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    Paterson Institute for Cancer Research Scientific Report 2010


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    Scientific Report 2010 Cover images Top Paterson Institute Multicolour immunofluorescence images of lung cancer cells spiked into human blood, see Figure 1 in the Clinical and Experimental Pharmacology report for Cancer Research (page 27) for details. Bottom Imaging podosomes on acute lymphoblastic leukaemia cells in culture, see the Children's Cancer Group report on page 49 for details. 1 | Paterson Institute for Cancer Research Scientific Report 2010


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    Contents Director’s Introduction 5 John Brognard 38 Stuart Pepper 61 Signalling Networks in Cancer Group Cancer Research UK GeneChip Microarray Service Research Highlights 2010 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 65 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 Tim Illidge 50 Seminar Series 2010 76 Targeted Therapy Group Nic Jones 22 Cell Regulation Group Catharine M.L. West 52 Postgraduate Education 78 Angeliki Malliri 24 Translational Radiobiology Group Cell Signalling Group Robert Hawkins 54 Operations 80 Caroline Dive and Malcolm Ranson 26 Medical Oncology: Clinical and Experimental Clinical and Experimental Pharmacology Group Immunotherapy Group Cancer Research UK’s 84 Local Engagement and Development Ivan Ahel 28 Gordon Jayson 56 Medical Oncology: Translational DNA Damage Response Group Anti-Angiogenesis Group Acknowledgement for Funding 86 of the Paterson Institute Donald Ogilvie 30 Drug Discovery Group Research Services Career Opportunities at the 87 Peter L Stern 32 Steve Bagley 58 Paterson Institute Immunology Group Advanced Imaging Facility Nullin Divecha 34 Duncan Smith 59 Contact Details 88 Inositide Laboratory Group Biological Mass Spectrometry Facility Tim Somervaille 36 Biological Resources Unit 60 Leukaemia Biology Group


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    Director’s introduction Welcome to the 2010 Paterson Institute Annual Scientific Report. This is the last report from me as Director of the Institute and therefore provides an opportunity not only to look back on the events and successes of the last year but also on the progress we have made over the last ten years and its Nic Jones aspirations for the future. The last ten years has been a period of great Institute. New laboratory facilities – such as change within the Institute. During this time the TRF1, TRF2 and the Drug Discovery Centre – research focus has been re-prioritised with a have been developed to allow expansion of our complete reorganisation of the research research base. The training programmes within programmes facilitated through the recruitment the Institute have expanded greatly and of many new group leaders – in fact over the increased in quality fulfilling an important remit last few years over twenty new group leaders of the Institute to train the researchers of the have joined the Institute and helped to ensure future. All of these changes and developments that the Institute is now internationally have lead to a great increase in international recognised for the high quality of research that it recognition and reputation, an increase in supports. The research services have also scientific output and quality and maximising the developed significantly and are now extensive, potential and opportunities of the core funding state-of-the-art and crucial to the success of the we receive from Cancer Research UK. Over the Figure 1 Architects cartoon showing the proposed new MCRC building 4 | Paterson Institute for Cancer Research Scientific Report 2010 Director’s Introduction | 5


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    last ten years, the Institute has been reviewed have been made. There is a very strong leaders that have really demonstrated significant twice by high level, international panels and in platform for which to build further and ensure output and success and who have gained both cases was praised for the positive that the Institute continues its upward trajectory. international recognition are expected to be developments that had taken place and the plans This will be the task for my successor! successful in this process. This was the case with for the future. Angeliki Malliri who was promoted to Senior Focussing on the last year, a number of positive Group Leader on the basis of her excellent Another significant development has been the developments have taken place. John Brognard work on the role of regulators of Rho-like creation of the Manchester Cancer Research joined us as a Junior Group Leader. John was a GTPases in cancer. Karim Labib was elected as Centre (MCRC) with the Paterson Institute at its postdoctoral fellow in the laboratory of Tony an EMBO member. Our research services also core. The MCRC, with its mission of co- Hunter at the Salk Institute in California and has continued to develop and, in particular, this year ordinating cancer research in Manchester, is a initiated an exciting research programme that saw the Histology service expand and increase very exciting and important development and of aims to identify and characterise novel kinases or its capabilities by providing access to tissue great benefit to the Institute. It provides the signalling networks that are altered in tumours microarrays. One of the major reasons why the means by which the Institute can contribute and are essential for driving tumourigenesis. It is research services are so excellent is the across the research spectrum from basic to exactly this type of research programme that can leadership provided by Jenny Varley, Assistant translational to clinical research and thereby gain provide novel targets for our Drug Discovery Director of Research, who oversees all the considerable ‘added value’. Within its brief five Centre which, over the last year, has reached its research services. Jenny has decided to retire in year tenure it has already made a big difference full complement of research staff. A number of 2011 – she will be missed but leaves behind a with significant advances in a number of research exciting discovery projects have been initiated strong legacy. areas (for example biomarker and early phase and the Centre has already had a major clinical trial research, radiation-related research, influence on the Institute by promoting Plans for the new MCRC building are now well breast and lung cancer research) and discussions around a number of potential targets advanced and we anticipate that work can begin development of research infrastructure (for and instilling a ‘drug hunting’ culture. Junior early in 2012 with a likely handover date in early example the new early-phase clinical trials unit, Group Leaders are core-funded for six years in 2014. The iconic building will allow essential one of the biggest worldwide). The Centre is the first instance to provide sufficient time to expansion of research activities with still very much in its infancy but provides a build up a dynamic, successful and productive accommodation for approximately 150 wonderful platform for future development. research programme. At the end of this period laboratory-based researchers and 90 clinical there is a rigorous evaluation to consider trials unit staff. The building has been designed Looking back over the last ten years, we can I promotion to Senior Group Leader and the to reflect and further embed the cross- think feel very satisfied of the achievements that prospect of long term support. Only those disciplinary research approach of the MCRC. The search for a new Director of the Paterson Institute has now been instigated and the expectation is that a new incumbent will be in place during the coming year. Being Director of a core-funded Institute is a very exciting and rewarding job. I have been privileged to lead the Institute for the last eleven years and to steer it through a period of great change and development. Institutes are critical to the success of CR-UK – they represent approximately 40% of CR-UK’s total research spend and are therefore vital to the delivery of the organisation’s research strategy. I am confident that the Paterson will continue to strengthen and thus play its role in realising the ambitions and goals of CR-UK. 6 | Paterson Institute for Cancer Research Scientific Report 2010 Director’s Introduction | 7


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    Research Highlights In this section we are highlighting some research publications Kojima, Y., Acar, A., Eaton, E.N., Mellody, K.T., experimentally generated CAFs from preexisting human mammary fibroblasts that have been Scheel, C., Ben-Porath, I., Onder, T.T., Wang, from 2010 which report significant advances in specific areas. Z.C., Richardson, A.L., Weinberg, R.A. and extracted from human breast carcinomas. These The selected papers demonstrate the breadth and the quality Orimo, A. cells recapitulate the tumour-promoting myofibroblastic phenotypes of CAFs prepared Autocrine TGF-β and stromal cell-derived of the research being undertaken by Cancer Research UK- factor-1 (SDF-1) signaling drives the evolution from breast cancer patients. During the course funded groups in the Paterson Institute. of tumor-promoting mammary stromal of tumour progression, resident fibroblasts progressively increase cell-autonomous and myofibroblasts. Proc Natl Acad Sci U S A 2010; 107: 20009- self-sustaining TGF-β and SDF-1 autocrine Bitton, D.A., Smith, D.L., Connolly, Y., Scutt, P.J. Daayana, S., Elkord, E., Winters, U., Pawlita, M., 20014. signaling that promotes their differentiation into and Miller, C.J. Roden, R., Stern, P.L. and Kitchener, H.C. tumour-promoting myofibroblastic CAFs. This An integrated mass-spectrometry pipeline Phase II trial of imiquimod and HPV therapeutic Much interest is currently focused on the autocrine signalling may prove to be an identifies novel protein coding-regions in the vaccination in patients with vulval intraepithelial emerging role of tumour-stroma interactions attractive drug target to block the evolution of human genome. neoplasia. essential for supporting tumour progression. tumour-promoting CAFs. PLoS One 2010; 5: e8949. Br J Cancer 2010; 102: 1129-1136 (featured Carcinoma-associated fibroblasts (CAFs), rich in article). myofibroblasts, are predominantly present in the Protein identification by mass spectrometry is a Highlighted in: stroma of human carcinomas and substantially Castillo-Lluva, S., Tatham, M.H., Jones, R.C., fundamental component of the modern Nature Medicine 2010; 16: 499. contribute to promoting tumorigenesis. Jaffray, E.G., Edmondson, R.D., Hay, R.T. and molecular biology toolkit. The approach works However, the precise cellular origins of these Malliri, A. by fragmenting peptides and then accurately Vulval intraepithelial neoplasia (VIN) is a cells and the molecular mechanisms by which SUMOylation of the GTPase Rac1 is required measuring the mass/charge ratio of each premalignant condition, which is frequently they evolve into tumour-promoting for optimal cell migration. individual fragment using a mass spectrometer. associated with type HPV 16 infection, and myofibroblasts remain unclear. Using a Nat Cell Biol 2010; 12: 1078-1085. The resultant mass spectrum provides a multifocal disease has high rates of surgical co-implantation tumour xenograft model, we diagnostic fingerprint that can be used to identify treatment failure. This study treated the high The Rho-like GTPase Rac1 is well known for its each peptide when compared against a grade VIN lesions of 19 women topically for 8 role in cytoskeletal rearrangements and cell computer-generated database of predicted weeks with imiquimod (an immunostimulatory migration. Rac activation is regulated through a spectra created from the set of known proteins. cream) followed by three monthly vaccinations number of mechanisms, including control of A limitation of the technique is that it is reliant with TA-CIN, a fusion of the HPV 16 L2 minor nucleotide exchange and hydrolysis, regulation of on a database of candidate proteins against capsid, E6 and E7 oncogenic proteins. The subcellular localization or modulation of which to search, preventing its use in finding rationale was that the imiquimod would altered protein-expression levels. In this study it was novel proteins. We therefore extended the the local balance between CD8 T cells, which shown that the small ubiquitin-like modifier technique by creating a much larger database can destroy the HPV infected premalignant cells, (SUMO) E3-ligase, PIAS3, interacts with Rac1 comprising all possible protein sequences that and regulatory T cells which suppress immune and enhances levels of active (GTP-bound) Rac, might be expressed in the human genome, activity, with the vaccination boosting the promoting cell migration in response to derived by translating the entire genome effective HPV 16 oncogene T cell immunity. A hepatocyte growth factor (HGF). Significantly, it sequence in all three forward and all three majority of women had objective clinical was demonstrated that Rac1 can be conjugated reverse strands. We then searched existing mass responses and no symptoms one year after to SUMO-1 in response to HGF treatment and spectrometry data against this much larger receiving the treatment and this therapeutic that SUMOylation is enhanced by PIAS3. database to identify sequences that matched to effect was associated with both increased Moreover it was shown that this modification novel regions outside known protein coding CD8/Treg ratios locally and boosted E6/E7 T cell increases the levels of GTP-bound Rac1 and genes. Through this approach we identified responses systemically. The potential for promotes membrane ruffling, cell migration and hundreds of novel proteins, allowing us not only increased vaccine immunogenicity by addition of invasion. SUMOylation of Rac1 seems to be to predict new genes, but also to predict novel an adjuvant has been established in preclinical required for maintaining, rather than inducing, isoforms of existing proteins. models (Karanam et al., Vaccine 2009; 27: 1040) activation, and may strengthen interactions with so a future goal is to test the adjuvanted vaccine guanine nucleotide-exchange factors or inhibit in combination with local immune stimulation to interactions with GTPase-activating proteins. further increase patient response rates. These results point to the existence of a novel mechanism, through SUMOylation, for regulating GTPases. 8 | Paterson Institute for Cancer Research Scientific Report 2010 Research Highlights | 9


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    Parker, C., Waters, R., Leighton, C., Hancock, J., predominantly observed in CD41+ cells, agent oxaliplatin by reducing the amount of response to environmental stress, we showed Sutton, R., Moorman, A.V., Ancliff, P., Morgan, M., contrasting with Sox7, mostly expressed in Flk1+ oxaliplatin taken up by the cells. This was that the activity and localisation of ATX1 is Masurekar, A., Goulden, N., Green, N., Revesz, cells. Conversely, Sox17 remained marginally because saracatinib prevented the drug controlled by changes in PtdIns5P levels through T., Darbyshire, P., Love, S. and Saha, V. expressed during blood specification. Overall, transporter responsible for carrying oxaliplatin PtdIns5P interaction with the PHD finger of Effect of mitoxantrone on outcome of children our data uncover contrasting effect and into cells, Organic Cation Transporter 2, from ATX1. Changes in PtdIns5P lead to a decrease with first relapse of acute lymphoblastic expression pattern for Sox18 and Sox17 at the functioning effectively, resulting in reduced in the levels of ATX1 and H3K4Me3 at the leukaemia (ALL R3): an open-label randomised onset of haematopoiesis specification. oxaliplatin uptake. Therefore, this work suggests promoter of a target gene and to a decrease in trial. that combining saracatinib with oxaliplatin in the its transcription. These data provide a Lancet, 2010; 376: 2009-2017 clinic should be treated with caution, with the mechanistic link between PtdIns5P and Pearson, S., Lancrin, C., Lacaud, G. and schedule the two drugs are given and the effect H3K4Me3 through the regulation of trithorax This reports the conclusion of a randomised Kouskoff, V. on oxaliplatin cytotoxicity being of upmost methylase activity which is often deregulated in study comparing the effect of mitoxantrone with The sequential expression of CD40 and Icam2 importance. human tumours. idarubicin in children with first relapse of acute defines progressive steps in the formation of lymphoblastic leukaemia (ALL). A clear survival blood precursors from the mesoderm germ Eustermann, S., Brockmann, C., Mehrotra, P.V., advantage of >20% was noted in those who layer. Ndamukong, I*., Jones, D.R.*, Lapko, H., Divecha, Yang, J.C., Loakes, D., West, S.C., Ahel, I. and received mitoxantrone and the randomisation Stem Cells, 2010; 28: 1089-1098. N.+ and Avramova, Z.+ Neuhaus, D. stopped early. This is one of the largest *joint first authors; +joint communicating Solution structures of the two PBZ domains improvements by a single modification to During embryogenesis, the haematopoietic authors from human APLF and their interaction with treatment ever reported in childhood ALL. programme is specified from the mesodermal Phosphatidylinositol 5-phosphate links poly(ADP-ribose). Curiously, the speed of clearance of disease, as germ layer through the formation of dehydration stress to the activity of ARABIDOPSIS Nat Struct Mol Biol 2010; 17: 241-243. measured by minimal residual disease (MRD) haemangioblast. This precursor gives rise to a TRITHORAX-LIKE factor ATX1. techniques was similar in both arms. Thus this haemogenic endothelium that later on mature to PLoS One 2010; 5: e13396. Posttranslational modification of proteins by serves a caveat for the use of MRD as a generate haematopoietic precursors. A major poly(ADP-ribosyl)ation in response to DNA surrogate marker of response in evaluating new hurdle in the quest to further understand blood In advanced breast tumours the expression of damage acts as an important cellular signal for agents. Though overall toxicity was less in formation is the lack of specific cell surface PIP4Kβ may be a prognostic indicator of patient the recruitment of DNA repair factors and thus mitoxantrone patients, they exhibited delayed markers to identify cells with discrete survival, suggesting that PIP4Kβ plays a role in for the efficient restoration of genome integrity. count recovery 5-12 months after receiving the developmental potential. In the present study, tumour progression. PIP4Kβ phosphorylates and Several eukaryotic DNA repair proteins, drug, suggesting a noxious effect on the we identify CD40 and Icam2, two markers controls the levels of PtdIns5P a including the histone chaperone Aprataxin and haematopoietic stem cell niche. Thus the clinical typically associated with the adult immunological phosphoinositide that interacts with proteins PNK-Like Factor (APLF) possess a specific data contends that the microenvironment plays a compartment, as expressed at the earliest stages possessing a PHD Zinc finger motif. poly(ADP-ribose)-binding zinc-finger (PBZ) major role in disease recurrence and the effect of blood specification. We show that the Post-translational modification of histone element in their structure that enables their of chemotherapy may be indirect by disrupting sequential expression of CD40 and Icam2 proteins is important in the epigenetic control of timely recruitment to the sites of DNA damage interaction between host and tumour cell rather delineates a transition in the acquisition of the gene expression patterns which modulates via direct interaction with poly(ADP-ribose). In than by direct cytotoxicity. blood potential from haemangioblast to proliferation and differentiation and organismal this study, we present the solution structures of haemogenic endothelium leading to the adaptation to the environment. Tri-methylation PBZ modules of APLF protein in the complex formation of haematopoietic progenitors. Taken at lysine 4 of histone H3 (H3K4Me3) occurs with the fragments of poly(ADP-ribose), Serrano, A.G., Gandillet, A., Pearson, S., Lacaud, together, our data identify novel cell surface at active promoters and can enhance gene revealing for the first time the structural basis of G. and Kouskoff, V. markers allowing us to further refine our transcription. ATX1 is an Arabidopsis how this novel type of zinc finger recognizes Contrasting effects of Sox17- and Sox18- understanding of the events marking progressive trithorax-like protein that controls H3K4Me3 poly(ADP-ribose sustained expression at the onset of blood haematopoietic commitment from the and has a PHD motif which binds PtdIns5P. In specification. mesoderm germ layer. Blood, 2010; 115: 3895-3898. We have previously shown that Sox7 is Morrow, C.J., Ghattas, M., Smith, C., Bonisch, H., transiently expressed at the onset of blood Bryce, R.A., Hickinson, D.M., Green, T.P. and specification and is implicated in the regulation of Dive, C. cell survival, proliferation and maturation of Src Family Kinase Inhibitor Saracatinib haematopoietic precursors. In this manuscript, (AZD0530) Impairs Oxaliplatin Uptake in we have assessed, using embryonic stem cell Colorectal Cancer Cells and Blocks Organic differentiation as a model system, whether Sox17 Cation Transporters. and Sox18, two close homologues of Sox7, may Cancer Res 2010; 70: 5931-5941. act similarly to Sox7 at the onset of haematopoietic development. Sox18-enforced The preclinical evaluation of drug combinations expression led to the enhanced proliferation of is crucial, not only to identify combinations that early haematopoietic precursors while blocking may have clinical utility but also to determine their maturation, a phenotype highly reminiscent whether a drug combination should be avoided. of Sox7-enforced expression. In striking In this study the effect of combining the novel contrast, Sox17-enforced expression dramatically Src family kinase inhibitor saracatinib with increased the apoptosis of these early colorectal cancer standard of care cytotoxic precursors. Similarly to Sox7, Sox18 was agents was investigated in colorectal cancer cell transiently expressed during early lines. The main finding was that saracatinib haematopoiesis, but its expression was impaired the efficacy of the DNA-damaging 10 | Paterson Institute for Cancer Research Scientific Report 2010 Research Highlights | 11


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    Research groups Paterson Institute for Cancer Research 12 | Paterson Institute for Cancer Research Scientific Report 2010 Research Groups - Paterson Institute for Cancer Research | 13


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    Applied Computational Biology and Bioinformatics Group http://www.paterson.man.ac.uk/bioinformatics The Applied Computational Biology and Bioinformatics (ACBB) group is focused on developing an improved understanding of the 98% of the human genome that does not code for proteins, and the role they play in cancer. To do this, the group applies a mixture of computer science, software engineering and mathematics to the analysis of high-throughput data arising Group Leader from proteomics, microarrays and high-throughput sequencing. Crispin Miller We then test our hypotheses at the bench in collaboration Postdoctoral Fellows with other research groups. Work in the group is increasingly James Bradford multidisciplinary, and projects span from wet-bench molecular John Hall (joint with Translational Radiobiology Group) biology through computational analysis to more clinically Hui Sun Leong Yaoyong Li focused studies. Software Architect Tim Yates Next generation sequencing the MBCF have been working closely together The human genome contains over 3 billion to develop automated pipelines that take data that some of these transcripts correspond to expressed, increasing the likelihood of spurious Bioinformatics Programmer residues. Generation of the first sequence from the sequencer and onto our computer Figure 1 Chris Wirth novel protein coding genes, which have yet to be matches by chance. Consequently, additional involved many years of collaboration between cluster, where the preliminary alignments and A) RNA-Seq data aligned to the genome and visualised in the X:Map identified using conventional gene-prediction filtering steps were necessary to reduce the sequencing centres around the world. Progress read-counting tasks that form the first steps of Bioinformatician genome browser. The image shows techniques. In collaboration with the Biological error rate to something more manageable. Jan Taylor (joint with Translational in the field has been remarkable, and recent any RNA-Seq data analysis take place. To do this the region of chromosome 17 Mass Spectrometry Facility a graduate student in Using these approaches, Danny has been able to Radiobiology Group) advances in technology now make it possible to we are making heavy use of the open source containing the gene TP53 (large the group, Danny Bitton, has been using Mass identify hundreds of novel proteins. perform equivalent amounts of sequencing in a software project BioConductor, which provides purple box). Each of the individual Scientific Officer Spectrometry to identify novel proteins. In a few weeks using a single high-throughput access to a wide range of statistical tools and tracks within the gene represents a Paul Scutt different transcript, as annotated by typical proteomics identification experiment, Formalin-fixed paraffin embedded (FFPE) tissue machine. This year, the Molecular Biology Core software libraries for handling the data from our ENSEMBL (Flicek et al., Nucleic Acids proteins are first cleaved into sets of peptides, We are continuing to collaborate with the MBCF Graduate Students Facility (MBCF) took delivery of one such sequencer. Tim Yates has continued to develop Res 2010; epub Nov 2), with the which are then submitted to the mass and the Translational Radiobiology Group in the Danny Asher Bitton machine, an AB SOLiD™, which is capable of our annotation database and genome browser, locations of exons identified by the spectrometer for analysis (Bitton et al., 2010). analysis of microarray data from FFPE tissue. Sharmin Naaz (joint with Stem generating hundreds of millions of short 50-mer X:Map (http://xmap.picr.man.ac.uk), and we have smaller red boxes in each track. Cell and Haematopoiesis Group) The peptides are further fragmented, and the This material is important because vast archives reads in a single machine run over a few days. been using our BioConductor package, White boxes correspond to UTRs. Andrzej Rutkowski (joint with The blue and green graphs overlayed mass/charge ratio of each of the fragments is of well-annotated clinical material have been Although these machines are often used for xmapcore, to supply the genome annotation we Immunology Group) represent RNA-Seq data for two cell measured. This yields a characteristic mass- preserved in this way. They present a challenge, DNA sequencing, they can also be used to need to interpret the RNA-Seq data. 2010 saw lines, MCF7 and MCF10A aligned to spectrum for each peptide that can then be because this method of tissue preservation was Systems Administrator sequence RNA from cells, an approach called our first paper using data from the platform, and the genome, such that the height of identified by searching against a database of developed before techniques such as Zhi Cheng Wang (joint with IT RNA-Seq. When these data are aligned to a we have many other projects currently underway the peaks correspond to the log2 of department) candidate mass spectra derived from a catalogue microarrays were developed, and fixation in reference copy of the genome they can provide in collaboration with other groups in the the number of reads matching at that location, and thus an estimate of known proteins. Although powerful, the formalin can lead to chemical modification and a highly sensitive measure of gene expression, Institute, these include work analysing ChIP Seq of transcript abundance. These data reliance on a database of known targets makes it increased degradation of the mRNA. However, since the number of reads aligning to a particular data and consideration of DNA level changes. can then be grouped by the exons impossible to identify novel peptides using this with the right protocols and data analysis locus provides an estimate of the amount of they match using xmapcore and the technique. We therefore generated a database methods, we are finding that it is possible to RNA originating from that region. Clearly, Proteomics R/BioConductor libraries to yield of candidate proteins by translating the entire extract useful data from archival samples. aligning hundreds of millions of short 50-mer Although only a small part of the genome codes expression levels (B). genome in all three forward and all three sequences to the 3 billion residues that make up for proteins, a substantial proportion is (B) Fold-change plot of log2 exon reverse reading frames, to yield a set of all the human genome, and then mapping them to transcribed, even if it does not result in protein level expression data, showing high possible proteins that could be expressed. By Publications listed on page 66 the location of known genes, is a computationally expression. Increasing numbers of non-coding correspondence for statistically searching mass spectrometry data against this intensive task, and a major focus of the group RNAs are being found to be functional, and a significant loci (black points). Purple expanded dataset, we have been able to identify has been to develop the analysis pipelines and significant proportion of work within the group clouds offset on the y-axis correspond to exons for which a set of additional peptides corresponding to strategies necessary to use this technology is focused on exploring their role in cancer. no-reads matched in the seq data in novel protein coding genes. A challenge with routinely as part of our studies. Chris Wirth However, the large amount of transcription one sample or the other (Bradford et this approach is that the vast majority of from the ACBB group, and Mark Wappett from observed in the genome raises the possibility al., 2010). candidate protein sequences would never be 14 | Paterson Institute for Cancer Research Scientific Report 2010 Applied Computational Biology and Bioinformatics Group | 15


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    Carcinogenesis Group this is being further examined. The crystal structures strongly suggest that both Atl1 and http://www.paterson.man.ac.uk/carcinogenesis MGMT bind to DNA, “flip” out the O6-alkylguanine from the base stack using an arginine “finger”, rotate the phosphodiester bond by means of a tyrosine residue, and accommodate the base in the binding pocket. In the case of MGMT, this allows alkyl group transfer and rapid dissociation of the MGMT, but ATL, unable to transfer the alkyl group, remains bound. The group’s work focuses on the mechanism of action of a Some organisms do not have an alkyltransferase The current evidence suggests that the bound Figure 1 ATL is a substrate for and/or recruits, group of chemical compounds called alkylating agents. Agents Electrophoretic mobility shift assay of gene, and we have established that these possess components of the nucleotide excision repair a different mechanism for dealing with of this type display a wide range of biological effects in living binding of increasing concentrations of Atl1 to short oligonucleotides O6-alkylguanine damage in their DNA. Because system which ultimately results in the elimination of the lesion from DNA. Establishing precisely organisms all of which are attributed to the introduction of containing guanine (G, left panel), O6-methylguanine (central panel) or of the possible implications to repair of alkylation how this occurs and what proteins are involved damage in DNA in man, we are currently various types of DNA damage. The ability of these agents to O6-pyridyloxobutylguanine (right attempting to establish the precise details of how is one of the major tasks of the group. panel). On the left is shown the kill cells is exploited in their use as anti-tumour agents in the structure of the target double this mechanism operates using the fission yeast CHEMORES stranded oligonucleotide and the S. pombe as a model organism. Group Leader treatment of certain types of cancer. Although a number of suggested structures of the Over the past few years we have been one of the members of a European Union Framework Geoff Margison different lesions can be generated in DNA, one of these, O6- complexes. Alkyltransferase-like proteins 6 programme-supported Consortium, the main Alkyltransferase genes are present in Postdoctoral Fellow alkylguanine, seems to be the most important. We are trying prokaryotes, archea and eukaryotes and the objective of which is to investigate the mechanisms of chemotherapy resistance (hence Vitaly Latypov to establish precisely how cells respond to this damage and the encoded proteins share an active site domain the acronym “CHEMORES”) in melanoma and characterized by a cysteine residue, usually within Scientific Officers impact that this has on the biological effects of these agents. the sequence PCHRV, that accepts the alkyl lung cancer. Dr Paul Lorigan from The Christie Gail McGown Hospital Foundation Trust is co-Principal Mary Thorncroft group from the O6-position of guanine. In some Investigator on Work Package 10, which focuses Mandy Watson Background biological effects of O6-methylguanine effects are organisms the cysteine residue is replaced by a on DNA repair aspects of chemotherapy Chemotherapeutic alkylating agents include manifested is the damage reversal protein tryptophan residue and we have previously resistance, of this programme. As we have Graduate Students drugs such as dacarbazine, which is used in the O6-methylguanine-DNA methyltransferase cloned yeast (S. pombe) and bacterial (E. coli) Andy Marriott (until Sept) reported previously, despite promising results treatment of malignant melanoma, and the (MGMT), which can transfer the methyl group genes encoding these proteins and named them from in vitro and preclinical studies of the MGMT Pat Senthong (Jointly with Dr Andy Povey, Health Sciences Cancer Research-UK drug Temozolomide, which from O6-methylguanine and restore the DNA to alkyltransferase-like (ATL) proteins. ATL genes inactivator, LM in combination with group at The University of is used in the treatment of melanoma and its pre-damaged state in a reaction that also are present in a number of organisms, but Temozolomide, the clinical trials of this Manchester) glioma. These and other agents such as results in the inactivation of the protein. MGMT interestingly, while budding yeast only have an combination in late stage melanoma have shown steptozotocin, used in the treatment of and other alkyltransferases can therefore protect alkyltransferase, and S. pombe only has an ATL, no clinical benefit. Prolonging the treatment Undergraduate Students pancreatic cancer, damage cellular cells against both the mutagenic and toxic effects E. coli expresses both alkyltransferase genes (ada schedule did not improve this situation James Ding (Aug-Sept) Sonia McNichol (until May) macromolecules via the formation of highly of alkylating agents. and ogt) and an ATL gene (eATL) so the suggesting that there may be other DNA Ali Bennett (until Sept) reactive methyl groups. Attack on the DNA evolution of these proteins and why some repair-based mechanisms of resistance to this Jo Kelly (from June) bases generates more than a dozen different In some patients, the very poor response of organisms seem to need both functions is itself agent. Studies we undertook many years ago Vitaly Sukhinin (from Sept) types of damage but one of the minor lesions, tumours to dacarbazine or Temozolomide intriguing. showed that nucleotide excision repair (NER) O6-methylguanine which constitutes just 6% of treatment has been attributed to this protective might act on the same type of damage as the total damage, appears to be the product that effect of MGMT. However, our attempts to One of the characteristics of the ATL proteins is MGMT in certain cell lines, but since then, the is responsible for the majority of the biological circumvent this, using the potent MGMT their ability to bind very strongly to alkylguanine vast majority of evidence had supported the effects. Thus, if DNA containing this lesion is inactivating drug, Lomeguatrib (LM), developed in lesions in DNA, and in our original in vitro studies critical role for MGMT in resistance. To pursue replicated, an O6-methylguanine-thymine mispair collaboration with Prof Brian McMurry and the this was shown to effectively block the repair of the possibility of alternative repair processes, we can occur, and further replication of this mispair late Dr Stanley McElhinney (and their group at the lesion by MGMT. In collaboration with David have been examining melanoma cell lines from can result in the formation of a permanent the Chemistry Department, Trinity College, Williams and his group (University of Sheffield) the Karolinska Institute and our own stocks for change in the form of an adenine-thymine Dublin) did not result in improved tumour binding to these lesions in short oligonucleotides evidence of such a pathway. We are using transition mutation. Such mutations are a responses. This was despite preclinical data has also been shown and in collaboration with biochemical assays of functional activity of DNA classical effect of the alkylating agents and have demonstrating that LM effectively sensitised John Tainer and his group (Scripps Research repair proteins along with highly sensitive been seen in oncogenes and tumour suppressor human cells and human tumour xenografts to Institute, La Jolla), crystal structures of Atl1 methods of quantifying DNA lesions generated genes in human tumours, suggesting that the killing effect of Temozolomide and other bound to short duplex oligonucleotides by treatment with Temozolomide. The idea is alkylating agents could be involved in human agents of that type, and clinical data showing that containing O6-methylguanine and the much larger that this pathway is likely to resemble the Atl1 cancer aetiology. The O6-methylguanine-thymine LM very effectively ablated MGMT activity in O6-pyridyloxobutylguanine were published last pathway in S. pombe, so the information and mispair can undergo an alternative fate if it is tumours. The reasons for the lack of year. O6-pyridyloxobutylguanine is generated in methodology that we have from this research is recognised by the post replication mismatch effectiveness of LM may include the existence of DNA by the metabolite of a tobacco-specific being applied to human cells. repair system, which can result in cell death or, if alternative repair pathways for O6-methylguanine, nitrosamine. We suspect that rather than cells survive, chromosomal rearrangement and this is being investigated. S. pombe being exposed to, or endogenously induced by DNA recombination. The most generating such agents, this reflects the size of Publications listed on page 66 critical factor in whether or not any of these the lesion-binding domain in ATL proteins, and 16 | Paterson Institute for Cancer Research Scientific Report 2010 Carcinogenesis Group | 17


