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    SCIENTIFIC REPORT 2012 cruk.org

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    Cover image Dr Andrew Porter, a Post-doctoral Research Fellow in the Cell Signalling Group. SCIENTIFIC REPORT 2012

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    CONTENTS SECTION 1 SECTION 2 DIRECTOR’S INTRODUCTION 04 THE UNIVERSITY OF MANCHESTER, RESEARCH HIGHLIGHTS 07 INSTITUTE OF CANCER SCIENCES THE PATERSON INSTITUTE FOR RESEARCH GROUPS CANCER RESEARCH Vaskar Saha 50 Children’s Cancer RESEARCH GROUPS Robert Hawkins 52 Crispin Miller 14 Medical Oncology: Clinical and Applied Computational Biology Experimental Immunotherapy and Bioinformatics Gordon Jayson 54 Geoff Margison 16 Medical Oncology: Translational Carcinogenesis Anti-Angiogenesis Karim Labib 18 Tim Illidge 56 Cell Cycle Targeted Therapy Iain Hagan 20 Catharine M. L. West 58 Cell Division Translational Radiobiology Nic Jones 22 Cell Regulation SECTION 3 Angeliki Malliri 24 RESEARCH SERVICES Cell Signalling Steve Bagley 62 Caroline Dive 26 Advanced Imaging and Flow Cytometry Clinical and Experimental Duncan Smith 63 Pharmacology Biological Mass Spectrometry Ivan Ahel 30 Biological Resources Unit 64 DNA Damage Response Garry Ashton 65 Donald Ogilvie 32 Histology Drug Discovery Mark Craven 66 Peter L. Stern 34 Laboratory Services Immunology Stuart Pepper 67 Nullin Divecha 36 Molecular Biology Core Facility Inositide Laboratory The Paterson Institute Tim Somervaille 38 RESEARCH PUBLICATIONS 68 Leukaemia Biology THESES 79 Richard Marais 40 Molecular Oncology EXTERNAL SEMINAR SPEAKERS 2012 80 John Brognard 42 POSTGRADUATE EDUCATION 82 Signalling Networks in Cancer OPERATIONS 84 Georges Lacaud 44 CANCER RESEARCH UK’S Stem Cell Biology RESEARCH ENGAGEMENT 88 Valerie Kouskoff 46 ACKNOWLEDGEMENT FOR FUNDING OF Stem Cell and Haematopoiesis THE PATERSON INSTITUTE 90 CAREER OPPORTUNITIES AT THE PATERSON INSTITUTE 91 CONTACT DETAILS 92 2 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH 3

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    Development of a the winner of the University of Manchester DIRECTOR’S INTRODUCTION pharmacodynamic Institute of Cancer Sciences/PICR postgraduate circulating tumour cell assay: student of the year award. Bill used chemical Spiked tumour cells (green) and white blood cells (blue) inhibitors that had been synthesised by the Drug recovered from a whole blood Discovery Group to down-regulate KDM1A and sample by ‘Isolation by Size of the success of the approach has resulted in this Epithelial Tumour Cells’ (ISET). A potential therapeutic strategy progressing drug target (red) can be towards clinical trials. quantified, providing a potential pharmacodynamic biomarker for novel targeted therapies. The tough financial climate, in which we find CD45: Blue, Cytokeratin: Green, ourselves, presented us with significant Drug Target: Red. Image challenges in 2012 and will continue to do so provided by Robert Sloane from It is a privilege to have taken on the Directorship of the the Clinical and Experimental over the next couple of years. A major focus Pharmacology Group. going forward is to use our core grant from Paterson Institute and my first year in post has been highly Cancer Research UK to leverage additional enjoyable. This report offers a chance to look back at the John Brognard in 2010 and the intense activity funding from external sources in order to carry in this area within the Clinical and Experimental out the full breadth of research that we wish to developments that have taken place and an opportunity to Pharmacology Group. undertake. Gill Campbell joined us during the discuss our ambitions for the future. A major highlight of the year as a Grants Advisor to help facilitate this. An ongoing goal is to promote translational Successful external funding applications year was the successful bid, made with colleagues at the research at the Paterson Institute. A major increased dramatically in 2012 and successful Professor Richard Marais Director of The Paterson University of Manchester and the Manchester Cancer activity in this respect is provided by the Clinical grants included a Wellcome Trust Senior Institute for Cancer Research and Experimental Pharmacology Group led by Investigator Award (Richard Marais) and Research Centre, to the UK Research Partnership Caroline Dive. This year the CEP Biomarker significant funding from Leukaemia and Investment Fund which resulted in an award of £12.8m Portfolio has expanded to include a Nucleic Lymphoma Research (Georges Lacaud, Tim Acids Biomarkers (NAB) team, led by CEP Somervaille, Peter Stern) and the Pancreatic towards our aim of implementing personalised medicine Deputy Ged Brady, which is dedicated to Cancer Research Fund (Caroline Dive and Ged for cancer patients in the North West. developing the current validated CEP blood- Brady). based NAB assays. To do so, NAB projects have been initiated to examine miRNA and In addition to the major boost provided by the The funds provide £4.1m towards completion of development in his new post at the Juntendo methylation patterns present in circulating free UKRPIF award, there have been further exciting the new Manchester Cancer Research Centre University School of Medicine in Tokyo. DNA, RNA profiling and mutation detection in developments in the core research facilities. A (MCRC) research building which is currently Successful quinquennial reviews were circulating tumour cells. CEP successfully successful bid was made to the MCRC for funds under construction across the road from the undertaken by Crispin Miller (Applied re-negotiated their contract with AstraZeneca, to purchase a multi-modal imaging system Paterson Institute and due for completion in Computational Bioinformatics and Biology) and worth £3.1million over three years, to continue which will include far-field super-resolution 2014, while the remaining £8.7m will go towards Iain Hagan (Cell Division). My own research their alliance covering blood borne serological imaging via gated Stimulation Emission specialised equipment , the majority of which team focuses on signalling through the RAS/RAF biomarker assays. Caroline was awarded the Depletion (STED) and two-photon confocal will be housed in the Paterson Institute core pathway and the role that this plays in the highly prestigious Pasteur-Weizmann/Servier microscopy. We shall be the first site in the UK to facilities and will support research across both development and progression of melanoma. Prize and Tribute. This well-deserved honour is have such a capability which will put us at the buildings. This will help us to progress the basic The group is now established in the Institute in recognition of the fantastic work by Caroline forefront of super-resolution imaging on live and translational research that is pivotal to the with eight new members in addition to five and her group in developing minimally-invasive cells and have a major impact on a range of development of further novel anti-cancer Postdoctoral Fellows who have joined me in biomarker tests for a range of cancers and projects across the Institute. We said farewell to treatments and to accelerate the development moving here from the Institute of Cancer treatments. Morgan Blaylock after five very successful years of approaches that will lead to improved Research in London. building up our Flow Cytometry Facility. Steve outcomes for patients. We have made a start on The Drug Discovery Group continue to make Bagley now oversees this service after its merger acquiring the necessary technologies with the A key objective at the Institute for the coming good progress and have two active phase with Advanced Imaging. Several new members installation of two next generation sequencing year is to recruit four new Junior Group Leaders. projects in collaboration with core Group of staff have been recruited who will work platforms which will greatly expand our These new appointments will help strengthen Leaders (Tim Somervaille and Ivan Ahel) and across Imaging, Flow Cytometry and Histology capabilities in this area. The remaining the Institute’s portfolio in translational research three pre-projects with core Group Leaders to provide an integrated service for users. purchases will be made over a three year period and will focus on our areas of priority, which (John Brognard, Ivan Ahel and Karim Labib). The Significant refurbishment work and updating of which will allow us to take advantage of rapidly include lung cancer, pancreatic cancer, increasing synergy between DDG and CEP was equipment has resulted in improvements to evolving technology across many of the women’s cancers and molecular pathology. enhanced this year by the joint appointment of Laboratory Services while the Biological relevant platforms. These themes are largely aligned with the an in vivo scientist who will be responsible for Research Unit has had a busy year relocating cancer research strategy of our local MCRC progressing the DDG portfolio. The advantages transgenic services to our new facility at the During the summer, Geoff Margison, leader of partners, the University of Manchester and The of embedding Drug Discovery within the University which will allow for much needed the Carcinogenesis group, retired from the Christie NHS Trust, a joint strategy that has been Institute have been clearly illustrated by another expansion of our experimental capability. Institute after an association extending back developed over the past year and has major highlight of the year which was the over forty years. He continues his research with personalised medicine at its core. Critically, the publication by Tim Somervaille’s Leukaemia It is also a pleasure to note some further an honorary position at the University of priority areas offer exceptional opportunities in Biology Group of their discovery of KDM1A as a individual achievements by members of the Manchester. Akira Orimo returned to Japan after Manchester due to local clinical need and candidate target for differentiation therapy in Institute. Alexia Eliades, a member of the Stem five years at the Institute as a Junior Group existing research activities. Lung cancer is a one of the most common types of acute Cell Haematopoiesis Group headed by Valerie Leader. He will continue to investigate the role particular priority on which we wish to focus, myeloid leukaemia. The first author of this study Kouskoff, was awarded an EMBO Post-Doctoral that non-cancerous stromal cells play in tumour building on the recruitment to the Institute of was PhD student Bill Harris who was deservedly Fellowship for her research into the molecular 4 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH DIRECTOR’S INTRODUCTION 5

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    DIRECTOR’S INTRODUCTION (CONTINUED) RESEARCH HIGHLIGHTS Addition of a novel KDM1A inhibitor to primary cells from a In this section we are highlighting some research publications patient with AML results in differentiation from leukaemic from 2012 which report significant advances in specific areas. blasts into macrophages. Cells The selected papers demonstrate the breadth and the quality shown in the right hand panel have been treated with the of the research being undertaken by the groups at the Institute. inhibitor whereas those in the left hand panel are untreated. Figure provided by William Harris Harris WJ, Huang X, Lynch JT, Spencer GJ, therapeutic strategy in other molecular and Tim Somervaille from the Hitchin JR, Li Y, Ciceri F, Blaser JG, Greystoke subtypes of myeloid malignancies. Further Leukaemia Biology Group. BF, Jordan AM, Miller CJ, Ogilvie DJ, lab-based studies are required to characterise Somervaille TC. KDM1A as a therapeutic target in AML, to The histone demethylase KDM1A sustains the optimise inhibitor dosing strategies in vivo and oncogenic potential of MLL-AF9 leukemia stem to determine whether it is optimally effective in cells. Cancer Cell, 2012, 17;21(4):473-87. combination with other differentiation inducing agents such as retinoic acid. This work also mechanisms of haematopoietic specification. carry out our research. Our annual Open Day Drugs targeting aberrant epigenetic provides a basis for early-phase clinical trials of Avinash Patel from Cell Division was awarded a was a great success and attended by over 100 mechanisms are under intense investigation KDM1A inhibitors. short term EMBO fellowship to participate in a visitors who enjoyed research demonstrations within the pharmaceutical industry as candidate highly successful phosphoproteomic by Institute scientists in several laboratories. The novel therapies in cancer. The Leukaemia collaboration with Boris Maček of the Proteome guests included Dr Elspeth Russell, who is the Biology Laboratory has been assessing the Weston R, Peeters H, Ahel D. centre in Tübingen. Allan Jordan, who leads the daughter of Edith and Ralston Paterson, two potential of targeting deregulated epigenetic ZRANB3 is a structure-specific ATP-dependent Chemistry team in the Drug Discovery Group pioneering doctors, after whom the Institute mechanisms in the treatment of acute myeloid endonuclease involved in replication stress was admitted as a Fellow of the Royal Society of takes its name. Our School’s Day was also highly leukaemia (AML) because few effective response. Genes and Dev, 2012, 26:1558–1572. Chemistry (FRSC). The designation FRSC is given successful with a number of sixth form students therapies currently exist for this disease. to a group of elected Fellows who have made spending a day in the lab. Scientists from the During DNA replication, cells are frequently major contributions to chemistry and represents Institute attended a number of events to meet Their study identified the histone demethylase, confronted with the challenge of overcoming the highest category of membership of the supporters including Race for Life, Relay for Life KDM1A, as an enzymatic target which, when potentially dangerous DNA lesions. When such Society. Ali Raaof, also a chemist within Drug and to give talks at fundraising committee inhibited either by knockdown or damage is encountered, a cell faces a choice of Discovery, organised a one day meeting, as part annual general meetings. It was also great to see pharmacological inhibition, induced repairing the damage, or bypassing it without of his activities within the Society of Chemical our staff getting involved with their own differentiation of murine leukaemia stem cells. the actual repair. The mechanism which cells Industry. The meeting took place at the fundraising efforts which included setting up an In collaboration with the Drug Discovery Unit, employ to repair DNA damage, and thus ensure Paterson Institute and was attended by a mixture impromptu hairdressing salon for a day, running potent inhibitors of KDM1A active in the continuity of DNA replication on an undamaged of academic and industrial chemists as well as in the Manchester 10K race, cake sales, nanomolar range were synthesised in-house, DNA template, is not well understood. Recent biological scientists. Poster prizes were sponsored walks and participating in the and these too were found to abrogate work by the DNA Damage Response Group awarded to Darren Roberts, a postdoctoral Manchester Shine event. clonogenic potential and induce differentiation published in the journal ‘Genes and scientist who works jointly with Peter Stern and of both murine AML cells and primary human Development’ provides new insights into this Andrew Renehan, who won the poster prize at The coming year promises to be very exciting patient leukaemia cells. Significantly, the process. the recent 8th World Congress on Peritoneal with the introduction of further innovative functional potential of normal haematopoietic Surface Malignancies for his work on gene technologies and the strengthening of our stem and progenitor cells was largely unaffected The DNA Damage Response Group has expression profiling and development of a cell research portfolio through the recruitment of by these compounds, providing strong identified a novel replication associated protein culture model of Pseudomyxoma peritonei. new Group Leaders. I look forward to the evidence for a potential therapeutic window. ZRANB3 (Zinc finger, RAN-Binding domain Romina Girotti from the Molecular Oncology challenges ahead and to continuing to develop Thus KDM1A (also known as LSD1) is a candidate containing 3), and proposed a role for it in the Group won a poster prize at the South American the Institute in a way that helps us to contribute target for differentiation therapy in MLL mutated repair of replication-blocking lesions. ZRANB3 Spring Symposium in Signal Transduction and to CR-UKs goals of finding new ways to AML, a common cause of both childhood and localises at DNA replications sites and interacts Molecular Medicine meeting for her work on diagnose and treat cancer. secondary, treatment-associated AML, which with the components of the replication mechanisms of resistance to BRAF inhibitors in tend to have a poor clinical outcome. machinery. It is recruited to damaged replication melanoma. forks by multiple mechanisms, which involve This study also revealed intriguing preliminary interactions with the replication factor PCNA, The past year has provided us with many evidence that KDM1A inhibition could be a K63-polyubiquitin chains, and branched DNA opportunities to engage with Cancer Research UK’s supporters whose generosity allows us to 6 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH RESEARCH HIGHLIGHTS 7

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    RESEARCH HIGHLIGHTS (CONTINUED) The coordinated action Par3 (green) and Syntrophin (blue) on Tiam1 (red) sets up a gradient of Rac activity along the cell membrane, and it is this gradient which is crucial for the correct formation of tight junctions, disruption of which is linked to the development and progression of tumours. The Cell Signalling Group has been able to identify and subsequently disrupt this Rac gradient by interfering with the expression of structures. Most importantly, the authors have Interestingly, reduced membrane β2-syntrophin Syntrophin, a novel regulator of A multitude of signalling pathways influence the treatment. We posit that understanding CTC Tiam1, and a potential marker for shown that ZRANB3 acts as a unique structure- was found to correlate with human prostate timing of cell division by regulating the balance biology will aid efforts to combat this disease. prognosis in prostate cancer. specific endonuclease, whose activity is cancer progression. Therefore, reduced Image taken 6 hours after of Wee1 kinase and Cdc25 phosphatase This study explored the presence and dependent on ATP hydrolysis. It cleaves membrane β2-syntrophin and/or formation of the tight junctions. activities towards the Cdk1/Cyclin B complex prognostic value of CTCs in 97 SCLC patients. branched DNA structures with unusual polarity, overexpression of Tiam1 and Rac, all frequently that promotes commitment to mitosis. While Using the CellSearch technology platform, we generating a break in the leading strand DNA observed in tumours, could contribute to the Image provided by Andrew the identity of most of the key players has been showed that CTCs were present in 85% of Porter from the Cell Signalling template. Collectively, these data support a role deregulation of adhesion and polarity and known for over 20 years, the “how”, “where”, patients and that CTC number was reduced Group for ZRANB3 in the replication stress response promote tumourigenesis. “when” and “why” of this activation remains after one cycle of therapy mirroring the initial and suggest how DNA repair might be unclear. This study describes how two highly patient response. With careful consideration of coordinated with DNA replication to maintain conserved protein kinases control Wee1 activity biostatistics, we showed that patients with ≥ 50 genome stability. De Piccoli G, Katou Y, Itoh T, Nakato R, to regulate the timing of mitotic commitment. CTCs in a 7.5ml blood sample at baseline had Shirahige K, Labib K. The NDR kinase Sid2/Mob1 is activated roughly significantly worse progression free and overall Replisome stability at defective DNA replication two thirds of the way through the G2 phase that survival than those with < 50 CTCs. We also Mack NA, Porter AP, Whalley HJ, Schwarz JP, forks is independent of S phase checkpoint precedes mitosis to promote the activity of the showed that apoptotic CTCs were evident in Jones RC, Khaja AS, Bjartell A, Anderson KI, kinases. Mol Cell, 2012, 45(5):696-704. NIMA kinase, Fin1, towards a “Cell Geometry 57% of patients; circulating tumour microemboli Malliri A. Network” (CGN). The CGN inhibits Wee1’s (CTM, clusters of CTCs) were present in 32% of β2-syntrophin and Par-3 promote an apicobasal Defects in DNA replication are thought to drive repression of Cdk1/Cyclin B. The involvement of patients and just one CTM in a patient’s blood Rac activity gradient at cell-cell junctions by the early stages of cancer development and are Sid2/Mob1 in controlling mitotic commitment sample heralded worse prognosis. The authors differentially regulating Tiam1 activity. Nat retained in some mature tumours. It is therefore was unanticipated as this kinase and its budding hypothesise that tumour cells within CTM might Cell Biol, 2012, 11:1169-1180. very important to understand the pathways that yeast counterpart have only been linked to the have a survival advantage as they have not allow cells to survive such ‘DNA replication control of mitotic exit and cytokinesis in detected apoptosis in CTM. Moreover, unlike the Epithelial cells are attached through junctional stress’, as drugs targeting these pathways might previous work. The links between the equivalent majority of viable single CTCs, cells within CTM complexes spaced along their lateral kill cancer cells preferentially. The S-phase kinases (Nek2, Lats/Mob1) and cancer make are not proliferative, based on Ki67 staining membranes. The most apical junctions, tight checkpoint pathway is central to this regulation translation of these findings from this model implying a relative resistance to the junctions, block the free passage of molecules and is conserved from yeasts to humans. system into the more complex controls of conventional chemotherapeutics used to treat between cells and the diffusion of molecules Defects in DNA replication lead to activation of human cell division an appealing proposition. SCLC. This translational research formed the from apical to basolateral membrane domains. the ATR kinase, which then activates the basis for ongoing studies where the team are The assembly and disassembly of junctional downstream kinase Chk1. Acting together, these now making strides to perform single CTC and complexes is modulated during development, kinases preserve the functional integrity of the Hou JM, Krebs MG, Lancashire L, Sloane R, CTM molecular analysis with a view to the wound healing and tissue homeostasis. DNA replication machinery in ways that are still Backen A, Swain RK, Priest LJ, Greystoke A, discovery of new drug targets and predictive Deregulated intercellular adhesion is a hallmark understood poorly. Previous work indicated Zhou C, Morris K, Ward T, Blackhall FH, Dive C. biomarkers. of cancer cells. The Rho-family member Rac, that the checkpoint kinases were needed to Clinical significance and molecular and its activator Tiam1, have been implicated in preserve the stability of the replisome at DNA characteristics of circulating tumor cells and the regulation of junctional complexes. replication forks. In contrast, this study in circulating tumor microemboli in patients with Lancrin C, Mazan M, Stefanska M, Patel R, This study now furthers our understanding of Molecular Cell shows that replisome stability is small-cell lung cancer. J Clin Oncol, 2012, Lichtinger M, Costa G, Vargel O, Wilson NK, the spatiotemporal control of Rac signalling at actually independent of the S-phase checkpoint 30(5):525-32. Möröy T, Bonifer C, Göttgens B, Kouskoff V, junctional complexes. The Cell Signalling Group pathway. It now seems likely that the Lacaud G. identified β2-syntrophin, part of a larger cell checkpoint kinases regulate replisome function Lung cancer is the leading cause of cancer- GFI1 and GFI1B control the loss of endothelial adhesion complex, as a binding partner of rather than replisome stability, and the related death worldwide. Small Cell Lung identity of hemogenic endothelium during Tiam1. Furthermore, they demonstrated that underlying mechanisms will be an important Cancer (SCLC) accounts for 10-15% of lung hematopoietic commitment. Blood, 2012, β2-syntrophin promotes the activation of Rac focus for future work in this area. cancer cases and is characterised by early 120(2):314-22. via Tiam1 and also limits the diffusion of Tiam1 development of widespread metastasis. Patients and Rac towards the apical domain. In parallel, with this disease initially respond well to Recent studies have established that most, if not Par-3, associated with tight junctions, inhibits Grallert A, Connolly Y, Smith DL, Simanis V, conventional chemotherapy but fatal disease all, blood cells are generated from specific types Tiam1. The combined actions of Par-3 and Hagan IM. relapse is almost universal and often rapid. of endothelial cells with haematopoietic β2-syntrophin establish a gradient of Rac The S. pombe cytokinesis NDR kinase Sid2 There are few biomarkers to guide patient potential, i.e. haemogenic endothelium cells. concentration and activity required for correct activates Fin1 NIMA kinase to control mitotic management and serial tumour biopsies are The transcription factor RUNX1 is a frequent formation and function of tight junctions and for commitment through Pom1/Wee1. Nat Cell very challenging. Circulating Tumour Cells target of gene rearrangements and mutations in the generation of apicobasal polarity. Biol, 2012, 14(7):738-45. (CTCs) are thus appealing as a readily sampled human acute myelogenous leukemia (AML) and source of tumour cells before, during and after acute lymphoblastic leukemia (ALL). Consistent 8 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH RESEARCH HIGHLIGHTS 9

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    with its initial implication in leukaemias, RUNX1 the protein. Recently, the Carcionogenesis In a xenograft model 5T4+ve, compared to is critical for normal haematopoietic Group used the human AGT as a model to 5T4-ve, B-ALL cells showed differential spread development and in particular for this process of interrogate a Schizosaccharomyces pombe to the omentum and ovaries following endothelial to haematopoietic transition (EHT). database and discovered a protein that intraperitoneal inoculation; this could also be However the precise identities and specific roles resembles AGT, but without the critical cysteine blocked by mAb5T4. Consistent with this, the of RUNX1 downstream effector genes remain residue. Originally, they demonstrated that this 5T4+ve B-ALL cells show increased invasion in largely unknown. alkyltranserase-like (ATL) protein, Atl1, binds to vitro concordant with increased LFA-1 and O6-alkylguanines and flags them for processing VLA-4 integrin expression, adhesion to In this study, the Stem Cell Biology Group by the nucleotide excision repair (NER) pathway. extracellular matrix and secretion of matrix reported the identification of the transcriptional Now the team show that binding affinity varies metalloproteases (MMP-2/-9) compared with repressors GFI1 and GFI1b as critical targets of according to the nature of the alkyl group and their negative counterparts. The xenograft RUNX1 and establish their crucial function in the relative ease of dissociation of Atl1 from DNA model system was used to show that 5T4+ve regulating this trans-differentiation from containing some O 6-alkylguanines allows B-ALL are susceptible to 5T4 specific endothelial to blood cells. GFI1 and GFI1B are completion of NER, whereas strong binding of superantigen antibody-dependent cellular able to trigger, in the absence of RUNX1, the Atl1 to other O 6-alkylguanines stalls toxicity providing support for targeted down-regulation of endothelial markers and the transcription and diverts the damage to the immunotherapy in high risk pre-B-ALL patients. formation of round cells, a morphological transcription-coupled NER pathway. These The results of this study are consistent with the change characteristic of this cell fate switch. findings raise the question of whether or not hypothesis that 5T4 is a marker of leukaemic Conversely, in Gfi1 and Gfi1b deficient embryos, O6-alkylguanine lesions that are poor substrates cells which are relatively resistant to the first generated blood progenitors maintain for human AGT might, by analogy, signal such chemotherapy including through an increased the expression of endothelial and cell adhesion lesions for repair by NER. ability to migrate to tissue sites which provide for molecules. These cells are therefore hampered disease relapse following treatment. If 5T4 in their ability to be released in the vasculature marks the most drug resistant B-ALL then 5T4 and to be disseminated in the yolk sac and in the Castro FV*, McGinn OJ*, Krishnan S, Marinov G, directed therapies could provide a rational and embryo-proper. Li J, Rutkowski AJ, Elkord E, Burt D, Holland M, effective way to treat such patients. Vaghjiani R, Gallego A, Saha V, Stern PL. The group demonstrates a critical and specific 5T4 oncofoetal antigen is expressed in high risk role of the GFI1 transcriptional repressors in the of relapse childhood pre-B acute lymphoblastic Dovedi SJ, Melis MH, Wilkinson RW, Adlard AL, generation of haematopoietic progenitors from leukemia and is associated with a more invasive Stratford IJ, Honeychurch J, Illidge TM. dependent upon the activation of CD8+ haemogenic endothelium. The results suggest and chemotactic phenotype. Leukemia, 2012, Systemic delivery of a TLR7 agonist in cytotoxic T-cells, as depletion of these immune that the EHT could be decoupled firstly into 26(7):1487-98. combination with radiation primes durable cells rendered the combination ineffective. repression of the endothelial identity controlled anti-tumor immune responses in mouse by Gfi1s repressors and in parallel into activation Although the overall prognosis in childhood models of lymphoma. Blood, 2012, Oct 18, Moreover, the CD8+ T cells provided a durable of the haematopoietic cell fate programme. acute lymphoblastic leukaemia (ALL) is good, doi: 10.1182. response capable of protecting against disease These new insights could result in new outcome after relapse is poor. Recurrence is recurrence. These data reveal that combination strategies to generate blood cells for frequently characterised by the occurrence of Radiotherapy plays an important part in the therapy with radiation and a TLR7 agonist is able regenerative medicine from embryonic stem disease at extramedullary sites such as the local control of many lymphomas and leads to to generate tumour-specific immunological cells or from induced pluripotent stem cells. central nervous system and gonads. extremely high response rates even in patients memory. These findings demonstrate the Subpopulations of blasts able to migrate to such that are refractory to conventional potential for novel therapeutic combination areas may have a survival advantage and give chemotherapy approaches. The cell death approaches involving radiotherapy and Latypov VF, Tubbs JL, Watson AJ, Marriott AS, rise to disease recurrence. In this collaboration caused by radiation therapy has the potential to immunotherapy for the treatment of cancer. McGown G, Thorncroft M, Wilkinson OJ, between the Immunology and Children’s stimulate immune responses against the cancer Senthong P, Butt A, Arvai AS, Millington CL, Cancer groups, gene expression profiling of cells. However, these immune responses tend Povey AC, Williams DM, Santibanez-Koref MF, diagnostic pre-B-ALL bone marrow samples to be weak and insufficient to improve a Tainer JA, Margison GP. revealed higher 5T4 oncofoetal antigen patient’s outcome. Combining radiation therapy Atl1 Regulates Choice between Global Genome transcript levels in cytogenetic high-risk with a drug that can stimulate the immune and Transcription-Coupled Repair of O 6- subgroups of patients. Flow cytometric analysis system has the potential to generate durable Alkylguanines. Mol Cell, 2012, 47(1):50-60. determined that bone marrow from relapse and effective anti-cancer immune responses patients have a significantly higher percentage capable of eradicating widespread malignant Alkylating agents are mutagenic, carcinogenic of 5T4 positive leukaemic blasts than healthy disease and reducing disease recurrence. and toxic and we are exposed both donors. 5T4 has also been shown to regulate exogenously, for example during certain types CXCL12 chemokine and Wnt signalling Using a synthetic agonist of TLR-7 which of cancer chemotherapy, and endogenously, pathways which are important in leukaemia activates a systemic immune response by the origins of which are currently not defined. growth and spread. For example, CXCL12 is mimicking a viral infection, the Targeted One of the most genotoxic lesions that is naturally produced by various tissues of the Therapy Group found that the anti-tumour generated by these agents is O 6-alkylguanine. In body and controls the distribution of different efficacy of radiation therapy can be enhanced in humans, and many other organisms, this types of immune cells but can also act as a pre-clinical models of lymphoma. Combination damage is repaired by O 6-alkylguanine-DNA “magnet“ for leukaemia cells. Only 5T4+ve therapy but not single-agent treatment resulted alkyltransferase (AGT), which stoichiometrically B-ALL cells show CXCL12 specific chemotaxis in long-term clearance of tumour and reverses the damage by transferring the alkyl in vitro and this can be blocked by a monoclonal enhanced survival. This response was group to a cysteine residue in the active site of antibody (mAb) to 5T4 but not HLA. 10 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH RESEARCH HIGHLIGHTS 11

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    THE PATERSON INSTITUTE FOR CANCER RESEARCH RESEARCH GROUPS Transcriptional profiling was used to identify genes up-regulated upon activation of stress signalling. The heat map depicts the expression of stress-responsive genes in a panel of breast tumours versus normal tissue samples, (blue colour indicates under-expression). The impaired expression of this set of genes occurs in a range of tumour types, suggesting a suppressive role for this pathway in tumour development. Image provided by Steve Lyons from the Cell Regulation Group. 12 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH THE PATERSON INSTITUTE FOR CANCER RESEARCH 13

