in the regulation of DNA repair, poly(ADP-ribose)-regulated factors which is controlled by the three PARPs Our laboratory is particularly interested in the responsive to DNA strand breaks (PARP1, identification of new pathways and protein PARP2 and PARP3). Poly(ADP-ribosyl)ation functions regulated by poly(ADP-ribosyl)ation. 28 | Paterson Institute for Cancer Research Scientific Report 2011 DNA Damage Response Group | 29

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    Drug Discovery Group http://www.paterson.man.ac.uk/drugdiscovery During 2011 we have expanded the Drug Discovery team, facility, we have also made significant investments in alternative hit finding strategies that we can Project Portfolio Most drug discovery projects do not progress all consolidated our informatics platform and, most importantly, use locally. These include Virtual Screening – the way to the clinic so our major task is to have progressed our discovery project portfolio. Five hit-to- described in detail in last year’s report - and, recently, functional fragment screening. The latter deliver quality data, as quickly as possible, to identify and stop the “losers” early and redeploy lead phase projects are now underway, targeting a variety of approach involves testing a collection of ~1200 our resources onto potential “winners”. During cancer targets. These activities are underpinned by multiple smaller compounds (mw~300) against our targets, at higher concentrations than used those 2011, we reached stop decisions on three projects and have started three others. Our collaborations both within and beyond the Manchester Cancer normally used in HTS. Using the Echo acoustic current portfolio includes five hit identification Group Leader Research Centre (MCRC). dispensing technology we have been able to run screens against five potential drug target projects against metabolic, DNA repair (x2), redox modulation and oncogene signalling Donald Ogilvie molecules using a small sample of a fragment targets. Two of these projects are approaching People Drug Discovery Targets library (kindly provided by the Beatson Institute consideration for progression to the lead Head of Chemistry With the increased funding stream for this We continue to review many cancer drug target Drug Discovery Unit). In just three months we identification stage. For the most advanced Allan Jordan programme from April 2011, following a major opportunities and, during 2011, have worked have identified early hits against all of these project, HTS screening has identified hit recruitment campaign, we have expanded the closely with several Paterson Institute group targets, some of which have proved elusive to compounds whose potency has been improved Head of Bioscience Ian Waddell team from nine to twenty four scientists. leaders (John Brognard, Tim Somervaille & Ivan other hit finding technologies. One advantage of ten fold by our chemists in the last few months. Alongside cell and molecular biologists we have Ahel) in the identification, validation and screening with fragments is that the compounds The in vitro pharmacology of these more potent Chemists also recruited synthetic, medicinal and prosecution of targets. It is very exciting to be are smaller and can fit into protein pockets compounds is currently being explored in cells Ali Raoof computational chemists and a drug metabolism able to access, and exploit pre-publication data inaccessible to larger (HTS) inhibitors. A by us and key expert collaborators. In support Alison McGonagle specialist. This multidisciplinary team allows us to and facilitate new basic discoveries by provision potential downside is that fragment hits generally of all of our projects, we have initiated a range of Amanda Lyons Bohdan Waszkowycz pursue projects at all stages of drug discovery of tool compounds. We have also carried out a have a lower affinity and require more target biology and technology-related Daniel Mould from target selection to lead optimisation. After broad high level review of breaking cancer optimisation for potency. We have also secured collaborations both within and beyond the James Hitchin much searching, we were particularly pleased to science and have prioritised key areas for more access to a focused ~10,000 compound kinase MCRC. Kate Smith secure Dr Ian Waddell, a highly experienced thorough target investigation. To support this we inhibitor collection from CRT-DL and will be Laura MacGuire academic and industrial scientist, as Head of are looking to strengthen our links to Crispin using this for screening selected targets in house. Cancer Research UK Niall Hamilton Rebecca Newton Drug Discovery Bioscience. Miller’s bioinformatics group through a jointly An additional, unanticipated hit finding activity We have actively participated in broader Cancer Stuart Jones funded post-doc who will focus on the drug has been to engage with pharmaceutical Research UK drug discovery activities and are Infrastructure discovery target selection process. companies regarding the opportunity to work now involved in collaborations, some of which Bioscientists Our Dotmatics chemoinformatics and electronic on validated hits (and sometimes associated have been described in this report, with most of Alex Boakes notebook platforms are now fully deployed and, Hit finding assay technologies) from their deprioritised the Cancer Research UK drug discovery units. Dominic James Emma Fairweather after significant efforts, are fully operational for Once a target has been selected for drug cancer projects, but only if they meet our strict During the summer of 2011 we underwent a Gemma Hopkins integration and visualisation of all kinds of data. discovery the next stage is to try and identify target criteria. successful annual review with the Drug Graeme Thomson The more specialised computational chemistry prototype small molecules, or “hits”, that interact Discovery Advisory Group. Helen Small platform allows us to integrate protein and with the target molecule. One approach is to Figure 1 Mandy Watson chemical ligand structural information for target conduct a high throughput screen (HTS), in Inhibitors of the enzyme PARG The Future Mark Cockerill represent a promising new approach Nicola Hamilton assessment and virtual screening. On the which a simple drug target-related assay is During 2011 we have expanded out team, technology hardware front, we have been interfaced with a collection of ~100,000 diverse to disrupting DNA repair pathways Nikki March progressed our project portfolio and, pleasingly, in tumour cells. Knowledge of the 3D Samantha Fritzl particularly impressed with the Echo acoustic chemicals (mw~400) in order to try and identify atomic structure of PARG enables increased the depth of our interactions with Sarah Holt dispensing technology and are planning to compounds (“hits”) which interfere with the DDU scientists to design novel Paterson Institute scientists. In 2012 we look upgrade this facility to improve throughput and functional activity of the target. During 2011 we compounds with the potential to forward to starting and stopping more projects also handle aqueous solutions. We have also completed a successful HTS against one of our block the action of the enzyme. The and progressing the best ones to the next stage. sought to improve existing laboratory equipment DNA repair enzyme targets in collaboration with figure shows the surface of the bacterial PARG catalytic site as by adding simple automation such as plate the Cancer Research Technology Drug Discovery determined using X-ray stackers. These cost-effective modifications have Laboratory (CRT-DL) laboratory in London. This crystallography by Ivan Ahel’s DNA Publications listed on page 69 allowed us to screen larger compound subsets has provided useful chemical start points which Damage Response Group, with efficiently. are now being investigated further by our several small drug-like compounds chemists. Since we do not have our own HTS that have been computationally modelled into the binding site. Using virtual screening methods, very large databases of commercial compounds can be analysed to find the most promising candidates for purchase and testing. 30 | Paterson Institute for Cancer Research Scientific Report 2011 Drug Discovery Group | 31

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    Immunology Group Figure 1 Expression of 5T4 by B-ALL blasts in http://www.paterson.man.ac.uk/immunology bone marrow samples from three relapse patients; magnification x 40, scale bar 5 µm. CXCL12 mediated chemotaxis in mouse immunocompromised mice. Even with the embryonic cells. Using WT and 5T4KO murine current successful B-ALL treatments, embryonic fibroblasts (MEFs), Owen McGinn has approximately 1 in 4 children will relapse. led a study which has established that CXCL12 Recurrence is frequently characterised by the Historically, a principle goal was to harness immunity to 5T4 binding to CXCR4 activates both the ERK and occurrence of disease at extramedullary sites oncofoetal antigen for cancer therapy. Two approaches are in AKT pathways within minutes but while these such as the central nervous system and gonads. pathways are intact they are non-functional in 5T4 expression may be a prognostic marker of late phase clinical trial: TroVax, a recombinant viral vaccine 5T4KO cells treated with CXCL12. However, in such spread but also could be mechanistically (Oxford BioMedica, UK) and Anyara, an antibody targeted the absence of 5T4 expression, CXCR7 is involved. We have also shown that 5T4 positive upregulated and becomes the predominant pre-B-ALL cells are susceptible to 5T4 specific super-antigen (Active Biotech, Sweden); 5T4 antibody targeting receptor for CXCL12, activating a distinct signal superantigen antibody-dependent cellular of a toxin is in clinical development (Pfizer). transduction pathway with slower kinetics cytotoxicity in vitro and in vivo providing support involving transactivation of the EGFR, eliciting for high risk pre-B-ALL 5T4 immunotherapy. Group Leader proliferation rather than chemotaxis. Thus the We have recently exploited our 5T4 knock out folate receptor 4 (FR4) antibody significantly surface expression of 5T4 marks the use of the 5T4 Inhibits Wnt/β-Catenin Signalling and Peter L. Stern (5T4KO) mouse to investigate how endogenous reduced the frequency of Tregs in WT mice, CXCR4 rather than the CXCR7 receptor, with Activates Noncanonical Wnt Pathways by expression of 5T4 influences 5T4 vaccine enhancing 5T4 specific IFN-γ while reducing IL- distinct consequences for CXCL12 exposure, Modifying LRP6 Subcellular Localization Postdoctoral Fellows Fernanda Castro (until May) induced T cell immunity and tolerance in 10 T cell responses but did not reveal IL-17 relevant to the spread and growth of a tumour. Wnt proteins can activate distinct signalling Owen McGinn comparison to wild type (WT) mice. The producing effectors. Treatment with FR4 antibody Consistent with this hypothesis we have pathways, but little is known about the Rasilaben Vaghjiani regulation of CXCL12/CXCR4 chemotactic after Adm5T4 vaccination altered the balance of identified some human tumour cell lines with mechanisms regulating pathway selection. In Julie Brazzatti (from November) responses by 5T4 molecules provides a effectors and provided modest protection Darren Roberts (from February) similar 5T4/CXCR7 reciprocity that is predictive collaboration with the Weidinger group in functional role for 5T4 in the spread of tumour against autologous B16m5T4 melanoma of their biological response to CXCL12. Dresden, we have shown that the Zebrafish Clinical Fellow cells from the primary site (PLoS ONE 5(4): challenge. We conclude that the efficacy of 5T4 homologue of mammalian 5T4, Wnt-activated Saladin Sawan e9982. doi:10.1371/journal.pone.0009982). and some other tumour associated antigen 5T4 oncofoetal antigen is expressed in high risk inhibitory factor 1 (Waif1) interferes with Further work has now shown that 5T4 (TAA) vaccines may be limited by the of relapse childhood pre-B acute lymphoblastic Wnt/β-catenin signalling and concomitantly Scientific Officer molecules integrate at least two distinct signalling combination of TAA specific Tregs, the deletion Jian Li leukemia (ALL) and is associated with a more activates noncanonical Wnt pathways. Waif1 pathways of high relevance to the growth and and/or alternative differentiation of CD4 T cells invasive and chemotactic phenotype inhibits β-catenin signalling in zebrafish and Graduate Students movement of developing or malignant cells. as well as the absence of distinct subsets of CD8 While mature hematopoietic cells lack the Xenopus embryos as well as in mammalian cells, Andrzej Rutkowski (with T cells. Comparison and adoptive transfer of expression of 5T4, open access microarray data and zebrafish waif1a acts as a direct feedback Crispin Miller) Regulation of autologous immunity to the effector T cells between WT and 5T4KO mice suggests that 5T4 is present during the early inhibitor of wnt8-mediated mesoderm and Georgi Marinov mouse 5T4 oncofoetal antigen; implications for can provide a useful platform to explore and stages of normal B cell development in particular neuroectoderm patterning during zebrafish immunotherapy improve immune therapeutic modalities in the at the pro- and pre-B cell phase. Building on our gastrulation. Waif1a /5T4 binds to the Wnt Undergraduate student Milena Kalaitsidou (until Effective vaccination against the 5T4 oncofoetal context of an autologous TAA which provides a collaboration with the Children’s Cancer Group coreceptor LRP6 and inhibits Wnt-induced LRP6 September) glycoprotein may be limited by the nature of the more realistic model for assessment of (Blood 118: 638-49, 2011), Fernanda Castro and internalisation into endocytic vesicles, a process T cell repertoire and the influence of immunogenicity, efficacy and safety (Cancer Owen McGinn have led the research showing that is required for pathway activation. Thus, immunomodulatory factors, in particular T Immunology, Immunotherapy that lymphoblasts from patients of the high risk Waif1a/5T4 modifies Wnt/β-catenin signalling by regulatory cells (Tregs). Work led by Fernanda doi:10.1007/s00262-011-1167). of relapse subset of childhood pre-B ALL regulating LRP6 subcellular localisation. In Castro identified mouse 5T4-specific T cell express higher levels of 5T4. Gene expression addition, Waif1a/5T4 enhances β-catenin- epitopes using a 5T4KO mouse and evaluated 5T4 oncofoetal glycoprotein, CXCL12 receptor profiling of 85 diagnostic pre-B ALL bone independent Wnt signalling in zebrafish embryos corresponding WT responses as a model to preference, signal transduction and biological marrow samples revealed higher 5T4 oncofoetal and Xenopus explants by promoting a refine and improve immunogenicity. We showed response antigen transcript levels in the cytogenetic high- noncanonical function of Dickkopf1. These results that 5T4KO mice vaccinated by replication CXCL12 is a pleiotropic chemokine capable of risk subgroup of patients (p < 0.001). The figure suggest that Waif1/5T4 modulates pathway defective adenovirus encoding mouse 5T4 eliciting multiple intracellular signal transduction shows immunofluorescence staining for 5T4 on selection in Wnt-receiving cells. Interestingly, β- (Adm5T4) generate potent 5T4 specific cascades and biological functions, via interaction DAPI labelled cytospins of bone marrow catenin -independent signalling can enhance interferon(IFN)-γ CD8 and CD4 T cell responses with either CXCR4 or CXCR7. Factors that samples from pre-B ALL relapse patients. Using motility and invasion of cancer cells raising the which mediate significant protection against 5T4 determine CXCL12 receptor choice, intracellular 5T4+ve and 5T4-ve cells derived from a high risk possibility that Waif1/5T4 promotes invasion of positive tumour challenge. 5T4KO CD8 but not signalling route and biological response are cytogenetics BCR-ABL+ pre-B ALL line, we have cancer cells by enhancing noncanonical Wnt CD4 primed T cells also produced IL-17. By poorly understood but are of central shown that 5T4 expression correlates with signalling (Developmental Cell doi: contrast, Adm5T4 immunized WT mice showed importance in the context of therapeutic increased invasion, adhesion and 10.1016/j.devcel.2011.10.015). no tumour protection consistent with only low intervention of the CXCL12 axis in multiple CXCR4/CXCL12 chemotaxis in vitro and avidity CD8 IFN-γ and no IL-17 T cell responses disease states. We have recently demonstrated differential infiltration of extramedullary sites and no detectable CD4 T cell effectors that 5T4 oncofoetal glycoprotein facilitates following intraperitoneal challenge of Publications listed on page 70 producing IFN-γ or IL-17. Treatment with anti- functional CXCR4 expression leading to 32 | Paterson Institute for Cancer Research Scientific Report 2011 Immunology Group | 33

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    Inositide Laboratory Group in the levels of PtdIns5P rather than a substantial decrease in PtdIns(4,5)P2 and from our previous http://www.paterson.man.ac.uk/inositide studies that PIP4Ks are important during stress signalling. Oxidative signalling plays a key role in aging and cancer, exemplified by the development of pathologies in mice genetically manipulated to mismanage reactive oxygen species. We find that oxidative stress leads to a rapid increase in the levels of PtdIns5P and that PtdIns5P signalling impinges on the oxidative stress sensitivity of the cell. Pin1 null mice show phenotypes of premature aging and in cancer Phosphoinositides are a family of lipid second messengers that development and MEFs derived from these mice show growth insensitivity to oxidative stress. are regulated in response to environmental changes by the Interestingly, we find that Pin1 null MEFs exhibit activities of a network of kinases and phosphatases. Alterations increased levels of PtdIns5P in response to hydrogen peroxide stimulation and that removal in phosphoinositide levels can regulate many different cancer- of this increased PtdIns5P, by overexpression of a relevant pathways including cell survival, proliferation, migration, Figure 1 PIP5K as a target for drug development PIP4K, re-instigates oxidative stress sensitivity. There are two pathways for PIP5K inhibitors were identified using a novel Pin1 is a proline-directed phosphorylation- cell substratum interactions and transcription. In cancer cells PtdIns(4,5)P2 synthesis however the high throughput in vitro assay for PIP5K (Cancer dependent regulator of protein conformation. In PtdIns(4,5)P2 is at the heart of phosphoinositide signalling as major synthetic pathway is through PIP5K. PIP4K regulates the levels of Research Technology). To investigate if these response to stress signalling we show that Pin1 Group Leader inhibitors could attenuate PIP5K activity in vivo, interacts with and regulates PIP4Ks which in turn Nullin Divecha it is the substrate for phosphatidylinositol-3-kinase (PI3K) and PtdIns5P. Diacylglycerol (DAG) activates protein kinase C (PKC) we have developed a novel assay based on the regulates the levels of PtdIns5P and the cellular phospholipase C (PIC) both of which are deregulated in which regulates the phosphorylation inducible cellular expression of fluorescent response to oxidative stress. How PtdIns5P Associate Scientist and intracellular activity of PIP5K. probes that interact specifically with and report regulates cellular processes is far from clear and David Jones human tumours (Figure 1). Furthermore, PtdIns(4,5)P2 is We expect that an inhibitor of PIP5K the levels of PtdIns(4,5)P2 in living cells. Using we are using lipid affinity purification and mass will inhibit both the PI3K and the Postdoctoral Fellows itself a regulator of cytoskeletal dynamics, cell survival and Phospholipase C (PLC) pathway. this and other assays we have assessed the spectrometry to identify proteins that interact Daniel Fitzgerald structure/function relationships of inhibitors in with and therefore are likely to function as Maria Carla Motta cell polarity. Figure 2 vivo in order to drive rational chemical synthesis downstream targets for PtdIns5P signalling. For Iman van den Bout The intracellular localization of wild of new compounds with enhanced affinity and example, the PHD finger is a cross-braced Zinc PIP5Ks and PtdIns(4,5)P2 addition), or when PtdIns(4,5)P2 was being type GFP-PIP5K (WT), a mutant specificity. Furthermore we have shown that finger, present in numerous chromatin regulating Scientific Officer unable to interact with Rac (RacM), PtdIns(4,5)P2 is present in the plasma membrane resynthesised (EGTA addition). Biochemical these inhibitors attenuate PtdIns(4,5)P2 synthesis proteins that can interact with and decode Yvette Bultsma a kinase inactive mutant (KD) and of and in the nucleus where its levels in these two analysis demonstrated that ionomycin/calcium in vivo and in doing so reduce growth factor histone modifications and can also interact with GFP-PIP5K mutated to reduce its Graduate Students compartments can be regulated distinctly. led to a rapid and sustained depletion in both interaction with Rac and block its induced activation of both the PLC and phosphoinositides. We found that the PHD Julian Blaser (co-supervised with PtdIns(4,5)P2 is synthesised by two different PtdIns4P and PtdIns(4,5)P2 and that the addition kinase activity (RacM/KD) were PI3Kinase pathway (Figure 1). These data suggest finger of TAF3, a regulator of the basal Tim Somervaille) determined in live cells using confocal that PIP5K inhibitors likely will have added families of kinases using two different substrates of EGTA led to a resynthesis of both transcription complex and cell differentiation, Rebecca Foulger microscopy. (Figure 1). There are three active isoforms of phospholipids. A reduction in intracellular therapeutic value compared to the inhibition interacts with phosphoinositidies and mutants Willem-Jan Keune Lilly Sommer PIP5K, α, β and γ which all localise to the plasma PtdIns(4,5)P2 is apparent from the delocalisation solely of PI3Kinase. that ablate this interaction compromise basal Figure 3 membrane, although specific isoforms can also from the membrane into the cytosol of the RFP- Cells were transfected with constructs transcription and muscle differentiation. Future localise to other subcellular compartments, such PH domain, a specific in vivo fluorescent reporter as indicated on the right and imaged PIP4K and PtdIns5P studies are aimed at identifying novel pathways as the golgi (PIP4Kβ), the nucleus (PIP4Kα and of PtdIns(4,5)P2, (Figure 3). Under conditions of live as controls (cont), after activation There are three isoforms of PIP4Ks of which α is regulated by PIP4Ks. of PLC and reduction in PtdIns(4,5)P2 cytosolic, β is cytosolic and nuclear and γ γ), focal adhesions (PIP5Kγ) and the cytokinetic low PtdIns(4,5)P2, GFP-PIP5K was delocalised (iono/Ca) or after the initiation of furrow (PIP4Kα). We have identified two into the cytosol and upon PtdIns(4,5)P2 localises to internal membrane compartments. Phosphoinositide and their therapeutic PtdIns(4,5)P2 resynthesis (EGTA). components that coordinate the localisation of resynthesis became relocalised to the plasma PIP5K inhibition was carried out by PIP4Ks homo and heterodimerise and the potential PIP5K to the plasma membrane. The first is the membrane (Figure 3). In order to implicate treating the cells with a specific heterodimerisation between α and β localises In order to define new targets for potential small molecular weight G protein Rac and the PtdIns(4,5)P2 resynthesis we treated cells with a PIP5K inhibitor (bottom two panels). the α isoform to the nucleus. The therapeutic development we have utilised a small Iono/ca leads to a dramatic decrease heterodimerisation is important as the majority second is the plasma membrane level of specific inhibitor of PIP5K (see PIP5K as a drug shRNAi lentiviral library targeting all the known in PtdIns(4,5)P2 levels shown by the PtdIns(4,5)P2. We identified and characterised a target) and monitored GFP-PIP5K localisation. of PIP4K activity associated with PIP4Kβ is genes involved in phosphoinositide metabolism. decrease in membrane localisation of binding site for Rac that is highly conserved Biochemical analysis demonstrated that the RFP-PH domain, whereas EGTA accounted for by its interaction with PIP4Kα. This library had been used to identify genes between all isoforms of PIP5K. Mutation of this treatment with the inhibitor prevented the addition stimulates PtdIns(4,5)P2 Using tissue microarrays of advanced human potentially involved in migration and in site, which attenuates interaction with Rac, resynthesis of PtdIns(4,5)P2 but not of PtdIns4P. resynthesis and relocalisation of the breast tumour samples, we have found that leukemogenesis. Hits have been validated and PH domain to the membrane. PIP4Kβ expression is both up and down reduces but does not abolish membrane Ionomycin treatment led to the delocalisation of future work is aimed at understanding how localisation of PIP5K (Figure 2). We also GFP-PIP5K from the membrane, however the regulated and interestingly, high expression these genes modulate phosphoinositide observed that a mutation, which attenuates relocalisation of PIP5K was prevented upon correlates with better patient survival (with metabolism to regulate cell responses. PIP5K activity, also leads to its reduced blocking the resynthesis of PtdIns(4,5)P2 (Figure Professor Landberg, Breakthrough Breast Cancer localisation at the membrane. Interestingly, a 3). The lack of RFP-PH domain at the plasma and University of Manchester). Studies in human combination of both mutations led to the membrane demonstrates that the inhibitor breast tumour cell lines are ongoing to define Publications listed on page 70 complete delocalisation of PIP5K from the prevented PtdIns(4,5)P2 resynthesis (Figure 3). signalling pathways regulated by PIP4Kβ plasma membrane (Figure 2). We also studied Future studies are aimed at understanding how expression. Our hypothesis is that PtdIns5P is a the intracellular localisation of PIP5K under changes in PtdIns(4,5)P2 regulate membrane key signalling intermediate as we have shown conditions where PtdIns(4,5)P2 was reduced, by localisation of PIP5K and how PIP5Ks localise to that genetic manipulation, to reduce the levels of PLC mediated hydrolysis (ionomycin/calcium other specific cellular locations. PIP4K in different organisms, leads to an increase 34 | Paterson Institute for Cancer Research Scientific Report 2011 Inositide Laboratory Group | 35