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    Cell Cycle Group A and Sho1. In addition, cells lacking Hof1 grew more slowly in the absence of a fourth factor, http://www.paterson.man.ac.uk/cellcycle Sla1 (previously shown to have a related function to Rvs167). Sho1 is a transmembrane protein that activates a signaling pathway culminating in the Hog1 MAP control hof1 cyk3 sho1 kinase, in response to osmotic stress. We found that other components of this pathway also become essential in the absence of Hof1, suggesting that hof1∆ cells might suffer from osmotic stress. It is interesting to note, however, Our group studies the mechanisms and regulation of rvs167 cyk3 hof1 rvs167 hof1 sho1 hof1 that previous work indicated that Sho1 might contribute to activation of the Mitotic Exit chromosome replication and cytokinesis. In animal cells and Network that controls cytokinesis, and so it B yeasts, cell division is driven by the assembly at the cleavage INN1-GFP INN1-GFP hof1-td INN1-GFP rvs167 INN1-GFP sho1 hof1-td INN1-GFP rvs167 hof1-td remains possible that Sho1 plays a more direct 100 role in cell division in cells lacking Hof1. site of a contractile ring of actin, type II myosin and many other % binucleate cells 80 factors. Work with fission yeast showed that the Cdc15 60 Rvs167 is the yeast orthologue of the Amphiphysin protein of higher eukaryotes and is protein is essential for assembly of the contractile ring and 40 20 a regulator of the actin cytoskeleton that plays a Group Leader contains an amino terminal F-BAR domain that induces 0 60' 90' 120' 60' 90' 120' 60' 90' 120' 60' 90' 120' 60' 90' 120' key role during endocytosis. Whereas Hof1 is a member of the F-BAR family, Rvs167 has a Karim Labib membrane curvature as well as an SH3 domain at the carboxyl Time after release from G1 arrest at 37°C / minutes conventional BAR domain at its amino terminus Postdoctoral Fellows terminus that recruits other cytokinesis factors to the cleavage C (an additional alpha helix distinguishes such INN1-GFP INN1-GFP INN1-GFP INN1-GFP INN1-GFP ‘N-BAR’ domains from the F-BAR sub-family) % cells with Inn1 rings/spots Giacomo de Piccoli site. The budding yeast Hof1 protein is orthologous to Cdc15 hof1-td rvs167 sho1 hof1-td rvs167 hof1-td and was one of the founding members of the Luis Garcia-Rodriguez Alberto Sanchez-Diaz but is not essential, and we have screened systematically for BAR domain family. Although Rvs167 is not Sugopa Sengupta closely related to Hof1 and has not previously other SH3 proteins that become essential for cytokinesis in the been shown to act during cytokinesis, both Scientific Officers Frederick van Deursen absence of Hof1. In this way we have identified a novel role proteins share an analogous structure, with an amino terminal BAR or F-BAR domain, and an Pedro Junior Nkosi during cytokinesis for Rvs167, the yeast orthologue of 0 60' 90' 120' 60' 90' 120' 60' 90' 120' 60' 90' 120' 60' 90' 120' SH3 domain at the carboxyl terminus. We have Graduate Students Amphiphysin in higher eukaryotes. Time after release from G1 arrest at 37°C / minutes found that cytokinesis is blocked upon Asli Devrekanli simultaneous inactivation of either Rvs167 and Magdalena Foltman Hof1, or Cyk3 and Hof1. Our data indicate that Tim Maculins The BAR domain (Bin1/Amphiphysin/Rvs167) ring at the cleavage site, in part through binding assembly of the contractile ring is defective in Marija Maric (from October) Figure 1 for cytokinesis (Sanchez-Diaz et al., Nat Cell Biol forms an alpha-helical dimer in the shape of a of F-BAR to the formin Cdc12. In addition to its cells lacking both Rvs167 and Hof1, as is Redundancy between budding yeast 2008; 10: 395). The carboxy terminal 60% of recruitment of Inn1 to the cleavage site. In banana, which is present in a diverse range of amino terminal F-BAR domain, Cdc15 also has SH3 proteins during cytokinesis. (A) Inn1 is very rich in proline and contains many addition we have shown that Inn1 can interact eukaryotic proteins. The best characterized role an SH3 domain at the carboxyl terminus that Cells of the indicated genotypes were examined 24 hours after PXXP motifs that are typically found in the directly via its proline-rich carboxy terminal of the BAR domain is to induce or sense recruits other cytokinesis proteins to the germination of spores. (B) Cells binding sites of SH3 proteins. As Inn1 is essential domain with Rvs167 Hof1, and Cyk3, in a membrane curvature by binding along the length cleavage site. The SH3 domain of Cdc15 is not combining the heat-sensitive hof1-td for cytokinesis in contrast to Hof1, we manner dependent upon the SH3 domains of of the dimer to phospholipids. In diverse essential, but a recent study found that this allele and deletion of the indicated speculated that recruitment of Inn1 (and the latter proteins. Hof1 becomes essential in eukaryotic species, proteins containing BAR reflects redundancy with the SH3 domain of a genes were synchronised in G1 probably other factors) to the cleavage site cells lacking the SH3 domain of Rvs167, and our domains have been shown to play a key role in related F-BAR protein called Imp2 (Roberts- phase at 24°C, and then monitored as they completed mitosis at 37°C. might be shared in a redundant fashion between data indicate that a network of SH3 proteins endocytosis and other aspects of membrane Galbraith et al., J Cell Biol 2009; 184: 113). (C) Recruitment of Inn1 to the Hof1 and other SH3 proteins. These need not collaborate to recruit key factors to the cleavage trafficking. In addition, some BAR domains are cleavage site was monitored in the necessarily have an F-BAR domain like Hof1 or site during cytokinesis in budding yeast (Targosz known to bind a range of small GTPases, It is not yet clear whether analogous F-BAR same experiment. Bzz1, as previous work showed that Hof1 et al., in preparation). Together with the previous indicating that BAR domains can have other proteins play a similar role to Cdc15 during becomes essential in the absence of another studies of Cdc15 and Imp2 in fission yeast, it roles as well as sensing or inducing membrane cytokinesis in animal cells. Nevertheless, the SH3 protein called Cyk3. now seems possible that diverse eukaryotes curvature. budding yeast orthologue of Cdc15 is known as Hof1 (‘Homologue of Fifteen’) and also might use a variety of SH3 proteins to recruit To address in a systematic and unbiased fashion cytokinesis factors to the cleavage site, and these The F-BAR domain is a related module found in contributes to cytokinesis, but the molecular the ability of other yeast SH3 proteins to play a SH3 proteins can have either F-BAR or N-BAR diverse eukaryotic proteins, and also forms function of Hof1 is understood less well, partly redundant role with Hof1 during cytokinesis, we domains that are capable of inducing membrane curved alpha-helical dimers. F-BAR domains due to the fact that Hof1 is not an essential created a series of diploid yeast strains lacking curvature. It is possible that the high potential can induce membrane curvature in vitro or in protein. Budding yeast also contains another one copy of the HOF1 gene and with only one for redundancy between such factors might have vivo, analogous to the action of BAR domains, closely related F-BAR protein called Bzz1, but copy of each of the other 23 genes encoding masked the role of orthologous factors during and have also been found to mediate key there is no evidence to suggest that this shares proteins with SH3 domains. Upon sporulation cytokinesis in previous studies of animal cells, and protein-protein interactions. One of the best an essential role during cytokinesis with Hof1. we separated the meiotic progeny by tetrad this issue might be addressed by more characterized F-BAR proteins is fission yeast analysis, and examined the growth of the systematic studies in the future. Cdc15, which is essential for cytokinesis. The We found previously that Hof1 is the major resultant colonies. In this way we found that F-BAR domain of Cdc15 is thought to play a key binding partner in budding yeast cell extracts of Hof1 becomes essential in the absence of any role in assembly of the contractile actomyosin the Inn1 protein, which we showed is essential one of three other SH3 proteins: Cyk3, Rvs167 Publications listed on page 67 18 | Paterson Institute for Cancer Research Scientific Report 2010 Cell Cycle Group | 19


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    Cell Division Group Cdc25 because it inappropriately prompts Polo to shut down Wee1. This line of work subsets of sites that influence Cut12’s ability to modify mitotic commitment in different ways. http://www.paterson.man.ac.uk/celldivision uncovered a direct relationship between polo Mutations to acidic residues, in an attempt to activity and Cut12 status; Polo activity was mimic the phosphorylated state in one subset, elevated when Cut12 function was enhanced mirror the ability of cut12.s11 to promote and severely reduced when Cut12 function was mitosis when Cdc25 activity is compromised. compromised. Furthermore, Polo association This suggests that the activation of Cut12 helps the SPB was modified by promotion of Cut12 to flip the mitotic switch into a “pro-mitotic function. Polo normally associates with the SPB state” that drives commitment to mitosis. for 30 minutes prior to mitosis. In cut12.s11 cells this association occurred earlier, starting an Initiation of mitotic commitment at the pole hour before mitosis. in humans Errors in chromosome transmission alter the balance of The investigations into the initiation of mitosis in Polo and the environmental control of human cells by the group of Prof. Jon Pines tumour suppressor and tumour promoter genes. This mitotic commitment (Gurdon Institute, Cambridge) have revealed imbalance favours changes in genome composition in the Phosphorylation of Polo on serine 402 promotes that active MPF first appears on the its association with the SPB thereby promoting centrosomes (spindle poles). This strongly ensuing cell divisions that can lead to cancer. Chromosome commitment to mitosis. The reliance of serine suggests that the networks we are studying in segregation during mitosis is initiated by the attachment of the 402 phosphorylation upon signalling from the yeast occur in human cells. In other words, key Sty1 MAPK kinase links changes in the decisions about whether to divide or not do not microtubules of the mitotic spindle to the chromosomes. extracellular environment to the timing of cell arise from the gradual accumulation of a “pro Group Leader Once all chromosomes have become attached to both spindle division. For example, Sty1-dependent mitosis” state, rather, they are taken at a discrete phosphorylation of serine 402 is promoted by a location, the spindle pole. This concentration of Iain Hagan poles the chromosomes split into two identical chromatids that reduction in the quality of nutrients to accelerate signalling to a limited subset of molecules at a Associate Scientist then move to the poles. Because the regulatory networks that division. Similarly MAPK driven phosphorylation discrete location facilitates rapid and highly at this site promotes re-entry into cell division sensitive cross talk between different signalling Agnes Grallert regulate mitotic progression are highly conserved, studying the after it has been blocked by a rapid heat shock. networks. Postdoctoral Fellows complexities of cell division in the relatively simple unicellular Marisa Alonso-Nuñez Loops within loops Lessons from yeast Ashapurna Biswas yeasts greatly accelerates the analysis of the more complex Our recent efforts have focused upon assessing The ability to manipulate genes at will in a simple Marisa Madrid Ye Dee Tay issue of the control of cell division in man. the impact of phosphorylation upon the control organism, whose primary purpose is to divide, is of Cut12 function and addressing the means by enabling us to explore the finer points of the Scientific Officer which events on the interphase SPB influence pathways that co-ordinate a successful cell We study cell division in the fission yeast Specifically, they suggest that the MPF amplifying division. This information informs studies in Kuan Yoow Chan the subsequent commitment to mitosis. Schizosaccharomyces pombe because it is a positive feedback loop is primed from the SPB. higher systems that, in turn, raise models that can Surprisingly, we have found that factors that Graduate Students simple, unicellular organism with excellent The core data that prompted the work leading be most readily tested in yeast. This re-iterative were previously considered to be strictly mitotic Elvan Boke genetics that is cheap to grow and divides to this view are reciprocal genetic interactions cycle of comparative studies ensures that great Dorota Feret are required for the interphase control of Cut12 rapidly. Commitment to mitosis in S. pombe is between cut12 and cdc25. The cut12.s11 gain of strides are being made in understanding the Avinash Patel function at the SPB to promote mitosis. regulated by the activity of a protein kinase function mutation suppresses loss of function molecular basis of cell division. Furthermore, assessment of the functional called MPF. MPF is composed of a catalytic mutations in cdc25. Conversely, mutational significance of phosphorylation on Cut12 at 25 sub-unit encoded by the cdc2+ gene and a enhancement of Cdc25 activity suppresses loss mitotic different sites has identified different regulatory sub-unit called Cyclin B. Prior to of Cut12 function. Consistently, combining mitosis MPF is inhibited via phosphorylation by conditional loss of function mutations within the protein kinase Wee1 on a residue (tyrosine cdc25 and cut12 in the same strain generates 15) that lies in the ATP-binding pocket of p34cdc2. synthetic lethality. Legend This phosphate can be removed by a protein phosphatase encoded by the cdc25+ gene. The Figure 1 G2 M SPB Cut12 and polo in mitotic commitment balance of activity between Cdc25 and Wee1 is In seeking ways to understand how an SPB nuclear envelope the critical factor in determining when MPF will component could compensate for loss of Cdc25, phosphate P be activated to drive mitotic commitment. Once a critical threshold level of MPF is activated a we exploited the key genetic relationship uncovered by Peter Fantes in 1979: removal of ?+ positive feedback loop is promoted to boost Wee1 function enables cells to survive without P P Cdc25 Cdc25 activity and suppress Wee1 activity, Cdc25. The molecular basis for this genetic Cdc2 Cdc2 thereby driving full-scale commitment to mitosis. observation is now very clear: without the kinase CyclinB CyclinB Fully activated MPF then activates a number of that puts the phosphate in the catalytic pocket Wee1 highly conserved kinases that are named after of Cdc2, there is no requirement for a the founder members of each group Polo, phosphatase to remove this phosphate. Thus, ? Aurora and NIMA. MPF activation and mitotic commitment will occur in the absence of Cdc25 when Wee1 is P Cut12, the spindle pole and mitotic inhibited. A second cue for the direction for our Plo1 Environmental commitment studies came from the key role played by Polo Sty1 cues Our studies of the spindle pole body (SPB) kinase in the MPF positive feedback loop in Cut12 component Cut12 have uncovered a critical role higher eukaryotes. We therefore considered the for events on the spindle pole in mitotic control. possibility that Cut12 suppresses ablation of 20 | Paterson Institute for Cancer Research Scientific Report 2010 Cell Division Group | 21


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    Cell Regulation Group mediating a general stress response. Like its mammalian counterpart, p38, Sty1 is http://www.paterson.man.ac.uk/cellregulation phosphorylated and activated by a variety of stress stimuli and inactivation of the kinase results in pleiotropic stress sensitivity. Sty1 phosphorylates a number of targets and co-ordinates an integrated response to stress by affecting the activities of proteins involved in a range of cellular activities; an example being the Atf1 transcription factor which directs the expression of many stress-induced genes. We MAP kinase signalling pathways are frequently deregulated in have developed a novel biochemical approach to identify targets of Sty1 and are currently cancer. However, distinct MAP kinase families regulate different characterising a number of previously and diverse cellular programmes. While oncogene-mediated regulation by ATF2 of specific JNK specific unidentified substrates for this kinase. These include proteins involved in processes such as activation of the ERK MAP kinase family is commonly Figure 1 phosphatases in hepatoblasts and HCC. transcription, cell cycle control and signal associated with cell survival and growth promotion, the p38 ATF2 B-cell specific knockout mice display significantly accelerated Eμ- In an independent mouse model we found that transduction. MAP kinase family can induce cell cycle arrest and apoptosis Myc induced B lymphoma onset in the absence of ATF2/7 in B-cells, cMyc (Eμ- For any MAP kinase, both the amplitude and compared to ATF2 heterozygote or Myc) dependent B lymphoma development was Group Leader and is generally regarded as tumour suppressive. In contrast, wild type controls (red, blue lines). significantly accelerated. Further experiments in duration of signalling is carefully controlled. (All mice were on an ATF7 knockout These parameters can greatly influence the Nic Jones the JNK MAP kinase family has been associated with both background.) vitro suggested that B-lymphoma cells deficient cellular response to the input signal and their for ATF2/7 are also less prone to increased tumour promoting as well as tumour suppressing activities. spontaneous and genotoxic drug-induced control is orchestrated through positive- and negative-acting signals, as well as through the Associate Scientists Wolfgang Breitwieser For example, in hepatocellular carcinoma (HCC), JNKs have apoptosis compared to ATF2 proficient tumour spatial distribution of the pathway’s components. cells. Chemical inhibition experiments further Caroline Wilkinson been found to be frequently hyperactive and in a mouse showed that DNA damage induced apoptosis in The negative-acting signals include phosphatases which dephosphorylate and thereby inactivate Postdoctoral Fellows model for HCC the germline loss of JNK1 resulted in reduced cMyc transformed B cells is also JNK dependent. the MAPK. Two classes of phosphatase act upon It is therefore likely that JNK mediated induction Yujun Di Saki Kondo tumour formation suggesting that JNK1 is a major tumour of apoptosis depends, at least in part, on ATF2 Sty1: the Pyp and the Ptc phosphatases which dephosphorylate P-tyr and P-thr residues Hayley Thirkettle driver in liver cancer. dependent gene expression. respectively. The tyrosine-specific phosphatase, Pyp1, is constitutively expressed and is largely Scientific Officers Stress responses in fission yeast responsible for maintaining low levels of active Keren Dawson Among the critical targets of MAP kinase knockout hepatoblasts resulted in early onset The Cell Regulation Group also uses fission yeast Figure 2 Sty1 under basal conditions. We have found that Steve Lyons activities are members of the AP-1 transcription and significantly accelerated development of The fission yeast stress-activated as a convenient model to gain insights into the factor, a dimeric transcription factor consisting of tumours in recipient livers compared to ATF2 Pyp1 is a phospho-protein that becomes MAP kinase pathway. The genes nature and regulation of stress responses. As Jun, Fos, and ATF family DNA binding proteins. wild type control cells. Furthermore, this model encoding some of the phosphatases hyper-phosphorylated upon stress. Many of the Graduate Students these processes are conserved in eukaryotes, it phosphorylation sites have been mapped and Malgorzata Gozdecka Depending on the cellular context the also allowed us to induce ATF2 deletion into that down-regulate Sty1 are induced upon stress in a Sty1/Atf1 dependent is anticipated that these insights will be relevant a kinase responsible for a subset of them has Emily Holmes composition of the dimeric complexes cells that were derived from established HCC. to mammalian cells. In Schizosaccharomyces Jacek Walczynski manner and hence form part of a been identified. We have generated both non- determines the regulation of growth, survival or When these cells were reintroduced into negative feedback loop. pombe, the Sty1 MAP kinase plays a key role in Lu Zhang apoptosis. Using genetic model systems our recipient mice as grafts, the ATF2-deleted phosphorylatable and phospho-mimic mutants group is exploring the functions and molecular tumour cells grew markedly faster compared to of Pyp1 and are using these to study how activities of MAP kinase pathways and in controls suggesting that tumours which had phosphorylation of the phosphatase affects its particular their targets among the ATF family, in developed in the presence of ATF2 gained ability to modulate Sty1 activation. stress and oncogenic transformation. significant growth potential once ATF2 has been inactivated. In vitro assays, for example colony Ptc4 is a PP2C family phosphatase and we have Exploring the role of ATF2 in cancer models formation in soft agar, confirmed the growth found that it regulates both the magnitude and Among the ATF family, ATF2 and ATF7 are advantage of ATF2/7 mutant tumour cells duration of Sty1 activation in response to closely related proteins with a wide spectrum of observed in animals. Interestingly, both double hydroperoxides, but not to other stress transcriptional targets. To understand potential knockout hepatoblasts and tumour cells had conditions. Furthermore, Ptc4 undergoes a roles for ATF2, and ATF7, we have adapted markedly elevated levels of activated JNK. In post-translational modification specifically in different mouse models of oncogenic addition experiments using chemical inhibitors response to hydroperoxides. The oxidative transformation. To explore their role in showed that while colony formation was largely stress-generated isoform has increased affinity oncogenic Ras-mediated transformation and independent of ERK and p38, it was strongly for Sty1 thus providing an explanation for the tumorigenesis in the liver, we adopted a model dependent on JNK activity. In conclusion, this stress-specific nature of Ptc4’s regulation of Sty1. which is based on the explantation of embryonic suggested that JNK is a crucial mediator of We are currently investigating the mechanism hepatocyte precursors (hepatoblasts), and their hepatoblast growth and HCC development and that underpins the modification of Ptc4 under oncogenic transformation in vitro followed by that ATF2/7 may be critical in limiting the these conditions. their reintroduction into recipient mice through activated levels of JNK, possibly through orthotopic transplantation. We found that regulating negative acting feedback mechanisms. introduction of activated H-Ras into ATF2/7 We are currently investigating the potential Publications listed on page 67 22 | Paterson Institute for Cancer Research Scientific Report 2010 Cell Regulation Group | 23


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    Cell Signalling Group Novel role of Tiam1-Rac signalling during bipolar spindle assembly that modifier (SUMO) E3-ligase, PIAS3, as a novel Rac interacting protein. We found that PIAS3 http://www.paterson.man.ac.uk/cellsignalling facilitates chromosome congression and interacts better with the GTP-bound form of mitotic progression Rac. We then showed that PIAS3 is required for Centrosome separation, critical for bipolar increased Rac activation and optimal cell spindle formation and subsequent chromosome migration in response to hepatocyte growth segregation during mitosis, occurs via distinct factor (HGF) signalling. Subsequently we prophase and prometaphase pathways. Kinesin- demonstrated that Rac1 can be conjugated to 5 (Eg5), a microtubule (MT) motor, pushes SUMO-1 in response to hepatocyte growth centrosomes apart during bipolar spindle factor treatment and that SUMOylation is assembly and its suppression results in enhanced by PIAS3. We also showed that the monopolar spindles and mitotic arrest. Forces GTP-bound form of Rac is a better substrate for Tumour initiation and progression result from inappropriate that antagonize Eg5 in prophase are unknown. SUMOylation. Furthermore, we identified non- We identified a new force-generating mechanism consensus sites within the polybasic region of activation of intracellular signalling cascades. Rho-like GTPases mediated by Tiam1, dependent on its ability to Rac1 as the main location for SUMO are molecular switches in signalling pathways that regulate activate Rac. We revealed that Tiam1 and Rac conjugation. We demonstrated that PIAS3- localize to centrosomes during prophase and mediated SUMOylation of Rac1 controls the cytoskeletal and junctional organisation, as well as gene prometaphase, and Tiam1, acting through Rac, levels of Rac1-GTP and the ability of Rac1 to transcription. In this way, Rho proteins influence cell ordinarily retards centrosome separation. stimulate lamellipodia, cell migration and invasion. Importantly, both Tiam1-depleted cells in culture The finding that a Ras superfamily member can morphology, adhesion, motility, as well as cell cycle progression and Rac1-deficient epithelial cells in vivo escape be SUMOylated provides an insight into the Group Leader and cell survival. Rho proteins are transforming in vitro and are the mitotic arrest induced by Eg5 suppression. regulation of these critical mediators of cell Moreover, Tiam1-depleted cells transit more behaviour. Moreover, our data revealed a role Angeliki Malliri essential for Ras-mediated in vitro transformation. Moreover, slowly through prometaphase and display for SUMO in the regulation of cell migration and Postdoctoral Fellows data have emerged to directly implicate Rho proteins in Figure 1 increased chromosome congression errors. invasion (Castillo-Lluva et al., 2010). Significantly, Eg5 suppression in Tiam1-depleted Sonia Castillo-Lluva tumour initiation and progression in vivo. Our group (A) MDCKII cells stably expressing cells rectifies not only their increased A distinct role for the Tiam1 family member, Helen Rushton GFP-tagged Tiam1 (green) stained Simon Woodcock (until March investigates the mechanisms by which certain regulators of the also for tubulin (red) and DNA (blue) centrosome separation but also their STEF, in regulating focal adhesions 2010) reveal Tiam1 localization to chromosome congression errors and mitotic The mechanisms underlying focal adhesion Rho protein Rac control cell cycle progression and cell centrosomes at prophase. (B) Cells delay. These findings identified Tiam1-Rac disassembly, required for optimal cell migration, Scientific Officer Gavin White adhesion and how their activities, as well as activity of Rac treated with low concentrations of the Kinesin-5 (Eg5) inhibitor- STLC- signalling as the first antagonist of centrosome are poorly understood. Microtubules are critical separation during prophase, demonstrated its mediators of this process; direct targeting of Graduate Students itself, are controlled. We are also identifying signalling events and expressing control siRNA (Scr RNAi) display monopolar mitotic requirement in balancing Eg5-induced forces focal adhesions by microtubules coincides with Natalie Mack and cellular processes downstream of Rac that modulate spindles while cells treated with STLC during bipolar spindle assembly in vitro and in their disassembly. Re-growth of microtubules, Chong Tan and expressing siRNA selectively vivo, and showed that proper centrosome induced by removal of the microtubule Hadir Marei (from October) tumour susceptibility and disease progression. targeting Tiam1 (RNAi #1 and #2) separation in prophase facilitates subsequent destabiliser nocodazole, activates the Rho-like have bipolar spindles. (C) Model: chromosome congression (Woodcock et al., GTPase Rac, concomitant with focal adhesion Suppression of Eg5 in control cells Tiam1 (for T-lymphoma invasion and metastasis a non-receptor tyrosine kinase implicated in leads to a monopolar spindle, 2010). disassembly. Recently we have shown that STEF protein) is a guanine nucleotide exchange factor malignant progression, potently induces resulting in the activation of the is responsible for Rac activation during (GEF) that selectively activates Rac. Mice epithelial–mesenchymal transition (EMT) by spindle assembly checkpoint (SAC). SUMOylation of the GTPase Rac1 is required microtubule re-growth. Importantly we also Conversely, Tiam1-depletion results in for optimal cell migration showed that STEF is required for multiple deficient for Tiam1 are resistant to the formation targeting AJs for disassembly. We recently a greater intercentrosomal separation of skin tumours induced by chemical carcinogens showed that direct phosphorylation of Tiam1 by It is well established that Rac1 induces targeting of focal adhesions by microtubules. As in the early stages of mitosis, leading and consequent oncogenic activation of the Src is required for the initial stages of Src- to mis-aligned chromosomes and cytoskeletal rearrangements required for cell a result, focal adhesions in STEF knock-down c-Ha-Ras gene. Nonetheless, the few skin induced EMT. Moreover, we identified a novel activation of the SAC. However, migration. Rac activation is regulated through a cells have a reduced rate of disassembly and are tumours arising in these mice progressed more post-translational mechanism of regulating Tiam1 Eg5 suppression within a Tiam1- number of mechanisms, including control of consequently enlarged. This leads to a reduced depleted cell now restores the inter- nucleotide exchange and hydrolysis, regulation of speed of migration in these cells (Rooney et al., frequently to malignancy than those in wild-type levels. We showed that Src phosphorylates centrosomal distance to that of a mice, suggesting that Tiam1 deficiency promotes Tiam1 on tyrosine 384 (Y384). This occurs subcellular localization or modulation of protein- 2010). ‘control’ Eg5 inhibitor-untreated malignant conversion (Malliri et al., Nature 2002; predominantly at AJs during the initial stages of mitotic cell, thus allowing efficient expression levels. We performed a screen for 417: 867). Src-induced EMT and creates a docking site on chromosome congression and proteins that interact with Rac under conditions Tiam1 for Grb2. We found that Tiam1 is therefore progression through mitosis. of migration and identified the small ubiquitin-like Publications listed on page 67 One mechanism by which Tiam1 and Rac constitutively associated with extracellular signal- suppress malignant progression is through regulated kinase (ERK). Following recruitment of promoting cell–cell adhesion. Over-expression of the Grb2-Sos1 complex, ERK becomes activated activated Rac or Tiam1 promotes the formation and triggers the localised degradation of Tiam1 of adherens junctions (AJs) and the at AJs through activating calpain proteases. accompanying induction of an epithelial-like Abrogating Tiam1 phosphorylation and phenotype in a number of mesenchymal cell degradation suppressed Src-induced AJ lines (Malliri and Collard, Curr Opin Cell Biol disassembly. As a consequence, cells expressing 2003; 15: 583). Moreover, Tiam1 is required for a non-phosphorylatable Tiam1 showed a marked both the formation as well as the maintenance decrease in wound closure in response to Src of cadherin-based adhesions (Malliri et al., J Biol (Woodcock et al., Mol Cell 2009; 33: 639). Chem 2004; 279: 30092). The oncoprotein Src, 24 | Paterson Institute for Cancer Research Scientific Report 2010 Cell Signalling Group | 25