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    Figure 1 APPLIED COMPUTATIONAL BIOLOGY Annmap (annmap.picr.man.ac. uk) is a genome browser based AND BIOINFORMATICS on the Google Maps API. Each grey box represents a gene, with www.paterson.man.ac.uk/bioinformatics individual transcripts shown as horizontal tracks within. Coding exons are black, UTRs, grey. Regions encoding protein domains are identified in yellow. Green lines represent target locations for Affymetrix Exon 1.0ST microarrays. Associated expression, and builds on previous work using is not clear what proportion of these newly with the genome browser is a Schizosaccharomyces pombe (fission yeast) in discovered non-coding RNAs are functional, Bioconductor package that We are interested in how genes influence the way tumours provides programmatic access which we identified sets of cis-acting rather than simply corresponding to non-coding RNAs that are differentially background ‘chatter’ in the genome, their grow and develop, and how the behaviour of genes differs to the same annotation data, allowing it to be brought into the expressed as fission yeast undergoes meiosis. prevalence, and their ability to act through a between tumour and normal cells. Whilst most genes encode statistical context provided by Bioconductor and the R We used strand specific sequencing of total diversity of mechanisms, raises the possibility RNA first to reannotate the genome to include that they may have a profound impact on our protein sequences, nearly forty percent of human genes are programming language. accurate representations of each gene’s understanding of genes and gene expression. transcribed but never translated into proteins. Our research is untranslated regions (UTRs), and then to explore We are using high throughput genomics tools, how patterns of RNA abundance changed over including deep sequencing and quantitative focussed on developing a better understanding of how these the course of meiosis. When we did this, we protein mass spectrometry, to identify novel non-coding genes function, and how their activity is altered in identified a set of non-coding RNAs opposite non-coding RNAs, to investigate the function of Group Leader protein coding genes that are critical regulators existing ones, and to search for those with cancer cells. We are doing this by applying a mixture of of sexual differentiation, leading us to speculate behaviour that is altered in tumour cells. This Crispin Miller computational biology, bench- and clinical-science, in an that they might act to control the activity of work therefore integrates experimental and Postdoctoral Fellows these key proteins. In collaboration with Cell computational approaches and makes use of Hui Sun Leong interdisciplinary programme centred on high throughput Division (p20), we were able to show that this both in vitro and clinical datasets. Janet Taylor2 genomics technologies that include deep sequencing and was indeed the case: over-expression of an Antisense Regulatory Transcript (ART) integrated Analysis of archival material Scientific Officer tandem mass spectrometry. ectopically into the genome phenocopied a Archival Formalin Fixed Paraffin Embedded Keren Dawson deletion of the corresponding protein-coding (FFPE) tissue is an immensely valuable source of Graduate Students In parallel, the group collaborates with many wider community. Underpinning all of our gene. We also showed that their function was information pertaining to cancer. Unfortunately, Elli Marinopolou3 dependent on components of the RNA the preservation process damages RNA, making other groups in the Institute and the wider research is a computational platform that Danish Memon interference (RNAi) pathway, an aspect of the it hard to use these samples as a source for Sharmin Naaz4 Manchester Cancer Research Centre (MCRC) to includes a High Performance Computing (HPC) María José Villalobos provide computational input into their research Linux cluster and associated Lustre file system. fission yeast genome that is conserved with H. systematic analysis of gene expression profiles, Quesada5 programmes (see for example the work by Programmers in the group have developed sapiens, and suggesting that similar and difficult, therefore, to use this material in Harris et al in the Leukaemia Biology group pipelines to process our deep sequencing and mechanisms might occur in human cells. Many global genomic studies. The development of Computational Biologist of these transcripts arise from overlapping 3’ successful methods for measuring global RNA described on p36). This last year has seen a proteomics data, and Bioconductor packages Yaoyong Li UTRs of convergent adjacent gene pairs abundance in these samples, and for significant increase in demand for support as that help support our downstream analysis. The Bioinformatics Analyst groups exploit the power of our microarray, goal is to take novel methods developed as part transcribing towards one another, and we were performing strict Quality Control, would be Phil Chapman1, 6 deep sequencing and mass spectrometry of our research, and turn them into tangible able to confirm that genes in this configuration extremely beneficial. We have been platforms. These fields are all moving rapidly, software tools when they will be used to can indeed co-regulate to modulate the collaborating with Translational Radiobiology Software Architect expression of their neighbours. (p58), to develop methods to support the and the complexity of the datasets they support frequent analyses across multiple Tim Yates analysis of RNA from FFPE samples. We have generate often demands novel analyses, datasets. Since much of our research is directed Bioinformatics resulting in many projects requiring substantial at generating a better understanding of the less Nearly 40% of known human genes are previously shown that it is possible to generate Programmer research-level computational biology. We have well-characterised regions of the genome (see non-coding, and whilst the majority have yet to meaningful gene expression data from archival Chris Wirth met this demand with the development of a below), the major computational focus of the be assigned a function, an increasing body of material, however in some samples, the quality model in which Postdoctoral Fellow-level group is on developing tools that allow fine- research is showing that they can interact with of RNA is too poor to be amenable for further Systems Administrator DNA, with proteins and with other RNAs to study. Recently we showed that miRNAs, a type Zhi Cheng Wang7 analysts develop extended collaborations with grained representations of gene structure to be other research groups, allowing them to integrated with statistical methods (Figure 2). modulate and control their function. Although it of short non-coding RNA, are less susceptible to 1 joined in 2012 become immersed in the research question, This then allows these annotations to be used to the effects of preservation in FFPE than longer Figure 2 2 joint with Translational and leading to a much deeper contribution than provide detailed context when interpreting high Underpinning much of our work mRNA transcripts, and can be used to generate Radiobiology would be possible with a more traditional throughput genomics datasets, and to help are genome annotation meaningful data from FFPE samples even when 3 joint with Stem Cell ‘analysis-as-service’ approach. This allows us bring the data that emerges from different databases that link genes to the the mRNA has deteriorated beyond the point of Biology RNA and protein molecules they utility (Hall JS, Taylor J et al. 2012 British Journal not only to make use of the latest techniques technologies together into a single integrated encode. The figure shows just a 4 joint with Stem Cell emerging from the computational biology analysis. The group therefore sits at the interface of Cancer 107(4):684-694). These, plus other tiny subset of the millions of Haematopoiesis research community, but also to develop the of biology, mathematics and computing, and relationships we need to record. advances in both biochemistry and 5 joint with Cell Division novel algorithms and software tools we need to comprises a highly interdisciplinary team that We use computer software to bioinformatics, are raising the prospect of 6 embedded by Drug perform these analyses (Figure 1). We are active incorporates both ‘wet’ and ‘dry’ science. help interpret these networks. generating useful classifiers and biomarkers Discovery contributors to the Bioconductor project – an from archival material, further unlocking the 7 joint with IT department international collaboration to develop open Novel non-coding regulators of potential of this enormously valuable resource. source software packages for the analysis of gene expression biological data, and this is the primary route by Current research in the group is investigating the Publications listed on page 68 which we make our software available to the role of non-coding RNAs in regulating gene 14 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH APPLIED COMPUTATIONAL BIOLOGY AND BIOINFORMATICS 15

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    University of Sheffield, we have synthesised CARCINOGENESIS short oligodeoxyribonucleotides (ODNs) containing O6-alkylguanines and used these as substrates for purified E. coli Ogt and MGMT proteins in the strategies outlined in Figure 1. We found that an O6-MeG-containing ODN inactivated both alkyltransferases with similar efficiency. However, while the O6-CMG- containing ODN was a very poor substrate for Ogt, it was a very effective inactivator of MGMT, with an IC50 (1.8nM) very close to that of the O6-MeG-containing ODN (0.93nM). That The consumption of red meat is a risk factor in human MGMT actually removed the carboxymethyl group from the O6-CMG-containing ODN was colorectal cancer (CRC) causation, possibly by enhancing the shown in enzyme-linked immunosorbent nitrosation of bile acid conjugates, which rapidly convert to assays (ELISAs) in which ODN were bound via terminal biotin linkers to streptavidin-coated DNA-damaging carcinogens. Indeed, the toxic and mutagenic microtitre plates. The O6-CMG lesions were DNA adduct, O6-carboxymethylguanine (O6-CMG), is detected using polyclonal antibodies that we raised to this adduct, and also using the S. frequently present in human DNA, increases in abundance in pombe alkyltransferase-like protein Atl1, which people with high levels of dietary red meat and may therefore we have previously described to bind strongly to Group Leader all DNA-O6-alkylguanines that we have so far be an important factor in CRC. Using synthetic assessed, including O6-CMG. We confirmed Geoff oligodeoxyribonucleotides containing O6-CMG, we now show Figure 1 Furthermore, colorectal tumours often occur in that the carboxymethyl group had been Margison1 Strategies for demonstrating that gastrointestinal regions expressing low MGMT removed from the O6-CMG by exploiting our that, contrary to our previous suggestion, the human DNA O 6CMG is a substrate for MGMT. activity, and low activity in normal colon tissue observation that this lesion blocked digestion by Postdoctoral Fellow At the top, glycocholic acid is Vitaly Latypov1 repair protein, O6-methylguanine-DNA methyltransferase nitrosated to has been associated with the presence of K-ras the restriction endonucleases, BsaJI and PstI, N-nitrosoglycocholic acid, a GC→ AT transition mutations in colorectal when located in the recognition sequences of Scientific Officers (MGMT), is very effectively inactivated by O6-CMG in vitro and process that is enhanced by red tumours. In addition, cytosine-methylation of these enzymes. Incubation of these ODN with Gail McGown1 this involves the transfer of the carboxymethyl group to its meat consumption. N-nitrosoglycocholic acid is CpG islands within the promoter region of the MGMT converted the lesion to guanine, and Mary Thorncroft 1 MGMT gene is associated both with reduced allowed digestion by these enzymes, clearly active site cysteine residue. O6-CMG is therefore an MGMT metabolised by cytochrome P450 enzymes and gives rise to a MGMT expression and with an increased demonstrating that repair was by damage Graduate Students Pat Senthong2 substrate and hence MGMT is likely to be an important carboxymethylating agent that frequency of GC→ AT transition mutations in reversal. Finally, in collaboration with Clare Eyers damages DNA to produce K-ras in CRCs. Indeed, adenomas containing a at the University of Manchester, mass Undergraduate Students protective factor in CRC. O 6-CMG (and O 6-MeG, not K-ras GC→ AT mutation have lower MGMT levels spectrometric analysis of tryptic digests of the Amy Hatch1 shown). When present in short (relative to adjacent normal tissue) than MGMT following incubation with the O6-CMG Sarah Pinder1 ODN, this can be detected by binding with anti- O 6-CMG adenomas without this mutation. As MGMT ODN showed the presence of the CM group in Emma Williams1 Background chromatid exchanges. More than a decade ago, antibodies or Atl1 (results not removes O6-alkylguanine lesions from DNA, S-carboxymethylcysteine within the active site Most sporadic colorectal cancers arise through we showed that almost all human colorectal 1 left in 2012 shown) and also blocks digestion these observations strongly support the cysteine residue contained in a tryptic peptide. an adenoma-carcinoma sequence, the DNA samples contain O6-MeG, the levels of with BsaJI. Purified active MGMT 2 Jointly with Dr Andy hypothesis that alkylating agents are involved in Control experiments showed methyl group molecular pathways of which have been well which vary 100-fold, with the highest occurring (in pink) removes the Povey, Health Sciences the aetiology of at least a proportion of CRC. transfer from ODN containing O6-MeG. group at the University characterised. Known risk factors for CRC in the sigmoid colon and rectum, where most carboxymethyl group to restore include dietary red and processed meat and sporadic tumors occur. In addition, others have guanine (G) in the ODN, which of Manchester These findings clearly demonstrate that now becomes digestible by While MGMT is known to have very broad while the mechanisms by which these factors shown that exfoliated colon cell DNA contains BsaJI giving rise to two small substrate specificity, our previous work using O6-CMG is as good a substrate for MGMT as modify CRC risk remain to be fully elucidated, O6-CMG, probably arising from N-nitrosation of fragments that can be resolved cell-free extracts of E. coli overexpressing the E. O6-MeG, and connect increased red meat one possibility is that such diets increase the bile cojugate, glycocholic acid, which also from undigested ODN using coli O6-alkylguanine-DNA alkyltransferase- consumption with low levels of MGMT as N-nitrosation reactions within the colon. generates O6-MeG in DNA. That the levels of agarose gel electrophoresis. The repaired ODN no longer binds encoding genes, ada or ogt and a human cell combined critical factors in human colorectal N-nitrosation of compounds containing amino O6-CMG in DNA from exfoliated colonic cells is anti- O 6-CMG antibodies or Atl1. line expressing endogenous MGMT suggested carcinogenesis. groups, such as bile acid conjugates, can result increased by diets high in red meat diet strongly Finally the inactivated that alkyltransferases do not act on O6-CMG in in the formation of alkylating agents that can be implies its role in CRC. carboxymethylated MGMT is DNA (Shuker and Margison GP. Cancer Res. Publications listed on page 68 potent mutagens, clastogens and carcinogens. digested with trypsin and the 1997, 57: 366-369). Given the compelling These genotoxic effects of alkylating agents can Processing of mutagenic lesions in DNA active site peptide, GNPVPILIPCHR, is shown to evidence for a role for MGMT in protecting be attributed largely to their ability to alkylate O6-MeG is eliminated from DNA by the DNA contain carboxymethylcysteine against CRC, this would suggest that O6-CMG is DNA and the biological properties of some of repair protein, O6-methylguanine DNA based on its mass to charge ratio. unlikely to be a significant factor in CRC risk, the DNA adducts formed, especially O6- methyltransferase (MGMT), in a stoichiometric despite the observation that its abundance alkylguanine lesions, are well-characterized. process that results in the transfer of the methyl increases in high-risk diet situations. Thus O6-methylguanine (O6-MeG) is a known group to Cys145 in the active site of the protein. toxic, mutagenic and carcinogenic base In human colorectal mucosa, MGMT activity is Revisiting the substrate specificity of MGMT modification in DNA which, in the absence of highly variable, partly a consequence of MGMT To address this apparent inconsistency, in repair, can induce GC→ AT transition mutations polymorphisms, which have been found to collaboration with David Williams at the and recombination events in the form of sister modify CRC risk depending upon the diet. 16 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH CARCINOGENESIS 17

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    Figure 1 loops that interact with lipid or protein targets. CELL CYCLE We found that lysine 31 in ‘loop 1’ of the G1-phase Origin of DNA replication C2-domain of Inn1 is conserved in all www.paterson.man.ac.uk/cellcycle orthologues of Inn1 and is essential for cytokinesis, suggesting that this site makes an Loaded Mcm2-7 complex important contact with a key target of Inn1. To (inactive) S-phase identify this factor, Asli Devrekanli screened CDK and DDK during her PhD studies for mutations in other Cdc45 budding yeast genes that could suppress the Mcm10 function lethal effects of the inn1-K31A mutation (Figure GINS 2). In this way she identified a large number of dominant mutations that are extremely efficient Our group studies the mechanisms and regulation of suppressors of inn1-K31A, allowing cells to grow P P in a very similar fashion to control cells. chromosome replication and cytokinesis, defects in which P 5 Remarkably, all these suppressor mutations 7 2- c4 mapped to the CHS2 gene, which encodes the drive cancer development. Drugs targeting these pathways are M cm 5 G IN S DNA replication forks I NS Cd 7 G 2- essential chitin synthase that makes the primary c4 cm effective against a variety of tumour cells, so that a better Cd P M septum during cytokinesis in budding yeast. Cdc45-MCM-GINS understanding of these areas of cell biology should aid the Most importantly, the suppressor mutations P P DNA helicase (active) map to two adjacent sites in the catalytic development of novel anti-cancer treatments. domain of Chs2, which flank a predicted binding site for the substrate, UDP-N- Group Leader Figure 2 acetylglucosamine. These findings suggested A highlight of our work with chromosome Frederick van Deursen showed that origin that the mechanism of suppression might Karim Labib replication this year was the identification of a unwinding was blocked very efficiently upon involve an alteration in the catalytic activity of novel role for the Mcm10 protein during the inactivation of Mcm10, but assembly of Cdc45- Chs2, further suggesting that Inn1 might Postdoctoral Fellows Giacomo de Piccoli initiation of chromosome replication. The MCM-GINS was just as efficient as in control normally act by regulating the catalytic activity Cecile Evrin Cdc45-MCM-GINS DNA helicase is needed to cells. It thus appears that Mcm10 is dispensable of Chs2 during cytokinesis. Luis Garcia-Rodriguez unwind the parental DNA duplex, and we for assembly of Cdc45-MCM-GINS and instead Alberto Sanchez-Diaz1 discovered a novel Mcm10-dependent step defines a novel step during helicase activation at Unlike Chs3 that makes the majority of the chitin Sugopa Sengupta1 during the activation of Cdc45-MCM-GINS at replication origins. in the yeast cell wall, Chs2 is normally inactive Scientific Officers DNA replication origins. In our cytokinesis when assayed in purified cell membranes, Frederick van Deursen studies, we showed that the Inn1 protein is an Future work will be needed to establish exactly indicating that its function is carefully regulated Pedro Junior Nkosi essential activator of chitin synthase in budding how Mcm10 helps activate Cdc45-MCM-GINS, in vivo and must be stimulated during yeast. Our work suggests how novel anti-fungal but we found that Mcm10 interacts preferentially Figure 1 cytokinesis is mediated by a contractile ring of cytokinesis by a previously unknown Graduate Students Mcm10 is required for activation therapies could be developed against important with the loaded double-hexamer of inactive actin, type II myosin and many other factors, mechanism. Critically, we found that the Tim Maculins of the Cdc45-MCM-GINS DNA Marija Maric human pathogens such as Candida albicans, Mcm2-7, and an exciting possibility would be which assembles under the plasma membrane suppressor alleles of Chs2 are constitutively helicase during the initiation of which affect immuno-compromised people that Mcm10 modulates the loaded Mcm2-7 chromosome replication. The at the cleavage site and is activated at the end of active in vitro. Moreover, we showed that Inn1 1 left in 2012 including cancer patients. complex in some way during the initiation Mcm2-7 complex is loaded at mitosis, causing ingression of the plasma associates with Chs2 from yeast cell extracts. reaction. Work from other groups indicated that replication origins during membrane and thus producing division of the Overall, these data indicate that the C2-domain Mcm10 defines a novel step during activation Mcm2-7 is initially loaded around double-strand G1-phase as an inactive double cytoplasm. Unlike animal cells, however, the of Inn1 regulates Chs2 activity during of the essential DNA helicase at eukaryotic DNA at origins, but it seems likely that it hexamer, which is then activated plasma membrane in yeasts is surrounded by a cytokinesis, in order to stimulate the formation in situ when cells enter S-phase. DNA replication origins subsequently encircles single-strand DNA at Previous work showed that the cell wall, which means that contraction of the of the primary septum behind the contracting In all eukaryotic cells, the major regulated step replication forks. This suggests that the Mcm2-7 initiation step involves actomyosin ring and ingression of the plasma actomyosin ring. Our findings also show how during the initiation of chromosome replication ring must transiently open and close during the recruitment of GINS and Cdc45 membrane must also be coupled to the cell-based screens could be developed for is the assembly at replication origins of the activation of the Cdc45-MCM-GINS helicase, to form the Cdc45-MCM-GINS deposition of primary and secondary septa. small-molecule inhibitors of Inn1 function. 11-subunit DNA helicase known as Cdc45- and Mcm10 is an excellent candidate for a factor complex. Our work indicates When cytokinesis is complete, digestion of the Such molecules should block cytokinesis in wild that Mcm10 is dispensable for MCM-GINS, which unwinds the parental DNA that facilitates this step. Intriguingly, earlier Cdc45-MCM-GINS assembly, primary septum induces cell separation and the type budding yeast cells, but should be much duplex at DNA replication forks. The Mcm2-7 studies searched for mutations in yeast genes but is needed for a novel step formation of two independent daughter cells. less effective against the suppressor alleles of complex forms the catalytic core of the that could suppress defects in Mcm10 function, during activation of the Cdc45- Inn1 associates with the actomyosin ring and is CHS2. Both Inn1 and Chs2 are conserved in helicase, and is loaded around double strand and identified mutations in the Mcm2 subunit of MCM-GINS helicase at required for membrane ingression and septum other budding yeasts including Candida DNA at origins during the G1-phase of the cell the Mcm2-7 complex, at sites predicted to be at replication origins. formation. We showed previously that the key albicans and Cryptococcus neoformans, cycle, as an inactive double-hexameric ring. an interface with adjacent subunits. It is possible Figure 2 to Inn1 function during cytokinesis is the indicating that small-molecule inhibitors of the When cells enter S-phase, activation of cyclin- that these mutations weaken an inter-subunit Inn1 activates Chs2 during recruitment to the bud-neck of an essential Inn1-Chs2 interface would provide a novel dependent kinase (CDK) and Cdc7 kinase (also interface in such a way as to mimic the action of cytokinesis in budding yeasts. ‘C2-domain’ at the amino terminus of the Inn1 therapy that should be effective against these known as Dbf-dependent kinase or ‘DDK’) leads Mcm10 during the initiation of chromosome Mutation of lysine 31 of Inn1 in protein, since mutations in the C2-domain important human pathogens. to the recruitment of Cdc45 and the four- replication. the C2-domain blocks block cytokinesis, whereas fusion of the cytokinesis, so that germinating protein GINS complex to the pre-loaded spores die as chains of a few C2-domain to other cytokinesis factors Publications listed on page 69 Mcm2-7 double-hexamer, producing the Inn1 regulates chitin synthase during cells. Mutations at a predicted supports cell proliferation in the absence of the Cdc45-MCM-GINS helicase that unwinds the cytokinesis in budding yeasts substrate-binding site in CHS2 endogenous INN1 gene. The critical issue was origin DNA and allows the establishment of two We originally discovered the budding yeast Inn1 suppress the lethal phenotypes thus to define how the C2-domain acts. replication forks (Figure 1). By developing a protein as a novel factor that is essential for of inn1-K31A , allowing cells to novel ‘degron’ allele of budding yeast Mcm10, cytokinesis. In animal cells and in yeasts, grow in a very similar fashion to C2-domains are formed by a sandwich of two the control. The scale-bars Beta-sheets, one side of which has protruding denote 20µm. 18 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH CELL CYCLE 19

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    Figure 1 discrete location facilitates rapid and highly CELL DIVISION sensitive cross talk between different signalling www.paterson.man.ac.uk/celldivision networks. Control of mitotic commitment by a “mitotic exit” kinase We have been studying the function of the fission yeast NIMA kinase, Fin1, in the control of the activity of the “Cell Geometry Network” (CGN). This network ensures that cells do not inhibit Wee1 kinase to promote cell division before they have reached a critical cell size. Errors in chromosome transmission alter the balance between Pom1 kinase inhibits the activity of the Cdr1 and Cdr2 kinases towards Wee1 so that the ultimate tumour suppressor and tumour promoting genes. This impact of Pom1 kinase is to delay mitotic imbalance favours changes in genome composition in the commitment by delaying time at which inhibition of Wee1 by Cdr1 and Cdr2 is relieved. ensuing cell divisions that can lead to cancer. Pom1 accumulates at cell tips, while the Cdr1/ Cdr2/Wee1 kinases associate with cortical nodes at the cell equator. In short cells the Chromosome segregation during mitosis is and suppresses Wee1 activities, thereby driving gradient of Pom1 kinase activity reaches and initiated by the attachment of the microtubules full-scale commitment to mitosis. Fully inhibits Cdr1/Cdr2 at the nodes thereby of the mitotic spindle to the chromosomes. activated MPF then activates a number of highly Group Leader blocking commitment to mitosis. When growth Once all chromosomes have become attached conserved kinases including members of the moves the Pom1 gradient away from the nodes, Iain Hagan to both spindle poles the chromosomes split Polo, Aurora and NIMA families of kinases. Figure 2 Pom1 inhibition of Cdr1/Cdr2 is relieved and into two identical chromatids that then move to Associate Scientist they inhibit Wee1 to promote mitosis (Figure 2). opposite poles. Because the regulatory Cut12, the spindle pole and mitotic Agnes Grallert networks that regulate mitotic commitment and commitment Fin1 activity restrains CGN activity towards Postdoctoral Fellows progression are highly conserved, studying the Our studies of the spindle pole body (SPB) Wee1, resulting in an acceleration of the timing Marisa Alonso-Nuñez1 complexities of cell division in the relatively component Cut12 have uncovered a critical role Ye Dee Tay of mitotic commitment. Fin1 activity towards simple unicellular yeasts greatly accelerates the for events on the spindle pole in mitotic control. Kuan Yoow Chan the CGN is promoted by a protein kinase called analysis of the more complex issue of cell Specifically, they suggest that the MPF Sid2. Sid2 is a member of the NDR (nuclear Scientific Officer division control in man. amplifying positive feedback loop is primed Dbf2-related) kinase family. Prior to our study, Ben Hodgson from the SPB. The foundations for this view lie in the activity of Sid2 had only been associated the reciprocal genetic interactions between Figure 1 - Cut12, Polo and the Graduate Students We use the fission yeast Schizosaccharomyces switch. We have used mass spectrometry to with events during mitotic exit where it controls cut12 and cdc25. The cut12.s11 gain of function mitotic commitment switch. identify 25 sites of phosphorylation on Cut12 Elvan Boke pombe to study cell division because it is a cytokinesis (the splitting of one cell into two). mutation suppresses loss of function mutations The dephosphorylation of Cdc2/ Avinash Patel simple, unicellular organism with excellent and are following this up by assessing the This demonstration that a key component of in cdc25. Conversely, enhancement of Cdc25 Cyclin B that promotes mitotic María-José Villalobos genetics that is cheap to grow and divides consequences of blocking or mimicking the mitotic exit machinery also controls mitotic commitment is accelerated Quesada2 activity suppresses loss of Cut12 function. rapidly. Our major activity asks how cells take through phosphorylation by the phosphorylation on these sites. We are also commitment suggests that there maybe a 1 the decision to divide? We also collaborate with polo kinase Plo1. This active Plo1 collaborating with the group of Boris Maček of greater level of co-ordination between mitotic left in 2012 In seeking ways to understand how an SPB also inhibits the Wee1 kinase that the Proteome Center, Tübingen, to use global 2 the Applied Computational Biology and entrance and exit pathways than has been joint with ACBB component could compensate for loss of puts these phosphates onto Bioinformatics Group to use fission yeast as a phosphoproteomic approaches to identify sites appreciated previously. Cdc25, we drew upon the observation that Cdc2. Crucially, this entire model organism in which to develop control only operates once of phosphorylation that are reduced by loss of removal of Wee1 function enables cells to approaches for the interrogation of genome Cdc2/CyclinB is active, making it polo function or enhanced in a cut12.s11 Lessons from yeast survive without Cdc25 as there is no a feedback control that ensures a function on a global scale with a particular focus background. We have also been assessing the The ability to manipulate genes at will in a requirement for a phosphatase to remove a rapid and complete transition on the role played by the function of consequences of targeting active Cdc2 or Plo1 simple organism whose primary purpose is phosphate from Cdk1 if the kinase that puts this from interphase into mitosis. non-coding RNAs in regulating cellular to different locations within the cell. simply to grow and divide is enabling us to phosphate there is absent. Because Polo kinase Recruitment of Plo1 to the differentiation and stress responses. spindle pole by Cut12 appears to Encouragingly, we can only trigger mitotic explore the finer points of the pathways that plays a key role in the MPF positive feedback be critical for this control. commitment when we target either kinase to co-ordinate growth with spatial and loop, we considered the hypothesis that Cut12 Mitotic commitment the spindle pole and no other location tested. environmental cues. This information informs suppresses ablation of Cdc25 as it Figure 2 - The Cell Geometry Commitment to mitosis is regulated by the studies in higher systems that, in turn, raise inappropriately prompts Polo to shut down Network (CGN). activity of a protein kinase called MPF. MPF is Work from the group of Professor Jon Pines models that can be most readily tested in yeast. Wee1. We found a direct relationship between Sequestration of the DRYK composed of a Cdk1 catalytic subunit and a (Dual-specificity tyrosine- (Gurdon Institute, Cambridge) has shown that This re-iterative cycle of comparative studies Polo activity and Cut12 status; Polo activity is regulatory subunit Cyclin B. Prior to mitosis MPF regulated kinase) Pom1 to cell active MPF first appears on human ensures that great strides are being made in elevated when Cut12 function is enhanced and tips (red) establishes a gradient of is inhibited via phosphorylation on a residue centrosomes, strongly suggesting that the understanding the molecular basis of cell severely reduced when Cut12 function is Pom1 activity. This gradient can (tyrosine 15) within Cdk1’s ATP binding pocket. networks we are studying in yeast occur in division and growth. compromised (Figure 1). inhibit the Cdr1/Cdr2/Wee1 This phosphate is removed by Cdc25 human cells. In other words, key decisions complexes that accumulate on phosphatase. The balance of Cdc25 and Wee1 centrally positioned nodes about whether to divide or not do not arise from Publications listed on page 69 We are now exploiting a range of approaches to activities determines when MPF will be activated (green) more efficiently in shorter the gradual accumulation of a “pro mitosis” identify the means by which a structural cells than it can in longer cells. to drive mitotic commitment. Once a critical state, rather, they are taken at a discrete location, component of the spindle pole can exert such a Thus, the CGN couples mitotic threshold level of MPF is reached a positive the spindle pole. This concentration of strong impact upon the mitotic commitment commitment to increased cell feedback loop is triggered that boosts Cdc25 signalling to a limited subset of molecules at a length. 20 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH CELL DIVISION 21

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    CELL REGULATION of ATF2 (ATF2-AA) fails to rescue the ATF2 loss of function mutant. Furthermore, we find that of apoptosis induced by growth factor withdrawal or treatment with genotoxic www.paterson.man.ac.uk/cellregulation expression of active JNK had similar suppressive drugs, e.g. doxorubicin or mitoxantrone. It activities during oncogenic transformation of has previously been shown that while forced hepatoblasts and that these activities strictly Myc expression induces proliferation of require ATF2/7. Therefore, tumour suppressive lymphoma and other cells it also sensitises functions involving JNK mediated pathways are cells to stress induced apoptosis. Our findings at least in part dependent on ATF2/7 therefore suggest that JNK and ATF2/7 may transcriptional activities. Using microarray be at least partially responsible for the apoptotic analysis we identified ATF2 target genes that arm of Myc activities and that this could may be involved in tumour suppression. In underlie the observed tumour suppressive meta-analysis of tumour expression data we activities of ATF2/7 in the mouse B lymphoma The Cell Regulation Lab is addressing molecular biological find that down-regulation of many ATF2/7 model. In an analysis of ATF2/7 dependent targets correlates with tumour status. transcription regulation we showed that questions centred around MAP kinase signalling pathways and transcriptional targets include other AP-1 downstream transcription factors, in particular members of the ATF2/7 roles in Myc transformed B cells and factors, including c-Jun and ATF3 as well as at B lymphoma least one member of BH3 domain containing AP-1 family. Over the years our group has identified and In samples of human B cell lymphoma cell lines, apoptotic regulators, Hrk (Walczynski et al., characterised components of these pathways involved in we found that ATF2 as well as JNK MAP kinase accepted for publication in Oncogene, 2013). are significantly up-regulated compared to development, homeostasis as well as cellular response to normal human B cell lines. This was particularly Oncogenic Ras regulated microRNAs growth and stress inducing stimuli. We also found that these the case in Burkitt’s lymphoma and other involved in suppression of transformation Group Leader lymphoma types, typically associated with the and drug induced apoptosis pathways have important and diverse roles in the initiation of activation of the c-Myc transcription factor. To Over the past few years it has become apparent Nic Jones tumorigenesis as well as in the response to therapeutic analyse the potential interaction between that non coding RNAs display important Associate Scientists activated c-Myc and the JNK-ATF2 signalling functions in the regulation of gene expression Wolfgang Breitwieser treatment. We have addressed these functions using pathway we developed a mouse model in and modulation of signalling pathways. In an Postdoctoral Fellows biochemical and genetic approaches and have employed which ATF2 and 7 were specifically deleted in approach to identify new mechanisms involved the B cell compartment and tested this in the in oncogenic Ras-mediated signalling, we Yujun Di genetic model systems including mouse and fission yeast. presence of B cell specific overexpression of identified a number of microRNAs that were Malgorzata Gozdecka Saki Kondo1 Importantly, these diverse model systems have shown a c-Myc (Eµ-Myc). As with the human B specifically down regulated in their expression in Hayley Thirkettle1 lymphoma samples, we found that mouse response to activated HRas. Upon further Jacek Walczynski1 remarkable degree of conservation in specific signalling lymphomas induced by Myc overexpression characterisation we found that a number of Scientific Officer pathways and have provided a powerful tool to address also showed strongly enhanced activation of these miRs, including miR99 and miR335 are JNK as well as of ATF2. Furthermore, B cell involved in the suppression of Hras mediated Steve Lyons fundamental questions about stress signalling. specific loss of ATF2/7 resulted in a significant transformation, for example by reducing colony Graduate Students acceleration of Eµ-Myc induced lymphoma formation in soft agar. Furthermore, ectopic Emily Holmes onset suggesting that the JNK-ATF2/7 pathway expression of miR335 leads to enhanced Alexander Thapa2 MAP kinases of the JNK and p38 family are Suppressive functions of ATF2/7 in is engaged in tumour suppressive activities in a apoptosis in response to genotoxic drugs such active in response to cytotoxic and genotoxic oncogenic Ras mediated cellular 1 left in 2012 Myc overexpression context. as cisplatin. To evaluate this role for miR335 in a stimuli, including ionising radiation, ROS, and transformation 2 clinically relevant setting we analysed ovarian joined in 2012 chemotherapeutic drugs. JNK and p38 can In tumorigenesis, ATF2 can exert pro- To further characterise these activities we tumour cell lines for the expression of miR335 activate apoptosis by directly modulating the tumorigenic but also anti-tumorigenic activities derived cell lines from Myc induced primary and found that while a number of these show mitochondrial apoptotic pathway as well as by depending on the tumour type (Gozdecka and lymphomas and induced the deletion of ATF2 in strong resistance to cisplatin induced cell death regulating the activities of downstream Breitwieser, 2012). Therefore one focus of our vitro using Cre/loxP mediated recombination. they also have very low expression of miR335. In transcription factors, including members of the research has been to decipher these context Here we found that while loss of ATF2/7 in B contrast re-expression of miR335 strongly AP-1 family. AP-1 is a dimeric complex dependent activities. In a current project we lymphoma cells did not affect growth in culture, re-sensitises cisplatin resistant cell lines to the composed of proteins belonging to the JUN, adapted a mouse model of hepatocellular they showed remarkably reduced levels of cisplatin-induced apoptosis. A typical mode of FOS, ATF, and MEF families of B-ZIP transcription carcinoma (HCC), whereby oncogenic HRas spontaneous apoptosis as well as reduced levels evasion to drug sensitivity by tumour cells is by factors. Different AP-1 complexes regulate a vast transformed hepatoblasts, proficient or deficient modifications in the methylation status of the number of target genes involved in cell growth, in ATF2/7 functions, are transplanted Figure 1 genome. Interestingly, we found that treatment differentiation, but also cell cycle arrest and orthotopically and develop primary liver Apoptotic (Caspase 3) staining of drug resistant cells with demethylating agents apoptosis. The transcription factors ATF2 and tumours. As a result, we found that ATF2/7 of Myc induced mouse lymphomas. led to enhanced expression of miR335 in ATF7 are highly homologous members of the double mutant hepatoblasts show a significantly correlation with their increased sensitisation to AP-1 family. Increasing evidence shows that stronger tendency to develop into HCC in cisplatin treatment. We therefore aim to further JNK- and p38-dependent pathways are recipient livers compared to ATF2 active test the possibility that at least one mechanism frequently modulated in tumours. Our long controls. We also found that active ATF2 induces of acquired drug resistance in tumours is by term goal has been to identify key biological cell death in transformed hepatoblasts in culture silencing the expression of microRNAs activities that are mediated by the JNK-ATF2/7 and reduces colony formation in soft agar. including miR335. We are also exploring the and p38-ATF2/7 signalling axes during cellular These activities appear to be dependent on the targets of mir335 involved in cisplatin induced stress and oncogenic transformation. activation of an upstream kinase (JNK) because cellular responses. expression of a phosphorylation deficient form Publications listed on page 69 22 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH CELL REGULATION 23