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    Leukaemia Biology Group http://www.paterson.man.ac.uk/leukaemia Figure 1 Figure 1 Figure 2 It has become apparent in the past several years that Survival curve of mice transplanted with MLL-AF9 AML cells transduced epigenetic dysfunction has a central role in the pathology of with non-targeting control (NTC) or myeloid cancers. This is illustrated by the discovery of Kdm1a knockdown (construct #4) lentiviruses. Knockdown of Kdm1a recurrently occurring mutations targeting genes that code for confers a significant survival advantage in these mice relative to key epigenetic regulators such as the methylcytosine control mice. hydoxylase TET2, the DNA methyltransferase DNMT3A, the Figure 2 Primary human MLL-AF9 AML blast Group Leader Polycomb-related complex 2 H3 K27 methyltransferase EZH2 cells treated with a novel Tim Somervaille and the Polycomb-related protein ASXL1. Mutations in IDH1 tranylcypromine analogue undergo terminal macrophage differentiation. Postdoctoral Fellows and IDH2 likely also affect the epigenome through neomorphic Left panel shows vehicle treated cells, right panel shows an-TCP Xu Huang generation of 2-hydroxyglutarate which inhibits TET2 and treated cells. James Lynch Jumonji-domain histone demethylases. Further, sub-types of Clinical Fellow Brigit Greystoke acute myeloid leukemia (AML) exhibit distinct and abnormal Scientific Officer patterns of DNA methylation. differentiation and apoptosis of LSCs from mice experimentally initiated MLL-AF9 AML with an- with experimentally initiated MLL-AF9 AML. TCP was sufficient to promote in vivo loss of Gary Spencer Similar findings were obtained in knockdown clonogenic potential and induction of Graduate Students Novel oncoproteins generated by recurrently highlights the necessity for identification and experiments using human MLL-AF9 cells lines differentiation of MLL-AF9 AML cells while William Harris occurring chromosomal translocations in myeloid evaluation of other therapeutic targets. and primary MLL AML cells from patients sparing the clonogenic potential of the residual Julian Blaser (co-supervised with leukaemia also induce epigenetic dysfunction. For treated at The Christie NHS Foundation Trust. normal bone marrow stem and progenitor cells. Nullin Divecha) example, the gene coding for the H3 K4 One such candidate is KDM1A (also known as KDM1A knockdown cells are unable to initiate In keeping with this, death from AML was Filippo Ciceri methyltransferase MLL, itself a key epigenetic AOF2, LSD1, KIAA0601 or BHC110), a flavin AML in syngeneic or xenogeneic transplant delayed in mice treated with the KDM1A Tim Somerville regulator, is mutated by translocation in about adenine dinucleotide (FAD) dependent lysine- assays. Exon array analysis demonstrates that inhibitor. 4% of human AML. This results in constitutive specific demethylase with monomethyl- and KDM1A is required to sustain expression of an transcription at MLL target genes through dimethyl-histone H3 lysine-4 and lysine-9 MLL-AF9 associated oncogenic program. Thus our data establish KDM1A as a realistic aberrant recruitment by MLL fusion partners of substrate specificity. It is a component of the new target for differentiation therapy in human complexes associated with transcription MLL supercomplex associated with transcription KDM1A may be pharmacologically inhibited myeloid leukaemia. This work will be published in elongation including pTEFb, PAFc and the activation. It is also a component of complexes using tranylcypromine (TCP), a monoamine 2012. DOT1L complex, which contains a H3 K79 associated with transcription repression such as oxidase inhibitor used in the clinic for many methyltransferase. By contrast, fusion of RUNX1 the CoREST-HDAC and CtBP co-repressor years to treat depression. Use of TCP, at (AML1) to RUNX1T1 (ETO), which occurs in 7% complexes and the nucleosome remodelling and concentrations close to the IC50 for KDM1A, Publications listed on page 70 of patients with AML, generates a novel deacetylation complex (NuRD). While KDM1A phenocopies knockdown in both murine and constitutive transcriptional repressor. expression has been correlated with poor primary human MLL-AF9 AML cells. However, prognosis in high-risk prostate and breast cancer the poor selectivity for KDM1A and the high These and other observations have led to and poor differentiation in neuroblastoma, to IC50 likely preclude its use as a useful therapy for speculation that regulators of the structure and date there is no information as to its functional AML. Instead, in collaboration with the Drug function of chromatin, such as enzymes which role in myeloid leukaemia, nor whether it Discovery Group at the Paterson Institute (led regulate turnover of DNA methylation, histone represents a viable therapeutic target. by Donald Ogilvie) we have synthesised a methylation or histone acetylation, might be recently patented tranylcypromine analogue (an- attractive therapeutic targets in myeloid In the past year, focusing on the subtype of TCP) reported to have substantially higher malignancy. To date only the DNA demethylating human AML associated with translocations selectivity and potency versus KDM1A. Active in agent azacitidine has provided a survival benefit targeting MLL, we have generated a significant the low nanomolar range, use of an-TCP also in phase III trials, in patients with high-risk body of data which suggests that KDM1A is a phenocopied KDM1A knockdown in both myelodysplasia. While this emphasises that critical regulator of MLL leukaemia stem cells murine and primary human MLL-AF9 AML cells. epigenetic therapies hold significant potential in (LSCs). Knockdown experiments demonstrate Furthermore, treatment of mice with the treatment of myeloid malignancy, it also that KDM1A is required to prevent 36 | Paterson Institute for Cancer Research Scientific Report 2011 Leukaemia Biology Group | 37

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    Signalling Networks in Cancer Group kinase activity utilising in vivo and in vitro kinase activity assays (Figure 2B). We will compare the http://www.paterson.man.ac.uk/signalling_networks activity of the kinases harbouring cancer mutations (engineered through site-directed mutagenesis) to WT, kinase dead (KD) and hyperactivated forms of the kinase (Figure 2B). Next we will determine phenotypic effects of expressing the WT, KD and mutant forms of the target kinase on proliferation, survival and transformed properties of appropriate tumour and normal cell lines. We will verify the function of the kinase using si/shRNA and evaluate the Signalling pathways dictate a range of important cellular role of the endogenous kinase in regulating cellular phenotypes associated with outcomes ranging from cell death, to replication, to cellular tumorigenesis. We will also investigate the migration. Genetic lesions that skew the balance of these molecular mechanisms utilised by the cancer mutants to promote tumorigenesis. For example, pathways towards abnormal growth, proliferation, and cell if the mutation is an activating mutation, we will survival are the fundamental mechanisms that cause normal identify cancer relevant substrates that are phosphorylated by the cancer mutants to cells to become premalignant. high probability of possessing a driver mutation, promote tumorigenesis. Finally we will assess the Figure 1 A high-throughput approach to find and if mutations are predicted to be cancer consequences of somatic mutations utilising cell Group Leader mutations based on bioinformatic analyses lines that harbour endogenous mutations in the Kinases are the key regulators of signalling cancer cells (Figure 1), providing a snapshot into the drivers of lung cancer. Using lung John Brognard pathways (similar to transistors in a circuit) and the future of personalised medicine. We will cancer cells where all mutations are (CanPredict, PMUT, polyphen2, mutationtaster, target kinase. The overall goal of these studies identified through cancer genomic snpeffect, puppasuite and SNPs and go). will be to identify common and convergent Postdoctoral Fellows dictate the activation or amplification of a given also be able to gain insight into when a mutant sequencing, we deplete cells of the signal that ultimately leads to cellular fate protein is a strong driver and should be targeted Additionally, we model many of the mutations pathways utilised by cancer cells to promote Shameem Fawdar mutated proteins to identify the Anna Marusiak decisions. When these kinases are hyperactivated with small molecule inhibitors. For example, we essential driver mutations. We then that score highly in our analysis to determine the tumorigenesis and identify convergent and or inactivated by genetic mutations they become are discovering that in some lung cancers use this as a guide to assess which structural consequences of the putative driver essential targets that could be exploited for the Scientific Officer the main drivers of tumorigenesis and thus serve activating K-ras mutations are essential for small molecule inhibitors should be mutations (Figure 2A). development of novel therapeutics. Eleanor Wendy Trotter used for the treatment of each as primary targets for the development of small maintenance of tumorigenic phenotypes, while in unique and individual cancer (ie molecule inhibitors. Cancer genomic sequencing other lung cancers with similar activating To characterise the mutant kinases, our general Graduate Students personalizing and tailoring treatment Zoe Edwards (since October) studies and genome-wide siRNA screens are mutations in K-ras they are dispensable, as to a specific cancer). strategy is to first assess the functional Publications listed on page 71 highlighting the amazing diversity in the kinases targeted depletion of the mutant K-ras does not consequences of somatic mutations on overall required to maintain tumorigenic phenotypes or dramatically effect cancer cell survival or drug resistance and emphasising the importance proliferation in these specific cancer cells. Thus that neglected or understudied kinases play in the background of mutations present in a specific Figure 2 the development and maintenance of a tumour. tumour dictates the relative importance of a Evaluation of novel loss-of-function Thus a major goal of our lab is to identify and putative driver mutation. In summary, with this mutations in a cancer associated elucidate novel kinase drivers that are essential approach we will be able to (1) identify novel kinase. A. Structurally modelling for tumour development and/or therapeutic kinases with activating mutations and determine cancer kinase mutations allows us to resistance. their importance in a larger cohort of lung predict which mutations will alter the conformation of a kinase in such a cancer samples and (2) determine the “linchpin” manner that it has greater or Drivers of Lung Cancer mutant proteins that are essential to maintain reduced catalytic activity. B. The lab combines cancer genomic sequencing tumorigenic phenotypes and demonstrate that Biochemical characterization of with siRNA screening technology to screen lung targeting these cancer drivers will result in mutations identified in cancer cancers and identify kinases that harbour specific killing of cancer cells (Figure 1). As we patients demonstrating a majority of mutations in this particular kinase activating mutations. This approach streamlines enter the age of personalised medicine where are loss-of-function mutations. the identification of mutationally activated kinases every cancer patient’s genome will be that are likely to be robust drivers of lung sequenced, these studies will illustrate how we tumorigenesis and therefore potentially can manage these data to deliver the right drugs druggable targets. Additionally, these studies to the right patients. allow us to begin to illuminate a basic understanding of how oncogenic mutations Novel Cancer Associated Kinases interact with other mutations in a specific Another primary objective of the laboratory is tumour or cancer cell and we will be able to to use bioinformatic tools to analyse kinases with identify if the mutant protein interactions will be somatic mutations reported in various cancer additive or synergistic in promoting tumorigenic kinome-sequencing studies for those most likely phenotypes. We will determine if inhibiting to contribute to tumorigenesis or drug multiple pathways that are aberrantly activated resistance. Candidate cancer-associated kinases by driver mutations in kinases in a specific cancer that our lab will study are determined based on will result in better and more specific killing of whether the kinase has limited characterisation, a 38 | Paterson Institute for Cancer Research Scientific Report 2011 Signalling Networks in Cancer Group | 39

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    Stem Cell Biology Group absence of RUNX1. We are also further documenting the abnormalities, which frequently result in the formation of chimeric transcription factors. The http://www.paterson.man.ac.uk/stemcellbiology regulation of the expression of core binding factors AML1/RUNX1 and CBFβ these genes by RUNX1 using are the most frequent targets of these genetic reporter assays. alterations. The t(8;21) translocation resulting in the AML1-ETO fusion and the inv(16) generating Generation of blood, endothelial the SHMMC-CBFβ fusion accounts together for and smooth muscle cells more than 20% of all AML cases. Animal models It is now well established that have indicated that the full length AML-ETO, clonal haemangioblast precursors expressed either upon viral transfer or as a generate blast colonies containing transgene, is not able by itself to induce vascular smooth muscle, leukaemia in mice. However an alternatively The major interest of our lab is to decipher the cellular and endothelial and haematopoietic spliced form of AML1-ETO, AML1-ETO9a (AE9a), potentials. However the order of has recently been shown to cause a rapid molecular mechanisms that control the development and restriction to these developmental potentials is development of leukaemia in mice following Figure 1 maintenance of the haematopoietic system. In this context, we Smooth muscle cells generated from not well understood. To enable us to track the retroviral transfer. Using an ES cell line with a cells committed to these different cell fates, we doxycycline AE9a IRES GFP inducible cassette, we study the functions of the transcription factor AML1/RUNX1 differentiated ES cells. The red staining corresponds to smooth have created reporter ES cell lines expressing first validated in vitro that the induced expression and of the transcriptional co-activator MOZ. muscle actin (SMA) whereas the green nuclear staining corresponds to fluorescent protein under the control of early of AE9a, thought to act as a dominant inhibitor genes specifically expressed in these different of AML1/RUNX1, blocked the generation of expression of H2B-venus under the lineages. We are currently investigating the haematopoietic cells during blast development. control of the SMA locus. AML1/RUNX1is one of the most frequent evidence for the presence of this bipotential pattern of development of these different Based on these results, we subsequently Group Leader targets of gene rearrangements and mutations in precursor in vivo. lineages and examining the influence of different generated a mouse line from these ES cells. The Georges Lacaud acute leukaemia. Similarly the gene MOZ signalling pathways on their generation. mice expressing AML1-ETO9a developed extra is involved in myeloid chromosomal We have recently demonstrated that the medullary haematopoiesis followed by the Postdoctoral Fellows translocations. Understanding the function of haemangioblast generates haematopoietic cells Expression and Function of Runx1/AML1 development of acute myeloid leukaemia. The Kiran Batta (from these transcription regulators during normal through the formation of a haemogenic isoforms disease latency was shorter when AML1-ETO9a September 2011) Julia Draper (from haematopoiesis should result in a better endothelium intermediate, providing the first Runx1/AML1 is expressed as multiple naturally expression was induced on a p53-/- background. February 2011) comprehension of how perturbations of their direct link between these two precursor occurring spliced isoforms that generate proteins Altogether these results indicate that we have Michael Lie-A-Ling functions lead to development of leukaemia. populations. This haemogenic endothelial cell with distinct activities on target promoters. We now established a new model of leukaemia Flor Perez-Campo population is transiently generated during blast have generated ES cells and mouse lines development, which will allow us to investigate Roshana Thambyrajah Generation of blood cells development and is also detected in gastrulating containing a reporter gene knock-in in the further the molecular and cellular events (from November 2011) There is a worldwide shortage of matched embryos. At the molecular level, we have different isoforms and produced knockouts associated with t(8;21) leukemogenesis. Scientific Officer donors for blood stem cell transfers for demonstrated that the transcription factor altering the specific expression of these isoforms. Rahima Patel leukaemia or lymphoma patients. The generation SCL/TAL1 is indispensable for the establishment We have previously demonstrated that the Function of the HAT activity of MOZ of blood cells upon in vitro differentiation of of this haemogenic endothelium cell population expression of these isoforms is differentially The Moz gene is involved in leukaemia in three Graduate Students embryonic stem cells or induced pluripotent from the haemangioblast whereas regulated during early haematopoietic independent myeloid chromosomal Monika Antkiewicz Magdalena Florkowska stem cells could represent a powerful approach RUNX1/AML1 is critical for the generation of development both in vitro and in vivo and that translocations fusing Moz to the partner genes (from September 2011) to generate the autologous cell populations haematopoietic cells from this haemogenic their expressions define specific stages of CBP, P300 or TIF2. All these genes encode Elli Marinopoulou required for these transplantations. In this endothelium. haematopoietic development. We are currently enzymes containing a histone acetyl transferase Milena Mazan context, it is important to further understand investigating the pattern of expression of the domain (HAT) suggesting that aberrant Olga Tsoulaki (until April 2011) the development of blood cells. Haemogenic endothelium and transcriptional different RUNX1 isoforms in the different modification of histones or other factors could targets of RUNX1/AML1 compartments of the adult hematopoietic provide the first step in the route to The earliest site of blood cell development in These results suggest that RUNX1 regulates the hierarchy. We are also further evaluating the oncogenicity. We specifically addressed the role the mouse embryo is the yolk sac where blood expression of a set of genes critical for the requirement for these different isoforms in adult of the HAT activity of MOZ during islands, consisting of haematopoietic cells development of the haematopoietic system from haematopoiesis. haematopoiesis by generating a mouse strain surrounded by a layer of angioblasts, develop at the haemogenic endothelium. To identify these that carries a single amino acid change in the approximately day 7.5 of gestation. The parallel genes, we have compared gene expression in cell Model of leukaemia HAT domain of MOZ. Analysis of these mice Figure 2 development of these two lineages in close populations generated from either Runx1 Embryoid bodies generated from ES Human acute leukaemia is characterised by the has revealed a profound defect in association provided the basis for the hypothesis deficient or Runx1 competent ES cells at cells containing red blood cells. presence of recurrent chromosomal haematopoiesis. The numbers of haematopoietic that they arise from a common precursor, a cell different stages of haematopoietic development. stem cells and their potential is dramatically called the haemangioblast. A conflicting theory We have also developed strategies to identify affected in homozygous mice. We also however associates the first haematopoietic cells genome-wide RUNX1 binding sites at these uncovered that the balance between to a phenotypically differentiated endothelial cell different stages of blood development by either proliferation and differentiation of blood with haematopoietic potential, i.e. a haemogenic chromatin immunoprecipitation or DamID (a progenitors is hampered in the absence of MOZ endothelium. Support for the haemangioblast method developed by Professor Bas van driven acetylation. We are currently investigating concept was initially provided by the Steensel, NKI, Amsterdam). These studies are further the molecular mechanisms affected in the identification during embryonic stem (ES) cells’ performed in collaboration with the Applied absence of the HAT activity of MOZ leading to differentiation of a clonal precursor, the blast Computational Biology and Bioinformatics Group such a phenotype. colony-forming cell (BL-CFC), which gives rise led by Dr Crispin Miller. We are currently after 4 days to blast colonies with both evaluating the potential of some of these endothelial, smooth muscle and haematopoietic identified transcriptional targets to rescue the Publications listed on page 71 potential. Recent studies have now provided development of the haematopoietic system in 40 | Paterson Institute for Cancer Research Scientific Report 2011 Stem Cell Biology Group | 41

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    Stem Cell and Haematopoiesis Group Figure 1 Schematic representation of the http://www.paterson.man.ac.uk/sch molecular circuitry controlling haematopoietic specification. The overarching goal of the Stem Cell and Haematopoiesis group is to further delineate and understand the network downstream of ETV2, we performed a precursors since neither GATA2 nor FLI1 comparative gene expression survey upon ETV2 expression is able to significantly rescue the transcriptional networks that orchestrate the formation of the re-expression, analysing the transcriptome of formation of haematopoietic progenitors. haematopoietic system during embryonic development. Many Etv2-deficient cells stimulated or not with doxycycline. The most prominent category of Haematopoietic specification: an ETV2-SCL transcriptional regulators implicated in haematopoietic genes up-regulated upon ETV2 induction linear module specification have been linked to leukemogenesis events, often belonged to the cardiovascular system, a The regulation of Scl transcription in developmental network including many genes, haematopoietic and endothelial lineages is as a result of aberrant expression. Through a better such as Ve-cadherin, Gata2, Scl or Hhex, all known controlled by a 5’-enhancer region containing Group Leader Valerie Kouskoff understanding of the function of these transcriptional to be implicated and important in vasculogenesis five Ets binding sites. Luciferase assays revealed and haematopoiesis. The presence of Scl, Fli1 and that ETV2 was able to efficiently activate the regulators at the onset of haematopoietic specification, we Gata2 within this list of genes was of particular transcription from this 5’-enhancer region Postdoctoral Fellows Alexia Eliades hope to gain insight into their potential role in the initiation interest given that these three factors form a whereas mutations in the five Ets binding sites Maud Fleury recursive loop controlling multiple blood genes contained within this enhancer region completely Sarah Lewis and maintenance of haematological malignancies. during development. Strikingly, however, Scl is the abolished the transcription driven by ETV2. only one of those three genes whose deficiency Furthermore, chromatin immuno-precipitation Scientific Officer Stella Pearson Molecular and cellular control of blood precursors but not in the emergence of results in a complete haematopoietic defect experiments showed that ETV2 occupancy was specification primitive erythropoiesis. More recently this similar to Etv2 deficiency. These observations led enriched on the 5’ enhancer of Scl when Graduate Students During embryonic life, the onset of transcription factor has been identified as a us to investigate the degree of interconnection compared to intergenic regions further away Guilherme Costa between the downstream programmes from the transcription start site of Scl. Taken haematopoiesis occurs soon after gastrulation master regulator controlling the transition from Sara Cuvertino and is one of the first programmes to be haemogenic endothelium to blood precursors. controlled by ETV2 and SCL. We assessed together, these data strongly suggest that ETV2 Andrzej Mazan specified from the mesodermal germ layer. The transcription factor SCL is critically required whether SCL expression might rescue to some directly controls the transcription of Scl through Mesoderm precursors within the primitive streak for blood cell formation as SCL deficiency led to extent the haematopoietic defect resulting from binding to Ets sites within the 5’ enhancer of Scl. become progressively committed to the blood a complete absence of both primitive erythroid ETV2 deficiency. This hypothesis was tested using Our study identifies the molecular switch that programme as they migrate toward the extra- and definitive blood precursors and early a doxycycline-inducible Scl transgene introduced drives blood specification, showing that the SCL- embryonic region of the developing embryo embryonic lethality. Further studies have revealed into the Etv2-deficient ES cells. Strikingly, FLI1-GATA2 recursive loop controlling the where they later form the yolk sac blood islands. a role for SCL in the formation of induction of SCL expression in Etv2-deficient expression of many haematopoietic genes is The first identified step toward haematopoietic haemangioblast and haemogenic endothelium cells was sufficient to fully rescue the production activated by ETV2 via Scl as an entry point commitment occurs when mesodermal cells up- precursors. A newly identified player in of all haematopoietic precursors. A time course (Figure 1). Our data also indicate that GATA2 regulate the tyrosine kinase receptor FLK1 haematopoietic specification, the Ets transcription analysis of haematopoietic progenitor formation and FLI1 are not able to initiate this recursive marking the formation of the haemangioblast, a factor ETV2, appears to be equally critical for revealed a pattern very similar to the one loop on their own. While our findings further bi-potential precursor giving rise to blood and haematopoiesis since its deficiency also leads to previously observed in wild-type cultures. Taken define how ETV2 controls haematopoietic endothelial cells upon further maturation. These the absence of all blood progenitors and early together, these findings demonstrate that SCL specification, the transcriptional control of haemangioblast precursors rapidly form embryonic lethality. acts downstream of ETV2 in the specification of endothelium specification by this factor remains specialised endothelium cells, termed the haematopoietic programme. Given that FLI1 to be explored given that vascular defects are haemogenic endothelium, that generate blood ETV2, an Ets factor at the top of the hierarchy and GATA2 were also found to be dependent much more profound in Etv2-deficient embryos precursors upon further maturation. The To further evaluate the role of ETV2 at the on ETV2 expression, we set out to test whether than in Scl-deficient embryos. It will be interesting transition from haemogenic endothelium to onset of blood specification, we generated an re-expression of either of these two to define whether, similar to blood specification, blood precursors is marked by the acquisition of Etv2-deficient ES cell line in which an Etv2 transcription factors would also be sufficient to the control of vascular development is also CD41, the αIIb integrin that defines the earliest transgene could be re-expressed upon rescue ETV2 deficiency. In a similar doxycycline regulated by a very limited set of ETV2 fully committed blood precursors. While the doxycycline induction using a Tet-on inducible inducible system, FLI1 or GATA2 were expressed transcriptional targets. cellular basis underlying the formation of the first system. The in vitro differentiation of this ES cell in ETV2-/- differentiating cells and replated in a blood cells from the mesoderm germlayer are line deficient for Etv2 expression revealed a clonogenic assay. However, unlike the rescue well characterised, the molecular mechanisms complete absence of all haematopoietic observed upon SCL induction, very few Publications listed on page 71 orchestrating this developmental process still progenitors in the absence of doxycycline, a haematopoietic progenitors were detected upon remain poorly understood. Several transcription phenotype similar to the one described for Etv2- FLI1 or GATA2 expression. Altogether, our data factors have been implicated at various steps of deficient embryos. Addition of doxycycline suggest that SCL is a limiting factor for blood this developmental programme. Early studies on during the in vitro differentiation allowed a specification in ETV2-deficient mesodermal the function of RUNX1 revealed its critical role complete rescue of haematopoietic in the formation of definitive haematopoietic development. In search of the molecular 42 | Paterson Institute for Cancer Research Scientific Report 2011 Stem Cell and Haematopoiesis Group | 43