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    Clinical and Experimental Pharmacology Group Kalena Marti Marti Danielle Shaw biomarkers and notably, the smallest percentage cells induced to die or to stop proliferating that Circulating tumour cells Clinical studies involving CTCs are currently are http://www.paterson.man.ac.uk/cep Scientific Officers can be detected. The imaging studies are underway in melanoma, lung, colorectal prostate Jessica Booth conducted with parallel collection of tumour and and pancreatic cancers. CTC number and in Damien Brown Fouziah Butt blood samples allowing circulating, tissue and some cases assessment of drug target levels, Martin Dawson imaging biomarkers to be correlated with the and/or potential biomarkers of drug Olive Denneny horizon of improved interpretation of multi responsiveness are being assessed. Using the Maciej Dolniak Martin Greaves modality biomarker data in the clinic. Further CellSearch technology platform, we Grace Hampson collaborations have recently been initiated to demonstrated in a cohort of 101 Non Small Cell Cassandra Hodgkinson utilise these ‘cell fate switch’ models further in Lung Cancer patients that those with ≥5 CTCs Paul Kelly Matthew Lancashire MRI/S imaging studies to investigate the effects per 7.5ml blood had a significantly worse Daniel Morris of tumour cell death or proliferation on the prognosis than those with fewer CTCs (Krebs et CEP validates and implements pharmacodynamic, prognostic Karen Morris Andrew Price extracellular and extravascular space. al., JCO in press). We observed that tumour cells within circulating tumour microemboli and predictive biomarkers working in tandem with the Early Tony Price Lyndsey Priest Preclinical pharmacology of PI-3K inhibition (clusters of CTCs) in lung cancer patients were Clinical Trials Unit at the Christie Hospital. This year witnessed Robert Sloane Nigel Smith There are compelling links between the activity out of cycle and appeared to avoid apoptosis of phosphatidylinositol-3 kinase (PI3K) and whereas some subpopulations of single CTCs the opening of the new £35M Cancer Treatment Centre Hannah Turpin common human cancers and great interest in were proliferative and others were apoptotic incorporating one of the largest early clinical trials units Graduate Students Cristina Martin-Fernandez targeting PI3K as an anti-cancer therapy. By (Hou et al., Am J Path, in press). We recently inducible over-expression of a dominant negative developed a multi-colour immunofluorescence worldwide. In parallel, CEP opened its third Good Clinical Elizabeth Hitchman Shaun Villa PI3K subunit or addition of commercially assay that can now be applied to explore the Practice (GCP) biomarker laboratory reflecting the current Scientific Administrator available PI3K inhibitors in colorectal cancer process of epithelial to mesenchyme transition in (CRC) cell lines, we demonstrated that PI3K CTCs (Figure 1). and future significant increases in clinical trials incorporating Aileen Jardine inhibition reduced cellular proliferation via delay biomarkers of cell death and of angiogenesis, mutation in G1 phase of the cell cycle without increased Progress on the CEP quality management apoptosis. This occurred in vitro and in vivo and system detection in circulating free DNA (cfDNA) and Circulating to the same degree in cells with mutant and Quality Assurance is vital to success of the three Tumour Cells (CTCs). Biomarker research highlights reported wild-type PIK3CA (the gene encoding the PI3K GCP laboratories and the new Biomarker subunit p110α). PI3K inhibition did not Laboratory in the expanded clinical trials Unit at this year include preclinical pharmacology of PI-3K inhibition, modulate the sensitivity of CRC cell lines to the the Christie Hospital. The CEP QA team was studies of CTC number in lung cancer, a proteomic approach CRC standard of care cytotoxic agents expanded this year and continues to be a driving oxaliplatin or 5FU, however there was a cell line force within the Paterson, the Manchester to define early changes in the micro-environment of a cell dependent increase in the sensitivity of cells to ECMC, the national ECMC network and the Group Leaders destined for apoptosis and pilot studies with colleagues at the SN38 (the active metabolite of Irinotecan). The Manchester Cancer Research Centre for quality Caroline Dive and Wolfson Molecular Imaging Centre (WMIC) to examine inducible dominant negative PI3K system was assurance (QA), regulatory affairs and expertise also exploited in a pilot phosopho-proteomics in biomarker method validation. Twenty eight Malcolm Ranson changes in imaging biomarkers that accompany apoptosis or study aimed at the identification of novel new biomarker projects were initiated this year, Disease Focus Team Leaders downstream targets of PI3K signalling. This has 25 protocols were written, reviewed and Fiona Blackhall cytostasis. revealed several potential new direct or indirect approved; 31 study reports were QA checked Guy Makin Andrew Renehan targets of PI3K signalling which are now being and signed off and 56 Standard Operating Preclinical models of induced tumour cell suggesting reduction of molecules normally verified. Procedures were issued. Significant QA input Staff Scientists was invested in three Jeff Cummings death and cytostasis as tools for circulating released by viable tumour cells into their Tim Ward biomarker discovery and characterisation of microenvironment might be an early Figure 1 major undertakings in imaging biomarkers consequence of triggering tumour apoptosis. A Multicolour immunofluoresence of 2010 a) the Associate Scientists H526 lung cancer cells spiked into implementation of a Kathryn Simpson We developed and validated colorectal cancer panel of cell death biomarkers is now under volunteer blood and recovered by Laboratory Information Christopher Morrow cell lines engineered to express a robust validation for future clinical implementation. ISET filtration onto a track edged Management System Clinical Lecturers doxycycline (dox) inducible constitutively active polycarbonate membrane perforated mutant of effector caspase 3. This allows In collaboration with Dr Kaye Williams and Dr by 8µM pores. Tumour cells were (LIMS), b) achieving Emma Dean Alastair Greystoke induction of synchronous apoptosis where Chris Cawthorne at the WMIC, pilot studies in stained for E-cadherin (green), white compliance to the changes in released/secreted proteins early and pre-clinical PET imaging, using [18F]FLT (a blood cells were stained for CD45 MHRA guidance Postdoctoral Fellows (red) and cell nuclei were stained document on GCP for Alison Backen late after ‘death switching’ are being identified biomarker of proliferation) or [18F]FDG (a with DAPI (blue). Ivona Baricevic-Jones using quantitative proteomics both in monolayer biomarker of tumour metabolism) are being laboratories, and c) Luke Harrison culture, and in the blood stream of mice bearing conducted in mice bearing colorectal cancer conducting a Sarah Holt (AZ secondment) comprehensive Jian Mei Hou ‘death switched’ tumour xenografts in tumour xenografts in which cytostasis or David Moore collaboration with Prof Tony Whetton’s apoptosis are synchronously dox-induced. programme to validate Darren Roberts proteomics team. Proteins released into cell Changes in tumour cell population dynamics can all the software systems Cong Zhou within CEP. media from ‘death switched’ cells in vitro were be induced in all or a defined percentage of cells Clinical Fellows also identified in the bloodstream of mice within the ‘switched’ tumour allowing us to Jenny Adamski Kyaw Aung bearing ‘death switched’ tumours. Interestingly, explore the impact of changes in cell death or early after dox addition and before loss of proliferation on these commonly used PET Publications listed on Sarah Hughes Leila Khoja plasma membrane integrity, most changes in imaging biomarkers. This approach is allowing us page 67 Matthew Krebs Gireesh Kumaran secreted/released proteins were decreases to define the dynamic range of the imaging 26 | Paterson Institute for Cancer Research Scientific Report 2010 Clinical and Experimental Pharmacology Group | 27


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    DNA Damage Response Group transiently delays chromosome condensation and nuclear envelope breakdown in response to Another class of proteins in focus of our research are macro-domain proteins (Figure 2). http://www.paterson.man.ac.uk/dnadamage a variety of stresses. The elucidation of the The macro-domain is another module with the function of the PBZ motif gave us a vital clue to capacity to bind poly(ADP-ribose) and we discover that the CHFR-dependent checkpoint is recently identified several human macro-domain regulated by PARPs and that the PBZ motif in chromatin-associated protein factors that are the CHFR protein is critical for checkpoint recruited to broken DNA ends in a poly(ADP- activation. Another PBZ-regulated protein that ribose)-dependent manner. These include a we are studying is a protein called Aprataxin- histone H2A variant called MacroH2A and PNK-like factor (APLF). APLF uses tandem PBZ chromatin remodeler called ALC1 (Amplified in repeats for direct interaction with poly(ADP- Liver Cancer; also know as CHD1L), as well as ribosyl)ated PARP1, which allows APLF’s timely several other uncharacterized macro-domain DNA is constantly exposed to damage and living organisms localization to the sites of DNA damage (Figure proteins. 1). We recently discovered that the role of APLF have evolved a variety of DNA repair mechanisms to maintain is to act as a histone chaperone to modulate genome stability. Inadequate or abnormal DNA repair can chromatin structure and facilitate DNA repair Publications listed on page 68 reactions in response to poly(ADP-ribose) cause diseases that in humans are associated with cancer, signalling (Mehrotra et al., Mol Cell 2011; in neurodegeneration, immunodeficiency or developmental press). abnormalities. Therefore, furthering our understanding of the Group Leader molecular pathways employed in the mammalian response to Figure 1 Ivan Ahel DNA damage potentially provides a basis for the development Recruitment of fluorescently labelled APLF protein to the laser-induced Postdoctoral Fellows of new therapies in the treatment of human disease. DNA break sites in the cell nucleus. The recruitment is blocked by Dragana Ahel treatment of cells with a specific Rolf Kraehaenbuehl (until Sept) Many cancer therapy procedures, such as poly(ADP-ribosyl)ation has been known to have PARP inhibitor (lower panel). Marianna Rossi radiotherapy and certain types of chemotherapy, a role in the relaxation of chromatin structure, as Scientific Officer work by overwhelming the capacity of the cell to well as in apoptotic signalling. The recent Ria Weston repair DNA damage, resulting in cell death. development of potent PARP1/2 inhibitors Most rapidly dividing cells - cancer cells - are provided powerful tools to study pathways Graduate Students preferentially affected by such treatments, regulated by poly(ADP-ribose), as well as Eva Barkauskaite (from October) Pawan Mehrotra providing the opportunity to use DNA damaging providing a very promising novel class of drugs agents to selectively kill cancer cells. In addition, for cancer treatment. Specifically, selective Visiting Scientist genomic instability is the driving force of cancer inhibition of the single-strand break repair Dea Slade development, which requires multiple DNA pathway using permeable PARP inhibitors has mutations resulting in the loss of cellular growth been proven highly effective against breast and control. In order to accelerate the accumulation ovarian cancers (Bryant et al., Nature 2005; 434: of these genetic changes, cancers often sacrifice 913). Thus, understanding the molecular basis of specific DNA repair pathways. This can make poly(ADP-ribose)-dependent DNA repair cancer cells additionally susceptible to DNA processes is likely to be of vital importance for damaging agents and/or to inhibitors that block selecting and developing efficient therapies. alternative repair pathways that allow cancers to thrive without the full DNA repair repertoire. Identification and characterization of novel Figure 2 For these reasons, studying the protein poly(ADP-ribose)-regulated factors 3D structural overlay of the 4 different macro-domain protein active components involved in the repair of damaged Our laboratory is particularly interested in the sites with bound ADP-ribose. DNA has been proven to be a valuable strategy identification of new pathways and protein in searching for novel approaches and targets in functions regulated by poly(ADP-ribosyl)ation. cancer therapy. Recently, in screening for proteins with the ability to bind poly(ADP-ribose), we discovered a Poly(ADP-ribosyl)ation in the regulation of poly(ADP-ribose)-binding zinc finger motif DNA repair (PBZ). PBZ is a structurally distinctive, atypical The role of poly(ADP-ribosyl)ation is best type of zinc finger that is associated with several understood in the regulation of DNA repair, proteins involved in response to DNA damage which is controlled by the two poly ADP ribose (Ahel et al., Nature 2008; 451: 81). One of the polymerases (PARPs) responsive to DNA strand human proteins containing a PBZ motif is a breaks (PARP1 and PARP2). Poly(ADP- protein called Checkpoint protein with FHA and ribosyl)ation arising at the sites of damaged RING domains (CHFR). CHFR is a ubiquitin DNA serves as a platform for the specific ligase frequently inactivated in human epithelial recruitment and scaffolding of DNA repair tumours, which acts as a key regulator of the complexes. In addition, damage-induced poorly understood early mitotic checkpoint that 28 | Paterson Institute for Cancer Research Scientific Report 2010 DNA Damage Response Group | 29


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    Drug Discovery Group chemistry group onto one of the cancer lead identification projects in the DL. In just six in silico to calculate how well they interact, in all possible conformations that they can adopt, with http://www.paterson.man.ac.uk/drugdiscovery months, our chemistry team designed and the biological target of interest. Those with the synthesised over 350 novel compounds. This has most promising calculated affinity can then be significantly advanced this project, and one of the purchased and screened in a real biological assay chemical series embellished by our medicinal to confirm this activity (or not). The virtual chemists has been selected for further nature of this approach allows the investigation optimisation by the DL team. In addition to the of a higher number and wider diversity of benefits for this cancer drug discovery project, chemical compounds than would be possible, or this collaboration also allowed our chemistry cost-effective, in a HTS screen. VS is not without capability to be fully established and immediately its limitations and can only be utilised when the deployed onto our first in house hit-to-lead atomic structure of the target protein is known. During 2010 we have opened and equipped a new Drug project in September. The second vital Despite this caveat, it offers a useful strategy for component of the DL collaboration has been the generation of starting points for new drug Discovery laboratory, recruited a highly skilled team and have the running of high throughput screens (HTS) discovery programmes which is complementary initiated the Manchester Cancer Research Centre drug for novel cancer drug targets initiated in our to HTS. laboratory. In HTS, a simple drug target-related discovery portfolio. Four hit-to-lead phase projects are now assay is interfaced with a collection of ~100,000 Our current drug discovery portfolio consists of underway targeting a variety of cancer targets. These activities diverse chemicals in order to try and identify four hit-to-lead projects, two of each directed at compounds (“hits”) which interfere with the metabolism and DNA repair biological targets. are underpinned by multiple collaborations and significant functional activity of the target. The first HTS In support of these projects, we have initiated a Group Leader investments in infrastructure and informatics. campaign with an MCRC target was completed range of target biology and technology-related in July 2010 and has identified useful start points collaborations both within and beyond the Donald Ogilvie for our initial hit-to-lead project. At the time of MCRC. Infrastructure These compounds, carefully selected to remove writing, a second HTS campaign is also underway Head of Chemistry Following completion of the building phase, on any potentially troublesome or toxic chemical against a second target and these efforts are The future Allan Jordan time and within budget, in December 2009, the features, also form the basis of our “virtual anticipated to generate preliminary data in In 2011, with the increased funding stream for refurbished laboratory was equipped with capital screening collection”, the use of which is December 2010. this programme, we will be doubling the size of Principal Chemists Niall Hamilton items and open for the arrival of the first new described in more detail below. In collaboration the team and progressing the project portfolio Stuart Jones recruits in January 2010. By the end of April, the with an expert chemoinformatics partner, we Whilst HTS campaigns can provide useful from hit identification to the more advanced lead team was up to full strength with five have completed the major task of formatting this chemical startpoints, we have diversified our hit- identification phase, in which the potential of a Principal Bioscientist bioscientists and five medicinal chemists. In the database for use in generating startpoints for finding efforts and are also employing virtual chemical series to develop a drug is explored Paul Depledge laboratory design, we have sought to integrate novel targets and are now actively employing this screening (VS) on projects within our portfolio. further. Senior Chemists closely the making, by chemists, and testing, by information to progress two drug discovery In VS, compounds are computationally assessed James Hitchin bioscientists, of novel compounds. A particularly projects. Ali Raoof important feature of this “make-test” workflow Nicola Rogers is the ability to handle accurately nanolitre In mid-2010, we began a phased move from Figure 1 Views of the laboratory and some of quantities of compounds in solution using paper-based laboratory records toward a newly Senior Bioscientists the equipment used in the drug Mark Cockerill state-of-the-art acoustic dispensing technology. established electronic laboratory notebook discovery process Dominic James This has significant benefits both for chemical (ELN) system. ELN integrates closely with both Graeme Thomson synthesis (less compound required) and our databases of chemical reagents and our biological data quality (accurate and consistent database of compounds for screening, allowing dilution and dispensing). increases in our efficiency and productivity alongside greater accuracy of recorded data. Informatics Moreover, when fully implemented, it will allow Apart from the facilities described above, we rapid searching of all our laboratory experiments have also made major investments in integrated across the group, giving every user rapid access informatics platforms to support the drug to stored historical data and technical discovery process. As a key part of this, we have information. This ELN functionality is being purchased in an in-house “Dotmatics” database developed in close conjunction with Dotmatics, to keep accurate records of the compounds and our early adoption allows the precise synthesised in the laboratory and all the tailoring of the software to meet our specific biological information accrued on them. This needs and requirements within the Drug informatics capability allows our project teams to Discovery group. access, process and visualise complex datasets and make optimum decisions around project Projects strategy and progression. In order to kick start our capabilities and project portfolio we have engaged in a major We have also generated an internal database of multifaceted collaboration with the Cancer some three million commercially available, drug- Research Technology Drug Discovery Laboratory like compounds which can be used to support (DL) in London. The first component of this and complement our internal synthetic efforts. collaboration was to deploy our new medicinal 30 | Paterson Institute for Cancer Research Scientific Report 2010 Drug Discovery Group | 31


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    Immunology Group MEFs while 5T4KO MEFs show only intracellular CXCR4 (Figure 1C) that can be rescued at the expression and chemotaxis. To document the basic components of trafficking, primary WT http://www.paterson.man.ac.uk/immunology cell surface by RAd-m5T4 (Figure 1D). Upon MEFs were treated for 24 hours with ligand binding, CXCR4 undergoes a cytochalasin D, brefeldin A or nocodazole to conformational change that facilitates activation disrupt the cytoskeleton, Golgi or microtubules of heterotrimeric G proteins and signalling respectively and the pattern of 5T4 and CXCR4 effectors at the plasma membrane. This initiates expression was determined before and after a signalling cascade resulting in downstream washout of the drugs. These experiments phosphorylation of proteins such as ERK1/2. showed that detection of plasma membrane These activities are dependent on CXCR4 co-localized 5T4/CXCR4 molecules is expression at the plasma membrane and cellular dependent on microtubules and the molecules events that reduce the latter can abrogate the are not obligatorily associated at the Golgi. 5T4 oncofetal glycoprotein was discovered while searching for biological effects. We have shown in MEFs that Disruption of the Golgi or the actin cytoskeleton in the absence of 5T4, the CXCR4 is no longer per se does not disrupt all 5T4/CXCR4 molecules with invasive properties likely to be shared by able to activate this pathway and the co-localization at the plasma membrane. It trophoblast and cancer cells. 5T4 expression has been shown phosphorylation status of ERK1/2 is not appears that CXCR4 and 5T4 molecules can responsive to CXCL12. form a stable interaction at the cell surface to influence adhesion, cytoskeletal organization and motility, facilitating the biological response to CXCL12. properties which might account for its association with poorer In the embryonic cells investigated it appears that cell surface expression of, and chemotactic The 5T4 gene is highly conserved across clinical outcome in some cancers. Our recent work reported response through, CXCR4 can be regulated by different vertebrate species and the the unexpected discovery that 5T4 molecules are required for Figure 1 5T4 expression. To examine the role of the transmembrane (TM) region is completely Group Leader Role of 5T4 expression in the extracellular, transmembrane and cytoplasmic conserved. Chemokine receptors, G-protein- Peter L. Stern functional expression of CXCR4 at the cell surface of CXC12/CXCR4 axis. (A) 5T4 gene domains of 5T4 molecules in CXCR4 surface coupled seven TM spanning proteins, are also embryonic cells (Southgate et al., 2010). dose related CXCL12 chemotaxis of MEFs. (B) 5T4KO MEF chemotaxis expression, a series of murine 5T4 gene plasmid highly conserved in evolution, with the Postdoctoral Fellows constructs were generated and cloned into a hydrophobic amino acids of TM domains forming Fernanda Castro is rescued by RAd-m5T4 infection. (C) CXCR4 (green) and 5T4 (red) retrovirus also encoding eGFP as a reporter α-helical structures which anchor the receptors Owen McGinn CXCR4 mediated chemotaxis is regulated by WT or 5T4KO undifferentiated ES cells infected Rasilaben Vaghjiani (from August) co-localize at cell surface (composite gene. 5T4KO MEFs were infected with the in the membrane. Initial interaction between 5T4 oncofetal glycoprotein with the different vectors. Expression of m5T4 yellow or co-localized areas in retroviral constructs and cells were examined for CXCL12 and the CXCR4 extracellular region Clinical Fellow One significant transcriptional change identified in differentiating 5T4KO-ES cells restored separate channel) in WT but not both eGFP expression and CXCR4 localization facilitates rapid binding and efficient extracellular Saladin Sawan in a microarray analysis of differentiating CXCL12 chemotaxis comparable to that of 5T4KO MEFs. (D) RAd-m5T4 by immunofluorescence. These data identified anchoring while the ligand-binding N-terminus embryonic stem (ES) cells, stratified by up- differentiating WT-ES cells. These data show that infected 5T4KO MEFs show co- localized surface expression 5T4 and the 5T4 transmembrane domain as sufficient and remains highly dynamic and searches for the Scientific Officers regulation of surface 5T4 expression, was the 5T4 expression is a necessary cofactor for Kate Mulryan (until April) CXCR4 (Southgate et al., 2010). necessary to enable CXCR4 cell surface binding cavities buried with the receptor TM down-regulation of transcripts for the dipeptidyl CXCR4 functional expression and CXCL12 helices; this second step interaction triggers Debbie Burt (seconded to CIMML) peptidase IV, CD26, a cell surface protease that chemotaxis in differentiating ES cells. One conformational changes in the CXCR4 TM to Jian Li (from October) cleaves the chemokine CXCL12. Interestingly, mechanism that might account for these results induce G-protein signalling (Kofuku et al., J Biol differentiating ES cells also showed an up- would be if 5T4 molecules facilitate stable cell Chem 2009; 284: 35240). Importantly, the Graduate Students regulation of CXCL12 transcription. CXCL12, membrane expression of CXCR4 molecules in Mariam Al-Muftah (joint with chemotactic response of both differentiated ES through its receptor CXCR4, regulates many differentiating ES cells. Undifferentiated WT-ES cells and MEFs can be blocked by some but not David Gilham) Georgi Marinov biological processes but also plays an important cells are 5T4-negative with CXCR4 expression all antibodies recognizing distinct parts and Andrzej Rutkowski (joint with role in tumorigenesis. Therefore, the inverse low and intracellular, however following epitopes of m5T4 molecules. It seems likely that Crispin Miller) correlation between 5T4 and CXCL12 with differentiation both molecules can be detected the inhibition results from allosteric effects on CD26 transcript levels during mouse ES cell at the cell surface with clear areas of co- CXCR4 altering the nature of ligand binding or Undergraduate student differentiation, and the known roles of these localization. By contrast, differentiated 5T4KO ES its consequences. It is possible that the 5T4 Crystal Chia (until May) molecules in cell migration/motility, suggested cells show only intracellular CXCR4 expression. transmembrane region specifically recognizes that particular regulatory processes are common However, when differentiating but not intramembrane residues of CXCR4 and to both ES cell differentiation and tumour undifferentiated 5T4KO-ES cells are infected with contributes not only to the stability of the metastasis. RAd-m5T4, CXCR4 can be detected at the cell CXCR4 expression in the plasma membrane but surface co-localized with 5T4 molecules. These possibly also to conformational changes in the To examine biological response to CXCL12, wild data are consistent with 5T4 molecules being receptor which govern responsiveness to ligand. type (WT) and 5T4 knockout (KO) ES cells necessary for the surface expression of the were tested for CXCL12 chemotaxis before and CXCR4 receptor and chemotaxis to CXCL12 in The regulation of CXCR4 surface expression by after differentiation. Both WT and 5T4KO differentiating ES cells. 5T4 molecules is a novel means to control undifferentiated ES cells showed no chemotaxis responses to the chemokine CXCL12 for towards CXCL12 but upon differentiation WT- 5T4 dependency for CXCR4-mediated example during embryogenesis but can also be ES cells were significantly more chemotactic, chemotaxis is also apparent in mouse embryo selected to advantage the spread of a 5T4 while 5T4KO-ES cells remained unresponsive. To fibroblasts (MEFs) as shown by: (1) a 5T4 gene- positive tumour from its primary site. The test whether 5T4 might play a role in CXCL12- dose influence on CXCL12 chemotaxis in WT, influence of 5T4 on CXCL12/CXCR4 responses dependent chemotaxis, undifferentiated and heterozygote and 5T4KO MEFs (Figure 1A); (2) in human cancer is now being explored. differentiating 5T4KO-ES cells were infected with the restoration of the chemotactic response of recombinant adenoviral vector encoding mouse 5T4KO MEFs by RAd-m5T4 (Figure 1B); and (3) 5T4 (RAd-m5T4) or RAd-GFP control vector. the co-localization of some CXCR4 molecules Publications listed on page 69 There was no change in chemotaxis of either with typical 5T4 cell surface expression in WT 32 | Paterson Institute for Cancer Research Scientific Report 2010 Immunology Group | 33


