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    SCIENTIFIC REPORT 2013 cruk.org

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    SCIENTIFIC REPORT Cover image Invading cancer cells (green) in muscle layers (red) are surrounded by collagen (red and white). Image provided by Haoran Tang of the Molecular Oncology group. 2013 MANCHESTER INSTITUTE

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    CONTENTS SECTION 1 SECTION 2 DIRECTOR’S INTRODUCTION 04 RESEARCH SERVICES RESEARCH HIGHLIGHTS 2013 07 Steve Bagley 48 CANCER RESEARCH UK Advanced Imaging and Flow Cytometry MANCHESTER INSTITUTE Duncan Smith 49 Biological Mass Spectrometry RESEARCH GROUPS Crispin Miller 14 Biological Resources Unit 50 Applied Computational Biology Garry Ashton 51 and Bioinformatics Histology Karim Labib 16 Mark Craven 52 Cell Cycle Laboratory Services Iain Hagan 18 Stuart Pepper 52 Cell Division Molecular Biology Core Facility Nic Jones 20 Wei Xing 53 Cell Regulation Scientific Computing Angeliki Malliri 22 Cell Signalling SECTION 3 Caroline Dive 24 Clinical and Experimental Pharmacology RESEARCH PUBLICATIONS 56 Ivan Ahel 28 THESES 64 DNA Damage Response SEMINAR SERIES 2013 66 Donald Ogilvie 30 POSTGRADUATE EDUCATION 68 Drug Discovery OPERATIONS 70 Peter L. Stern 32 Immunology CANCER RESEARCH UK’S LOCAL Nullin Divecha 34 ENGAGEMENT AND DEVELOPMENT 76 Inositide Laboratory ACKNOWLEDGEMENT FOR FUNDING 78 Tim Somervaille 36 CAREER OPPORTUNITIES 79 Leukaemia Biology CONTACT DETAILS 80 The Cancer Research UK Manchester Institute is located in The Paterson Building Richard Marais 38 Molecular Oncology John Brognard 40 Signalling Networks in Cancer Georges Lacaud 42 Stem Cell Biology Valerie Kouskoff 44 Stem Cell Haematopoiesis 2 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE 3

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    DNA-damage based therapy and is a good Towards the end of the year, the renewal of DIRECTOR’S INTRODUCTION strategic fit with the personalised medicine funding for the Manchester Cancer Research agenda that runs through all of our priority Centre (MCRC) was announced by CRUK. This areas. partnership between the University of Manchester, Cancer Research UK and The In July, Peter Stern retired after twenty-four Christie NHS Foundation Trust provides years as a Senior Group Leader in the Institute. infrastructure and training support that During this time he made valuable contributions facilitates interactions between scientists and to the field of cancer immunology, in particular clinicians. The next five years of funding will with his work in harnessing the immune system play a crucial role in driving the personalised to attack the 5T4 protein which is highly cancer medicine agenda in Manchester and the expressed in many carcinomas. He will remain North West. The MCRC building that is currently It has been a highly eventful year for the Institute with a new actively involved in research for some time being constructed opposite the Paterson through an honorary position with the Building is due for completion in Autumn 2014 identity, a major centre award, Group Leader recruitment and University’s Institute of Cancer Sciences. and is set to provide space for both the the continuation of our drive to extend the capabilities of our expansion of CRUK MI as well as recruitment of Karim Labib joined the MRC Protein further University of Manchester cancer core research services. Our two largest research teams Phosphorylation and Ubiquitylation Unit in research groups. An exciting development is underwent very successful Quinquennial Reviews and Dundee, as well as becoming Professor of that Manchester has been shortlisted for further Genome Integrity at the College of Life Sciences discussions with CRUK regarding Major Centre towards the end of the year, Tim Somervaille was promoted at the University of Dundee. Nullin Divecha will status which would greatly accelerate Professor Richard Marais Director of The Cancer to Senior Group Leader. take up a position at the University of achievement of our research aims. Research UK Manchester Southampton in 2014 while Ivan Ahel moved to Institute the William Dunn School of Pathology at the It is a pleasure to note a number of prizes and Our rebranding to the Cancer Research UK key role in the ground-breaking TRACERx University of Oxford. Two of our Post-doctoral awards to members of the Institute. Eva Manchester Institute (CRUK MI), that took place (Tracking Cancer Evolution through Treatment) Research Fellows, Giacomo de Piccoli and Barkauskaite, was the recipient of the Institute’s in October, acknowledges the core funding that lung cancer study announced by CRUK in 2013. Dragana Ahel, were both awarded Career Dexter Award for Young Scientists. This honour we receive from the charity and recognises the This £14 million study spanning nine years will Development Fellowships from Cancer was in recognition of her highly successful generosity of our supporters. We renamed the be performed by a network of teams around the Research UK to start their own research groups structural and mechanistic work on poly(ADP- site where we are located as The Paterson UK with the aim of tracking how lung tumours studying the regulation of genome stability at ribose) glycohydrolase, which has culminated in Building to ensure that we continue to celebrate develop and evolve as patients receive The University of Warwick and The University of an impressive set of publications in just over the legacy of Edith and Ralston Paterson and the treatment. Oxford respectively. three years during her PhD studies in the DNA impact that they had on cancer research and Damage Response group. In addition, Eva was treatment in Manchester. During the year, the Following a successful tenure review, Tim In the summer, we welcomed Wei Xing, who awarded a prize for the best talk at 7th Professor Caroline Dive finalisation of the management structure of the Somervaille has been promoted to Senior joined us to lead the new Scientific Computing International PhD Student Cancer Conference Deputy Director of The Institute was completed with the appointment Group Leader and will now continue to build on Facility, which will provide a High Performance hosted by students from the CRUK London Cancer Research UK of Caroline Dive as Deputy Director and Stuart the platform of basic and translational research Manchester Institute Computing and data management service Research Institute as well as receiving the Ruth Pepper and Caroline Wilkinson taking on the into acute myeloid leukaemia that he has allowing us to fully exploit the complex datasets Bowden Scholarship from the British Federation roles of Chief Laboratory Officer and Chief established over the last six years. Tim’s success that we obtain from the technological platforms of Women Graduates. Operating Officer respectively. to date was also recognised through the award that are now available in our core research of International Researcher of the Year by The facilities. These facilities will serve scientists in Professor Richard Marais was awarded the Major research highlights from 2013 included Christie NHS Foundation Trust. both the new MCRC research building as well as second Griem Lectureship in Molecular and the identification of driver mutations for throughout the Paterson Building and were Cellular Oncology from University of Chicago non-small cell lung cancer; a study which Recruitment was one of the major goals for the given a significant boost towards the end of Comprehensive Cancer Center (UCCCC) and uncovered a novel mechanism by which year and resulted in the appointment of three 2012 with an £8.7M investment from the UK will travel there in 2014 to receive the award and melanoma develops resistance to BRAF new Junior Group Leaders who will join the Research Partnership Investment Fund (UKRPIF). deliver a research lecture; Ged Brady, Dominic targeted drugs; the identification of critical Institute in 2014. Claus Jorgensen, starts in early To help with their development and plan for Rothwell and Debbie Burt of the Clinical and oncogenic factors in acute myeloid leukaemia; 2014 and will study tumour-stromal signalling in expansion, I commissioned a review of these Experimental Pharmacology group, received the development of a unique approach to study pancreatic cancer; he will be joined later on in facilities, which was conducted by an external first prize for their poster presentation at the 9th small cell lung cancer using patient-derived the year by Michela Garofalo who will establish a panel chaired by Professor Owen Sansom from International Symposium on Minimal Residual circulating tumour cell explant mouse models; group to examine the role of micro-RNAs in the CRUK Beatson Institute. This was a highly Cancer, held in Paris. Ivan Ahel’s success during and the elucidation of key mechanistic insights lung cancer, and Esther Baena whose team will valuable exercise and we shall continue to his time at the Institute was