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    Stromal-Tumour Interaction Group However, emerging evidence now proposes a different scheme in which metastatic spread is cells co-cultured in collagen gels. Tenascin C, HGF and SDF-1, which are secreted by CAFs, also http://www.paterson.man.ac.uk/stromal/ not totally dependent on the acquisition of play a role in mediating CAF-stimulated invasion genetic alterations within carcinoma cells. The of cultured carcinoma cells. It remains, however, tumour microenvironment also serves as an unclear as to how CAFs influence each step of important determinant that encourages the metastatic cascades and what paracrine carcinoma cells in the primary tumour to signalling from CAFs is essential for facilitation of become motile and invasive, and to disseminate metastatic dissemination of carcinoma cells in into distant organs. Interaction of carcinoma cells vivo and what molecular alteration(s) is provoked with the tumour-associated stroma facilitates the in such metastatic carcinoma cells. Studying invasion-metastasis cascade. crosstalk between tumour cells and mesenchymal cells during tumour progression Human tumours are highly complex tissues and the non- Tumour-associated stromal fibroblasts play a could help understand the nature of biology of a significant role in regulating migratory and bulk of human carcinomas and facilitate the neoplastic cell compartment of tumours, which is often termed invasive behaviours in carcinoma cells. This is development of novel stroma-targeted the “stroma”, is itself quite complex histologically. Carcinoma supported by evidence indicating that stromal therapeutic approaches. myofibroblasts are frequently present at the cells initially recruit and/or activate these various stromal non- invasive front of human carcinomas. In addition, it neoplastic cells, including fibroblasts, myofibroblasts, immune has been shown that CAFs increase the Publications listed on page 71 migratory and invasive propensity of the cancer cells, endothelial cells, bone marrow-derived cells. The resulting Group Leader stromal cells reciprocate by fostering carcinoma cell growth Akira Orimo and survival and neoangiogenesis during the course of tumour Figure 1 Co-evolution of stromal fibroblasts Postdoctoral Fellow progression. with carcinoma cells during tumour progression Resident stromal Urszula Polanska fibroblasts within the tumour Studying the heterotypic interactions between carcinoma cells, once it is acquired, they display increasingly acquire two autocrine Graduate Student Ahmet Acar the neoplastic cells and the supporting stroma is this trait independent of further signalling from signalling loops involving TGF-β and essential for understanding the nature of the the carcinoma cells. We have reported that SDF-1 during the series of tumour progression. These autocrine signalling bulk of carcinoma mass. We study how the normal human mammary fibroblasts, when co- loops are very likely conferred by the evolution of stromal fibroblasts facilitates tumour inoculated with breast carcinoma cells into interaction with nearby carcinoma cells to become invasive and metastatic during immunodeficient mice, convert stably into cells during the series of tumour the course of tumour progression. tumour-promoting myofibroblasts within the progression and they mediate resulting tumours (Kojima et al., 2010, Proc. Natl. transdifferentiation of stromal fibroblasts into tumour-promoting Evolution of stromal myofibroblasts in tumours Acad. Sci. USA., 107, 20009-20014). During CAF myofibroblasts. Apposed Neoplastic epithelial cells coexist in carcinomas tumour progression, these fibroblasts carcinoma cells are also subject to with a stroma composed of various types of progressively elevate two autocrine signalling acquire invasive and metastatic mesenchymal cells as well as extracellular matrix loops mediated by the TGF-β and SDF-1 propensities through the interaction (ECM), both of which create the complexity of cytokines in self-stimulating and cross- with CAFs during tumour progression. The molecular mechanism(s) the tumour microenvironment. communicating fashions, thereby enhancing both mediating the metastasis-promoting their transdifferentiation into myofibroblasts and ability of carcinoma cells remains Noticeable numbers of myofibroblasts, which are the associated tumour-promoting capability. Taken unclear. characterised by their production of α-smooth together, these findings indicate that the muscle actin (α-SMA), have been observed establishment of cross-communicating TGF-β repeatedly in the stroma of the majority of and SDF-1 autocrine signalling gives rise to Figure 2 How do CAFs influence the tumour invasive human breast cancers. However, the myofibroblast differentiation and mediates the invasion-metastasis cascade? specific contributions of these cells to tumour evolution of residual fibroblasts into tumour- CAFs likely provide nearby carcinoma progression are poorly defined. Stromal promoting myofibroblasts. cells with an invasive propensity in fibroblasts and myofibroblasts, collectively termed vivo that results in an increase in carcinoma-associated fibroblasts (CAFs), were Stroma-derived signalling crucial in promoting tumour cell intravasation. Carcinoma cells educated by CAFs within extracted from various human carcinomas. CAFs, tumour metastasis primary tumours may stably or in comparison with their control fibroblasts, The tumour invasion-metastasis cascade is a transiently maintain their phenotypes when co-injected with carcinoma cells into complex multistep process that includes localised during extravasation and colonisation immunodeficient mice, are known to substantially invasion of carcinoma cells, entrance into the in distant organs. The molecular promote carcinoma growth and neoangiogenesis. systemic circulation, survival during mechanism(s) underlying the potential CAF-induced tumour CAFs retain their myofibroblastic properties and transportation, extravasation and colonisation in invasion-metastasis cascade remains tumour-promoting phenotypes, after they have establishing micro and macro metastases in unclear. been passaged for ten population doublings distant organs. It has long been assumed that (PDs) in vitro in the absence of ongoing contact dissemination of metastatic carcinoma cells with carcinoma cells. Accordingly, even though depends largely on their cell-autonomous effects, the CAFs appear to have initially acquired their due to epigenetic and/or genetic alterations that unique phenotypes under the influence of accumulate within these malignant cells. 44 | Paterson Institute for Cancer Research Scientific Report 2011 Stromal-Tumour Interactions Group | 45

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    Research groups The University of Manchester School of Cancer and Enabling Sciences 46 | Paterson Institute for Cancer Research Scientific Report 2011 Research Groups - The University of Manchester School of Cancer and Enabling Sciences | 47

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    Children’s Cancer Group Figure 1 SD1 cells seeded onto Cell Tak and probed for (a) LAMP1 or (b) talin and co-stained for actin using Alexa Fluor 555 (Red) and Dapi (Bue). Preparations also show microvesicles. (c) TEM image at x2900 Our group investigates the biological mechanisms responsible for the variations in the therapeutic response in children with Ashish Masurekar has developed a technique to We reported earlier this year that lymphoblasts acute lymphoblastic leukaemia (ALL). We conduct a number capture leukaemia specific MVs from patient from patients with high risk ALL express of international clinical trials which provides us with the data samples. Suzanne Johnson and Zahaan Pinto are adhesion molecules, such as ICAM1 and LFA-1, now exploring the effect of MVs on stromal cells and produce central nervous system (CNS) and clinical material for hypotheses based laboratory in vitro and in vivo. leukaemia in NSG mice (Blood 2011;118:638). investigations. Mark Holland used a semi quantitative discovery Jizhong Liu has been investigating the converse, approach to examine the plasma membrane Group Leader i.e. how the microenvironment protects the proteome of these cells. Almost half the proteins Vaskar Saha Clinical protease expression, suggesting unknown and leukaemic cell from chemotherapy. He first unique to the plasma membrane of the cells We have previously reported on the perhaps complex mechanisms of inactivation. created an organotypic, orthotropic model of the transgressing into the CNS were associated with Postdoctoral Fellows degradation and inactivation of the key anti- Further analysis showed for the first time, that bone marrow microenvironment using adhesion, invasion and cytoskeleton re- Clare Dempsey leukaemic drug L-Asparaginase by lysosomal inadequate L-Asparaginase activity during mesenchymal cells obtained from bone marrow organisation and included ICAM1 and LFA-1. Mark Holland Suzanne Johnson cysteine proteases expressed by lymphoblasts. induction significantly affects disease clearance in aspirates. Under culture conditions these cells Pathway analyses suggested a model by which Jizhong Liu This led us to devise protease resistant L- standard risk patients. This suggests that showed differentiation into mesenchymal lineage. cellular adhesion proteins ICAM1 and LFA-1 Asparaginase and show that the glutaminase modification of dosage schedule and the addition This model is able to support the leukaemic cells activated RAC2 leading to the development of Clinical Fellow activity of the drug was essential for cytotoxicity of other drugs have the potential to further in co-culture without the addition of exogenous an organised cytoskeleton and diapedesis. Seema Ashish Masurekar (Blood, 2011, 117:1614). Until this discovery, improve outcome in childhood ALL and that in factors. Conditioned medium from this stromal Alexander showed that inhibition / silencing of Scientific Officer resistance to L-Asparaginase was thought future, the monitoring of L-Asparaginase activity cell culture model provided broad-spectrum RAC2 abrogated in vitro invasion and that the Seema Alexander primarily to be either intrinsic (due to unknown during therapies will become incorporated into chemoprotection to a wide variety of cancer cell invasive cell lines had a well-organised mechanisms) or as a result of neutralising national trials. lines. The protective soluble factors include filamentous actin structure. Using a NSG mouse Graduate Student antibodies developed post-exposure. Our miRNA enriched exosomes that regulate the model they were then able to show that while Stephanie Harrison findings suggested that lysosomal proteases could The results of the international clinical trial in PI3K/AKT and aerobic glycolysis pathways. This non-invasive cell lines produced leukaemia in Clinical MRes Students contribute to an inadequate therapeutic effect. relapsed ALL (ALLR3), reported last year, produces a quiescent state in leukaemic cells, mice, the invasive cell lines led not only to Caroline Fong continue to show some of the best outcomes in leading to a low tonic expression of pAKT and leukaemia but the establishment of CNS disease, Zahaan Pinto Ashish Masurekar, along with Caroline Fong and the world (Lancet 2010; 376:209). A new trial, ROS. Stimulation of ROS generation using PEITC which was partially inhibited by silencing RAC2. Adiba Hussain investigated this in a prospective IntReALL is due to open next year that will now or piperlongumine is cytotoxic to multidrug Overall the work suggests a model by which Clinical Trials Manager fashion in the national frontline trial, ALL2003. include 20 different countries making it the resistant leukaemic cells. While the cells are cells migrate to extramedullary sites, adhere to Catriona Parker / Adiba Hussain They serially analysed ~500 patients for largest such study worldwide. resistant to L-Asparaginase, the drug is the host cells then are able to invade across the Administrator Asparaginase activity and antibody levels and additionally cytotoxic when given along with blood and CSF brain barriers into the central Charlotte O’Horo / Parisa correlated this with known risk factors and Laboratory PEITC. This may be due to the glutaminase nervous system. Stephanie Harrison, a new PhD Mahjoob surrogate markers of outcome. They found that We wanted to understand how leukaemic activity of the drug. We are verifying this in vivo student who joined the group in October, is now 70% of patients had good activity throughout lysosomal proteases interacted with L- and if reproducible will plan to test this in an further investigating the role of ICAM1 and LFA- their treatment. Those who did not mostly had Asparaginase. We initially identified these Figure 2 early phase clinical trial. 1 in CNS disease. Clare Dempsey has been non-detectable levels. This is an important lysosomes at the periphery of the cell localised working with them to devise suitable in vivo finding. The pegylated version of the drug used in to vesicles contiguous with the cell membrane models. the UK is expensive. Based on our previous (JCI 2009;119:1964). We subsequently identified experience, we use 1000 u/m². Most groups that these vesicles are shed as microvesicles We are also pleased to report that Shekhar worldwide use 2000-3000 u/m². Our results (MVs). Suzanne Johnson showed that the MVs Krishnan and Seema Alexander successfully show that this is unnecessary and that increasing stain positive for actin, talin and vinculin (Figure defended their doctoral dissertations in 2011 the dose is unlikely to improve activity in those 1A and B), are motile and show a complex and Catriona Parker gave birth to a bouncing who do not achieve adequate levels. We also anucleate structure (Figure 1C). MVs taken up by baby boy in September. Charlie moved on in found that 10% of patients with good initial other lymphoblasts, develop an activated October and we wish her a very successful activity, subsequently showed low activity. Most phenotype typical of the cell of origin (Figure 2). career in her new management role. We of these patients had developed anti- Bone marrow stromal cells actively take up MVs, welcome Parisa Mahjoob-Afag into the role of asparaginase antibodies. Of the remaining 20%, suggesting horizontal transfer of RNA, DNA and department administrator. all had inadequate activity initially, though half protein. Thus this suggests a mechanism by which showed adequate activity later on. None of leukaemic cells regulate the microenvironment these patients had detectable antibodies, nor and is possibly a general mechanism by which Publications listed on page 72 could we correlate this with the levels of cancer cells establish a cancer specific niche. 48 | Paterson Institute for Cancer Research Scientific Report 2011 Children’s Cancer Group | 49

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    sterile environment (Figure 1). Materials are investigators wishing to exploit any form of cell Medical Oncology: Clinical and Experimental moved into the isolator through a separate therapy in the clinical setting. Immunotherapy Group transfer unit which means that the area required to be maintained to this high level is significantly Pre-Clinical Research reduced from that of a standard clean room. Two The group continues to focus upon isolators have been generated with a shared understanding and improving adoptive T cell transfer unit which are sterilised by perfusion therapy for cancer. In particular, our focus has with hydrogen peroxide gas and permits up to been upon engineering T cells with anti-tumour four different T cell products to be manipulated specificity through genetic modification. Two per day. In addition to the use of closed culture routes have been explored – co-expression of and selection systems, this means that a higher the α and β chains of a tumour antigen specific T throughput of cell products is possible which cell receptor (TCR) or the expression of an Immunotherapy utilises the immune system to control and should translate to cost efficiencies. antibody-fusion receptor (Chimeric Antigen Receptor, CAR) on the T cell surface which potentially eradicate cancer. Whilst the group is involved in Dr Ryan Guest has overseen the development circumvents the normal T cell recognition a number of innovative approaches in clinical trials, at a pre- of the CTU which was accredited for the process and directs the functional activity of the production of Investigational Medicinal Products T cell against cell surface protein antigens. clinical level we focus on developing adoptive cell therapy. (IMP) and to produce ‘Specials’ (one-off patient products) in November, 2011. The first patient Both approaches have their own advantages and product produced by the Unit was a T cell limitations. However, one key advantage of the Clinical Trials impact upon the ability to translate academic product generated from a Malignant Melanoma CAR approach is that many different signalling The lack of an approved Cell Therapy Unit bench-based observations through to clinical Tumour (Tumour Infiltrating lymphocyte, TIL). TILs domains can be engineered downstream of the for much of the year has meant there was no testing due to the requirement to comply Group Leader are generated from enzymatically digested antibody targeting domain. In this way, many recruitment to cell therapy trials – now the unit with GMP even in first into man early phase Robert Hawkins tumour and cultured for a period of three weeks varied signalling pathways can be activated is open (see below) the trial targeting CD19 clinical trials. prior to a rapid expansion using donor though a single CAR binding event. The most with engineered T cells should re-open shortly. Consultant/Senior Lecturer lymphocytes, antibodies and cytokines. The commonly used CAR-based signalling Fiona Thistlethwaite Trials in melanoma also form a major part of Producing drugs in compliance with GMP is successful generation of TILs is of major combinations have been CD3ζ linked with the our future plans. The initial patient treated with difficult. Producing individual patient cell products importance; this confirms that complicated CD28 cytosolic domain. Upon antigen binding, Senior Fellow tumour infiltrating lymphocytes (TIL) was is extremely demanding. For example, handling David Gilham handling procedures can be efficiently carried the CD3ζ domain drives ‘signal 1’ which is successful and we hope to obtain NHS funding of the cell product is only allowed with a out in an isolator and that closed culture systems effectively activation of the T cells’ effector for this treatment as well as NIHR funding for continuously monitored category A environment Research Fellow are suitable for expansion of these T cells. One functions while the CD28 domain provides Ryan Guest (Cellular trials to assess improvements to the treatment. (determined by a particle measurement of less further patient tumour has been processed with ‘signal 2’ which is a survival signal typified by the Therapeutics Unit) In addition, we are working with our EU than 1 >5μm particle present in 1m3 of air). To the T cells isolated in readiness for rapid T cell producing cytokines such as IL-2 and up- partners to develop trials targeting NY-ESO1 achieve this in our previous clinical trials of ATTRACT Clinical Fellow/ expansion. regulating anti-apoptotic gene expression to assess the potential benefits of using adoptive T cell therapy, all work was carried out Marie Curie Fellow resulting in survival and proliferation of the selected cells. in a dedicated suite of laboratories maintained Symeon Eleftheriades With the CTU now on-line, the immediate activated T cell. The combination of two signalling by the National Blood Service (NBS) sited at intention is to continue TIL therapy for domains into a single CAR have been termed Scientific Officers On-going analysis of previous immunotherapy Plymouth Grove, Manchester. A major issue was Melanoma and also to resume the gene- ‘2nd generation CARs while those CARs Vicky Sheard trials continues to throw up clues as to the the lack of flexibility of the use of such Natalia Kirillova (Cellular modified T cell adoptive therapy targeted B-cell containing the CD3ζ effector domain are most appropriate biomarkers to use to select laboratories; each single patient product required Therapeutics Unit) lymphoma which has been on-hold waiting for termed ‘1st generation CARs. patients who are likely to benefit from the exclusive use of one of the labs hence Sam Mowbray (Cellular cell production to re-commence. Importantly, the Therapeutics Unit) treatment. This could be very useful for future meaning low throughput and high cost Unit is well equipped including flow cytometry Despite this clear separation of function, we have Lidan Christie (Cellular trials and these biomarkers are increasingly associated with each cell product generated. (Miltenyi MACSQuant) and clinical grade cell previously found that mouse and human T cells Therapeutics Unit, until July) being incorporated into cancer vaccine studies. Nonetheless, 23 patients were treated with cells selection technology (Clinimacs) which is bearing a 1st generation CAR specific for CD19 Other targeted therapy trials are increasingly generated in these clean rooms (17 gene Graduate Students Figure 1 designed to provide a flexible service for produce IL-2 when co-cultured with B cell looking at angiogenic pathways other than the modified; 6 non-modified, regulatory T cell Hannah Gornall lymphoma cells. This activity is independent of Vickie Hambleton VEGF pathway. depleted). However, the NBS took the decision the activity of the native CD28 receptor but is to close the clean rooms in Manchester as a dependent upon the binding of the CD2 co- Clinical Fellow Cellular Therapeutics Unit (CTU) part of a large scale move of stem cell activities Rob Brown stimulatory receptor to its ligand on tumour cells A recent major effort of the group has been the from Manchester to Liverpool. To continue cell (Cheadle et al., 2011, Gene Ther). Importantly, development of a unit capable of delivering therapy clinical studies, an alternative cell Marie-Curie Training Fellow this may explain why T cells engrafted with a 1st Vania Baldan clinical grade cells to treat patients. Since the production unit was required. generation CD19-specific CAR persist and incorporation of the provisions of the European maintain anti-tumour activity for an extended Project Managers Union Clinical Trials directive into UK law, there After consultation with the Regulators (MHRA), Nikki Price period of time in mouse models of systemic B has been a requirement to deliver medicines and we initiated the development of a new Cellular Helena Kondryn cell lymphoma. This observation indicates that therapies produced in accordance with the Therapeutics Unit (CTU) at the University the ligands of the CD2 receptor present on the principles of Good Manufacturing Process Incubator Building in Grafton Street. In order to tumour target cell can play a role in determining (GMP). In effect, this means that all medicines avoid the lack of flexibility of large clean-rooms, a engineered T cell response and suggests that and therapies have to be produced to a level design based upon the use of isolators was manipulation of the CD2 signalling pathway may equivalent of that in the pharmaceutical industry. followed. In this scenario, the category A enhance engineered T cell function. This includes validation, quality control, environment is maintained within the sealed continuous environmental monitoring and isolator with sealed glove units allowing the rigorous product testing. This has had a major operator to manipulate the cells within the Publications listed on page 72 50 | Paterson Institute for Cancer Research Scientific Report 2011 Medical Oncology: Clinical and Experimental Immunotherapy Group | 51