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    Figure 1 CELL SIGNALLING Multiple mechanisms exist to regulate Rac activity. The Rac www.paterson.man.ac.uk/cellsignalling GTPase cycles between inactive GDP-bound and active GTP- bound states. Rac activation is facilitated by the action of GEFs (such as Tiam1), which promote GDP dissociation from Rac and allow GTP to bind instead. Through the association with GAPs the intrinsic GTPase activity of Rac is accelerated thereby inactivating Rac. Through association with Tumour initiation and progression result from the inappropriate RhoGDIs Rac can be activity of intracellular signalling cascades. Rho-like GTPases sequestered in its inactive state. Activated Rac can also be are molecular switches in signalling pathways that regulate removed through ubiquitylation- induced degradation (mediated cell morphology, adhesion, motility, as well as cell cycle by HACE1 following a migration stimulus) or it can be maintained progression and survival. Data has emerged to directly following its modification by Role of the Rac activator Tiam1 in cell- Src-induced AJ disassembly and inhibited SUMO (mediated by PIAS3). implicate Rho proteins in tumourigenesis. We investigate cell adhesion. cell migration (Woodcock et al. Mol Cell. Tiam1 (for T-lymphoma invasion and metastasis 2009; 33: 639). the mechanisms by which certain regulators of the protein) is a Rac specific GEF. Mice deficient for Group Leader Rho-like GTPase Rac control cell cycle progression and Tiam1 are resistant to the formation of skin To better our understanding of the contribution Angeliki Malliri tumours. However, the few tumours arising in of Tiam1-Rac signalling to tumourigenesis, we cell adhesion and how their activities, as well as activity of these mice progressed more frequently to further investigated its function at cell-cell Postdoctoral Fellows Natalie Mack 2 Rac itself, are controlled. malignancy (Malliri et al., Nature 2002; 417: 867). adhesions. A screen we performed for Tiam1 Andrew Porter One mechanism by which Tiam1 and Rac interacting proteins identified β2-syntrophin as Lynsey Vaughan1 suppress malignant progression is through one of its binding partners. β2-syntrophin is a Helen Whalley Rac1 cycles between a GDP- and a GTP-bound mediated SUMOylation of Rac1 controls promoting cell–cell adhesion. Over-expression component of the dystroglycan adhesion state. When GTP-bound, it interacts with various Rac1-GTP levels and the ability of Rac1 to of activated Rac or Tiam1 promotes the complex. Our study (Mack et al. Nat Cell Biol. Scientific Officer effector molecules that elicit downstream stimulate lamellipodia, cell migration and formation of adherens junctions (AJs) and an 2012; 14: 1169) unearthed a novel role for this Gavin White responses. Multiple mechanisms control Rac1 invasion (Castillo-Lluva et al. Nat Cell Biol. epithelial-like phenotype in a number of complex in regulating the assembly of adherens Graduate Students activity including control of nucleotide binding 2010; 12:1078). mesenchymal cell lines (Malliri & Collard, Curr junctions (AJ) and tight junctions (TJ) and the Hadir Marei and hydrolysis by Guanine nucleotide Exchange Opin Cell Biol 2003; 15: 583). Moreover, Tiam1 is generation of apicobasal polarity through Erinn-Lee Ogg Factors (GEFs) and GTPase Activating Proteins Rac1 activity is also regulated through required for both the formation as well as controlling Tiam1-Rac signalling. The Chong Tan2 (GAPs) respectively, regulation of subcellular ubiquitylation and subsequent degradation. maintenance of cadherin-based adhesions mechanism we uncovered entails the Anna Woroniuk 1 localization and modulation of Rac1 protein However, the E3 ubiquitin ligase responsible for (Malliri et al., J Biol Chem 2004; 279: 30092). generation of a Rac activity gradient in the 1 joined in 2012 levels. More recently, regulation by post- Rac1 degradation following activation by a The oncoprotein Src, a non-receptor tyrosine membrane region encompassing these 2 left in 2012 translational modification has emerged as a migration stimulus was unknown. Recently, we kinase implicated in malignant progression, junctions, with lower Rac activity at apical TJ significant means of regulating Rac activity. identified this to be the tumour suppressor potently induces epithelial–mesenchymal and higher Rac activity sub-apically. This HACE1. We showed that HACE1 and Rac1 transition (EMT) by targeting AJs for disassembly. gradient depends upon the ability of β2- Post-translational modifications of Rac1 interaction is enhanced by HGF signalling and We recently showed that Src phosphorylates syntrophin to stimulate Tiam1-Rac signalling at during cell migration that HACE1 catalyses the poly-ubiquitylation of Tiam1 on tyrosine 384 predominantly at AJs the sub-apical end and of the polarity To gain further insight into the regulation of Rac Rac1 at lysine 147 following its activation by HGF, during the initial stages of Src-induced EMT determinant Par3 to inhibit Tiam1-Rac signalling during cell migration, we performed a screen resulting in its proteasomal degradation. triggering the localised degradation of Tiam1 at at the apical end. By targeting constitutively for proteins that interact with Rac following HACE1-depletion is accompanied by increased AJs by calpain proteases. Abrogating Tiam1 active Rac to TJs which disrupts the gradient, we treatment of cells with a motility-inducing total Rac1 levels and accumulation of Rac1 in phosphorylation and degradation suppressed demonstrated that the gradient of Rac activity is factor, Hepatocyte Growth Factor (HGF). This membrane ruffles. Moreover, HACE1-depletion required for optimal TJ assembly and the revealed the small ubiquitin-like modifier enhances cell migration independently of Figure 2 generation of apicobasal polarity. Finally, we Model depicting the differential (SUMO) E3-ligase, PIAS3, as a novel Rac growth factor stimulation, which may have localisations of Par-3 and found that reduced membrane β2-syntrophin interacting protein. PIAS3 interacts better with significance for malignant conversion. These β2-syntrophin and their correlates with human prostate cancer GTP-bound Rac and is required for increased findings identified HACE1 as an antagonist of differential effects on Tiam1-Rac progression. We conclude that β2-syntrophin Rac activation and optimal cell migration in cell migration through its ability to degrade activity at cell-cell junctions. and Par-3 finely-tune Rac activity along cell-cell response to HGF. Subsequently we active Rac1 (Castillo-Lluva et al. Oncogene junctions controlling TJ assembly and the demonstrated that Rac1 can be conjugated to 2012). Jointly the above two studies suggest that establishment of apicobasal polarity. SUMO-1 in response to HGF and that the SUMOylation and ubiquitylation of Rac1 act Furthermore, we propose that deregulation of GTP-bound form of Rac is a better substrate for coordinately to fine-tune Rac1 activity in β2-syntrophin, Par-3, or Tiam1, would disrupt SUMOylation. Furthermore, we identified migrating cells, promoting Rac activity at sites the Rac activity gradient, and in turn disrupt TJs non-consensus sites within the polybasic region where the cell membrane is advancing, while and apicobasal polarity, thereby promoting of Rac1 as the main location for SUMO antagonising Rac1 at sites where membrane tumourigenesis. conjugation. We demonstrated that PIAS3- protrusion needs to cease. Publications listed on page 70 24 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH CELL SIGNALLING 25

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    Kalena Marti Marti CLINICAL AND EXPERIMENTAL Robert Metcalf Project Project Name and Description Biomarkers Studied Danielle Shaw No PHARMACOLOGY Laura Cove Smith CEP073 CONVERT (Concurrent ONce-daily VErsus DNA analysis from whole blood samples. twice-daily RadioTherapy): A 2-arm www.paterson.man.ac.uk/cep Scientific Officers randomised controlled trial of concurrent CTC enumeration with assessment of Jenny Antonello1 Ki-67 and BCL-2 expression. Stephen Bramley1 chemo-radiotherapy comparing twice- Jessica Booth daily and once-daily radiotherapy Cytology on tumour biopsies. Damien Brown schedules in patients with limited stage Debbie Burt small cell lung cancer (SCLC) and good Fouziah Butt performance status. John Castle1 Samson Chinien CEP074 CHEMORES (Tumour Chemotherapy Genomic and proteomic analysis of Jakub Chudziak 1 Resistance): Biomarker analysis of patient tumour and serum/plasma CEP validates and implements pharmacodynamic, prognostic Martin Dawson Molecular mechanisms of drug resistance samples. and predictive biomarkers working in tandem with the Christie Olive Denneny Shital Dulabh in lung cancer. Enumeration of CTCs. Cancer Treatment Centre that incorporates one of the largest Suzanne Faulkner Joseph Halstead1 CEP153 RADAR (Resistance and Damage to Measurement of the following by ELISA Radiotherapy in Lung Cancer): Lung Cancer in patient plasma specimens: Ang1, early clinical trials units worldwide. This year we have focussed Grace Hampson Biomarkers of Response and Toxicity to Ang2, FGFb, HGF, PDGFbb, VEGFA, Cassandra Hodgkinson on enumeration and molecular analysis of circulating tumour Paul Kelly Radiotherapy. VEGFC, IL8, KGF, PlGF, VEGFR1, VEGFR2, Matthew Lancashire Tie2, M30, M65, EGF, E Selectin, VCAM1, cells; developed a suite of circulating nucleic acids biomarker Daniel Morris IL1b, Il6, IL10, IL12, TNFa, OPN, CA-IX, assays, examined a large panel on angiogenesis associated Karen Morris Jackie Pierce CYRFA. Group Leader circulating proteins at baseline in patients receiving drug Andrew Price Measurement of Ki-67 in FFPE tumour Caroline Dive Tony Price blocks by IHC. regimens containing Avastin and the associated requirement Lynsey Priest CEP155 MEKRT: Phase I trial of the MEK Inhibitor Measurement of K-RAS, pMAPK, & Ki-67 Deputy Group Leader Robert Sloane Ged Brady of biomarker statistics. Nigel Smith AZD6244 in combination with thoracic in tumour biopsy specimens using IHC. radiotherapy in NSCLC. Phase I Lead Graduate Students Assessment of circulating free DNA in Malcolm Ranson We have also built an expanded pipeline of The translational research in the CTC team is Danielle Potter blood specimens for K-RAS mutations. Shaun Villa Lung Cancer Lead ongoing clinical trials in lung cancer patients increasingly integrated with that in the Nucleic Measurement of the following in patient Fiona Blackhall incorporating circulating biomarkers. The Acids Biomarkers team as we seek to undertake Laboratory Manager plasma specimens using ELISA: M30, Biostatistics Lead highlight of 2012 was the award to Professor molecular profiling of purified CTCs. Martin Greaves M65, OPN, VEGF, PlGF, VEGF-R1 & R2. Andrew Renehan Dive of the prestigious Pasteur-Weizmann/ Scientific Assistant CEP209 An investigation of blood-borne biomarkers Enumeration of CTCs. Servier International Research Prize and Tribute The Nucleic Acids Biomarkers (NAB) Team Senior Staff Scientists Aileen Jardine of early stage lung cancer using differential for her laboratory’s research on non-invasive The CEP Biomarker Portfolio has expanded to Jonathan Tugwood1 analysis of pulmonary venous and arterial Stephen Walker2 biomarkers to aid the treatment of cancer include a Nucleic Acids Biomarkers (NAB) team Administration Assistants blood (with Dr Philip Crosbie). patients. led by CEP Deputy Ged Brady. Over the last year, Judith Thorpe2 CEP238 STOMP: Small cell lung cancer Trial of Enumeration of CTCs. Staff Scientists the NAB team has established a range of Lisa Waters1 Jeff Cummings Olaparib (AZD2281) as Maintenance Building a portfolio of lung cancer trials circulating biomarker assays including miRNA Plasma biomarkers (to be confirmed). Dominic Rothwell 1 joined in 2012 Programme. incorporating biomarkers (Lead: Fiona profiling of plasma samples and RNA profiling of Blackhall) single isolated cells. NAB assay development 2 left in 2012 cfDNA (to be confirmed). Clinical Lecturers We have expanded our lung cancer trial takes into consideration the requirement for CEP241 VanSel-1: A Cancer Research UK Phase I Enumeration of CTCs. Emma Dean Alastair Greystoke portfolio this year to evaluate newly validated simple sample processing amenable to dose escalation trial of the oral VEGFR and Depending on tissue available, Cliona Kirwan1 biomarker assays and in particular we have upcoming multi-site clinical trials. Projects using EGFR inhibitor, Vandetanib in combination Matthew Krebs measurement of the following markers initiated new collaborations within the CR-UK next generation sequencing (NGS) of paired with the oral MEK inhibitor, Selumetinib in in CTCs and/or tumour biopsy Experimental Cancer Medicine Centre (ECMC) lung cancer patients’ blood and tumour solid tumours (dose escalation) and NSCLC Associate Scientists specimens: Phospho ERK, Total ERK, Chris Morrow network (see Table 1). In collaboration with samples are underway within CEP and in (expansion cohort). Ki67, Phospho EGFR, Total EGFR, Kathryn Simpson Corrinne Faivre-Finn, we are examining the collaboration with Peter Campbell at the Cancer Phospho AKT, Total AKT. impact of radiotherapy on circulating tumour Genome Project group at the Wellcome Trust Service Manager Measurement of the following by ELISA cell (CTC) numbers in the CONVERT and MEKRT Sanger Institute. A major focus of the NAB team David Moore trials. Having demonstrated the high prevalence has been to establish routine molecular analysis in patient plasma specimens: VEGFR2, Postdoctoral Fellows of CTCs in small cell lung cancer (SCLC) - Hou of CTCs that complements and expands on VEGF-A, M30, M65. Mahmood Ayub et al JCO 2012, see research highlights - we are CTC analytical approaches established within Alison Backen Assessment of cfDNA for mutations in now evaluating their utility in several clinical CEP. Currently CEP makes extensive use of the Ivona Baricevic-Jones K-RAS & EGF-R. trials. In a new exciting collaboration with Veridex CellSearch system which delivers Becky Bola1 CEP263 MAPPING: Double blind randomised phase Enumeration of CTCs. Jian Mei Hou Immunogen and with a validated assay to reliable and informative enumeration of EpCam III study of maintenance Pazopanib versus Radoslaw Polanski measure CD56 (a neuroendocrine marker) in and Cytokeratin positive CTCs from patient placebo in NSCLC patients non progressive Cong Zhou SCLC CTCs, we are deploying this assay on the blood samples. Over the last few months the after first line chemotherapy. North Trial evaluating a CD56 targeted therapy. NAB team established a single cell isolation and Clinical Fellows Exciting data are emerging in collaboration with whole genome amplification (WGA) approach CEP264 ImmunoGen NORTH trial: The NORTH Enumeration of CTCs with assessment Kyaw Aung clinical trial is designed to assess the impact of CD56 expression. Louise Carter Dr Phil Crosbie on the detection of CTCs in the applicable to individual CTCs enriched by the pulmonary vein of patients with resectable Veridex CellSearch system. New collaborations on patient outcomes of adding the Leila Khoja non-small cell lung cancer (NSCLC). have also been established with companies experimental agent, IMGN901, to standard treatment for newly diagnosed SCLC. 26 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH CLINICAL AND EXPERIMENTAL PHARMACOLOGY 27

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    CLINICAL AND EXPERIMENTAL PHARMACOLOGY (CONTINUED) Debbie Burt and Jakob Chudziak using the DEPArrayTM platform for CTC detection and manipulation. significantly longer progression free survival developing a multi-marker (PFS) than seen with conventional treatment immunofluorescence assay to investigate the alone. The data also suggest that patients with levels of androgen receptor (AR) protein high levels of both Ang1 and Tie2 at baseline expression together with epithelial and should not be given Avastin. Further prospective mesenchymal markers in prostate cancer CTCs Figure 1 developing novel CTC/blood biomarker toxicity in those patients where benefit is trials are now under discussion to confirm the to evaluate CTC heterogeneity and inform on Schematic of the workflow used technology platforms; in particular the unlikely. ICON 7 was a randomised two-arm predictive biomarker utility of Ang1 and Tie2 for CTC utility for AR directed therapy. A second for isolation and genetic analysis acquisition of the Silicon Biosystems DEParray Phase III trial in which patients with advanced Avastin treatment and the TAG team will example is the development of serum of individual CRC CTCs. Images are of 5 putative CTCs stained for platform, which purifies single CTCs, has added ovarian cancer were treated either with conduct ‘reverse translation’ research to biomarker assays to support development of a cytokeratin (CK-PE) and DAPI. enormous strength to our ability to deliver our bevacizumab or placebo, in addition to standard investigate the molecular mechanism(s) that pan-Erb inhibitor. Highly sensitive ELISAs are Sanger sequencing data ambitious goals. The single cell isolation chemotherapy. Using the Aushon Biosystems underpin these clinical observations. enabling circulating levels of the ErbB ligands to obtained from each of the 5 approach has been applied to over 20 clinical (Boston, US) multiplex ELISA platform and CEP be measured in cancer patients’ plasma with the putative CTCs and WBCs from samples and has led to the identification of validated assays, we examined 15 proteins The CEP/AstraZeneca Serological Alliance hypothesis that the baseline levels of these the same patient is shown where red arrows indicate the location mutations found in CTCs and not in healthy associated with angiogenesis (Ang1, Ang2, Since 2006 an Alliance has been in place ligands may predict for response to inhibitors of of the heterozygous PIKC3A white blood cells (WBC) from the same patient FGFb, GCSF, HGF, IL8, KGF, PDGFbb, PlGF, Tie2, between AstraZeneca (AZ) and CEP to advise Erb pathway signalling. mutation observed. (Figure 1). Currently the single cell WGA VEGFA, VEGFC, VEGFD, VEGFR1 and VEGFR2) on, analyse, report, and interpret data obtained approach is being adapted to allow Next collected at baseline from a subset of 91 ICON7. on circulating biomarker assays validated for Our biomarker alliance was expanded and Generation Sequencing (NGS) of a large Real progress with biomarker statistics has been clinical use in CEP’s Good Clinical Practice renewed in September 2012 (£3.1M) under the number of clinically relevant tumour ‘driver’ made this year by Dr Andrew Renehan and Dr Laboratories (GCPL). The AZ/CEP Alliance has guidance of Carl Barrett (AZ Boston). The new genes. Cong Zhou in CEP along with the recent grown yearly to encompass a broad range of Alliance remit allows a more diverse range of recruitment to the University of Manchester of biomarkers (cell death, invasion, angiogenesis, biomarkers to be investigated, from nucleic acid Predictive biomarkers in ovarian cancer for Professor Carlo Benzuini. Together they CTCs, cfDNA) supporting the exploratory based work with Ged Brady’s NAB team, the anti-angiogenic drug Avastin conducted a thorough statistical analysis and biomarker requirements of AstraZeneca’s through tissue biomarkers, to further bespoke CEP works in tandem with the translational identified Ang1 and Tie2 as candidate predictive oncology portfolio. Over the past two years, in biomarker work including preclinical projects angiogenesis (TAG) group led by Professor biomarkers for Avastin in this patient cohort. The addition to an ongoing high throughput that map to our focus on SCLC. Gordon Jayson. One of the most important information provided by these two biomarkers biomarker analysis component, the Alliance has conundrums regarding anti-angiogenic translates to a clinical decision strategy that drawn upon the wider expertise of CEP to Publications listed on page 70 therapies such as Avastin is the absence of a Avastin should be added to conventional deliver several bespoke biomarkers for clinical predictive biomarker(s) to identify patients most chemotherapy for patients with high Ang1 levels trial deployment. As one example we are likely to benefit, and to avoid drug-induced and low Tie2 levels at baseline leading to 28 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH CLINICAL AND EXPERIMENTAL PHARMACOLOGY 29

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    cancer therapy. For this, we have been another evolutionary widespread module with DNA DAMAGE RESPONSE screening for proteins that have the ability to the capacity to bind poly(ADP-ribose) and we www.paterson.man.ac.uk/dnadamage respond to DNA damage in a manner that is recently identified several human macrodomain blocked by treatment with clinically relevant protein factors that are recruited to broken DNA PARP inhibitors such as olaparib. Our goal is to ends in a poly(ADP-ribose)-dependent manner. characterise some of the obtained candidate These include a histone H2A variant called proteins and elucidate their exact biochemical MacroH2A and several other uncharacterised functions in DNA repair, as well as their mode of macrodomain proteins. regulation in response to DNA damage. Recently, in screening for proteins with the Structural and functional analysis of ability to bind poly(ADP-ribose), we discovered a poly(ADP-ribose) glycohydrolase (PARG) and poly(ADP-ribose)-binding zinc finger motif its validation as a target for cell-permeable Many cancer therapy procedures, such as radiotherapy and (PBZ). PBZ is a structurally distinctive, atypical inhibitor design type of zinc finger that is associated with several Available data indicates that inhibiting PARG some types of chemotherapy, work by overwhelming the proteins involved in response to DNA damage might offer a promising and beneficial approach capacity of the cell to repair DNA damage, resulting in cell (Ahel et al, Nature, 2008). One of the human in the treatment of cancer and cardiovascular proteins containing a PBZ motif is a protein conditions. However, unlike the case of PARP death. Most rapidly dividing cells - cancer cells - are called Checkpoint protein with FHA and RING inhibitors, progress in developing permeable, preferentially affected by such treatments, providing the domains (CHFR). CHFR is an ubiquitin ligase small-molecule PARG inhibitors has been frequently inactivated in human epithelial limited, partly due to the lack of functional and opportunity to use DNA damaging agents to selectively kill tumours, which acts as a key regulator of the structural data for the human PARG protein. cancer cells. In addition, genomic instability is the driving force poorly understood early mitotic checkpoint that Recently, we solved the crystal structures of Group Leader transiently delays chromosome condensation several PARG enzymes from bacteria and lower of cancer development, which requires multiple DNA and nuclear envelope breakdown in response to eukaryotes, which gave the first insight into the Ivan Ahel mutations resulting in loss of cellular growth control. In order a variety of stresses. The elucidation of the basic principles of PARG structure and its Postdoctoral Fellows function of the PBZ motif gave us a vital clue to mechanism of catalysis (Slade et al, Nature, Dragana Ahel to accelerate the accumulation of genetic changes, cancers discover that the CHFR-dependent checkpoint 2011; Dunstan et al, Nat Commun, 2012) (Figure Benito Banos1 Rosa Morra often sacrifice specific DNA repair pathways. This can make is regulated by PARPs and that the PBZ motif in 1). These structures revealed that the PARG CHFR protein is critical for checkpoint activation. catalytic centre is a diverged type of Roko Zaja1 cancer cells additionally susceptible to DNA damaging agents Another PBZ-regulated protein we are studying macrodomain and demonstrated that they are Scientific Officer and/or to inhibitors that block alternative repair pathways. For is a protein called Aprataxin-PNK-like factor likely to prove useful in guiding structure-based Ria Weston (APLF). APLF uses tandem PBZ repeats for direct discovery of new classes of PARG inhibitors. these reasons, studying the protein components involved in interaction with poly(ADP-ribosyl)ated PARP1, Despite these advances, structural information Graduate Students Eva Barkauskaite the repair of damaged DNA has proven to be a valuable which allows APLF’s timely localisation to the on human PARG is still lacking. Our goal is to sites of DNA damage. We recently discovered solve the structures of human PARG in complex Michael Tallis strategy in searching for novel approaches and targets in that the role of APLF is to act as a histone with substrate analogues and inhibitors which in 1 joined in 2012 cancer therapy. chaperone to modulate chromatin structure combination with solution and cell biology and facilitate DNA repair reactions in response studies, should address the mechanism, to poly(ADP-ribose) signalling (Mehrotra et al, structure and regulation of human PARG, as well Poly(ADP-ribosyl)ation in regulation of platform for specific recruitment and scaffolding Mol Cell, 2011). as providing a foundation for the development DNA repair of DNA repair complexes. In addition, the of small, cell-permeable PARG inhibitors. Poly(ADP-ribosyl)ation is a post-translational damage-induced poly(ADP-ribosyl)ation has a Another class of DNA damage response protein modification that controls several role in relaxation of chromatin structure and in proteins on which we focus our research is the Publications listed on page 71 nuclear processes known to be important for apoptotic signalling. The recent development of macrodomain proteins. The macrodomain is genome stability, including DNA repair, potent PARP inhibitors provides powerful tools regulation of chromatin structure, cell cycle to study pathways regulated by poly(ADP- Figure 1 checkpoint activity, transcription, apoptosis and ribose), as well as providing a promising novel Active site of Tetrahymena mitosis. Poly(ADP-ribose) is a highly negatively class of drugs for cancer treatment. Specifically, thermophila PARG enzyme with charged polymer that is formed from repeating selective inhibition of the DNA break repair bound ADP-ribose (in red). ADP-ribose units linked via glycosidic ribose- pathway using permeable PARP inhibitors has ribose bonds, and is synthesised by the proven highly effective against certain breast poly(ADP-ribose) polymerase (PARP) family of and ovarian cancers (Bryant et al, Nature 2005). enzymes using a vital cellular cofactor NAD+ as Thus, understanding the molecular basis of a substrate. The reversion of poly(ADP-ribosyl) poly(ADP-ribose)-dependent DNA repair ation is performed by the hydrolytic action of an processes is likely of vital importance for enzyme called poly(ADP-ribose) glycohydrolase selecting and developing efficient therapies. (PARG), which specifically targets ribose-ribose bonds and cleaves poly(ADP-ribose) into Identification and characterisation of novel ADP-ribose monomers. The role of poly(ADP- poly(ADP-ribose)-regulated factors ribosyl)ation is best understood in the regulation Our laboratory is particularly interested in of DNA repair, which is controlled by the three identification of novel DNA repair pathways and PARPs responsive to DNA strand breaks (PARP1, protein functions regulated by poly(ADP-ribosyl) PARP2 and PARP3). Poly(ADP-ribosyl)ation ation in order to identify components of these arising at the sites of damaged DNA serves as a pathways that can be exploited as targets for 30 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH DNA DAMAGE RESPONSE 31

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    Figure 1 Project portfolio DRUG DISCOVERY Nicola Hamilton working at the Our current portfolio includes one lead Echo access station www.paterson.man.ac.uk/drugdiscovery identification project (DNA repair) and four hit identification projects against metabolic, epigenetic, redox modulation and oncogene signalling targets. For the most advanced project, HTS screening has identified hit compounds whose in vitro potency has been improved a thousand fold by our chemists. The pharmacology of these more potent compounds is currently being explored in cells, both internally and through key expert During 2012 we have further enhanced the Drug Discovery collaborators. Additionally, another of these projects is close to meeting our requirements team in key areas, specifically by developing our bioinformatics for progression to the lead identification stage. platform and strengthening our chemistry efforts. Most In support of all of our projects, we have initiated To address this question, and to investigate a range of target biology and technology- significantly, we have progressed one of our DNA repair possible early hit matter, we have invested time related collaborations both within and beyond projects into the lead identification stage, while the rest of our in developing a fragment-based ligandability the MCRC. assessment of novel targets. In this approach, portfolio has also advanced. In addition we currently have four we screen our fragment library of around 1200 Cancer Research UK hit-to-lead projects aimed at a variety of cancer targets. These compounds (kindly donated by our sister drug We remain actively involved in the broader Group Leader discovery unit at the Beatson Institute) against Cancer Research UK drug discovery activities activities are underpinned by multiple collaborations both novel targets in a representative biological assay and interact with most of the Cancer Research Donald Ogilvie within and beyond the Manchester Cancer Research Centre. system. The resultant hit profiles allow us to UK drug discovery units. During the summer of Head of Chemistry assess the likelihood of finding new hit matter 2012 we underwent a successful annual review Allan Jordan against the target. Those with higher hit rates with the Drug Discovery Advisory Group. People issues. All of this combines to make us more and with a unique “fingerprint” of hits (as Head of Bioscience One of the most challenging aspects of drug efficient and effective in our search for new depicted in Figure 2) are proposed to be more The future Ian Waddell discovery is target selection. We have therefore drugs delivering patient benefit. amenable to finding potent and selective hits During 2012 we have expanded our team and Chemists strengthened our links to Crispin Miller’s Applied through additional hit finding approaches such progressed our project portfolio into the lead Ali Raoof Computational Biology and Bioinformatics Drug discovery targets as High Throughput Screening (HTS). Through identification stage. Increasingly, new project Alison McGonagle group by jointly recruiting Phil Chapman, a We continue to review many cancer drug target this we intend to judge the relative chemical risk opportunities are arising out of our Amanda Lyons skilled bioinformatician with drug hunting opportunities and, during 2012, have worked of new projects, compared to those already collaborations with Paterson Institute scientists. Bohdan Waszkowycz experience. Phil will focus on the target closely with several Paterson Institute Group under active investigation. We have spent some In 2013 we look forward to progressing at least Colin Hutton1 Daniel Mould selection process and will help build our Leaders (John Brognard, Tim Somervaille, Ivan time building confidence in this approach by one more internal project to the next stage of Eleanor French target-related interactions with groups inside Ahel and Karim Labib) in the identification, comparing our ligandability assessments the drug discovery process and maintaining our James Hitchin the Paterson Institute and with other Cancer validation and prosecution of putative drug against our historical successes (and failures) focus on the ones most likely to deliver patient Kate Smith Research UK funded drug discovery groups. As targets. In 2012 we have also focussed on with HTS and are ready to deploy this platform, benefit We would also hope to announce at Kristen Goldberg1 our portfolio expands we have further expanding our interaction with industrial drug to make real decisions as to whether new least one partnership with a large Niall Hamilton Rebecca Newton strengthened our synthetic and medicinal hunting companies. By collaborating early with targets displace those in our existing portfolio, pharmaceutical company. Stuart Jones chemistry team by welcoming Kristin and Colin, large and medium-sized pharmaceutical early in 2013. two new team members with significant companies and within the Biotech arena we Publications listed on page 71 Bioscientists industry experience. We have also said goodbye hope to find partners who will help us take our Alex Boakes to Laura, one of our synthetic chemists who left projects through the development stage and Dominic James us to pursue a different career path. deliver benefit to patients more quickly, Figure 2 Emma Fairweather “Ligandability” profiling of our Gemma Hopkins leveraging extra value from our existing funding. current active and early portfolio. Graeme Thomson Infrastructure Each bar in the heatmap Helen Small In terms of infrastructure, the biggest change Hit finding represents a fragment “hit” Mandy Watson has been the addition of an Access Workstation Once a target has been selected for drug against the target. Light green Mark Cockerill to our existing Echo acoustic dispensing discovery the next stage is to try and identify bars represent weaker hits and Nicola Hamilton darker bars represent the Nikki March platform. The Access system (shown in Figure 1) prototype small molecules, or “hits”, that interact stronger hits. This allows a fast, Phil Chapman1,2 has allowed us to fully automate compound and with the target molecule. However, not all visual representation of hit Samantha Fritzl reagent dispensing for our in vitro and cellular interesting oncology targets are amenable to patterns, to determine outline Sarah Holt assays. The advantages of this increased interaction with small molecules. Whilst certain “ligandability”. For example, 1 efficiency are several fold; first the accurate target classes are known to be hard to drug, closely matching our joined in 2012 experimental observations, our 2 joint with ACBB dispensing allows us to save on valuable such as transcription factors, for many of the epigenetics and DNA repair compounds and expensive reagents, secondly it targets we discuss with local group leaders, targets appear to be more gives us the ability to effectively screen medium there is no prior precedent in terms of chemically tractable than those sized (15-20K) compound libraries. Most anticipated “ligandability”, i.e. whether a small in our dehydrogenase areas of importantly, the system frees our highly skilled molecule can interact with the target in a potent interest. biologists from routine tasks allowing them to and selective fashion in order to cause the concentrate on more challenging biological desired biological response. 32 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH DRUG DISCOVERY 33