acknowledged with into the control of mitosis. investigate the molecular mechanisms driving implement recommendations from the review the award from CRUK of their 2013 Future tumorigenesis in prostate cancer. The addition throughout the coming year. In addition to Leaders’ Prize which was presented to him at the The Drug Discovery Unit, led by Donald Ogilvie, of prostate cancer to our areas of interest is in several major items acquired during 2013 with NCRI conference in Liverpool. Drug Discovery and the Clinical Experimental Pharmacology light of a successful major funding application in the UKRPIF investment, we also purchased a won the S-Lab Effective Laboratory Award while Group, led by Caroline Dive, performed collaboration with Queen’s University, Belfast, to super resolution gSTED microscope with funds Daniel Mould won the Society of Chemical extremely well at their respective Quinquennial establish a Prostate Cancer UK Movember from the MCRC, which has significantly Industry North West Industrial placement reviews. These groups have a particularly strong Centre of Excellence. The programme provides enhanced our imaging capabilities. student of the year for his undergraduate project translational focus that lies at the heart of our £5m over five years (split equally across the two with this group. strategy at the CRUK MI. The expertise of the sites) with the aim of improving prostate cancer CEP group in circulating tumour cells will play a survival rates via personalised delivery of 4 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE DIRECTOR’S INTRODUCTION 5

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    DIRECTOR’S INTRODUCTION (CONTINUED) RESEARCH HIGHLIGHTS A migrating breast cancer cell in a 3D collagen gel. Grey In this section we highlight some research publications from represents collagen; green represents active integrin and the 2013 which report significant advances in specific areas. The cell nucleus is shown in blue. selected papers demonstrate the breadth and the quality of Image provided by Haoran Tang the research being undertaken by the groups at the Cancer (Molecular Oncology) Research UK Manchester Institute. Grallert A, Patel A, Tallada VA, Chan KY, Bagley BRAF inhibitors, such as vemurafenib, target S, Krapp A, Simanis V, Hagan IM. mutations in the BRAF protein, found in around Centrosomal MPF triggers the mitotic and half of all melanomas. Treatment with morphogenetic switches of fission yeast. vemurafenib is initially effective, however Nature Cell Biology, 2013; 15:88-95. patients often develop resistance to this drug after a short time. Here, the Molecular Mitotic commitment is driven by the activation Oncology Group show that using drugs which of the Cdk1-Cyclin B protein kinase complex. target a different molecular pathway can halt the The Cell Division Group wanted to ask whether growth of BRAF inhibitor-resistant cancer cells. the initial appearance of Cdk1-Cyclin B on They developed vemurafenib-resistant human centrosomes indicates that the decision melanoma cell lines and measured the to divide is driven by events at spindle poles. phosphorylation state of receptor tyrosine Using fission yeast as a model system, they kinases present. It was observed that, while generated a fusion between the single chain EGFR phosphorylation was increased, the Elli Marinopoulou from the Stem Cell Biology Our highly successful programme of public llama antibody GBP (GFP Binding Protein) that negative regulator of EGFR was inactivated, group won the Young Investigator Award at the engagement continued throughout the year. has a very high affinity for Green Fluorescent indicating a role for EGFR in vemurafenib EMBO Workshop, “RUNX Transcription Factors We welcomed Eve Hart into the role of Research Protein, and a mutant version of Cdk1 that was resistance. Increased SRC activation was also in Disease and Development ”. The first North Engagement Manager in June, replacing James unique amongst all kinases in the cell because it detected in the resistant samples and it was West Bio-Pharma Postdoctoral Symposium was Dunphy who took up a new post as CRUK could be inhibited by a specific ATP analogue. further discovered that drug resistant cell lines held in March at AstraZeneca’s Alderley Park and Senior Research Engagement Manager for the Because GFP is widely used to study protein are more invasive than sensitive cell lines, but was well attended by numerous postdoctoral north of the UK. Between them, James and Eve distribution in fission yeast, they were able to that invasion could be blocked by inhibiting the scientists from AstraZeneca and the Universities co-ordinated over 1000 visitors to CRUK MI generate an array of strains in which the EGFR pathway or SRC, suggesting that EGFR/ of Manchester, Liverpool, Leeds and Sheffield. during 2013 during numerous lab tours and 12 chimeric kinase was targeted to a variety of SRC activation not only increases resistance to James Lynch of the Leukaemia Biology Group other events including the annual open day subcellular structures. Targeting was initiated in BRAF inhibitors but also increases ability to won the prize for best talk while Urszula which welcomed 150 visitors and tours/talks for the presence of ATP analogue before removal invade and metastasise. The authors posit that Polanska (Stromal-Tumour Interactions group) major donors, CRUK shop managers and race of the analogue released a pulse of kinase combining both BRAF inhibitors and EGFR won the prize for best poster. The best oral for life volunteers. For the first time, the Institute activity at each location. Of the many locations inhibitors or treating resistant cells with SRC abstract presentation at the Melanoma participated in the Manchester Science Festival tested, the only one at which Cdk1-Cyclin B inhibitors alone could provide an effective Research Congress was awarded to Malin and we developed a Social Media presence with activation promoted mitosis was the spindle new treatment for certain BRAF mutated Pedersen of the Molecular Oncology group. the development of a Facebook site and the pole, suggesting that this organelle does indeed melanoma patients. Hadir Marei from the Cell Signalling group was opening of a twitter account. act as a site that integrates signal from diverse awarded a prize for the best presentation at transduction pathways to generate the coherent University of Manchester, Faculty of Medical and The coming year promises a number of exciting decision to initiate division. Huang X, Spencer GJ, Lynch JT, Ciceri F, Human Sciences, Postgraduate Showcase while developments, including further Group Leader Somerville TD, Somervaille TC. Shameem Fawdar from the Signalling Networks recruitment in Melanoma Immunology and Enhancers of Polycomb EPC1 and EPC2 in Cancer group won a poster prize at CRUK Molecular Pathology, as we continue to Girotti MR, Pedersen M, Sanchez-Laorden B, sustain the oncogenic potential of MLL Postdoctoral Researcher Career Development strengthen the research portfolio of the Institute Viros A, Turajlic S, Niculescu-Duvaz D, Zambon leukemia stem cells. Day. Other notable achievements included the and develop the necessary research facilities to A, Sinclair J, Hayes A, Gore M, Lorigan P, Leukemia, 2013 Oct 29. doi: 10.1038/ election of Ian Waddell to the BACR and Richard drive forward our translational research agenda. Springer C, Larkin J, Jorgensen C, Marais R. leu.2013.316. Marais as President of the EACR; Angeliki Malliri Inhibiting EGF Receptor or SRC Family Kinase was awarded a research grant by the MRC to Signaling Overcomes BRAF Inhibitor Resistance While genetic mutations initiate acute myeloid study the regulation of the Rac activator Tiam1 in Melanoma. leukaemia (AML), epigenetic dysfunction plays a by ubiquitylation. Cancer Discovery, 2013; 3: 158-167. critical but incompletely understood role in 6 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH HIGHLIGHTS 7