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    Inositide Laboratory Group PtdIns4P PtdIns5P phosphoinositides or with neither (Figure 3). These mutants will be crucial in defining the http://www.paterson.man.ac.uk/inositide relationship between nuclear phosphoinositides, PIP5K PIP4K histone interaction and the regulation of basal PKC CRT359 - transcription. DAG PtdIns(4,5)P2 PIP3 We have also investigated if PtdIns5P can PLC PI3K modulate the levels of trimethylation of lysine4 of histone H3 (H3K4Me3) at the promoters of active genes. In plants the protein product of the ATX1 gene can modulate H3K4Me3 at Cell survival, proliferation migration specific gene promoters to regulate Figure 1 Figure 2 Phosphoinositides are a family of lipid second messengers that developmental gene transcription programmes. In collaboration with Prof. Z. Avramova (UNL are regulated in response to environmental changes by the Figure 1 in human breast tumour cell lines also show that Center for Biotechnology and Plant Science expression of PIP4Kβ may regulate tumour cell activities of a network of kinases and phosphatases. Alterations There are two pathways for PtdIns(4,5)P2 synthesis however the growth. As PIP4Ks phosphorylate PtdIns5P, we Initiative, UNL, Lincoln, NE, USA) we show that the activity of ATX1 can be controlled by in phosphoinositide levels can regulate many different major pathway is probably through PIP5K. PIP4K probably regulates the presume that changes in the expression of PtdIns5P levels through the ability of PtdIns5P to PIP4Kβ lead to changes in the levels of nuclear cancer-relevant pathways including cell survival, proliferation, levels of PtdIns5P. CRT359 is an PtdIns5P. However, PIP4Kβ has 2000 times less interact with the PHD finger of ATX1. Changes inhibitor of PIP5K which should inhibit in PtdIns5P lead to a decrease in the levels of migration, cell substratum interactions and transcription. In the PI3K and the phospholipase activity than PIP4Kα, which has led us to H3K4Me3 at the promoter of a downstream (PLC) pathway. Diacylglycerol (DAG) re-evaluate the intracellular localisation of the Group Leader cancer PtdIns(4,5)P2 is at the heart of phosphoinositide activates protein kinase C (PKC) two isoforms. Unexpectedly, we have found that target gene and to a decrease in its transcription. ATX1 is a plant homologue of the mammalian Nullin Divecha signalling as it is the substrate for phosphatidylinositol-3-kinase which regulates the phosphorylation of PIP5K. PIP4Kα and PIP4Kβ interact and that their trithorax gene family, which often undergo interaction can relocalise PIP4Kα from the Associate Scientist (PI3K) and phospholipase C (PIC) both of which are Figure 2 cytosol to the nucleus (Figure 2). Furthermore chromosomal translocations and deregulation leading to human leukaemias. David Jones deregulated in human tumours (figure 1). Furthermore, Myc-PIP4Kα was expressed alone or the ability of PIP4Kβ to regulate PtdIns5P levels together with HA-PIP4Kβ and cells appears to largely depend on the activity of Postdoctoral Fellows PtdIns(4,5)P2 is itself a regulator of cytoskeletal dynamics, cell were fixed and stained for Myc PIP4Kα. PIP5K as a target for drug development. Daniel Fitzgerald and HA. When expressed alone As PtdIns(4,5)P is important in the regulation of Maria Carla Motta survival and cell polarity. Myc-PIP4Kα localizes to the cytosol, 2 cancer relevant pathways, inhibition of Iman van den Bout however, co expression with How PtdIns5P regulates nuclear processes is far PtdIns(4,5)P2 synthesis may be useful to inhibit HA-PIP4Kβ leads to enhanced from clear although some PHD finger-containing PIP5Ks and PtdIns(4,5)P2 To define how PtdIns(4,5)P2 might regulate cancer cell growth (Figure 1). PIP5K inhibitors, Scientific Officer localization of PIP4Kα both at the proteins are downstream nuclear targets for Yvette Bultsma PtdIns(4,5)P2 is present in the plasma membrane nuclear function we have identified nuclear membrane and in the nucleus, where were identified using a high throughput PIP5K phosphoinositides. The PHD finger is a cross- and in the nucleus where its levels in these two proteins that interact specifically with this lipid it co-localises with PIP4Kβ. assay and have been subsequently developed by braced Zinc finger that can interact with Graduate Students compartments can be regulated separately. (collaboration with Dr. C. D’Santos CRI rational chemistry (Cancer Research Julian Blaser (co-supervised with Figure 3 phosphoinositides and can also interact with PtdIns(4,5)P2 can be synthesised by two different Cambridge). 168 proteins harbouring Technology). We have developed a 96 well in Tim Somervaille) GST-PHD fingers were assessed for methylated and non-methylated histone proteins families of kinases using two different substrates phosphoinositide-binding domains were vivo assay to measure PtdIns(4,5)P in living cells, 2 Xiaowen Hu interaction with either to modulate gene transcription. For example, Willem-Jan Keune (Figure 1). It is likely that PIP5Ks are the major identified. We identified a subset of proteins phosphoinositides (PI) or with a which will be used to asses the the PHD finger of TAF3 interacts with both Lilly Sommer regulators of PtdIns(4,5)P2 synthesis while the with known phosphoinositide-binding such as the Histone-H3 peptide containing structure/function relationships of inhibitors in PtdIns5P and methylated histones and TAF3 PIP4Ks regulate PtdIns5P levels and perhaps a pleckstrin homology (PH) or plant lysine4, which was methylated vivo to enhance their specificity and potency. (H3K4Me). Wild type (WT) PHD interacts with and regulates the activity of the minor pool of PtdIns(4,5)P2. homeodomain (PHD) modules, although the finger interacts with PI and H3K4Me, basal transcriptional machinery. We have vast majority of the nuclear proteins that while Mutant M1only interacts with identified mutants within the PHD finger of TAF3 There are four isoforms of PIP5K (α, β and γ interacted with PtdIns(4,5)P2 contained methylated peptides and M2 only Publications listed on page 69 that interact only with methylated histone or and L) of which α, β and γ are active while L is lysine/arginine-rich patches (K/R-(Xn=3-7)-K-X- interacts with PI. M3 no longer inactive but can interact with and may regulate K/R-K/R). For example, toposiomerase IIα helps interacts with either. the localisation and activity of the other PIP5K to unwind DNA by creating transient breaks in isoforms. Using various techniques to study the the DNA. Toposiomerase IIα interacts with intracellular localisation of PIP5K, we find that PtdIns(4,5)P2 and the interaction with while all isoforms of PIP5Ks localise at the phosphoinositides regulates its in vitro plasma membrane, they also localise to other decantenation activity. subcellular compartments, such as the Golgi, focal adhesions or the cytokinetic furrow. These PIP4K and PtdIns5P studies suggest that the synthesis of PtdIns(4,5)P2 There are three isoforms of PIP4Ks (α, β and γ) at these specific sites may be required to of which α is cytosolic, β is cytosolic and nuclear regulate specific signalling pathways. How PIP5Ks and γ localises to internal membrane localise to specific cellular locations is not clear compartments. We have developed an antibody but is probably dependent on a combination of to PIP4Kβ and in collaboration with Prof posttranslational modification together with Landberg (Breakthrough Breast Biology Group isoform-specific interacting proteins. Mass and The University of Manchester) have spectrometry has shown that PIP5Ks are interrogated tissue microarrays of advanced modified by phosphorylation at multiple sites breast tumour samples. PIP4Kβ expression is and we have used affinity purification and yeast both up- and down-regulated in human breast two hybrid analysis to identify novel interacting tumours and interestingly, expression is a proteins. prognostic indicator of patient survival. Studies Figure 3 34 | Paterson Institute for Cancer Research Scientific Report 2010 Inositide Laboratory Group | 35


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    Leukaemia Biology Group Fi gur e 1A Fi gur e 1B http://www.paterson.man.ac.uk/leukaemia Human acute myeloid leukaemias (AML) are considered to be hierarchically organised neoplasms in which the cells of the malignant clone exhibit heterogeneous proliferative potentials. At the apex of the hierarchy is a population of leukaemia stem cells (LSC) which has the highest proliferative, or self-renewal, as absence of Dicer1 or Rb1, can lead to non-cell dependent on the presence of the human MSCs. potential. These cells sustain the malignant disease but critically, Figure 1 Knockdown of a chromatin autonomous haematological disorders such as While the haematopoietic cells are murine in Group Leader if not eradicated by chemotherapy or bone marrow regulatory gene induces differentiation of human MLL-AF9 myelodysplasia or myeloproliferation. To further origin, osteoblasts, osteocytes and adventitial investigate the role of the haematopoietic reticular cells are human. This model will be Tim Somervaille transplantation, they initiate leukaemic relapse and consequent myelomonocytic leukaemia cells. microevironment in normal human used to investigate candidate key regulators of Control THP1 cells normally grow in Postdoctoral Fellows treatment failure. Understanding the genes and cellular clumps in semi-solid culture (A) but haematopoiesis and leukaemogenesis, we have the haematopoietic microenvironment which following knockdown of a chromatin developed a heterotopic ossicle mouse model of have been identified through whole genome Xu Huang pathways that LSCs use to undergo self-renewal is the main regulatory gene using a lentivirally the human haematopoietic microenvironment. transcriptional profiling of human bone marrow James Lynch focus of the Leukaemia Biology Laboratory. expressed shRNA, they differentiate into macrophages (B). Human mesenchymal stromal cells (MSCs) stromal cells. The model will also be used to Clinical Research Fellow loaded on to a tricalcium phosphate determine the extent to which human leukaemia Brigit Greystoke hydroxyapatite scaffold and implanted sub- cells might be dependent on the presence of Proteins that regulate chromatin are important prevent apoptosis and, as shown in Figure 1, cutaneously on to the back of a mouse, undergo human microenvironmental cells, rather than Scientific Officer mediators of self-renewal in stem cells in general. some appear to be required to prevent terminal differentiation in vivo into osteoblasts and murine ones, which is especially relevant given Gary Spencer Through post-translational modifications of differentiation. In the months ahead, validation of osteocytes to form bone and haematopoietic the typically poor levels of engraftment seen Graduate Students histone proteins, regulation of chromatin density the roles of these genes as LSC regulators in microenvironmental cells that support when human AML LSCs are transplanted in to Julian Blaser (co-supervised with and targeted assembly of transcription primary human AML will be performed in haematopoiesis. As shown in Figure 2, eight mice. Nullin Divecha) complexes to promoters, they regulate further xenograft experiments and additional weeks following implantation, both bone and Filippo Ciceri expression of key genes that specify cell fate and studies will be performed to identify mechanisms haematopoiesis can be seen within the William Harris function. For example, the chromatin by which these genes promote or sustain self- heterotopic ossicle, both of which are Publications listed on page 70 remodelling factor ISWI is essential for self- renewal. A particular emphasis will be placed on renewal of germ-line stem cells in Drosophila; the genes with enzymatic activity that could function polycomb protein BMI1 is essential for as therapeutic targets. maintaining self-renewal of haematopoietic stem cells; and the histone acetyltransferase HTATIP A similar screening strategy is being pursued for Figure 2 (TIP60) is a key regulator of embryonic stem cell regulators of phosphoinositide signalling in Heterotopic ossicles. Human identity. Relatively little is known, however, of the collaboration with Nullin Divecha of the Inositide mesenchymal stromal cells were role such proteins play in LSCs. To address this Laboratory. Proliferation of primary myeloid loaded on to a tricalcium phosphate hydroxyapatite scaffold (HA/TCP) question, we have generated a library of lentiviral leukaemia blast cells is known to be dependent and implanted sub-cutaneously in an vectors that express shRNAs that target for upon signalling through the phosphoinositide-3- immune deficient mouse. Eight knockdown over 250 human genes known to kinase pathway, however the role of other weeks later, bone (B) and regulate chromatin. These include SWI/SNF, components of the phosphoinositide pathway in extramedullary haematopoiesis (Ma) ISWI, NuRD and INO80 chromatin remodelling survival, proliferation and differentiation of is observed. Osteoblasts, osteocytes and adventitial reticular cells forming complex components, histone methylases and myeloid leukaemia cells is not known. As for the a haematopoietic microenvironment demethylases, histone acetyltransferases and chromatin library, to date this approach has are human, haematopoiesis is deacetylases, Polycomb and Trithorax complex revealed a number of interesting candidate murine. genes, histone arginine methylases and regulators of LSC function and these are deiminases, and genes that regulate DNA currently being validated in further experiments. methylation. Using both myeloid leukaemia cell As before, a number of these “hits” have lines and primary human leukaemia cells enzymatic activity and could be targeted obtained from the Manchester Cancer Research therapeutically. Centre’s Biobank, studies performed to date have identified a number of genes that appear to The bone marrow microenvironment is a critical be required for self-renewal of human LSCs. regulator of self-renewal of both normal and Some genes appear to be required for cell cycle leukaemic haematopoeitic stem cells. Indeed, progression, others appear to be required to abnormalities in bone marrow stromal cells, such 36 | Paterson Institute for Cancer Research Scientific Report 2010 Leukaemia Biology Group | 37


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    Signalling Networks in Cancer Group (since July 2010) endogenous mutations in the target kinase. The overall goal of these studies will be to identify http://www.paterson.man.ac.uk/signalling_networks common and convergent pathways utilized by tumour cells to promote tumorigenesis and identify convergent and essential targets that could be exploited for the development of novel therapeutics (for example MLC-2 in Figure 1). PKC signalling and tumorigenesis The protein kinase Cs (PKC) are an important family of ten signal-transducing enzymes. There are three subfamilies of PKCs defined on the Protein phosphorylation regulates most intracellular processes, mode of regulation in response to lipid signalling - conventional PKCα, βI/II, and γ, contain a Ca2+- and serves a critical role in signal transduction from the sensitive C2 domain and tandem DAG-sensitive membrane to the nucleus. Now, more than ever, in-depth C1a-C1b domains; novel PKCδ, ε, η, and θ contain tandem DAG-sensitive C1a-C1b analysis is critical for understanding the function of domains and a C2 domain that does not sense phosphorylation in both normal and cancer cells, where the kinases harbouring cancer mutations Ca2+; atypical PKCλ and ζ contain a single C1 Figure 1 domain, insensitive to DAG, and are evidence that perturbation in phosphorylation of several Shedding light on a novel signalling (engineered through site-directed mutagenesis) predominantly regulated by scaffolding protein pathway. The recent discovery that to wild type (WT), kinase dead (KD) and Group Leader networks plays a causal role in cancer has accumulated at an LKB1 can activate NUAK1, leading hyperactivated forms of the kinase. Next we will interactions. The PKC family has been intensely investigated for over 25 years in the context of John Brognard accelerated pace as a result of genomic sequencing of to inactivation of MYPT1 and increased MLC-2 phosphorylation, determine phenotypic effects of expressing the cancer. Historically, this arises from the discovery WT, KD and mutant forms of the target kinase Scientific Officer tumours. Further highlighting the importance of signalling suggests this pathway may be important in cancers with LKB1 LOF on proliferation, survival and transformed of PKC as the receptor for the tumour- promoting phorbol esters, such as TPA, which Eleanor Wendy Trotter pathways in cancer is the development of small molecule mutations. LOF mutations in other properties of appropriate tumour and normal suggested that activation of PKC by phorbol kinases, such as DAPK3, could be a cell lines. We will verify the function of the inhibitors specifically targeting activated kinases in cancer means for a tumour cell to reach the kinase using si/shRNA and evaluate the role of esters promoted tumorigenesis induced by same end point. carcinogens. However, this interpretation is now patients (B-RAF and ALK), which are achieving unprecedented the endogenous kinase in regulating cellular open to question, since long-term treatment phenotypes associated with tumorigenesis. We success in the clinic for historically unresponsive cancers, such will also investigate the molecular mechanisms with phorbol esters is known to initiate degradation of PKC, thus down-regulating its as metastatic melanoma and non-small cell lung cancer. These utilized by the cancer mutants to promote activity. tumorigenesis. For example, if the mutation proof-of-principle drugs are ushering in a new era of occurs in only one allele and results in loss-of- Given this debate regarding the necessity of personalized cancer treatment, portending a future where function (LOF), we will determine if the mutant activation or inactivation of PKC family of kinases kinase acts as a dominant negative (DN) to tumours will be classified and treated based on their genetic suppress the function of the WT allele. in cancer, the discovery of non-synonymous point mutations in many PKC family members, aberrations and each cancer patient will be administered a Alternatively, if the mutation is an activating primarily in colorectal cancer (CRC) and non- mutation, we will identify cancer relevant cocktail of specific inhibitors (or possibly activators) tailored to Figure 2 substrates that are phosphorylated by the cancer small cell lung cancer (NSCLC), provides an Model describing potential opportunity to address this very pertinent their specific cancer. mechanism whereby inactivating PKC mutants to promote tumorigenesis. Finally we question. We will characterize mutations mutations contribute to K-Ras will assess the consequences of somatic observed in the PKC family of isozymes in mediated tumorigenesis. mutations utilizing cell lines that harbour Novel cancer associated kinases Candidate cancer-associated kinases that our lab cancer utilizing live-cell imaging techniques. The technology to sequence a cancer patient’s will study are determined based on several Additionally, we have discovered that all PKC genome is accelerating at an amazing pace, criteria: (1) the kinase must have limited mutations we will be studying in CRC and however our understanding of the many genetic characterization and a high probability of NSCLC occur in the context of activating K-Ras aberrations that contribute to the cancer possessing a driver mutation (Greenman et al., mutations. K-Ras is a substrate for PKC, where phenotype lags far behind the development and Nature 2007; 446: 153); (2) mutations should be phosphorylation of K-Ras by PKC alters its application of this technology. A primary predicted to be cancer mutations based on subcellular targeting and causes K-Ras to objective of the laboratory is to identify novel bioinformatic anaylses (CanPredict and PMUT); promote apoptosis. We will determine if LOF kinases with functional mutations in cancer (3) mutations should occur at evolutionarily mutations in PKC can promote survival of colon patients that are essential for tumorigenesis. As conserved amino acids. The main goal of these cancer cells harbouring K-Ras mutations, by an initial step in identifying novel or understudied preliminary studies was to identify kinases where suppressing a K-Ras-induced apoptosis feedback protein kinases with candidate cancer driver all or almost all of the observed somatic loop (Figure 2). In the context of other mutations, we used bioinformatic tools to mutations are predicted to be functional driver mutations present in a given tumour, evaluation analyze kinases with somatic mutations reported mutations. of PKC cancer mutants should begin to shed in various cancer kinome sequencing studies for some light on the role this family of kinases play those most likely to contribute to tumorigenesis To characterize the mutant kinases, our general in tumour progression and provide guidance for (primarily lung tumorigenesis). This analysis has strategy is to first assess the functional therapeutic treatment. led to the identification of several novel kinases consequences of somatic mutations on overall as rational targets, including MLK4, MYO3B, kinase activity utilizing in vivo and in vitro kinase ANKK1, and SgK085. activity assays. We will compare the activity of Publications listed on page 70 38 | Paterson Institute for Cancer Research Scientific Report 2010 Signalling Networks in Cancer Group | 39


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    Stem Cell Biology Group was recently found to induce acute myeloid leukaemia (AML) in a mouse retroviral http://www.paterson.man.ac.uk/stemcellbiology transduction-transplantation model. Using an ES cell line with a doxycycline AE9a IRES GFP inducible cassette, we first validated in vitro that the induced expression of AE9a, thought to act as a dominant inhibitor of AML1/Runx1, blocked the generation of hematopoietic cells during blast development. Based on these results, we subsequently generated a mouse line from these ES cells. The major interest of our lab is to decipher the cellular and To restrict the induction of AE9a expression to the haematopoietic system, we transplanted molecular mechanisms that control the development and antibodies and large quantities of cells. Instead bone marrow cells of these mice into sub- Figure 1 maintenance of the haematopoietic system. In this context, we Detection of induced AML-ETO9a the chromatin associated protein is fused to the lethally irradiated recipients and subsequently fed expression (+Dox) by DNA adenosine methylase (Dam) of E. coli them with either normal food or food containing study the functions of the transcription factor AML1/RUNX1 immunofluorescence. which will create a methyl tag at GATC genomic doxycycline. The mice expressing AML1-ETO9a and of the transcriptional co-activator MOZ. AML1/RUNX1 sequences close to the binding sites of the fused developed extramedullary haematopoiesis protein. These sequences can be specifically followed by the development of acute myeloid is one of the most frequent target of gene rearrangements isolated by DpnI digests and amplified. We have leukaemia. The disease latency was shorter Group Leader and mutations in acute leukaemia. Similarly the gene MOZ is now performed these analyses and the products when AML1-ETO9a was induced on a P53-/- generated have been sequenced in our institute background. This result is consistent with the Georges Lacaud involved in myeloid chromosomal translocations. by the Molecular Biology Core Facility on an AB two-hit model of leukemia development in Postdoctoral Fellows Understanding the function of these transcription regulators SOLiD™ high throughput sequencing machine. which one genetic alteration will affect We are currently analyzing these results and haematopoietic differentiation (such as AML- Cristina Ferreras (until July) during normal haematopoiesis should result in a better validating them in collaboration with James ETO) whereas another is required to alter signal Christophe Lancrin Michael Lie-A-Ling comprehension of how perturbations of their functions lead to Bradford and Crispin Miller (Applied transduction cascades associated with cell Flor Perez-Campo Computational Biology and Bioinformatics proliferation (such as mutations to Flt3, c-Kit or development of leukaemia. Group). We are also planning to extend the Ras). Accordingly, transplantations of the Scientific Officer leukaemic cells led to development of AML in DamID approach to mice to survey Runx1 Rahima Patel (from May) Generation of blood cells demonstrated that the transcription factor binding sites at different stages of secondary recipients with a shorter latency. Graduate Students The earliest site of blood cell development in SCL/TAL1 is indispensable for the establishment haematopoietic development and at different Monika Antkiewicz the mouse embryo is the yolk sac where blood of this haemogenic endothelium cell population levels of the haematopoietic hierarchy. Altogether these results indicate that we have Elli Marinopoulou (from islands, consisting of haematopoietic cells from the haemangioblast whereas now established a new model of leukaemia October) development, which will allow us to investigate surrounded by a layer of angioblasts, develop at RUNX1/AML1 is critical for the generation of Model of leukaemia Olga Tsoulaki approximately day 7.5 of gestation. The parallel haematopoietic cells from this haemogenic To study the aetiology of leukaemia, we initiated further the molecular and cellular events development of these two lineages in close endothelium. the development of a murine model of AML associated with t(8;21) leukaemogenesis. association provided the basis for the hypothesis based on a Runx1 translocation. For this project, that they arise from a common precursor, a cell Transcriptional targets of RUNX1/AML1 and we used AML1-ETO9a (AE9a), a natural alternate called the haemangioblast. A conflicting theory identification of RUNX1 binding sites splice variant of the AML1-ETO transcript. AE9a Publications listed on page 70 however associates the first haematopoietic cells These previous studies indicate that RUNX1 is to a phenotypically differentiated endothelial cell likely to regulate the expression of a critical set with haematopoietic potential, i.e. a haemogenic of genes at this stage of development. To Figure 2 Blood cell analysis of control (A) and endothelium. Support for the haemangioblast identify these genes, we have compared gene leukaemic (B) mice. concept was initially provided by the expression in haemangioblast-derived cell identification during embryonic stem (ES) cell populations generated from either Runx1 differentiation of a clonal precursor, the blast deficient or Runx1 competent ES cells. These colony-forming cell (BL-CFC), which gives rise microarray analyses of Runx1-/- and Runx1+/- after 4 days to blast colonies with both transcriptomes identified a relatively large endothelial and haematopoietic potential. number of genes differentially expressed. It Recent studies have now provided evidence for would be therefore pertinent to correlate these the presence of this bipotential precursor in vivo. data with the detection of Runx1 binding by chromatin immunoprecipitation (ChIP) to We have recently demonstrated that the identify direct transcriptional targets. One haemangioblast generates haematopoietic cells limitation of this approach is the large number of through the formation of a haemogenic cells required and the requisite for a ChIP grade endothelium intermediate, providing the first antibody. direct link between these two precursor populations. This haemogenic endothelial cell As an alternative approach, we have generated population is transiently generated during blast constructs based on the DamID strategy, development and is also detected in gastrulating developed by Bas van Steensel, NKI, Amsterdam, embryos. At the molecular level, we have which alleviate the need for highly specific 40 | Paterson Institute for Cancer Research Scientific Report 2010 Stem Cell Biology Group | 41


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    Stem Cell and Haematopoiesis Group progenitors and might modulate the dynamic of the Myc-Mad-Max regulatory network. Based cell-cycle progression of haematopoietic progenitors rather than increasing apoptosis or http://www.paterson.man.ac.uk/sch on this hypothesis, we were interested to gain interfering with differentiation. insight into the potential role of Mxd4 in embryonic haematopoiesis and to study the To explore further the cell cycle alterations significance of its down-regulation at the time of detected upon Mxd4 induction, we determined haematopoietic commitment. This was the protein levels of key cell cycle regulators in performed using an ES cell doxycycline-inducible cells induced or not to express Mxd4. These expression system to analyze the effect of analyses revealed that the protein levels of the enforced Mxd4 expression upon haematopoietic cell cycle kinases Cdk4 and Cdk6 were markedly specification in vitro. Data obtained using this decreased in Mxd4-expressing cells. This was inducible ES cell system revealed that modulation accompanied by an increase in protein level of The balance between proliferation and differentiation during of Mxd4 expression interfered with the the cell cycle inhibitor p27. Furthermore, total colony-forming ability of the earliest precursors c-Myc, phospho-c-Myc as well as phospho-Rb haematopoietic development in the embryo is a complex generated. Further analyses indicated that the levels were also consistently reduced in process, the detailed molecular mechanisms of which remain enforced expression of Mxd4 during the Mxd4-expressing cells. Altogether, these results emergence of haematopoietic progenitors was correlated with the cell cycle profile observed to be fully characterized. Many studies suggest that leukaemia detrimental to the production of mature blood upon Mxd4 induction with an increase in the cell may result from the re-initiation of this embryonic programme cells and that this effect was already apparent cycle inhibitor p27 and decrease in the cell cycle when Mxd4 expression was maintained only for activators Cdk4, Cdk6 and phospho-Rb, all effect during adult life or from the inappropriate expression of a short period. possibly mediated via the modulation of the specific genes controlling the proliferation and differentiation of Myc-Max complex known to directly regulate Group Leader The reduction in colony numbers seen upon p27 (Figure 1). Valerie Kouskoff embryonic haematopoietic precursors. The main goal of our Mxd4 induction suggested that Mxd4 might play laboratory is to further our understanding of the a part in survival, proliferation or differentiation In light of Mxd4 function as an antagonist of Postdoctoral Fellows of the developing progenitors. However, the c-Myc, we propose that the sharp Maud Fleury transcriptional networks that orchestrate the formation of the observed reduction in clonogenicity was not down-regulation of Mxd4 in newly formed Katalin Boros haematopoietic system during embryonic development. accompanied by significant changes in lineage haematopoietic progenitors is necessary to allow Scientific Officer marker expression, suggesting no direct Myc to promote proliferation and expansion of Stella Pearson implication of Mxd4 in the differentiation the population prior to further differentiation. Embryonic haematopoiesis is a dynamic process definitive haematopoiesis, show impaired process. In contrast, the possible implication of Further studies will be needed to ascertain how Graduate Students that originates from mesodermal precursors and primitive haematopoiesis, and die at E9.5. While Mxd4 in the survival and/or proliferation of Mxd4 function to antagonise c-Myc in this Guilherme Costa ultimately gives rise to the haematopoietic stem the widespread role of Myc in normal and Sara Cuvertino (from October) progenitors came from the analysis of cell context. Their interaction might be direct cells that are the source of blood cell production tumourigenic development has been extensively number counts upon culture, which was through transcriptional control of the same Sarah Lewis Andrzej Mazan throughout adult life. The development of blood studied, less is known about its functional significantly lower in Mxd4-induced cultures. To target genes or indirect by binding to different cells in the embryo takes place in multiple antagonists, the Mad protein family. Like Myc, the address whether this decrease could be linked to target promoters. The reported ability of Mxd4 anatomical locations and incorporates a rapid four Mad proteins (Mad1, Mxi1, Mad3 and a decrease in cell proliferation or an increase in to bind partners other than Max raise the succession of cell fate specification and Mxd4) require heterodimerization with Max in apoptosis, we performed cell cycle analysis on intriguing possibility that it might exert its effect proliferation events. The earliest precursors are order to bind DNA. Mad-Max heterodimers progenitors expressing or not Mxd4. A in more than one way in specific cell context multi-potential haemangioblasts, with the capacity recognize the same consensus sequences as significant reduction in the S-phase population and perhaps play a role in other, as yet to give rise to blood, endothelial and smooth Myc-Max, but with opposing transcriptional was observed in the Mxd4-expressing cells, undetermined functions during embryonic muscle. Differentiation of endothelial and blood effects: while Myc is generally a transcriptional concomitant with an increase in the G0/G1 and haematopoietic development. cells from the haemangioblast leads to the activator, Mad proteins are transcriptional G2 subsets. Increase in apoptosis was not formation of yolk sac blood islands, the source of repressors. Most of the information we have on detected by either BrdU or AnnexinV staining the first haematopoietic cells. Yolk sac the role of Myc-Max-Mad interaction in assays. Altogether, these results suggest that Publications listed on page 70 haematopoiesis can be modelled in vitro by the embryonic haematopoiesis is from the Myc enforced expression of Mxd4 influences the differentiation of mouse embryonic stem (ES) aspect. Since Mad proteins are antagonists of cells. A number of regulators of haematopoietic Myc function, we were intrigued when Mxd4 specification have already been identified. Some was detected in a genome-wide expression Figure 1 of these regulators, such as Etv2 or Scl are analysis showing a tightly regulated pattern of critical for the proper development of all blood expression during embryonic haematopoietic cells, while others, such as Runx1 or the EPO development. We therefore set out to study the signalling network are essential only for definitive role that Mxd4 might play in haematopoietic haematopoiesis and do not impair the formation differentiation, with the aim of establishing a of primitive erythrocytes. better understanding of the balance between proliferation and differentiation during this In a global gene expression profiling screen for process, and the specification of cell lineage type. novel haematopoietic regulators, we recently identified Mxd4 as being tightly regulated at the Our initial findings established that Mxd4 onset of haematopoietic specification. Mxd4 is a expression followed a distinctive and tightly transcription factor belonging to the Myc-Max- regulated pattern during embryonic Mad transcriptional network. The function of haematopoiesis in vitro, suggesting that the c-Myc is essential for embryonic haematopoiesis: expression level of this transcription factor might c-Myc deficient embryos do not generate play a role in the development of the blood 42 | Paterson Institute for Cancer Research Scientific Report 2010 Stem Cell and Haematopoiesis Group | 43