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    IMMUNOLOGY We have now shown that in 5T4 positive SKOV-3 human ovarian tumour cells, the canonical Wnt www.paterson.man.ac.uk/immunology pathway is inhibited and the non-canonical Wnt pathway is active, potentiated by Dkk-1. In SKOV-3 5T4 KD cells, the levels of secreted Dkk-1 and phosphorylated JNK are reduced whereas β-catenin-dependent signalling and levels of phosphorylated LRP6 are increased. We have developed a mouse model which will enable us to determine whether this perceived Figure 1 that functional CXCR4 and 5T4 interactions may “switch” in Wnt pathway activity has an effect on The 5T4 intracellular (IC) region be achieved via additional molecules, likely in vivo tumour growth and metastasis. Since the is necessary for the dendritic We have established that 5T4 oncofoetal glycoprotein arborisation. A-C, Lentiviral including actin binding proteins. Consistent with 5T4 intracellular domain is necessary and this, 5T4 is found extensively in clathrin-coated sufficient for the sensory input-dependent expression is associated with epithelial mesenchymal vectors carrying EYFP ( A ), 5T4-IRES-EYFP (B) and 5T4-ΔIC- pits, where it could form such a functional dendritic shaping in the 5T4+ interneuron transition, CXCL12 chemokine and Wnt signalling pathway IRES-EYFP (C) under the control of CMV promoter and expressed complex with CXCR4 prior to ligand stimulation granule cells of the olfactory bulb (Figure 1), we with CXCL12. 5T4 influence on chemotaxis was speculate that it may interact with Wnt signalling modulation in embryonic and human cancer cells. 5T4 in interneurons in the olfactory further investigated exploiting 5T4 positive molecules to regulate the dendritic arborisation bulb. Expression of the 5T4 promotes CXCL12/CXCR4 chemotaxis but when absent, construct gave more branched (Sup5T4) and negative (Sup) sub-lines (Yoshihara et al 2012). dendrites in the interneurons established from the SupB15 B-acute CXCL12 uses the alternative receptor CXCR7 which promotes than the control EYFP, while lymphoblastic leukaemia. Only the Sup5T4 cells Gene expression profiling and development proliferation or anti-apoptosis. 5T4 also inhibits Wnt/β-catenin expression of the IC domain- deletion construct (5T4-ΔIC) exhibit chemotaxis to CXCL12 and this of a cell culture model of pseudomyxoma Group Leader correlated with the ability of Sup5T4 cells to peritonei (PMP) canonical while concomitantly activating the non-canonical showed a similar level of desensitise and internalise CXCR4 more slowly PMP is a rare neoplastic process characterised Peter L. Stern branching numbers in the Wnt signaling pathway associated with increased motility. It is dendrites to the control EYFP. and to a lesser extent than Sup cells, leading to by progressive intra-abdominal dissemination of Postdoctoral Fellows IRES; internal ribosome entry prolonged signalling from the receptor (Castro mucinous tumour, and generally considered Owen McGinn likely that the integrated 5T4 regulation of these pathways acts site, EYFP; enhanced yellow et al 2012). resistant to systemic chemotherapy. In fluorescent protein. Rasilaben Vaghjiani2 Julie Brazzatti to promote cancer spread as well as functional migration in collaboration with Sarah O’Dwyer and Andrew 5T4 inhibits canonical but favours non- Renehan (Peritoneal Tumour Service) and also Darren Roberts3 development. New 5T4 antibody based therapies are in canonical Wnt signalling in human supported by NORD/Christie Trust Charitable Clinical Fellow development to target this functional tumour associated cancer cells funds, we have determined the gene expression Saladin Sawan In the Wnt canonical pathway, a Wnt ligand profile of PMP in comparison to normal colonic molecule. binds to a Frizzled receptor/LRP5/6 complex mucosa and established immortalised cell lines Scientific Officers leading to translocation of β-catenin from the suitable for laboratory based investigations into Jian Li2 Cheryl Petit 1 membrane to the nucleus where it regulates the PMP. Primary cultures of cells from PMP biopsies 5T4 and CXCR4/ CXCR7 mediated biological of 5T4 versus CXCR7 predictive of specific expression of target genes involved in cell cycle were established and subsequently responses in human cancer cells biological responses to CXCL12. Furthermore Graduate Student regulation through partnerships with TCF/LEF. immortalised with a SV40 T antigen lentiviral Georgi Marinov CXCL12 is a pleiotropic chemokine capable of 5T4 KD in 5T4 positive SCLC cells led to loss of This pathway is often mutated in cancer, leading vector. They demonstrate epithelial eliciting multiple signal transduction cascades chemotactic responsiveness (McGinn et al to oncogenic transformation and increased morphology, expression of mucin 2, cytokeratin Undergraduate Students and functions, via interaction with either CXCR4 2012). Alteration of the receptor preference Eleni Panagioti proliferation. The non-canonical pathways do 20 and other PMP markers. For expression or CXCR7. Factors that determine CXCL12 equilibrium of CXCL12 could dramatically Cathrine Richnagel not involve β-catenin signalling and are profiling, cells from PMP and normal colonic receptor preference, intracellular signalling change cellular behaviour in response to the activated by Wnt ligands binding to the Frizzled epithelium were harvested from each of three 1 joined in 2012 route and biological response, are poorly chemokine. In a primary tumour, 5T4 surface or ROR family receptors, producing a calcium fresh frozen surgical biopsies using laser capture 2 understood but are of central importance in the expression by cells at the periphery would left in 2012 flux in the Ca2+ pathway and/or activating microscopy. Isolated RNA was hybridised to context of therapeutic intervention of the provide for chemotaxis to CXCL12 secreting 3 with Andrew Renehan downstream effectors such as JNK in the planar Affymetrix Human Exon 1.0 ST arrays and CXCL12 axis in multiple disease states. We have endothelial cells and metastatic spread whereas cell polarity (PCP) pathway. The non-canonical differentially expressed genes identified. In recently demonstrated that 5T4 oncofoetal in the centre of the tumour, preferential CXCR7 pathway regulates gene expression through comparison to normal colonic mucosa, 34 and glycoprotein facilitates functional CXCL12/ expression could detect lower levels of NFAT and actin cytoskeleton rearrangement 27 genes were significantly altered, at least two CXCR4 mediated chemotaxis in mouse chemokine and promote cell growth. leading to increased motility. Activation of log2 fold lower or higher in PMP respectively (p< embryonic cells. Using wild type (WT) and 5T4 non-canonical signalling, through the 0.05 after adjustment for multiple testing). Many knockout (5T4KO) murine embryonic fibroblasts 5T4 and CXCR4 trafficking archetype non-canonical ligand Wnt5a or of these down- or upregulated genes have been (MEFs), we have now established that CXCL12 Investigation of the trafficking mechanisms of through disregulation of the canonical Wnt associated with cancer biology (20, including 5 binding to CXCR4 activates both the ERK and 5T4 and CXCR4 in E6/E7 immortalised MEFs suppressor Dkk-1, is hypothesised to lead to a tumour suppressors), metabolism/ion transport AKT pathways within minutes, but while these suggested that the two molecules employ more invasive cancer phenotype; increased (11) or cell migration (5). Several of these genes pathways are intact they are non-functional in distinct trafficking pathways for transport to the serum levels of these proteins has been are current targets for drug development. The 5T4KO cells treated with CXCL12. Importantly, in cell surface, constitutive and ligand induced correlated with poor cancer survival outcomes. PMP gene profiles are being validated by various the absence of 5T4 expression, CXCR7 is endocytosis/turnover. 5T4 shows dependence We have previously shown in zebrafish expression analyses in primary biopsies as well upregulated and becomes the predominant on intact microtubules and actin cytoskeleton embryogenesis that 5T4 expression acts as an as by using our immortalised PMP cell lines. This receptor for CXCL12, activating a distinct signal for endocytosis, whereas CXCR4 requires intact inhibitor of the canonical Wnt pathway in work has provided important tools for transduction pathway with slower kinetics microtubules for expression at the cell surface. Wnt-receiving cells (Dev. Cell 21: 1129-1143, preclinical development of improved therapies involving transactivation of the EGFR, eliciting By overexpressing suitably tagged 5T4 and 2011). 5T4 interacts with LRP6 and interferes for PMP. proliferation rather than chemotaxis. Consistent CXCR4 molecules in 293T cells, FRET with Wnt-dependent internalisation of LRP6, with these observations we have identified 5T4/ techniques were used to investigate their without affecting LRP/Frizzled interaction. 5T4 Publications listed on page 71 CXCR7 reciprocity in human small cell lung molecular interactions. Together with the competition with Dkk1 for LRP6 binding carcinoma (SCLC) cell lines with the expression results from a proteomics study it seems likely provides Dkk1 to help activate the non- 34 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH canonical Wnt/PCP pathway. IMMUNOLOGY 35

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    Figure 1 Figure 2 Figure 3 INOSITIDE LABORATORY www.paterson.man.ac.uk/inositide Phosphoinositides are a family of lipid second messengers that are regulated in response to environmental changes by a Figure 1 The phosphatidylinositol-5- to undergo an epithelial to mesenchymal modifications to regulate transcription and transition (EMT). This transition is important for chromatin structure. We found that the PHD network of kinases and phosphatases. Alterations in phosphate kinase (PIP4K) and tumour cells to leave the primary tumour site finger of TAF3, a component of the basal phosphatidylinositol-4- phosphoinositide levels can regulate many different cancer- phosphate kinase PIP5K and to metastasise to distant organs. In other transcription complex that regulates pathway contribute to cell types targeted RNAi studies indicate that transcription and cell differentiation, interacts relevant pathways including cell survival, proliferation, phosphatidylinositol(4,5) decreased expression of PIP4Kα specifically with phosphoinositides. We identified mutants bisphosphate (PtdIns(4,5) P2) migration, cell substratum interactions and transcription. In synthesis, however the major reduces tumour cell growth, while having a that no longer interact with PtdIns5P and used relatively minor effect on normal cell growth. these to demonstrate that changes in nuclear cancer cells PtdIns(4,5)P2 is at the heart of phosphoinositide synthetic pathway is likely through PIP5K. PIP4K Our studies are aimed at unravelling how PtdIns5P target the transcription of a subset of Group Leader signalling and can be synthesised by two different kinase regulates the levels of expression of different isoforms of PIP4K TAF3 regulated genes through its interaction Nullin Divecha phosphatidylinositol-5- controls cell fate and if specific inhibitors of with the PHD finger. Mutants that do not interact pathways (Figure 1). De-regulation of the PIP5K and PIP4K phosphate (PtdIns5P). PIP4K might be useful to target cancer cell with PtdIns5P are still able to interact with Associate Scientist Diacylglycerol (DAG) activates David Jones pathway leads to different outcomes in cancer signalling protein kinase C (PKC) which growth. components of the basal transcription regulates the phosphorylation machinery and to interact with modified histone Postdoctoral Fellows suggesting that each pathway also controls specific and intracellular activity of PIP5K. How PIP4Ks signal is still not clear, and we tails. We are using TAF3 as a model to Maria Carla Motta downstream targets. We expect that an inhibitor of PIP5K will inhibit both the PI3K hypothesised that PtdIns5P is a likely key understand how changes in nuclear PtdIns5P Iman van den Bout 1 signalling intermediate controlled by PIP4K to directly regulate gene transcription. and the Phospholipase C (PLC) regulate specific downstream pathways. Scientific Officer pathway. Yvette Bultsma PIP5Ks and PtdIns(4,5)P2 to treatment of cells with phorbol ester (a PKC Oxidative signalling plays a key role in aging and Measuring phosphoinositides and their PtdIns(4,5)P2 is present in the plasma membrane activator), after energy depletion or hydrogen Figure 2 cancer, and in response to oxidative stress, cells interaction with proteins Graduate Students and in the nucleus and can be synthesised by peroxide (H2O2) treatment. Specific inhibitors of Diagram showing how glucose engage adaptive responses in order to control The Inositide Laboratory has developed Julian Blaser2 deprivation and oxidative stress cell fate. We discovered that oxidative stress strategies to be able to measure two different families of kinases utilising two kinase pathways defined a stress dependent Rebecca Foulger regulate S413 phosphorylation different substrates (Figure 1). There are three pathway that requires the activity of the cellular leads to a rapid and reversible increase in the phosphoinositides in mammalian cells and Willem-Jan Keune1 leading to a decrease in the Lilly Sommer1 active isoforms of PIP5K, α, β and γ which all energy sensor AMP-Kinase (AMPK) and PKC to kinase activity of PIP5Kβ. PKC levels of PtdIns5P and that PtdIns5P signalling tissues and in model organisms such as localise to the plasma membrane, although regulate S413 phosphorylation. Furthermore we directly phosphorylates PIP5Kβ. impinges on the survival capacity of cell. The zebrafish and Drosophila. In collaboration with 1 left in 2012 specific isoforms also localise to other discovered that PKC can directly phosphorylate AICAR is a direct activator of PKB, FOXO and NRF2 pathways are Professor Martin Lowe (University of AMPK leading to enhanced S413 evolutionarily highly conserved regulators that Manchester) we showed that reduced 2 co-supervised with subcellular compartments, such as the golgi S413. Studies using phosphomimetic mutations phosphorylation. Tim Somervaille (PIP5Kβ), the nucleus (PIP5Kα and γ), focal revealed that unlike Rac and PtdIns(4,5)P2 , control responses to oxidative insults. Oxidative expression of the OCRL gene led to an increase adhesions (PIP5Kγ) and the cytokinetic furrow which regulate the localisation of PIP5Kβ, S413 Figure 3 stress induced increases in PtdIns5P maintain in PtdIns(4,5)P2 in fish. Mutations in OCRL, a (PIP5Kα). The targeting and regulation of PIP5Ks phosphorylation decreases PIP5Kβ activity both Oxidative stress rapidly and PKB activation and increase cell survival. phosphoinositide phosphatase, in humans lead are still not exactly understood. Previously, we in vitro and PtdIns(4,5)P2 synthesis in vivo. Our reversibly increases the levels of Increased PtdIns5P also stimulates the to a rare X-linked recessive disorder with life PtdIns5P. PtdIns5P stimulates expression of genes downstream of the NRF2 expectancy rarely exceeding 40. The identified the small molecular weight G protein studies suggest that S413 phosphorylation is a the levels of PtdIns(3,4,5) P 3 and Rac and the plasma membrane level of critical switch which regulates PtdIns(4,5)P2 pathway that modulate the accumulation of development of these techniques will enable PtdIns(3,4) P2 which in turn PtdIns(4,5)P2 as critical components that synthesis in response to the cells energy status control PKB activation and cell detrimental reactive oxygen species (ROS) in further analysis of phosphoinositides in cancer coordinate the localisation of PIP5K to the and in response to oxidative stress (Figure 2). survival. PtdIns5P also enhances response to oxidative stress. We found that cells relevant zebrafish models. Over the years we plasma membrane. Mass spectrometric the transcription of genes that that have higher levels of PtdIns5P have lower have also developed novel techniques to control the accumulation of analysis of PIP5Kβ identified twelve PIP4K and PtdIns5P accumulation of toxic ROS. Stress induced analyse the interaction of phosphoinositides toxic reactive oxygen species phosphorylation sites on PIP5Kβ, two of which There are three isoforms of PIP4Ks of which α is (ROS). This may be through PtdIns5P appears to regulate both cell survival with protein domains. This year we collaborated are on tyrosine residues, nine on serine and one cytosolic, β is cytosolic and nuclear and γ regulation of the NRF2 protein. and management of ROS levels, possibly with Dr. Mark Petronski’s group (LRI, Clare Hall on a threonine residue revealing potential novel localises to internal membrane compartments. Together these responses through the regulation of two distinct Laboratories) who identified a novel protein that pathways that regulate PIP5Kβ. All the identified We developed a specific antibody to PIP4Kβ and represent an adaptive change to subcellular targets: PKB in the cytoplasm and in controls cell abscission during mitosis. The C2 oxidative stress. the nucleus, NRF2-mediated gene transcription. domain within this protein targets it to the sites are conserved between human and mouse interrogated tissue microarrays of advanced PIP5Kβ while six are present in Drosophila but human breast tumour samples. We show that membrane and mutations in the C2 domain only one is present in the yeast homologue. down-regulation of PIP4Kβ correlates with Using lipid affinity purification and mass that ablate membrane interaction also attenuate The majority of phosphosites that we detected worse overall patient survival (with Professor spectrometry we have identified proteins that progression through mitosis. We showed that are present in the C-terminal domain of the Goran Landberg, University of Manchester). interact with and therefore function as the C2 domain confers interaction with the PIP5Kβ isoform. We developed an antibody that Studies in human breast tumour cell lines show downstream targets for PtdIns5P signalling such phosphoinositides PtdIns4P and PtdIns(4,5)P2 specifically recognises S413 phosphorylation that down-regulation of PIP4Kβ expression as PHD (Plant Homeo Domain) finger and that mutation within this domain ablates its and together with mutants that mimic or block decreases the expression of the tumour containing proteins. The PHD finger is a interaction with phosphoinositides. the phosphorylation of S413, we showed that suppressor protein E-cadherin, which regulates cross-braced Zinc finger that can interact with, S413 phosphorylation is increased in response cell adhesion, and increases the cells propensity and decode changes in, histone tail Publications listed on page 72 36 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH INOSITIDE LABORATORY 37

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    Figure 1 - Domain structure LEUKAEMIA BIOLOGY of LSD1 A) Linear representation and www.paterson.man.ac.uk/leukaemia B) surface structure of LSD1 highlighting the SWIRM domain (orange), the amine oxidase domains (green) and the tower domain (blue). Image (B) was generated using PyMOL (v1.5) based on X-ray structure PDB 2DW4. Figure 2 - Protein components of the EP400-centred complex and interacting proteins Figure 1 Figure 2 High throughput sequencing analysis of haematological Chr – chromodomain; Br – malignancy genomes has revealed that mutations in genes bromodomain; SANT – histone binding module; SWI/SNF – Foundation Trust, an option now being actively and perhaps counterintuitive role for the EP400 explored by our group in a formal collaboration complex in leukaemia cells: it prevents excess coding for epigenetic regulators recur frequently. These and ATPase nucleosome remodelling activity; PHD – with Oryzon Genomics. accumulation of MYC in transformed AML cells other observations have emphasised the critical importance of plant homology domain; HAT - through promotion of MYC turnover. histone acetyltransferase; PI-3K Identification of genes and cellular pathways the structure and function of chromatin in the biology of blood – phosphoinositide-3-kinase. which are required for the proliferation or To determine why this was observed in AML cancers, and hold promise for the development of epigenetic survival of LSC, but which are of less importance cells and not normal HSPC, we performed in normal HSPC is key to the development of chromatin immunoprecipitation followed by therapies. Using microarray data from a mouse model of novel leukaemia-selective therapies. In separate next-generation sequencing (ChIPseq) using Group Leader human acute myeloid leukaemia (AML), we identified the work, and in keeping with the increasing antibodies raised against EPC1 and MYC. These Tim appreciation of the critical role in leukaemia of studies were performed with the assistance of histone demethylase LSD1 as a candidate regulator of proteins that regulate the structure and function Yvonne Hey in the Molecular Biology Core Somervaille leukaemia stem cell (LSC) potential. In collaboration with the of chromatin, Xu Huang performed a lentiviral Facility, and Yaoyong Li and Crispin Miller in the Postdoctoral Fellows screen in human AML cells, targeting some 280 Applied Computational Biology and Xu Huang Drug Discovery Unit, we synthesised small molecule inhibitors genes coding for chromatin regulators for Bioinformatics Group. The two proteins James Lynch of LSD1 and demonstrated their in vitro and in vivo efficacy in knockdown. In addition to MLL itself and menin, exhibited strong genome wide co-localisation which is a critical oncogenic co-factor for MLL at promoters in proportion to gene expression Clinical Fellows inducing LSC differentiation of both murine and primary oncogene mediated transformation, the screen in AML cells, but not in normal HSPC, Brigit Greystoke Daniel Wiseman1 human leukaemia cells. identified EPC1 and EPC2 (for enhancer of suggesting a strong physical co-localisation in polycomb) as essential to prevent apoptosis of the former, but not the latter, cell setting. In Scientific Officer AML stem cells. EPC is conserved and essential separate ChIPseq analyses we have also Gary Spencer LSD1 (also known as KDM1A) (Figure 1) was the some decades. However, its relatively low in yeast, fly and mouse and its gene product discovered a defect in histone H3 and H4 tail Graduate Students first histone demethylase discovered and is a selectivity and potency versus LSD1 render it forms part of the EP400 chromatin regulatory acetylation at promoter regions in AML cells William Harris2 member of the flavin adenine dinucleotide inappropriate for clinical trials. complex, variants of which include the TIP60 which is not observed in normal HSPC. Indeed Julian Blaser3 (FAD)-dependent amine oxidase family of histone acetyltransferase complex and a MYC the genome wide distribution of EPC1 in AML Filippo Ciceri demethylases. It is found associated with protein To address this issue, and in collaboration with binding complex. Given the lack of information cells was a virtual mirror image of that of the Tim Somerville complexes that function as repressors of the Drug Discovery Unit at the Paterson as to any role for EPC in normal or malignant histone H4 lysine 5 acetylation mark, raising the 1 joined in 2012 transcription, including CoREST and NuRD and Institute, we synthesised some novel haematopoiesis, we investigated this novel possibility that EP400 complex components act 2 demethylates monomethyl- and dimethyl- tranylcypromine analogues recently reported in dependency further. Intriguingly, while at promoters in MLL leukaemia cells to inhibit left in 2012 3 histone H3 lysine 4 (H3K4Me1 & H3K4Me2) the patent literature by Oryzon Genomics, a knockdown of Epc1 or Epc2 induced apoptosis histone acetyltransferase activity. This is relevant co-supervised with Nullin Divecha marks, which are associated with active Spanish biotechnology company. These of LSC, the functional potential of Epc because, when EPC1 is knocked down, the transcription states. It is also found in complexes compounds exhibit significantly higher potency knockdown normal HSPC was spared. Analyses version of MYC that accumulates is significantly associated with active transcription and may and selectivity versus LSD1 than the parental of Epc knockdown AML cells using exon arrays, acetylated. Indeed, double knockdown of EPC1 demethylate monomethyl- and dimethyl- compound tranylcypromine. We found that in performed with the assistance of the Molecular and the histone acetyltransferase GCN5 histone H3 lysine 9 (H3K9Me1 & H3K9Me2) vitro and in vivo pharmacologic targeting of Biology Core Facility, revealed a transcriptional reduced accumulation of MYC and marks, which are associated with repressed LSD1 in AML cells using these analogues, which signature of MYC activation prior to induction of ameliorated apoptosis. transcriptional states. Having identified are active in the nanomolar range, phenocopied apoptosis. This prompted analysis of MYC expression of Lsd1 as correlated with LSC Lsd1 knockdown in both murine and primary protein levels in Epc knockdown AML cells Our data establish EPC1 and EPC2 as potential in a mouse model of MLL leukaemia, human AML cells exhibiting MLL translocations. which revealed robust accumulation of MYC 48 components of a complex which serves to William Harris performed Lsd1 knockdown By contrast, once again, the clonogenic and hours following initiation of knockdown. MYC is prevent MYC accumulation in myeloid experiments in murine MLL-AF9 AML cells and repopulating potential of normal HSPC was a key transcription factor that acts genome wide leukaemia cells and their sensitisation to discovered that leukaemia cells underwent spared. Our data establish LSD1 as a key effector as an amplifier of the transcription of expressed apoptosis, thus sustaining oncogenic potential differentiation and apoptosis whereas normal of the differentiation block in MLL leukaemia genes. Next, to determine whether regulation of (Figure 2). Therapeutic targeting of the EPC/ haematopoietic stem and progenitor cells which may be selectively targeted to MYC turnover was dependent solely on EPC, or EP400 complex may facilitate restoration of the (HSPC) were spared. These results were therapeutic effect in myeloid leukaemias is instead dependent on multiple members of natural tumour suppressor mechanisms that phenocopied using the monoamine oxidase exhibiting translocations targeting the mixed the EP400 complex, knockdown of other prevent cellular transformation by MYC. This inhibitor tranylcypromine, which inhibits the lineage leukaemia gene. This work, which was complex members was performed. In each work will be published in 2013. activity of LSD1 at an IC50 of approximately published in April 2012 in Cancer Cell, lays the case there was robust accumulation of MYC 20uM. Tranylcypromine is a licensed therapy for groundwork for early phase clinical trials of LSD1 protein followed by induction of AML cell death. Publications listed on page 72 depression that has been in the formulary for inhibitors in AML at The Christie NHS These data indicate a previously unappreciated 38 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH LEUKAEMIA BIOLOGY 39

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    Figure 1 As an alternative approach to improving our We have shown that the RAF-inhibitor paradox MOLECULAR ONCOLOGY The RAS-RAF-MEK-ERK pathway knowledge of melanoma, we are also using underlies the development of non-melanoma is depicted. NRAS is activated www.paterson.man.ac.uk/molecularoncology downstream of receptor tyrosine next generation sequencing to reveal the skin lesions (keratoacanthomas and squamous kinases (RTK) and it activates landscape of mutations that occur in individual cell carcinomas) in about a third of patients BRAF, which in turn activates human melanoma samples. Over the last year, treated with these drugs. Notably, this is not MEK and MEK activates ERK, we have focussed on acral melanoma in because the BRAF drugs act as tumour driving cell growth and survival. particular, a rare form of melanoma that promoters per se; rather they act by accelerating develops on the non-hair bearing skin of the the growth of pre-existing, pre-malignant hands and feet. These sites were thought to be tumours in susceptible patients. The drugs in protected from the damaging effects of UV effect place a growth-selective advantage on light, but our sequencing revealed that some these pre-existing tumours, and this knowledge acral melanomas present a UV light led us to discover that anti-proliferative agents Our group aims to develop new therapeutic strategies for DNA-damage “signature”. This suggests that UV such as 5-fluorouracil can be used to treat these light also appears to play a role in the aetiology lesions in patients for whom surgery is not an cancer based on improved understanding of cancer biology. of some acral melanomas. We are also option. In particular, we focus on melanoma and use a range of expanding our studies to examine the genetics of other rare forms of melanoma to allow us to Although BRAF drugs have provided a paradigm approaches including biochemistry, cell biology, molecular gain further insight into melanoma biology so shift in the treatment of melanoma, biology, mass spectrometry and next generation sequencing. that we can develop new therapeutic strategies unfortunately the responses to these drugs are for the treatment of all forms of this disease. generally short-lived and most patients will fail We also make extensive use of model systems and tumour on treatment after a relatively short period of samples to understand the biology of each tumour so that we A major breakthrough in melanoma treatment disease control. Furthermore, about 20% of Group Leader occurred with the development of drugs that patients do not respond to BRAF drugs despite can begin to develop tailored treatments for individual patients. inhibit BRAF. These drugs inhibit the RAS-RAF- the presence of a BRAF mutation. We have Richard Marais This approach is called “personalised medicine” because MEK-ERK pathway in cells in which BRAF is therefore continued to develop new BRAF drugs Postdoctoral Fellows mutated. Importantly, these drugs can achieve and are testing if these are effective in patients Franziska Baenke1 treatment is tailored to each patient’s tumour, rather than using impressive clinical responses in patients whose whose tumours are resistant to the pre-existing Kelly Brooks1 Romina Girotti2 a one size fits all approach. Thus, our aim is to implement a tumours express the mutant forms of BRAF, but drugs. We are also studying how resistance are ineffective in patients whose tumours develops in patients undergoing treatment. Koorosh Korfi1 personalised medicine approach for melanoma to improve express wild-type BRAF. Curiously, one of the Matthew Martin2 Berta Lopez Sanchez- treatment outcomes for patients and we anticipate that the unexpected side-effects of BRAF drugs is that My Group relocated to Manchester in October Laorden2 they activate rather than inhibit the RAS-RAF- 2012. We are a multi-disciplinary team that Grazia Saturno2 lessons we learn in melanoma will be applicable to other MEK-ERK pathway when RAS is mutated. This provides an excellent training environment for HaoRan Tang1 cancer types. effect occurs because RAF drugs stabilise the scientists and clinicians alike. Our philosophy is Bioinformaticians formation of complexes between ARAF, BRAF that patient care should be based on in-depth Simon Furney2 and CRAF in the presence of active RAS. The knowledge of cancer biology and we work at Mikis Gavrilidis1 Melanoma is a potentially deadly form of skin and nutrients and allowing the tumours to complexes contain drug-bound, and drug-free the basic-clinical interface to translate our basic cancer. In the UK, melanoma affects over grow more rapidly. Agents that target VEGF RAF molecules and the drug-bound partners research findings into patient benefit. At the Scientific Officer 12,000 new patients and kills over 2,000 people overcome this effect and cooperate with hyper-activate the drug-free partners, thereby Paterson Institute for Cancer Research we aim Clare McManus1 each year. Work over the last decade has metformin to suppress the growth of the BRAF hyper-activating the signalling pathway and to continue to investigate the biology of Eamonn Morrison1 demonstrated that the RAS-RAF-MEK-ERK tumours. These data highlight the importance stimulating the growth of the cells (Figure 2). melanoma and to use the knowledge from Graduate Student signalling pathway plays a critical role in this of understanding the biology of each tumour if Thus, while BRAF drugs inhibit the signalling those studies to develop and implement a Kate Hogan1 disease. RAS is a small G-protein that is activated effective therapies are to be developed for pathway in cells in which BRAF is mutated, they platform for personalised medicine for downstream of receptor tyrosine kinases and individual patients. activate the pathway in cells when NRAS is melanoma patients. 1 joined in 2012 RAF, MEK and ERK are cytosolic protein kinases mutated and this effect is called the “RAF- 2 Moved to the Paterson that control cell growth and survival (Figure 1). Melanoma develops from specialised cells in Institute from the ICR inhibitor paradox”. Publications listed on page 73 There are three RAS (HRAS, KRAS, NRAS) and the skin called melanocytes. These cells three RAF (ARAF, BRAF, CRAF) genes in humans. provide skin and hair tone, but more Notably, NRAS is mutated in about 20% of importantly, they protect us from the damaging Figure 2 The RAF paradox. (A) In the melanomas, and BRAF is mutated in a further effects of ultraviolet (UV) light radiation. UV light presence of mutated BRAF, the 45% of cases. is present in sunlight and is produced by tanning MEK/ERK pathway is hyper devices, and it is the only known environmental activated even though RAS is not We have found that the different genetic forms risk factor for melanoma. The most common active. (B) In BRAF mutant cells, of melanoma display different biochemical form of melanoma occurs on hair bearing skin BRAF drugs block BRAF activity and inhibit the activity of MEK properties. For example, the anti-diabetic drug that is intermittently exposed to UV light and ERK. (C) When cells in metformin drives the growth of melanoma in (recreational sun exposure) and we have which NRAS is mutated are which BRAF is mutated, but inhibits the growth developed transgenic models for melanoma treated with BRAF drugs, of melanoma in which NRAS is mutated. This is that allow us to investigate gene-gene and although BRAF is inhibited, it is because metformin increases the production of gene-environment interactions that drive driven into a complex with CRAF and hyper-activates CRAF, vascular endothelial growth factor (VEGF) in melanoma development. We are currently thereby driving paradoxical BRAF mutant, but not in NRAS mutant using these models to identify the genes that hyper-activation of MEK and ERK. melanoma cells. Critically, VEGF encourages interact with BRAF and NRAS to drive melanoma the development of new blood vessels into the development and to examine the role of UV growing tumours, increasing the flow of oxygen light. 40 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH MOLECULAR ONCOLOGY 41