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    RESEARCH HIGHLIGHTS (CONTINUED) biochemical studies showed that the product of this gene is a PARP-interacting macrodomain Lung cancer is the most common cause of protein, capable of removing the mono(ADP- cancer-related death in the UK. Although the Tumour tissue visualised at x400 magnification; Definiens Tissue ribose) from PARP-modified proteins. The data implementation of kinase-specific targeted Studio was used to classify suggests that the c6orf130 protein causes a therapy has improved the overall survival rate of tumour (orange) and stroma defect in these patients’ cells that ultimately lung cancer patients in recent years, we are still (blue) in tumour tissues stained leads to cell death and neurodegeneration. faced with the enormous challenge of for CD44. Image 1: subset of the The authors propose that the c6orf130 protein deciphering the mutated drivers of lung cancer total tissue; Image 2: colour mask applied during training of the either directly reverses protein mono(ADP- in about 50% of cases. In an attempt to elucidate two data subsets; Image 3: ribosyl)ation or completes the reversal of the low frequency novel drivers of lung cancer, separation of tissue and stroma protein poly(ADP-ribosyl)ation following the the Signalling Networks in Cancer group, with the mask removed. PARG reaction, and so renamed this protein supported by the Bioinformatics and Drug Terminal ADP-Ribose protein Glycohydrolase Discovery Groups, undertook a functional Image supplied by Francesca Trapani, Clinical and disease pathology. The Leukaemia Biology replisome components that bind to histone (TARG1). Overall, the study reveals that the genomics approach to screen a panel of six Experimental Pharmacology. Group hypothesised that a lentiviral knockdown complexes released from chromatin in cell function of TARG1 is important for normal previously sequenced adenocarcinoma cell screen of genes coding for proteins which extracts of yeast. Gene expression patterns cellular proliferation and cellular response to lines (datasets accessible on COSMIC and CCLE regulate the structure or function of chromatin demonstrated that the Mcm2 helicase subunit DNA damage. databases). A targeted siRNA genetic would reveal novel and hitherto unappreciated and FACT complex (facilitates chromatin dependency screen was designed to deplete all critical regulators of leukaemogenic potential. transcription) bind together to parental histone somatically mutated genes in the cancer cell This approach identified the EP400 complex complexes that have been released from Fawdar S, Trotter EW, Li Y, Stephenson NL, lines. Three targets conferring a survival components EPC1 and EPC2 as critical chromatin. FACT is not recruited to Mcm2-7 at Hanke F, Marusiak AA, Edwards ZC, Ientile S, advantage to the cancer cells were identified: oncogenic co-factors in AML. EPC1 and EPC2 replication origins before initiation but is present Waszkowycz B, Miller CJ, Brognard J. FGFR4, MAP3K9 and PAK5. The mutations in were required for the clonogenic potential of at DNA replication forks, indicating that FACT Targeted genetic dependency screen facilitates each of the kinases were further functionally human AML cells of multiple molecular and Mcm2 help to retain parental histones identification of actionable mutations in FGFR4, and structurally validated to be gain-of-function subtypes. Epc1 or Epc2 knock down (KD) transiently at the fork before deposition onto MAP3K9, and PAK5 in lung cancer. mutations by their enhanced activity towards induced apoptosis of murine MLL-AF9 AML nascent DNA just behind the replisome. This Proc Natl Acad Sci U S A, 2013; 110(30):12426-31. cells and abolished leukaemia stem cell study illustrates that the eukaryotic replication Mitotic cells in cysts – stained potential. By contrast, normal haematopoietic and transcription machineries use similar with actin (red), Hoescht (blue) stem and progenitor cells (HSPC) were spared. histone-binding modules to process parental and Tubulin (green). In keeping with these distinct functional histones in order to preserve chromatin integrity consequences, Epc1 or Epc2 KD induced during chromosome replication. The activation Image provided by Andrew Porter, Cell Signalling. divergent transcriptional consequences in of oncogenes is believed to cause defects in murine MLL-AF9 granulocyte-macrophage replication, which drive tumour development, progenitor-like (GMP) cells versus normal GMP, so a deeper understanding of the mechanics of with a signature of increased MYC activity in chromosome replication may help optimise leukaemic but not normal cells. This was novel strategies that better exploit replication caused by accumulation of MYC protein. defects in tumour cells. Pharmacological inhibition of MYC:MAX dimerisation, or concomitant MYC KD, reduced apoptosis following EPC1 KD, linking the Sharifi R, Morra R, Appel CD, Tallis M, Chioza B, accumulation of MYC to cell death. Therefore, Jankevicius G, Simpson MA, Matic I, Ozkan E, EPC1 and EPC2 are components of a complex Golia B, Schellenberg MJ, Weston R, Williams which directly or indirectly serves to prevent JG, Rossi MN, Galehdari H, Krahn J, Wan A, MYC accumulation and AML cell apoptosis, thus Trembath RC, Crosby AH, Ahel D, Hay R, sustaining oncogenic potential. Ladurner AG, Timinszky G, Williams RS, Ahel I. Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in Foltman M, Evrin C, De Piccoli G, Jones RC, neurodegenerative disease. Edmondson RD, Katou Y, Nakato R, Shirahige K, The EMBO Journal, 2013; 32: 1225–1237. Labib K. Eukaryotic replisome components cooperate to Adenosine diphosphate (ADP)-ribosylation is process histones during chromosome an evolutionarily conserved, reversible post- replication. translational protein modification, catalysed Cell Reports, 2013; 3(3):892-904. by poly(ADP-ribose) (PAR) polymerases (PARPs). The hydrolysis of PAR chains is catalysed by All eukaryotic cells create a near-perfect copy PAR glycohydrolase (PARG). ADP-ribosylation of their chromosomes in order to survive cell regulates a wide range of cellular processes, division. However, chromosome replication including DNA repair, so the timely modulation severely disrupts the chromatin, the integrity of of this dynamic post-translational modification which is preserved by the transfer of parental is critical. In this study, defects in the c6orf130 histones and the deposition of new histones. gene were identified in patients with severe In this study the Cell Cycle Group screened for neurodegeneration. X-ray structures and 8 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH HIGHLIGHTS 9

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    RESEARCH HIGHLIGHTS (CONTINUED) tumour, which led the Clinical and Experimental inhibitor AZD3965 in Small Cell Lung Cancer. Pharmacology Group to establish a model Clinical Cancer Research, 2013 Nov 25. [Epub system, entitled the “death-switch”, to control ahead of print] the pro-proliferative signalling pathway MEK/ SF3B1 mutations were associated with the timing and extent of cell death induced in a ERK. This approach provides a stratification differential alternative splicing of protein coding tumour. Colorectal cancer cells were Small cell lung cancer (SCLC) patients have poor strategy to select patients that may benefit from genes, including ABCC5 and UQCC, and of the manipulated to express and activate a key prognosis and there has been little a MEK inhibitor or alternatively encourage the long non-coding RNA CRNDE, highlighting a component of the apoptosis pathway, which improvement in treatment for 30 years. SCLC development of inhibitors specific to FGFR4, novel potential mechanism of tumourigenesis induced widespread and synchronous tumours rapidly proliferate and are hypoxic, MAP3K9 and PAK5 to be used in the clinic. in melanoma. apoptosis. Using proteomic methodologies, suggesting a reliance on glycolysis which could they identified and validated four proteins be targeted by inhibiting lactate transport with released into the bloodstream from cells monocarboxylate transporter (MCT) inhibitors. Furney SJ, Pedersen M, Gentien D, Dumont AG, Walczynski J, Lyons S, Jones N, Breitwieser W undergoing apoptosis. The death switch was Whilst AZD3965 (MCT1 inhibitor) is in Phase I Rapinat A, Desjardins L, Turajlic S, Piperno- Sensitisation of c-MYC-induced B-lymphoma found to induce regression of tumours with clinical trials, activity in SCLC has not been Neumann S, de la Grange P, Roman-Roman S, cells to apoptosis by ATF2. the release of the previously established assessed and predictive biomarkers are Stern MH, Marais R. Oncogene, 2013; Feb 18. doi: 10.1038/ biomarker, cytokeratin 18. This model was also unproven. Preclinical research undertaken by SF3B1 mutations are associated with alternative onc.2013.28. used to evaluate a novel imaging probe which the Clinical and Experimental Pharmacology splicing in uveal melanoma. could be applied in positron emission Group suggests that hypoxic SCLC cells which Cancer Discovery, 2013; 3(10):1122-9. A common feature in B lymphoma, as found in tomography (PET) to image tumour cell death. do not express MCT4 (an alternate lactate many other tumour types, is the aberrant activity These findings mark an important development transporter), are sensitive to AZD3965. Analysis Uveal melanoma, the most common eye of the c-MYC gene. It has been known for some in the assessment of anti-cancer therapies that of SCLC tissue microarray of 78 SCLC biopsies malignancy, causes severe visual morbidity and time that hyperactive c-MYC not only regulates can be used in clinical trials. demonstrated that 21% express MCT1 but not is fatal in approximately 50% of patients. Primary tumour cell growth, but also induces apoptosis. MCT4, predicting response to AZD3965. MCT1 uveal melanoma can be cured by surgery or A project led by Jacek Walczynski of the Cell expression also correlated with reduced survival radiotherapy, but the metastatic disease is Regulation Group found that