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    Stromal-Tumour Interaction Group http://www.paterson.man.ac.uk/stromal/ Human tumours are highly complex tissues and the non-neoplastic cell compartment of tumours, which is often Figure 1 others indicate that tumour-associated stroma colonization to form macroscopic metastases. It and CAFs exhibit no detectable genetic has long been assumed that dissemination of termed the “stroma”, is itself quite complex histologically. Coevolution of stromal fibroblasts with carcinoma cells during tumour alterations, as gauged by comparative genomic metastatic carcinoma cells depends largely on Carcinoma cells initially recruit and/or activate these various progression. Resident stromal fibroblasts within the tumour hybridisation (CGH) and single nucleotide their cell-autonomous effects, due to epigenetic polymorphism (SNP) array analyses, and show and/or genetic alterations that accumulate within stromal non-neoplastic cells, including fibroblasts, increasingly acquire two autocrine alterations in epigenetic modifications of the these malignant cells. However, emerging signalling loops involving TGF-β and myofibroblasts, immune cells, endothelial cells, bone SDF-1 during the series of tumour genome, such as DNA methylation. We note evidence now proposes a different schema in progression. These autocrine that our CAFs show no detectable aneuploidy as which metastatic spread is not totally dependant Group Leader marrow-derived cells etc. The resulting stromal cells signalling loops mediate determined by karyotype analysis, no anchorage- on the acquisition of additional genetic Akira Orimo reciprocate by fostering carcinoma cell growth and survival transdifferentiation of stromal fibroblasts into tumour-promoting independent growth in culture, and no alterations within carcinoma cells. The tumour tumourigenicity in vivo. Moreover, some of the microenvironment also serves as an important Postdoctoral Fellows during the course of tumour progression. CAF myofibroblasts. (Kojima et al., 2010). CAFs begin to senesce after 15 PDs in culture, determinant that encourages carcinoma cells in Yasushi Kojima similar to the behaviour of normal human the primary tumour to become motile and Urszula Polanska stromal fibroblasts. Alternatively, the stabilization invasive, and to disseminate into distant Studying the heterotypic interactions between in sites of wound healing, both of which contain the neoplastic cells and the supporting stroma is considerable numbers of myofibroblasts. of their phenotype may depend on some type organs. Interaction of carcinoma cells with the Scientific Officer Kieran Mellody believed to be essential for understanding nature of positive-feedback signalling of the sort created tumour-associated stroma facilitates the of a bulk of carcinoma mass. We focus on Stromal fibroblasts and myofibroblasts, by autocrine signalling loops. invasion-metastasis cascade. Graduate Student studying 1) how tumour-associated stromal collectively termed carcinoma-associated Ahmet Acar We find that normal human mammary Tumour-associated stromal fibroblasts play a fibroblasts become altered and co-evolve with fibroblasts (CAFs), were extracted from various tumour cells during the course of tumour human carcinomas. CAFs, in comparison with fibroblasts, when co-inoculated with breast significant role in regulating migratory and progression, 2) how the stromal fibroblasts their control fibroblasts, when coinjected with carcinoma cells into immunodeficient mice, invasive behaviours in carcinoma cells. This is facilitate tumour progression, and 3) what carcinoma cells into immunodeficient mice, are convert stably into tumour-promoting supported by evidence indicating that stromal specific stroma-derived signal is crucial in known to substantially promote carcinoma myofibroblasts within the resulting tumours. myofibroblasts are frequently present at the promoting tumour invasion and metastasis. growth and neoangiogenesis. During tumour progression, these fibroblasts invasive front of human carcinomas. In addition, progressively elevate two autocrine signalling it has been shown that CAF myofibroblasts Tumour-promoting roles of Evolution of tumour stromal myofibroblasts loops mediated by the TGF-β and SDF-1 extracted from human carcinomas increase the carcinoma-associated fibroblasts (CAFs) in tumour cytokines in self-stimulating and cross- migratory and invasive propensity of the cancer Neoplastic epithelial cells coexist in carcinomas CAFs retain their myofibroblastic properties and communicating fashions, thereby enhancing both cells co-cultured alongside them in collagen gels. with a stroma composed of various types of tumour-promoting phenotypes, after they have their transdifferentiation into myofibroblasts and Tenascin C, HGF and SDF-1, which are secreted mesenchymal cells as well as extracellular matrix been passaged for ten population doublings the associated tumour-promoting capability. by CAFs, also play a role in mediating CAF- (ECM), both of which create the complexity of (PDs) in vitro in the absence of ongoing contact Taken together, these findings indicate that the stimulated invasion of cultured carcinoma cells. the tumour microenvironment. with carcinoma cells. Accordingly, even though establishment of cross-communicating TGF-β It remains, however, unclear as to what paracrine Noticeable numbers of myofibroblasts, which are the CAFs appear to have initially acquired their and SDF-1 autocrine signalling gives rise to signalling from CAFs is essential for facilitation of characterized by their production of α-smooth unique phenotypes under the influence of myofibroblast differentiation and mediates metastatic dissemination of carcinoma cells in muscle actin (α-SMA), have been observed carcinoma cells, once it is acquired, they might the evolution of residual fibroblasts into vivo and what molecular alteration(s) is provoked repeatedly in the stroma of the majority of display this trait independent of further signalling tumour-promoting myofibroblasts. in such metastatic carcinoma cells. Studying invasive human breast cancers. However, the from the carcinoma cells. Unanswered by these crosstalk between tumour cells and specific contributions of these cells to tumour observations are (i) how do CAFs acquire and Stroma-derived signalling crucial in promoting mesenchymal cells during tumour progression progression are poorly defined. Myofibroblasts maintain their activated, tumour-enhancing tumour metastasis could help understand nature of biology of a also exist in areas of wound healing and chronic phenotypes and (ii) might CAFs harbour genetic The tumour invasion-metastasis cascade is a bulk of human carcinomas and facilitate the inflammation, and are often portrayed as and/or epigenetic alterations that act to confer complex multistep process that includes localised development of novel stroma-targeted “activated fibroblasts” that play crucial roles in the unique phenotypes? invasion of carcinoma cells, entrance into the therapeutic approaches. wound repair; myofibroblasts possess greatly systemic circulation, survival during increased contractile ability, promote Some reports indicate that stromal regions transportation, extravasation, the establishment angiogenesis, and stimulate epithelial cell growth microdissected from human breast cancers of micrometastases in distal tissues and Publications listed on page 70 through the production of ECM and the exhibit a high frequency of genetic alterations, secretion of growth factor and cytokines. The such as chromosomal regions of loss of striking histological resemblance is observed heterozygosity (LOH) and somatic mutations between tumour stroma and the stroma present (for example in the TP53 gene). However, 44 | Paterson Institute for Cancer Research Scientific Report 2010 Stromal-Tumour Interaction 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 2010 Research Groups - The University of Manchester School of Cancer and Enabling Sciences | 47


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    Children’s Cancer Group in vitro and in vivo models to test this hypothesis. Discovery-based proteomics and murine models suggest that leukaemic blast cells with aberrant peripheral localisation of lysosomes and expressing RAC2, the adhesion dyad of LFA-1 and ICAM1 and the signalling molecule CD70, have the ability to cross the blood brain and CSF barrier in NSG mice. Examination of primary leukaemic blast cells suggest that there is a higher prevalence of ICAM1/CD70+ cells in a CD10+/CD19+ selected population in relapsed compared to de novo disease and normal volunteers. Investigations of the mechanism of Our group investigates the biological mechanisms responsible transgression suggest leukaemic blast cells are for the variations in the therapeutic response in children with able to migrate via transcellular and paracellular methods (Figure 2). Our data suggests that key acute lymphoblastic leukaemia (ALL). We conduct a number to the development of extramedullary disease of international clinical trials which provides us with the data maybe the nature of the adhesion molecule(s) expressed on the surface of the cell. Thus and clinical material for hypotheses based laboratory heterotypic contact between host and tumour investigations. This year our studies have produced significant may be responsible not only for disease recurrence but also for the development of an Group Leader advances in the field of childhood ALL. invasive phenotype. If this can be verified, then toxicity, rather than disease control. We have Vaskar Saha Figure 1 Randomised drug effect on shown that in the absence of glutaminase activity, this becomes a potential target for therapy. Clinical trials are given on the first two days of therapy. progression-free survival by patient cell kill is markedly reduced. However, only Postdoctoral Fellows We have continued to refine our approach to We reported on the results of randomisation of Increased gastrointestinal and hepatic toxicity characteristics. P values for minimal glutaminase activity is required to Clare Dempsey mitoxantrone with idarubicin in the ALL R3 trial were seen in the first eight weeks in the interaction test the hypothesis that creating suitable in vitro and in vivo models to Mark Holland restore the cytotoxic effect of the drug, understand the disease process and the Suzanne Johnson (Parker et al., 2010). This trial runs in all idarubicin arm. In contrast those in the the randomised treatment effect varies between the subgroups. suggesting that there is a synergy between the response to therapy. Overall our clinical and Jizhong Liu paediatric oncology centres in UK, Ireland, mitoxantrone arm, who were not transplanted, two enzymatic activities. Thus we have created a Hazard ratios indicate that all laboratory data suggest that the tumour Netherlands, Australia and New Zealand. The showed a delay in haematopoietic recovery subgroups show a treatment effect new drug which potentially is less toxic and may Clinical Research Fellows microenvironment plays a major role in estimated 3-year progression-free survival (PFS) while on maintenance therapy more than four favouring mitoxantrone. HR = have a longer half-life than the currently available Shekhar Krishnan was 35.9% (95% CI, 25.9 – 45.9) in the months later. This suggests that mitoxantrone hazard ratio. MRD = minimal protecting ALL cells. Currently therapy which Ashish Masurekar native product. targets both tumour and host cells provides the idarubicin group versus 64.6% (54.2 – 73.2) in affects the normal haematopoietic stem cell residual disease. Cyto = cytogenetic Scientific Officer the mitoxantrone group (p=0.0004). The (HSC) niche. While in vitro and in vivo models subgroups. best result, with considerable morbidity. If we Seema Alexander Last year we reported on the clinical are able to disrupt the cross talk between host differences in progression-free survival (PFS) have long suggested that the bone marrow presentation and outcome of children with ALL Adiba Hussein and leukaemic cell, cheaper, simpler and less were mainly related to a decrease in disease microenvironment is protective of leukaemic who suffered CNS relapse (Krishnan et al., 2010). events (progression, second relapse, disease- cells, this is the first clinical study to suggest that toxic therapy may become a distinct possibility. Graduate Student Our clinical data suggested that CNS disease Eva Diffner related deaths; HR 0.56, 0.34 – 0.92, p=0.007) this is a highly significant factor in the eradication occurred from systemic seeding from leukaemic rather than an increase in adverse treatment of leukaemic cells. This has thus become a major cells with the ability to transgress blood-brain Undergraduate Student effects (treatment death, second malignancies; focus for our laboratory investigations. Publications listed on page 71 Caroline Fong and blood-CSF barriers. We have created both HR 0.52, 0.24 – 1.11, p=0.11) (Figure 1). This is Clinical Trials Manager the largest improvement ever achieved by a In an international collaborative study, we have Catriona Parker single treatment modification in childhood ALL. also published the outcome of 326 children with Figure 2 Mitoxantrone is a cheap and readily available Philadelphia chromosome positive childhood Podosomes present on the ventral Administrator drug and thus the results of the trials offers ALL, treated in nine different countries, prior to surface of the acute lymphoblastic Charlotte O’Horo possibilities for children world wide. the introduction of tyrosine kinase inhibitors leukaemia cell line, grown on a (Arico et al., 2010). The seven year overall CellTak coated coverslip. Cells were fixed with paraformaldehyde and The trial produced another important finding. survival of 44.9% was better than reported probed for Cortactin (green), The detection of minimal residual disease (MRD) previously (36%) for this high risk group of polymerised actin (red) and DNA after a block of treatment has been widely patients. The data suggested, as previously, the (blue) using fluorescence reported to be of prognostic value in patients benefit of matched donor transplantation in immunocytochemistry. Podosomes undergoing treatment for cancer. In some maintaining remission. are a structural change observed in transcellular migration. studies, MRD is being used as a surrogate marker for outcome. In this trial, there was no Laboratory difference in MRD levels between the two We have further refined the drug L-asparaginase, randomised arms. Thus if MRD and not PFS had from our report of its degradation by leukaemic been used as a surrogate endpoint, lysosomal proteases last year (Offman et al., mitoxantrone would not have been further 2010). Using molecular modelling and molecular evaluated. Thus the study serves as a caveat to dynamics performed by our collaborator Dr Paul the use of surrogate markers as endpoints in Bates at the London Research Institute, we have clinical trials. So if the effect of mitoxantrone is developed a protease resistant recombinant unrelated to disease clearance, what then is the L-asparaginase with activity comparable to the mechanism by which it controls disease wild type. L-asparaginase also has glutaminase recurrence? Both mitoxantrone and idarubicin activity. The latter is thought to contribute to 48 | Paterson Institute for Cancer Research Scientific Report 2010 Children’s Cancer Group | 49


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    Targeted Therapy Group V/propidium iodide staining) and chromium release assays. ROS scavengers do not affect T-cell responses can be induced with anti-CD40 against irradiated lymphoma cells. In these homotypic adhesion (HA), lysosomal membrane studies the potential importance of MΦ in permeability or cathepsin release; all critical cellular vaccination has been demonstrated. determinants of the Type II mAb-mediated cell Depletion of MΦ using clodronate-encapsulated death pathway. Moreover, pharmacological liposomes has been shown to considerably inhibitors of actin polymerisation (cytochalasin D, enhance primary vaccination efficacy in the latrunculin B), vacuolar ATPase (concanamycin A) presence of adjuvant anti-CD40 mAb. Our and cathepsin activity (cathepsin inhibitor III) all results demonstrate that in order to induce a block the induction of ROS by Type II mAb, protective immune response, additional host suggesting that ROS generation lies downstream immune stimulation is required and that of cathepsin release. The mitochondrial depletion of MΦ populations can improve The overarching goal of the Targeted Therapy Group is to respiratory chain is an important source of ROS; tumour cellular vaccination strategies. Recent however, whilst loss of ΔΨm occurs in response work done by Simon Dovedi has demonstrated define the optimal way to combine radiotherapy (RT) with to Type II mAb, we do not believe mitochondria for the first time that the efficacy of external immunotherapy in the treatment of cancer by enhancing our are the source of ROS in our system. Through a beam radiotherapy (EBRT) can be significantly collaboration with Peng Huang and Helene enhanced by combination with R848, a clinically understanding of the underlying mechanisms of action. The Pelicano at the MD Anderson, University of established TLR7 agonist to elicit a CD8+ T-cell specific objectives of the group are i) to investigate the Texas, experiments have revealed that Type II dependent anti-tumour immune response anti-CD20 mAb induce HA, cell death and ROS leading to long-term tumour-free survival. We mechanisms of action of radioimmunotherapy ii) to investigate in respiratory deficient Raji sub-clones (ρ- cells).. are currently expanding this study to include Group Leader how RT-induced tumour cell death is recognized and Mahsa Azizyan (MSc student) is now working to additional syngeneic models of lymphoma. Work confirm the source of ROS and the potential is ongoing to elucidate the role of the DC, B cell Tim Illidge processed by different antigen presenting cells (APC) in the contribution to this mechanism of cell death. and MΦ in the generation of protective anti- Postdoctoral Fellows tumour microenvironment and how this impacts on the This work has been accepted for oral tumour immunity post-EBRT. presentation at the Keystone Symposium- Ellie Cheadle subsequent adaptive immune response; iii) to investigate the ‘Antibodies as Drugs’ on February 2011 and will Monique Melis in collaboration with Kathryn Simon Dovedi Jamie Honeychurch role of bone marrow derived myeloid cells in tumour Figure 1 be presented by PhD student Waleed Alduaij. Simpson (Clinical and Experimental Pharmacology Group) has developed a Graduate Students regrowth after RT. We aim to translate our experimental Raji Burkitt’s lymphoma cells were preincubated with PBS or the ROS Immune response to RT induced dying Doxycycline regulated Caspase-3 death switch in Waleed Alduaij Monique Melis research findings into developing early phase clinical trials and scavenger Tiron and subsequently tumour cells a number of tumour models. In vitro, treated with the anti-CD20 mAb Our recent work in this area has focused on Doxycycline induced apoptosis was verified by MRes Student over the last year our research highlights have included the GA101 for 4 hours. Fluorescence understanding the nature of the host immune Annexin V/Propidium Iodide staining and microscopy of the lysosomal protease Khimara Naidoo areas outlined below. Cathepsin B staining (red) was then response to RT induced tumour cell death. We western blotting for cleaved caspase 3 and performed. DNA was counter- have focused our attentions on two types of cleaved PARP. Up to 80% apoptosis was MSc Student stained with DAPI (blue). GA101 APC, namely macrophages (MΦ) and Dendritic observed at 24 hours which could be inhibited Mahsa Azizyan induces marked Cathepsin B release Novel mechanisms of antibody induced independent of Bcl-2 over-expression and cells (DC). This work, done by Jamie by the pan-caspase inhibiter Q-VD. Apoptotic into the cytosol and surrounding Honeychurch has demonstrated that by cell death was confirmed by electron Senior Lecturer cell death caspase activation. This mAb induced cell death points of cellular adhesion in the Yong Du We have focused on investigations into a new appears to be executed by lysosomes which presence of absence of the ROS manipulating MΦ within the tumour microscopy. Death was associated with release of form of monoclonal antibody (mAb) induced cell disperse their contents into the cytoplasm and scavenger Tiron. microenvironment protective anti-tumour CD8 danger signals including HMGB1. In vivo, death in B-cell lymphomas and leukaemias in surrounding environment. Doxycycline treatment resulted in pronounced collaboration with Prof Mark Cragg's group in tumour regression and tumour eradication was Southampton. Using both lymphoma cell lines Building on these observations we have gone on observed in C57BL/6 immunocompetent mice. and primary chronic lymphocytic leukemia (CLL) to further characterize the mechanisms involved However, no tumour eradication was observed cells we demonstrated for the first time the in this cell death and have defined an important in SCID/NOD immunodeficient mice. importance of lysosome-mediated cell death for role for reactive oxygen species (ROS) in the cell Preliminary data show that there is increased antibody therapy (Ivanov et al., J Clin Investig death pathway. Type II anti-CD20 mAb infiltration of CD11b and F4/80 cells into the 2009; 119: 2143). We have followed up these (tositumomab, GA101) but not Type I anti-CD20 tumour microenvironment after Doxycycline initial observations by investigating the mAb (rituximab) or control mAb induce the treatment and induction of large amounts of mechanisms of action of a novel third generation production of ROS in the Raji cell line, Raji cells tumour cell apoptosis. We are currently humanized type II anti-CD20 mAb called GA101 over-expressing Bcl-2 (confirming independence investigating the role of the immune system and which has a modified Fc region aimed at from apoptotic pathways) and primary human what regulates immunogenicity of cell death in improving antibody directed toxicity (ADCC). In CLL samples. In addition, both whole IgG and vivo. The “death switch” which we have addition to this potential mechanism of action, F(ab)2 fragments of Type II mAb can induce ROS, developed allows us to explore the relationship we have observed that GA101 can initiate providing further evidence that the induction of between the amount of apoptotic cell death and profound non-apoptotic cell death in a range of cell death by Type II mAb is independent of the the host immune response and how this changes B-lymphoma cell lines and primary B-cell Fc arm of the mAb. ROS scavengers such as with depletion of selective APC. malignancies. We have gone on to demonstrate tiron, ascorbic acid and tempol sequester that GA101-induced cell death is dependent superoxide and protect lymphoma cells from upon homotypic adhesion, can be abrogated by Type II anti-CD20 mAb-induced cell death as Publications listed on page 71 inhibitors of actin polymerization and is measured by flow cytometry (Annexin- 50 | Paterson Institute for Cancer Research Scientific Report 2010 Targeted Therapy Group | 51


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    Translational Radiobiology Group large (CV 74.3%) and not associated with tumour stage or grade. Analysis of multiple biopsies from the same patient showed that the HS is more robust (CV 22.7%; range: 1.6-46.0%) than CA9 mRNA expression alone (CV 58.0%; range: 31.6-154.5%; n=13; Figure 1). Comparison with other measures of hypoxia showed TLDA HSs correlated with microarray-derived HSs (n=15; r=0.91, p<0.01) and the level of pimonidazole binding measured using immunohistochemistry (0.22, p=0.021 and n = 24). To maximise clinical utility, the customised Since the discovery of radiation at the end of the twentieth TLDA has been validated for use with FFPE HNC samples. The correlation between century, it has become one of the most important modalities housekeeper gene expression in matched frozen for the curative treatment of cancer. Radiotherapy schedules developmental axis plays a role in glandular cell and FFPE HNC samples supports its use with this sample type (r=0.71; p<0.0001; n=28). In developed somewhat empirically over the years, to maximise Figure 1 Biological network analysis of FFPE differentiation of the cervix, through the summary, we have shown that the TLDA HS is a tumour kill while minimising damage to surrounding healthy samples identifies hepatic nuclear transcriptional regulation of multiple target genes robust and reliable measure of hypoxia in HNC factor (HNF) regulated transcription (Figure 1). This work shows that gene samples (Amanda Williamson, Guy Betts, Joely tissue. As there is a direct relationship between radiation dose in adenocarcinoma. A) Analysis expression signatures can be derived from old Irlam). identifies a putative developmental and tumour control, the development of side-effects in a FFPE material that are sufficiently robust to a) be axis centred on HNF. B) qRT-PCR Group Leader validation of HNF genes. Data are applied to independent dataset in a different Normal tissue radiosensitivity Catharine M.L. minority constrains the potentially curative dose that can be for 7 SCC and 6 AC samples. TP63 tissue type; b) be used to design assays that can RAPPER - Radiogenomics: Assessment of safely prescribed to the majority of patients. The ability to is a control expressed only in SCC. be successfully applied using an independent Polymorphisms for Predicting the Effects of West platform with potential for clinical use, and c) Radiotherapy - has now recruited 2,957 samples Postdoctoral Fellows predict a patient’s likely response to radiotherapy would enable identify potential transcriptional regulation from patients enrolled in national radiotherapy John Hall (joint with Applied individualised dose prescriptions and decrease the mortality pathways important in biology. trials. The Translational Radiobiology Group Computational Biology and co-ordinates the sample collection (Rebecca Bioinformatics) and morbidity associated with cancer. The Translational Tumour hypoxia Elliott, Sophie Perry, Helen Valentine) with Amanda Williamson Radiobiology Group aims to exploit high throughput Hypoxia in tumours limits the efficacy of genotyping and analysis carried out in Cambridge Clinical Research Fellows radiotherapy. However, its accurate (Drs Gill Barnett and Alison Dunning). Although Guy Betts technologies to develop molecular profiles that predict a measurement is difficult and no technique has ~2,000 samples have been genotyped using the Ahmad Mirza cancer patient’s response to radiotherapy. been incorporated into routine clinical practice. Illumina Human CytoSNP-12 GWAS chip, results Navin Mani Towards this goal, we derived a hypoxia will not be available until the middle of 2011. metagene using microarray gene expression However, a candidate gene study was carried Scientific Officers Some tumours respond well to radiotherapy, modification of RNA in FFPE tissue prevents its Joely Irlam-Jones analysis. A meta-analysis showed that expression out to test all single nucleotide polymorphisms Helen Valentine whereas others do not. The underlying biology routine use in expression profiling studies. of a 25-gene signature was highly prognostic in (SNPs) reported in the literature to be linked that accounts for differences in response to Working with the Applied Computational multiple cancers (Buffa et al., 2010). Customised with the development of radiotherapy toxicity. A Graduate Students radiotherapy is poorly understood. There is Biology and Bioinformatics Group (Hui Sun TaqMan Low Density Arrays (TLDAs) were multivariate analysis of 99 SNPs in 1,167 DNA Stephanie Donaldson (joint with evidence that intrinsic sensitivity to radiation, Leong) a unique pipeline was developed developed and used to investigate hypoxia samples from breast and prostate cancer Imaging Science) hypoxia and proliferation are important. employing an ensemble of new techniques. We Lucile Armenoult (MSc student, metagene expression in head and neck cancer patients included clinical co-variables that might The University of Limoges) showed that robust genome-wide signatures (HNC) samples and cell lines. Median influence the development of toxicity. Only one Rohan Iype (MRes student) Tumour radiosensitivity with potential for clinical exploitation can be Figure 2 expression of the metagene (Hypoxia Score; HS) SNP in ATM emerged as significant after Work carried out by the group several years ago reliably obtained from FFPE material. Affymetrix Inter- and intra-tumour variation in was higher in 2 HNC cell lines (CAL-27 and Bonferroni correction for multiple comparisons. Undergraduate Students TLDA Hypoxia Score (A) and CA9 showed measurements of primary human Human Exon 1.0 ST arrays were used to profile SSC-25) cultured in hypoxia (0.1% O2) versus Sophie Benoliel gene expression (B). Biopsies were tumour radiosensitivity determined as surviving 19 cervical squamous cell carcinoma (SCC) and taken from 3 or 4 locations within normoxia (n=3; p<0.05). In frozen HNC Dimple Jain Kathryn O’Shea fraction after 2 Gy (SF2) in an in vitro clonogenic 9 adenocarcinoma (AC) samples. The gene the same HNC sample before samples variation in HS between patients was Publications listed on page 72 assay was an independent prognostic factor for signature was tested on an independent fresh- analysis. Bioinformatician radiotherapy outcome. Ongoing work by the frozen non-small cell lung cancer (NSCLC) Jan Taylor (joint with Applied group is deriving a gene expression signature series. Analysis revealed 1,062 genes higher in Computational Biology and associated with tumour radiosensitivity (John SCC compared with AC, and 155 genes higher Bioinformatics; from October 2010) Hall). A challenge has been whether old in AC (FDR p<0.01; absolute fold-change >2). formalin-fixed paraffin-embedded (FFPE) blocks This signature of 1,217 genes was capable of Scientific Administrators linked to unique SF2 data can be profiled to correctly separating 58 NSCLC samples into Rebecca Elliott generate gene expression signatures associated SCC and AC subtypes. Twenty-six genes from Sophie Perry with measurements of primary human tumour the signature were then shown to correctly radiosensitivity. Over the last few years the partition cervix samples using Quantigene 2.0 Molecular Biology Core Facility has developed Plex (a multiplex bead-based alternative to qRT- and refined a series of workflows that have PCR) into the appropriate histological groups. In supported the laboratory aspects of this the AC a network of genes centred on HNF approach. FFPE samples contain a wealth of transcription factors (HNF1B, HNF4A and information pertaining to clinical disease, HNF4G) and GATA6 was also identified, particularly cancer. Degradation and chemical suggesting for the first time, that a HNF-GATA 52 | Paterson Institute for Cancer Research Scientific Report 2010 Translational Radiobiology Group | 53


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    CARs containing the CD3ζ receptor protein-protein interactions (Bridgeman et al., Medical Oncology: Clinical and Experimental incorporate into the endogenous T cell 2010a). This method conclusively proved that Immunotherapy Group receptor complex. the CD3ζ CAR is indeed present within the The expression of CARs on the T cell surface TCR complex and, importantly, that this enables the potent re-direction of T cell function. interaction remains critical for the optimal However, there is little information concerning function of the CAR (Bridgeman et al., 2010b). the biochemical and structural interactions that the CAR has with endogenous host proteins on Improved model systems to investigate CAR T the T cell. In particular, does the expression of a cell function CAR affect normal T cell function? To investigate We have put a considerable effort into this, we determined the potency of cytokine developing models that reflect the immunological response of cell lines following mitogenic setting of the cancer patient. Most targets are Immunotherapy utilises the immune system to control and activation and found that cells expressing a CAR tumour associated antigens where the particular containing the CD3ζ protein showed an protein is expressed at high level on tumours potentially eradicate cancer. Whilst the group is involved in increased sensitivity to mitogenic stimulation. but is also expressed at lower levels on normal a number of innovative approaches in clinical trials, at a We explored the reasons for this and identified healthy tissue. These models seek to answer that the expression of the CD3ζ CAR resulted whether a tumour specific response can be pre-clinical level we focus on developing adoptive cell therapy. in an increased level of TCR expression on the generated against tumour associated antigens Figure 1 transduced T cell. This modulation of cell surface and whether the patient’s own immune response Targeting of MART-1+ melanoma TCR complexes appeared to be due to the can be triggered to respond to tumour but not Clinical trials pazopanib. In upper gastrointestinal cancer our cells by T cells expressing the F5 TCR. incorporation of the CAR into the TCR to over-react by targeting those proteins We are undertaking two Phase I trials of gene- phase II study of an anti-CTLA4 antibody (A) F5TCR transduced co-cultured for 4 hours with Mel624 melanoma complex. Site-directed mutagenesis studies expressed on healthy tissue. modified cell therapy and both have shown (tremelimumab) was published (Ralph et al., Group Leader tumour activate by the cell surface confirmed that charged transmembrane amino- interesting clinical effects. The initial data were 2010) and the remarkable durable remission in Robert Hawkins translocation of CD107a while mock acids within the CAR were driving this A key model is CD19 which is found on normal presented at the International Cellular Therapy one of the patients continues now at over 3 control T cells fail to respond to the interaction. However, proving the interaction B cells as well as on B-cell malignancies. When of Cancer Symposium chaired by Professor years. tumour cells. (B) F5 TCR+ T cells Consultant/Senior Lecturer through the analysis of co-precipitating proteins using a CAR consisting of the CD3ζ receptor Fiona Thistlethwaite (Mat leave Hawkins and organised by ATTACK in can prevent the growth of 7 day established HLA-A2+ Mel624 cells proved difficult using standard techniques. To this only, tumour is eradicated and mice remain B cell 2010) Montpellier in September 2010. The trial Laboratory aspects of clinical trials but not HLA-A2- Mel888 tumour end, we have developed a novel bead-based depleted until the CAR T cells eventually targeting CEA caused unexpected respiratory To facilitate development of further trials two Senior Fellow cells. flow cytometry based method to examine disappear at around one month after transfusion toxicity. The scientific basis of this side effect is new laboratories have been developed. The David Gilham (Cheadle et al., 2010) suggesting the CAR T cells being further examined but it correlated with Clinical Immune and Molecular Monitoring become exhausted during this time because of Research Fellows high levels of T cells and high levels of cytokine Laboratory (CIMML) is fully functional and has a their continuous killing of re-populating normal Dominic Rothwell (CIMML) release suggesting specific T cell activation. range of GCLP immune and molecular assays set healthy B cells. More recently experiments have Eyad Elkord (until Dec ) Whilst the side effects were self limiting the up. It is a generally available facility and links with Ryan Guest (GMP Cell confirmed that using a CAR with greater DSMB decided the trial should close rather than a number of clinical groups (eg Haematology, Processing Unit) signalling power results in longer term survival of attempt to modulate toxicity. In a trial targeting Radiobiology). The Cellular Therapeutics unit the CAR T cells and also long term depression Postdoctoral Fellow CD19 on B-cell malignancies one patient has had was opened in February 2010 and has gone of B cell numbers in the treated mice. This is Vivien Hanson (until Oct) a durable partial remission. through a process of setup and validation prior encouraging since it suggests that the engineered to submission of an MHRA licence application. Scientific Officers T cells can protect against CD19 expressing cells Phase II and subsequent Phase III trials of Trovax Approval is expected early in 2011 and trials Debbie Burt (with Immunology) over a long period of time. (targeting 5T4 –discovered by Peter Stern, targeting melanoma and other cancers are (CIMML) Natalia Kirillova (GMP Cell Immunology Group) in renal cancer have now expected to start after that. Working with the Engineered T cells for the treatment of Processing Unit) been fully reported – overall, there is no benefit melanoma group and with input from Mark malignant melanoma. Sam Mowbray (GMP Cell for adding vaccine to standard of care but there Dudley (NCI) we plan to set up adoptive cell Processing Unit) Through collaborations with the NCI (Dr Steven appears to be a benefit of Trovax with therapy treatment for melanoma. Lidan Christie (GMP Cell Rosenberg/Dr Paul Robbins) we are also testing interleukin-2 (IL2) compared to IL2 alone in Processing Unit) T-cell receptor (TCR) engineered T cells. We Vicky Sheard good prognosis patients (Amato et al., 2010). In Pre-clinical research have TCRs that target both MART-1 and NY- Marzieh Kamjoo (until Nov) this context we have updated our analysis of A key aim is to re-direct the effector functions of ESO1 which are HLA-A2 restricted epitopes renal cancer patients treated with high-dose IL2 T cells towards tumour cells by gene-modifying Graduate Students which is expressed to high level on many after prospectively assessing histological features. primary T cells to express targeting receptors. Grazyna Lipowska-Bhalla melanomas and some other tumours. For The outcome for these patients is excellent with Our predominant focus has been upon the use Erik Alcantar Orozco example, primary human T cells transduced to Mariam Al-Muftah (joint with around 25% obtaining a complete remission and of Chimeric Antigen Receptors (CAR) which use express the F5 TCR de-granulate in the presence Immunology) most of these appear durable (Shablak et al., in antibody-type domains to target the T cell to cell of tumour cells expressing MART-1 and HLA-A2 Hannah Gornall (from Oct) press). We now plan an international surface protein antigens (reviewed in Bridgeman and these cells can prevent the growth of these randomised study to formally confirm the et al., 2010c). However, to target intracellular Clinical Fellows tumour cells but fail to affect the growth of cells potential advantages over other available proteins, natural T cell receptors (TCR) are used Alaaeldin Shablak (to Sept) which lack HLA-A2 thereby confirming Smita Sharma (CIMML) treatments for this group of patients. – this involves the expression of the α and β therapeutic specificity (Figure 1). We are further chains of a tumour-specific TCR thereby optimising this approach prior to potential future Marie-Curie Training Fellow Targeted therapies are currently the mainstay of bestowing the T cell with new tumour specificity. Vania Baldan (from Oct) trials. renal cancer treatment and we play a leading Over the past year, we have been working with role in many trials. Notably the Phase III study of both types of gene-modified T cell as well as ATTACK Project Managers Nikki Hudson (until Nov) pazopanib in renal cancer (Sternberg et al., natural T cells to develop adoptive cellular Publications listed on page 73 Helena Kondryn (from Nov) 2010) is a potentially practice-changing study in therapy. renal cancer and lead to the licensing of 54 | Paterson Institute for Cancer Research Scientific Report 2010 Medical Oncology: Clinical and Experimental Immunotherapy Group | 55