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    Medical Oncology: Translational Anti-Angiogenesis Group Heparan sulphate (HS) is an essential regulator of the on a precise structure opens opportunities for sprouting and tube formation in cells with Figure 1 biological activity of the majority of angiogenic cytokines and Endothelial HS 6-O sulphation levels the design of more potent inhibitors of reduced 6-O sulphation. Although FGF2 or VEGF chemokines. However, the key HS structural features involved regulate vasculogenesis in vivo. A. angiogenic cytokines. binding to HS or to receptors on cells was Human CD34 and SMAα staining of unaffected by reduced 6-O sulphation, FGF2 and in the inhibition of cytokine/receptor complexes remain HUVEC spheroids at day 21 after implantation. B. Average number of Endothelial HS 6-O sulphation levels modulate VEGF-induced receptor phosphorylation was unclear. Using our unique synthetic chemistry programme we human CD31-positive blood vessels FGF2- and VEGF-dependent endothelial cell compromised (manuscript in preparation), per standardized area of a plug. functions suggesting that 6-O-sulphation of N-substituted have started to unravel HS structural requirements for Ovarian cancer is one of only a few diseases that glucosamine residues plays an important role in inhibition of angiogenic cytokine-specific functions. We have respond to single agent VEGF inhibitors, such as the regulation of angiogenesis in vivo. Group Leader bevacizumab. Recent data showed an Gordon Jayson identified precise HS structures that are capable of inhibiting improvement in progression-free and overall Clinical significance of HS multiple angiogenic cytokines. In addition we have focussed on survival of ovarian cancer patients treated with In preclinical animal models of cancer, endothelial Senior Research Fellow bevacizumab in combination with chemotherapy. progenitor cells (EPCs) have been identified as Egle Avizienyte defining the role of HS in human ovarian cancer angiogenesis. To determine the role of HS in ovarian cancer, catalysts of vasculogenesis in primary or We are currently evaluating HS specific sulphation epitopes we evaluated expression of HS sulphation metastatic tumours. However, it is unclear how Postdoctoral Fellows Claire Cole patterns in ovarian tumours and normal ovaries. much they contribute to tumour vasculogenesis Steen Hansen and their functional relevance in ovarian cancer tissue and We found an increase in staining for 6-O in humans. As a number of angiogenic cytokines Gavin Miller circulating endothelial cells and their progenitors from ovarian sulphation specific epitopes in tumour that regulate EPC functions critically depend on endothelium, while 6-O sulphation in normal HS, we aim to investigate HS sulphation patterns Clinical Fellows Danielle Shaw cancer patient blood. ovaries was often detected in the perivascular and their functional significance in EPCs from the Laura Horsley region of blood vessels. To test functional blood of ovarian cancer patients and healthy Kalena Marti-Marti significance of 6-O sulphation in endothelial cells, controls. We developed a flow cytometry Evaluation of synthetic structurally-defined HS charge density and domain organization are the we down-regulated 6-O sulphotransferases 1 or protocol for evaluation of EPC and circulating Scientific Officer oligosaccharides key determinants. Our unique organic synthesis 2 (6-OST-1 and 6OST-2) and discovered that endothelial cell (CEC) staining with sulphation Graham Rushton Most angiogenic growth factors and chemokines, programme allows us to address these Figure 2 reduced 6-O sulphation in S-domains by 25-40% specific anti-HS antibodies. We discovered a including Fibroblast Growth Factors (FGFs), questions. Previously we showed that longer Percentage of CECs and EPCs significantly compromised endothelial tube higher proportion of EPCs bearing Vascular Endothelial Growth Factor 165 oligosaccharides that were uniformly 2-O and N- positive for staining with antibodies formation and sprouting in vitro and CD31+CD146+CD45-CD133+ (VEGF165), Stromal Cell-Derived Factors (Sdfs) sulphated (ISNS) had superior inhibitory recognizing HS sulphation specific vasculogenesis in vivo (Figure 1). In addition, immunophenotype that stained with anti-HS and interleukin-8 (IL-8), depend on HS for their properties when compared to 2-O (IS) epitopes. NS antibody recognizes 2- O, N- and 6-O sulphates and HS4C3 wild-type HS in neighbouring endothelial or antibody specifically recognizing 6-O, N- and 3-O biological activity. HS, which is a major sulphated oligosaccharides of the same length antibody recognizes N-, 6-O and 3-O smooth muscle cells did not rescue sulphation when compared to the number of component of HS proteoglycans, contains (Cole et al., PLoS One, 2010). Specifically, ISNS sulphates. compromised FGF2- or VEGF-dependent CECs (CD31+CD146+CD45-CD133-) (Figure repeating disaccharide units which are composed dodecasaccharide showed significant activity 2). These data indicate that specific HS sulphation from D-glucuronic acid (GlcA) or L-iduronic acid against FGF2- and VEGF-induced endothelial cell patterns might be important in EPC functions, (IdoA) linked to N-substituted-glucosamine. In migration, tube formation and signalling, while specifically in their response to cytokines that cells, initial N-sulphation is usually followed by poorly inhibiting endothelial cell proliferation in regulate EPC homing and differentiation in conversion of some GlcA to IdoA, sulphation at vitro. In addition, we showed that ISNS tumours. the 2-O position in iduronic acid and at the 6-O dodecasaccharide was well tolerated by animals and 3-O positions of glucosamine. Heavily at a high dose, was detected in tumour sulphated HS regions (S-domains) that are xenografts at biologically active concentration, Publications listed on page 73 usually separated by poorly sulphated regions did not affect coagulation cascade components (NA-domains) facilitate numerous signalling and reduced tumour xenograft microvessel events by providing interaction sites for a density by 40% (unpublished data). number of growth factors and their receptors, By varying the degree of sulphation along chemokines and extracellular matrix proteins. synthetic HS oligosaccharides, we have Although the significance of HS in regulating demonstrated unique inhibition of angiogenic angiogenic cytokine functions is well appreciated, cytokines, which was not observed when testing the structural requirements for such regulation homogeneously sulphated species. Our data remain unclear. Some studies suggest that show for the first time that HS structural sulphation at specific positions in HS is specificity is more important than charge density important, while other studies indicate that in regulating biological activity. Such dependency 52 | Paterson Institute for Cancer Research Scientific Report 2011 Medical Oncology: Translational Anti-Angiogenesis Group | 53

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    Targeted Therapy Group to demonstrate that the source of mAb induced ROS is extra-mitochondrial as Type II anti-CD20 against subsequent tumour rechallenge by the induction of a tumour-specific memory response. mAb induce cell death and ROS in respiratory We are currently expanding this study to include deficient Raji sub-clones (ρ- cells), which lack additional syngeneic models of solid tumours. functional mitochondria. ROS generation was Further work is ongoing to elucidate the role of instead found to be mediated by NADPH immune effector cells such as DC, helper-T cell, oxidase localised in the cell membrane using B cell, NK cell and macrophage in the generation pharmacological inhibitors. The potential role of of protective anti-tumour immunity post NADPH oxidases in this pathway was confirmed combination TLR7 and EBRT. using siRNA knockdown of one of the key B-cell associated NADPH oxidases, NOX2, which Monique Melis in collaboration with Kathryn resulted in a significant reduction in mAb- Simpson (Clinical Experimental Pharmacology The overarching goal of the Targeted Therapy Group is to induced cell death. These findings provide Group) has developed a number of doxycycline further insights into a previously unrecognized (Dox)-dependent caspase-3 “death switch” enhance the efficacy of RT (radiotherapy) in the treatment of role for NADPH oxidase-derived ROS in syngeneic murine tumour models. Tumour cells cancer with immunotherapy. The research programme aims mediating nonapoptotic PCD evoked by mAbs transfected with the “death switch” can be in B-cell malignancies. This newly characterised conditionally induced to undergo rapid, to further increase our understanding of the mechanisms cell death pathway may potentially be exploited synchronous apoptosis, and provides a unique underlying the host immune response to irradiated tumour to eliminate malignant cells which are refractory and useful tool to compare the immunogenicity to conventional chemotherapy and of classical apoptotic cell death with that induced cells and by using radio- immunotherapy combinations to immunotherapy. This work is currently under by RT or chemotherapy. In particular, this model Group Leader increase the anti-tumour immune response and outcome review at the journal, Blood. can be used to assess immune responses to cell death in vivo in established tumours. Following Tim Illidge in cancer. Immune response to RT induced dying induction of the caspase-3 death switch, tumours tumour cells rapidly regress and >50% disappear leading to Postdoctoral Fellows Recent work done by Simon Dovedi has long term remission and in vivo immunity. Tumour Ellie Cheadle The specific objectives of the group are i) to dependent, lysosome-mediated process using demonstrated for the first time that the efficacy clearance was observed in immuno-competent Simon Dovedi investigate the mechanisms of action of radio- both lymphoma cell lines and primary chronic Jamie Honeychurch of external beam radiotherapy (EBRT) can be mice but not in immuno-deficient mice. This immunotherapy and to define optimal lymphocytic leukemia (CLL) cells (Ivanov et al., J significantly enhanced by combination with the indicates that an intact immune system is combination strategies to take forward to early Clin Investigation, 2009). More recently we have PhD students selective TLR7 agonist R848. Our data required for long term tumour clearance. Waleed Alduaij phase clinical trial design ii) to investigate how been further investigating the mechanisms of demonstrate that intra-venous administration of Interestingly, when cells undergoing caspase-3 Clara Chan RT-induced tumour cell death is recognised and action involved in this death pathway. We have R848 can lead to the activation of dendritic cells death switch induced apoptosis were used as Monique Melis processed by different antigen presenting cells focused our work on a novel third generation Figure 1 (DC) and lymphocytes and induce the prophylactic cellular vaccination they were (APC) in the tumour microenvironment and humanised type II anti-CD20 mAb called Schematic diagram illustrating the MRes Student expression of several pro-inflammatory cytokines unable to protect against subsequent tumour how this impacts on the subsequent adaptive GA101, which has entered a large clinical trial proposed sequence of events in the Lauren Sidon measurable in the serum. Furthermore, our data challenge. In contrast, cells dying after RT and immune response. This is a translational research development programme. We have discovered proposed cell death pathway evoked by type II anti-CD20 and anti-HLA demonstrate that combination therapy with Adriamycin treatment were capable of inducing BSc Pathology Students programme where the aim is to take our that on binding the CD20 antigen GA101 DR mAb. mAb ligation results in HA EBRT and weekly systemic dosing of R848 leads a protective immune response in a prophylactic Charlotte Pollard experimental research findings into the clinic and initiates large amounts of non-apoptotic PCD in and actin reorganisation followed by to the induction of a tumour-specific CD8+ T tumour vaccination approach, but not in a where the laboratory research is influenced by a range of B-lymphoma cell lines and primary B- lysosomal membrane cell response capable of increasing the survival of therapeutic setting when treating established clinical insights and developments. cell malignancies. Our recent studies performed permeabilisation, release of mice bearing established tumours. Importantly, a tumours. This work suggests that there are by Jamie Honeychurch and Waleed Alduaij have cathepsins and generation of ROS via NAPDH oxidase which ultimately proportion of mice treated with this novel important differences between tumour cells This last year has been highly successful for the demonstrated that the induction of PCD by a culminates in non-apoptotic cell therapeutic combination are able to completely dying after RT and Adriamycin compared to Targeted Therapy Group and the highlights have range of anti B cell mAbs against HLA DR death. reject the primary tumour and are protected classical apoptotic cells. It appears that the included both Waleed Alduaij and Monique Melis antigens and the type II anti-CD20 mAb such as supposedly immunogenic apoptotic cell death being awarded PhD degrees. Waleed Alduaij was GA101 (obinutuzumab), directly correlates with induced by RT for example, is on its own awarded the highly prestigious Royal Society of their ability to produce reactive oxygen species insufficient to generate an immune response Medicine Oncology Section - Sylvia Lawler Prize (ROS) in human B-lymphoma cell lines and in a therapeutic setting. Finally, this study open to all clinicians and scientists in training for primary B-cell chronic lymphocytic leukemia demonstrates that the addition of the best PhD project in oncology. Both Waleed cells, and that inhibition of ROS using various immunotherapy such as immunostimulatory anti- and Monique gave oral presentations at the ROS scavengers blocks cell death. ROS CD40 monoclonal antibody may enhance the 2011 Keystone cancer meeting. Oral generation is also independent of Bcl2 or therapeutic response towards dying tumour cells. presentations have also been given at blockade of caspase activity confirming that ROS international leading research meetings including production (and subsequent cell death) is In summary, the observations are important for ASCO, ASTRO and ASH the UK National apoptosis-independent. Moreover, ROS were the understanding of cell death induced immune Cancer Research Institute meeting as well as generated downstream of mAb-induced actin responses, which suggest that the generation some notable high impact publications. cytoskeletal reorganisation and lysosome of a successful host immune response towards membrane permeabilisation as confirmed tumour cell death is dependent on several Novel Mechanisms of antibody induced through use of specific pharmacological factors, including the amount of cell death cell death inhibitors. induced, the immunogenicity of the cell death We recently demonstrated that certain mAbs and the tumour micro-environment. (type II anti-CD20 and anti-HLA DR mAbs) Through a collaboration with Professor Peng potently evoked a direct Programmed Cell Huang and Helene Pelicano at the MD Death (PCD) pathway through an actin- Anderson, University of Texas we have been able Publications listed on page 74 54 | Paterson Institute for Cancer Research Scientific Report 2011 Targeted Therapy Group | 55

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    Translational Radiobiology Group analyses showed that the presence of tumour necrosis was a significant independent predictor of benefit from CON with the risk of dying 57% lower compared to RT alone (HR 0.43, 95% CI 0.25-0.73, p=0.002). This trend was not observed when there was no evident tumour necrosis (HR 1.64, 95% CI 0.95-2.85, p=0.08). The work has shown that simple pathology reporting of tumour necrosis predicts which bladder cancer patients are likely to benefit from hypoxia- modifying treatment in combination with 333 patients enrolled in the BCON phase III radiotherapy. Ongoing work is comparing the Figure 1 The goals of the Translational Radiobiology Group are to a: Graph showing expression of TP63 randomised trial comparing radical radiotherapy predictive ability of necrosis with other measures (RT) with and without carbogen and of hypoxia including our gene signature. derive and validate molecular profiles that predict a cancer probeset data from Affymetrix Exon arrays. Un-degraded cell line RNA nicotinamide (CON). Tumour necrosis was patient’s response to radiotherapy. We aim to develop discriminates between SCC and AC samples. The difference was seen in scored on whole tissue sections as absent or Normal tissue radiosensitivity present. Necrosis was identified in 121 of the RAPPER (Radiogenomics: Assessment of signatures associated with tumour radiosensitivity, tumour young (median age 11.5±1.7 years) 231 patients (52%). For the subset of BCON Polymorphisms to Predict the Effects of but not old (median age 17.5±1.2 hypoxia and normal tissue radiosensitivity. In order to facilitate years) FFPE blocks. b: Graph showing patients for whom tissue material was available, Radiotherapy) and our long-running hsa-miR-205 (similar specificity to 5-year overall survival estimates were 41% for collaboration with researchers at Cambridge signature derivation, associated research investigates TP63) expression measured using the RT arm and 48% for the CON arm (log University has now yielded results for a genome Group Leader approaches for unlocking information contained in clinical TaqMan qPCR on the same samples. hsa-miR-205 expression rank p=0.14). When stratified using tumour wide association study (GWAS) of the genetic necrosis, the 5-year overall survival rates were variation underlying a patient’s risk of toxicity Catharine M.L. samples. discriminates between SCC and AC, regardless of the age of the block. 48% (RT) and 39% (CON) (log rank p =0.32) in following radiotherapy. The Stage 1 GWAS patients with no evident tumour necrosis and involved 2.4x106 genotyped and imputed SNPs West in 1,853 patients who underwent radiotherapy Some tumours respond well to radiotherapy, samples failed to discriminate between AC and 34% (RT) and 56% (CON) (log rank p=0.004) Postdoctoral Fellows whereas others do not. The underlying biology SCC, whether using our gene signature or a in patients with tumour necrosis. Multivariate for breast (n=1,217) or prostate (n=636) cancer. John Hall that accounts for differences in response to single well-characterised gene (Figure 1a). This The quantile-quantile (Q-Q) plots showed no Amanda Williamson obvious bias due to population stratification or radiotherapy is poorly understood. There is may explain why most microarrayed FFPE evidence that intrinsic sensitivity to radiation, cohorts in the literature are generally <10 years Figure 2 genotype error. No single nucleotide Bioinformatician Janet Taylor (joint with Applied hypoxia and proliferation are important. old. As such, the majority of archived FFPE Tumour necrosis predicts benefit from polymorphisms (SNPs) were identified as being Computational Biology & specimens, including those with the longest hypoxia modifying therapy in patients associated with an overall measure of late (2 Bioinformatics) with bladder cancer enrolled in the years) radiotherapy toxicity in all 1,853 patients. Profiling of archival formalin fixed paraffin clinical follow-up, remain incompatible with high- UK phase III BCON trial. 2a: A embedded tumours throughput technologies. MicroRNAs are A number of potential associations were found Clinical Fellows bladder tumour with a large area of There are estimated to be almost one billion thought to be more stable in FFPE blocks. We necrosis (pale pink). 2b: In 110 with tissue specific toxicity at P-values <10-7 with Jonathan Bernstein Guy Betts tumour samples archived world-wide, the reasoned that our cohort would be an ideal patients with no tumour necrosis, more SNPs associated with toxicity in prostate Navin Mani majority formalin-fixed paraffin-embedded series to investigate whether miRNA stability addition of carbogen and than in breast patients. We are conducting a Ahmad Mirza (until July) nicotinamide (CON) to radiotherapy rapid replication of the top 90 SNPs in 463 (FFPE). A wealth of biological information is overcomes near total mRNA degradation (RT) did not improve overall survival. associated with these samples, but degradation associated with prolonged storage. TaqMan real- prostate samples from a group in Spain and a Scientific Officers 2c: In 121 patients with tumour inherent to the processes of fixation and time qPCR for hsa-miR-205, a microRNA necrosis, the hypoxia-modifying further ~500 cases from the USA. Some SNPs Joely Irlam-Jones Helen Valentine embedding results in fragmented and modified expressed in SCC in a similar manner to TP63 therapy improved survival. apparently replicate but caution is required since RNA that is poorly compatible with modern discriminates between AC and SCC, regardless they have lower minor allele frequencies than MRes Students high-throughput RNA techniques. We showed of sample age (Figure 1b). This finding the study was designed to detect and so might Rohan Iype (until July) represent false positives. Kenneth Oguejiofor (from previously that FFPE tumour samples in demonstrates that mature miRNAs are less September) conjunction with Exon array profiling can be sensitive to the degradation incurred by mRNA Junaid Iqbal Wahid (from used to derive a gene signature in FFPE, without and should be a preference in the profiling of September) the requirement for matched fresh-frozen older FFPE blocks. Ongoing work confirmed the Publications listed on page 74 Scientific Administrator samples. The signature was applied to an result using Affymetrix miRNA v2 arrays, which Rebecca Elliott independent fresh-frozen cohort and successfully have complete miRBase coverage and are ideal stratified histologically distinct subtypes: for miRNA profiling in FFPE samples. Future adenocarcinoma (AC) and squamous cell work aims to exploit this new knowledge to carcinoma (SCC) (Hall et al., 2011, Br J Cancer, derive a miRNA signature associated with 104:971-81). RNA degradation in FFPE blocks is tumour radiosensitivity. not a single event, but a continuum of events that persist beyond the processing of the blocks. Presence of tumour necrosis predicts benefit Profiling of >150 human tumour samples from hypoxia modifying therapy revealed that the age of the FFPE block at RNA Over the past year we have investigated the extraction correlated inversely with the technical ability of tumour necrosis to predict benefit success of the Exon arrays (R2= -0.72; age vs. following radiotherapy with and without percentage detection above background hypoxia-modifying treatment in high grade [DABG]). Biological signals decrease with urothelial cell carcinoma of the urinary bladder. increasing sample age. In our cohort, older Tissue samples were obtained for 231 of the 56 | Paterson Institute for Cancer Research Scientific Report 2011 Translational Radiobiology Group | 57

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    Research Services http://www.paterson.man.ac.uk/research Over the last few years the Institute’s recruitment programme has been very successful. For the Research Service units, the corresponding increase in scientific activity has meant that the demand for scientific services has steadily increased to a point where service units have needed to expand again to match the needs of users. In the case of the Biological Resources Head of Research Services Unit, this has meant an ambitious expansion into new space biochemical processes. This background • Purchase of a second system via a joint Stuart Pepper made available at the University, whereas for Laboratory fluorescence is often the limiting factor in our venture between Breakthrough Breast Cancer studies when trying to discern fluorescent labels and Cancer Research UK. This system is faster Services the expansion has taken the form of an extra post attached to a process of interest. It has been than the original and allows for batches of 384 and extra equipment. Mass Spectrometry has acquired a new estimated that auto-fluorescence can be slides to be imaged so to allow greater equivalent to 34,000 fluorescent molecules per throughput of data. ultra-high pressure nano LC system which, along with internal cell, sources of which include endogenous development work, is allowing an increased throughput of NAHD, flavins and flavoproteins. • The original system has been redeveloped to allow fluorescence imaging, with the aim to be samples through the existing equipment. A macro confocal has been acquired and able to image up to six fluorescent tagged developed for the visualisation of large areas of proteins simultaneously. This will make possible tissue. This is in contrast to a conventional the development of providing biochemical Along with the expansion of existing services we have also microscope where only portions of the total signatures of biopsy material. been able to procure equipment that has allowed new tissue can be imaged. The confocal properties allow a three dimensional volume of tissue to be Generally image analysis of histological data has developments. The Advanced Imaging Facility (AIF) acquired a rendered and the localisation of multiple not been as well developed as fluorescent macro confocal imaging system as well as a new histology processes studied. Rather than using a camera, imaging mainly due to the complex nature of the this system captures data via a data, the lack of viable software tools and the scanning platform. The histology service has benefitted from a spectrophotometer, which permits the exclusion size of the data (each scanned image is 12 x 25 new Bondmax immuno-histochemistry (IHC) platform and the of auto-fluorescence consequently increasing metres in size). Analysis had been carried out contrast and clarity. Although these techniques with software developed in the microscopy Molecular Biology Core Facility (MBCF) had a new 5500 clonal are still in development the system has already community but validation of these techniques sequencer installed. The following sections give further details enabled us to examine patient material that has been somewhat lacking thus calling into previously has been difficult to study. question the final result. The facility has of these developments along with a variety of other technical purchased a fully validated software solution, advances that have taken place over the last year. Histology Scanning which can describe the data with up to 130 Over the last four years a new development for different measures and then relate those the facility has been integrating automated measures to each other so to numerically Advanced Imaging Facility microscopes, taking the total up to 161 histology scanning into the laboratory so that describe, and with the goal of developing Steve Bagley, Achille Dunne individuals, working with 28 research groups tissue on glass slides could be scanned in batches methods to mathematically model, the data. and generating 21 terabytes of data. in a tightly controlled manner. At the time, the The remit of the Imaging Facility is to provide techniques were rudimentary and the Induction of DNA Damage Microscopy state of the art imaging tools for the visualisation Over the past year the facility has brought in technology in its infancy, however the system Working in conjunction with the requirements of of cancer cells (from molecular interactions new equipment, redeveloped older equipment quickly became became over-subscribed as the the DNA Damage Response Group (Ivan Ahel) through to tissue- wide responses), develop new and introduced a range of novel techniques, process of using immuno-histochemically labelled a system was developed three years ago for cell imaging methods and to train scientists to apply which are detailed below. tissues was accelerated. The facility has based assays of the mammalian DNA damage these to their research. The facility is one that collaborated with the Institute’s Histology service response pathway using a spinning disk confocal responds to the requirements of the researchers Macro Confocal Imaging with in order to fine tune the process of sample microscope. An additional laser induced DNA and develops solutions to aid in providing visual Spectrophotometric Detection preparation for scanning and numerical analysis. damage to a sample which could be strictly and numerical clues for both fundamental and When imaging primary tissue from patients, Recently two developments have improved how regulated so that monitoring of the repair translational oncology. Over the last year, 39 new endogenous fluorescence (auto-fluorescence) histological research can now be carried out processes was possible. Recently this technology users have been trained in the use of from the sample itself masks our ability to study within the Institute: has been modified in order to produce a laser 58 | Paterson Institute for Cancer Research Scientific Report 2011 Research Services | 59