lymphomas that Polanski R, Hodgkinson C, Fusi A, Nonaka D, time. Taken together, these data support clinical refractory to treatment. Furthermore, the role of have highly active c-MYC also often show Priest L, Kelly P, Trapani F, Bishop P, White A, evaluation of AZD3965 in a SCLC enriched ultra-violet radiation in uveal melanoma strongly active JNK kinase, as well as activated Critchlow SE, Smith PD, Blackhall FH, Dive C, cohort, coupled with assessment of hypoxia, aetiology is unclear. Researchers from the ATF2, a stress-induced transcription factor and Morrow CJ. MCT1 and MCT4 expression as a predictive Molecular Oncology Group conducted phosphorylation target of JNK. Furthermore, in Activity of the Monocarboxylate Transporter 1 signature for AZD3965 response. single-nucleotide polymorphism arrays and a mouse model of c-MYC driven B lymphoma, whole-genome sequencing of 12 primary uveal the B cell specific inactivation of ATF2 led to Breast cancer cells (green) on an melanomas and matched normal DNA to significantly reduced apoptotic cell death extra-cellular matrix (ECM) array, understand the somatic genomics of uveal triggered by c-MYC and to significantly more which is used to study cell melanoma comprehensively. Whole genome aggressive behaviour of lymphomas. In adhesion and spreading on sequencing revealed a remarkably low mutation addition, JNK and ATF2 were shown to direct various types of ECM. This burden in the tumour genomes and the the induction of apoptosis in response to picture shows a single spot of the array. Actin cytoskeleton is in red. researchers did not observe an ultraviolet chemotherapeutic drugs but only once B radiation DNA damage signature. They lymphocytes have progressed towards Image provided by Haoran Tang identified SF3B1 mutations in three samples and lymphoma stages by the actions of c-MYC. (Molecular Oncology). a further 15 mutations in an extension cohort of Thus, ATF2 is an effector of c-MYC induced 105 samples. Interestingly, these mutations were apoptosis through activation by JNK. associated with good prognosis. SF3B1 encodes a component of the spliceosome, and the authors conducted RNA sequencing of the Simpson KL, Cawthorne C, Zhou C, tumour samples to investigate differences in Hodgkinson CL, Walker MJ, Trapani F, Kadirvel splicing between the SF3B1 mutant and M, Brown G, Dawson MJ, MacFarlane M, wild-type tumours. Sequencing revealed that Williams KJ, Whetton AD and Dive C. A caspase-3 `death-switch’ in colorectal cancer Normal human appendix cells for induced and synchronous tumor stained with an antibody to the apoptosis in vitro and in vivo facilitates the marker CDX2 (brown). CDX2 is development of minimally invasive cell death used as a marker in colorectal cancer prognosis. The tissue biomarkers. has been counterstained with Cell Death & Disease, 2013; 4: e613. haematoxylin to show the nuclei of cells (blue). The ability to determine the success of anti-cancer therapies is critical; however, for Image provided by Darren Roberts (Immunology) many cancers this is only possible by invasive surgery, biopsies or expensive imaging techniques. Consequently, the ability to detect drug action in the bloodstream of the patient has become an attractive option. Many cancer therapies work by inducing cell death in the 10 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH HIGHLIGHTS 11

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    THE CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH GROUPS MDCK cells, grown in a collagen matrix, where they form hollow spheres, called cysts. When the growth factor HGF is added to the cysts, they start to sprout, with actin-rich projections emerging from the surface of some of the cells. After one or two days, some of these cells undergo EMT, and re-orientate their axis of cell division, forming long chains of cells which can start to hollow out to form hollow tubules. This recapitulates stages in normal kidney development, and is a useful model for studying EMT and oriented cell division, and the way in which these might contribute to tumour progression. Actin is cyan, and nuclei are red. Image supplied by Andrew Porter, Cell Signalling. 12 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE THE CANCER RESEARCH UK MANCHESTER INSTITUTE 13

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    6 joint with Cell Division Since most of our downstream data analysis is data from genetic dependency screens APPLIED COMPUTATIONAL BIOLOGY 7 embedded within Drug performed using R and Bioconductor, these (Fawdar et al., Proc Natl Acad Sci U S A, 2013). AND BIOINFORMATICS 8 Discovery Unit embedded within Clinical tools bring the data into the R programming environment, where they can be analysed We have been collaborating with the www.cruk.manchester.ac.uk/bioinformatics and Experimental using the comprehensive statistical toolkits Translational Radiobiology Group (part of Pharmacology that accompany this programming language, the University of Manchester Institute of 9 joint with IT department complementing other work in the ACBB Cancer Sciences) on the development and group that has focused on bringing genome application of RNA signatures of tumour annotation data into the same environment hypoxia. (Eustace et al., Clin Cancer Res., 2013; (see http://annmap.cruk.manchester.ac.uk/, Ramachandran , et al., Eur J Cancer, 2013 ; and for example). Hall et al., P Int J Radiat Oncol Biol Phys., 2013 ). Many of these studies required the analysis of The Applied Computational Biology and Bioinformatics Group Collaborative Analysis microarray data generated from Formalin fixed Additional collaborative support is provided by Paraffin Embedded Tissue (FFPET) samples, is interested in the computational analysis of genome-wide Figure 1 computational analysts that work closely with which presents a challenge because the RNA is datasets arising from deep sequencing and quantitative Integrating strand specific RNA other research groups. Where demand is degraded and chemically modified, sequencing data with global sufficient, these can be provided by full time requiring new bioinformatics strategies. proteomics experiments. It collaborates widely within the quantitative protein mass embedded posts; both the CEP and the DDU spectrometry to identify gene Institute and has its own research programme focused on expression changes in fission Groups embed analysts in this way. In addition, RNA Biology Research Group yeast cells.Rows in the heat map we have established an exciting collaboration Although less than 2% of the human genome developing a better understanding of the transcriptional correspond to individual coding with the CEP group (page 24) to develop encodes protein sequences, the majority is systems that control gene expression within cells. It is regions for which quantitative proteomics data were also bioinformatics approaches for the analysis of transcribed, raising the possibility that many of Group Leader whole genome and transcriptome data derived these non-coding RNAs may play an important particularly interested in the role played by long non-coding available. Columns correspond from single cell genomics experiments. but currently unknown role in the cell. We are Crispin Miller to individual samples in a time RNAs (lncRNAs) in regulating these processes and how these course. A wild type and mutant integrating bench-, clinical- and computational Postdoctoral Fellows strain were compared. Sense: While much of our work is focused on science to identify novel lncRNAs that play an Jing Bi are disrupted in tumours. coding sequence expression genome-wide datasets, we are also interested important role in cancer. Hui Sun Leong levels. Antisense: antisense in statistical methods, and were able to Janet Taylor2, 3 expression levels corresponding This year, the Institute has invested further in Support Team, to complement a new and to these coding regions. Protein: contribute to work with the Signalling Networks Some of our work uses the model system Scientific Officer deep sequencing, with the purchase of a new separate RNA Biology Research Group focused protein abundance. Blue: low in Cancer Group (page 40) by providing the fission yeast (Schizosaccharomyces pombe) to Keren Dawson Illumina HiSeq 2500 and a MiSeq. These on regulatory RNAs (see below). abundance, red high abundance. mathematical modelling necessary to analyse investigate the basic functions of non-coding platforms offer exciting opportunities, RNAs (Figure 1), since many of the core Graduate Students pathways known to be involved in their action but come with an increased computational A major aspect of the support team’s work has Elli Marinopolou4 burden, not only in the sheer volume of been to develop pipelines that streamline the are conserved with human cells. Our interest in Danish Memon Sharmin Naaz5 information (each run of the 2500 can require pre-processing and alignment of deep non-coding RNAs and transcription has led to María José Villalobos terabytes of