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    Figure 1 in the laboratory to determine if it is required to SIGNALLING NETWORKS IN CANCER maintain lung cancer cell survival and A. B. C. www.paterson.man.ac.uk/signallingnetworks proliferation and to elucidate what downstream mechanisms are utilised to promote these phenotypes. To characterise the mutant kinases, our general strategy is to first assess the functional consequences of somatic mutations on overall Figure 2 kinase activity utilising in vivo and in vitro kinase A. B. C. activity assays. We compare the activity of the kinases harbouring cancer mutations Signalling pathways dictate a range of important cellular (engineered through site-directed mutagenesis) to WT, kinase dead (KD) and hyperactivated outcomes ranging from cell death, to replication, to cellular forms of the kinase. Next we determine migration. Genetic lesions that skew the balance of these phenotypic effects of expressing the WT, KD and mutant forms of the target kinase on pathways towards abnormal growth, proliferation, and cell proliferation, survival and transformed survival are the fundamental mechanisms that cause normal properties of appropriate tumour and normal cell lines. We then verify the function of the cells to become premalignant. Kinases are the key regulators kinase using si/shRNA and evaluate the role of of signalling pathways (similar to transistors in a circuit) and Figure 1 Structural analysis of mutations found that this approach works extremely well the endogenous kinase in regulating cellular Group Leader in identifying loss of function (LOF) mutations phenotypes associated with tumorigenesis. We dictate the activation or amplification of a given signal that in a candidate tumour and candidate tumour suppressing kinases. also investigate the molecular mechanisms John Brognard suppressing kinase. ultimately leads to cellular fate decisions. When these kinases MLK4 is a novel kinase that is utilised by the cancer mutants to promote Postdoctoral Fellows In a second approach we use genetic tumorigenesis. For example, if the mutation is Shameem Fawdar are hyperactivated or inactivated by genetic mutations they frequently mutated in lung and colon cancer. A homology dependency screens to identify mutationally an activating mutation, we will identify cancer Anna Marusiak become the main drivers of tumorigenesis and thus serve as model of the MLK4 kinase activated drivers of lung cancer. In this approach relevant substrates that are phosphorylated by domain was produced using we deplete cells of all somatically mutated the cancer mutants to promote tumorigenesis. Clinical Fellow primary targets for the development of small molecule SwissModel, based on the genes in an effort to identify gain-of-function Finally we will assess the consequences of Andrew Hudson1 structure of MLK1 (Fig 1A; inhibitors. Cancer genomic sequencing studies and coloured based on homology). (GOF) mutations that are likely to be robust somatic mutations utilising cell lines that Scientific Officers Comparison of binding site drivers of lung tumorigenesis and potentially harbour endogenous mutations in the target Natalie Stephenson1 genome-wide siRNA screens are highlighting the amazing predictions (Fig 1B) and the druggable targets. In our knockdown screen we kinase. The overall goal of these studies will be Eleanor Wendy Trotter diversity in the kinases required to maintain tumorigenic location of mutations (Fig 1C) show most mutations occur monitor for alterations in cell survival and to identify common and convergent pathways proliferation under the premise that depletion of utilised by cancer cells to promote lung Graduate Students Zoe Edwards phenotypes or permit drug resistance and emphasise the close to areas predicted to be a mutationally activated driver will result in tumorigenesis and identify convergent and involved in substrate binding. Ewelina Testoni1 importance that neglected or understudied kinases play in robust increase in cell death and inhibition of essential targets that could be exploited for the Figure 2 proliferation. Top targets are further development of novel therapeutics for the 1 joined in 2012 the development and maintenance of a tumour. Thus a major Analysis of the structures of characterised in detail, initially monitoring treatment of lung cancer patients. MLK4 mutants shows that most goal of our lab is to identify and elucidate novel kinase drivers mutants interrupt polar contacts impacts of mutations on kinase activity. This approach has worked well to date and we have This next generation of personalised medicine is that are essential for tumour development, required for within the kinase domain (Fig 2A) and are predicted to be loss-of- identified three novel kinases with GOF becoming a reality in NSCLC. Normal cellular maintenance of tumorigenic phenotypes, or play a role in function mutations. Interestingly, mutations in lung cancer, and have expanded growth relies on the interaction of networks of mutant G291E causes a large these initial results and observe that two of kinases that turn cellular processes ‘on’ and ‘off’. therapeutic resistance, focusing primarily on lung cancer. perturbation of the structure these kinases are mutationally activated in 3-5% A small percentage of NSCLC patients have a close to a region required for coordinating binding to ATP (Fig of all lung cancers. This screening approach has specific genetic change that generates an 2B). Mutant W296stop removes a the potential to identify both therapeutic targets “always on” version of a kinase (EML4-ALK is an Genetic Drivers of Lung Cancer bioinformatic tools to evaluate the functional large proportion of the substrate and biomarkers. example of a genetically activated kinase). Cells Approximately 70% of all non-small cell lung impact of somatic mutations in novel or binding region (Fig 2C). with this mutated kinase are unable to turn ‘off’ cancer (NSCLC) patients present with late stage understudied kinases identified in lung cancer Our final strategy involves an unbiased kinome certain cellular processes and therefore grow disease (stage III-IV) and there is a pressing need genomic screens. We use the following wide evaluation of all kinases focusing on out of control to form a tumour. Patients with to develop better therapies for these patients. bioinformatics applications to assess the amplification and somatic mutations in this defective kinase benefit significantly from This remains a major challenge as the functional impact of somatic mutations: squamous cell lung cancer. Utilising online data treatment with a small molecule inhibitor underlying genetic causes of nearly half of all CanPredict, PMUT, polyphen2, mutationtaster, portals such as cBio the lab hones in on kinases (Crizotinib) that targets the activated kinase to NSCLCs remain unknown. Recent success with snpeffect, puppasuite and SNPs and go. Kinases that are both amplified and somatically mutated turn ‘off’ the pathway. The major aim of our therapies targeting mutationally activated EGFR where a majority of mutations are predicted to in an effort to identify kinases where increased research is to identify new druggable targets by and constitutively active EML4-ALK serve as be “likely cancer”, “pathological” or likely to have expression or mutational activation drives lung screening lung cancers to find the next set of proof of principle that drugs targeting a “high” functional impact are further evaluated tumorigenic phenotypes. We start with the hyperactivated genes that represent an Achilles genetically activated drivers of lung cancer in the lab. Additionally, we model many of the kinome wide approach, a candidate kinase is heel for tumours of the lung and can result in result in better, more durable therapeutic mutations that score highly in our analysis to identified that is frequently amplified or mutated better therapies for lung cancer patients. responses in patients. The lab utilises three determine the structural consequences of the in lung cancer, and regions of mutations are strategies to identify genetic drivers of lung putative driver mutations concomitant with highlighted. This kinase is then studied in depth Publications listed on page 74 cancer. In the first approach we use experiments in the lab (Figure 1 and 2). We have 42 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH SIGNALLING NETWORKS IN CANCER 43

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    Figure 1 blood progenitors generated in Gfi1 and Gfi1b STEM CELL BIOLOGY Model of Activities of P1 and P1 + P2 + HSC double knockout embryos maintain the P2 Promoters in Adult www.paterson.man.ac.uk/stemcellbiology Haematopoiesis. P1 - P2 - expression of endothelial genes and cannot be LSK released from their cell layer within the yolk sac (Lin- Sca1 high MPP cKit high) and therefore fail to disseminate into embryonic tissues. Taken together, our findings strongly CD48+ Progenitors suggest a new and unexpected role of GFI1 and GFI1B transcription factors in mediating the loss PreCFUe Pre- of endothelial identity in the generation of MegE PreGM CLP haematopoietic progenitors from the haemogenic endothelium. CFUe Pre- MegE GMP Transcription factors bind to specific sequences in DNA Red blood RUNX1 isoforms Previous studies have indicated a critical and control how genes are transcribed into RNA and, as a cells MkP Macrophages Granulocytes requirement for RUNX1 at the onset of consequence, indirectly control the translation of RNA haematopoietic development and that RUNX1/ AML1 is expressed as multiple, naturally into functional proteins. Genes encoding the AML1/RUNX1 Megakaryocytes occurring spliced isoforms that generate transcription factor and its cofactor CBFβ, are frequently Platelets proteins with distinct activities on target promoters. Deletion of Runx1 in adult rearranged or mutated in human leukaemias such as acute haematopoietic cells results in significant cells. We identified the Gfi1 and Gfi1b genes as myeloid leukaemia (AML) and acute lymphoblastic leukaemia direct targets of RUNX1. Gfi1 and Gfi1b genes defects, including an expansion of peripheral Group Leader blood monocytes and granulocytes, a (ALL). Consistent with its implication in leukaemias, RUNX1 encode two highly homologous nuclear zinc reduction in lymphocytes and Georges finger proteins that function as transcriptional has also been shown to be critical for haematopoietic repressors. A C-terminal domain containing six thrombocytopenia. Stringent regulation of Lacaud Runx1 expression during haematopoiesis is development. Similarly the transcriptional co-activator MOZ C2-H2-type zinc finger motifs mediates the DNA therefore vital. The transcription of Runx1 is Postdoctoral Fellows binding activity of these proteins while their Julia Draper is involved in three independent myeloid chromosomal repressional activity requires the N-terminal under the control of 2 promoters: the Distal (P1) and Proximal (P2) promoters, encoding isoforms Kiran Batta Roshana Thambyrajah translocations fusing MOZ to the partner genes CBP, P300 SNAIL/GFI-1 (SNAG) domain. GFI1 deficiency RUNX1C and RUNX1B respectively. RUNX1B and leads mainly to neutropenia and reduction in Michael Lie-A-Ling or TIF2 in human leukaemia. Our group studies the function self renewal capacity of the haematopoitic stem RUNX1C differ solely in their N-terminal Flor Perez-Campo2 domains, RUNX1C being 14 amino acids longer Guilherme Costa1 of RUNX1 and MOZ in haematopoietic development and cells. In contrast, its paralogue Gfi1b is mostly and beginning with MASDS and RUNX1B expressed in erythroid and megakaryocytic Scientific Officer maintenance in order to better understand how alterations lineages and Gfi1b knockout results in beginning with MRIPV. Rahima Patel of these functions lead to leukaemogenesis. embryonic lethality at E14.5 due to a deficiency To study their expression during adult Graduate Students in erythroid and megakaryocyte development. haematopoiesis, we utilised a dual P1-GFP/ Monika Stefanska Several studies have shown that the expression P2-hCD4 reporter mouse line to sort adult Milena Mazan Novel functions of GFI1 transcriptional We have recently established a new model of of Gfi1 and Gfi1b are controlled by negative Elli Marinopoulou3 haematopoietic cell populations by flow repressor in the generation of blood cells haematopoietic development based on the in feedback loops and that GFI1 and GFI1b can Magdalena Florkowska cytometry and investigate their haematopoietic There is a worldwide shortage of matched vitro differentiation of embryonic stem cells. We repress expression of each other in a cross potential. We found that P1-GFP is expressed in 1 joined in 2012 donors for blood stem cell transfer of leukaemia demonstrated that haematopoietic cells are regulatory fashion. all Lin-cKit+ progenitors as well as mature 2 left in 2012 or lymphoma patients. The generation of blood generated from the haemangioblast through macrophages, granulocytes, B cells, subsets of cells upon in vitro differentiation of embryonic the formation of a haemogenic endothelium We first validated the differential expression of 3 joint with ACBB T cells and immature erythroblast populations. stem (ES) cells or induced pluripotent stem (iPS) intermediate. During this process, the both Gfi1 and Gfi1b in the presence/absence of By comparison, P2-hCD4 expression is highly cells could represent a powerful approach to haemogenic endothelial cells loose their RUNX1 and then demonstrated a direct binding heterogeneous, being upregulated in subsets of generate the autologous cell populations endothelial identity by altering their flat, of RUNX1 to these loci by chromatin Lin-cKithigh Sca1high (LSK) CD48+ progenitors and required for these transplantations. In this adherent appearance into the characteristic immunoprecipitation (ChIP) and Chip-seq. To in lymphoid and granulocyte-monocyte (GM) context, it is important to further understand the round shape of mobile haematopoietic investigate the potential functions of GFI1 and progenitors. The pre-megakaryocyte-erythroid development of blood cells. precursor cells. This haemogenic endothelial GFI1b in the endothelial to haematopoietic progenitor (PreMegE) can also be separated into cell population is transiently generated during transition, we evaluated their ability to rescue P2-hCD4- “pro-erythroid” and P2-hCD4+ The earliest site of blood cell development in these blast cultures and is also detected in defects observed in Runx1-/- differentiation “pro-megakaryocyte” subsets.To elucidate the the mouse embryo is the yolk sac where blood gastrulating embryos. Further experiments cultures. We demonstrated that in the absence functional roles of the Runx1 isoforms, we islands, consisting of haematopoietic cells showed that the transcription factor RUNX1/ of RUNX1, GFI1 and GFI1B are able to trigger the utilised P1 null mouse models. The absence of surrounded by a layer of angioblasts, develop at AML1 is critical for generation of haematopoietic loss of endothelial identity of haemogenic RUNX1C results in aberrant T cell development, approximately day 7.5 of gestation. The parallel cells from this haemogenic endothelium. endothelium. Upon Gfi1 or Gfi1b expression, reduced long-term competitive repopulation development of these two lineages in close these cells down-regulate the expression of and an alteration of myelo-lymphoid potential association provided the basis for the hypothesis These results suggest that RUNX1 regulates the endothelial genes and undergo the in engrafted bone marrow cells. These data that they arise from a common precursor, a cell expression of a set of genes critical for the morphological changes observed in the support a hypothesis of distinct roles for the two called the haemangioblast. A conflicting theory development of the haematopoietic system transition from endothelium adherent cells into RUNX1 isoforms during adult haematopoiesis. however associates the first haematopoietic from the haemogenic endothelium. To identify haematopoietic round cells. However these cells to a phenotypically differentiated these genes, we have compared gene cells are unable to acquire the full competence Publications listed on page 74 endothelial cell with haematopoietic potential, expression in cell populations generated from to generate haematopoietic colonies. i.e. a haemogenic endothelium. either Runx1 deficient or Runx1 competent ES Conversely, we established that fully committed 44 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH STEM CELL BIOLOGY 45

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    Ve-Cadherin is a transcriptional STEM CELL AND HAEMATOPOIESIS VEGF target of SOX7 Hemogenic Blood www.paterson.man.ac.uk/sch endothelium precursor The expression of Ve-Cadherin in embryonic haematopoiesis is associated with its expression Haemangioblast SOX7 in the haemogenic endothelium. Understanding the regulation of Ve-Cadherin expression is of crucial importance to gain SOX7 insight into the poorly explored molecular SOX7 Ve-Cadherin X Ve-Cadherin mechanisms that drive haemogenic endothelium specification from the haemangioblast. In order to shed light on the Figure 1 haemogenic endothelium cells which then transcriptional activity of SOX7, we focused on At the early stages of vertebrate ontogeny, blood and A model for the action of Sox7 during haematopoietic further mature into blood precursors. The the expression of Ve-Cadherin a potential target haemangioblast-derived haemogenic of SOX7 at the onset of haematopoiesis. The endothelial cells develop from a common mesodermal development. endothelium has been characterised as a promoter of mouse Ve-Cadherin has been progenitor, the haemangioblast. Upon commitment, the population of cells expressing endothelial cell described as a region of 2,500 base pairs, surface markers, such as TIE2, PECAM-1 or sufficient to drive the transcription of this gene haemangioblast generates blood precursors through Ve-Cadherin along with the tyrosine kinase in endothelial cells. In order to define whether populations of endothelial cells with haemogenic properties. receptor cKIT. Upon differentiation, this SOX7 may bind and activate this promoter, we population gradually loses its endothelial first identified potential SOX DNA binding motifs identity and inversely, gains a haematopoietic in this promoter sequence. To explore whether Although several transcription factors have been haemangioblast to haemogenic endothelium immuno-phenotype, with the rapid up- SOX7 regulates the activation of Ve-Cadherin Group Leader implicated in this developmental process, the and blood precursors still remain poorly regulation of the alpha-IIb integrin CD41 through these motifs, we generated reporter precise mechanisms governing cell fate understood. It is known that the transcription followed by a slower acquisition of the pan- plasmids containing either full-length or Valerie decisions towards the generation of blood factor SCL is critical for the generation of haematopoietic marker CD45. The careful truncated promoter fragments to perform Kouskoff precursors remain largely unknown. The haemogenic endothelium cells and that these analysis of SOX7 expression in the context of transient transactivation assays. Interestingly, research performed in our group aims at further cells require RUNX1 expression to generate these cell surface markers revealed that this SOX7 induced an eight-fold increase in Postdoctoral Fellows understanding the transcriptional networks that definitive haematopoietic progenitors. Studies transcription factor is strictly associated with the transcriptional activity regardless of the length Alexia Eliades Maud Fleury orchestrate this developmental process. Many in our laboratory over the past few years have expression of endothelial markers and sharply of the promoter region used, demonstrating not Eva Garcia-Alegria1 transcriptional regulators implicated in been aimed at understanding the role of the down-regulated during further commitment to only that the Ve-Cadherin promoter was Josh Lilly1 haematopoietic specification during transcription factor SOX7 during this early haematopoiesis. activated by SOX7, but also that the most embryogenesis are also linked to developmental process. proximal region studied, containing only one Scientific Officers leukemogenesis events, often as a result of The enforced expression of Sox7 maintains SOX binding motif, was sufficient for this effect. Stella Pearson Kirstie Wallace1 aberrant expression. Through a better SOX7 is transiently expressed at the onset of haemogenic endothelium precursors We next undertook a series of experimental understanding of the function of these haematopoietic development To understand the functional role played by assays to evaluate the interaction of SOX7 with Graduate Students transcriptional regulators at the onset of This transcription factor belongs to the SOX7 during this differentiation process, we this motif. Through both in vitro and in vivo Sara Cuvertino haematopoietic specification, we hope to gain SRY-related HMG-box family and shares similar took advantage of a mouse embryonic stem cell assays, we were able to demonstrate that SOX7 Andrzej Mazan insight into their potential role in the initiation protein structure with SOX17 and SOX18, two line containing a doxycycline inducible Sox7 binds the most proximal SOX DNA binding motif Sharmin Naaz2 and maintenance of haematological other members of the F subgroup. Early in cDNA. This line allowed us to counteract the on the promoter of Ve-Cadherin. Together, 1 joined in 2012 malignancies. murine development, Sox7 transcripts can be bona fide transient expression of Sox7 during these data reveal that SOX7 regulates the 2 joint with ACBB detected in the parietal endoderm and in haematopoietic development and to assess the expression of Ve-Cadherin by binding to its The first haematopoietic and endothelial cells of vascular tissues. Compelling evidence supports impact of maintaining the expression of this promoter, hence identifying Ve-cadherin as a vertebrates emerge extra-embryonically within the idea that SOX7, together with SOX17 and transcription factor beyond its normal time transcriptional target of SOX7. the yolk sac. Long-standing observations early SOX18, plays an important role during vertebrate frame. Unlike the control culture, FLK1+ in the 20th century initially suggested that both cardiovascular development. The role of SOX F haemangioblast cells differentiated in the SOX7 is a critical regulator of haemogenic lineages originated from a common precursor, proteins in haematopoiesis is far less presence of doxycycline failed to down- endothelium at the onset of haematopoiesis the haemangioblast. However, the existence of understood. Recent studies have demonstrated regulate the endothelial programme and In summary, our data reveal that SOX7 is this progenitor was only demonstrated much that SOX17 regulates the proliferation of showed little sign of commitment toward specifically expressed in haemogenic later in vitro using the differentiation of mouse neonatal and foetal haematopoietic stem cells, haematopoiesis. The continued expression of endothelium cells at the onset of embryonic stem cells as a model system and whereas we have recently established the Sox7 appears to inhibit the full differentiation of haematopoietic development and that SOX7 more recently through in vivo studies. The involvement of SOX7 and SOX18 at the earliest haemangioblast, maintaining an immature controls the expression of Ve-Cadherin, a gene differentiation of embryonic cells is a powerful stages of blood development. In these previous endothelial-like state. Given that Sox7 is critically associated with the endothelial nature method to explore the molecular and cellular studies, a genome-wide expression analysis of endogenously expressed in the transient of the haemogenic precursors. It is tempting to mechanisms that regulate embryonic differentiated embryonic stem cells revealed haemogenic endothelium population, and that propose a model in which SOX7 acts upstream haematopoiesis. Using this model system, that Sox7 was up-regulated at the onset of enforcing its expression beyond its normal time of endothelial-related genes in the haemogenic elegant studies have revealed that the haemangioblast development. To further frame maintained an endothelial cell endothelium before the haematopoietic haemangioblast generates blood precursors characterise the pattern of Sox7 expression in population, we performed further experiments differentiation has been initiated (see Figure 1). through the formation of an intermediate early haematopoiesis, we studied its detailed demonstrating that the differentiation blockage Thereafter, the down-regulation of this specialised endothelium termed haemogenic kinetics of transcription during haemangioblast induced by Sox7-enforced expression led to the transcription factor is necessary for the dynamic endothelium. The precise molecular differentiation. Upon culture in the presence of maintenance of haemogenic endothelium process of blood precursor emergence from mechanisms that underlie the specification of VEGF, FLK1+ haemangioblasts give rise to progenitors. the haemogenic endothelium. Publications listed on page 75 46 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH STEM CELL AND HAEMATOPOIESIS 47

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    THE UNIVERSITY OF MANCHESTER INSTITUTE OF CANCER SCIENCES RESEARCH GROUPS Normal human colon stained with an antibody to the marker CDX2 (brown). CDX2 is used as a marker in colorectal cancer prognosis. The tissue has been counterstained with haematoxylin to show the nuclei of cells (blue). Image provided by Darren Roberts from the Immunology Group. 48 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH THE UNIVERSITY OF MANCHESTER INSTITUTE OF CANCER SCIENCES 49

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    organelles including mitochondria, lysosomes, CHILDREN’S CANCER endoplasmic reticulum and polyribosomes (Figure 2). We have also shown that human leukaemia cells are able to produce MVs in vivo which are internalised by mouse stromal cells in the bone marrow. In vitro internalisation of the leukaemic cell MVs by BMSCs appears to effect key signalling pathways involved in metabolism. Thus tumour derived MVs may modulate the host cells to create an environment that favours the survival of leukaemic cells over normal haematopoietic precursors in targeted niches. Our group conducts investigations into the biological Clare Dempsey and Seema Alexander have mechanisms responsible for the variations in the therapeutic created a number of xenograft mouse models response in children with acute lymphoblastic leukaemia (ALL). to study pathogenesis and understand the effects of therapy. Collaboration with Professor We conduct a number of international clinical trials, which Stern (Paterson Institute) using such a model provide us with the data and clinical material for hypotheses showed that a subpopulation of leukaemic blast cells expressing 5T4 displayed invasive and based laboratory investigations. chemotactic behaviour, suggesting a model of recurrent disease (Castro et al., 2012). Stephanie Group Leader Harrison is investigating the role of the adhesion Clinical international trial for relapsed patients, designed Figure 1 two-way communication. We believe molecule LFA-1, a molecule we have previously Vaskar Saha Childhood ALL is a rare disease with high cure by us and funded by the FP7 program, is due to Progression free survival of microvesicles (MV) produced by the leukaemic identified using a discovery based proteomic EsPhALL patients in the UK; rates. Our interests are in refractory and relapsed launch in 2013 (http://www.intreall-fp7.eu/). cell to be one such mechanism by which the approach, in chemo-resistance, adhesion and Postdoctoral Fellows Kaplan-Meier analysis of Imatinib Clare Dempsey disease. Thus most of the clinical trials in which versus no Imatinib showed no leukaemic cell modulates its environment. invasion. She has identified a panel of high and Yujun Di 1 we participate are done as part of international Laboratory significant difference in PFS Suzanne Johnson has characterised leukaemia low LFA-1 expressing cell lines and measured Suzanne Johnson collaborations. In the largest such collaborative Investigating the biological mechanisms of between those patients who cell-derived microvesicles, showing that they received Imatinib and those who their response to the bacterial protein Jizhong Liu study ever performed, we showed that contrary therapeutic failure in collaboration with Dr have distinct gene expression and protein did not. Leukothera (collaboration with Dr Scott to conventionally held beliefs, some children Krstic-Demonacos (University of Manchester, profiles compared to the parent cell. Genes Kachlany, New Jersey); which binds LFA-1 and Graduate Student Stephanie Harrison with ALL who do not respond to initial therapy Faculty of Life Sciences), we have shown that involved in redox status, histone regulation and induces cell death. can be salvaged by allogeneic stem cell steroid sensitivity in ALL is controlled through a lipid metabolism are upregulated in the MV. MVs Clinical MRes Student transplantation. Moreover as these children can series of feedback loops operating in differential are enclosed in a lipid bilayer and lipidomic We are pleased to report that Ashish Masurekar Tasos Ioannou be identified by typical cytogenetic changes, temporal patterns that will, at least in part, analyses (in collaboration with Professor successfully defended his PhD thesis, Steph this study offers practical therapeutic strategies determine cellular levels of GR, AP-1, Erg and Goodacre, Manchester Institute of Harrison made the transition to 2nd year PhD Clinical Trials Manager Catriona Parker (Schrappe et al., 2012). In another international Bim, ultimately contributing to cell fate (Chen et Biotechnology) revealed a unique profile from student and Wayne Pinto gained his MRes. We study (EsPhALL), we were involved in designing al., 2012). However, emerging evidence from MVs produced under hypoxic conditions. bid farewell to Adiba Hussain, and Mark Holland Administrator and conducting a randomised trial of the our clinical trials suggests that recurrence Electron microscopy has identified a who moved on to pastures new, and welcome Parisa Mahjoob-Afag tyrosine kinase inhibitor (TKI) imatinib in children cannot be explained only by intrinsic resistance. heterogeneous population of MVs including Dr Yujun Di and Tasos Ioannou to our group. 1 with Philadelphia positive ALL. This is the first To investigate extrinsic mechanisms, Jizhong exosomes and larger MVs derived from both cell joined in 2012 such randomised trial of a TKI in such a rare sub Liu has created an in vitro organotypic, lines and ALL patient cells which contain Publications listed on page 75 group. The results do not show a significant orthotropic model of the bone marrow difference in outcome between those who microenvironment, developed using received and did not receive imatinib (Figure 1). mesenchymal cells obtained from bone Figure 2 However, the study is perhaps underpowered to marrow aspirates (BMSCs). Conditioned media Transmission electron polyribosomes establish the difference in survival. Imatinib was from this system is able to confer chemo- micrograph showing a large cytoplasmic protrusions well tolerated in the context of intensive therapy, protection in ALL cells by activating prosurvival microvesicle derived from an acute lymphoblastic leukaemia and there appears to be trend in its favour and signalling pathways. Our analyses suggest that cell (x 2900 magnification). thus the randomisation was discontinued BMSCs dynamically regulate mitochondrial heterolysosome Image shows intact membrane (Biondi et al., 2012). In the international trial for redox status and antiapoptotic gene expression, with cytoplasmic protrusions relapsed patients (ALLR3) we demonstrated a which confer resistance to chemotherapy in and intra-vesicle organelles. mitochondria benefit of the drug Mitoxantrone over leukaemia cells. Targeting this process using Idarubicin. This is one of the largest differences drugs that modulate the redox adaptation ever observed by a single drug intervention in reverses the chemoresistance leading to cell the context of combination chemotherapy and death. This has potential implications for future microvesicle the randomisation was stopped. The trial therapeutic strategies not only in childhood ALL endoplasmic continues to enrol patients to answer secondary but in other cancers. These data earned Jizhong reticulum objectives and with over 400 now recruited, is a highly rated award for his poster at the AACR one of the largest of trials of its kind worldwide. meeting in Chicago in April. parent cell Further investigations include the detection of clone specific molecular biomarkers to risk Our interest in the interaction between the bone stratify patients for transplantation and marrow microenvironment and the leukaemic evaluation of the new agent Clofarabine. A new cell has led us to hypothesise a model of 50 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH CHILDREN’S CANCER 51