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    line that approaches the diffraction limit for fine mapping of sites of Sumoylation. This research opportunity to respond; we are now awaiting Institute facility. We will no longer have the control of DNA damage induction. Work is on- and development project has lead to Institute the summary from the Home Office which will requirement for a quarantine area at the going into 2012 to develop the system further scientists having access to a unique biochemical be used to formulate the necessary changes to Paterson site as all aspects of this work will so to simultaneously study multiple proteins at and mass spectrometry workflow able to deliver the UK legislation 1st Jan 2013. be carried out at the University sharing their the site of DNA repair. biological answers previously beyond the field. re-derivation area. This approach has enabled the Cell Cycle group Transgenic Services Installation of Local Data Servers to answer sumo-specific questions about their This year has seen a slight reduction in demand Experimental Services The analysis of microscopy data is problematic target of interest. with five embryonic stem cell (ES cell) The experimental area has continued to support due to the size of the data and the amount of microinjections and two constructs for all aspects of the in vivo requirements for the data generated within the laboratory. As the During 2011, the demand for large scale global pronuclear microinjections performed. The Institute’s twelve scientific project licences; this number of techniques has increased to protein profiling approaches has increased pronuclear microinjection experiments have has involved surgical and non surgical techniques, automatically scan and assess data so has our significantly. These analyses are demanding due produced over 230 implanted embryos for each Home Office record keeping and full daily health requirement for temporary storage. This year to the massive complexity of protein mixtures line with an expression rate of 13-17% and welfare checks. saw the introduction of 32TB of local temporary that represent a typical sample. The facility has transgenic in live young. network-attached storage to hold microscopy supported this demand by strengthening The blood analyser in the facility had been data and to allow space for image analysis. workflows in both online nano LC separations Eight new transgenic lines have also been heavily used for the last twenty years and had and offline HPLC pre-fractionation. Both new successfully imported via surgical embryo reached the end of its economic life so earlier approaches are designed to maximise the transfer whilst one cryopreserved line has this year a new F-820 Sysmex analyser was Biological Mass Spectrometry Facility capability of our existing LTQ-Orbitrap. In been exported to Amsterdam. purchased, the previous equipment only allowed Duncan Smith, Yvonne Connolly, John Griffiths August 2011, the Institute invested in the next us to carry out total blood counts whereas the generation of ultra-high pressure nano LC Cryopreservation has continued with new machine also allows us to complete The role of this facility is to enable PICR systems. This system enables higher resolution approximately 5,000 embryos/ 13 strains differential and reticulocyte analysis. This has scientists to access cutting-edge proteomic separations ‘off the shelf ’ with the use of higher being preserved this year and the sperm been a useful tool and since it was installed it workflows to enhance their research capabilities. efficiency nanocolumns. This commercial cryopreservation developments continue to has been used to track leukaemia development. The facility spans areas of activity from routine configuration involves shifting from conventional be implemented enhancing sperm motility service provision, project design and data 15cm long to 50cm long columns that increases post thawing. The experimental area of the animal facility will interpretation through to the bespoke resolution by a factor of approximately three- also be expanded over a period of time as the collaborative development of novel workflows fold. This delivers significant improvements in the New developments are also being introduced transgenic area becomes vacant, this will allow us designed to answer previously intractable number of peptide identifications from a for in-vitro Fertilisation (IVF) where increased to increase the procedural activity that currently biological questions. We have a constantly complex sample. Furthermore, we have rates of fertilisation are beginning to be realised has 960 individually ventilated cages but with an evolving portfolio of capabilities to facilitate the demonstrated that separations on a 1.5M long even for the more difficult strains. Continual acquired 2300 extra cages we will be able to qualitative and quantitative characterisation of nano column delivers performance way beyond improvements with the sperm cryopreservation accommodate greater throughput. proteins and their post-translational the commercial product’s capacity and pushes and IVF will ensure that this will offer a modifications (PTMs). complex mixture analysis to the very limits of viable addition to embryo cryopreservation our hardware’s capabilities. longer term. Cancer Research UK GeneChip Microarray Our current portfolio of PTM analyses has been Service boosted with the successful development and In addition, the facility has developed a novel At the end of 2010 we had identified that there Stuart Pepper, Gill Newton, Julie Watson, implementation of site mapping of nitrosylated peptide fractionation workflow exploiting the was a real need to expand the existing BRU Jodie Whitaker tyrosine, di-methylated lysine and tri-methylated unique physiochemical properties of facility at the Institute. Due to the scientific lysine. In addition, the facility has developed a polygraphitic carbon designed to reduce the research need, a favourable agreement between The microarray service based in the PICR completely novel approach to the high efficiency complexity of samples prior to online analysis the PICR, Manchester Cancer Research Centre Molecular Biology Core Facility has now been (Griffiths et al, J.Chrom, in press). The second and the University of Manchester led to the running for a decade, and is still heavily used by stage of development of this project, designed to offer of additional space located at the University groups across the whole of CR-UK. The service address issues of phosphopeptide retention and giving us the additional space required to has been used by around 200 CR-UK scientists fractionation, is now well underway and should provide high quality and high health status and the total number of projects has now deliver novel capabilities to phosphoproteome transgenic animals. passed 500, with 54 new projects submitted analysis to PICR groups in 2012. this year. In early summer, an extensive programme of refurbishment works began at the newly For several years the facility has been supporting Biological Resources Unit acquired area to accommodate in the region of expression profiling of archival paraffin 5,600 newly purchased, individually ventilated embedded samples. This approach presents The Paterson Institute animal facility has run at cages for transgenic activity and associated problems, both in extracting the RNA for full capacity throughout 2011 with a steady techniques for the microinjection service. In this microarray analysis and in analyzing the data as throughput for both the transgenic and scheme of building works there was also the the data quality is generally not as good as experimental areas. We currently have over 150 need to update the cage wash area with new obtained with standard RNA extractions. This lines in production and these have been sterilising equipment to support the day to day year, we collaborated with Catharine West’s maintained under the centralised service project husbandry. As the year draws to a close, the area group and demonstrated the power of licence with animals transferring to the required has just completed Home Office trials and will expression profiling on archival samples by scientific project licence for experimental use. soon be operational. classifying cervical cancers. In this case, exon arrays were used to generate the most detailed The public consultation on the UK In early 2012 the existing transgenic lines will expression analysis of archival samples published implementation of Directive 2010/63EU closed move to the new area and over a period of time to date. on the 5th September and this gave us the all lines will be decanted out of the existing 60 | Paterson Institute for Cancer Research Scientific Report 2011 Research Services | 61

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    The service at PICR is based on the Affymetrix at low levels is impressive; given high quality cell replacement for the BD FACS Vantage. As part compliment each other, offering high throughput platform, however there are now two other preparations and reagents, as few as 50 of our constant internal annual review of our routine, troubleshooting and antibody validation companies that offer a solution for archival molecules per cell may be detected. The Core service we have decided to retire the BD FACS service availability together with unrivalled profiling. This year we have set up a collaboration currently has four bench top cytometers Vantage after 18 years solid service. The Jazz is standardisation and reproducibility. The majority with James Hadfield at the CR-UK Cambridge including one plate based bead reader. These are the first of its kind, a small form sorter capable of the research groups have made use of this Research Institute in order to carry out a all user-operated systems which we offer basic of two-way sorting of eight parameters and is service, with numerous antibodies successfully platform comparison between Affymetrix, training in a group setting which is supplemented based on the already proven InFlux platform. The optimised. IHC and its use in the assessment of Illumina and Agilent arrays so that we can be with one to one training for specific applications. ethos of this sorter is to remove the complexity tissue quality during acquisition and the sure that CR-UK scientists will be using the best found in modern day high end sorters and to subsequent stabilisation of tissue are areas we available platform for this approach. FACS Calibur - 3 colour single laser (blue) provide a very basic but pure system capable of are also currently investigating. FACS Calibur - 4 colours dual laser being operated by all. The Jazz is a dual laser There has been a new development to the (blue and red) two-way sorter capable of measuring six specific Tissue microarrays enable the high throughput service this year with the addition of microRNA colours and is a welcome addition to our fleet of standardised analysis of a large number of tissue profiling using the Affymetrix platform to provide FACS Array - 4 colours, dual laser sorters. We have been really pleased by the size, samples. Both the ATA27 and MTA1 tissue a simple, cost effective alternative to using (green and red) accuracy, modularity, design, speed and purity of microarray platforms have been heavily used this RNAseq. Expression profiling of miRNA from LSRII - 17 colours, quadruple laser the system. The stability and simplicity of this year. TMAs from disease groups including breast, archival samples has been described in the (UV, violet, blue and red) system has allowed us to implement this melanoma, prostate (cores and chips), bladder, literature though as yet this is not a routine machine as our fluorescent protein sorter freeing lymphoid, small cell and non small cell lung application on clonal sequence platforms. Cell sorters time on the high end sorters for our more cancer have all been constructed incorporating One of the properties of the larger flow complex procedures. well in excess of 2000 donor blocks. In addition, Over the last few years the service has cytometers is the ability to electronically deflect mouse model and cell pellet control microarrays supported numerous projects using the linear cells with preset, defined properties into a In addition to training 47 new flow cytometry have been constructed. Construction of the amplification system from NuGEN Inc. This has separate collection tube. For cell purification, users and providing ongoing advice, further prostate core TMA and ensuring true been very successful for samples in the 0.5 to flow cytometry is especially well suited for training and guidance on experimental design to representation of the disease was particularly 1.0ng range but has been limited to samples applications requiring high purity. Because the 200+ active users, we have presented and challenging. The quality of all the TMAs where there is sufficient material to extract and multiple fluorochromes (e.g. up to fourteen demonstrated to more than 200 members of constructed has been exceptional. purify the RNA through a column. This year we distinct fluorescent probes reacting with different the public on the uses of flow cytometry and its have used alternate amplification systems that cell associated molecules) can be assessed impact on the field of oncology. The unit also houses the MCRC Biobank which have allowed us to work with pools of twenty simultaneously, cell sorting by flow cytometry can has now been up and running for more than cells obtained by FACS and generate good separate complex mixtures of cells on the basis three years and to date, around 2500 tissue quality expression data. We are currently of multiple marker expression. The sorting suite Histology samples have been processed for use in developing the service to be able to support currently houses three cell sorters which are Garry Ashton, Caron Abbey, Michelle subsequent research with 40 projects approved. single cell profiling and hope to have this able to retrieve up to four specifically defined Greenhalgh (MCRC, Tissue Biobank) Blood and bone marrow from haematological operational during 2012. populations so that cells may be recovered for David Millard (Histology /Tissue Biobank), malignancy patients is also processed and stored. further study including re-culture, RNA or DNA Deepti Wilks (Haematological Malignancy One of our key aims is to focus on ensuring extraction or use in functional cell assays. The cell Biobank) sample quality. Samples are also being processed Flow Cytometry Facility sorters are operated solely by the Flow and microtomy performed for Cancer Research Morgan Blaylock, Michael Hughes, Jeff Barry Cytometry team on a daily basis: The histology unit has seen exceptional demand UK’s Stratified Medicine Programme (SMP), a during 2011 in all areas of the services offered. national pilot study involving the collection, The Cytometry Core Facility at the Paterson BD FACS Jazz – 2 way sorting 6 colours, dual The recruitment and retention of existing staff processing and genetic testing of tissue from six provides state-of-the-art instrumentation, laser (blue and red) has allowed for the continued development of cancer types. The ultimate aim of the programme education and expert technical assistance to the unit. With the introduction of new is to link genetic data with clinical data and to BD FACS Aria II u - 4 way sorting 12 colours, investigators for the successful performance of equipment all samples are now 2D bar-coded. triple laser (violet, blue and red) flow cytometry based studies. The goal of the Generally both frozen and fixed / processed facility is both to support current research BD InFlux - 4 way sorting, 14 colours, equipped samples of mouse, human and cell lines are applications and to continuously extend the with 5 lasers (UV, violet, blue, red and orange) routinely prepared for both H&E morphological repertoire of flow cytometric methods available, analysis and for further downstream analysis. In providing the tools to help our researchers Other services addition, special stains such as Alcian blue, understand, treat and prevent cancer in its Our facility offers a full range of educational and Gordon and Sweets, MSB and Oil Red O have many forms. cytometric services. We are able to provide all been used to support current research advice on a wide variety of cytometry related projects. A more specific example being the use The use of flow cytometry in the Paterson subjects including experimental design, selection of Pearls and Turnbulls blue special stains to Institute can broadly be divided into two broad of reagents, data analysis, presentation, investigate the proposed hypothesis that the categories; analytical cytometry and cell sorting. interpretation, we also act as a beta test site for inhibition of UROD (uroporphyrinogen novel cytometry equipment and applications, we decarboxylase), a radio-sensitising target in head Analytical Cytometry also advise on data presentation. The latter is and neck cancer results in an increase in ferrous The ability of flow cytometers to evaluate cells becoming more and more important as journals Fe2+ iron which upon radiation is converted to at an extremely rapid rate (e.g. up to 20,000 require cytometric data to be more transparent. Fe3+ which subsequently enhances radiation events per second) makes this technology ideally induced cell death. suited for the reliable and accurate quantitative Technical developments analysis of selected physical properties of cells of This year we were fortunate to be the first in This year saw the introduction of a Leica interest. The sensitivity of these instruments for the world to acquire the new and so far Bondmax immunohistochenistry (IHC) platform detecting the presence of molecules expressed unreleased BD FACS Jazz™ sorter as a direct which together with the existing Biogenix i6000 62 | Paterson Institute for Cancer Research Scientific Report 2011 Research Services | 63

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    provide information at the genetic level which The Lab Services department continues to are in charge of replacement and ordering as profiling generally known as RNAseq. This may identify patients at risk of specific disease or provide three main roles within the Institute: and when is necessary. The department works approach relies on generating very large most likely to benefit from a particular therapy. closely with all groups and helps out where numbers of sequence reads from cDNA libraries The pilot study will test the logistics of the • Washing and sterilizing of glassware followed necessary, be it tracing and confirming delivery and then using the relative frequency of reads approach and determine whether this is by re-stocking of lab supplies of goods with suppliers, and dealing with missing, that map to each transcript to estimate relative feasible and reproducible on a larger scale at • Production of sterile pipette tips and damaged or wrong items. We also assist or abundance. This application is very demanding in the national level. Eppendorfs manage the moving of heavy equipment or that large numbers of reads are required to give • Production of sterile water and PBS furniture, and the setting up of various meeting coverage of the whole transcriptome. On the In addition there has been some exciting work in rooms for numerous events. new machine we are able to generate other areas. A technique which allows both the The department’s autoclaves are also available approximately one billion reads per slide, extraction of high quality RNA and protein from to sterilize material sent up from labs and we compared to approximately 0.3 billion reads the same tissue sample in addition to allowing supply the Institute with its requirements for Molecular Biology Core Facility on the older system. the morphology of the sample to be reported liquid media and agar plates. We can produce Stuart Pepper, Chris Clark, Yvonne Hey, Rheanna has been developed. The use of the proximity over 1000 litres of liquid media a month and Makorie, Julie Watson There are two approaches to RNAseq; the most ligand assay for the detection and quantification liaise with labs in producing new media types common approach is to use a polyA selection to of proteins, protein interactions and as required. The Molecular Biology Core Facility has a team enrich for RNA molecules that code for modifications in fixed cells and tissue samples of six people who between them support a proteins. This simplifies the complexity of the continues to be evaluated. Lab Services provides each lab with a broad range of services and equipment, including sample being analysed but still yields more Laboratory Aide to perform a range of the CR-UK microarray service (described complete data than microarrays as the entire In addition, an upgrade to the laser capture housekeeping duties tailored to the specific separately). The regular sample processing coding sequence of a transcript is interrogated. microdissection system (LMD6000) with needs of the lab. These duties can also include services have all been busy this year with the Microarray platforms generally have either a fluorescent capabilities is imminent. This together equipment monitoring such as Safety Cabinet team processing 13,350 samples for plasmid single, or a fairly small number of probes to with our recent work on improving sample Airflows. DNA extraction and over 32,500 samples for assay each transcript. An alternate approach to acquisition and ultimately quality using the DNA sequencing. These services continue to RNAseq is to sequence all the RNA from a system will prove very useful over the next year. A new role taken on this year is the offer a good turnaround such that plasmid DNA sample without enrichment. The major problem maintenance of the Institute’s two X-ray can be prepared and then sequenced on the with this approach is that a large percentage of film processors. same day. RNA in most cells is ribosomal RNA, and so Laboratory Services >95% of all reads can end up mapping to Mark Craven, Andy Burns, Tony Dawson, Over the last two years the facility has also ribosomal genes which in most cases are not of Corinne Hand, John Higgins, Frances Hockin, Logistics offered a new cell line authentication service major biological interest. This problem has to Amy Moloney, Christine Whitehurst Maurice Cowell, Sedia Fofana, Antony Griffin, based on STR profiling. For this service we have some extent been countered by using Phil Jackson, Andrew Lloyd, Jonathan Lloyd constructed a database of STR profiles for many techniques to remove ribosomal sequences Following an internal review during 2011, the commonly used cell lines so that for most cell though it is still the case that 25-40% of samples department has undergone some changes; we The Logistics facility provides a comprehensive lines we are now able to give a positive can end up mapping to ribosomal genes. This have taken on an additional post and are in the and vital role in supporting the research carried identification. Over the last year we have year some new products have been released process of adding an additional glass washer and out at the Institute. Duties include the accurate processed 60 cell lines samples spread across that have significantly improved the removal of replacing an older autoclave. receipting, checking, booking in and efficient several research groups. The STR profiling service ribosomal sequences and we are now seeing less distribution of goods ordered by numerous is one of the diagnostic services on offer; we than 5% of reads mapping to ribosomal genes. personnel in the Institute as well as the also run monthly Mycoplasma screening to check This improvement, coupled with the increase in collection and removal of waste. They are also the status of cell lines in use in the building, last the number of reads generated per run, is accountable for the collection and refilling of year we processed nearly 1400 samples with, making total RNA sequencing much more cost liquid nitrogen containers and delivery of dry ice. thankfully, very few positives being detected. effective. Over the last few years it has become Ordering and distribution of the Central Stores clear that the traditional description of RNA into stock via the intranet E-mail “Order Stock Items” Alongside the diagnostic services the core facility three classes (ribosomal- transfer- and function is also our responsibility and it is our offers more technical support for applications messenger- RNA) has missed many classes of duty to ensure adequate stock levels are based on quantitative PCR. We have two ABI RNA, such as miRNA or snoRNA, which are maintained at all times. Included in this is the 7900 machines and an Eppendorf epMotion now being analysed in detail. During the next media and enzymes stored in the Institute robot to allow work in 384 well format. During few years RNAseq will be a valuable technique freezers in Phase 5 (Sigma, Invitrogen, Roche, the last year we have had sixty one people both to analyse in detail the RNA species that Promega, New England Biolabs, Fisher kits and using this machine requiring support that varies we know about, and possibly discover new Qiagen), again the Logistics department is from minimal training on the machine to full categories that have not yet been recognised. responsible for the ordering, distribution and training on assay design, machine operation stock levels of these items. and data analysis. At the beginning of the year, the Promega The biggest development area this year has been freezer went onto a swipe card system, where in our clonal sequencing service. During July and once user cards had been set up they are able August we had a new AB 5500 system installed to access the freezer via their swipe card and and this instrument has already proved to be a take the product they require which then is major improvement on previous machines. One automatically replenished by Promega the major application of interest to the Institute is following week. The Institutes usage of gas the use of clonal sequencing for expression cylinders is looked after by the porters who 64 | Paterson Institute for Cancer Research Scientific Report 2011 Research Services | 65