disk space to analyse) but also in the sequencing data. These are parallelised across an emphasis on genome annotation, and the Quesada6 computational power required to process them. our HPC system, and handle many of the development of computational tools to support routine tasks associated with Illumina data, de novo re-annotation of samples from RNA- Computational Biologist Scientific Computing freeing valuable time to conduct downstream sequencing and protein mass spectrometry Yaoyong Li In order to address the computational burden analysis and data interpretation. A significant data. Our discovery of novel protein coding Bioinformatics Analyst associated with our new sequencing platforms, part of this work has been to evaluate different genes has added almost 1% to the protein Phil Chapman7 we have established a new Scientific Computing aligners and to develop an understanding of the coding complement of fission yeast. Catriona Tate1, 8 Publications are now emerging that describe Team, led by Wei Xing (page 53), to provide the influence different parameter settings have on next generation of High Performance the performance of these tools. these loci in more detail (see, for example, Software Architect Tim Yates Computing (HPC) necessary to deal with future Dhani et al., Mol Biol Cell., 2013). deep sequencing, proteomics and imaging Pipelines for DNA-, RNA- and ChIP-seq have all Bioinformatics data. Wei has already helped manage the been built, and will continue to be refined in We are currently applying similar approaches to Programmer the ones we pioneered in fission yeast to the installation of additional processing and storage order to keep track with a fast moving field. The Chris Wirth capacity, and over the next year will be team has also developed proteomics tools to analysis of human cell lines and clinical Systems Administrator coordinating the development of a new Cancer complement those provided for deep datasets, allowing us to identify novel long Zhi Cheng Wang9 Genomics Data Centre to further extend our sequencing. These bring peptide-level tandem intergenic non-coding-coding RNAs computing capabilities. mass spectrometry datasets into a format that (lincRNAs) of potential relevance in cancer. 1 joined in 2013 We are in the process of characterising these supports integrated analysis with RNA- 2 left in 2013 further at the bench. Computational Biology Support sequencing data, in part by mapping them into a 3 joint with Translational As computational biology becomes central to common genome-level coordinate system. Radiobiology Publications listed on page 56 increasing numbers of groups within the This further extends approaches we developed 4 joint with Stem Cell Institute, our collaborations have grown to support the integration of genomics data (e.g. Biology significantly. The close of 2013 saw a Bitton et al., Genetics, 2011; Bitton et al., PLoS 5 joint with Stem Cell restructuring of the ACBB group through the ONE, 2010; and Bitton et al., BMC Haematopoiesis formation of a distinct Computational Biology Bioinformatics, 2008). 14 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE APPLIED COMPUTATIONAL BIOLOGY AND BIOINFORMATICS 15

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    to set the rate of fork progression, making CELL CYCLE the helicase unwind DNA much more quickly than it would normally have been able to. Until now, it has not been possible to address 3 3 these issues in eukaryotes, as the connections Transfer of between helicase and polymerases have not 4 4 been understood. We reported this year that Parental histones 3 4 the Dpb2 subunit of the leading strand DNA polymerase epsilon plays a key role in this 4 4 regard (Sengupta et al., Curr Biol, 2013), as it Deposition connects the polymerase to the Cdc45-MCM- Replisome Chromosome replication is still understood poorly in (Mcm2, FACT, of new GINS DNA helicase, by binding directly to the GINS component of the latter (Figure 2). eukaryotic cells, but is intimately linked to our understanding of etc.?) histones Previous work from the lab of Hiro Araki showed the development and treatment of human cancer. Defects in that GINS and DNA polymerase epsilon are replication arise early in cancer development, so that agents 3 4 recruited together to replication origins as a complex, during the initiation of replication. We perturbing chromosome replication are often able to kill 4 4 found that the amino terminal domain of Dpb2 cancer cells preferentially. A multi-protein machine known as 3 3 (Dpb2NT) is required for this recruitment. Interestingly, if cells express Dpb2NT as the the replisome is assembled at DNA replication forks, and the 4 4 only form of Dpb2, this is sufficient to allow Group Leader recruitment of GINS and assembly of active mechanisms and regulation of the replisome form the focus of Cdc45-MCM-GINS, but leads to the production Karim Labib1 our research. During the last year, we identified conserved of a replisome that lacks DNA polymerase Postdoctoral Fellows epsilon. Cells are viable under such conditions Giacomo de Piccoli1 histone-binding activities within the replisome, which might (presumably because another DNA polymerase Cecile Evrin1 Luis Garcia-Rodriguez1 contribute to the retention of parental histones at sites of Figure 1 Our goal for the future will be to identify all the can make the leading strand in the absence of The eukaryotic replisome remaining histone binding activities within the Pol epsilon), but growth is very poor, indicating Tim Maculins1 replication, in order to maintain epigenetic histone marks that contains multiple histone- eukaryotic replisome and then map and mutate that the connection between DNA polymerase binding activities, which are Scientific Officers control gene expression. We also discovered how the likely to cooperate with each the relevant domains, so that by combining epsilon and the helicase is important for normal Frederick van Deursen1 other, in re-depositing parental these mutations we will be able for the first time replisome function. Future studies will explore Pedro Junior Nkosi1 replicative DNA helicase is connected to the leading strand histones onto nascent DNA to assess the contribution of the replisome to why this is the case. Graduate Student DNA polymerase, making a physical link that is likely to play an during chromosome replication. the preservation of parental chromatin during chromosome replication. Publications listed on page 56 Marija Maric1 important role in regulating the progression and function of 1 left in 2013 the eukaryotic replisome. Figure 2 Dpb2 connects the leading strand DNA G1-phase Mcm2-7 Dpb2 incorporates DNA polymerase to the Cdc45-MCM-GINS DNA (Inactive) polymerase epsilon into the helicase within the eukaryotic replisome Copying chromatin during DNA replication (Figure 1). Until now, nothing was known about eukaryotic replisome, by In all species, a DNA helicase is required to Eukaryotic chromosomes contain a single how this feat is actually achieved during direct association with the GINS component of the unwind the parental DNA duplex at DNA molecule of DNA that is packaged into chromosome replication. Cdc45-MCM-GINS DNA replication forks, so that DNA polymerases can nucleosomes by association with histones. helicase. S-phase then synthesise the leading and lagging strands. The density of nucleosomes along each We are searching systematically for histone- CDK and DDK Seminal work with E. coli showed that the Cdc45 chromosome is an important determinant of binding activities in the eukaryotic replisome, Pol2 replicative helicase and DNA polymerases are Dpb4 gene expression, as are post-translational and this year we described the first stage of this (interaction Sld5 Dpb2 Dpb3 connected to each other by other factors, to of Dpb2NT with Psf1 modifications of the extended tails at the amino work (Foltman et al., Cell Rep, 2013). Using a GINS form a large assembly known as the replisome. Psf1 is critical Psf2 Psf3 termini of the histone proteins. In addition to simple assay in which histones are released into Assembly of the replisome allows the rate of for this step) making a near-perfect copy of the DNA double a yeast cell extract by digestion of the DNA unwinding to be co-ordinated with the rate helix during chromosome replication, cells chromosomal DNA, Magda Foltman discovered of DNA synthesis, thus minimising the exposure need to ensure that the complex patterns of that the Mcm2 subunit of the replicative DNA of single-strand DNA that might otherwise be a nucleosome density, and post-translational helicase was able to pick up the displaced (Polε)ε) (Pol dangerous target for nucleases or Dpb3 modifications to histone tails, are conserved histones. Interestingly, Mcm2 binds to histones Dpb4 Pol2Pol2 c4 -P7 recombination enzymes in the cell. More Cd m2 when the chromosomes are duplicated, in together with another replisome component Dpb2 surprisingly, it turns out that replisome assembly c 5 M order to preserve patterns of gene expression. called FACT, which is known to help process GINS is a critical determinant of the rate of fork Unwinding of the DNA duplex displaces parental histones during transcription. Magda Ctf4 progression. The DNA polymerases can only histones, and a long-standing model supposes found that the extended amino-terminal tail of act on a single-strand template generated by Dpb2 tethers Pri that parental histones must then be retained Mcm2 contains a histone-binding motif that is Pol α Primase the helicase, so that it would be natural to think Pol ε to the locally at DNA replication forks, so that they can conserved from yeast to humans. In budding that the helicase might determine the rate of rest of the be re-deposited immediately onto the nascent yeast, this motif is important to preserve the replisome fork progression. But the polymerase is DNA, thus preserving the parental chromatin nature of sub-telomeric chromatin, although it inherently faster than the helicase, and features throughout each chromosome is not required for DNA synthesis per se. replisome assembly allows the polymerase 16 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE CELL CYCLE 17