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    Renal Cancer Data Manger MEDICAL ONCOLOGY: CLINICAL AND Dionne Lawrence project as joint-chief investigator and the overview of the trial in illustrated in figure 1. Major Histocompatibility Complex proteins. However, it is also clear that the interactions of EXPERIMENTAL IMMUNOTHERAPY Project Managers This trial is scheduled to start in 2013 (Q2). T cell-tumour proteins outside of the initial Nikki Price CAR-target interaction are important. Indeed, Helena Kondryn 2. Does selecting cells with better repopulating the optimal activity of CAR-T cells is dependent ability and optimised production processes upon interactions including that of the CD2 1 joined in 2012 (see below – pre-clinical section) produce receptor with its cognate ligands (Cheadle 2 left in 2012 better cell survival and better clinical et al, 2012). 3 Cellular Therapeutics Unit responses? This trial will be undertaken in malignant melanoma where high clinical Importantly, we are seeking to develop new response rates have been seen. The trial is model systems to test the power of gene planned as a randomised phase II study and modified T cells. To this end, our collaborative The Medical Oncology Clinical and Experimental should commence in 2013 (Q3) once the studies with the Immunology group (Professor pre-clinical validation of the processes has Peter Stern) targeting the mouse 5T4 antigen Immunotherapy Group undertakes translational research to been achieved. has identified some of the issues that relate to develop immunotherapy for cancer. This is based around immunological tolerance (Castro et al., 2012). The ATTACK consortium (Co-ordinator Robert We have generated CAR’s specific for the clinical expertise with immunotherapy in renal cancer and is Hawkins) comprises 17 partners with many mouse 5T4 protein and mouse T cells engrafted developing into work in gastro-intestinal cancer, lymphoma major clinical centres in Europe and these trials with these CAR’s show specific functional will be the first multi-centre engineered T-cell activity against mouse 5T4 expressing targets. and melanoma. The major focus is on the development of trials in Europe. As well as the clinical centres Our future studies will investigate the potency of Figure 1 adoptive cellular therapy and a major recent infrastructure The ATTACK clinical trial in there are four companies (three SMEs and one these 5T4-specific T cells and investigate what Group Leader large company). One of the SMEs is Cellular role immune tolerance and expression of the development is the recently licensed Good Manufacturing oesophago-gastric cancer. Therapeutics Ltd – a spinout company from the natural antigen has upon the in vivo function, Robert Patients will be screened for Process (GMP) Cellular Therapy Unit base in the UMIC building HLA-A2 tissue type and to assess University/this Group. The inclusion of relevant persistence and trafficking of CAR T cells. Hawkins if their tumour expresses commercial companies accords with the EU on the main Medical School site. This enables us to produce NY-ESO-1. Patients who are focus on developing novel therapies in a It seems self-evident that the long-term Consultant/Senior eligible will undergo Lecturer clinical grade cells for trials and has also led to a spinout leukapheresis to obtain large sustainable way that should both improve persistence of therapeutic T cells in the patient number of T-cells. After health outcomes and provide employment and is likely to correlate with potential clinical Fiona Thistlethwaite company. The group lead several EU consortia in the field and activation, the T-cells are economic value. response. However, many of the methods used Senior Fellow much of our research links with other leading EU centres for genetically modified using a lentivirus to produce cells to generate CAR T cells results in the David Gilham Pre-clinical research polarisation of the T cells towards a this type of work. engineered to target NY-ESO-1. Patients are treated with Previous studies in the group have differentiated phenotype which tends to result Research Fellow Ryan Guest3 pre-preconditioning demonstrated the potential of gene-modified T in reduced in vivo persistence. To counter this, chemotherapy to deplete cells to target and eliminate cancer cells which we have investigated whether cytokines can Clinical/translational research therapy. The Cellular Therapy Unit (CTU) has normal host T-cells - this results Scientific Officers have led to several early phase clinical trials modulate gene-modified T cell phenotype. Vicky Sheard renal cancer begun making clinical cell products and the first in the in vivo homeostatic testing this approach in patients with advanced Indeed, the combination of IL-7 and IL-15 during Anthony Oojageer A number of major clinical trial results were patients have been treated with tumour expansion of the returned engineered T cells. The cancer. Chimeric Antigen Receptors (CAR) the culture of T cells drives these T cells towards Natalia Krillova3 released in 2012 with the renal cancer clinical infiltrating lymphocytes. This along with other Sam Mowbray2,3 engineered T cells are also endow T cells with antibody-type specificity a less differentiated phenotype and group playing a major role. These included the immunotherapy research by the group was Asiya Arshad1,3 supported with ectopic thereby allowing T cells to directly target tumour consequently, results in an improved comparative trials of Sunitinib and Pazopanib featured on a BBC Newsnight programme in cytokines. The trial is designed to Gary Shaw1,3 cells through the recognition of cell surface engraftment potential of T cells in mouse assessing both patient preference (PISCES) and August 2012. The opening of the CTU has also assess the RECIST response rate proteins thus avoiding many of the mechanisms models. This work has formed the basis of a Clinical Fellows efficacy (COMPARZ). These were presented at enabled a phase I trial targeting CD19 in B-cell to the treatment and it is anticipated to include up to 28 tumours employ to avoid immune cell successful European Union Framework 7 Rob Brown ASCO and ESMO respectively and malignancies to reopen. This trial had previously Victoria Galvis1 patients. recognition including the down regulation of programme ATTACK where we will test the demonstrated similar efficacy and a clear patient shown very encouraging responses at the Shien Chow1 effect of IL-7/IL-15 on T cells engrafted with a preference for Pazopanib. The renal team have lowest dose and it is hoped that the further NY-ESO-1 T cell receptor in comparison to T Graduate Students also established a major data base for audit and cohorts planned will produce further clinical cells cultured in standard methods in a Hannah Gornall research of renal cancer management. The benefit. In addition, the EU has awarded a major randomised phase II Malignant Melanoma Victoria Hambleton initial research output was presented at ESMO clinical trial grant (€ 6M) to target NY-ESO-1 (FP7 Milena Kalaitsidou clinical trial setting. Our future studies are assessing the value of CAIX expression as a ATTACK: Adoptive T-cell Therapy to Achieve focusing upon the selection of T cells for biomarker to predict response to high-dose Cancer Killing). NY-ESO-1 is a cancer testis Marie Curie Training specific chemokine receptors to investigate Fellow interleukin-2 in addition to our previously antigen which is strongly expressed in a whether the specific selection of T cell subsets Vania Baldan published selection criteria (Shablak et al., 2011). proportion of many common cancers. The use prior to genetic modification further improves In addition, this database has facilitated the of T-cells to target NY-ESO-1 has been tested in MRes in Oncology the in vivo potency of gene-modified T cells. setting up of collaborative projects with the melanoma and in synovial sarcoma with Students Christie pathology department and the Clinical excellent clinical response rates and no Christopher Mansbridge2 Publications listed on page 75 Lyndon Ridges-Jones2 Genetics department at St Mary’s Hospital to significant on-target toxicity. This background Elizabeth O’Donovan1 assess the potential value of further histological has allowed us to set up this project to test two and genetic biomarkers. major hypotheses: Cell therapy 1. Is adoptive T-cell therapy active in a common The year has seen a major step forward in the epithelial malignancy where NY-ESO-1 is clinical /translational development of cellular over-expressed? Dr Thistlethwaite leads this 52 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH MEDICAL ONCOLOGY: CLINICAL AND EXPERIMENTAL IMMUNOTHERAPY 53

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    Figure 1 VEGF165 showed a better response rate in MEDICAL ONCOLOGY: TRANSLATIONAL ISNSmono6S inhibits FGF2 CD34-negative population when compared to signalling in tumour vasculature. ANTI-ANGIOGENESIS Immunostaining with antibodies CD34-positive cells. Together these data show recognising endothelial cell the morphological and functional differences marker CD31 and between CD34-negative and CD34-positive phosphorylated FRS2 of populations within EPCs. endometrial carcinoma xenografts over-expressing Clinical studies exogenous FGF2 in mice treated with saline, ISNSmono6S or (collaboration with Clinical and Experimental sunitinib. Pharmacology, led by Professor Caroline Dive) Figure 2 Angiogenesis has been validated as a target for Angiogenesis has been validated as a target in ovarian cancer Assessment of endothelial the treatment of ovarian cancer in four functions in CD34-negative randomised trials; two in the first line setting and therapy through clinical trials in which the VEGF inhibitor (CD34-) and CD34-positive two in recurrent disease. One of the trials carried (CD34+) EPC populations. bevacizumab, in combination with cytotoxic chemotherapy, CD34- EPCs show angiogenic out in chemo-naïve patients was ICON7, which randomly allocated patients to receive resulted in improved progression-free survival. The majority of phenotypes when tested for tube formation on Matrigel, carboplatin and paclitaxel or the same regimen angiogenic cytokines, including VEGF, depend on heparan sprouting and tube formation supplemented with the anti-VEGF antibody, within three-dimensional fibrin bevacizumab. The results showed that in sulphate (HS) for their activity, thus presenting an opportunity matrix. CD34+ EPCs show no patients with advanced disease (FIGO stage III or angiogenic activity in these to target cytokine-HS interactions and thereby inhibit cytokine assays. IV) there was a statistically significant Group Leader improvement in progression free survival and activity. Our organic HS synthesis programme generated revealed early data that were suggestive of an Gordon inhibitory HS sequences where differentially 6-O-sulphated Phenotypic and functional evaluation of EPCs overall survival advantage. Jayson from ovarian cancer patients and healthy sequences target specific angiogenic cytokines. In addition to controls We carried out a translational research project Senior Research Fellow Egle Avizienyte HS structure-function studies, we focus on defining the Through a number of studies it has been shown through investigation of the angiogenic that EPCs play an important role in promoting cytokine concentrations in the plasma of Postdoctoral Fellows phenotypes and functions of circulating endothelial progenitor physiological and pathological angiogenesis, patients before treatment in ICON7; the aim Claire Cole cells (EPCs) isolated from ovarian cancer patients which might although their exact role in tumour being to identify the patients who will most Cristina Ferreras angiogenesis remains controversial. EPCs are benefit from bevacizumab. During the clinical lead to identification of new cellular targets for anti-angiogenic usually defined as cells present in bone marrow, trial Professors Dive and Jayson collaborated Clinical Fellows Laura Horsley therapies. umbilical cord blood or peripheral blood that with Dr Alison Backen to validate, to the Kalena Marti Marti (joint express endothelial, but not hematopoietic, cell standards of GCLP, a multiplex ELISA system that with CEP) surface markers and show high proliferative was capable of measuring the concentration of Danielle Shaw Significance of 6-O-sulphation in HS growth studies, ISNSmono6S potential. However, the definition of EPC 16 angiogenic growth factors simultaneously in oligosaccharides dodecasaccharide significantly reduced phenotype and function still poses a major small volumes of plasma. Our analysis showed Scientific Officer Graham Rushton (collaboration with Dr. John Gardiner, FGF2-induced microvessel density and the challenge in the field. that patients with high plasma concentrations of Manchester Interdisciplinary Biocentre) average vessel size. Correspondingly, there was Ang1 and low Tie2 were those who benefitted a reduction in phosphorylation levels in FRS2, We established an EPC ex vivo expansion the most from bevacizumab. Ang1 is implicated Our previous work showed that a synthetic HS FGFR1 adaptor molecule, within the tumour procedure where we routinely detect the in maintaining a quiescent endothelium and dodecasaccharide, consisting of six repeating vessels (Figure 1). In contrast, a synthetic tri- outgrowth of endothelial cell colonies from the potentially, our data suggest that in patients with disaccharide units that were composed of sulphated dodecasaccharide (ISNS6S) was a peripheral blood mononuclear cell population high plasma concentrations of Ang1 the tumour iduronate 2-O-sulphate linked to N-sulphated poor inhibitor of FGF2- and VEGF165- of healthy females and ovarian cancer patients. vasculature is more dependent on VEGF and glucosamine (ISNS) was a potent inhibitor of dependent endothelial cell functions. We have identified two distinct EPC populations that is why these patients benefit most from FGF2- and VEGF165-induced endothelial cell Significantly, testing the effects on chemokine- based on the expression of the progenitor stem bevacizumab. We will attempt to verify the responses in vitro (Cole et al., PLoS One, 2010). induced endothelial cell migration revealed that cell marker CD34. No differences were found in findings in an independent sample set over the We also showed that 6-O-sulphate levels in ISNSmono6S, but not ISNS, potently inhibited the expression of endothelial cell surface next year. endothelial cells play a major role in regulating Sdf-1α-induced endothelial cell migration, markers CD31, CD146, CD105 and VEGFR2 responses to FGF2 and VEGF (Ferreras et al., J whereas IL-8-induced endothelial cell migration within both populations, while the expression of These results are important not only because Biol Chem, 2012). To understand the was inhibited by ISNS dodecasaccharide, but the haematopoietic marker CD45 was bevacizumab can induce toxicity and is significance of the number and position of not ISNSmono6S variant. Such specificity was undetectable in CD34-negative and CD34- expensive but also because new anti- 6-O-sulphates in HS we generated a synthetic also reflected in inhibition of SDF-1α-induced positive cell populations. CD34-negative cells angiogenic agents that target Angiopoietin are ISNS dodecasaccharide with either one 6-O- phosphorylation of Erk and IL-8-induced formed an endothelial tube network when in late stage development. Conceivably, these sulphate moiety at the non-reducing end of the phosphorylation of Stat3, where ISNSmono6S plated on Matrigel, were able to sprout and data will allow us to use such tests to select the molecule (ISNSmono6S) or a uniformly 6-O was more potent in inhibiting SDF-1α-induced formed endothelial tubes in a three- right anti-angiogenic agents for the right patient sulphated molecule (ISNS6S). The inhibitory Erk phosphorylation, while ISNS inhibited dimensional fibrin matrix (Figure 2). The CD34- thereby reducing cost and toxicity while properties of ISNSmono6S were enhanced IL-8-induced Stat3 phosphorylation. This positive cell population showed no sprouting improving the survival of our patients. leading to a more pronounced inhibition of suggests that the number of 6-O-sulphate and tube formation either on Matrigel or in a FGF2- and VEGF165-dependent endothelial cell residues and their position are of major three-dimensional matrix (Figure 2). Evaluation Publications listed on page 76 proliferation, migration, sprouting and tube importance in differentially inhibiting specific of the chemotactic capacity of EPCs toward formation. During in vivo tumour xenograft cytokines. 54 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH MEDICAL ONCOLOGY: TRANSLATIONAL ANTI-ANGIOGENESIS 55

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    initial responses that are only short lived. These TARGETED THERAPY results provide new insights that may have important implications for further development of strategies that result in long term tumour clearance after initially effective anti-cancer treatment. We are currently investigating these models further to assess immune responses to cell death in vivo in established tumours. Novel Mechanisms of antibody induced cell death Monoclonal antibodies (mAbs) have revolutionised the treatment of B-cell This last year has been highly successful for the Targeted malignancies. Although Fc-dependent Therapy Group and the highlights have included important mechanisms of mAb-mediated tumor clearance have been extensively studied, the research findings leading to publication in two key research ability of mAbs to directly evoke programmed areas within the programme, namely combining RT with cell death (PCD) in the target cell and the underlying mechanisms involved remain immunotherapy and mechanisms of action of antibody action. under-investigated. We recently demonstrated Earlier in the year we provided new insights into the role of that certain mAbs (type II anti-CD20 and anti-HLA DR mAbs) potently evoked PCD Reactive Oxygen Species (ROS) in the mechanism of induced through an actin-dependent, lysosome- Group Leader tumor cell death with the anti-CD20 antibody (GA101, Figure 1 protective anti-tumour immunity post mediated process (Ivanov et al J Clin Tim Illidge Treatment with radiation therapy combination treatment with TLR7 and Investigation 2009, Alduaij et al Blood 2011). Obinutuzumab) in (Honeychurch et al 2012) and followed this (RT) in combination with a radiotherapy. More recently we have demonstrated that the Postdoctoral Fellows synthetic TOLL-like receptor Jamie Honeychurch with a novel approach for the treatment of cancer using (TLR)-7 agonist leads to the induction of PCD by these mAbs, including the Host immune recognition of treatment type II anti-CD20 mAb GA101 (obinutuzumab), Ellie Cheadle Simon Dovedi radiotherapy in combination with a TOLL-like receptor (TLR)-7 activation of antigen presenting cells (APCs). Following activation induced tumour cell death directly correlates with their ability to produce Grazyna Lipowska-Bhalla agonist, which was also published in Blood as a plenary paper APCs present tumour associated We have also investigated in vivo the host reactive oxygen species (ROS) in human antigens and the co-stimulatory immune response to dying tumour cells. Whilst B-lymphoma cell lines and primary B-cell MD Student with associated editorial (Dovedi et al 2012). Tim Illidge was factors required to bring about apoptosis is generally considered non- chronic lymphocytic leukemia cells. ROS Clara Chan the activation of cytotoxic T awarded the Faculty of Medical and Human Sciences cells. These activated T cells are immunogenic, recent evidence suggests that scavengers abrogated mAb-induced PCD MRes Student then able to selectively kill some anti-cancer therapies that induce indicating that ROS are required for the Benjamin Sanderson Researcher of the Year. tumour cells. apoptosis can elicit anti-tumour immune execution of cell death. ROS were generated BSc Pathology Student responses. In collaboration with Clinical downstream of mAb-induced actin cytoskeletal Conor McKenna Combining radiation therapy with agent treatment resulted in long-term clearance Experimental Pharmacology, led by Professor reorganisation and lysosome membrane immunotherapy approaches of tumour and enhanced survival (Figure 1). Caroline Dive, we have developed a number of permeabilisation. ROS production was Effective anti-cancer treatments often result in Combination therapy led to an increase in the doxycycline (Dox)-dependent caspase-3 “death independent of mitochondria and unaffected the induction of large amounts of tumour cell frequency of CD8+ cytotoxic T-cells present in switch” syngeneic murine tumour models. In by BCL-2 overexpression. Instead, ROS death. RT is an effective cancer treatment that the circulation. Our investigations revealed that these models a doxycycline inducible, generation was mediated by NADPH oxidase. plays an important part in the local control of the efficacy of this combination therapy was constitutively active caspase-3 (‘death switch’) These findings provide further insights into a many tumours and often leads to high response dependent upon the activity of these CD8+ was constructed in murine tumour cell lines to previously unrecognised role for NADPH rates in patients. RT induced tumour cell death cytotoxic T-cells as their depletion completely explore the impact of the host immune oxidase-derived ROS in mediating nonapoptotic has the potential to stimulate immune abrogated the therapeutic response. Moreover, response to rapid, synchronous and substantial PCD evoked by mAbs in B-cell malignancies. responses against the cancer cells. However, the combination of RT and systemic TLR7 tumour cell apoptosis. In vitro, activation of the This newly characterised cell death pathway these immune responses are usually too weak therapy led to the induction of long-term death switch triggers up to 80% apoptotic cell may potentially be exploited to eliminate to lead to durable anti-tumour responses that immunological memory, which was able to death that is accompanied by release of ‘danger malignant cells which are refractory to might subsequently lead to improvements in protect against disease recurrence. These data signal’ molecules such as HMGB1 and HSP90, conventional chemotherapy and patients’ outcome. However, combining RT with revealed that combination therapy with 12-24 hours post-induction. In vivo, death immunotherapy. These observations are novel agents that are capable of stimulating the radiation and a TLR7 agonist was able to switch induction provoked rapid, pronounced important for the understanding of cell death immune system has the potential to generate generate tumour-specific immunological tumour xenograft regression in immune- induced immune responses, which suggest that durable and effective anti-cancer immune memory. These findings demonstrate the competent and immune-deficient mice but the generation of a successful host immune responses that are able to eradicate widespread potential for novel therapeutic combination sustained tumour eradication was observed response towards tumour cell death is malignant disease and reduce disease approaches involving RT and immunotherapy only in immune-competent mice. Moreover, dependent on several factors, including the recurrence. for the treatment of cancer. We are currently mice which were tumour free after death switch amount of cell death induced, the expanding this study to include additional induction in their tumours were protected (< immunogenicity of the cell death and the By using a synthetic agonist of TOLL-like syngeneic models of solid tumours. In addition, 60%) from rechallenge by the same tumour tumour micro-environment. receptor family (TLR-7) which activates a we are also evaluating a series of novel small cells. These data suggest that sustained tumour systemic immune response by mimicking a viral molecule TLR7 agonists with the aim to test eradication after substantial tumour apoptosis Publications listed on page 77 infection our data demonstrate that the these combinations in the clinic. Further work is requires an anti-tumour host immune response anti-tumour efficacy of RT can be enhanced in ongoing to elucidate the role of immune which prevents tumour relapse. In many pre-clinical models of lymphoma. The effector cells such as DC, helper-T cell, B cell, patients, cancer therapies produce encouraging combination of TLR-7 and RT but not single- NK cell and macrophage in the generation of 56 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH TARGETED THERAPY 57

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      TRANSLATIONAL RADIOBIOLOGY   '% $&)!*!$&#!&!!+()!**$'&         The Translational Radiobiology Group explores approaches for predicting how a cancer patient is likely to respond to   radiotherapy with a goal of developing biomarkers for the       ')"                                      future individualisation of treatment. The group studies     tumour hypoxia, tumour radiosensitivity and normal tissue radiosensitivity. Figure 1 molecular mechanisms underlying were negative. In 148 patients with outcome Gene by gene comparison of the radioresistance in order to develop methods to data, those with HPVα9-positive tumours had 26-gene signature in laryngeal identify patients with radioresistant tumours and better local control compared with α7 patients Group Leader and bladder cancer. Left: Median Can hypoxia biomarkers predict benefit primary outcome measures were regional expression of each gene is to develop new drugs targeting radioresistance in univariate (p<0.004) and multivariate (HR 1.54, Catharine from hypoxia-modifying therapy? control (ARCON) and overall survival (BCON). plotted for 157 ARCON laryngeal to combine with radiotherapy. Radiosensitivity is 95% CI 1.11-1.76, p=0.021) analyses. There was In head and neck and bladder cancer tumour Laryngeal tumours classified as hypoxic and 185 BCON bladder cancer a broad term applied to individuals, tissues and no difference in the median SF2 of α9 and α7 M.L. West hypoxia is associated with a poor prognosis. The (TLDA-HS high) showed greater benefit from patients. Right: Fold increase in cells – it is genetically determined. The main cervical tumours (n=63). In the cell lines, there gene expression between lowest Postdoctoral Fellows addition of hypoxia-modifying agents to ARCON than non-hypoxic tumours (TLDA-HS mechanism of action of radiation is via was also no statistically significant difference in five and highest five patient radiotherapy improves outcome and hypoxic low). Five-year regional control was 81% induction of unrepaired double strand DNA SF2 between the α9 and α7 positive cells. John Hall samples (■ =bladder;▲=larynx). Amanda Williamson status can predict treatment benefit, but there is (accelerated radiotherapy alone) versus 100% breaks. Defects in DNA damage response Janet Taylor1 no universal measure of hypoxia in the clinic. (CON) for TLDA-HS high (log rank P=0.009). pathways can have a large impact on Normal tissue radiosensitivity We are investigating whether a hypoxia- There was no significant improvement in radiosensitivity. However, the molecular The group is also interested in developing Clinical Fellows Jonathan Bernstein associated 26-gene signature reflects the five-year regional control for ARCON patients mechanisms underlying differences in biomarkers that predict a cancer patient’s risk of Navin Mani hypoxic status of a tumour and predicts benefit with TLDA-HS low. TLDA-HS did not predict radiosensitivity between different types of toxicity following radiotherapy. The group from hypoxia-modifying treatment. The benefit from CON in bladder cancer. Gene by tumours and tumours of the same histological co-ordinates the national RAPPER Scientific Officers 26-gene signature was derived from three gene analysis of expression of the 26-gene type are not well understood. The group is (Radiogenomics: Assessment of Joely Irlam-Jones independent head and neck microarray datasets signature showed a similar profile in both cancer interested in deriving gene signatures that reflect Polymorphisms to Predict the Effects of Helen Valentine and showed prognostic significance in multiple types (Spearman ρ=0.73, p<0.0001; Figure 1A) radiosensitivity in cervix and head and neck Radiotherapy) study (Rebecca Elliott, Helen Graduate Student cancers (head and neck, lung, breast). Multiplex but differential expression was much greater in cancers. Human papillomavirus (HPV) are Valentine), which involves a long-running Guy Betts markers such as gene signatures better reflect laryngeal cancer (Figure 1B). The median fold associated with the aetiology of both cancer collaboration with researchers at Cambridge the complex cellular response to hypoxia. Work increase was 57.2 (range 5.7-8.3 x 103; laryngeal) types and work by John Hall and Jan Taylor over University. Blood samples are collected from Masters Students carried out by Guy Betts showed that a multiplex and 43.3 (range 6.2-1.3 x 103; bladder). For the past year explored whether HPV genotype patients enrolled in late-phase clinical trials in Kenneth Oguejiofor Junaid Iqbal Wahid marker approach can account for intra-tumour TLDA-HS fold increase was 429.6 and 66.5 for influenced tumour radiosensitivity. patients undergoing potentially curative heterogeneity of hypoxia better than use of laryngeal and bladder cancer respectively. The radiotherapy. Total accrual at the end of Scientific Administrator single markers such as CA9 or pimonidazole 26-gene hypoxia signature predicts benefit from Most cervical carcinoma are HPV positive with November 2012 was 4,891 with enrolment Rebecca Elliott (Betts et al 2012; Eur J Cancer). hypoxia-modifying treatment in laryngeal HPVα9 (primarily HPV16) and HPVα7 (primarily running 9% ahead of target. The excellent cancer but not bladder cancer. In laryngeal HPV18) genotypes accounting for >80%. As recruitment to the study reflects the patients with α7 tumours tend to have poorer 1 joint with Applied Over the past year Amanda Williamson, Navin cancer inter-tumour variation in TLDA-HS is commitment of the UK radiotherapy Computational Biology & Bioinformatics Mani and Joely Irlam-Jones showed that the 6.5-fold greater than in bladder cancer. The prognosis, we investigated the relationship community with samples collected in 48 26-gene signature predicts benefit from large dynamic range may permit more distinct between HPV genotype with outcome centres. Analysis of a genome wide association hypoxia-modifying therapy in head and neck categorisation of patients with clinically relevant following radiotherapy and intrinsic study in the first 1,853 patients was completed in but not bladder cancer. Samples were available tumour hypoxia. The predictive ability of the radiosensitivity. HPV inno-LiPA25 was used to 2012. The work was helped by the from 157 T2-T4 laryngeal cancer and 185 T1-T4a TLDA-HS now needs testing prospectively in a genotype cervix tumour biopsies (n=197). Radiogenomics Consortium, which the group bladder cancer patients enrolled on the ARCON clinical trial of hypoxia modifying therapy in Papillocheck and qRT-PCR was used to helped to establish in 2009. Samples from the and BCON phase III randomised trials of head and neck cancer. genotype cervix cancer cell lines (n=16). Local Radiogenomics Consortium were used in a radiotherapy alone or with carbogen and progression-free survival following radiotherapy rapid replication of the top single nucleotide nicotinamide (CON) respectively. Customised HPV genotype does not influence tumour alone was assessed using log-rank and Cox polymorphisms (SNPs) identified. The work has TaqMan Low Density Arrays (TLDAs) were used radiosensitivity proportionate hazard analyses. Intrinsic shown there are many SNPs associated with to assess expression of the 26-genes using A long sought goal in radiotherapy related radiosensitivity was measured as surviving radiotherapy toxicity and that larger cohorts quantitative real-time PCR. The median research is to derive not only a reliable approach fraction at 2 Gy (SF2) using clonogenic assays. must be studied to identify them with certainty. normalised expression of the 26 genes was for assessing tumour hypoxia but also an assay Of the 197 tumours, 106 (53.8%) were positive used to derive a hypoxia score (TLDA-HS). that measures tumour intrinsic radiosensitivity. for HPV16, 28 (14.2%) for HPV18, 8 (4.8%) for Publications listed on page 77 Patients were classified as TLDA-HS low As radioresistance limits the number of cancer multiple genotypes, 9 (4.6%) for HPV45, 23 (≤median) or TLDA-HS high (>median). The patients cured, there is a need to understand the (11.7%) for other HPV genotypes and 22 (11.2%) 58 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH TRANSLATIONAL RADIOBIOLOGY 59

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    PATERSON INSTITUTE RESEARCH SERVICES, PUBLICATIONS AND ADMINISTRATION Retinal Pigment Epithelial cells (RPE1) were arrested at the G2/M transition using the CDK1 inhibitor RO-3306, then released for 20 minutes to allow the cells to go into mitosis. Cells were fixed and stained with DAPI (blue), tubulin antibody (green), and phospho-histone H3 antibody to stain the cells which have successfully entered mitosis (red). Image provided by Helen Whalley from the Cell Signalling Group. 60 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH PATERSON INSTITUTE 61

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    the facility comprises a range of flow cytometry imaged and analysed in a semi-automatic RESEARCH SERVICES sorters (BD Influx, BD Aria II and Jazz) and manner to mathematically describe the effect of www.paterson.man.ac.uk/research/ analysers (LSR Foretessa, Caliburs for 4 and 3 potential drug candidates. colour, and a FACS array). In the coming year an additional sorter and analyser will be installed to New methods are being developed for the address equipment demands. Within imaging imaging and assessment of biomarkers in a cell there are five large microscope systems population to model the heterogeneity of gene (Deltavision, time-lapse, low light imaging, expression. Systems are being developed for spinning disk confocal and macro-confocal), scanning populations of primary cells and then two scanners for digitising histology slides and numerically describing the data; in the coming two high content screening systems. Alongside year these techniques will be extended by using the equipment are IT solutions for image imaging flow cytometry, three dimensional The remit of the Research Services is to provide technology processing and analysis (Imaris, Huygens and culture and high resolution imaging. Definiens). A third histology scanner will be platforms that will support the research programmes of the added in 2013 and additional high content Systems have been put in place during 2012 to Institute. During the past year, there have been a number of screening systems and a super resolution allow as much of the equipment as possible to microscope for molecular imaging will also be be run in a standardised, calibrated manner. As significant changes as well as notable achievements in these introduced in response to the demands for the amount of work that requires good clinical facilities. The head of our Flow Cytometry service, Morgan translational and molecular imaging. practice (GCP) standardisation increases, the facility has responded by implementing daily Blaylock, moved on to a new position having spent the last five In the Flow Cytometry laboratory a member of and weekly checks as well as incorporating a years building up our in house FACS service to a very high staff has been appointed to help calibrate paper trail on all of the equipment. Training for Head of Research Services equipment and to assist researchers with sorting researchers has been increased so that standard. Morgan’s contribution to the Institute will be greatly samples quickly and efficiently. Usually sorting is investigations can be designed incorporating Stuart Pepper missed as he brought a great deal of expertise to his role. used to find sub-populations of cells but there is the new techniques that are available and with a also a requirement to sort cells singularly so that view to statistically analysing the results. variation in gene expression and other Head of Research Services from the Institute to our new facility in the parameters can be enumerated. Development The data load for the facilities grew again this Stuart Pepper University of Manchester’s Incubator building. over this year is ongoing to allow single cell sorts year to 17TB per annum, and the amount of raw An ambitious program of re-derivation of for molecular biology methods, which will be data that was then processed for analysis also A common theme this year has been to look at mouse lines is currently underway. increasingly in demand over the coming year increased dramatically. A new member of staff the remit of each service and see how different and will be achieved in conjunction with the will be appointed early in 2013 as a joint technology areas are best supported. One result Significant funding from the UK Research Molecular Biology Core Facility. With the histology/imaging post; the role will be helping of this process has been the merging of Partnership Investment Fund, that was awarded appointment of a new support post in the to develop numerical analysis of histological Advanced Imaging and FACS into a single unit. towards the end of 2012, is set to dramatically imaging facility, the main focus during 2013 is data and to fine tune the digitisation process. This change will give a more robust team expand the range of technology platforms towards automated imaging so that numerical structure to manage technology platforms that across the Research Services over the next analysis can be achieved at a faster rate. Development projects over the coming year support a broad variety of cell imaging couple of years. As the year comes to an end we include: the merging of data from histology with applications. The need for close cooperation are already beginning to plan how to use this The Institute has been using automated systems other sources; investigating novel histology between different technical areas has also been funding to maximum effect to support our to scan primary tissue for five years as this digitisation methods; sorting of single cells for recognised by the appointment of a post to link research programmes. process removes the possibility of user-induced clonal sequencing; development of protocols between Histology and Advanced Imaging to error and expedites analysis. This process for imaging flow cytometry; the introduction of provide a seamless user experience for employs pyramidal data formats, which is robotics and improved confocal optics for high quantitative analysis of histological specimens. Advanced Imaging and Flow common with satellite and mapping data so the content screening and the development of Cytometry Facilities tissue can be visualised at a range of procedures for the modelling of variations in cell The theme of collaboration has been evident in Steve Bagley, Jeff Barry, Mike Hughes, Abi magnifications. Over the last year software has populations and photon based super-resolution other facilities too; the Molecular Biology Core Johnson1, Kang Zeng1 been introduced for the mathematical microscopy by gated Stimulated Emission Facility has worked closely with the Clinical and 1 joined in 2012 modelling of multiple labels in primary tissue Depletion in order to visualise events at a Experimental Pharmacology (CEP) and Applied and tissue microarrays. In conjunction with the molecular level. Computational Biology and Bioinformatics During 2012, the management of the facility Histology Facility, procedures are being put in (ACBB) Groups to develop a workflow that that visualises and analyses cells in high detail, place to allow histological preparations to be allows robust expression profiling of single cells, and the facility that both sorts and analyses standardised for computerised analysis rather Biological Mass Spectrometry Facility while the Mass Spectrometry team have been thousands of cells quickly, have been merged in than relying on human inspection. This form of Duncan Smith, Yvonne Connolly, John Griffiths involved in a number of collaborations which order to provide a seamless array of tools for the translational imaging and mathematical have led to several publications. Other major researcher. Within the facility there have been a modelling is to be expanded in 2013 in order to The key role of the facility is to enhance the initiatives this year have been undertaken in the range of new developments and tools have allow more data to be processed at an increased research output of the Institute by facilitating Biological Resources Unit ( BRU) and Laboratory been introduced which will provide faster turnover. access to Proteomic workflows for all groups Services. Lab Services have had a new autoclave methods of translating analogue systems (the onsite. The facility spans areas of activity from and glass washer installed as well as a laboratory cell) to digital data. To advance development within the Drug routine service provision, project design and refurbishment that will allow a new solution Discovery Group, multi-well plates are now data interpretation through to the bespoke provision service to be developed. The BRU has To help answer the questions that our taken through high content screening so that collaborative development of novel workflows been busy relocating transgenic breeding work researchers pose, the flow cytometry section of responses to potential drugs and combinational designed to answer previously intractable drugs can be numerated. Multi-well plates are biological questions. 62 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH RESEARCH SERVICES 63