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    Research publications Crispin Miller (page 14) A phase I study of the safety and tolerability of Karim Labib (page 18) Nic Jones (page 22) Applied Computational Biology and olaparib (AZD2281, KU0059436) and dacarbazine Cell Cycle Group Cell Regulation Group Bioinformatics Group in patients with advanced solid tumours. Br J Cancer, 104, 750 5. Other Publications Refereed Research Papers Refereed Research Papers Labib, K. (2011) Ackermann, J., Ashton, G., Lyons, S., James, D., Bitton, D. A., Wood, V., Scutt, P. J., Grallert, A., Yates, Lopez, S., Margison, G. P., McElhinney, R. S., Building a double hexamer of DNA helicase at Hornung, J. P., Jones, N. and Breitwieser, W. (2011) T., Smith, D. L., Hagan, I. M. and Miller, C. J. (2011) Cordeiro, A., McMurry, T. B. and Rozas, I. (2011) eukaryotic replication origins. EMBO J, 30, 4853-5. Loss of ATF2 function leads to cranial motoneuron Augmented annotation of the Schizosaccharomyces Towards more specific O6-methylguanine-DNA degeneration during embryonic mouse pombe genome reveals additional genes required methyltransferase (MGMT) inactivators. Bioorg Med Labib, K., and De Piccoli, G. (2011) development. PLoS One, 6, e19090. for growth and viability. Genetics, 187, 1207-17. Chem, 19, 1658-65. Surviving chromosome replication: the many roles of the S-phase checkpoint pathway. Philos Trans R Soc Di, Y., Holmes, E. J., Butt, A., Dawson, K., Mironov, Hall, J. S., Leong, H. S., Armenoult, L. S., Newton, G. Uboldi, S., Bernasconi, S., Romano, M., Marchini, S., Lond B Biol Sci, 366, 3554-61. A., Kotiadis, V. N., Gourlay, C. W., Jones, N. and E., Valentine, H. R., Irlam, J. J., Moller-Levet, C., Fuso Nerini, I., Damia, G., Ganzinelli, M., Marangon, Wilkinson, C. R. (2011) Sikand, K. A., Pepper, S. D., Miller, C. J. and West, C. E., Sala, F., Clivio, L., Chiorino, G., Digiandomenico, H2O2 stress-specific regulation of S. pombe MAPK M. (2011) S., Rocchi, M., Capozzi, O., Margison, G., Watson, A., Sty1 by mitochondrial protein phosphatase Ptc4. Caccuri, A., Pastore, A., Fossati, A., Mantovani, R., Iain Hagan (page 20) Exon-array profiling unlocks clinically and biologically Cell Division Group EMBO J, doi: 10.1038/emboj.2011.438. [Epub ahead relevant gene signature from formalin-fixed Grosso, F., Tercero, J., Erba E. and D'Incalci, M. of print]. paraffin-embedded tumour samples. Br J Cancer, (2011) Refereed Research Papers 104, 971 81. Characterization of a new trabectedin resistant Bitton, D. A., Wood, V., Scutt, P. J., Grallert, A., Yates, myxoid liposarcoma cell line that shows collateral sensitivity to methylating agents. Int J Cancer, T., Smith, D. L., Hagan, I. M. and Miller, C. J. (2011) Angeliki Malliri (page 24) Holland, M., Castro, F. V., Alexander, S., Smith, D., Augmented annotation of the Schizosaccharomyces Cell Signalling Group Liu, J., Walker, M., Bitton, D., Mulryan, K., Ashton, G., doi:10.1002/ijc.26340. [Epub ahead of print]. pombe genome reveals additional genes required Blaylock, M., Bagley, S., Connolly, Y., Bridgeman, J., for growth and viability. Genetics, 187, 1207-17. Refereed Research Papers Miller, C., Krishnan, S., Dempsey, C., Masurekar, A., Zhang, H., Xie, C., Spencer, H. J., Zuo, C., Higuchi, Daskalos, A., Oleksiewicz, U., Filia, A., Nikolaidis, G., Stern, P., Whetton, A. and Saha, V. (2011) M., Ranganathan, G., Kern, P. A., Chou, M. W., Tamm, T., Grallert, A., Grossman, E. P., Alvarez- Xinarianos, G., Gosney, J. R., Malliri, A., Field, J. K. RAC2, AEP, and ICAM1 expression are associated Huang, Q., Szczesny, B., Mitra, S., Watson, A. J., Tabares, I., Stevens, F. E. and Hagan, I. M. (2011) and Liloglou, T. (2011) with CNS disease in a mouse model of pre-B Margison G. P. and Fan, C. Y. (2011) Brr6 drives the Schizosaccharomyces pombe spindle UHRF1-mediated tumor suppressor gene childhood acute lymphoblastic leukemia. Blood, 118, Obesity and hepatosteatosis in mice with enhanced pole body nuclear envelope insertion/extrusion inactivation in nonsmall cell lung cancer. Cancer, 638-49. oxidative DNA damage processing in mitochondria. cycle. J Cell Biol, 195, 467-84. 117, 1027-1037. Am J Pathol, 178, 1715-27. Bitton, D. A., Grallert, A., Scutt, P. J., Yates, T., Li, Y., Bitton, D. A., Grallert, A., Scutt, P. J., Yates, T., Li, Y., Other Publications Bradford, J. R., Hey, Y., Pepper, S. D., Hagan, I. M. and Sabharwal, A., Waters, R., Danson, S., Clamp, A., Bradford, J. R., Hey, Y., Pepper, S. D., Hagan, I. M. and Mack, N. A., Whalley, H. J., Castillo-Lluva, S. and Miller, C. J. (2011) Lorigan, P., Thatcher, N., Margison, G.P. and Miller, C. J. (2011) Malliri, A. (2011) Programmed fluctuations in sense/antisense Middleton, M. R. (2011) Programmed fluctuations in sense/antisense The diverse roles of Rac signaling in tumorigenesis. transcript ratios drive sexual differentiation in S. Predicting the myelotoxicity of chemotherapy: the transcript ratios drive sexual differentiation in S. Cell Cycle, 10, 1571-81. pombe. Mol Syst Biol, 7, 559. use of pretreatment O6 methylguanine-DNA pombe. Mol Syst Biol 7, 559. methyltransferase determination in peripheral blood mononuclear cells. Melanoma Res, 21, 502-8. Other Publications Caroline Dive (page 26) Geoff Margison (page 16) Active Patents Pines, J., and Hagan, I. (2011) Clinical and Experimental Pharmacology Carcinogenesis Group The Renaissance or the cuckoo clock. Philos Trans R Baer, J., Freeman, A.A., Newlands, E.S., Watson, A.J., Soc Lond B Biol Sci, 366, 3625-34. Refereed Research Papers Rafferty, J.A., and Margison, G.P. Refereed Research Papers Coward, J., Kulbe, H., Chakravarty, P., Leader, D.A., Potentiation of temozolomide in human tumour Khan, O. A., Gore, M. Lorigan, P., Stone, J., Boke, E., and Hagan, I. M. (2011) Vassileva, V., Leinster, D.A., Thompson, R., Schioppa, cells. (USP: 5731304) through CR Technology Ltd. Greystoke A., Burke, W., Carmichael, J., Watson, A. Polo, Greatwall, and Protein Phosphatase PP2A T., Nemeth, J.A., Vermeulin, J., Singh, N., Avril, N.E., J., McGown, G., Thorncroft, M., Margison, G. P., Jostle for Pole Position. PLoS Genet, 7, e1002213. Cummings, J., Rexhepaj, E., Jirstrom, K., Gallagher, Califano, R., Larkin, J., Wellman, S. and Middleton, W.M., Brennan, D.J., McNeish, I.A., and Balkwill, F.R. M. R. (2011) (2011) Interleukin-6 as a therapeutic target in human ovarian cancer. Clin Cancer Res, 17(18), 6083-96. 66 | Paterson Institute for Cancer Research Scientific Report 2011 Research publications | 67

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    Coward, J., Kulbe, H., Chakravarty, P., Leader, D.A., Assessment of circulating biomarkers for potential A pilot study to explore circulating tumour cells in Smith, U., Sipido, K., Dive, C., and Nicod, L. (2011) Cummings, J., Zhou, C., and Dive, C. (2011) pharmacodynamic utility in patients with lymphoma. pancreatic cancer as a novel biomarker. Br J Cancer, Alliance for biomedical research in Europe. EMBO Application of the beta-expectation tolerance Br J Cancer, 104, 719-725. doi: 10.1038/bjc.2011.545. [Epub ahead of print]. Mol Med, 3, 505-506. interval to method validation of the M30 and M65 ELISA cell death biomarker assays. J Chromatogr B Harrison, L.R., Micha, D., Brandenburg, M., Simpson, Klymenko, T., Brandenburg, M., Morrow, C.J., Dive, Smith, U., Sipido, K., Dive, C., and Nicod, L. (2011) Analyt Technol Biomed Life Sci, 879, 887-893. K.L., Morrow, C.J., Denneny, O., Hodgkinson, C., C., and Makin, G. (2011) Medical societies unite to support research in Yunus, Z., Dempsey, C., Roberts, D., Blackhall, F., The novel Bcl-2 inhibitor ABT-737 is more effective Europe. Eur J Immunol, 41, 1187-1188. Danson, S.J., Johnson, P., Ward, T.H., Dawson, M., Makin, G., and Dive, C. (2011) in hypoxia, and is able to reverse hypoxia-induced Denneny, O., Dickinson, G., Aarons, L., Watson, A., Hypoxic human cancer cells are sensitized to BH-3 drug resistance in neuroblastoma cells. Jowle, D., Cummings, J., Robson, L., Halbert, G., mimetic-induced apoptosis via downregulation of Mol Cancer Ther, 10(12), 2373-83. Ivan Ahel (page 28) Dive, C., and Ranson, M. (2011) the Bcl-2 protein Mcl-1. J Clin Invest, 121(3), 1075-87. DNA Damage Response Group Phase I pharmacokinetic and pharmacodynamic Kraan, J., Sleijfer, S., Strijbos, M.H., Ignatiadis, M., study of the bioreductive drug RH1. Ann Hitchman, E., Hodgkinson, C., Roberts, D., Ashton, Peeters, D., Pierga, J.Y., Farace, F., Riethdorf, S., Refereed Research Papers Oncol, 22(7), 1653-60. G., Yunus, Z., Byers, R., Ward, T., Womack, C., and Fehm, T., Zorzino, L., Tibbe, A.G., Maestro, M., Chen, D., Vollmar, M., Rossi, M.N., Phillips, C., Dive, C. (2011) Gisbert-Criado, R., Denton, G., de Bono, J.S., Dive, Kraehenbuehl, R., Slade, D., Mehrotra, P.V., von Dean, E.J., Cummings, J., Roulston, A., Berger, M., Effect of prolonged formalin fixation on C., Foekens, J.A., and Gratama, J.W. (2011) Delft, F., Crosthwaite, S.K., Gileadi, O., Denu, J.M., Ranson, M., Blackhall, F., and Dive, C. (2011) immunohistochemical staining for the proliferation External quality assurance of circulating tumor cell and Ahel, I. (2011) Optimization of circulating biomarkers of obatoclax- marker Ki67. Histopathology, 59(6), 1261-3. enumeration using the CellSearch((R)) system: A Identification of macrodomain proteins as novel induced cell death in patients with small cell lung feasibility study. Cytometry B Clin Cytom, 80, 112-118. O-acetyl-ADP-ribose deacetylases. J Biol Chem, 286, cancer. Neoplasia, 13, 339-347. Holt, S.V., Brookes, K.E., Dive, C., and Makin, 13261-13271. G.W. (2011) Krebs, M.G., Sloane, R., Priest, L., Lancashire, L., Gandhi, L., Camidge, D.R., Ribeiro de Oliveira, M., Down-regulation of XIAP by AEG35156 in Hou, J.M., Greystoke, A., Ward, T.H., Ferraldeschi, Mehrotra, P.V., Ahel, D., Ryan, D.P., Weston, R., Bonomi, P., Gandara, D., Khaira, D., Hann, C.L., paediatric tumour cells induces apoptosis and R., Hughes, A., Clack, G., Ranson, M., Dive, C., and Wiechens, N., Kraehenbuehl, R., Owen-Hughes, T., McKeegan, E.M., Litvinovich, E., Hemken, P.M., Dive, sensitises cells to cytotoxic agents. Oncol Rep, 25, Blackhall, F.H. (2011) and Ahel, I. (2011) C., Enschede, S.H., Nolan, C., Chiu, Y.L., Busman, T., 1177-1181. Evaluation and Prognostic Significance of Circulating DNA repair factor APLF is a histone chaperone. Mol Xiong, H., Krivoshik, A.P., Humerickhouse, R., Tumor Cells in Patients With Non-Small-Cell Lung Cell, 41, 46-55. Shapiro, G.I., and Rudin, C.M. (2011) Hou, J.M., Krebs, M., Ward, T., Sloane, R., Priest, L., Cancer. J Clin Oncol, 29(12), 1556-63. Phase I Study of Navitoclax (ABT-263), a Novel Bcl- Hughes, A., Clack, G., Ranson, M., Blackhall, F., and Peterson, F.C., Chen, D., Lytle, B.L., Rossi, M.N., 2 Family Inhibitor, in Patients With Small-Cell Lung Dive, C. (2011) Krebs, M.G., Hou, J.M., Sloane, R., Lancashire, L., Ahel, I., Denu, J.M., and Volkman, B.F. (2011) Cancer and Other Solid Tumors. J Clin Oncol, 29, Circulating tumor cells as a window on metastasis Priest, L., Nonaka, D., Ward, T.H., Backen, A., Clack, Orphan macrodomain (human C6ORF130) is an 909-916. biology in lung cancer. Am J Pathol, 178, 989-996. G., Hughes, A., Ranson, M., Blackhall, F.H., and Dive, o-acyl-ADP-ribose deacylase: solution structure and C. (2011) catalytic properties. J Biol Chem, 286(41), 35955-65. Greystoke, A., O'Connor, J.P., Linton, K., Taylor, Khoja, L., Backen, A., Sloane, R., Menasce, L., Ryder, Analysis of Circulating Tumor Cells in Patients with M.B., Cummings, J., Ward, T., Maders, F., Hughes, A., D., Krebs, M., Board, R., Clack, G., Hughes, A., Non-small Cell Lung Cancer Using Epithelial Marker- Slade, D., Dunstan, M.S., Barkauskaite, E., Weston, Ranson, M., Illidge, T.M., Radford, J., and Dive, C. Blackhall, F., Valle, J.W., and Dive, C. (2011) Dependent and -Independent Approaches. J Thorac R., Lafite, P., Dixon, N., Ahel, M., Leys, D., and (2011) Oncol, Dec14. [Epub ahead of print]. Ahel, I. (2011) The structure and catalytic mechanism of a Lancashire, L.J., Roberts, D.L., Dive, C., and poly(ADP-ribose) glycohydrolase. Nature, 477, Renehan, A.G. (2011) 616-620. The development of composite circulating biomarker models for use in anti-cancer drug clinical Tumbale, P., Appel, C.D., Kraehenbuehl, R., development. Int J Cancer, 128, 1843-1851. Robertson, P.D., Williams, J.S., Krahn, J., Ahel, I., and Williams, R.S. (2011) Wedge, D.C., Allwood, J.W., Dunn, W., Vaughan, Structure of an aprataxin-DNA complex with A.A., Simpson, K., Brown, M., Priest, L., Blackhall, insights into AOA1 neurodegenerative disease. F.H., Whetton, A.D., Dive, C., and Goodacre, R. Nat Struct Mol Biol, 18(11), 1189-95. (2011) Is Serum or Plasma More Appropriate for Intersubject Comparisons in Metabolomic Studies? An Assessment in Patients with Small-Cell Lung Donald Ogilvie (page 30) Cancer. Anal Chem, 83(17), 6689-97. Drug Discovery Group Other Publications Refereed Research Papers Nicod, L.P., Kamel, N., Ward, B., Decramer, M., Roughley, S. D., and Jordan, A. M. (2011) Sibille, Y., Lambrecht, B., Dive, C., Smith, U., and The medicinal chemist's toolbox: an analysis of Sipido, K.R. (2011) reactions used in the pursuit of drug candidates. J ERS is founding member of a new Alliance for Med Chem, 54, 3451-79. Biomedical Research in Europe. Eur Respir J, 38, 237-238. The image shows a word cloud taken from all the abstracts of the Institute’s research output for 2011. The 150 words that appeared most often are indicated, with font size being proportional to frequency. The text has been filtered to remove the more common words. 68 | Paterson Institute for Cancer Research Scientific Report 2011 Biomolecular modelling | 69 Research publications

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    Peter Stern (page 32) Stern, P.L. and Hole, N. John Brognard (page 38) Immunology Group (1999). 5T4 antigen from human trophoblasts. Signalling Networks Group Publication information US5869053. Refereed Research Papers Refereed Research Papers Castro, F.V., Al-Muftah, M., Mulryan, K., Jiang, H.R., Ward, C.M. and Stern P.L. Brognard, J., Zhang, Y. W., Puto, L. A. and Hunter, T. Drijfhout, J.W., Ali, S., Rutkowski, A.J., Kalaitsidou, (2005) 5T4 antigen expression as an indicator of (2011) M., Gilham, D.E., and Stern, P.L. (2011) stem cell differentiation. Publication information. Cancer-associated loss-of-function mutations Regulation of autologous immunity to the mouse US2005260591. implicate DAPK3 as a tumor-suppressing kinase. 5T4 oncofoetal antigen: implications for Cancer Res, 71, 3152-61. immunotherapy. Cancer Immunol Immunother, Nov 30, [Epub ahead of print]. Nullin Divecha (page 34) Other Publications Inositide Laboratory Brognard, J., and Hunter, T. (2011) Holland, M., Castro, F.V., Alexander, S., Smith, D., Liu, Protein kinase signaling networks in cancer. Curr J., Walker, M., Bitton, D., Mulryan, K., Ashton, G., Refereed Research Papers Opin Genet Dev, 21, 4-11. Blaylock, M., Bagley, S., Connolly, Y., Bridgeman, J., Alderliesten, M.C., Klarenbeek, J.B., van der Luit, Miller, C., Krishnan, S., Dempsey, C., Masurekar, A., A.H., van Lummel, M., Jones, D.R., Zerp, S., Divecha, Active Patents/Filings: Stern, P., Whetton, A., and Saha, V. (2011) N., Verheij, M., and van Blitterswijk, W.J. (2011) Kozikowski, A.P., Dennis, P.A., Brognard, J. and RAC2, AEP, and ICAM1 expression are associated Phosphoinositide phosphatase SHIP-1 regulates Sun, H. with CNS disease in a mouse model of pre-B apoptosis induced by edelfosine, Fas ligation and Akt Inhibitors, Pharmaceutical Compositions, and childhood acute lymphoblastic leukemia. Blood, 118, DNA damage in mouse lymphoma cells. Biochem J, uses thereof. US Patent: 7,378,403, 2008. 638-649. 440(1), 127-35. (WO/2004/022569). Valerie Kouskoff (page 42) Stem Cell and Haematopoiesis Group Sawan, S., Burt, D.J., Stern, P.L., Holland, C., and Dominguez, V., Raimondi, C., Somanath, S., Bugliani, Newton A.C., Gao, T. and Brognard. J. Elkord, E. (2011) M., Loder, M.K., Edling, C.E., Divecha, N., da Silva- Compositions and Methods for Treating Diseases Refereed Research Papers Circulating Regulatory T Cells in Endometrial Xavier, G., Marselli, L., Persaud, S.J., Turner, M.D., Associated with PHLPP. US Patent Pending Boros, K., Lacaud, G., and Kouskoff, V. (2011) Cancer: A Role for Age and Menopausal Status. Rutter, G.A., Marchetti, P., Falasca, M., and Maffucci, 60/667,709, filed: March 31, 2006. Transcription factor Mxd4 controls proliferation of Immunol Invest, 40, 62-75. T. (2011) (WO/2006/105490). first blood precursors at onset of hematopoietic Class II phosphoinositide 3-kinase regulates development in vitro. Exp Hematol, 39(11), Kagermeier-Schenk B., D. Wehner, G. Özhan-Kizil, exocytosis of insulin granules in pancreatic beta cells. 1090-100. H. Yamamoto, J. Li, K. Kirchner, C. Hoffmann, P.L. J Biol Chem, 286, 4216-4225. Georges Lacaud (page 40) Stern, A. Kikuchi, A. Schambony and G. Weidinger Stem Cell Biology Group Ferreras, C., Lancrin, C., Lie, A.L.M., Kouskoff, V., (2011) Lewis, A.E., Sommer, L., Arntzen, M.O., Strahm, Y., and Lacaud, G. (2011) The transmembrane protein Waif1/5T4 inhibits Morrice, N.A., Divecha, N., and D'Santos, C.S. Refereed Research Papers Identification and characterization of a novel Wnt/b-catenin signaling and activates noncanonical (2011) Boros, K., Lacaud, G., and Kouskoff, V. (2011) transcriptional target of RUNX1/AML1 at the onset Wnt pathways by modifying LRP6 subcellular Identification of nuclear phosphatidylinositol 4,5- Transcription factor Mxd4 controls proliferation of of hematopoietic development. Blood, 118, 594-597. localisation. Developmental Cell, 21(6), 1129-43. bisphosphate-interacting proteins by neomycin first blood precursors at onset of hematopoietic extraction. Mol Cell Proteomics, 10, M110.003376. development in vitro. Exp Hematol, 39(11), 1090- Holley, R.J., Pickford, C.E., Rushton, G., Lacaud, G., Other Publications Gallagher, J.T., Kouskoff, V., and Merry, C.L.R. (2011) 100. Daayana, S., Winters, U., Stern, P. L. and Kitchener, Other Publications Influencing hematopoietic differentiation of mouse H. C. (2011) Keune, W., Bultsma, Y., Sommer, L., Jones, D., and embryonic stem cells using soluble heparin and Ferreras, C., Lancrin, C., Lie, A.L.M., Kouskoff, V., Clinical and immunological response to Divecha, N. (2011) heparan sulfate saccharides. J Biol Chem, 117, 83-87. and Lacaud, G. (2011) photodynamic therapy in the treatment of vulval Phosphoinositide signalling in the nucleus. Adv Identification and characterization of a novel intraepithelial neoplasia. Photochem Photobiol Sci, 10, Enzyme Regul, 51, 91-99. Mazzarella, L., Jorgensen, H.F., Soza-Ried, J., Terry, transcriptional target of RUNX1/AML1 at the onset 802-9. A.V., Pearson, S., Lacaud, G., Kouskoff, V., of hematopoietic development. Blood, 118, 594-597. Merkenschlager, M., and Fisher, A.G. (2011) Garcon N., Stern, P., Cunningham, T. and Stanberry, L. (2011) Tim Somervaille (page 36) Holley, R.J., Pickford, C.E., Rushton, G., Lacaud, G., ES cell-derived hemangioblasts remain epigenetically Leukaemia Biology Group Gallagher, J.T., Kouskoff, V., and Merry, C.L.R. (2011) plastic and require PRC1 to prevent neural gene Understanding modern vaccines. expression. Blood, 117, 83-87. Influencing hematopoietic differentiation of mouse Elsevier:http://www.sciencedirect.com/science/journa Refereed Research Papers embryonic stem cells using soluble heparin and l/22107622. Arnold, C.P., Tan, R., Zhou, B., Yue, S.B., Schaffert, S., heparan sulfate saccharides. J Biol Chem, 117, 83-87. Stern, P.L. (2011) Biggs, J.R., Doyonnas, R., Lo, M.C., Perry, J.M., Akira Orimo (page 44) Renault, V.M., Sacco, A., Somervaille, T., Viatour, P., Mazzarella, L., Jorgensen, H.F., Soza-Ried, J., Terry, Stromal-Tumour Interaction Group Cervarix could prevent cancer-related disease. Brunet, A., Cleary, M.L., Li, L., Sage, J., Zhang, D.E., A.V., Pearson, S., Lacaud, G., Kouskoff, V., Doctors Magazine 8th April 2011, page 30. Blau, H.M., Chen, C., and Chen, C.Z. (2011) Merkenschlager, M., and Fisher, A.G. (2011) Refereed Research Papers MicroRNA programs in normal and aberrant stem ES cell-derived hemangioblasts remain epigenetically Polanska, U. M., Acar, A. and Orimo, A. (2011) Active Patents and progenitor cells. Genome Res, 21, 798-810. plastic and require PRC1 to prevent neural gene Experimental Generation of Carcinoma-Associated Stern, P.L. and Hole, N. expression. Blood, 117, 83-87. Fibroblasts (CAFs) from Human Mammary (1989). Improvement relating to antigens. Publication information EP0336562. Fibroblasts. J Vis Exp, Oct 25;(56), pii: 3201. doi: 10.3791/3201. 70 | Paterson Institute for Cancer Research Scientific Report 2011 Research publications | 71