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    Figure 1 Recruitment of Protein Phosphatase 1 to the CELL DIVISION Cut12, Polo and the mitotic SPB controls mitotic commitment commitment switch. www.cruk.manchester.ac.uk/celldivision The dephosphorylation of Cdc2/ Protein Phosphatase 1 (PP1) is recruited by Cyclin B that promotes mitotic defined docking motifs to both direct targets commitment is accelerated and scaffolds from which it can through phosphorylation by dephosphorylate neighbouring proteins. The the polo kinase Plo1. This active cut12 mutations that suppress cdc25-22 reside Plo1 also inhibits the Wee1 kinase within a perfect match for a PP1 docking site. We that puts these phosphates onto Cdc2. Crucially, this entire employed a range of approaches to show that control only operates once PP1 recruitment to this site in Cut12 sets the Cdc2/CyclinB is active, making requirement for Cdc25 in fission yeast. it a feedback control that ensures Strikingly, cells could tolerate the otherwise a rapid and complete transition The inappropriate proliferation of cancer cells can arise from from interphase into mitosis. lethal ablation of the cdc25+ gene when the PP1 docking site had been removed from Cut12. We unchecked cell division, a failure to engage cell death pathways Recruitment of Plo1 to the spindle pole by Cut12 appears are yet to identify the target for the Cut12-PP1 or a simultaneous defect in both. Understanding how the to be critical for this control. complex however we did find an inverse correlation between the degree of PP1 diverse external and internal cues are integrated to co-ordinate recruitment to Cut12 and the activity of the cell division and death therefore sits at the heart of our need to feedback loop kinase Plo1. Furthermore, PP1 recruitment to Cut12 constitutes yet another understand the basic biology of cancer. Because the regulatory level of feedback control because PP1 docking networks that control cell division are highly conserved, was inhibited by phosphorylation of the docking Group Leader site by Cdk1-Cyclin B and the NIMA kinase Fin1. understanding how the relatively simple unicellular yeasts take Thus, elevation of Cdk1-Cyclin B kinase activity Iain Hagan the decision to divide greatly accelerates the analysis of the this library of GFP fusions. The catalytic pocket triggers a loop that reduces PP1 recruitment to Associate Scientist of each kinase was modified to incorporate Cut12 to boost Plo1 activity to further enhance Agnes Grallert more complex issue of cell division controls in man. mutations that conferred sensitivity to inhibition Cdk1-Cyclin B activity towards the PP1 docking by ATP analogues. We could then restrain the site. This loop will greatly accelerate the mitotic Postdoctoral Fellows We study cell division in the fission yeast (SPB), strongly endorse this view. Gain of activities of the targeted kinases with inhibitory commitment switch. Ye Dee Tay Kuan Yoow Chan Schizosaccharomyces pombe because it is a function mutations in the SPB component ATP analogues until removal of these analogues simple, unicellular organism with excellent Cut12 compensate for an otherwise lethal loss by a simple exchange of culture medium A novel approach for the study of Wee1 Scientific Officer genetics that is cheap to grow and divides of Cdc25 function arising from the conditional provided a burst of kinase activity at the function in vivo Ben Hodgson subcellular location of interest. We used this The sensitisation of Plo1 to inhibition by ATP rapidly. Commitment to mitosis is regulated by cdc25-22 mutation. This influence appears to the activity of the Cdk1-Cyclin B protein kinase stem from Cut12’s impact upon the activity of approach to release each kinase activity at analogues that we used to study Plo1 function in Graduate Student María-José Villalobos complex. Prior to mitosis the complex is the fission yeast polo kinase Plo1. The cut12 diverse subcellular locations including nuclear the SPB control of mitotic commitment relied Quesada1 inhibited through the phosphorylation by Wee1 mutations that suppress cdc25-22 boost Plo1 pores, centromeres and the cell cortex. The upon a novel methionine to phenylalanine kinase of a residue within the ATP binding kinase activity, while none can be detected only location at which the release of either mutation within its ATP binding pocket. The 1 joint with RNA pocket of the Cdk1 catalytic subunit. The timing when Cut12 function is lost. Enhancement kinase activity triggered mitosis was the SPB. same position is occupied by methionine in the Biology Group Figure 2 Moreover, of the three SPB components tested, catalytic pocket of Wee1. Because Wee1 is only with which the Cdc25 removes this phosphate of Plo1 activity by mutation of Cut12 would Model illustrating the inverse marginally sensitised to analogue inhibition by dictates the timing of Cdk1-Cyclin B activation be expected to increase feedback loop the drive into mitosis was greatest when the correlation between the degree and so the timing of mitotic commitment. The inhibition of Wee1, thereby reducing the chimeric kinases had been recruited by the canonical sensitising mutations, we of PP1 recruitment to Cut12 balance between Wee1 and Cdc25 activities need for Cdc25 activity. and the activity of the feedback Cut12-GFP. introduced the M > F mutation that had worked therefore determines when a cell will divide. loop kinase Plo1 during late so effectively with Plo1 into Wee1. The mutation Once a critical threshold level of Cdk1-Cyclin B Triggering mitotic commitment from G2/mitosis. gave a profound enhancement of Wee1 activation is exceeded a positive feedback loop the fission yeast SPB sensitivity to inhibition by ATP analogues. Proof simultaneously boosts Cdc25 activity and We have taken a direct approach to follow up of principle experiments then established that suppresses Wee1 activity to convert the hitherto this correlative evidence, suggesting that this allele provides a powerful tool for the gradual accumulation of Cdk1-Cyclin B activity centrosomes/SPBs play a key role in mitotic dissection of the conserved pathways that into an all or nothing bi-stable switch that drives commitment, by asking the simple question control mitotic commitment and execution. the cell into mitosis. The protein kinase Polo “What happens to the mitotic commitment plays a key positive role in this feedback loop. decision when a pulse of Cdk1-Cyclin B or Plo1 Lessons from yeast activity is released at a particular cell location?” The ability to manipulate genes at will in a Mitotic commitment and the We fused each kinase to a single chain llama simple organism, whose primary purpose is to centrosome/spindle pole body antibody called GFP binding protein (GBP). GBP divide, is enabling us to explore the finer points Centrosomes nucleate the two microtubule has a very high affinity for the Green Fluorescent of the pathways that co-ordinate a successful arrays that inter-digitate to form the mitotic Protein (GFP) that is widely used in cell biology cell division. This information informs studies in spindle. The initial appearance of active because its intrinsic fluorescence reveals the higher systems that, in turn, raise models that Cdk1-Cyclin B on human centrosomes and location of any fusion protein. A large number can be most readily tested in yeast. This iterative modelling of Cdk1-Cyclin B activation in frog of fission yeast strains have been modified to cycle of comparative studies ensures that great egg extracts suggests that the trigger for mitotic generate fusions between individual genes of strides are being made in understanding the commitment stems from this organelle. Our interest and sequences encoding GFP. We molecular basis of cell division. studies of a component of the fission yeast therefore used GBP to direct Plo1 and Cdk1 centrosome equivalent, the spindle pole body kinases to a wide range of structures defined by Publications listed on page 56 18 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE CELL DIVISION 19