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    Medical Oncology: Translational formation rather than proliferation (Cole et al., 2010b). In the case of VEGF165 these overall survival in several tumour types. The effect is modest and it is therefore appropriate Anti-Angiogenesis Group observations were associated with reduced to identify predictive biomarkers that will allow phosphorylation of VEGF receptor tyrosines patient selection thereby reducing toxicity and implicated in cell migration rather than expense while allowing combination regimens proliferation. In addition, reduced containing VEGF inhibitors to be developed. phosphorylation of FAK and assembly of F-actin at the cellular periphery was observed when We have developed a programme in which cells were treated with the most biologically imaging and circulating biomarkers are being active oligosaccharide (Figure 1). used to identify those patients who will benefit In a programme supported by Cancer Research from anti-angiogenic agents. In addition to CR- UK and the MRC we have explored in vivo UK phase I clinical trials (e.g. GSAO) we are Several randomised trials have demonstrated a survival activity of size and sulfation-defined testing the predictive value of Dynamic Contrast oligosaccharide that showed the highest Enhanced Magnetic Resonance Imaging (DCE- advantage in epithelial malignancies in patients who are inhibitory activity in endothelial cell-based assays. MRI), Diffusion Weighted Imaging (DWI) and treated with conventional therapy supplemented with VEGF We demonstrated that the oligosaccharide blood-borne, protein and cellular biomarkers in a achieves appropriate concentrations in tumours large trial in patients with metastatic colorectal inhibitors. However, the effect is modest, and new improved in vivo and that this is associated with significant cancer with liver metastases who are being anti-angiogenic agents are required. Heparan sulfate reductions in FGF receptor activity (data treated with cytotoxic chemotherapy and unpublished). bevacizumab. The complex data set that proteoglycans are essential for the biological activity of the emerges from this study will be evaluated in Group Leader majority of angiogenic growth factors. In previous work we FGFs are well known angiogenic growth factors collaboration with Professor Cindy Billingham but their contribution to the epithelial (University of Birmingham). Gordon Jayson demonstrated that heparin oligosaccharides were anti- component of cancer is less clear. We therefore Senior Fellow angiogenic in vivo. This year we have completed the chemical undertook a comprehensive evaluation of FGF In a second programme, we lead the and FGF receptor expression in epithelial ovarian translational research programme associated Egle Avizienyte synthesis of two families of heparan sulfate oligosaccharides, cancer and demonstrated that FGFR2 isotype with the MRC trial, ICON7; a randomised trial Postdoctoral fellows demonstrating structure-function relationships against different switching occurs upon transformation and that comparing carboplatin and paclitaxel with or Claire Cole the ligands for the new receptor are expressed without bevacizumab (an anti-VEGF antibody) in Steen Hansen endothelial phenotypes that are essential for angiogenesis. in ovarian cancer cell lines and tissue. the first line treatment of ovarian cancer. The Gavin Miller Knockdown of FGFR2 inhibited proliferation in trial demonstrated a significant but small Clinical Fellows Heparan sulfate oligosaccharides as oligosaccharides, containing 8-10 vitro and in vivo and increased the sensitivity of advantage in progression free survival, Danielle Shaw anti-angiogenic agents monosaccharide residues, inhibited angiogenesis cell lines and tumours to cisplatin chemotherapy highlighting the importance of translational Laura Horsley (Collaborator: Dr. John Gardiner, Manchester in different models in vivo. However, the in vitro and in vivo, respectively. However, research. The first translational research Kalena Marti-Marti Interdisciplinary Biocentre) oligosaccharide species involved had disparate results were seen when FGFR1 experiments have been conducted in the Heparan sulfate (HS) is a linear anti-coagulant potential as they were derived expression was reduced suggesting that selective Paterson Institute. Additional research Scientific Officer Graham Rushton glycosaminoglycan that consists of multiple from heparin and a scalable synthesis was FGFR tyrosine kinase inhibitors might be clinically programmes will occur around the world over copies of a disaccharide that contains N- required if the strategy was to be advanced. We superior (Cole et al., 2010a). the next year with the aim of identifying substituted glucosamine and a hexuronic acid; therefore established an organic chemistry predictive biomarkers that will allow us to select either iduronic or glucuronic acid, depending on programme that resulted in the first inexpensive Angiogenesis biomarkers patients who are most likely to benefit from anti- the C5 configuration. HS can be sulfated at and scalable synthesis of iduronate (Hansen et (Collaborators: Profs. Caroline Dive, Alan angiogenic agents. various positions of each disaccharide although al., Org Lett 2009; 11: 4528) and subsequently Jackson, Geoff Parker; Paterson Institute and The the most common sites are iduronic acid 2-O- led to the elucidation of the complete controlled University of Manchester) sulfate, glucosamine N-sulfate and glucosamine synthesis of heparan sulfate oligosaccharides The addition of VEGF inhibitors to conventional Publications listed on page 74 6-O-sulfate. Sulfation tends to be clustered, containing up to 12 saccharide residues with therapy has improved progression free and/or creating domains of high anionic charge that variable sulfation patterns. facilitate the interaction with protein ligands, the prototype of which are the Fibroblast Growth Structure-function studies were performed using Figure 1 FGF2- and VEGF165-induced Factors (FGFs). endothelial cells. Two families of HS peripheral accumulation of activated oligosaccharide were investigated: one was FAK and F-actin is inhibited by HS is covalently bound to a core protein to sulfated at the iduronic acid 2-O- position only oligosaccharide. (A) Lack of generate the HS proteoglycans, which have been while the other was sulfated at both the iduronic peripheral accumulation of FAK implicated in binding and activating several acid 2-O- position and glucosamine N- position. phosphorylated on tyrosine 397 and F-actin in serum-starved HUVECs. angiogenic cytokines, including FGF, VEGF (except Oligosaccharides were generated that contained Cells were co-stained with anti- VEGF121) and chemokines such as IL-8 and SDF- between 4 and 12 saccharide residues and their phospho-FAK (pTyr397) antibody 1. The fact that the dependency on HS extends potential to inhibit endothelial cell proliferation, and phalloidin-AlexaFluor568. (B beyond molecules that signal through tyrosine migration and tube formation were evaluated. and D) Peripheral FAK kinase receptors highlights a potential new phosphorylated at tyrosine 397 and F-actin are detected after 10 min approach to the development of anti-angiogenic The data demonstrated that the ability of the stimulation with FGF2 (B) or VEGF165 agents. oligosaccharides to inhibit endothelial (D). (C and E) Oligosaccharide phenotypes increased with saccharide length and prevents peripheral localization of In 2005 (Hasan et al., Clin Cancer Res 2005; 11: sulfation. Interestingly, the compounds were phosphorylated FAK and F-actin in 8172) we demonstrated that heparin most potent in assays of migration and tube response to FGF2 (C) or VEGF165 (E). Scale bar, 75 μm. 56 | Paterson Institute for Cancer Research Scientific Report 2010 Medical Oncology: Translational Anti-Angiogenesis Group | 57


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    Research Services modified. Hypoxia at the microscope, depriving a population of single cells or tissue of an study ineffective. To accomplish this task even a 5% increase in detection sensitivity or efficiency http://www.paterson.man.ac.uk/research adequate oxygen supply, allows for the modelling has a benefit to the investigations carried out in and study of tumour development and growth the laboratory, subsequently all of the systems where oxygen concentrations are lower than within the facility are constantly modified to that of the surrounding area. Hypoxic tumour improve efficiency. The requirement for low-light cells are usually resistant to radiotherapy and imaging is essential when trying to resolve the chemotherapy and so by having the ability to interaction of structural and molecular study hypoxia and its effect in the live cellular interactions within the cell by techniques such as environment under the lens our understanding Förster Resonance Energy Transfer or split-GFP. of protein expression and the tumour micro- To realise this aim a new camera has been environment is enhanced. purchased to push both our light detection limits There have been a number of significant developments in our and spatial resolution to greater levels than To compare the efficacy of newly-developed formerly possible. The new camera system Research Services in 2010. Appointments have been made in drugs or the effect of the environment on cell permits not only presenting the data in the form the Biological Mass Spectrometry and Histology Facilities, and populations there is a requirement to study of intensity but also in units of photons which populations of various cell types in differing leads to the analysis of both location and we are in the process of recruiting an additional scientific environments, and how they respond over time. quantity. officer to work in the Molecular Biology Core Facility. The Over the last fifteen years the accuracy of positioning devices has permitted multiple Twelve publications have been published with support for these posts demonstrates the importance that the investigations to be carried out simultaneously guidance and data derived from the facility over Head of Research Services Institute places on all of its support facilities. One notable which has led to the use of microscopy data to the last year. In addition to training 52 new move from the stand-alone image to statistically microscope users and the ongoing advice, Jenny Varley advance during 2010 has come from the implementation of relevant streams of data. Consequently new experimental design and training of the 121 workflows for high throughput sequencing (HTS) using the AB technologies are being developed to image not active users, there have been presentations and just multiple but thousands of areas, each area demonstrations to over two hundred and fifty SOLiDtm system. The techniques for HTS are evolving with different cellular or environmental members of the public on the techniques used incredibly rapidly but are providing vast amounts of data which conditions, and then to image over long time within the laboratory to study oncology in frames. High-throughput imaging is being addition to external demonstrations at Cancer would have been unthinkable just a few years ago. Coupling developed within the Institute along with the Research UK fundraising events. Over the next HTS, particularly for transcript analysis, with proteomic data is requirement for novel tools for the analysis of year the facility, as ever, remains responsive to the data. Capturing these large data sets is a the imaging and analysis requirements of the proving to be particularly fruitful. relatively routine process but being able to researchers within the Institute whilst developing derive meaningful numbers becomes a logistical novel techniques for elucidating tissues, problem which the facility is endeavouring to populations, single cells and molecular All the Research Services provide invaluable support to all solve. interactions in tandem with other technologies. researchers in the Institute. Whilst it is easy to pick out There is an ongoing requirement in the facility to modern new technologies and equipment for praise, we increase detection levels so that single molecular Biological Mass Spectrometry Facility should never forget the vital importance of the support given events can be revealed. Signals from molecular Head: Duncan Smith interaction of what is termed ‘low-light’ can be by teams such as Logistics and Laboratory Services. Without detected easily but the cell may not respond as Our remit is to provide cutting-edge LCMS such services supporting all groups the Institute would be it once did due to the effects of photo-toxicity workflows to Paterson groups for a multitude of (the light used to illuminate the cell causing protein characterisation needs. This unable to function. damage to cell processes) thus rendering the characterisation predominantly involves protein identification, analysis of the type and position of Advanced Imaging Facility of fluids (micro-fluidics) has been introduced. post-translational modifications (PTM) and Head: Steve Bagley Micro-fluidic systems allow for the analysis of peptide based protein quantification. We have a how single or populations of cells respond to a large portfolio of routine services plus a research Microscopes within the facility are utilised to switch of fluids or a concentration gradient. This and development pipeline designed to maintain impart visual and numerical evidence of localised technique can be utilised by shifting the the portfolio’s position at the cutting-edge of information concerning functional properties of concentration of fluids through a cell population cancer research related proteome analyses. the cell and to consider the spatial and temporal to facilitate the study of how a cell responds to relationships linking development, phenotype and rapid changes in the environment such as drug This year has seen recruitment of two new staff biochemical interactions. Over the previous year concentrations. Three research groups within into the facility. In January Simon Perkins was the facility has been developing new the Institute are now utilising these techniques in appointed as the team’s informatician followed methodologies to image more efficiently and diverse studies from examining organelle by John Griffiths who joined us a senior mass with more utility for the researchers within the processes within the cell to the chemotaxic spectrometrist in August. Both the new Institute. response in populations of cells. In addition, appointments underline the Institute’s another live cell system, currently in commitment to enhancing both routine service Techniques used in the laboratory for live cell development and which will be introduced in provision and research and development (R&D) assays have been re-examined and as a the middle of the coming year, will allow for the within the facility. The new appointees increase consequence precise control and manipulation gaseous environment around cells to be our ability to support the growing demand for 58 | Paterson Institute for Cancer Research Scientific Report 2010 Research Services | 59


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    Transgenic services superovulated specific pathogen free (SPF) This year has seen ten new transgenic lines being females mated to stud males with transfer of produced by embryonic stem cell microinjection collected embryos, thus ensuring that the SPF and we are currently awaiting germline status of the facility is maintained. transmission for a further five microinjected lines. Experimental services In vitro fertilisation (IVF) from cryopreserved Technical support has been provided by three sperm has historically presented many problems licenced technicians for a number of established due to low fertilisation rates and this has been a and newer research groups facilitating the particular problem in C57 backcrossed strains. scientific goals of the Institute. Planning in vivo This year however we have established the sessions, assigning stocks, preparing experimental technique successfully on one new line and cage labels and observation sheets has all been expect this will increase considerably in 2011. facilitated before licenced procedures Further IVF improvements maybe considered in commence. A range of techniques has been the near future by using a triple gas incubator delivered for both surgical and non surgical and associated media which gives a potential requests and these have included subcutaneous 20% increase in fertilisation rates for some C57 implantation of slow releasing hormone pellets, backcrossed strains. We have also started to parenteral injections, gavage, blood sampling with develop Non Surgical Embryo Transfer (NSET) and without recovery, whole body irradiation, this year with promising results comparable with local irradiation to subcutaneous tumour and in other NSET-experienced establishments, to date vivo imaging. we have achieved a live birth rate/total embryos implanted of ~25% and this technique will Health and welfare for animals under procedure continue to be developed through 2011 which remains paramount and extensive daily health will lead to not only a refinement but a major checks including body weight, tumour reduction in animal numbers. measurement, dental assessment and abdominal palpations have routinely been carried out. service provision and are critical to our ability to 2011 is set to be an exciting and demanding Cryopreservation of transgenic strains continues maintain R&D activity in an increasingly busy year for the facility. We expect of significant to increase each year with ~30 strains/10,000 New in vivo techniques developed have included: facility. Our R&D activity encompasses growth in routine service demand and a step- embryos being archived in 2010. Cryopreserved biochemistry, liquid chromatography, mass change in the demand for complex quantitative embryos are always tested in in vitro culture and • Intra-mammary injections spectrometry and bioinformatics. proteome and phosphoproteome analyses assessed for viability before cessation of a • Bilateral irradiation within the Institute. transgenic line. Sperm cryopreservation has also • Supra-spinal injections In the area of protein quantification, we have been employed for the archiving of transgenic added dimethyl labelling to our existing toolbox. strains with a further 13 lines processed and we The British Journal of Cancer published the This chemical labelling strategy facilitates MS Biological Resources Unit are expecting this to continue at a higher rate Guidelines for the welfare and use of animals in based quantification in the same fashion as throughout 2011. cancer research earlier this year which updates SILAC without the necessity to metabolically Once again in 2010 the Paterson animal facility the second edition of the UKCCR guidelines label cells in culture. This opens the potential to has continued to run at full occupancy with all With the introduction of a new cryogenic liquid (Workman et al., 2010). The guidelines focus on perform high quality protein quantification in 3000-plus cages in operation and this has led to nitrogen freezer for archiving all our transgenic animal welfare and the use of animals in cancer both preclinical and clinical environments. the identification of a real need to future-proof strains we now have more control over freezing research demonstrating the need to incorporate to ensure that we continue to deliver a high profiles, this new freezer supercedes the near the 3Rs: replacement, reduction and refinement, In the area of PTM analysis, we have developed quality research service. Although the Institute 20-year old programmable freezer (which has and they also include detailed information on a an approach to facilitate phospho-site specific has not seen a huge increase in project licence given sterling service having cryopreserved over number of models such as orthotopic and quantification (in collaboration with the Cell holders there has been expansion of existing 52,000 embryos). metastatic tumour systems. Regulation Group). Moreover, we are actively groups and three major users were granted developing novel workflows to enhance tenure this year which has led to more complex The rederivation process of new strains obtained selectivity of both phosphopeptide and requirements. In addition two new groups have as live mice in 2010 at the Paterson has included Cancer Research UK GeneChip Microarray isopeptide enrichment strategies critical to been recruited to the Institute whose research the following: Service. pushing the global phosphoproteomic and focus has extensive requirements for in vivo Head: Stuart Pepper Ublproteomic fields forward. studies. • ACTFlp • Flk-1 For the last nine years the Molecular Biology The new informatics research capability of our September 2010 saw the publication of the • Gfil/EGFP-k1 Core Facility at the Paterson Institute has hosted facility has been instrumental in supporting all European Union Directive 2010/63 which will • GfilB/EGFP-k1 a microarray service which has been made our current research projects. Some key current lead to rigorous debate over the coming months • p16/p19 available to all Cancer Research UK-funded projects include the active development of an to ensure that the changes to UK legislation in • PDX1 Cre groups. Over that time the service has built up exciting global phosphoproteomic pipeline (in January 2013 are achieved seamlessly. It is clear a broad customer base and supported collaboration with the Applied Computational that this will be the most significant change since On arrival these lines are housed in the numerous CR-UK groups – a total 189 individual Biology & Bioinformatics and Cell Division the Animal Scientific Procedures Act (1986) was dedicated Quarantine area using flexible film researchers have used the service, submitting Groups), automation of phosphosite assignment implemented over 20 years ago. isolators which run at negative pressure. The 455 projects between them. and novel label free quantification analysis rederivation process subsequently involves pipeline. 60 | Paterson Institute for Cancer Research Scientific Report 2010 Research Services | 61


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    Over the last few years there has been a identified and quantified by flow cytometry. We currently houses three cell sorters which are BCSCs can be re-sensitised to radio- and marked shift in the samples that are handled can assess cell phenotype by looking for able to retrieve up to four specifically-defined chemotherapy, indicated by an increase in DNA routinely. In the early years of microarrays most expression of cell surface, cytoplasmic or nuclear populations so that cells may be recovered for damage. work was based on cell lines as the amount of antigens; cellular DNA or RNA content; cell further study including re-culture, RNA or DNA RNA required for microarray labelling was quite cycle analysis; fluorescent protein expression; extraction or use in functional cell assays. The We have also continued our close involvement high; now we frequently handle samples where functional aspects of the cell such as enzyme cell sorters are operated solely by the Flow with John Bridgeman and David Gilham. less than 10ng of RNA is available. This has activity; apoptotic status; mitochondrial Cytometry team on a daily basis: Together we have developed a novel flow opened up opportunities for laser capture membrane potential; ion flux or pH. In addition, cytometric immunoprecipitation method capable microdissection (LCM) of specific populations for any population identified on an analytical flow • Vantage SE - 2 way sorting, 5 colours, dual of investigating protein-protein interactions array analysis and we have seen a lot more of cytometer can be retrieved by using a cell sorter laser (blue and red) specifically the analysis of the T-cell receptor these types of projects arriving over the last year. which has the ability to physically separate cells • FACS Aria II u - 4 way sorting 12 colours, which has yielded a number of publications for of interest from a biologically complex triple laser (violet, blue and red) the facility (Bridgeman et al., 2010a). The service has continued to support profiling of population. Some of the more common • InFlux - 4 way sorting, 14 colours, equipped archival formalin fixed samples, another area research applications include immunology; cell with 5 lasers (UV, violet, blue, red and orange) where we are seeing an increase in the number cycle and cell growth; cell function and activation; Histology of projects. Profiling of archival samples has cell differentiation; apoptosis; toxicology and Other services Head: Garry Ashton moved from an exploratory phase where most fluorescent protein detection. Our facility offers a full range of educational and papers where concerned with validating the cytometric services. We are able to advise on a Again the year has been exceptionally busy with technique to a position now where it is The use of flow cytometry in the Paterson wide variety of cytometry related subjects several key developments taking place. The becoming generally accepted that expression Institute can be divided into two broad including experimental design, selection of recruitment and training of another scientific profiling of archival samples is a valid, if difficult, categories; analytical cytometry and cell sorting. reagents, data analysis, presentation, officer has allowed the unit to continue to offer approach. interpretation, we also act as a beta test site for a comprehensive and flexible service whilst also Analytical cytometry novel cytometry applications and we also advise allowing us to continue to focus on our With the advent of clonal sequence platforms The ability of flow cytometers to evaluate cells on data presentation. The latter is becoming development. there is a lot of speculation about the future for at an extremely rapid rate (e.g. up to 20,000 more and more important as journals require microarrays. Our service has been consistently events per second) makes this technology ideally cytometric data to be more transparent. Use of the Leica LMD6000 laser capture busier over the last two years then at any time suited for the reliable and accurate quantitative microdissection (LCM) system has increased. previously, and the number of new projects analysis of selected physical properties of cells of Technical developments Projects focusing on the role of heparan sulfate being discussed has not slowed down. This interest. The sensitivity of these instruments for This year as part of our internal annual review of (HS) in ovarian cancer, the effects of intermittent would suggest that at least for the next year or detecting the presence of molecules expressed our instrument stock we upgraded the energy restriction on gene expression in breast two at least microarrays will still be in big at low levels is impressive; given high quality cell computing systems which control the Caliburs tissue in women at increased risk of breast demand. preparations and reagents, as few as 50 which has greatly improved the data acquisition cancer and the targeting of tumour cells/normal molecules per cell may be detected. The facility and stability of the systems. Additionally we have bronchial epithelium for subsequent nucleic acid currently has four bench top cytometers upgraded the FACS Aria to the FACS Aria II; this extraction in lung cancer patients are examples Flow Cytometry Facility including one plate-based bead reader. These has drastically improved the workflow and of current studies. Head: Morgan Blaylock are all user-operated systems for which we offer stability of the system allowing us to provide a basic training in a group setting which is much improved product to our users. In anticipation of future demand from the The Flow cytometry Facility at the Paterson supplemented with one to one training for expansion of tissue biomarkers and the provides state-of-the-art instrumentation, specific applications: We have been involved in the continuation and requirements of the Manchester Cancer education and expert technical assistance to development of a number of projects this year. Research Centre (MCRC) Biobank, in early 2010 investigators for the successful performance of • FACS Calibur - 3 colour single laser (blue) We have been collaborating extensively with a the lab took delivery of the new ATA27 flow cytometry based studies. The goal of the • FACS Calibur - 4 colours dual laser number of researchers in the Institute including automated tissue microarray platform. The facility is both to support current research (blue and red) Dr Gillian Farnie and Pam Willan who have been system allows high throughput, accurate TMA applications and to continuously extend the • FACS Array - 4 colours, dual laser (green investigating the resistance of breast cancer stem construction of tumour-specific and custom repertoire of flow cytometric methods available, and red) cells (BCSCs) to DNA damage by radio- or arrays. A large amount of time has been spent providing the tools to help our researchers treat, • LSRII - 17 colours, quadruple laser (UV, chemotherapy. There is evidence the BCSCs optimising this process resulting in the prevent and understand cancer in its many violet, blue and red) preferentially survive this type of anti-cancer production of extremely high quality TMAs giving forms. therapy and Gillian and Pam have been true representation. One example is the Cell sorters addressing whether the amount of DNA construction of a small fifty-patient breast tissue Flow cytometry can be viewed as a specialised One of the properties of the larger flow damage caused by therapy is similar in BCSCs vs. array where both tumour and matched normal form of fluorescence microscopy and is a means cytometers is the ability to electronically deflect non-BCSCs by flow cytometry. They treat cells cores were transplanted to study the role of of measuring the physical and chemical cells with preset, defined properties into a with radio- or chemotherapy, and stain the cell ATF2 and stress activated protein kinases (SAPK) characteristics of cells or particles using separate collection tube. For cell purification, surface markers to define the BCSC population. in breast cancer progression. fluorescent markers. As the cells flow past a flow cytometry is especially well suited for Cells are then stained for an intra-nuclear focused laser beam of appropriate wavelength, applications requiring high purity. Because protein which is a marker of double strand DNA The Manchester Cancer Research Centre the probes fluoresce and emit light; this is multiple fluorochromes (e.g. up to fourteen breaks and a DNA stain to allow cell cycle Biobank has now been running for two and half collected by detectors which in turn translate distinct fluorescent probes reacting with different analysis, and are then analysed on the LSRII. years. Solid tumour with paired normal tissue is the light signals into electronic signals cell associated molecules) can be assessed Their results have shown that the BCSCs have collected and processed centrally by the facility. proportional to the amount of light collected. simultaneously, cell sorting by flow cytometry can lower levels of DNA damage after radio- and Blood and bone marrow from haematological Any aspect of a cell which can be labelled or separate complex mixtures of cells on the basis chemotherapy compared to the non-BCSCs. malignancy patients is also processed. To date detected with a fluorescent marker can be of multiple marker expression. The sorting suite They have also shown that after pre-treatment samples from over 1600 patients has been of breast cancer cells with differentiating agents banked. 2010 has seen a significant rise in the 62 | Paterson Institute for Cancer Research Scientific Report 2010 Research Services | 63