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    Elkord, E., Sharma, S., Burt, D.J., and Hawkins, Gordon Jayson (page 52) R.E. (2011) Medical Oncology: Translational Anti-Angiogenesis Expanded subpopulation of FoxP3(+) T regulatory Group cells in renal cell carcinoma co-express Helios, indicating they could be derived from natural but Refereed Research Papers not induced Tregs. Clin Immunol, 140, 218-22. Banerji, A., Naish, J.H., Watson, Y., Jayson, G.C., Buonaccorsi, G.A., and Parker, G.J. (2011) Gore, M.E., Hariharan, S., Porta, C., Bracarda, S., DCE-MRI model selection for investigating Hawkins, R., Bjarnason, G.A., Oudard, S., Lee, S.H., disruption of microvascular function in livers with Carteni, G., Nieto, A., Yuan, J., and Szczylik, C. metastatic disease. J Magn Reson Imaging, 35(1), (2011) 196-203. Sunitinib in metastatic renal cell carcinoma patients with brain metastases. Cancer, 117, 501-509. Goncalves, V., Jayson, G., and Tarrier, N. (2011) A longitudinal investigation of posttraumatic stress Grunwald, V., Karakiewicz, P.I., Bavbek, S.E., Miller, disorder in patients with ovarian cancer. J Psychosom K., Machiels, J.P., Lee, S.H., Larkin, J., Bono, P., Rha, Res, 70, 422-431. S.Y., Castellano, D., Blank, C.U., Knox, J.J., Hawkins, R., Anak, O., Rosamilia, M., Booth, J., Pirotta, N., and Griffiths, R.W., Zee, Y.K., Evans, S., Mitchell, C.L., Bodrogi, I. (2011) Kumaran, G.C., Welch, R.S., Jayson, G.C., Clamp, An international expanded-access programme of A.R., and Hasan, J. (2011) everolimus: Addressing safety and efficacy in patients Outcomes after multiple lines of chemotherapy for with metastatic renal cell carcinoma who progress platinum-resistant epithelial cancers of the ovary, after initial vascular endothelial growth factor peritoneum, and fallopian tube. Int J Gynecol Cancer, receptor-tyrosine kinase inhibitor therapy. Eur J 21, 58-65. Cancer, Jul 29. [Epub ahead of print] Jayson, G. (2011) Harrop, R., Shingler, W.H., McDonald, M., Treasure, VEGF inhibitors and advanced ovarian cancer. Lancet Other Publications Other Publications P., Amato, R.J., Hawkins, R.E., Kaufman, H.L., de Oncol, 12(12), 1082-3. Shimoda M., Mellody, K. and Orimo, A. (2011) Krishnan S, Masurekar, A. and Saha, V. (2011) Belin, J., Kelleher, M., Goonewardena, M., and The SDF-1-rich tumour microenvironment provides Identifying Targets for New Therapies in Children Naylor, S. (2011) Jonker, D.J., Rosen, L.S., Sawyer, M.B., de Braud, F., a niche for carcinoma cells., Chapter 13, 245-55, with Acute Lymphoblastic Leukemia. In: Saha V, MVA-5T4-induced immune responses are an early Wilding, G., Sweeney, C.J., Jayson, G.C., McArthur, “Tumor-associated fibroblasts and their matrix” edited Kearns P, editors. New Agents for the Treatment of marker of efficacy in renal cancer patients. Cancer G.A., Rustin, G., Goss, G., Kantor, J., Velasquez, L., by Norbert E. Fusenig and Margareta Mueller (ed.), Acute Lymphoblastic Leukemia. New York: Springer; p. 25-37. Immunol Immunother, 60, 829-37. Syed, S., Mokliatchouk, O., Feltquate, D.M., Kollia, Springer. G., Nuyten, D.S., and Galbraith, S. (2011) Khan, S., Burt, D.J., Ralph, C., Thistlethwaite, F.C., A phase I study to determine the safety, Kearns, P. and Saha. V. (2011) Hawkins, R.E., and Elkord, E. (2011) pharmacokinetics and pharmacodynamics of a dual Vaskar Saha (page 48) Nucleoside Analogues. In: Saha V, Kearns P, editors. Tremelimumab (anti-CTLA4) mediates immune VEGFR and FGFR inhibitor, brivanib, in patients with Children’s Cancer Group New Agents for the Treatment of Acute Lymphoblastic responses mainly by direct activation of advanced or metastatic solid tumors. Ann Oncol, 22, Leukemia. New York: Springer; 2011. p. 167-87. effector cells rather than by affecting T regulatory 1413-1419. Refereed Research Papers cells. Clin Immunol 138, 85-96. Holland, M., Castro, F.V., Alexander, S., Smith, D., Liu, Ledermann, J.A., Hackshaw, A., Kaye, S., Jayson, G., J., Walker, M., Bitton, D., Mulryan, K., Ashton, G., Robert Hawkins (page 50) Shablak, A., O'Dwyer, J., Hawkins, R., and Board, Gabra, H., McNeish, I., Earl, H., Perren, T., Gore, M., Blaylock, M., Bagley, S., Connolly, Y., Bridgeman, J., Medical Oncology: Clinical and Experimental R. (2011) Persic, M., Adams, M., James, L., Temple, G., Merger, Miller, C., Krishnan, S., Dempsey, C., Masurekar, A., Immunotherapy Group Management of a New Isolated Metastasis during M., and Rustin, G. (2011) Stern, P., Whetton, A., and Saha, V. (2011) Sunitinib Treatment in Renal Cell Carcinoma Randomized Phase II Placebo-Controlled Trial of RAC2, AEP, and ICAM1 expression are associated Refereed Research Papers Patients: A Lesson from Two Cases. Urol Int, 86, Maintenance Therapy Using the Oral Triple with CNS disease in a mouse model of pre-B Castro, F.V., Al-Muftah, M., Mulryan, K., Jiang, H.R., 245-248. Angiokinase Inhibitor BIBF 1120 After childhood acute lymphoblastic leukemia. Blood, 118, Drijfhout, J.W., Ali, S., Rutkowski, A.J., Kalaitsidou, Chemotherapy for Relapsed Ovarian Cancer. J Clin 638-649. M., Gilham, D.E., and Stern, P.L. (2011) Shablak, A., Sikand, K., Shanks, J.H., Thistlethwaite, Oncol, 29(28), 3798-804. Regulation of autologous immunity to the mouse F., Spencer-Shaw, A., and Hawkins, R.E. (2011) Offman, M.N., Krol, M., Patel, N., Krishnan, S., Liu, J., 5T4 oncofoetal antigen: implications for High-dose interleukin-2 can produce a high rate of Mitchell, C.L., O'Connor, J.P., Roberts, C., Watson, Saha, V., and Bates, P.A. (2011) immunotherapy. Cancer Immunol Immunother, Nov 30, response and durable remissions in appropriately Y., Jackson, A., Cheung, S., Evans, J., Spicer, J., Harris, Rational engineering of L-Asparaginase reveals [Epub ahead of print]. selected patients with metastatic renal cancer. J A., Kelly, C., Rudman, S., Middleton, M., Fielding, A., importance of dual activity for cancer cell toxicity. Immunother, 34, 107-112. Tessier, J., Young, H., Parker, G.J., and Jayson, G.C. Blood, 117, 1614-1621. Cheadle, E.J., Rothwell, D.G., Bridgeman, J.S., (2011) Sheard, V.E., Hawkins, R.E., and Gilham, D.E. (2011) Other Publications A two-part phase II study of cediranib in patients van Delft, F.W., Horsley, S., Colman, S., Anderson, Ligation of the CD2 co-stimulatory receptor Gilham, D.E. (2011) with advanced solid tumours: the effect of food on K., Bateman, C., Kempski, H., Zuna, J., Eckert, C., enhances IL-2 production from first-generation Effective adoptive T-cell therapy for cancer in the single-dose pharmacokinetics and an evaluation of Saha, V., Kearney, L., Ford, A., and Greaves, M. (2011) chimeric antigen receptor T cells. Gene Ther. doi: absence of host lymphodepletion. Immunotherapy 3, safety, efficacy and imaging pharmacodynamics. Clonal origins of relapse in ETV6-RUNX1 acute 10.1038/gt.2011.192. [Epub ahead of print]. 177-9. Cancer Chemother Pharmacol, 68, 631-41. lymphoblastic leukemia. Blood, 117, 6247-6254. 72 | Paterson Institute for Cancer Research Scientific Report 2011 Research publications | 73

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    O'Connor, J.P., Rose, C.J., Jackson, A., Watson, Y., Assessment of circulating biomarkers for potential Barnett, G.C., Coles, C.E., Elliott, R.M., Baynes, C., Other Publications Cheung, S., Maders, F., Whitcher, B.J., Roberts, C., pharmacodynamic utility in patients with Luccarini, C., Conroy, D., Wilkinson, J.S., Tyrer, J., West, C. M., and Barnett, G. C. (2011) Buonaccorsi, G.A., Thompson, G., Clamp, A.R., lymphoma. Br J Cancer, 104, 719-725. Misra, V., Platte, R., Gulliford, S.L., Sydes, M.R., Hall, Genetics and genomics of radiotherapy toxicity: Jayson, G.C., and Parker, G.J. (2011) E., Bentzen, S.M., Dearnaley, D.P., Burnet, N.G., towards prediction. Genome Med 3, 52. DCE-MRI biomarkers of tumour heterogeneity Harrington, K.J., Billingham, L.J., Brunner, T.B., Pharoah, P.D., Dunning, A.M., and West, C.M. (2011) predict CRC liver metastasis shrinkage following Burnet, N.G., Chan, C.S., Hoskin, P., Independent validation of genes and polymorphisms West, C (2011) bevacizumab and FOLFOX-6. Br J Cancer, 105, Mackay, R.I., Maughan, T.S., Macdougall, J., McKenna, reported to be associated with radiation toxicity: a Molecular targeting for improving the outcome of 139-145. W.G., Nutting, C.M., Oliver, A., prospective analysis study. Lancet Oncol, Epub 2011 radiotherapy. In Recent Advances Research Updates: Plummer, R., Stratford, I.J., and Illidge, T. (2011) Dec 12. Medical Physics and Radiobiology. Eds LG Marcu & E Rustin, G., Reed, N., Jayson, G.C., Ledermann, J.A., Guidelines for preclinical and early phase clinical Bezak. Researchman Publishers PVT Ltd (Kerala, Adams, M., Perren, T., Poole, C., Lind, M., Persic, M., assessment of novel radiosensitisers. Br J Cancer, Donaldson, S.B., Betts, G., Bonington, S.C., Homer, India) pp 81-90. Essapen, S., Gore, M., Calvert, H., Stredder, C., 105(5), 628-39. J.J., Slevin, N.J., Kershaw, L.E., Valentine, H., West, Wagner, A., Giurescu, M., and Kaye, S. (2011) C.M., and Buckley, D.L. (2011) Active Patents A phase II trial evaluating two schedules of Lowry, L., Smith, P., Qian, W., Falk, S., Benstead, Perfusion Estimated with Rapid Dynamic Contrast- West, C. M., Miller, C., Buffa, F., and sagopilone (ZK-EPO), a novel epothilone, in patients K., Illidge, T., Linch, D., Robinson, M., Enhanced Magnetic Resonance Imaging Correlates Harris, A. (2011) with platinum-resistant ovarian cancer. Ann Oncol, Jack, A., and Hoskin, P. (2011) Inversely with Vascular Endothelial Growth Factor WO Application No: PCT/EP2010/070583 22(11), 2411-6. Reduced dose radiotherapy for local control in Expression and Pimonidazole Staining in Head-and- Applicant: CR Technology Ltd. Hypoxia tumour non-Hodgkin lymphoma: A randomised Neck Cancer: A Pilot Study. Int J Radiat Oncol Biol markers. Zweifel, M., Jayson, G.C., Reed, N.S., Osborne, R., phase III trial. Radiother Oncol, 100, 86-92. Phys, 81(4),1176-83. Hassan, B., Ledermann, J., Shreeves, G., Poupard, L., Lu, S.P., Balkissoon, J., Chaplin, D.J., and Rustin, G.J. Naidoo, K., Clay, V., Hoyland, J.A., Swindell, R., Egelmeer, A.G., Velazquez, E.R., de Jong, J.M., (2011) Linton, K., Illidge, T., Radford, J.A., and Additional Publications Oberije, C., Geussens, Y., Nuyts, S., Kremer, B., Phase II trial of combretastatin A4 phosphate, Byers, R.J. (2011) Rietveld, D., Leemans, C.R., de Jong, M.C., Rasch, carboplatin, and paclitaxel in patients with platinum- YY1 expression predicts favourable outcome C., Hoebers, F., Homer, J., Slevin, N., West, C., and Meyer, S., Bristow, C., Wappett, M., Pepper, S., resistant ovarian cancer. Ann Oncol, 22(9), 2036-41. in follicular lymphoma. J Clin Pathol, 64, Lambin, P. (2011) Whetton, A.D., Hanenberg, H., Neitzel, H., 125-129. Development and validation of a nomogram for Wlodarski, M.W., Ebell, W., and Tonnies, H. (2011) Other Publications prediction of survival and local control in laryngeal Fanconi anemia (FA)-associated 3q gains in leukemic Hasan, J. and G. C. Jayson, G. C. (2011) Other Publications carcinoma patients treated with radiotherapy alone: transformation consistently target EVI1, but do not In “Emerging Therapeutic Targets in Ovarian Cancer”. Alduaij, W., and T. M. Illidge, T. M. (2011) a cohort study based on 994 patients. Radiother affect low TERC expression in FA. Blood, 117, Eds Kaye B, Brown R, Gabra H, Gore M. Springer The future of anti-CD20 monoclonal antibodies: are Oncol, 100, 108-115. 6047-6050. Science, New York 2011, ISBN 978 1 4419 7215 6, we making progress? Blood 117, 2993-3001. eISBN 978 1 4419 7216 3. Gee, H.E., Buffa, F.M., Camps, C., Ramachandran, A., Gilson, D., Illidge, T. and Ford, D. (2011) Leek, R., Taylor, M., Patil, M., Sheldon, H., Betts, G., Shaw, D. and G. C. Jayson, G. C. (2011) Current developments in specialty training. Clin Homer, J., West, C., Ragoussis, J., and Harris, A.L. Angiogenesis: a target for the treatment of ovarian Oncol (R Coll Radiol) 23, 431-3. (2011) cancer. Treatment Strategies Oncology 2(2), p72-78. The small-nucleolar RNAs commonly used for Illidge, T., and Morschhauser, F. (2011) microRNA normalisation correlate with tumour Radioimmunotherapy in follicular lymphoma. Best pathology and prognosis. Br J Cancer, 104, Tim Illidge (page 54) Pract Res Clin Haematol 24, 279-93. 1168-1177. Targeted Therapy Group Mayes, S., Brown, N. and Illidge, T. M. (2011) Hall, J.S., Leong, H.S., Armenoult, L.S., Newton, G.E., Refereed Research Papers New antibody drug treatments for lymphoma. Valentine, H.R., Irlam, J.J., Moller-Levet, C., Sikand, Alduaij, W., Ivanov, A., Honeychurch, J., Expert Opin Biol Ther 11(5), 623-40. K.A., Pepper, S.D., Miller, C.J., and West, C.M. Cheadle, E., Potluri, S., Lim, S.H., Shimada, K., (2011) Chan, C.H., Tutt, A., Beers, S.A., Glennie, M.J., Exon-array profiling unlocks clinically and biologically Cragg, M.S., and Illidge, T.M. (2011) Catharine West (page 56) relevant gene signatures from formalin-fixed Novel type II anti-CD20 monoclonal antibody Translational Radiobiology Group paraffin-embedded tumour samples. Br J Cancer, (GA101) evokes homotypic adhesion and 104, 971-981. actin-dependent, lysosome-mediated cell death in Refereed Research Papers B-cell malignancies. Blood, 117, 4519-29. Barnett, G.C., West, C.M., Coles, C.E., Pharoah, Lyons, A.J., West, C.M., Risk, J.M., Slevin, N.J., Chan, P.D., Talbot, C.J., Elliott, R.M., Tanteles, G.A., C., Crichton, S., Rinck, G., Howell, D., and Shaw, Erenpreisa, J., Cragg, M.S., Anisimov, A.P., and Symonds, R.P., Wilkinson, J.S., Dunning, A.M., R.J. (2011) Illidge, T.M. (2011) Burnet, N.G., and Bentzen, S.M. (2011) Osteoradionecrosis in Head-and-Neck Cancer Has a Tumor cell embryonality and the ploidy number Standardized Total Average Toxicity Score: A Scale- Distinct Genotype-Dependent Cause. Int J Radiat 32n: is it a developmental checkpoint? Cell and Grade-Independent Measure of Late Oncol Biol Phys, Jun 25. Cycle, 10, 1873-1874. Radiotherapy Toxicity to Facilitate Pooling of Data from Different Studies. Int J Radiat Oncol Biol Phys, Greystoke, A., O'Connor, J.P., Linton, K., 82,1065-74. Taylor, M.B., Cummings, J., Ward, T., Maders, F., Hughes, A., Ranson, M., Illidge, T.M., Radford, J., and Dive, C. (2011) 74 | Paterson Institute for Cancer Research Scientific Report 2011 Research publications | 75

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    External Seminar Speakers 2011 John Rouse Breakthrough Breast Cancer Research Unit The seminar series that we run is vital for the Institute, connecting MRC Protein Phosphorylation Unit, Dundee Seminar Series 2011 world-class researchers across the broad spectrum of cancer Elmar Schiebel Mohamed Bentires-Alj research. 2011 was another successful year for scientific interaction Center for Molecular Biology, University of Friedrich Miescher Institute for Biomedical Research, with an excellent set of internationally renowned speakers visiting the Heidelberg, Germany Switzerland Institute. In its third year, the Breakthrough Breast Cancer Research Almut Schulz Marina Glukhova Unit seminar series continues to produce an outstanding range of London Research Institute Curie Institute, France speakers. The postdoctoral researchers at the Institute also give Chris Smith Nancy Hynes weekly seminars which are very well attended and help to integrate Department of Biochemistry, University of Friedrich Miescher Institute for Biomedical Research, Cambridge Switzerland the entire cancer research efforts of the Institute. Bas Van Steensel Clare Isacke Asifa Akhtar Eyal Gottlieb The Netherlands Cancer Institute, Amsterdam Breakthrough Breast Cancer Research Centre, Max Planck Institute of Immunobiology and The Beatson Institute, Glasgow London Epigenetics, Germany Daniel St Johnson Tessa Holyoake The Gurdon Institute, Cambridge Christoph Klein Rene Bernards The Beatson Institute, Glasgow Department of Pathology, University of Regensburg, The Netherlands Cancer Institute, Amsterdam David Taussig Germany Cristina Lo Celso Barts Cancer Institute, Queen Mary University Cédric Blanpain Imperial College London of London Seth Schor Free University of Brussels (ULB) Unit of Cell and Molecular Biology, School of Pentao Lui David Tuveson Dentistry, University of Dundee Vania Braga Wellcome Trust Sanger Institute, Cambridge Cambridge Research Institute Imperial College London Andy Sims Florian Markowetz Frank Uhlmann Breakthrough Breast Cancer Research Unit, Tony Carr Cambridge Research Institute London Research Institute Edinburgh Centre for Genome Damage and Stability, University of Sussex Chris Marshall Henning Walczak Mathijs Voorhoeve Institute of Cancer Research, London Imperial College London Department of Biochemistry, Duke - National John Diffley University of Singapore London Research Institute Katrin Ottersbach Cambridge Institute for Medical Research Ivan Dikic Institute of Biochemistry II, Goethe University, Mark Petronczki Frankfurt, Germany London Research Institute Jesus Gil Jonathon Pines Imperial College London The Gurdon Institute, Cambridge 76 | Paterson Institute for Cancer Research Scientific Report 2011 External Seminar Speakers 2011 | 77