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    DEG Expression LFC>2 Identification of novel targets of the CELL REGULATION S. pombe MAP kinase Sty1 www.cruk.manchester.ac.uk/cellregulation The SAPK pathways are highly conserved between yeast and mammalian cells, and in Schizosaccharomyces pombe the MAPK Sty1 is a key regulator of the stress response. Sty1 is activated following the exposure of cells to a wide variety of environmental stress conditions. Similarly to the mammalian SAPKs, Sty1 is activated through dual phosphorylation by an upstream MAPKK. Due to the high level of conservation between the mammalian and Stress Signalling Pathways describe the molecular activities yeast signalling pathways, S.pombe provides an ideal model to investigate the function and emanating from (generally adverse) external stimuli that are regulation of the SAPK signalling pathways. received by cells via surface or internal receptors and Upon activation Sty1 mediates the appropriate transmitted via relays of protein kinases. Depending on the stress response through the phosphorylation type of stimulus and cellular context, activation of these of downstream target proteins, the best characterised of which is the transcription factor pathways may lead to induction of diverse cellular Atf1. Whilst only a small number of Sty1 targets programmes, ranging from growth to growth arrest, are currently known, the large number of Group Leader cellular processes regulated by the Sty1 pathway differentiation, or cell death. suggests that there are likely to be a number of, Nic Jones as yet uncharacterised, Sty1 target proteins. To Associate Scientists One such signalling pathway is described by the ATF2: an effector transcription factor of fully understand the stress response, it is vital Wolfgang Breitwieser stress activated protein kinase (SAPK) pathway. stress kinase signalling that we identify targets of SAPKs. Formally, this has been categorised as a specific One focus of research in the Cell Regulation Scientific Officer branch of the wider acting MAPK (mitogen group is the AP-1 transcription factor ATF2, results in accelerated disease onset. Further in In order to identify novel targets of Sty1 Steve Lyons activated protein kinase) pathways and involves which is an effector substrate of the SAPKs, JNK vitro analysis revealed that ATF2 responds to phosphorylation we performed a SILAC Graduate Students a series of protein kinases which act as signalling and p38. Our research work has demonstrated oncogene induced cellular stress by inducing screen in collaboration with the laboratory of Figure 1 Emily Holmes molecules that transmit information via that ATF2 has essential functions during programmed cell death. In addition, JNK and Boris Maček at the University of Tübingen. Gene Level Analysis: A heat Alexander Thapa ATF2 were shown to direct the induction of Intriguingly, we found that following exposure phosphorylation of their protein substrates. development of the embryonic liver, heart and map depicting differentially Thus, SAPKs (including members of the JNK brain, and that this is dependent on its activation expressed genes in hepatoblasts apoptosis in response to chemotherapeutic to oxidative stress, proteins upstream of Sty1 in and p38 subfamilies) are activated by upstream by the SAPK signalling cascade. in response to ATF2 activity. drugs but only once B lymphocytes have the MAPK pathway are phosphorylated in a Expression array experiments progressed towards lymphoma stages by Sty1-dependent manner. Furthermore, one acting layers of kinases comprising SAPKKs (e.g. were carried out in triplicate MAP2K3, 4, 6, and 7) and SAPKKK (e.g. MAP3K1, High levels of phospho-ATF2 have been the actions of cMYC. Thus, ATF2 is an effector such protein is a potential direct target of Sty1 (columns) of control or ATF2 2, etc). At the base of this hierarchy lie cellular detected in both human melanoma and expressing cells. Over 250 of cMYC induced apoptosis through phosphorylation. Further investigation revealed effector proteins that include other kinases, prostate carcinoma samples, and a role for ATF2 up-regulated (red bars) and over activation by JNK. that Sty1-dependent phosphorylation of apoptosis regulators, and transcription in driving progression of these tumours has 30 down-regulated (blue bars) upstream components appears to promote genes were identified. Tumour suppressive transcriptional Sty1 phosphorylation, thus forming a positive factors. Together, the actions of the effector been suggested in the literature. For example, molecules, in response to activation by the recent findings indicate that the SS18-SSX2 programmes by ATF2 feedback loop to increase the level of Sty1 Figure 2 SAPKs determine the cellular response to a fusion protein, found in human synovial The S. pombe Sty1 MAPK An independent project has focused on the activity following oxidative stress exposure. specific stimulus. sarcomas, derives its oncogenicity from the signalling pathway. The Sty1 role of SAPK pathway components in ability to interact with ATF2. Conversely, low kinase is activated by a kinase hepatocellular carcinoma. Here, we Publications listed on page 57 cascade. The activated MAPK demonstrated that JNK dependent activation Stress Kinase pathways in cancer levels of ATF2 expression in human breast Sty1 then phosphorylates a Recent advances in cancer genome analyses tumours have also been reported. of ATF2 is critical in blocking the oncogenic range of target proteins to have highlighted the frequency at which stress mediate the stress response. transformation of hepatocyte precursors signalling pathways are deregulated, or Functions of JNK and ATF2 in B lymphoma (hepatoblasts), as well as in suppressing their mutated, in various tumour types. For example Experimental analyses using mouse tumour tumourigenicity after orthotopic transplantation the signalling kinase MEKK1 (MAP3K1) is mutated models carried out by our group as well as by into recipient livers. In addition, we defined a in 9% of breast invasive and endometrial others, have uncovered diverse roles for ATF2 in JNK and ATF2 dependent transcriptional carcinoma and 6% in prostate adenocarcinoma tumourigenesis. Accordingly, ATF2 was shown programme that acts in a tumour suppressive (source: cbioportal.org), while its substrate, to contribute to melanoma development manner. Further analysis revealed that this MAP2K4, is mutated in 6-7% of breast and colon through its role in a melanocyte-specific gene programme is frequently found inactivated or cancers. The nature of these mutations activation programme. In contrast, ATF2 genetically altered in a variety of human tumour suggests that the pathway is aberrantly activated deficient mice are sensitised to carcinogen- types, including breast, lung, pancreatic and in some types of cancer, but inactivated to a far induced skin tumourigenesis, underlining the hepatocellular carcinoma. This analysis greater extent in many other types. Thus, stress tumour context dependent activities of the therefore confirmed that the experimental signalling pathways are thought to be pro- stress signalling pathway. In a mouse model of tumour models reflect human cancer oncogenic or tumour suppressive depending MYC oncogene induced B-cell lymphoma scenarios. Further analysis of SAPK dependent on the cancer context. development, we showed that ATF2 deficiency effectors, as well as other components of stress signalling pathways, will therefore be the focus of future research. 20 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE CELL REGULATION 21