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    number of applications to use Biobank samples. production of media and agar plates. We also In total, there have been almost thirty supply the research labs with a member of the applications (six of which have been for TMAs), department, a Lab Aide, to perform certain lab with an approval rate for projects of over 90%. duties in order to assist the smooth running of Applications have been received from a variety the labs. Each day we collect the dirty glassware of sample types, including lung, breast and liver. and deliver clean glassware and plastics to the labs. The routine and troubleshooting immunohistochemistry (IHC) service offered by As the number of groups within the Institute has the facility has seen unprecedented demand risen over the last year, we have had to process recently. In addition the number of research larger amounts of glass and plastics on a daily groups using the facilities to perform their own basis. To absorb this additional workload it has IHC has risen sharply. The lab now houses been necessary to adjust the amount of Lab several epitope retrieval stations and the i6000 Aide time assigned to individual labs. We have automated IHC platform which gives groups the taken on an additional member of staff to help flexibility to optimise and validate their own with this increased throughput. The number of specific IHC projects. The i6000 platform is labs requesting bulk media has increased and we regularly oversubscribed and as a result we soon have increased the range of media we offer hope to purchase a second fully enclosed IHC whilst keeping to our production deadlines. platform. Both systems will compliment each other, offering high-throughput antibody validation service availability and unrivalled Logistics standardisation and reproducibility. Both these Head: Maurice Cowell systems will become invaluable with the availability of any new image analysis software. The Logistics department provides a Molecular Biology Core Facility Aside from technology platforms such as the comprehensive and vital role in supporting the Head: Stuart Pepper SOLiD and quantitative PCR (qPCR) systems, Specialised techniques have been employed to research carried out at the Institute. This group the core facility also provides routine screening process hydroxyapatite/tricalcium phosphate encompasses a wide range of duties including Over the last year the main focus for services. For some time now the facility has biomaterial that has been implanted the accurate receipting, checking, booking in and development has been on our new clonal offered a regular cell line mycoplasma screen but subcutaneously in NOD/SCID IL2Rγ-/- efficient distribution of goods ordered by sequence platform. At the start of the year we this year we have expanded to include a cell line immunocompromised mice in a study with the numerous Personnel in the Institute. The had just completed our purchase of an Applied authentication service. The standard technology Leukaemia Biology Group defining and evaluating collection and removal of waste, be it general Biosystems SOLiDtm platform and were starting for authentication is to use short tandem repeat biological mechanisms in bone marrow stroma rubbish, yellow bags or GM waste is also the to work on developing services. Clonal profiling, which is the same approach used in responsible for the regulation of normal and porters responsibility. They are accountable for sequencers are bringing a true revolution to forensic labs to identify individuals. As part of malignant bone marrow haematopoietic stem the collection of liquid nitrogen containers from biological research as they have made genome this service we have created a database of and progenitor populations using a humanised laboratories, transporting to the loading bay for sequencing accessible to small core facilities as reference profiles that allows confident validation implantation mouse model. refilling and returning to the labs. This is done well as the major sequencing centres such as of test samples. There has been a high uptake three times a week and dry ice is delivered Sanger. Aside from sequencing entire genomes for this service resulting in more than 400 Work has continued with the Stromal Tumour twice a week. these systems are also open platforms which can samples being analysed. Having completed the Interaction Group, where IHC has been used in be adapted for many applications, including first round of screening we will now offer this order to understand the paracrine and/or Ordering and distribution of the Central Stores transcriptome analysis and chromatin IP studies. service on a regular basis so that new cell lines autocrine mechanisms responsible for trans- stock via the intranet E-mail “Order Stock Items” can be authenticated. It is gratifying that the vast differentiation of normal fibroblasts into tumour- function has been updated to become more Our main interest has been looking at the majority of our cell lines are correct. promoting stably maintained myofibroblast rich user friendly and it is our duty to ensure potential to use sequencing of RNA (RNAseq) carcinoma-associated fibroblasts (CAFs). We adequate stock levels are maintained at all times. to yield both quantitative and qualitative At the end of last year we were facing a have also continued studies with the Children’s Included in this are the media and enzymes information. For the last decade microarrays bottleneck on our qPCR platform, the Appplied Cancer Group where IHC has also been used in stored in the Institute freezers (Sigma, Invitrogen, have been the leading platform for expression Biosystems 7900 system. We were fortunate to the development a mouse model for acute Roche, Promega, New England Biolabs, Fisher kits profiling, however RNAseq offers some potential be able to buy a second 7900 system to support lymphoblastic leukaemia (ALL). and Qiagen), again the Logistics department is advantages over microarrays. Firstly, to design a qPCR this year thereby avoiding delays to responsible for the ordering, distribution and microarray it is necessary to have some advance research projects. Along with continued demand stock levels of these items. The Institutes’ usage knowledge of the transcripts to be detected to use the 7900s for expression profiling, we Laboratory Services of gas cylinders is looked after by the porters whereas RNAseq can be used to characterize have seen an increase in the number of groups Head: Mark Craven who are in charge of replacement and ordering novel transcripts. A further benefit of RNAseq is who want to study micro RNA expression and as and when necessary. The department works in the characterization of mutations which may so the extra capacity on the qPCR platform has The Laboratory Services department supports closely with all groups and helps out where cause loss of detection on a microarray. In the been particularly beneficial. all the research labs by supplying them with necessary, be it tracing and confirming delivery of first year we have supported several research sterile glass and plastic ware, sterile fluids and goods with suppliers, and dealing with missing, groups in projects exploring human, mouse and media. We will also sterilize non routine items damaged or wrong items. We also assist or yeast transcriptomes with exciting results. The sent up from the labs. We use two industrial manage the movement of heavy equipment or first publication from this service is already out glass washers and two autoclaves to provide this furniture, and the setting up of various meeting (Bradford et al., 2010) with more on the way for service together with a clean room for the rooms for numerous events. next year. 64 | Paterson Institute for Cancer Research Scientific Report 2010 Research Services | 65


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    Research publications Crispin Miller (page 14) Sabharwal, A., Corrie, P.G., Midgley, R.S., Palmer, C., Karim Labib (page 18) Daskalos, A., Oleksiewicz, U., Filia, A., Nikolaidis, G., Applied Computational Biology and Brady, J., Mortimer, P., Watson, A.J., Margison, G.P. Cell Cycle Group Xinarianos, G., Gosney, J.R., Malliri, A., Field, J.K. and Bioinformatics Group and Middleton, M.R. (2010) Liloglou, T. (2010) A phase I trial of lomeguatrib and irinotecan in Other Publication UHRF1-mediated tumor suppressor gene Refereed Research Papers metastatic colorectal cancer. Cancer Chemother Labib, K. (2010) inactivation in nonsmall cell lung cancer. Cancer, Bitton, D.A., Smith, D.L., Connolly, Y., Scutt, P.J. and Pharmacol, 66, 829-835. How do Cdc7 and cyclin-dependent kinases trigger epub Nov 8. Miller, C.J. (2010) the initiation of chromosome replication in An integrated mass-spectrometry pipeline identifies Watson, A.J., Sabharwal, A., Thorncroft, M., eukaryotic cells? Genes Dev, 24, 1208-1219. Rooney, C., White, G., Nazgiewicz, A., Woodcock, novel protein coding-regions in the human genome. McGown, G., Kerr, R., Bojanic, S., Soonawalla, Z., S.A., Anderson, K.I., Ballestrem, C. and Malliri, A. PLoS One, 5, e8949. King, A., Miller, A., Waller, S., Leung, H., Margison, (2010) G.P. and Middleton, M.R. (2010) The Rac activator STEF (Tiam2) regulates cell Tumor O6-methylguanine-DNA methyltransferase Nic Jones (page 22) migration by microtubule-mediated focal adhesion Bradford, J.R., Hey, Y., Yates, T., Li, Y., Pepper, S.D. and Cell Regulation Group Miller, C.J. (2010) inactivation by oral lomeguatrib. Clin Cancer Res, disassembly. EMBO Rep, 11, 292-298. A comparison of massively parallel nucleotide 16, 743-749. Refereed Research Papers sequencing with oligonucleotide microarrays for Woodcock, S.A., Rushton, H.J., Castaneda-Saucedo, Li, S., Ezhevsky, S., Dewing, A., Cato, M.H., global transcription profiling. BMC Genomics, 11, Other Publications E., Myant, K., White, G.R., Blyth, K., Sansom, O.J. and Scortegagna, M., Bhoumik, A., Breitwieser, W., 282. Kaina, B., Margison, G.P. and Christmann, M. (2010) Malliri, A. (2010) Braddock, D., Eroshkin, A., Qi, J., Chen, M., Kim, J.Y., Targeting O6-methylguanine-DNA methyltransferase Tiam1-Rac signaling counteracts Eg5 during bipolar Jones, S., Jones, N., Rickert, R. and Ronai, Z.A. Buffa, F.M., Harris, A.L., West, C.M. and Miller, C.J. with specific inhibitors as a strategy in cancer spindle assembly to facilitate chromosome (2010) (2010) therapy. Cell Mol Life Sci, 67, 3663-3681. congression. Curr Biol, 20, 669-675. Radiation Sensitivity and Tumor Susceptibility in ATM Large meta-analysis of multiple cancers reveals a Phospho-Mutant ATF2 Mice. Genes Cancer, 1, 316- common, compact and highly prognostic hypoxia Margison, G. (2010) 330. metagene. Br J Cancer, 102, 428-435. O6-methylguanine in DNA: bad penny? Cell Cycle, Caroline Dive (page 26) 9, 441-442. Clinical and Experimental Pharmacology Nemoto, N., Udagawa, T., Ohira, T., Jiang, L., Hirota, Southgate, T.D., McGinn, O.J., Castro, F.V., K., Wilkinson, C.R., Bahler, J., Jones, N., Ohta, K., Rutkowski, A.J., Al-Muftah, M., Marinov, G., Active Patents Refereed Research Papers Wek, R.C. and Asano, K. (2010) Smethurst, G.J., Shaw, D., Ward, C.M., Miller, C.J. O6-Substituted guanine derivatives, a process for Board, R.E., Wardley, A.M., Dixon, J.M., Armstrong, The roles of stress-activated Sty1 and Gcn2 kinases and Stern, P.L. (2010) their preparation and their use in treating tumour A.C., Howell, S., Renshaw, L., Donald, E., Greystoke, and of the protooncoprotein homologue Int6/eIF3e CXCR4 mediated chemotaxis is regulated by 5T4 cells. McMurry, T.B.H., McElhinney, R.S., McCormick, A., Ranson, M., Hughes, A. and Dive, C. (2010) in responses to endogenous oxidative stress during oncofetal glycoprotein in mouse embryonic cells. J.E., Elder, R.H., Kelly, J., Margison, G.P., Rafferty, J.A., Detection of PIK3CA mutations in circulating free histidine starvation. J Mol Biol, 404, 183-201. PLoS One, 5, e9982. Watson, A.J. and Willington, M.A. DNA in patients with breast cancer. Breast Cancer International Patent Application PCT/IE94/0031, Res Treat, 120, 461-467. Shah, M., Bhoumik, A., Goel, V., Dewing, A., published (WO 94/29312, 70 pages) through CR Breitwieser, W., Kluger, H., Krajewski, S., Krajewska, Technology Ltd. (Chem. Abs. 1995, 122, 239458e). Geoff Margison (page 16) M., Dehart, J., Lau, E., Kallenberg, D.M., Jeong, H., Brookes, K., Cummings, J., Backen, A., Greystoke, A., Carcinogenesis Group Eroshkin, A., Bennett, D.C., Chin, L., Bosenberg, M., Ward, T., Jayson, G.C. and Dive, C. (2010) Pyrimidine derivatives and guanine derivatives, and Issues on fit-for-purpose validation of a panel of Jones, N. and Ronai, Z.A. (2010) their use in treating tumour cells. McMurry, T.B.H., ELISAs for application as biomarkers in clinical trials Refereed Research Papers A Role for ATF2 in Regulating MITF and Melanoma McElhinney, R.S., McCormick, J.E., Donnelly, D.J., of anti-Angiogenic drugs. Br J Cancer, 102, 1524- Duthie, S.J., Grant, G., Pirie, L.P., Watson, A.J. and Development. PLoS Genet, 6, e1001258. Murray, P., Carola, C., Elder, R.H., Kelly, J., Margison, 1532. Margison, G.P. (2010) G.P., Watson, A.J., Rafferty, J.A., Willington, M.A. and Folate deficiency alters hepatic and colon MGMT Willington, M.R. Cole, C., Lau, S., Backen, A., Clamp, A., Rushton, G., and OGG-1 DNA repair protein expression in rats Angeliki Malliri International Patent Application PCT/IE96/00084, (page 24) Dive, C., Hodgkinson, C., McVey, R., Kitchener, H. but has no effect on genome-wide DNA Cell Signalling Group published (WO97/20843, 129 pages) through CR and Jayson, G.C. (2010) methylation. Cancer Prev Res 3, 92-100. Technology Ltd. (Chem.Abs., 1997,127, 108802t). Inhibition of FGFR2 and FGFR1 increases cisplatin Refereed Research Papers Saad, A.A., Kassem, H., Povey, A.C. and Margison, sensitivity in ovarian cancer. Cancer Biol Ther, 10, Potentiation of temozolomide in human tumour Castillo-Lluva, S., Tatham, M.H., Jones, R.C., Jaffray, G.P. (2010) 495-504. cells. Baer, J., Freeman, A.A., Newlands, E.S., Watson, E.G., Edmondson, R.D., Hay, R.T. and Malliri, A. Expression of O6-Alkylguanine-DNA Alkyltransferase (2010) A.J., Rafferty, J.A., and Margison, G.P. Dean, E.J., Ward, T., Pinilla, C., Houghten, R., Welsh, in Normal and Malignant Bladder Tissue of Egyptian SUMOylation of the GTPase Rac1 is required (USP: 5731304) through CR Technology Ltd. K., Makin, G., Ranson, M. and Dive, C. (2010) Patients. J Nucleic Acids, 2010, 840230. for optimal cell migration. Nat Cell Biol, 12, 1078-1085. 66 | Paterson Institute for Cancer Research Scientific Report 2010 Research Publications | 67


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    Buonaccorsi, G.A., Clamp, A.R., Hasan, J., Byrd, L., Peter Stern (page 32) Ward, C.M, and Stern, P.L. (2005) Backen, A., Dive, C. and Jayson, G.C. (2010) Immunology Group 5T4 antigen expression as an indicator of stem cell Identification of early predictive imaging biomarkers differentiation. Publication information and their relationship to serological angiogenic Refereed Research Papers US2005260591. markers in patients with ovarian cancer with residual Daayana, S., Elkord, E., Winters, U., Pawlita, M., disease following cytotoxic therapy. Ann Oncol, 21, Roden, R., Stern, P.L. and Kitchener, H.C. (2010) 1982-1989. Phase II trial of imiquimod and HPV therapeutic Nullin Divecha (page 34) vaccination in patients with vulval intraepithelial Inositide Laboratory Moreira-Leite, F.F., Harrison, L.R., Mironov, A., neoplasia. Br J Cancer, 102, 1129-1136. Roberts, R.A. and Dive, C. (2010) Refereed Research Papers Inducible EGFR T790M-Mediated Gefitinib Ralph, C., Elkord, E., Burt, D.J., O'Dwyer, J.F., Bultsma, Y., Keune, W.J. and Divecha, N. (2010) Resistance in Non-small Cell Lung Cancer Cells Austin, E.B., Stern, P.L., Hawkins, R.E. and PIP4Kbeta interacts with and modulates nuclear Does Not Modulate Sensitivity to PI103 Provoked Thistlethwaite, F.C. (2010) localization of the high-activity PtdIns5P-4-kinase Autophagy. J Thorac Oncol, 5, 765-777. Modulation of lymphocyte regulation for cancer isoform PIP4Kalpha. Biochem J, 430, 223-235. therapy: a phase II trial of tremelimumab in Morrow, C.J., Ghattas, M., Smith, C., Bonisch, H., advanced gastric and esophageal adenocarcinoma. Dominguez, V., Raimondi, C., Somanath, S., Bugliani, Bryce, R.A., Hickinson, D.M., Green, T.P. and Dive, Clin Cancer Res, 16, 1662-1672. M., Loder, M.K., Edling, C.E., Divecha, N., da Silva- C. (2010) Xavier, G., Marselli, L., Persaud, S.J., Turner, M.D., Src Family Kinase Inhibitor Saracatinib (AZD0530) Sawan, S., Burt, D.J., Stern, P.L., Holland, C. and Rutter, G.A., Marchetti, P., Falasca, M. and Maffucci, Impairs Oxaliplatin Uptake in Colorectal Cancer Elkord, E. (2010) T. (2010) Cells and Blocks Organic Cation Transporters. Circulating Regulatory T Cells in Endometrial Class II phosphoinositide 3-kinase regulates Cancer Res, 70, 5931-5941. Cancer: A Role for Age and Menopausal Status. exocytosis of insulin granules in pancreatic beta cells. Immunol Invest, epub Sep 1. J Biol Chem, epub Dec 2. Pires, I.M., Ward, T.H. and Dive, C. (2010) Oxaliplatin responses in colorectal cancer cells Southgate, T.D., McGinn, O.J., Castro, F.V., Halstead, J.R., Savaskan, N.E., van den Bout, I., Van are modulated by CHK2 kinase inhibitors. Br J Rutkowski, A.J., Al-Muftah, M., Marinov, G., A small molecule inhibitor of XIAP induces Horck, F., Hajdo-Milasinovic, A., Snell, M., Keune, Pharmacol, 159, 1326-1338. Smethurst, G.J., Shaw, D., Ward, C.M., Miller, C.J. apoptosis and synergises with vinorelbine and W.J., Ten Klooster, J.P., Hordijk, P.L. and Divecha, N. and Stern, P.L. (2010) (2010) cisplatin in NSCLC. Br J Cancer, 102, 97-103. Other Publications CXCR4 mediated chemotaxis is regulated by 5T4 Rac controls PIP5K localisation and PtdIns(4,5)P Cummings, J., Raynaud, F., Jones, L., Sugar, R. and oncofetal glycoprotein in mouse embryonic cells. synthesis, which modulates vinculin localisation and Dive, C., Smith, R.A., Garner, E., Ward, T., George- Dive, C. (2010) PLoS One, 5, e9982. neurite dynamics. J Cell Sci, 123, 3535-3546. Smith, S.S., Campbell, F., Greenhalf, W., Ghaneh, P. Fit-for-purpose biomarker method validation for and Neoptolemos, J.P. (2010) application in clinical trials of anticancer drugs. Other Publications Lewis, A.E., Sommer, L., Arntzen, M.O., Strahm, Y., Considerations for the use of plasma cytokeratin 18 Br J Cancer, 103, 1313-1317. Brabin, L., Kitchener, H.C. and Stern, P.L. (2010) Morrice, N.A., Divecha, N. and D'Santos, C.S. as a biomarker in pancreatic cancer. Br J Cancer, 102, 577-582. Implementation of prophylactic HPV vaccination: (2010) Cummings, J., Ward, T.H. and Dive, C. (2010) progress and future challenges. Expert Rev Obstet Identification of nuclear phosphatidylinositol 4,5- Fit-for-purpose biomarker method validation in Gynecol, 5, 591-603. Hampson, G., Ward, T.H., Cummings, J., Bayne, M., bisphosphate-interacting proteins by neomycin anticancer drug development. Drug Discov Today, Tutt, A.L., Cragg, M.S., Dive, C. and Illidge, T.M. extraction. Mol Cell Proteomics, epub Nov 3. 15, 816-825. Stern, P.L. (2010) (2010) Progress in vaccination against cancer (PIVAC-10), Ndamukong, I., Jones, D.R., Lapko, H., Divecha, N. Validation of an ELISA for the determination of Murukesh, N., Dive, C. and Jayson, G.C. (2010) Cambridge, UK. IDdb Meeting Report, September and Avramova, Z. (2010) rituximab pharmacokinetics in clinical trials subjects. Biomarkers of angiogenesis and their role in the 27-30. Phosphatidylinositol 5-phosphate links dehydration J Immunol Methods, 360, 30-38. development of VEGF inhibitors. Br J Cancer, 102, stress to the activity of ARABIDOPSIS 8-18. Stern, P.L. (2010) Kraan, J., Sleijfer, S., Strijbos, M.H., Ignatiadis, M., TRITHORAX-LIKE factor ATX1. PLoS One, 5, Should Australia change to the bivalent vaccine? e13396. Peeters, D., Pierga, J.Y., Farace, F., Riethdorf, S., Roberts, D.L., Dive, C. and Renehan, A.G. (2010) Sexual Health, 7, 238-241. Fehm, T., Zorzino, L., Tibbe, A.G., Maestro, M., Biological Mechanisms Linking Obesity and Cancer Gisbert-Criado, R., Denton, G., de Bono, J.S., Dive, Other Publications Risk: New Perspectives. Annu Rev Med, 61, Stern, P.L. and Einstein, M.H. (2010) C., Foekens, J.A. and Gratama, J.W. (2010) Divecha, N. (2010) 301-316. From HPV infection to oncogenesis; a brief review External quality assurance of circulating tumor cell Lipid kinases: charging PtdIns(4,5)P2 synthesis. Curr of the complex immunobiological events. Current Biol, 20, R154-157. enumeration using the CellSearch((R)) system: A Cancer Therapy Reviews, 6, 110-116. feasibility study. Cytometry B Clin Cytom, epub Nov 10. Ivan Ahel (page 28) Divecha, N. (2010) DNA Damage Response Group Active Patents Methods to assess changes in the pattern of Stern, P.L. and Hole, N. (1989) nuclear phosphoinositides. Methods Mol Biol, 645, Lancashire, L.J., Roberts, D.L., Dive, C. and Refereed Research Papers Improvement relating to antigens. Publication 165-177. Renehan, A.G. (2010) Eustermann, S., Brockmann, C., Mehrotra, P.V., Yang, information EP0336562. The development of composite circulating J.C., Loakes, D., West, S.C., Ahel, I. and Neuhaus, D. 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    Omed, A., Lawrance, J.A., Murphy, G., Laasch, H.U., Enhancing fraction measured using dynamic Other Publications multiple protein-protein interactions and its Wilson, G., Illidge, T., Tipping, J., Zivanovic, M. and contrast-enhanced MRI predicts disease-free survival Mehanna, H., Paleri, V., West, C.M. and Nutting, C. application to T-cell receptor analysis. Cytometry A, Jeans, S. (2010) in patients with carcinoma of the cervix. Br J Cancer, (2010) 77, 338-346. A retrospective analysis of selective internal 102, 23-26. Head and neck cancer--Part 1: Epidemiology, radiation therapy (SIRT) with yttrium-90 presentation, and prevention. BMJ, 341, c4684. Bridgeman, J.S., Hawkins, R.E., Bagley, S., Blaylock, microspheres in patients with unresectable hepatic Donaldson, S.B., West, C.M., Davidson, S.E., M., Holland, M. and Gilham, D.E. (2010b) malignancies. Clin Radiol, 65, 720-728. Carrington, B.M., Hutchison, G., Jones, A.P., Mehanna, H., West, C.M., Nutting, C. and Paleri, V. The optimal antigen response of chimeric antigen Sourbron, S.P. and Buckley, D.L. (2010) (2010) receptors harboring the CD3zeta transmembrane Other Publications A comparison of tracer kinetic models for T1- Head and neck cancer--Part 2: Treatment and domain is dependent upon incorporation of the Elkord, E., Alcantar-Orozco, E.M., Dovedi, S.J., Tran, weighted dynamic contrast-enhanced MRI: prognostic factors. BMJ, 341, c4690. receptor into the endogenous TCR/CD3 complex. D.Q., Hawkins, R.E. and Gilham, D.E. (2010) application in carcinoma of the cervix. Magn Reson J Immunol, 184, 6938-6949. T regulatory cells in cancer: recent advances and Med, 63, 691-700. Robson, T. and West, C. (2010) therapeutic potential. Expert Opin Biol Ther, 10, Radiation and the genome: from risks to Cheadle, E.J., Hawkins, R.E., Batha, H., O'Neill, A.L., 1573-1586. Farnell, D.J., Mandall, P., Anandadas, C., Routledge, J., opportunities for therapeutic exploitation. Br J Dovedi, S.J. and Gilham, D.E. (2010) Burns, M.P., Logue, J.P., Wylie, J.P., Swindell, R., Radiol, 83, 635-637. Natural expression of the CD19 antigen impacts Illidge, T.M. (2010) Livsey, J., West, C.M. and Davidson, S.E. (2010) the long-term engraftment but not antitumor Radioimmunotherapy of lymphoma: a treatment Development of a patient-reported questionnaire West, C. and Rosenstein, B.S. (2010) activity of CD19-specific engineered T cells. J approach ahead of its time or past its sell-by date? J for collecting toxicity data following prostate Establishment of a radiogenomics consortium. Immunol, 184, 1885-1896. Clin Oncol, 28, 2944-2946. brachytherapy. Radiother Oncol, 97, 136-142. Radiother Oncol, 94, 117-118. Dangoor, A., Lorigan, P., Keilholz, U., Schadendorf, Khan, A.R., Dovedi, S.J., Wilkinson, R.W. and Gee, H.E., Camps, C., Buffa, F.M., Patiar, S., Winter, West, C., Rosenstein, B.S., Alsner, J., Azria, D., D., Harris, A., Ottensmeier, C., Smyth, J., Hoffmann, Pritchard, D.I. (2010) S.C., Betts, G., Homer, J., Corbridge, R., Cox, G., Barnett, G., Begg, A., Bentzen, S., Burnet, N., Chang- K., Anderson, R., Cripps, M., Schneider, J. and Tumor infiltrating regulatory T cells: tractable targets West, C.M., Ragoussis, J. and Harris, A.L. (2010) Claude, J., Chuang, E., Coles, C., De Ruyck, K., De Hawkins, R. (2010) for immunotherapy. Int Rev Immunol, 29, 461-484. hsa-mir-210 is a marker of tumor hypoxia and a Ruysscher, D., Dunning, A., Elliott, R., Fachal, L., Hall, Clinical and immunological responses in metastatic prognostic factor in head and neck cancer. Cancer, J., Haustermans, K., Herskind, C., Hoelscher, T., Imai, melanoma patients vaccinated with a high-dose 116, 2148-2158. T., Iwakawa, M., Jones, D., Kulich, C., Langendijk, poly-epitope vaccine. Cancer Immunol Immunother, Catharine West (page 52) J.H., O'Neils, P., Ozsahin, M., Parliament, M., 59, 863-873. Translational Radiobiology Group Hannigan, A., Smith, P., Kalna, G., Lo Nigro, C., Polanski, A., Rosenstein, B., Seminara, D., Symonds, Orange, C., O'Brien, D.I., Shah, R., Syed, N., P., Talbot, C., Thierens, H., Vega, A., West, C. and El-Hariry, I., Powles, T., Lau, M.R., Sternberg, C.N., Refereed Research Papers Spender, L.C., Herrera, B., Thurlow, J.K., Lattanzio, Yarnold, J. (2010) Ravaud, A., von der Maase, H., Zantl, N., Harper, P., Barnett, G.C., Coles, C.E., Burnet, N.G., Pharoah, L., Monteverde, M., Maurer, M.E., Buffa, F.M., Mann, Establishment of a Radiogenomics Consortium. Int J Rolland, F., Audhuy, B., Barthel, F., Machiels, J.P., P.D., Wilkinson, J., West, C.M., Elliott, R.M., Baynes, J., Chu, D.C., West, C.M., Patridge, M., Oien, K.A., Radiat Oncol Biol Phys, 76, 1295-1296. Patel, P., Kreuser, E.D. and Hawkins, R.E. (2010) C. and Dunning, A.M. (2010) Cooper, J.A., Frame, M.C., Harris, A.L., Hiller, L., Amplification of epidermal growth factor receptor No association between SNPs regulating TGF-beta1 Nicholson, L.J., Gasco, M., Crook, T. and Inman, G.J. West, C.M.L., Barnett, G.C., Dunning, A.M., Elliott, gene in renal cell carcinoma. Eur J Cancer, 46, secretion and late radiotherapy toxicity to the (2010) R.M. and Burnet, N.G. (2010) 859-862. breast: Results from the RAPPER study. Radiother Epigenetic downregulation of human disabled Genetic predictors of normal tissue response to Oncol, 97, 1295-1296. homolog 2 switches TGF-beta from a tumor radiotherapy. In: Pharmacogenetics: making cancer Gilham, D.E., Lie-A-Ling, M., Taylor, N. and Hawkins, suppressor to a tumor promoter. J Clin Invest, 120, treatment safer and more effective. Eds Newman, R.E. (2010) Brown, M., Sillah, K., Griffiths, E.A., Swindell, R., 2842-2857. W.G. pp 127-135. Springer Dordrecht, Heidelberg, Cytokine stimulation and the choice of promoter West, C.M., Page, R.D., Welch, I.M. and Pritchard, London, New York are critical factors for the efficient transduction of S.A. (2010) Sillah, K., Williams, L.R., Laasch, H.-U., Saleem, A., mouse T cells with HIV-1 vectors. J Gene Med, 12, Tumour budding and a low host inflammatory Watkins, G., Pritchard, S.A., Price, P.M., West, 129-136. response are associated with a poor prognosis in C.M.L. and Welch, I.M. (2010) Robert Hawkins (page 54) oesophageal and gastro-oesophageal junction Computed tomography overestimation of Medical Oncology: Clinical and Experimental Gore, M.E., Hariharan, S., Porta, C., Bracarda, S., cancers. Histopathology, 56, 893-899. esophageal tumor length: implications for Immunotherapy Group Hawkins, R., Bjarnason, G.A., Oudard, S., Lee, S.H., radiotherapy planning. World J Gastroinest Oncol, 2, Carteni, G., Nieto, A., Yuan, J. and Szczylik, C. Buffa, F.M., Harris, A.L., West, C.M. and Miller, C.J. 197-204. Refereed Research Papers (2010) (2010) Amato, R.J., Hawkins, R.E., Kaufman, H.L., Sunitinib in metastatic renal cell carcinoma patients Large meta-analysis of multiple cancers reveals a Silva, P., Slevin, N.J., Sloan, P., Valentine, H., Ryder, D., Thompson, J.A., Tomczak, P., Szczylik, C., with brain metastases. Cancer, epub Sep 22. common, compact and highly prognostic hypoxia Price, P., West, C.M. and Homer, J.J. (2010) McDonald, M., Eastty, S., Shingler, W.H., de Belin, J., metagene. Br J Cancer, 102, 428-435. Use of multiple biological markers in radiotherapy- Goonewardena, M., Naylor, S. and Harrop, R. Khan, S., Burt, D.J., Ralph, C., Thistlethwaite, F.C., treated head and neck cancer. J Laryngol Otol, 124, (2010) Hawkins, R.E. and Elkord, E. (2010) Burrows, N., Resch, J., Cowen, R.L., von 650-658. Vaccination of Metastatic Renal Cancer Patients Tremelimumab (anti-CTLA4) mediates immune Wasielewski, R., Hoang-Vu, C., West, C.M., with MVA-5T4: A Randomized, Double-Blind, responses mainly by direct activation of T effector Williams, K.J. and Brabant, G. (2010) Thurlow, J.K., Pena Murillo, C.L., Hunter, K.D., Buffa, Placebo-Controlled Phase III Study. Clin Cancer cells rather than by affecting T regulatory cells. Clin Expression of hypoxia-inducible factor 1 alpha in F.M., Patiar, S., Betts, G., West, C.M., Harris, A.L., Res, 16, 5539-5547. Immunol, epub Nov 3. thyroid carcinomas. Endocr Relat Cancer, 17, 61-72. Parkinson, E.K., Harrison, P.R., Ozanne, B.W., Partridge, M. and Kalna, G. (2010) Bridgeman, J.S., Blaylock, M., Hawkins, R.E. and Ralph, C., Elkord, E., Burt, D.J., O'Dwyer, J.F., Austin, Donaldson, S.B., Buckley, D.L., O'Connor, J.P., Spectral clustering of microarray data elucidates the Gilham, D.E. (2010a) E.B., Stern, P.L., Hawkins, R.E. and Thistlethwaite, Davidson, S.E., Carrington, B.M., Jones, A.P. and roles of microenvironment remodeling and immune Development of a flow cytometric co- F.C. (2010) West, C.M. (2010) responses in survival of head and neck squamous immunoprecipitation technique for the study of cell carcinoma. J Clin Oncol, 28, 2881-2888. 72 | Paterson Institute for Cancer Research Scientific Report 2010 Biomolecular modelling | 73 Research publications