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    numerous mouse embryonic fibroblast lines. comprehensive and flexible service. We Cryopreservation has continued throughout specialise in the histological preparation and 2012 with approximately 4,200 embryos being analysis of a wide range of samples including archived. Sperm cryopreservation has also mouse model tissues, whole mouse embryo complimented the banking of strains during this preparations, cell preparations and human period. biopsies. The samples are either frozen or embedded in paraffin. Several special stains Experimental Services have been used including Pearl’s and Turnbull’s Blue together with the Quincke’s reaction for The experimental area has been supported by ferrous/ferric iron, Massons Trichrome for four experienced licensed technicians who have collagen, Masons Fontana, PAS and Neutral Red. delivered a varied range of non-surgical and In addition the use of special stains and the surgical procedures for 11 research groups. The subsequent downstream extraction of nucleic requirement for orthotopic models has acids from these tissues have also been increased this year; refinements have been evaluated. made for the delivery of cells via intra-femoral injection and skills adapted and increased for The Stem Cell Biology Group has recently found the melanoma models. that the first blood cells are generated from specific types of endothelial cells with As always, we have ensured that the highest haematopoietic potential and that the quality of care has been provided, and that transcription factors Gfi1 and Gfi1b are critical for We recently acted as a beta test site for a new Production and Experimental Services with a Home Office legislation has been adhered to, by the development of these cells. Using serial nLC MS (nano-liquid chromatography mass total of 15 Project Licences in place with primary performing daily health and welfare checks frozen sections of developing embryos, spectrometry) interface project named ‘Kevlar’. and secondary availability. The renewal of the along with extensive monitoring for animals together with immunohistochemistry (IHC), the This project has involved optimising the Transgenic Service Licence over the summer under procedure. group is trying to determine the spatial and interface between ultra-high performance nano has ensured production of progeny for the next temporal expression of these two genes, and separations and electrospray mass five years for the scientific programmes. The experimental area of the animal facility will investigate their potential co-expression, at both spectrometry. As a direct result of these be required to expand in the foreseeable future different sites of emergence of blood cells and optimisations, we have improved the sensitivity Transgenic Services as the transgenic area is decanted to the in the context of other markers of endothelial of our Orbitrap based workflows by eight-fold. Incubator facility; the planning for this will begin and blood cells. The beta test period involved the facility acting The transfer of the Paterson Institute transgenic in 2013. in an expert consulting and directing role. The lines to the additional site at the University of As the expansion of tissue biomarkers continues conclusion of this period resulted in the gift of a Manchester’s Incubator Building began at the The UK implementation of Directive 2010/63EU to gather pace, the construction of tissue full production unit of ThermoFisher Scientific’s end of the summer with the requirement to will take place on the 1st January, 2013, which microarrays (TMAs) has also seen a large ‘EASYSpray’ apparatus allowing the Institute to re-derive approximately 100 lines via embryo will lead to some local changes for the increase with high throughput, and accurate benefit from this technology on a permanent transfer; to date we have achieved 28 viable management of the Certificate of Designation TMA construction of tumour-specific and basis. productions and this will continue throughout which will become the Licence 2C for the custom arrays giving true representation. Even 2013. During this period the breeding and establishment. The requirement for a Training more challenging has been the continued During 2012, members of the facility were production at the Paterson Institute facility has and Competency Officer will ensure that all development of a truly representative prostate authors of seven peer reviewed publications been maintained to ensure that the research Personal Licence Holders are assigned the including main authorship on three papers goals of the scientific groups are not disrupted. correct category of authorisation and that in directly from our research and development house processes are streamlined. During 2013 pipeline. The first describes the development The arrival of Professor Richard Marais, as both we will be working towards the required and utilisation of enhanced two dimensional head of the Molecular Oncology Group as well retrospective review for all animals under peptide separation technologies in complex as Director of the Institute, required an extensive procedure to ensure that we best capture the proteome analysis (Griffiths et al). The second re-derivation programme of the group’s required information for statistical return. describes a novel approach to the LCMSMS transgenic lines from The Institute of Cancer analysis of SUMOylation (Chicoree et al). Research. During this period we transferred 20 Another study, (also Chicoree et al) describes live lines into our quarantine area and Histology the chemical derivitisation of Ubiquitin remnant maintained minimal breeding programmes due Garry Ashton, Caron Abbey, Michelle isopeptides to facilitate the generation of to space restrictions whilst transferring clean Greenhalgh (MCRC, Tissue Biobank) diagnostic ions enabling their screening in embryos into the facility’s transgenic area. David Millard (Histology /Tissue Biobank), advanced LCMS analysis. These three These programmes are now expanded and to Deepti Wilks (Haematological Malignancy workflows are now directly accessible by PICR date we have 31 lines actively producing Biobank) research groups. progeny for scientific use. As the Histology Facility continues to be This year has also seen a steady demand for developed, the range of services offered Biological Resources Unit embryonic stem cell (ES Cell) microinjections continues to grow. As a result the unit has seen with no less than 20 clones processed. exceptional demand this year from both existing Biological Resources Assistance has also been given to the Stem Cell groups and new groups, resulting in all services Biology Group for the re-derivation of offered being used extensively. The continued The animal facility has been fully occupied embryonic stem cell lines via blastocyst professional development and retention of staff throughout 2012 both for Transgenic immuno-surgery for the production of has ensured that we continue to offer a 64 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH RESEARCH SERVICES 65

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    core TMA. A new evaluation process has been The unit continues to process formalin-fixed The team also provides a centralised Legionella introduced allowing the more accurate paraffin- embedded (FFPE) and frozen samples monitoring service covering every tap in the reporting of TMAs and a second platform has for the MCRC Biobank. To date, samples from building. recently been introduced to cope with demand. over 3600 patients have been collected, with the largest number of samples from lung and Laboratory Services also supports the Institute The Leica Bondmax automated IHC platform, prostate cancer. There has also been a in other ways including monitoring and together with the open Biogenix i6000, significant increase in collections of colorectal restocking First Aid supplies, maintaining the continues to offer a high throughput, routine, and breast cancer samples as well as supply of clean lab coats and managing the two troubleshooting and antibody validation service. melanoma. Blood, bone marrow and plasma Institute X-ray film processors. Taken together Demand on all these services has seen a sharp (at various disease status time points) from over the changes made to Laboratory Services rise. Many antibodies have now been validated 250 haematological malignancy patients has during 2012 have allowed a small team to using the BondMax. In addition dual IHC and in also been collected. To date, over 50 research operate very efficiently, and support the Institute situ hybridisation have also been performed on projects have been approved. Samples have research programs across a range of services. In the platform. With increasing demand it is also been released for method validation 2013 the refurbishment programme will be anticipated that a second platform will be studies. The number and types of samples finished and the team will be able to further purchased early in 2013. The antibody validation processed for the Cancer Research UK Stratified extend the range of media and solutions service together with a high throughput image Medicine Programme has also increased. provided to the Institute. analysis service will continue to play a pivotal Genetic results are now routinely being fed back role in tissue biomarker research. to clinicians for these cancer disease types. Extensive quality control measures and the Molecular Biology Core Facility and Cancer In addition, we have continued to focus on the results from the Stratified Medicine Programme Research UK Microarray Service optimisation of pre capture variables and demonstrate the high quality of all the samples Stuart Pepper, Chris Clark, Yvonne Hey, methods of cell identification for laser capture processed by the unit. Rheanna Makorie2, Gill Newton, Julie Watson2, some drop in demand for this service we are still microdissection. Immunofluorescence and John Weightman1, Jodie Whittaker seeing more new projects then many other chromogenic IHC have both been used to A technique that we developed that allows both 1 joined in 2012, 2left in 2012 facilities. identify specific cell immunophenotypes. The the extraction of multiple cellular components use of holding buffers during capture and from the same piece of tissue, in addition to The Molecular Biology Core Facility provides a For expression profiling of individual genes, sample acquisition have been evaluated. allowing the morphology of the sample to be range of services to support the research groups rather than entire transcriptomes, the facility has Results have been encouraging, allowing the reported, has been used on several projects. In on site. These include diagnostic services, such two ABI7900 machines with a choice of 96 or extraction of good quality RNA and DNA. In addition studies looking at the possible use of as cell line authentication and mycoplasma 384 well formats. These systems, in conjunction addition, we have also successfully extracted molecular-friendly fixation and processing, screening, and research services for expression with our Probe Library, continue to be heavily good quality nucleic acids from both together with improvements in tissue profiling and DNA sequence analysis. used and provide a very cost effective route for macrodissected tissue and whole samples using stabilisation at time of sample acquisition, are small experiments on small numbers of morphological analysis as a guide to disease ongoing. Over the last year there has been an increase in samples. status. the use of Next Generation Sequencing (NGS) to support a wide variety of projects. In the last The need for cell line authentication has Laboratory Services year 19 NGS projects have been completed become widely accepted in the research Mark Craven, Andy Burns, Tony Dawson, including sequencing of total RNA, PolyA RNA, community. We have had a service up and Corinne Hand, John Higgins, Frances Hockin, microRNA, human exomes, Chromatin running for three years and have seen good Amy Moloney, Christine Whitehurst, Immunoprecipitation (ChIP) samples and yeast uptake by research groups. All cell lines in use in genomes. During this we year we have also the building can now be regularly authenticated The Laboratory Services area has undergone been part of a collaboration with the ACBB and by Short Tandem Repeat (STR) profiling, which is some major improvements during 2012, and CEP Groups to develop a workflow for the gold standard method for checking cell line our extensive refurbishment plan will continue sequencing RNA from single cells. This protocol identity. Combined with regular mycoplasma into 2013. At the start of the year the capabilities was initially tested on expression arrays and then screening this gives researchers the necessary of the service were enhanced by the addition of modified to be compatible with NGS. This confidence in the cell lines they are working a new glass washer and the replacement of an workflow is now available to groups on site and with. old autoclave with an updated model. This has will be of great interest for projects studying allowed the team to maintain the provision of circulating tumour cells or cancer The regular plasmid DNA preparation and sterile glassware and solutions at times of peak heterogeneity. Sanger sequencing services have continued to demand. function well. Whilst demand for minipreps has Alongside the demand for NGS we have also dropped over the last year we have seen a This year there has also been a shift in the team seen a continued need for microarray based continued high demand for DNA sequencing. operation to enhance the robustness of the expression analysis. The Cancer Research UK As more validation work from NGS projects service provided. The old system of having an microarray service, based in MBCF, has feeds through to the Sanger sequence service it individual lab aide allocated to each research processed around 500 arrays this year and we is likely that our 3130xl sequence instrument will group is being replaced by a centralised solution have seen 50 new projects either completed or no longer have sufficient throughput. This provision service. To facilitate development of initiated. With the advent of NGS for quantitative machine has given reliable service in the facility this service a lab space has been completely expression profiling it has been predicted that for over a decade but looking ahead to next year refurbished to provide a clean environment for microarray expression profiling will be we may need to expand our capacity for this making solutions and media, and a cold room is superseded; however, whilst we have seen service. being developed to allow bulk storage of media. 66 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH RESEARCH SERVICES 67

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    RESEARCH PUBLICATIONS Crispin Miller (page 14) Alkylpurine-DNA-N-glycosylase confers resistance Wilkinson, O.J., Latypov, V., Tubbs, J.L., Millington, Applied Computational Biology and to temozolomide in xenograft models of C.L., Morita, R., Blackburn, H., Marriott, A., Bioinformatics Group glioblastoma multiforme and is associated with McGown, G., Thorncroft, M., Watson, A.J., poor survival in patients. J Clin Invest 122, 253-266. Connolly, B.A., Grasby, J.A., Masui, R., Hunter, Refereed Research Papers C.A., Tainer, J.A., Margison, G.P., and Williams, Betts, G.N., Eustace, A., Patiar, S., Valentine, H.R., Crosbie, P.A., Watson, A.J., Agius, R., Barber, P.V., D.M. (2012) Irlam, J., Ramachandran, A., Merve, A., Homer, J.J., Margison, G.P., and Povey, A.C. (2012) Alkyltransferase-like protein (Atl1) distinguishes Moller-Levet, C., Buffa, F.M., Hall, G., Miller, C.J., Elevated N3-methylpurine-DNA glycosylase DNA alkylated guanines for DNA repair using cation-pi Harris, A.L., and West, C.M. (2012) repair activity is associated with lung cancer. Mutat interactions. Proc Natl Acad Sci U S A 109, 18755- Prospective technical validation and assessment of Res 732(1-2):43-6. 18760. intra-tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous Latypov, V.F., Tubbs, J.L., Watson, A.J., Marriott, cell carcinoma. Eur J Cancer 49, 156-165. doi: A.S., McGown, G., Thorncroft, M., Wilkinson, O.J., Karim Labib (page 18) 10.1016/j.ejca.2012.07.028. Epub 2012 Aug 27. Senthong, P., Butt, A., Arvai, A.S., Millington, C.L., Cell Cycle Group Povey, A.C., Williams, D.M., Santibanez-Koref, M.F., Hall, J.S., Taylor, J., Valentine, H.R., Irlam, J.J., Tainer, J.A., and Margison, G.P. (2012) Refereed Research Papers Eustace, A., Hoskin, P.J., Miller, C.J., and West, Atl1 regulates choice between global genome and De Piccoli, G., Katou, Y., Itoh, T., Nakato, R., C.M. (2012) transcription-coupled repair of O(6)-alkylguanines. Shirahige, K., and Labib, K. (2012) Enhanced stability of microRNA expression Mol Cell 47, 50-60. Replisome stability at defective DNA replication facilitates classification of FFPE tumour samples forks is independent of s phase checkpoint kinases. Millington, C.L., Watson, A.J., Marriott, A.S., exhibiting near total mRNA degradation. Br J Cancer Margison, G.P., Povey, A.C., and Williams, D.M. Mol Cell 45, 696-704. Iain Hagan (page 20) 107, 684-694. Cell Division Group (2012) Devrekanli, A., Foltman, M., Roncero, C., Sanchez- Harris, W.J., Huang, X., Lynch, J.T., Spencer, G.J., Convenient and efficient syntheses of Diaz, A., and Labib, K. (2012) oligodeoxyribonucleotides containing o Refereed Research Papers Hitchin, J.R., Li, Y., Ciceri, F., Blaser, J.G., Greystoke, Inn1 and Cyk3 regulate chitin synthase during Funaya, C., Samarasinghe, S., Pruggnaller, S., Ohta, B.F., Jordan, A.M., Miller, C.J., Ogilvie, D.J., and (6)-(carboxymethyl)guanine and o (6)-(4-oxo-4-(3- cytokinesis in budding yeasts. J Cell Sci. Sep 6. [Epub pyridyl)butyl)guanine. Nucleosides Nucleotides M., Connolly, Y., Muller, J., Murakami, H., Grallert, Somervaille, T.C. (2012) ahead of print]. A., Yamamoto, M., Smith, D., Antony, C., and The Histone Demethylase KDM1A Sustains the Nucleic Acids 31, 328-338. Tanaka, K. (2012) Oncogenic Potential of MLL-AF9 Leukemia Stem Kilkenny, M.L., De Piccoli, G., Perera, R.L., Labib, K., Transient Structure Associated with the Spindle Pole Cells. Cancer Cell 12;21(6):856. O’Hanlon, K.A., Margison, G.P., Hatch, A., and Pellegrini, L. (2012) Fitzpatrick, D.A., Owens, R.A., Doyle, S., and Jones, Body Directs Meiotic Microtubule Reorganization in A conserved motif in the C-terminal tail of DNA S. pombe. Curr Biol. 22(7):562-74. G.W. (2012) polymerase alpha tethers primase to the eukaryotic Molecular characterization of an adaptive response replisome. J Biol Chem 287, 23740-23747. Geoff Margison (page 16) to alkylating agents in the opportunistic pathogen Grallert, A., Connolly, Y., Smith, D.L., Simanis, V., Carcinogenesis Group Aspergillus fumigatus. Nucleic Acids Res 40, and Hagan, I.M. (2012) Sanchez-Diaz, A., Nkosi, P.J., Murray, S., and Labib, The S. pombe cytokinesis NDR kinase Sid2 activates 7806-7820. K. (2012) Refereed Research Papers Fin1 NIMA kinase to control mitotic commitment Abdu, K., Aiertza, M.K., Wilkinson, O.J., Grasby, J.A., The Mitotic Exit Network and Cdc14 phosphatase through Pom1/Wee1. Nat Cell Biol 14, 738-745. Uboldi, S., Bernasconi, S., Romano, M., Marchini, S., initiate cytokinesis by counteracting CDK Senthong, P., Povey, A.C., Margison, G.P., and Fuso Nerini, I., Damia, G., Ganzinelli, M., Marangon, Williams, D.M. (2012) phosphorylations and blocking polarised growth. E., Sala, F., Clivio, L., Chiorino, G., Di Giandomenico, EMBO J 31, 3620-3634. Synthesis of oligodeoxyribonucleotides containing S., Rocchi, M., Capozzi, O., Margison, G.P., Watson, Nic Jones (page 22) a conformationally-locked anti analogue of A.J., Caccuri, A.M., Pastore, A., Fossati, A., Cell Regulation Group O6-methyl-2’-deoxyguanosine and their van Deursen, F., Sengupta, S., De Piccoli, G., Mantovani, R., Grosso, F., Tercero, J.C., Erba, E., and Sanchez-Diaz, A., and Labib, K. (2012) recognition by MGMT and Atl1. Chem Commun D’Incalci, M. (2012) Other Publications (Camb) 48, 11214-11216. Mcm10 associates with the loaded DNA helicase at Characterization of a new trabectedin-resistant replication origins and defines a novel step in its Gozdecka, M., and Breitwieser, W. (2012) myxoid liposarcoma cell line that shows collateral activation. EMBO J 31(9):2195-206. The roles of ATF2 (activating transcription factor 2) Agnihotri, S., Gajadhar, A.S., Ternamian, C., Gorlia, sensitivity to methylating agents. Int J Cancer 131, in tumorigenesis. Biochem Soc Trans 40, 230-234. T., Diefes, K.L., Mischel, P.S., Kelly, J., McGown, G., 59-69. Thorncroft, M., Carlson, B.L., Sarkaria, J.N., Margison, G.P., Aldape, K., Hawkins, C., Hegi, M., and Guha, A. (2012) 68 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH RESEARCH PUBLICATIONS 69

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    Moore, D.J., Greystoke, A., Butt, F., Wurthner, J., Weston, R., Peeters, H., and Ahel, D. (2012) Growcott, J., Hughes, A., and Dive, C. (2012) ZRANB3 is a structure-specific ATP-dependent A Pilot Study Assessing the Prognostic Value of endonuclease involved in replication stress CK18 and nDNA Biomarkers in Severe Sepsis response. Genes Dev 26, 1558-1572. Patients. Clin Drug Investig 32, 179-187. Mustafi, R., Dougherty, U., Shah, H., Dehghan, H., Donald Ogilvie (Page 32) Gliksberg, A., Wu, J., Zhu, H., Joseph, L., Hart, J., Drug Discovery Group Dive, C., Fichera, A., Threadgill, D., and Bissonnette, M. (2012) Refereed Research Papers Both stromal cell and colonocyte epidermal Hamilton, N.M., Dawson, M., Fairweather, E.E., growth factor receptors control HCT116 colon Hamilton, N.S., Hitchin, J.R., James, D.I., Jones, cancer cell growth in tumor xenografts. S.D., Jordan, A.M., Lyons, A.J., Small, H.F., Carcinogenesis 33, 1930-1939. Thomson, G.J., Waddell, I.D., and Ogilvie, D.J. (2012) Rudin, C.M., Hann, C.L., Garon, E.B., Ribeiro de Novel steroid inhibitors of glucose 6-phosphate Oliveira, M., Bonomi, P.D., Camidge, D.R., Chu, Q., dehydrogenase. J Med Chem 55, 4431-4445. Giaccone, G., Khaira, D., Ramalingam, S.S., Ranson, M.R., Dive, C., McKeegan, E.M., Chyla, B.J., Dowell, Harris, W.J., Huang, X., Lynch, J.T., Spencer, G.J., B.L., Chakravartty, A., Nolan, C.E., Rudersdorf, N., Hitchin, J.R., Li, Y., Ciceri, F., Blaser, J.G., Greystoke, Busman, T.A., Mabry, M.H., Krivoshik, A.P., B.F., Jordan, A.M., Miller, C.J., Ogilvie, D.J., and Humerickhouse, R.A., Shapiro, G.I., and Somervaille, T.C. (2012) Angeliki Malliri (page 24) Greystoke, A., Dean, E., Saunders, M.P., Cummings, Gandhi, L. (2012) The histone demethylase KDM1A sustains the Cell Signalling Group J., Hughes, A., Ranson, M., Dive, C., and Renehan, Phase II study of single-agent navitoclax (ABT-263) oncogenic potential of MLL-AF9 leukemia stem A.G. (2012) and biomarker correlates in patients with relapsed cells. Cancer Cell 21, 473-487. Refereed Research Papers Multi-level evidence that circulating CK18 is a small cell lung cancer. Clin Cancer Res 18, Castillo-Lluva, S., Tan, C.T., Daugaard, M., biomarker of tumour burden in colorectal cancer. 3163-3169. Hitchin, J., Hamilton, N., Jordan, A., Lyons, A. and Sorensen, P.H., and Malliri, A. (2012) Br J Cancer 107, 1518-1524. Ogilvie, D. (2012) The tumour suppressor HACE1 controls cell Stovold, R., Blackhall, F., Meredith, S., Hou, J., Dive, A novel scalable and stereospecific synthesis of migration by regulating Rac1 degradation. Hou, J.M., Krebs, M.G., Lancashire, L., Sloane, R., C., and White, A. (2012) 3a- and 3b-amino-5a-andro-stan-17-ones and Oncogene May 21. doi: 10.1038/onc.2012.189. Backen, A., Swain, R.K., Priest, L.J., Greystoke, A., Biomarkers for small cell lung cancer: 3a- and 3b-amino-5a-pregnan-20-ones. Zhou, C., Morris, K., Ward, T., Blackhall, F.H., and neuroendocrine, epithelial and circulating tumour Tetrahedron Lett, 53, 2868-2872. Mack, N.A., Porter, A.P., Whalley, H.J., Schwarz, J.P., Dive, C. (2012) cells. Lung Cancer 76, 263-268. Jones, R.C., Khaja, A.S., Bjartell, A., Anderson, K.I., Clinical Significance and Molecular Characteristics of Circulating Tumor Cells and Circulating Tumor Vaughan, A.A., Dunn, W.B., Allwood, J.W., Wedge, and Malliri, A. (2012) Microemboli in Patients With Small-Cell Lung D.C., Blackhall, F.H., Whetton, A.D., Dive, C., and Peter Stern (page 34) beta2-syntrophin and Par-3 promote an apicobasal Immunology Group Rac activity gradient at cell-cell junctions by Cancer. J Clin Oncol. 30(5):525-32 Goodacre, R. (2012) differentially regulating Tiam1 activity. Nat Cell Biol Liquid chromatography-mass spectrometry Khoja, L., Backen, A., Sloane, R., Menasce, L., Ryder, calibration transfer and metabolomics data fusion. Refereed Research Papers 14, 1169-1180. Castro, F.V., Al-Muftah, M., Mulryan, K., Jiang, H.R., D., Krebs, M., Board, R., Clack, G., Hughes, A., Anal Chem 84, 9848-9857. Blackhall, F., Valle, J.W., and Dive, C. (2012a) Drijfhout, J.W., Ali, S., Rutkowski, A.J., Kalaitsidou, A pilot study to explore circulating tumour cells in Zhou, C., Simpson, K.L., Lancashire, L.J., M., Gilham, D.E., and Stern, P.L. (2012) Caroline Dive (page 26) pancreatic cancer as a novel biomarker. Br J Cancer Walker, M.J., Dawson, M.J., Unwin, R.D., Regulation of autologous immunity to the mouse Clinical and Experimental Pharmacology 106, 508-516. Rembielak, A., Price, P., West, C., Dive, C., 5T4 oncofoetal antigen: implications for and Whetton, A.D. (2012) immunotherapy. Cancer Immunol Immunother 61, Refereed Research Papers Khoja, L., Lorigan, P., Zhou, C., Lancashire, M., Statistical Considerations of Optimal Study Design 1005-1018. Carter, L., Metcalf, R., Blackhall, F.H., Dive, C., and Booth, J., Cummings, J., Califano, R., Clack, G., for Human Plasma Proteomics and Biomarker Krebs, M.G. (2012) Hughes, A., and Dive, C. (2012b) Discovery. J Proteome Res 11(4):2103-13. Castro, F.V., McGinn, O.J., Krishnan, S., Marinov, Biology and clinical relevance of circulating tumour Biomarker Utility of Circulating Tumor Cells in G., Li, J., Rutkowski, A.J., Elkord, E., Burt, D.J., cells. J Thorac Dis 4, 453-455. Metastatic Cutaneous Melanoma. J Invest Holland, M., Vaghjiani, R., Gallego, A., Saha, V., Dermatol. Dec 6. doi: 10.1038/jid.2012.468. [Epub and Stern, P.L. (2012) Cummings, J., Zweifel, M., Smith, N., Ross, P., Ivan Ahel (page 30) 5T4 oncofetal antigen is expressed in high risk of ahead of print]. DNA Damage Response Group Peters, J., Rustin, G., Price, P., Middleton, M.R., relapse childhood pre-B acute lymphoblastic Ward, T., and Dive, C. (2012) Krebs, M.G., Hou, J.M., Sloane, R., Lancashire, L., leukemia and is associated with a more invasive and Evaluation of cell death mechanisms induced by Refereed Research Papers chemotactic phenotype. Leukemia 26(7):1487-98. Priest, L., Nonaka, D., Ward, T.H., Backen, A., Clack, Dunstan, M.S., Barkauskaite, E., Lafite, P., Knezevic, the vascular disrupting agent OXi4503 during a G., Hughes, A., Ranson, M., Blackhall, F.H., and phase I clinical trial. Br J Cancer 106, 1766-1771. C.E., Brassington, A., Ahel, M., Hergenrother, P.J., McGinn, O.J., Marinov, G., Sawan, S., and Stern, Dive, C. (2012) Leys, D., and Ahel, I. (2012) Analysis of circulating tumor cells in patients with P.L. (2012) Dean, E., Greystoke, A., Ranson, M., and Structure and mechanism of a canonical poly(ADP- CXCL12 receptor preference, signal transduction, non-small cell lung cancer using epithelial marker- ribose) glycohydrolase. Nat Commun 3, 878. Dive, C. (2012) dependent and -independent approaches. J biological response and the expression of 5T4 Biomarkers of cell death applicable to early clinical Thorac Oncol 7, 306-315. oncofoetal glycoprotein. J Cell Sci Sep 6. [Epub trials. Exp Cell Res 318, 1252-1259. Matic, I., Ahel, I., and Hay, R.T. (2012) ahead of print]. Reanalysis of phosphoproteomics data uncovers ADP-ribosylation sites. Nat Methods 9, 771-772. 70 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH RESEARCH PUBLICATIONS 71

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    Yoshihara, S.I., Takahashi, H., Nishimura, N., Nullin Divecha (page 36) Other Publications A., Ashworth, A., Henri Stern, M., and Reis-Filho, Naritsuka, H., Shirao, T., Hirai, H., Yoshihara, Y., Mori, Inositide Laboratory Lynch, J.T., Harris, W.J., and Somervaille, T.C. (2012) J.S. (2012) K., Stern, P.L., and Tsuboi, A. (2012) LSD1 inhibition: a therapeutic strategy in cancer? A whole-genome massively parallel sequencing 5T4 Glycoprotein Regulates the Sensory Input- Refereed Research Papers Expert Opin Ther Targets 16, 1239-1249. analysis of BRCA1 mutant oestrogen receptor- Dependent Development of a Specific Subtype of Ding, Y., Ndamukong, I., Zhao, Y., Xia, Y., negative and -positive breast cancers. J Pathol Newborn Interneurons in the Mouse Olfactory Riethoven, J.J., Jones, D.R., Divecha, N., and 227(1):29-41. Bulb. J Neurosci 32, 2217-2226. Avramova, Z. (2012) Richard Marais (page 40) Divergent Functions of the Myotubularin (MTM) Molecular Oncology Group Rae, J., Viros, A., Hayward, R., Bennett, D.C., Other Publications Homologs AtMTM1 and AtMTM2 in Arabidopsis Dhomen, N., Longley, B.J., Reis-Filho, J.S., and Stern, P.L., van der Burg, S.H., Hampson, I.N., thaliana: Evolution of the plant MTM family. Plant J Refereed Research Papers Marais, R. (2012) Broker, T.R., Fiander, A., Lacey, C.J., Kitchener, H.C., 70 (5):866-78. Andreadi, C., Cheung, L.K., Giblett, S., Patel, B., Jin, (V600E)Braf::Tyr-CreERT2::K14-Kitl mice do not and Einstein, M.H. (2012) H., Mercer, K., Kamata, T., Lee, P., Williams, A., develop superficial spreading-like melanoma: Therapy of human papillomavirus-related disease. Jones, D.R., Foulger, R., Keune, W.J., Bultsma, Y., McMahon, M., Marais, R., and Pritchard, C. (2012) keratinocyte Kit ligand is insufficient to “translocate” Vaccine 30 Suppl 5, F71-82. and Divecha, N. 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A. (2012) 76 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH RESEARCH PUBLICATIONS 77