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    Postgraduate Education http://www.paterson.man.ac.uk/education A well-supported graduate programme is of fundamental PhD studentships All our CR-UK funded studentships are of four years method development and application take place on both sites in all projects, with mutual benefit as each importance to a research institute such as the Paterson, both duration, and consist of an approved research Fellow brings newly acquired knowledge to each to train the researchers of tomorrow, and for the valuable project in one of our research groups. Some students have joint supervisors in different groups, site. Regular meetings take place between the Fellows, their supervisors, as well as other staff contribution made by our students to the labs they are fostering collaborations and giving the students members involved in the project, ensuring true working in. In 2011, we welcomed another eight graduate exposure to different disciplines. Recruitment is highly competitive, with hundreds of applications collaboration and a ‘joined-up’ approach. students from around the world to join our PhD programme, competing for around 8-10 places each year. Education Committee 2011 Postgraduate Education working in fields as diverse as yeast genetics, stem cells and Interviews are typically conducted over a two-day period in early January. Our goal is for every student to have a project that is both achievable and intellectually demanding. Manager clinical research. It was also particularly gratifying to see that Projects and students are monitored by the Julie Edwards over the course of the year, eight students (including three who All our students benefit from access to advanced Education Committee which makes sure that the state-of-the-art facilities including advanced imaging, proposed plan of research is suitable, and that things completed in 2010 along with five current students) published biological mass spectrometry, microarrays, flow are progressing as they should, throughout the first author articles in journals as diverse as the Journal of cytometry, histology and next generation sequencing. Our research groups offer PhD course of the studentship. Various assessments throughout the PhD programme, including regular Clinical Oncology and Molecular Systems Biology. During the studentships and projects covering the entire talks, progress meetings and written reports are an course of the year, a total of eleven PhD students and five breadth of research within the institute. essential key in ensuring successful completion of the PhD programme. Clinical Fellows were awarded their PhDs. Fellowships in Clinical Pharmacology Research In order to help train the next generation of clinical Valerie Kouskoff (Chair from April replacing Jenny pharmacologists with expertise in oncology, in 2007 Varley) The Paterson Graduate Programme The Paterson runs an external seminar series the Paterson Institute, in collaboration with the Fiona Blackhall Postgraduate Tutor We aim for each student to receive high quality featuring talks from many of the key players in MCRC and AstraZeneca, established a fellowship Richard Cowan training in scientific research through an cancer research, and students are expected to scheme in Clinical Pharmacology Research. The Julie Edwards Crispin Miller intellectually demanding but achievable research attend all of these external seminars. The fellowships are open to applicants who have Dave Gilham programme. Each project is peer-reviewed in speakers are internationally renowned scientists obtained, or are close to obtaining, their Ian Hampson advance and monitored throughout the course and we consider it essential that our students Completed Certificate of Specialist training Karim Labib of their studies, via a mixture of talks, written are exposed to outstanding work from the (CCST) in Medical Oncology. Angeliki Malliri reports and progress and planning meetings. leaders in different disciplines, which will give Crispin Miller These are designed not only to provide formal them a broad understanding of many aspects Each clinical Pharmacology Research Fellow Donald Ogilvie points at which progress (of both the student of cancer research and basic biology. In addition undertakes a three-year PhD project, which Vaskar Saha and the project) can be monitored, but also to we hold a series of weekly postdoctoral provides training in biomarker discovery, method Tim Somervaille help develop the presentation skills that are so research seminars which the students attend, development/validation, and in clinical trial Catharine West fundamental to the majority of careers in and they have the opportunity to present their methodology. During tenure, at the Caroline Wilkinson science and elsewhere. Graduate training is own work in lab meetings within the institute. Christie/Paterson, the post holders receive clinical monitored by an Education Committee, which supervision from Malcolm Ranson, and laboratory- Student Representatives features Group Leaders, senior clinicians and The annual Paterson Colloquium, held in based training from Caroline Dive in CEP (in Tim Maculins (until December) scientists, and student representatives (see September, is an excellent opportunity for our collaboration with MCRC colleagues); at Emily Holmes below). Each student is assigned an advisor new intake of students to meet other AstraZeneca they receive training in clinical trials Hadir Marei (from December) (similar to a personal tutor on an established PhD students, members of the management, regulatory interaction, translational undergraduate programme) whose role is Institute, including Group Leaders, postdoctoral research through project management and to provide impartial support and advice, fellows, and scientific officers. This forum attendance at investigator meetings. Clinical training while further support is also available from provides up to date science presentations both includes one research clinic per week, training in the postgraduate tutor and a student orally and posters given by Group Leaders, 2nd clinical trial design and methodology, ICH-GCP, EU welfare group. year PhD students and Postdoctoral scientists. Directives and research governance. Biomarker 78 | Paterson Institute for Cancer Research Scientific Report 2011 Postgraduate Education | 79

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    Operations During the course of 2011, the Operations department underwent significant restructuring resulting in the creation of two new positions, namely the Scientific Operations Manager and Head of Research Services, which have been taken up by Caroline Wilkinson and Stuart Pepper respectively. Stuart has the additional responsibility of overseeing IT services while the personal assistance to Richard Marais in his testing. In addition, team members have attended post as Director of The Paterson Institute as relevant courses to help improve their skills and Director of Operations Logistics and Estates departments now fall under the leadership well as overseeing the management of the keep their knowledge up to date with current Pippa McNichol of the Finance and Purchasing Manager, Margaret Lowe. In administration team. working practices and changing legislation. September, Ruth Perkins joined us as the Administration Estates Finance & Purchasing Services Coordinator, replacing Becky Allen. During these Steve Alcock, Graham Hooley, Tony Woollam Margaret Lowe, David Jenkins, Denise Owen, Muhammad Raja, Debbie Suthern changes, everyone in the Operations department has worked This year has been particularly challenging for the hard to ensure that we have maintained the high standards of Estates team with a significant amount of capital The Finance Department is responsible for work carried out in the Institute. The main scheme providing a comprehensive purchasing, travel and service that underpin the smooth running of the Institute. implemented was the upgrade to the main electrical finance service to the Research Groups and Service supply and its associated main distribution Units within the Paterson Institute. The procurement switchgear; it had been identified that the existing team continues to work with the research groups Administration Department community. A list of speakers for 2011 can be transformer rating was exceeded during the to identify savings in consumable spending. Early in Amy Weatheritt, Becky Allen, Ruth Perkins, found at www.paterson.man.ac.uk/seminars summer months and the main switchgear was 2011, the University introduced E-Marketplace Steven Morgan obsolete causing it to fail on more than one which is an enhancement tool with allows the Director’s Office occasion. comparison of products across suppliers. The The administration department has been EA to the Director catalogues on E-Marketplace include up-to-date and working hard to provide a consistent level of Amy Weatheritt To carry out the physical work in a safe manner was accurate product and pricing details in line with support to the Institute during a dramatic logistically challenging; in order to keep the agreed contracts. Easy price comparisons give an period of change. The department will be In February 2011 Nic Jones was appointed as disruption to the Institute’s work to a minimum, the instant cost saving as well as greater use of reviewed and possibly restructured once the Chief Scientist for Cancer Research UK, project was carried out over three weekends in preferred suppliers resulting in a longer term saving new Director is in place in 2012 in order for it stepping down as Director of the Paterson May accounting for several hundred man hours. As a on contract negotiations. The Central Procurement to best serve the Directorate and Institute as Institute. The office has been working hard to result of this work, the Institute now has a more team is continually adding to the list of suppliers efficiently as possible. support Nic in this new role alongside his secure electrical supply. Other small schemes carried available on the system. In addition to this, they continuing position as Director of the MCRC. out to improve facilities for the research groups advise on capital purchases working with the Over the year, and in conjunction with staff at Prior to the appointment of Richard Marais as included the refurbishment of a small lab which researchers to ensure legal legislation is adhered CR-UK, the department has helped to facilitate Director of The Paterson Institute, the specifically houses work with radio-isotopes. to and that the best possible price is obtained for several high profile visits and has provided department has provided assistance and the equipment. administration support to the Manchester support to the Paterson Senior Management The Estates team also concentrates on sustainability Cancer Research Centre (MCRC) in the early Team who have been leading the Institute in and reducing the Institute’s carbon footprint as It is crucial that the management information stages of the new MCRC building planning. The the interim period. The office has also stepped much as possible. Whenever a scheme is required, provided is current and accurate at all times to department has assisted in the management of in to provide support to the Manchester Breast energy saving devices are utilised and more efficient assist Group Leaders in managing their budgets and several conferences and events over the course Centre and Breakthrough Unit until a full time heating/cooling and ventilation systems are installed. for the Director to have a full overview of the of the year, including the Paterson Colloquium administrator is appointed. We are in the process of putting together a report Institute’s finances. We also assist the Group Leaders at its new location in Lancaster. on one area which has intensive energy usage, (IT with costing grant applications and provide full post- The Director’s Office looks forward to the main server room). Once completed, it will identify award administration support. The 2011 seminar series has been a great challenges 2012 will bring. Despite inevitable different ways to reduce the energy this room success and the 2012 series is already in place. changes following the appointment of a new consumes, with varying investment required. Health & Safety This will aim to provide a varied programme of Director, the Administration Department and Colin Gleeson national and international speakers, serving to Director’s Office will continue to provide a high The Estates team has been proactive throughout foster collaboration and encourage positive level of support to the Institute. Siana Peters the year, attending to many legislative requirements The second phase of the Institute-wide Risk interaction within the wider scientific will join the Institute in early 2012 to provide including Legionella best practices and fire alarm Management Survey was carried out. This 80 | Paterson Institute for Cancer Research Scientific Report 2011 Operations | 81

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    comprised a check made by management, that guidance, legislation and best practice. This year Institute, in the form of our Internet pages, has also Cancer Research Technology safety related tasks in the areas for which they are we have successfully recruited 34 highly skilled been revamped this year to help reflect the ever Martyn Bottomley responsible, had been completed. The Risk individuals in order to compliment and further evolving work of the Institute. Management exercise allows Institute managers to enhance the work of the Institute. Cancer Research Technology (CRT) is a specialist demonstrate that they have allocated resources to Some aspects of the infrastructure are now nearing oncology-focused development and health and safety related tasks and have made It has been a busy and challenging year with the the end of their useful life and to ensure we meet commercialisation company wholly owned by appropriate management checks that those tasks main focus being on the implementation of a new the needs of the next generation of research Cancer Research UK. CRT aims to maximise patient have been completed. online absence management system. The new requirements we will need to commission new benefit from publicly funded research worldwide by system allows for employees and managers to easily elements to our service. As 2011 draws to a close, advancing research discoveries into development A heavily revised Institute health and safety policy request and record leave and sickness. This enables plans are under way to purchase a new storage with pharmaceutical and biotechnology parties. document was developed. The document contains more accurate and useful information to be solution that will provide sufficient capacity for the At CRT we bridge the gap between cutting edge the Institute’s health and safety policy statement, the recorded. The benefits of effective absence recording Institute’s varied data storage needs. As always, academic research and industrial development of organisational responsibilities for health and safety to the Institute will be a reduction in costs, the creative solutions will be developed to ensure that cancer therapeutics and diagnostics. We achieve this and an arrangements section. The latter details how ability to manage trends in absences and to we maintain the high standards of IT that users have by working closely with prestigious international the Institute achieves its health and safety policy rehabilitate staff back into the workplace. become accustomed to whilst continuing to work in research institutes, such as the Paterson Institute aims. A new Institute code of practice for working a highly cost effective manner. and funding bodies to develop, protect and with biological agents was also developed. The Joint partnership working with the unions has commercialise oncology related discoveries. Core development of both documents involved continued throughout the year which has resulted in Scientific Operations Manager activities of business development and drug consultation with staff to ensure that they were the agreement of several new policies including the Caroline Wilkinson discovery are supported by specialists, integrated in user friendly. Recruitment and Selection Policy and the Presence the business with expertise in patents, legal, finance of Children and Young Persons policy. Moving This new post provides support to the Director in and marketing. Our exclusive focus in oncology Safety inspections of Institute laboratories were forward the main focus for the next year will be the order to facilitate the day-to-day running of the provides an unrivalled depth of knowledge and undertaken, with focus on the use of human blood successful implementation of the online Institute. Over the last year, this has meant working experience in cancer-specific translational and tissues and/or the use of sharps. We also probationary process system and a review of the closely with the Senior Management Team and then development and commercialisation. By undertook an audit of those laboratories that use current policies and procedures. towards the end of 2011, with the new Director arrangement with The University of Manchester, radio-chemicals to ensure they were compliant with Richard Marais to help facilitate his transition to CRT owns and is responsible for the development the legislation and local rules. Surveys were also Information Technology the Institute. and commercialisation of intellectual property undertaken of the acquisition and/or use of certain Malik Pervez, Brian Poole, Steve Royle, Ryan Smith, arising from Cancer Research UK funded research controlled chemicals, pathogens and toxins. This was Zhi Cheng Wang, Matthew Young The Scientific Operations Manager provides reports at The University of Manchester (including the to ensure that the Institute has the necessary for a number of external sources, including Cancer Paterson Institute). permissions and registrations required under the law This year the IT service has balanced the demands Research UK and the University of Manchester, as for these activities. There was also an Institute-wide of increasing the breadth of support offered to well as editing publications such as the Annual Our relationship with the Paterson Institute reflects portable appliance testing programme scientists at the Institute, whilst maintaining existing Scientific Report, writing updates for the intranet the specific requirements of the scientist, the encompassing some 9000 pieces of plug-in services at the same high standards that users have and external website and producing content for the Institute itself, Cancer Research UK and the electrical equipment! come to rely on. The IT service has maintained an Institute newsletter. Further responsibilities include individual project. To effectively facilitate these excellent service desk which offers rapid response organising Quinquennial and mid-term reviews, requirements and interactions, Martyn Bottomley, a Induction training for new starters was carried out rates to ensure that problems are rectified as soon liaising with library staff at the University of CRT Business Manager is based on-site dedicated to and supplemented with biological agent and as possible. The team has also maintained the Manchester to secure access to appropriate working closely with the staff at the Institute and hazardous chemical training presentations. continuous improvement philosophy that is required electronic journals, maintaining detailed databases of also The University of Manchester. Martyn offers Additionally a lentivirus workshop was held due to to maintain a modern IT infrastructure. The success the Institute’s publications, arranging a weekly access to oncology focused expertise in technology the expanding number of people in the Institute of such a strategy is illustrated by the stability of the seminar series presented by our post-doctoral evaluations, patent applications and management, using this technology. A series of fire extinguisher Institute’s systems which are rarely unavailable. research fellows and organising the Institute funding for development, commercialisation, drug training sessions, with hands-on experience of using In terms of development there have been a number colloquium. In 2011, this event moved to its new discovery, market intelligence, and project extinguishers was arranged. This proved popular of new solutions implemented which are aimed at venue at the University of Lancaster. As ever, the management. He also works closely with UMIP, The with some seventy people trained in fire either improving support for high performance colloquium provided an ideal platform for University of Manchester technology transfer extinguisher use. computing or allowing more flexible work patterns. interactions and discussions between the Institute’s organization. CRT continues to work very closely We introduced a new remote access system which scientists and we look forward to returning to with the Drug Discovery Laboratories based at the We have also made considerable progress in the has provided a straightforward interface that will Lancaster in 2012. PICR to facilitate the development of small development of an intranet based genetic allow all staff to access information from anywhere molecules drug therapies to satisfy the unmet modification risk management system. This in the world. Support for tablet devices such as the All grant submissions submitted by our scientists are clinical needs of cancer patients. CRT is currently comprises bespoke genetic modification risk Apple iPad, and greater use of the in house WiFi screened for the appropriate ethical approvals as actively managing a broad portfolio of development assessment forms with an integral document network are allowing staff to access scientific well as the ability of the Institute to accommodate programmes and exciting licensing opportunities management system. We anticipate that this will be publications and personal data in increasingly the proposed programme of work. Along with originating from the PICR that continue to attract rolled-out in early 2012. flexible ways. Stuart Pepper, the Head of Research Services, the commercial partners (ranging from enabling SOM manages all aspects of space usage in the technologies through to drugs in late stage Clinical Human Resources In collaboration with the Applied Computational Institute as well as overseeing the equipment Trials). We look forward to building on our Rachel Powell, Laura Humes, Julie Jarratt Biology and Bioinformatics Group (ACBB), a new budget. This year, we managed to secure the successes and continuing to work closely with the Lustre system has been added to the existing High purchase of an ex-demo macro-confocal PICR to advance discoveries to beat cancer in the Over the past year the HR Department has Performance cluster. This has provided immediate microscope which resulted in a significant reduction years ahead. continued to successfully develop and deliver a high benefits including increased processing capacity and in cost and is proving to be an excellent addition quality professional HR service to the Institute, productivity, greatly improving the ability of ACBB to to our Imaging Facility. Finally, we also carried out a providing advice to managers and staff on all handle large data sets such as those generated by detailed space audit of the Institute that will help in employment-related matters such as policy Next Generation Sequencing. The public face of the planning research activities over the next couple of years. 82 | Paterson Institute for Cancer Research Scientific Report 2011 Operations | 83

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    Cancer Research UK’s Local Engagement and Development 2011 has been a fantastic year of local engagement and development activity in Manchester. Researchers have been involved in over 50 events, with 25,000 Cancer Research UK supporters being reached, and a further 600 people having the opportunity to visit the Institute. LEAD Manager In addition to the monthly lab tour Commission for this to be increased in the programme, schools’ day and open day, an future and for a greater availability of grant James Dunphy extra event was held to commemorate the funding for scientists and researchers working five year anniversary of Manchester Cancer in this important area.” Research Centre. Key people from the founding partner organisations attended along In the autumn, a Manchester United and with approximately 75 fundraisers from Cancer England footballer swapped his team strip for a Research UK, Breakthrough Breast Cancer and lab coat when he enjoyed a visit. Ashley Young The Christie. The event was a great success visited the Paterson Institute, thanks to Cancer and attendees enjoyed hearing about Research UK’s relationship with the England advancements that had been made over the Footballers Foundation. Cancer Research UK last five years as well as plans for the future. has been chosen as an official charity partner of the Foundation for the next four years. The Paterson has also welcomed a variety of Ashley met Cancer Research UK’s chief high profile visitors over the last year. Mark scientist, Professor Nic Jones, before taking a Hunter MP, Sajjad Karim MEP and Tony Lloyd tour of the labs. MP visited in the summer to learn about the research taking place in the Institute. They met The 26-year-old winger said: “Everyone knows with Professor Caroline Dive to see some someone who has been affected by cancer in research in action and found out more about some way. Visiting the Paterson Institute for the future policy priorities for Cancer Research Cancer Research has been a real privilege. I UK. It was a great opportunity for them to hadn’t realised that such amazing research see some of the world class research that is work, saving so many lives, is taking place in being carried out in Manchester and Manchester – right on my door step." highlighted why it is so important to support the vital research which could make a Representatives of the Paterson Institute have significant difference to the 36,100 people continued to support their local fundraisers diagnosed with cancer in the North West with inspirational speeches and voluntary help every year. at Race for Life, Relay for Life and Shine events, throughout the year. The Paterson was utilised Speaking after the visit, Sajjad Karim MEP said: as a pit stop for the second successful Shine “Each year, there are an estimated 2.45 million event, with researchers manning the teas and Paterson scientists with MPs Tony Lloyd and Mark Hunter new cancer cases and 1.23 million deaths from coffees throughout the night. This year, around Footballer Ashley Young with Paterson Institute Group Leader and cancer in the EU. The Paterson Institute and 3500 people took part, in the night-time half Chief Scientist of Cancer Research UK, Professor Nic Jones other cancer research centres in the UK or full marathon and it is hoped this event will currently receive a large proportion of their raise over £1million for the charity. In addition funding from charitable sources, with very little to this support, the Institute has once again funding coming from the European Union. fundraised for the charity with the Keswick to Therefore as a Member of the European Barrow and Relay for Life teams raising a Parliament I will be pushing the European combined total of over £3000. 84 | Paterson Institute for Cancer Research Scientific Report 2011 Cancer Research UK’s Local Engagement and Development | 85

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    Acknowledgement for Funding of the Paterson Institute Career Opportunities at the Paterson Institute The total funding of the Paterson Institute for 2011 was £17.6m. The The Paterson Institute is located alongside The Christie NHS Foundation major source of this funding (62.3%) was through a core grant from Trust, and has a strong programme of basic and translational research. Cancer Research UK (CR-UK). The actual value of this award in 2011 There are very close links with clinical and translational research groups was £10.96m. This is divided between the various scientific groups and throughout the Christie Hospital site. service units within the Institute to enable them to carry out their research. In addition to this the CR-UK awarded us £2.16m to run the The Manchester Cancer Research Centre (MCRC) Postdoctoral applicants of high calibre are regularly was created nearly six years ago with partners sought. Although post docs will be encouraged to Drug Discovery unit (12.3%). including the Paterson Institute, The Christie apply for their own fellowships, funded positions are Hospital NHS Foundation Trust, The University of available for outstanding candidates. Interested Manchester and Cancer Research UK. This is an applicants should contact the Group Leaders extremely exciting development which is enhancing directly, with details of their area of interest and PATERSON INSTITUTE FUNDING 2011 all aspects of cancer research, education and recent experience. treatment. The Institute offers excellent laboratory facilities and outstanding core facilities, including In addition to postgraduate and postdoctoral molecular services, a microarray platform, opportunities, the Institute is still seeking to recruit 16.1% proteomics, flow cytometry, histology, the production outstanding candidates to the positions of Junior and of knock-in/knock-out animal models, real-time PCR, Senior Group Leaders. The packages provided are next generation sequencing and advanced imaging. extremely attractive and commensurate with the Details of all groups and facilities are given experience of the applicant, with significant funding CR-UK Core Grant 9.3% throughout this report, and can guide interested for personnel, recurrent expenditure and CR-UK Programme Grant parties to the appropriate contacts. equipment. Junior Group Leaders are appointed for HEFCE an initial six-year period with a review at five years Opportunities exist at a number of levels in the for consideration for promotion to Senior Group Other Sources Institute. We have a well-established programme of Leader, with Senior Group Leaders appointed to 12.3% 62.3% degrees by research which is described in the non-time limited positions. section on Postgraduate Education. We encourage applications from suitable qualified graduates to Specific vacancies can be found on our web pages apply to join either the PhD or MD programmes. (http://www.paterson.man.ac.uk/jobs/index.asp), but Graduates with a first or 2.1 honours degree in a suitably qualified and enthusiastic individuals should biological science can apply each year to train for a contact the Institute at any time to enquire about four-year PhD in one of our research laboratories. career possibilities. The infrastructure of the Paterson Institute is funded • European Commission First year students will complement their laboratory by HEFCE generated income at a cost of £1.6m • ECMC skills by attending a small number of specialised (9.3%) • BBSRC postgraduate taught and training courses allowing • Leukaemia & Lymphoma Research Fund them to gain a sound knowledge base of the latest The final 16.1% of the Institute’s funding is received • Novartis developments in cancer treatment and research. from a number of additional sources. The research • Qiagen The Institute also has a well-developed process for carried out through these additional projects • Chugai ensuring suitable pastoral care and mentoring for enhances and supports the research undertaken by • Abbott Laboratories all students. the core funding. • Cambridge University • GlaxoSmithKline These sources are as follows: • Christie Hospital NHS Foundation Trust • AstraZeneca We are immensely grateful to all our sponsors. • Roche 86 | Paterson Institute for Cancer Research Scientific Report 2011 Career Opportunities at the Paterson Institute | 87

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    Contact details Paterson Institute for Cancer Research ISSN 1479-0378 Wilmslow Road Copyright © 2011 Cancer Research UK Manchester M20 4BX United Kingdom Tel +44(0) 161 446 3156 Fax +44(0) 161 446 3109 www.paterson.man.ac.uk An electronic version of this report can be found at: www.paterson.man.ac.uk 88 | Paterson Institute for Cancer Research Scientific Report 2011

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    Paterson Institute for Cancer Research Wilmslow Road Manchester M20 4BX Tel +44(0) 161 446 3156 www.paterson.man.ac.uk

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