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    Modulation of lymphocyte regulation for cancer Hawkins, R.E., Gilham, D.E., Debets, R., Eshhar, Z., Inhibition of carboplatin-induced DNA interstrand Jayson, G., Gardiner, J. and Hansen, S. therapy: a phase II trial of tremelimumab in Taylor, N., Abken, H., Schumacher, T.N. and cross-link repair by gemcitabine in patients receiving Synthesis route for multioligomeric heparan sulfate advanced gastric and esophageal adenocarcinoma. Consortium, A. (2010) these drugs for platinum-resistant ovarian cancer. molecules, PCT Application: PCT/ GB2009/ 000300, Clin Cancer Res, 16, 1662-1672. Development of adoptive cell therapy for cancer: a Clin Cancer Res, 16, 4899-4905. 2009-02-04. clinical perspective. Hum Gene Ther, 21, 665-672. Rossi, J.F., Negrier, S., James, N.D., Kocak, I., Mitchell, C.L., O'Connor, J.P., Jackson, A., Parker, Prenant, C., Bailey, J., Gillies, J., Chimon, G., Smith, Hawkins, R., Davis, H., Prabhakar, U., Qin, X., G.J., Roberts, C., Watson, Y., Cheung, S., Davies, K., N., Jayson, G.C. and Zweit, J. Mulders, P. and Berns, B. (2010) Gordon Jayson (page 56) Buonaccorsi, G.A., Clamp, A.R., Hasan, J., Byrd, L., [18F] Fluoroacetaldehyde, a potential new reagent A phase I/II study of siltuximab (CNTO 328), an Medical Oncology: Translational Backen, A., Dive, C. and Jayson, G.C. (2010) for direct radiofluoroalkylation of proteins and anti-interleukin-6 monoclonal antibody, in metastatic Anti-Angiogenesis Group Identification of early predictive imaging biomarkers peptides (under review in Journal of Labelled renal cell cancer. Br J Cancer, 103, 1154-1162. and their relationship to serological angiogenic Compounds and Radiopharmaceuticals). Patent by Refereed Research Papers markers in patients with ovarian cancer with residual The University of Manchester: U.S. Patent Appln Rothwell, D.G., Crossley, R., Bridgeman, J.S., Sheard, Brookes, K., Cummings, J., Backen, A., Greystoke, A., disease following cytotoxic therapy. Ann Oncol, 21, No.60/902060. V., Zhang, Y., Sharp, T., Hawkins, R.E., Gilham, D. and Ward, T., Jayson, G.C. and Dive, C. (2010) 1982-1989. McKay, T.R. (2010) Issues on fit-for-purpose validation of a panel of Additional Publications Functional expression of secreted proteins from a ELISAs for application as biomarkers in clinical Mitchell, C.L., O'Connor, J.P., Roberts, C., Watson, bicistronic retroviral cassette based on FMDV 2A trials of anti-Angiogenic drugs. Br J Cancer, 102, Y., Jackson, A., Cheung, S., Evans, J., Spicer, J., Harris, can be position-dependent. Hum Gene Ther, 21, A., Kelly, C., Rudman, S., Middleton, M., Fielding, A., Refereed Research Papers 1524-1532. Betsou, F., Lehmann, S., Ashton, G., Barnes, M., 1631-1637. Tessier, J., Young, H., Parker, G.J. and Jayson, G.C. (2010) Benson, E.E., Coppola, D., DeSouza, Y., Eliason, J., Buonaccorsi, G.A., Rose, C.J., O'Connor, J.P., Glazer, B., Guadagni, F., Harding, K., Horsfall, D.J., Shablak, A., O'Dwyer, J., Hawkins, R. and Board, R. Roberts, C., Watson, Y., Jackson, A., Jayson, G.C. and A two-part Phase II study of cediranib in patients (2010) with advanced solid tumours: the effect of food on Kleeberger, C., Nanni, U., Prasad, A., Shea, K., Parker, G.J. (2010) Skubitz, A., Somiari, S. and Gunter, E. (2010) Management of a New Isolated Metastasis during Cross-visit tumor sub-segmentation and registration single-dose pharmacokinetics and an evaluation of Sunitinib Treatment in Renal Cell Carcinoma safety, efficacy and imaging pharmacodynamics. Standard preanalytical coding for biospecimens: with outlier rejection for dynamic contrast-enhanced defining the sample PREanalytical code. Cancer Patients: A Lesson from Two Cases. Urol Int, epub MRI time series data. Med Image Comput Comput Cancer Chemother Pharmacol, epub Dec 1. Dec 2. Epidemiol Biomarkers Prev, 19, 1004-1011. Assist Interv, 13, 121-128. Rustin, G.J., van der Burg, M.E., Griffin, C.L., Sternberg, C.N., Davis, I.D., Mardiak, J., Szczylik, C., Guthrie, D., Lamont, A., Jayson, G.C., Kristensen, G., Workman, P., Aboagye, E.O., Balkwill, F., Balmain, A., Cole, C., Lau, S., Backen, A., Clamp, A., Rushton, G., Bruder, G., Chaplin, D.J., Double, J.A., Everitt, J., Lee, E., Wagstaff, J., Barrios, C.H., Salman, P., Dive, C., Hodgkinson, C., McVey, R., Kitchener, H. Mediola, C., Coens, C., Qian, W., Parmar, M.K. and Gladkov, O.A., Kavina, A., Zarba, J.J., Chen, M., Swart, A.M. (2010) Farningham, D.A., Glennie, M.J., Kelland, L.R., and Jayson, G.C. (2010a) Robinson, V., Stratford, I.J., Tozer, G.M., Watson, S., McCann, L., Pandite, L., Roychowdhury, D.F. and Inhibition of FGFR2 and FGFR1 increases cisplatin Early versus delayed treatment of relapsed ovarian Hawkins, R.E. (2010) cancer (MRC OV05/EORTC 55955): a randomised Wedge, S.R. and Eccles, S.A. (2010) sensitivity in ovarian cancer. Cancer Biol Ther, 10, Guidelines for the welfare and use of animals in Pazopanib in locally advanced or metastatic renal 495-504. trial. Lancet, 376, 1155-1163. cell carcinoma: results of a randomized phase III trial. cancer research. Br J Cancer, 102, 1555-1577. J Clin Oncol, 28, 1061-1068. Cole, C.L., Hansen, S.U., Barath, M., Rushton, Other Publications G., Gardiner, J.M., Avizienyte, E. and Jayson, G.C. Hasan, J. and Jayson, G.J. (2010) Waddington, S.N., Crossley, R., Sheard, V., Howe, (2010b) Novel anti-angiogenic therapies in ovarian cancer. S.J., Buckley, S.M., Coughlan, L., Gilham, D.E., Synthetic heparan sulfate oligosaccharides inhibit In: Emerging therapeutic targets in ovarian cancer. Hawkins, R.E. and McKay, T.R. (2010) endothelial cell functions essential for angiogenesis. Eds Kaye, S., Brown, R., Gabra, H. and Gore, M. pp Gene Delivery of a Mutant TGFbeta3 Reduces PLoS One, 5, e11644. 51-72. Springer New York Markers of Scar Tissue Formation After Cutaneous Wounding. Mol Ther, 18, 2104-2111. Goncalves, V., Jayson, G. and Tarrier, N. (2010) Murukesh, N., Dive, C. and Jayson, G.C. (2010) A longitudinal investigation of psychological Biomarkers of angiogenesis and their role in the Other Publications disorders in patients prior and subsequent to a development of VEGF inhibitors. Br J Cancer, 102, Bridgeman, J.S., Hawkins, R.E., Hombach, A.A., diagnosis of ovarian cancer. J Clin Psychol Med 8-18. Abken, H. and Gilham, D.E. (2010c) Settings, 17, 167-173. Building better chimeric antigen receptors for Zee, Y.K., Chan, S.W., Harris, J. and Jayson, G.C. adoptive T cell therapy. Curr Gene Ther, 10, 77-90. Jonker, D.J., Rosen, L.S., Sawyer, M.B., de Braud, F., (2010) Wilding, G., Sweeney, C.J., Jayson, G.C., McArthur, The ethical and scientific case for phase 2C clinical Buning, H., Uckert, W., Cichutek, K., Hawkins, R.E. G.A., Rustin, G., Goss, G., Kantor, J., Velasquez, L., trials. Lancet Oncol, 11, 410-411. and Abken, H. (2010) Syed, S., Mokliatchouk, O., Feltquate, D.M., Kollia, Do CARs Need a Driver's License? Adoptive Cell G., Nuyten, D.S. and Galbraith, S. (2010) Zee, Y.K., O'Connor, J.P., Parker, G.J., Jackson, A., Therapy with Chimeric Antigen Receptor- A phase I study to determine the safety, Clamp, A.R., Taylor, M.B., Clarke, N.W. and Jayson, Redirected T Cells Has Caused Serious Adverse pharmacokinetics and pharmacodynamics of a dual G.C. (2010) Events. Hum Gene Ther, 21, 1039-1042. VEGFR and FGFR inhibitor, brivanib, in patients with Imaging angiogenesis of genitourinary tumors. Nat advanced or metastatic solid tumors. Ann Oncol, Rev Urol, 7, 69-82. Elkord, E., Alcantar-Orozco, E.M., Dovedi, S.J., Tran, epub Dec 3. D.Q., Hawkins, R.E. and Gilham, D.E. (2010) Active Patents T regulatory cells in cancer: recent advances and Ledermann, J.A., Gabra, H., Jayson, G.C., Spanswick, Jayson, G., Gardiner, J. and Hansen, S. therapeutic potential. Expert Opin Biol Ther, 10, V.J., Rustin, G.J., Jitlal, M., James, L.E. and Hartley, J.A. Production of L-Iduronate containing 1573-1586. (2010) polysaccharides, Int. publication: WO 2006/129075 A1: 2006-12-07. 74 | Paterson Institute for Cancer Research Scientific Report 2010 Research publications | 75


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    External Seminar Speakers 2010 Matthias Peter Breakthrough Breast Cancer Research Unit 2010 produced another set of excellent speakers for our external Swiss Federal Institute of Technology, Zurich Seminar Series 2010 seminar series. As in 2009 the Paterson seminars were Jordan Raff Gabriela Dontu complemented by seminars in the Breakthrough Breast Cancer Sir William Dunn School of Pathology, University of Department of Academic Oncology, Guy’s Hospital Research Unit series which produced an outstanding range of Oxford Mitch Dowsett speakers covering a wide range of topics. The Institute also has Peter Ratcliffe Royal Marsden Hospital weekly postdoctoral seminars which continue to be very well Nuffield Department of Clinical Medicine, University of Oxford Barry Gusterson attended. Head of Pathology & Gene Regulation and Forensic Joel Richter Medicine & Science, University of Glasgow King's College London Christoph Ballestrem Kristian Helin Lars Holmberg Wellcome Trust Centre for Cell-Matrix Research, Biotech Research & Innovation Centre (BRIC), Pablo Rodriguez-Viciana Division of Cancer Studies, King’s College London The University of Manchester University of Copenhagen, Denmark University College London School of Medicine Chris Boshoff Penny Jeggo Peter Ten Dyke Rudolf Kaaks University College London Cancer Institute Sussex Centre for Genome Damage and Stability, Queen Mary, University of London Division of Cancer Epidemiology, German Cancer University of Sussex, Brighton Research Centre Michael Boutros Bart Van Haesenbrook German Cancer Research Centre, Heidelberg, Peter Laslo Queen Mary, University of London (QMUL) Yibin Kang Germany Leeds Institute of Molecular Medicine, University of Department of Molecular Pathology, Princeton Leeds Ashok Venkitaraman University Keith Caldecott MRC Research Centre, Cambridge Sussex Centre for Genome Damage and Stability, Alison Lloyd Lucio Miele University of Sussex, Brighton University College London Juan Jose Ventura Ergon Professor of Medicine and Pharmacology Wellcome Trust Centre for Stem Cell Research, Director, University of Mississippi Cancer Institute, Jason Carroll Ultan McDermott Cambridge University of Mississippi Medical Center Cambridge Research Institute Wellcome Trust Sanger Institute, Cambridge John York Salvatore Pece Matthew Collin Claus Nerlov Duke University Medical Center, Durham USA University of Milan, Italy Cambridge Research Institute MRC Centre for Regenerative Medicine, University of Edinburgh Philip Zegerman John Stingl Simon Cook Gurdon Institute, Cambridge University CRUK Cambridge Research Institute The Babraham Institute, Cambridge Jim Norman The Beatson Institute, Glasgow Adrian Harris University of Oxford, Weatherall Institute of Klaus Pantel Molecular Medicine, Oxford University Medical Center Hamburg-Eppendorf, Germany Nick Hastie MRC Human Genetics Unit , Edinburgh Philippe Pasero Institute of Human Genetics, Montpellier, France 76 | Paterson Institute for Cancer Research Scientific Report 2010 External Seminar Biomolecular Speakers 2010 | 77 modelling


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


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    Operations 2010 has been a year of consolidation. After the shock of the financial situation and subsequent decrease in the Institute’s budget from CR-UK in 2009, staff across the Institute rose admirably to the challenge of keeping costs down and driving hard deals with all of the Institute’s suppliers. As a consequence at the end of the third quarter, the deficit has Director of Operations decreased to £96K. Pippa McNichol At the beginning of the year the heavy snow £1167 was raised for Cancer Research UK. The and bad weather caused the Institute to shut Paterson team had lost the last two matches to down for two days, which had never happened the Beatson so there was a lot at stake. It is for helping to organise the 2010 colloquium which The Director’s Office will continue to provide a before. Consequently, when it was re-opened very gratifying that the Paterson team won the went well and looks forward to arranging the 2011 supportive role to the Institute and its Group the Major Incident Plan was updated to match 4-2. All their training had at last paid off. colloquium at its new location in Lancaster. Leaders and aid the MCRC through what is encompass all the lessons learned. The IT expected to be an extremely busy but exciting department worked hard to introduce video- The long awaited new intranet for staff – The Director’s Office period over the next year. conferencing facilities in time for the annual PICRboo was launched in December. EA to the Director and Director of Operations: budget session with CR-UK. Amy Weatheritt Estates Just before Christmas it was announced that Manager: Steve Alcock The HR department worked miracles to ensure the Director, Professor Nic Jones would be The 2010 Paterson seminar series was a great that CR-UK’s new pay and grading system for stepping down as Director of the Institute to success with over 40 speakers visiting the Institute 2010 has been a challenging time for the Estates non-scientific and managerial staff was take up the very prestigious position of Chief including two distinguished seminar talks taking place team. There has been a considerable amount of implemented in a timely manner. Scientist for CR-UK. The news has left the in November. The 2011 seminar series is currently capital work carried out in the Institute. The Institute a little shell-shocked but absolutely being arranged and it is hoped this will be just as schemes include: reconfiguring an additional GCP lab Two new staff joined the Operations team delighted for Nic. An interim Senior productive. The seminar series is vital for the for CEP; office remodelling for two group leaders during 2010: Becky Allen as the Administration Management team is being established to run Institute, particularly for the students and and a write up area for their staff; refurbishing the Services Co-ordinator and Muhammad Raja as the Institute until a replacement director can be postdoctoral fellows in helping them to become area vacated by Nic Jones into a two person office the Finance Assistant. Both these posts were found, so 2011 will be a year of great exposed to world-class research. It brings together and write up area. The Building Management vacant posts rather than new posts. challenges. and connects researchers and provides an System has recently been upgraded to Schnyder’s opportunity for interaction for both the speaker and latest platform which will improve the controls and CR-UK organised a large new fundraising event Admin and Reception Services the staff at the Institute. A list of speakers for 2010 legislative record keeping capabilities. Refurbishment in Manchester in April – Shine – a midnight Manager: Amy Weatheritt can be found on pages 76/77. of the photocopying room was recently undertaken walking marathon. Paterson staff undertook to accommodate the institute scanner and printers, demonstrations of science at the start of the Over the year the admin department has Recruitment has been a high priority for the office plus many other small schemes to improve facilities walk at Manchester Central and showed the evolved and is now providing a productive and which has worked closely with HR. It was good to for the scientific groups. walkers how to extract DNA from effective administrative service to the Institute. welcome John Brognard who was appointed as a strawberries. The Paterson Institute was the six The appointment of a permanent Security Junior Group Leader of the Signalling Networks in A popular area of concern is sustainability, and mile mark and so another group of staff Coordinator and Receptionist and Cancer Group to the Institute. Recruitment is reducing the Institute’s carbon footprint as much as volunteered to provide the walkers with drinks Administration Services Coordinator has ongoing to fill another Group Leader position. possible is one of the Institute’s goals. Whenever a and encouragement. The atmosphere was enabled the department to ensure a high Furthermore the organisation of Angeliki Malliri’s new scheme is required, energy-saving devices are brilliant and the event was a huge success for quality service and provide support across the tenure, viva examinations and high profile meetings utilised and more efficient heating/cooling and CR-UK. groups within the Institute. The department has have also kept the department busy. ventilation systems are installed. One recent assisted with many different events over the scheme involved the removal of eight ageing The Paterson Football team hosted a return year, from fundraising to conferences and has It has also been a busy yet exciting year for the individual DX air conditioning units, replaced with a match for the Beatson Football team (our sister expanded its service to provide professional MCRC, and the Director’s Office has been single VRF (variable refrigerant flow) system which Institute) in October. The event was turned assistance in the arranging of events internally supporting the operations and administration of the will move heat taken from the labs (via the into a Family Fun Day for Paterson staff and and externally. The department was responsible Centre. As the new build progresses the Director’s refrigerant gas) and redistribute it in the office areas Office will be assisting the Director of Operations that the system serves. The VRF heating/cooling throughout the building process. 80 | Paterson Institute for Cancer Research Scientific Report 2010 modelling | 81 BiomolecularOperations


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    system is very efficient and has lowered energy assessment workshop was held, whereby individuals The main focus for the next twelve months is the At CRT we bridge the fundamental gap between consumption and improved the working discussed and developed risk assessments of actual successful implementation of the Absence cutting edge academic research and industrial environment of the areas it serves. techniques employed in the laboratory. Feedback Management system and the online probationary development of cancer therapeutics and diagnostics. form the workshop was positive and this format will process system. We achieve this by working closely with prestigious The Estates team have been pro-active throughout certainly be repeated next year. international research institutes, such as the Paterson the year with positive responses from the Estates Information Technology Institute for Cancer Research, and funding bodies to user group. The Estates team members have Statutory required performance testing of fume Manager: Malik Pervez develop, protect and commercialise oncology attended relevant courses to improve their skills and cupboards, safety cabinets and other local exhaust related discoveries. Core activities of business keep their knowledge up to date with current ventilation equipment was completed by a cost With continuous demand for superior, faster, cutting development and drug discovery are supported by working practices and changing legislation. effective and time efficient one-hit testing schedule edge technology to support the research specialists, integrated in the business with expertise by the chosen contractor. Other statutory required programme, the IT department has stepped up to in patents, legal, finance and marketing. Our Finance and Purchasing inspections, such as examination of liquid nitrogen the challenge of delivering an excellent IT exclusive focus in oncology provides an unrivalled Manager: Margaret Lowe pressure vessels, have also been completed as have infrastructure. depth of knowledge and experience in cancer- equipment maintenance regimes. The rolling specific translational development and The Finance Department is responsible for programme of formal safety inspections of During 2010, the introduction of a new desktop commercialisation. By arrangement with The providing a comprehensive purchasing, travel and laboratories and other areas has also been carried operating system and upgrading back-end systems University of Manchester, CRT owns and is finance service on a daily basis to the Research out and action-point reports fed back to those have provided the response time, stability and responsible for the development and Groups and Service Units within the Paterson concerned with follow up to check actions are security required to support pioneering research. commercialisation of intellectual property arising Institute. The procurement team continues to work completed. The constant need for additional data storage has from Cancer Research UK-funded research at The with the groups to identify savings in consumable provided a further challenge and the University of Manchester (including the Paterson). spending. In addition to this they advise on capital The health and safety pages of the local intranet implementation of an online archive system has purchases, working with the researchers to ensure have been updated and this will continue in to next alleviated the need to purchase additional storage. Our relationship with the Institute reflects the legal legislation is adhered to and that the best year. The need to process large data sets commands specific requirements of the scientist, the Paterson possible price is obtained for the equipment. enormous computing power. The task for the IT Institute, Cancer Research UK and the individual Human Resources department this year was to balance the computing project. To effectively facilitate these requirements In the current economic climate is it crucial that the Manager: Rachel Powell power demanded by researchers whilst at the same and interactions CRT has a Business Manager management information provided is current and time reducing the carbon footprint of the solution (Martyn Bottomley) based at the Paterson Institute accurate at all times to assist Group Leaders in Over the past year the HR Department has required. This meant detailed planning and design dedicated to working closely with the staff at the managing their budgets and for the Directors to continued to successfully deliver a high quality which resulted in the successful implementation of a PICR. We are here to offer access to oncology have a full overview of the Institute finances. professional service to the Institute. The focus has new computer cluster without increasing our carbon focused expertise in technology evaluations, patent been on developing a more customer-focused footprint. Information is the lifeblood of any applications and management, funding for Group Leaders are also assisted with costing up approach to enhance and support the aims and organisation. Sharing information effectively and development, commercialization, drug discovery, grant applications and receive full post-award objectives of the Institute. quickly requires organisations to have systems in market intelligence, and project management. CRT administration support. place that are responsive and easy to access. To also works closely with the recently formed Drug The department has continued its drive for maximise the flow of information a new Intranet Discovery Laboratories to facilitate the development Health and Safety efficiency by further improving the recruitment and was developed that provided functionality and of small molecules drug therapies to satisfy the Manager: Colin Gleeson selection process and evaluating where vacancies interactions that were lacking in previous systems. unmet clinical needs of cancer patients. CRT is are advertised to ensure that that they are cost This was launched in December 2010. 2010 has currently actively managing a broad portfolio of An Institute-wide Risk Management survey was effective and providing value for money. This year provided all organisations with a challenging financial development programmes and robust licensing devised and completed. The survey listed, in a 31 highly skilled scientists and support staff have climate which has led to the need to reduce costs. opportunities originating from the Paterson that checklist format, safety-related tasks and duties been recruited to compliment and further enhance The introduction of specific technologies such as continue to attract commercial partners (ranging which are essential to the safe running of a the work of the Institute. video conferencing has been pursued to maximise from enabling technologies through to drugs in late laboratory or department. The purpose of the scientists’ time whilst reducing travelling costs for stage Clinical Trials). We look forward to building on survey was to ensure that individual laboratories It has been a busy and challenging 12 months with routine meetings. our successes and continuing to work closely with and departments had named individuals to carry the department heavily involved in the transfer of the Institute to advance discoveries to beat cancer. out the safety-related tasks and duties and to the non scientific staff onto a new pay and grading In a year that has presented many challenges for IT, indicate whether the selected persons are trained structure. This was successfully achieved through the team has yet again succeeded in delivering very and competent to carry out their allotted tasks. effective communication and consultation with staff. good core systems, new, innovative solutions that Analysis of the survey results is continuing but it has are cost effective and environmentally friendly as already identified some training needs for key tasks, Joint partnership working with the unions has well as maximising value for money. for certain individuals and/or laboratories. The continued throughout the year which has resulted in survey will also help ensure that there is no loss of the agreement of several new and revised policies, Cancer Research Technology management control when key staff leave the including the Sickness Absence policy, Secondment Manager: Martyn Bottomley Institute as their safety-related tasks are recorded policy and the launch of a new Mentoring and can be passed on to others. Framework. The aim of the Mentoring Framework Cancer Research Technology (CRT) is a specialist is to allow for the transfer of skills and experience oncology focused development and Health and safety training has been provided at the within a supportive environment. This is available commercialisation company wholly owned by monthly Induction along with more detailed when an individual is taking on a new role or would Cancer Research UK. CRT aims to maximise information in training sessions on work with like to increase their knowledge in a specific area. patient benefit from publicly funded research biological agents, hazardous chemicals, radiation worldwide by advancing research discoveries into safety and risk assessment. Also a successful risk development with pharmaceutical and biotechnology parties. 82 | Paterson Institute for Cancer Research Scientific Report 2010 modelling | 83 BiomolecularOperations


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    Cancer Research UK’s Local Engagement and Development 2010 has proved to be another busy year of local engagement and development activity in Manchester. Researchers have been involved in fifty engagement events, with 30,000 Cancer Research UK supporters being reached, and a further 500 having the opportunity to visit the Institute. In addition to the monthly lab tour programme and open day, an extra event LEAD Manager was held to commemorate the opening of the new Drug James Dunphy Discovery laboratories. This was attended by fifty supporters who had been involved in helping to promote and organise Cancer Research UK’s Race for Life events across the northwest. Researchers have continued to support their CR-UK’s inaugural night-time walking marathon. local fundraisers with inspirational speeches A team of researchers hosted an interactive conducted at key Race for Life, Run 10k, Relay area at the start, another team manned the for Life and Committee events throughout the official pit stop at the Institute and a further year. In addition to this the Institute has once ten representatives took part in the walk. This again actively fund-raised for the charity with pilot event was a resounding success with the Keswick to Barrow and Relay for Life 7500 walkers taking part and raising over £1.8 teams raising £3000. A fun day was also million for Cancer Research UK. The organised as part of the Paterson Football importance of the Institute involvment in this Club versus Beatson Football Club game and event was highlighted by a participant: £1200 was raised from on the day activities and sponsorship. “I was a participant in SHINE on Saturday night and just wanted to thank the staff of the Institute An important element of local engagement for turning out and supporting us. The guys that I activity in Manchester involves making met at Central were very patient and explained connections with local schools and colleges to what the money is used for brilliantly. Then when inspire and inform their students about the we got to the Institute it was lovely to be cheered potential to become cancer researchers of the on. It was so nice to actually meet the people future. This years School’s day was the biggest who spend their work lives trying to help people yet with thirteen local schools sending 66 with this nasty disease. I would be grateful if you could pass on my thanks to all and tell them how Paterson scientists outside the Institute during the Shine night students to the Institute to gain a practical time walk insight into the work undertaken here. The much their time was appreciated on Saturday feedback was excellent: and how much their work is appreciated every day” “Being in the labs and being able to do real techniques confirmed that I definitely do want Shine will be returning to Manchester in 2011 to work in a lab in the future”. and the success of the activity undertaken in Manchester will be replicated at the new The Institute was heavily involved in the Glasgow and London events. promotion and on the night activity for Shine, 84 | Paterson Institute for Cancer Research Scientific Report 2010 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 2010 was £15.48M. The The Paterson Institute is located alongside The Christie NHS Foundation major source of this funding (69.2%) was through a core grant from Trust, and has a strong programme of basic and translational research. Cancer Research UK (CRUK). The actual value of this award in 2010 was There are very close links with clinical and translational research groups £10.7m. This is divided between the various scientific groups and service throughout the Christie Hospital site. units within the Institute to enable them to carry out their research. In addition to this the CRUK awarded us £1m to run the Drug Discovery The Manchester Cancer Research Centre (MCRC) Postdoctoral applicants of high calibre are regularly was created nearly five years ago with partners sought. Although post docs will be encouraged to unit (6.72%). 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 PATERSON INSTITUTE FUNDING 2010 extremely exciting development which is enhancing directly, with details of their area of interest and 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 13.63% 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 10.45% Details of all groups and facilities are given experience of the applicant, with significant funding CRUK Core Grant throughout this report, and can guide interested for personnel, recurrent expenditure and parties to the appropriate contacts. equipment. Junior Group Leaders are appointed for CRUK Programme Grant an initial six-year period with a review at five years HEFCE Opportunities exist at a number of levels in the for consideration for promotion to Senior Group 6.72% OTHER SOURCES Institute. We have a well-established programme of Leader, with Senior Group Leaders appointed to degrees by research which is described in the non-time limited positions. section on Postgraduate Education. We encourage 69.20% 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 (10.45%) • BBSRC postgraduate taught and training courses allowing • Leukaemia & Lymphoma Research Fund them to gain a sound knowledge base of the latest The final 13.63% of the Institute’s funding is received • Novartis developments in cancer treatment and research. from a number of additional sources. The research • DXS The Institute also has a well-developed process for carried out through these additional projects • Chugai ensuring suitable pastoral care and mentoring for all enhances and supports the research undertaken by • Abbott Laboratories students. the core funding. • GeminX These sources are as follows: We are immensely grateful to all our sponsors. • AstraZeneca • Roche 86 | Paterson Institute for Cancer Research Scientific Report 2010 Career Opportunities atBiomolecular Institute | 87 the Patersonmodelling


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    Contact details Paterson Institute for Cancer Research ISSN 1479-0378 Wilmslow Road Copyright © 2010 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 Electronic version of this report can be found at: www.paterson.man.ac.uk 88 | Paterson Institute for Cancer Research Scientific Report 2010 Biomolecular modelling | 89


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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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