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    Dibb, M., Han, N., Choudhury, J., Hayes, S., Other Publications Valentine, H., West, C., Ang, Y.S., and Sharrocks, West, C.M., Dunning, A.M., and Rosenstein, PICR THESES A.D. (2012) B.S. (2012) The FOXM1-PLK1 axis is commonly upregulated in Genome-wide association studies and prediction oesophageal adenocarcinoma. Br J Cancer 107, of normal tissue toxicity. Semin Radiat Oncol 1766-1775. 22, 91-99. Hall, J.S., Taylor, J., Valentine, H.R., Irlam, J.J., Active Patents Eustace, A., Hoskin, P.J., Miller, C.J., and West, WO Application No: PCT/EP2010/070583 C.M. (2012) Applicant: CR Technology Ltd. Hypoxia tumour Enhanced stability of microRNA expression markers. facilitates classification of FFPE tumour samples exhibiting near total mRNA degradation. Br J Ahmet Acar Lilly Sommer Stromal-Tumour Interactions Group Inositide Laboratory Cancer 107, 684-694. Additional Publications The role of Notch Signalling in Human Breast Nuclear phosphoinositides - Novel interactors Hughes, C., Iqbal-Wahid, J., Brown, M., Shanks, Carcinoma-associated Fibroblasts and the regulation of the transcriptional Refereed Research Papers J.H., Eustace, A., Denley, H., Hoskin, P.J., West, C., coactivator TAF3 Griffiths, J.R., Perkins, S., Connolly, Y., Zhang, L., Clarke, N.W., and Gardner, P. (2012) Julian Blaser Holland, M., Barattini, V., Pereira, L., Edge, A., FTIR microspectroscopy of selected rare diverse Leukaemia Biology Group/ Inositide Laboratory Chong Tan Ritchie, H., and Smith, D.L. (2012) sub-variants of carcinoma of the urinary bladder. J The utilisation of shRNA screens to investigate Cell Signalling Group The utility of porous graphitic carbon as a stationary Biophotonics Nov 2. doi: 10.1002/jbio.201200126. Elvan Boke the role of phosphoinositide modulator genes A novel regulation of Tiam1 protein stability by phase in proteomics workflows: Two-dimensional [Epub ahead of print]. in ALL ubiquitylation and its role in hepatocyte growth chromatography of complex peptide samples. J factor-induced cell scattering Chromatogr A. Lo, L., Valentine, H., Harrison, J., Hayes, S., Welch, I., Elvan Boke Pritchard, S., West, C., and Ang, Y. (2012) Cell Division Linton, K., Howarth, C., Wappett, M., Newton, G., Tissue factor expression in the metaplasia- An interplay between PP1 and PP2A Lachel, C., Iqbal, J., Pepper, S., Byers, R., Chan, W.J., adenoma-carcinoma sequence of gastric cancer in phosphatases coordinates mitotic progression and Radford, J. (2012) a European population. Br J Cancer 107, 1125-1130. in fission yeast Microarray Gene Expression Analysis of Fixed Archival Tissue Permits Molecular Classification and Lyons, A.J., West, C.M., Risk, J.M., Slevin, N.J., William Harris Identification of Potential Therapeutic Targets in Chan, C., Crichton, S., Rinck, G., Howell, D., and William Harris Leukaemia Biology Group Diffuse Large B-Cell Lymphoma. J Mol Diagn Shaw, R.J. (2012) Regulation of Self Renewal in Human MLL-AF9 14(3):223-32. Osteoradionecrosis in head-and-neck cancer has a Acute Myeloid Leukaemia Stem Cells distinct genotype-dependent cause. Int J Radiat Potier, D.N., Griffiths, J.R., Unwin, R.D., Walker, Oncol Biol Phys 82, 1479-1484. Sarah Hughes M.J., Carrick, E., Willamson, A.J., and Whetton, Clinical and Experimental Pharmacology A.D. (2012) Mirza, A., Foster, L., Valentine, H., Welch, I., West, A pre-clinical and clinical evaluation of the An assessment of peptide enrichment methods C.M., and Pritchard, S. (2012) specific endothelin-A antagonist zibotentan in employing mTRAQ quantification approaches. Anal Investigation of the epithelial to mesenchymal prostate cancer Chem 84, 5604-5610. transition markers S100A4, vimentin and Snail1 in gastroesophageal junction tumors. Dis Esophagus Timurs Maculins Timurs Maculins Weekes, M.P., Antrobus, R., Talbot, S., Hor, S., Oct 19. doi: 10.1111/j.1442-2050.2012.01435.x. Cell Cycle Simecek, N., Smith, D.L., Bloor, S., Randow, F., and [Epub ahead of print]. Regulation of the replisome by the ubiquitin Lehner, P.J. (2012) ligase SCFDia2 Proteomic Plasma Membrane Profiling Reveals an Talbot, C.J., Tanteles, G.A., Barnett, G.C., Burnet, Essential Role for gp96 in the Cell Surface N.G., Chang-Claude, J., Coles, C.E., Davidson, S., Avinash Patel Expression of LDLR Family Members, Including the Dunning, A.M., Mills, J., Murray, R.J., Popanda, O., Cell Division Group LDL Receptor and LRP6. J Proteome Res 11, Seibold, P., West, C.M., Yarnold, J.R., and Symonds, Interrogation of fission yeast polo kinase with 1475-1484. R.P. (2012) quantative phosphoproteomics and chemical A replicated association between polymorphisms genetics Other Publications near TNFalpha and risk for adverse reactions to Evans, C., Noirel, J., Ow, S.Y., Salim, M., Pereira- Lilly Sommer radiotherapy. Br J Cancer 107, 748-753. Andrzej Rutkowksi Medrano, A.G., Couto, N., Pandhal, J., Smith, D., Immunology Group Pham, T.K., Karunakaran, E., Zou, X., Biggs, C.A., Zhou, C., Simpson, K.L., Lancashire, L.J., Investigation of a novel locus encoding a and Wright, P.C. (2012) Walker, M.J., Dawson, M.J., Unwin, R.D., putative paralogue of 5T4 oncofoetal An insight into iTRAQ: where do we stand now? Rembielak, A., Price, P., West, C., Dive, C., glycoprotein Anal Bioanal Chem 404, 1011-1027. and Whetton, A.D. (2012) Statistical Considerations of Optimal Study Design Horimoto, Y., Polanska, U.M., Takahashi, Y., and for Human Plasma Proteomics and Biomarker Orimo, A. (2012) Discovery. J Proteome Res 11(4):2103-13. Emerging roles of the tumor-associated stroma in promoting tumor metastasis. Cell Adh Migr 6, Chong Tan 193-202. 78 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH THESES 79

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    EXTERNAL SEMINAR SPEAKERS 2012 The seminar series that we run is vital for the Institute, David Neuhaus Breakthrough Breast MRC Laboratory of Molecular Cancer Research Unit connecting world-class researchers across the broad spectrum Biology, Cambridge Seminar Series 2012 of cancer research. We had another successful year for Alexandra Newton Suresh Alahari scientific interaction with an excellent set of internationally University of California at San Diego, USA LSU Health Science Center New Orleans, USA renowned speakers visiting the Institute. In its fourth year, The Mark O’Connor Richard Clarkson Breakthrough Breast Cancer Research Unit seminar series AstraZeneca Cardiff University continues to produce an outstanding range of speakers. The Peter Parker Andrew Ewald postdoctoral researchers at the Institute also give weekly London Research Institute Johns Hopkins University School of seminars which are very well attended and help to integrate the Medicine, USA Gislene Pereira entire cancer research efforts of the Institute. German Cancer Research Centre Ingunn Holen (DKFZ), Heidelberg The University of Sheffield Dario Alessi Jorrit Enserink Erik Sahai Stephen Hursting MRC Protein Phosphorylation Unit, University Institute of Microbiology, Oslo University London Research Institute University of Texas at Austin, USA of Dundee Hospital, Norway JJ Schuringa Elad Katz Peter Andrews Opher Gileadi University of Groningen, The Netherlands Breakthrough Breast Cancer Research Unit, The University of Sheffield Nuffield Department of Clinical Medicine, University of Edinburgh University of Oxford Charles Swanton Buzz Baum London Research Institute Richard Pestell MRC Laboratory for Molecular Cell Biology, Ulrike Gruenberg Director of the Kimmel Cancer Centre at University College London Department of Biochemistry, University Nic Tapon Jefferson, USA of Oxford London Research Institute Olivier Bernard Matthew Smalley Institut Gustave Roussy, France Kamil Kranc Ali Tavassoli European Cancer Stem Cell Research Institute, University of Glasgow University of Southampton Cardiff University Christoph Borner Institute of Molecular Medicine and Cell James Larkin Helle Ulrich Marc Tischkowitz Research, University of Freiburg, Germany The Royal Marsden Hospital London Research Institute University of Cambridge Bob Brown Massimo Lopes Christopher Ward Christine Watson Imperial College London Institute of Molecular Cancer Research, The University of Manchester University of Cambridge University of Zurich, Switzerland Julian Carretero Heidi Welch Zena Werb University of Valencia, Spain Laura Machesky The Babraham Institute University of California, San Francisco, USA The Beatson Institute for Cancer Research Alessandro Costa London Research Institute Thomas Milne The Weatherall Institute of Molecular Medicine, Aled Clayton University of Oxford Cardiff University School of Medicine Jacques Neefjes Ruud Delwel The Netherlands Cancer Institute, Amsterdam Erasmus Medical Center, The Netherlands 80 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH EXTERNAL SEMINAR SPEAKERS 2012 81

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    POSTGRADUATE EDUCATION www.paterson.man.ac.uk/education A well-supported graduate programme is of fundamental applications competing for approximately 4-8 Education Committee 2012 places each year. Interviews are typically Our goal is for every student to have a project importance to a research institute such as the Paterson, both to conducted over a two-day period in January. that is both achievable and intellectually train the researchers of tomorrow, and for the valuable demanding. Projects and students are All our students benefit from access to monitored by the Education Committee which contribution made by our students to the laboratories that they advanced state-of-the-art facilities including makes sure that the proposed plan of research is are working in. In 2012, we welcomed another eight graduate advanced imaging, biological mass suitable, and that progress follows an spectrometry, microarrays, flow cytometry, appropriate timeline throughout the course of students from around the world to join our PhD programme, histology and next generation sequencing. Our the studentship. Various assessments Julie Edwards research groups offer PhD studentships and throughout the PhD programme, including Postgraduate working in fields as diverse as yeast genetics, stem cell biology projects covering the entire breadth of research regular talks, progress meetings and written Education Manager and clinical research. During the course of the year, PhDs were within the Institute. reports are an essential key in ensuring awarded to nine graduate students and one clinical fellow. successful completion of the PhD programme. Fellowships in Clinical Pharmacology Research Valerie Kouskoff (Chair, Education Committee) The Paterson graduate programme players in cancer research, and students are In order to help train the next generation of Anne Armstrong1 We aim for each student to receive high quality expected to attend all of these external clinical pharmacologists with expertise in Fiona Blackhall training in scientific research through an seminars. The speakers are internationally oncology, the Paterson Institute, in Richard Cowan intellectually demanding but achievable renowned scientists and we consider it essential collaboration with the MCRC and AstraZeneca, Julie Edwards research programme. Each project is peer- that our students are exposed to outstanding established a fellowship scheme in Clinical Dave Gilham reviewed in advance and monitored throughout work from the leaders in different disciplines, Pharmacology Research in 2007. The Ian Hampson Crispin Miller fellowships are open to applicants who have Sacha Howell1 Postgraduate Tutor the course of their studies, via a mixture of which will give them a broad understanding of seminars, written reports, and progress and many aspects of cancer research and basic obtained, or are close to obtaining, their Karim Labib until September planning meetings. These are designed not only biology. In addition, we hold a series of weekly Completed Certificate of Specialist training Was Mansoor1 to provide formal points at which progress (of postdoctoral research seminars that the (CCST) in Medical Oncology. Angeliki Malliri both the student and the project) can be students attend, while students themselves are Crispin Miller (Postgraduate Tutor until monitored, but also to help develop the asked to give talks at key points during their PhD Each clinical Pharmacology Research Fellow September) presentation skills that are so fundamental to the studies. They also have opportunities to present undertakes a three-year PhD project, which Donald Ogilvie majority of careers in science and elsewhere. their work at lab meetings within the Institute. provides training in biomarker discovery, Vaskar Saha2 Graduate training is monitored by an Education method development/validation, and in clinical Tim Somervaille Committee, which features Group Leaders, The annual Paterson Institute Colloquium, held trial methodology. During tenure, at the Christie Ian Waddell (Postgraduate Tutor senior clinicians and scientists, and student in September, is an excellent opportunity for our NHS Foundation Trust/Paterson Institute, the from October) representatives (see below). Following the new intake of students to meet other post holders receive clinical supervision from Catharine West Ian Waddell Malcolm Ranson, and laboratory- based training Caroline Wilkinson Postgraduate Tutor directive from the University of Manchester established PhD students, as well as other from October following a QAA (Quality Assurance Agency) members of the Institute, including Group from Caroline Dive in CEP (in collaboration with Richard Marais1 (Ex-Officio Member) audit, students registering from September 2011 Leaders, Postdoctoral Fellows, and Scientific MCRC colleagues); at AstraZeneca they receive onwards have also been appointed a second Officers. This forum provides up to date training in clinical trials management, regulatory Student Representatives co-supervisor who is able to provide additional scientific presentations by Group Leaders and interaction, translational research through Emily Holmes2 advice and consultation on academic and second year PhD students together with poster project management and attendance at Hadir Marei non-academic matters. Each student is also presentations by Postdoctoral Fellows, Scientific investigator meetings. Clinical training includes Danish Memon assigned an advisor (similar to a personal tutor Officers and other PhD students. one research clinic per week, training in clinical 1 on an undergraduate programme) whose role is trial design and methodology, ICH-GCP, EU Joined in 2012 2 to provide impartial support and advice. Further PhD studentships Directives and research governance. Biomarker Stepped down in 2012 support is also available individually from the All our CR-UK core funded studentships are four method development and application take Education Committee Chair Postgraduate Tutor, years in duration, and consist of an approved place on both sites in all projects, with mutual Postgraduate Manager, or collectively as the research project in one of our research groups. benefit as each Fellow brings newly acquired Education Committee Administration Group. Some students have joint supervisors in different knowledge to each site. Regular meetings take groups, fostering collaborations and exposing place between the Fellows, their supervisors, as The Paterson Institute runs an external seminar the students to different disciplines. Recruitment well as other staff members involved in the series featuring talks from many of the key is highly competitive, with hundreds of project, ensuring true collaboration and a ‘joined-up’ approach. 82 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH POSTGRADUATE EDUCATION 83

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    OPERATIONS Finance & Purchasing Following analysis of the performance of the Margaret Lowe, David Jenkins, Denise Owen, health and safety management systems, Muhammad Raja, Debbie Trunkfield additional Key Performance Indicators (KPIs) were introduced which will provide better The Finance Department is responsible for information about the performance of the providing a comprehensive purchasing, travel management system. The KPIs selected include and finance service to the Research Groups and pro-active and reactive elements of the Service Units within the Paterson Institute. The management system. procurement team continues to work with the research groups to identify savings in A number of compliance surveys were consumable spending. undertaken to ensure that the Institute meets its legal duties for biosafety-associated legislation; The Operations Department provides the necessary In May, the University introduced a new Travel this included schedule five of the Anti-Terrorist Management System, Egencia, to cover all Crime and Security Act, Misuse of Drugs infrastructure and services to facilitate the running of the corporate travel needs. This enables the legislation, the Human Tissue Act and the Institute. Finance and purchasing, as well as Estates and university team to capture all the necessary data Chemical Weapons Convention. to allow them to have up-to-date management Logistics, fall under the leadership of Margaret Lowe, while information and identify trends for the Central The Paterson Institute was visited by two Stuart Pepper oversees IT as well as Health and Safety; Rachel Procurement Office to negotiate route/volume enforcing authorities in 2012. In April the Greater deals with airlines and hotels. Egencia has a Manchester Fire and Rescue Service carried out Powell is head of HR and Caroline Wilkinson is responsible for all price match promise on all hotels and flights. a fire safety audit. Encouragingly, no specific Margaret Lowe Head of Finance aspects of Scientific Administration. This year we said goodbye Our procurement team has worked closely with recommendations were made in relation to fire staff to ensure their needs are met and all safety legislation. In July, the Environment to Amy Weatheritt but welcomed Siana Peters to the bookings are authorised immediately. Agency carried out a radiation audit which Administration team. Gillian Campbell was recruited to the found the Institute to be compliant and did not In addition to the everyday purchasing and lead to any remedial actions. Scientific Administration team, in the newly created post of finance procedures, we have continued to Grants Advisor, to help the Institute’s scientists to secure support the research groups by providing Training has been provided for new starters in effective and efficient professional advice when the form of induction training, and for additional external funding which will support the full breadth of costing new research proposals and laboratory workers who are working with research that we would like to undertake. administering existing grants. The Institute has biological agents and hazardous chemicals. For been successful in securing several new this purpose, a new training record form was Stuart Pepper external grant awards that will be activated developed and is being utilised by staff. Head of Research Services Administration Department Estates in 2013. Siana Peters (EA to the Director), Ruth Perkins, Steve Alcock, Graham Hooley, Tony Woollam Finally a review of respiratory protective Steven Morgan Health & Safety equipment is being undertaken, in light of A number of small projects have been carried Colin Gleeson updated guidance from the Health and Safety Siana Peters joined the Institute in early 2012 to out in order to improve certain areas and ensure Executive. This will continue into 2013. provide personal assistance to Richard Marais in that the building remains fit for purpose. A new intranet based genetic modification (GM) his post as Director of The Paterson Institute as Improvements to the Laboratory Services area risk management system was developed, tested Human Resources well as overseeing the management of the are ongoing which will allow for the expansion and then introduced throughout the Institute in Rachel Powell, Laura Jones, Julie Jarratt administration team. The department has of these facilities in order to incorporate the new 2012. This comprises of risk assessment forms assisted with the organisation of several events media preparation service. Other improvements for assessing GM work and an integrated Over the past year the Human Resources Rachel Powell over the course of the year, including the included the conversion of an old storage area document management system. The new Department has continued to successfully Head of Human Resources Paterson Colloquium and quinquennial reviews, into a tissue culture facility and the system should enable efficient risk provide a high quality professional and as well as running the main Institute reception refurbishment of laboratory and office space to management of GM work and facilitate our pro-active HR service to the Institute. This and coordinating catering requirements. accommodate the new Director and his group. compliance with the Genetically Modified includes providing advice and guidance to Administrative support is also provided for the Organisms (Contained Use) Regulations. managers and staff on all employment-related external seminar series. The 2012 series has The Estates team endeavour to identify matters such as recruitment, policy guidance, been a great success and the 2013 programme sustainable solutions when implementing new A number of pro-active initiatives were legislation and best practice. is already in place. This will aim to provide a schemes together to help reduce the Institute’s undertaken throughout the year to help varied programme of national and international carbon footprint whenever possible. safeguard health and safety. These included the The last twelve months has seen the successful speakers, serving to foster collaboration and The team has been pro-active throughout the annual self–assessment biosafety survey, the appointments of 51 individuals who will encourage positive interaction within the wider year, attending to many legislative requirements formal laboratory inspection programme, a endeavour to complement and enhance the Caroline Wilkinson scientific community. including Legionella best practices and fire review of UV personal protective equipment work of the Institute. Throughout the year we Scientific Operations alarm testing. A continual objective is to acquire and a well-being back care programme, which always look for ways to improve our processes Manager Details can be found at: new skills and refresh existing ones and with this and the service that we provide to staff. This included sessions in Alexander technique and www.paterson.man.ac.uk/Seminars/. in mind, team members have attended a Pilates. Some remedial actions were generated year, for example, we have improved our new number of relevant courses to keep their and completed successfully. The back care starter process. We ensure that all new knowledge up to date with current working programme received highly positive feedback employees attend an HR induction on their first practices and changing legislation. from the participants. day. It is important that an individual is given the best start and that their time at the Institute is an 84 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH OPERATIONS 85

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    kits and Qiagen). This year we have had a patient benefit from publicly funded research Bio-Rad fridge/freezer installed which operates worldwide by advancing research discoveries on a password and scanning system. The into development with pharmaceutical and department works closely with all the research biotechnology parties. groups and helps out where necessary, for example by tracing and confirming delivery of At CRT we bridge the gap between cutting edge goods with suppliers. The team also manages academic research and industrial development the moving of heavy equipment or furniture, of cancer therapeutics and diagnostics. We and the reconfiguration of meeting rooms for achieve this by working closely with prestigious numerous events. international research institutes, such as the Paterson Institute, and with funding bodies to Scientific Operations develop, protect and commercialise oncology Caroline Wilkinson, Gillian Campbell related discoveries. Core activities of business development and drug discovery are supported Scientific administration is overseen by the by specialists, integrated in the business with Scientific Operations Manager, Caroline expertise in patents, legal, finance and Wilkinson, who provides support to the Director marketing. Our exclusive focus in oncology informed and enjoyable experience from the a strategy for mobile devices and during the year in order to facilitate the day-to-day running of provides an unrivalled depth of knowledge and beginning. we procured in excess of 50 mobile devices in the Institute. The Scientific Operations Manager experience in cancer-specific translational the form of tablets and smart phones to give provides reports for, and liaises with, a number development and commercialisation. By We have continued our joint partnership service users as much freedom and flexibility of sources, including Cancer Research UK and arrangement with The University of Manchester, working with the unions which has resulted in with their off-site computing as possible. A the University of Manchester, as well as editing CRT owns and is responsible for the the agreement of several new policies, including further programme of work was developed to publications such as the Annual Scientific development and commercialisation of the Staff Training and Development Policy, and set up and support staff based at a satellite office Report and Institute Newsletter and writing intellectual property arising from Cancer the revision of existing ones such as the on the main University campus. updates for the intranet and external website. Research UK funded research at The University Mentoring Framework and the Flexible Working Talks and tours are also provided for a packed of Manchester (including the Paterson Institute). Policy. Over the next year the main focus for the Plans to upgrade the high performance cluster programme of fundraiser events throughout the HR department will be the successful (HPC) have been developed and will be year. Our relationship with the Paterson Institute implementation of the online probationary implemented in the first quarter of 2013. The reflects the specific requirements of the process system and a review of the current changes will provide the computing power that Further responsibilities include organising scientist, the Institute itself, Cancer Research UK contribution review process. is necessary to analyse the large data sets being Quinquennial and mid-term reviews, liaising and the individual project. To effectively facilitate generated by the latest technological platforms. with library staff at the University of Manchester these requirements and interactions, Martyn Information Technology to secure access to appropriate electronic Bottomley, a CRT Business Manager, is based Malik Pervez, Brian Poole, Steve Royle, Zhi Logistics journals, maintaining detailed databases of the on-site dedicated to working closely with the Cheng Wang, Matthew Young Maurice Cowell, Sedia Fofana, Antony Griffin, Institute’s publications, arranging a weekly staff at the Institute and also The University of Phil Jackson, Andrew Lloyd, Jonathan Lloyd seminar series presented by our post-doctoral Manchester. Martyn offers access to oncology The IT department provides a balance of quick research fellows and organising the Institute focused expertise in technology evaluations, response user-facing services and strategic The Logistics facility plays a vital role in colloquium. For the second year running, the patent applications and management, funding developments to ensure that the IT supporting the research carried out at the Colloquium was held at Lancaster University for development, commercialisation, drug infrastructure remains fit for purpose as the Institute. This includes the receipting, checking, with over 100 Institute scientists enjoying two discovery, market intelligence, and project Institute develops. During 2012, much of the booking in and distribution of goods ordered by days of scientific talks and poster sessions. management. He also works closely with UMIP, strategic development has been to enhance the staff as well as the collection and removal of All grant submissions submitted by our The University of Manchester technology support for mobile computing, along with waste. The team also provides assistance with researchers are screened for the appropriate transfer organisation. CRT continues to work upgrades to both the hardware and software moving equipment and supplies and therefore ethical approvals as well as the ability of the very closely with the Drug Discovery that comprises the core IT infrastructure of the helps facilitate internal rearrangements and the Institute to accommodate the proposed Laboratories based at the PICR to facilitate the building. arrival of new groups. A number of larger liquid programme of work. This year, the new post of development of small molecules drug therapies nitrogen vessels are now in operation, Grants Advisor was created to facilitate the to satisfy the unmet clinical needs of cancer The most significant IT challenge was the ever increasing the usage of nitrogen and its procurement of external funding by Group patients. CRT is currently actively managing a increasing demand for storage capacity. In order transportation to the labs. This is performed Leaders through identifying relevant broad portfolio of development programmes to address this need a new storage system has three times a week with dry ice deliveries taking opportunities, assisting with grant preparation and exciting licensing opportunities originating been procured and installed. The new unified place twice a week. Gas cylinders are also and overseeing an internal grant review process. from the PICR that continue to attract storage solution is highly resilient and scalable monitored and replaced as necessary. Savings Gillian Campbell was recruited to this post in commercial partners (ranging from enabling and will allow highly cost effective expansion of have been made following an audit of gas usage May 2012 and also participates in other aspects technologies through to drugs in late stage storage in the future. which allowed us to reduce cylinder rental of scientific administration. clinical trials). We look forward to building on charges. our successes and continuing to work closely During 2012, we also improved the IT Business Cancer Research Technology with the PICR to advance discoveries that will continuity strategy to reflect the new storage Researchers can order central stores stock items Martyn Bottomley allow us to beat cancer in the years ahead. solution and ensure that we have the required via the intranet which are then distributed by the resilience if the need arises. We also re- Logistics team who also ensure that adequate Cancer Research Technology (CRT) is a evaluated and subsequently optimised the stock levels are maintained at all times. Included specialist oncology-focused development and back-end systems to improve performance, in this system are the enzymes and media commercialisation company wholly owned by stability and security. As the need for mobile stored in the Institute freezers (Sigma, Invitrogen, Cancer Research UK. CRT aims to maximise computing continues to increase, we developed Roche, Promega, New England Biolabs, Fisher 86 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH OPERATIONS 87

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    The Drug Discovery Chemists CANCER RESEARCH UK’S get ready to run. RESEARCH ENGAGEMENT The Paterson Institute has had a fantastic year of supporting fundraisers, thanking them and joining them in a range of activities. Representatives from the Institute have been Manchester Run. They formed part of the involved in over 50 events, reaching 1400-strong team who took part, raising an approximately 25,000 supporters. The Institute estimated £180,000 for Cancer Research UK. has welcomed record numbers of the public Kate Smith explained their reasons for taking The Relay for Life team made up through its doors, with over 500 people visiting part: of some of the Institute’s Cancer Research UK’s Post-doctoral Fellows and PhD Research Engagement a variety of laboratory tours, open days, open students. Manager evenings and other public events. This also “We all felt we should do something to raise included an event to launch the new Cancer money for CR-UK as we know only too well James Research UK brand to local staff, researchers how vital fundraising is to the charity. It was also Dunphy and fundraising groups. our way of getting out and thanking all the fundraisers who work so hard for us and allow The Institute has enthusiastically entered teams our team to carry out such fantastic work here in into a wide variety of fundraising events and Manchester. The total raised as a team was challenges, raising thousands of pounds for £3200 and we thoroughly enjoyed taking our charity. This includes; running, 24 hour and 40 lab coats out of the lab and onto the streets of mile walking, mountain climbing, cake-baking, Manchester!” hairdressing and moustache-growing. The Paterson Institute also entered a team in the A team of 13 chemists and biologists from the local Relay for Life event. The relay is a 24 hour Drug Discovery Group participated in the Great event where at least one member of the team Shameem Fawdar (a Postdoctoral Fellow in the has to walk around the rugby pitch at any time. daughter Celia and grand-daughter, Phoebe Signalling Networks in Cancer During the day the team engage with who came along with me. We found the visits to Group) demonstrates gel loading supporters by showcasing local research in an the research laboratories both interesting and to Celia Russell, one of the interactive way. This year was the fourth year a informative. My parents would have been very grand-daughters of Drs Ralston and Edith Paterson. Watching the team has been entered and they raised over proud of the enormous strides made in research demonstration is Dr Elspeth £2300 for CR-UK. at the Institute. Elspeth Russell ( nee Paterson ).” Russell, the daughter of the Patersons along with one of her One of the most important events of the year is The Paterson was once again a designated grand-daughters, Phoebe. the annual open day. The open day provides the ‘pit-stop’, used to support CR-UK fundraisers Institute with the opportunity to give supporters who were walking 26 miles for the Shine event. and the general public a research update along This year 15 volunteers from the Institute worked with demonstrations of the science that is through the night, providing hot drinks, food undertaken, whilst thanking them for their and encouragement to the 2000 participants endeavours. This year was the most successful who passed by. Head of Research Services, open day yet, with over 120 visitors enjoying a Stuart Pepper, was in charge on the night: presentation from Professor Richard Marais about the Institute and his research into “Helping these committed fundraisers on their melanoma followed by a tour of several labs. way can be both uplifting and quite emotional Some very special guests were also invited and as people often want to share a few words they took the time to write in after their visit: about their experiences “. “As the daughter of Ralston and Edith Paterson It is hoped that the 3000 people who took part (the original Directors of the Institute), I in the 13 mile and 26 mile walks will raise over particularly enjoyed the Open Day as did my £500,000 for the charity. 88 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH CANCER RESEARCH UK’S RESEARCH ENGAGEMENT 89

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    ACKNOWLEDGEMENT FOR FUNDING OF CAREER OPPORTUNITIES AT THE THE PATERSON INSTITUTE PATERSON INSTITUTE The total funding of the Paterson Institute for 2012 was £18.7m. The The Paterson Institute is located alongside The Christie NHS major source of this funding was awarded by Cancer Research UK Foundation Trust, and has a strong programme of basic and (CR-UK) via a core grant of £10.7m plus additional strategic funding of translational research. There are very close links with clinical and £2.5m. This funding enables the various scientific groups and service translational research groups throughout the Christie Hospital site. units within the Institute to carry out their research. The Manchester Cancer Research Centre (MCRC) sought. Although post docs will be encouraged to was created nearly six years ago by its partners The apply for their own fellowships, funded positions PATERSON INSTITUTE FUNDING 2012 University of Manchester, The Christie Hospital NHS are available for outstanding candidates. Interested Foundation Trust and Cancer Research UK. This is applicants should contact the Group Leaders an extremely exciting development which is directly, with details of their area of interest and 17.8% enhancing all aspects of cancer research, recent experience. education and treatment. The Institute offers excellent laboratory facilities and outstanding core In addition to postgraduate and postdoctoral KEY facilities, including molecular services, a microarray opportunities, the Institute is still seeking to recruit platform, proteomics, flow cytometry, histology, outstanding candidates to the positions of Junior CR-UK Core Grant the production of knock-in/knock-out animal and Senior Group Leaders. The packages provided 57.4% models, real-time PCR, next generation are extremely attractive and commensurate with CR-UK Strategic Funding sequencing and advanced imaging. Details of all the experience of the applicant, with significant 11.2% groups and facilities are given throughout this funding for personnel, recurrent expenditure and HEFCE report, and can guide interested parties to the equipment. Junior Group Leaders are appointed appropriate contacts. for an initial six-year period with a review between Other Sources five and six years for consideration for promotion to Opportunities exist at a number of levels in the Senior Group Leader, with Senior Group Leaders Institute. We have a well-established programme appointed to non-time limited positions. 13.6% of degrees by research which is described in the section on Postgraduate Education. We encourage Specific vacancies can be found on our web pages applications from suitable qualified graduates to (http://www.paterson.man.ac.uk/jobs), but suitably apply to join either the PhD or MD programmes. qualified and enthusiastic individuals should The infrastructure of the Paterson Institute is funded • Leukaemia & Lymphoma Research Fund Graduates with a first or 2.1 honours degree in a contact the Institute at any time to enquire about by HEFCE generated income at a cost of £2.9m. • Novartis biological science can apply each year to train for a career possibilities. • Qiagen four-year PhD in one of our research laboratories. The balance of the Institute’s funding is received • Chugai The University of Manchester offers a wide range of from a number of additional sources. The research • Abbott Laboratories training for new and existing students which carried out through these additional projects • Cambridge University provides opportunities to acquire skills that will enhances and supports the research undertaken by • GlaxoSmithKline complement the research programme and help the core funding. • Christie Hospital NHS Foundation Trust achieve personal and career development goals. At • Association for International Cancer Research the Paterson, we also ensure that postgraduate These sources are as follows: • Medical Research Council students are provided with high quality, relevant and • Kay Kendal Leukaemia Fund appropriate training alongside development • Astra Zeneca • Wellcome Trust opportunities. The Institute also has a well- • Roche • Parsortix developed process for ensuring suitable pastoral • European Commission care and mentoring for all students. • BBSRC We are immensely grateful to all our sponsors. Postdoctoral applicants of high calibre are regularly 90 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH CAREER OPPORTUNITIES AT THE PATERSON INSTITUTE 91

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    CONTACT DETAILS Paterson Institute for Cancer Research ISSN 1479-0378 Director: Professor Richard Marais Copyright © 2012 Cancer Research UK Tel: +44(0) 161 446 3156 Fax: +44(0) 161 446 3109 Edited by: Caroline Wilkinson Gillian Campbell Address Paterson Institute for Cancer Research Wilmslow Road Manchester M20 4BX United Kingdom e-mail: paterson.man.ac.uk web site: www.paterson.man.ac.uk Tel +44(0) 161 446 3156 www.paterson.man.ac.uk Electronic version of this report can be found at: www.paterson.man.ac.uk Cancer Research UK Cancer Research UK is a registered charity in England and Wales (1089464), Scotland (SC041666) and the Isle of Man (1103). Registered address: Angel Building, 407 St John Street, London, EC1V 4AD. Tel 44(0) 20 1234 5678 www.cruk.org 92 SCIENTIFIC REPORT 2012 THE PATERSON INSTITUTE FOR CANCER RESEARCH

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

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