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    CELL SIGNALLING grow very slowly (Malliri et al., Nature 2002). To better understand the role of TIAM1 in www.cruk.manchester.ac.uk/cellsignalling promoting tumour growth we have examined its role in the cell cycle. We revealed that TIAM1 and RAC localise to centrosomes during prophase and prometaphase, and TIAM1, acting through RAC, ordinarily retards centrosome separation. TIAM1-depleted cells transit more slowly through mitosis and display increased chromosome congression errors. Significantly, suppression of the microtubule motor Kinesin-5/Eg5 in TIAM1-depleted cells rectifies Tumour initiation and progression result from the inappropriate not only their increased centrosome separation but also their chromosome congression errors activity of intracellular signalling cascades. RHO-like GTPases and mitotic delay (Woodcock et al., Curr Biol. are molecular switches in signalling pathways that regulate cell 2010). Subsequent to this study, we have found that TIAM1 is phosphorylated by Cyclin B/CDK1 morphology, adhesion, motility, as well as cell cycle in mitosis. This phosphorylation, while not progression and survival. Data has emerged to directly Figure 1 RAC GTPase cycles between RAC activity is also regulated through required for TIAM1 localisation to centrosomes, ubiquitylation and subsequent degradation. is essential for its role in regulating centrosome implicate RHO proteins in tumourigenesis. We investigate inactive GDP-bound and active Recently, we identified the tumour suppressor separation. Currently, we are investigating the GTP-bound states. RAC the mechanisms by which certain regulators of the RHO-like activation is facilitated by the HACE1 to be the E3 ubiquitin ligase responsible mechanism by which phosphorylation of TIAM1 Group Leader action of GEFs, which promotes for RAC degradation following activation by a influences its role at centrosomes. GTPase RAC control cell cycle progression and cell adhesion GDP dissociation from RAC and migration stimulus. We showed that HACE1 Angeliki Malliri and how their activities, as well as activity of RAC itself, allows GTP to bind instead. In turn, GEFs are stimulated by and RAC1 interaction is enhanced by HGF TIAM1 antagonises malignant progression Postdoctoral Fellows signalling and that HACE1 catalyses the poly- Despite their slower growth, tumours arising Zoi Diamantopoulou1 are controlled. receptor tyrosine kinases (RTK), integrins (αβ), and G-protein ubiquitylation of RAC1 at lysine 147 following in Tiam1-deficient mice progressed more Andrew Porter coupled receptors (GPCR). its activation by HGF, resulting in its proteasomal frequently to malignancy (Malliri et al., Nature Lynsey Vaughan RAC1 is active in melanoma human melanoma samples demonstrated Through the association with degradation. HACE1-depletion is accompanied 2002). One mechanism by which TIAM1 and Helen Whalley GAPs, the intrinsic GTPase Aberrant expression of RAC or RAC activators in widespread overexpression and hyperactivation by increased total RAC1 levels and accumulation RAC suppress malignant progression is through activity of RAC is accelerated, Scientific Officer human cancer, and the effect of conditional of RAC1. We also revealed overexpression of the of RAC1 in membrane ruffles. Moreover, promoting cell-cell adhesion. We further thereby inactivating RAC. Gavin White ablation of rac1 or Rac activators in mouse RAC activator TIAM1 (T-lymphoma invasion and Through association with HACE1-depletion enhances cell migration investigated the function of TIAM1 and RAC at tissues on tumour formation and progression, metastasis protein) in nodular forms of RhoGDIs (GDI) RAC can be independently of growth factor stimulation, cell-cell adhesions. We identified β2-syntrophin, Graduate Students have implicated RAC1 in tumourigenesis. While melanoma (Dalton et al., J Invest Dermatol. sequestered in its inactive state. (Castillo-Lluva et al., Oncogene 2013). Jointly, a component of the dystroglycan adhesion Hadir Marei Activated RAC can also be Erinn-Lee Ogg RAC mutations were not readily detected in 2013). Thus, we conclude that RAC1 the above two studies suggest that complex, as a TIAM1 binding partner. Our study removed through ubiquitylation- Anna Woroniuk human cancer using conventional sequencing, deregulation is a common event in the genesis SUMOylation and ubiquitylation of RAC1 act (Mack et al., Nat Cell Biol. 2012) unearthed a induced degradation (mediated subsequent exome sequencing has revealed of melanoma, which co-operates with aberrant by HACE1 following a migration co-ordinately to fine-tune RAC1 activity in novel role for this complex in regulating tight 1 joined in 2013 migrating cells, promoting RAC activity at sites junctions and the generation of apicobasal oncogenic RAC alleles. In particular, ~9% of RAS signalling to drive malignant progression. stimulus) or it can be maintained cases of melanoma developing in sun-exposed following its modification by where the cell membrane is advancing, while polarity through the formation of a RAC SUMO (mediated by PIAS3). antagonising RAC at sites where membrane activity gradient in the membrane region sites possess RAC1 mutated at a common Post-translational modifications of Rac1 amino acid, Proline 29. Conversion of this during cell migration protrusion needs to cease. encompassing these junctions. residue to Serine alters the conformation of the Recently, regulation by post-translational switch I loop of RAC1 and activates the protein. modification has emerged as a significant TIAM1-RAC signalling regulates bipolar Malignant progression can entail the loss of RAC1 mutation can coincide with gain-of- means of regulating RAC activity. To gain spindle assembly, chromosome congression cell-cell adhesion. The oncoprotein Src, a function of BRAF or NRAS mutation in further insight into the regulation of RAC and mitotic progression dependent on non-receptor tyrosine kinase, targets adherens Figure 2 phosphorylation of TIAM1 by Cyclin B/CDK1 junctions (AJ) for disassembly. Previously, we melanoma, suggesting that RAC1 co-operates during cell migration, we performed a screen RAC contributes to several with MAPK signalling to induce melanoma. for proteins that interact with RAC following Mice deficient for Tiam1 are resistant to the showed that Src phosphorylates TIAM1, cancer hallmarks that treatment of cells with a motility-inducing promote tumour formation formation of skin tumours induced by chemical inducing its cleavage by Calpain and its To investigate possible co-operation between factor, Hepatocyte Growth Factor (HGF). This and progression. carcinogens and the few resulting tumours depletion from AJs. Abrogating TIAM1 RAC and RAS in melanoma formation, we revealed the small ubiquitin-like modifier phosphorylation by Src suppressed AJ combined expression of an active RAC1 allele (SUMO) E3-ligase, PIAS3, as a novel RAC disassembly (Woodcock et al., Mol Cell 2009). (RAC1G12V) with oncogenic RAS (HRAS G12V) in interacting protein. Subsequently, we We have now found that TIAM1, like RAC1, is zebrafish melanocytes. The combination was demonstrated that RAC1 can be conjugated ubiquitylated and degraded upon treatment of significantly more potent at inducing tumour to SUMO-1 by PIAS3 in response to HGF. PIAS3 cells with HGF. We have mapped ubiquitylation nodules than either mutant gene alone. interacts better with GTP-bound RAC and the sites and identified the responsible E3 ligase. Surprisingly, expression of RAC1G12V alone did GTP-bound form of RAC is a better substrate for Moreover, we show that interfering with TIAM1 not perturb zebrafish melanocyte development, SUMOylation. Furthermore, we demonstrated ubiquitylation retards the scattering and invasion morphology, proliferation or migration, but in that PIAS3-mediated SUMOylation of RAC1 of cells through delaying AJ disassembly. combination with HRASG12V did induce controls RAC1-GTP levels and the ability of precocious proliferation and migration of Rac1 to stimulate lamellipodia, cell migration Publications listed on page 57 transformed melanocytes, promoting tumour and invasion (Castillo-Lluva et al., Nat Cell formation. Immunohistochemical staining of Biol. 2010). 22 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE CELL SIGNALLING 23

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    Francesca Trapani1 sample using CellSearch technology. Palpable CLINICAL AND EXPERIMENTAL Cong Zhou tumours were detected within 4 months of PHARMACOLOGY Clinical Fellows implantation with doubling times ranging from Kyaw Aung 5–21 days for a high proportion of patient www.cruk.manchester.ac.uk/cep Louise Carter samples. CDX derived from chemosensitive Leila Khoja2 patients were slower growing than those Kalena Marti Marti derived from chemorefractory patients and the Robert Metcalf number of CellSearch detected CTCs in patients Danielle Shaw Laura Cove Smith whose parallel blood samples gave rise to CDX were all >400/7.5ml. Scientific Officers Jenny Antonello The pathology, cytology and Mahmood Ayub1 The CEP group places emphasis on the discovery, Jessica Booth2 immunohistochemistry (IHC) of CTC tumours and micro-metastases mirrored that of the development and validation of circulating biomarkers to Stephen Bramley Debbie Burt corresponding clinical diagnostic specimens. facilitate drug development and to aid cancer patient treatment Fouziah Butt John Castle Mitotic and apoptotic cells were frequently observed in CDX. Detailed histological decision making. Our scientific focus is lung cancer and this Samson Chinien A examination of serial lung sections revealed Jakub Chudziak year we enhanced our portfolio of clinical trials incorporating Martin Dawson micrometastases comprised of human SCLC Olive Denneny cells in the alveolar wall. CDX tumours have now Circulating Tumour Cells (CTCs) as biomarkers. A highlight is Suzanne Faulkner been repeatedly passaged and on-going studies the enumeration and characterisation of CTCs within the Joseph Halstead Grace Hampson will show how closely their response to standard Group Leader of care chemotherapy reflects the pioneering TRACERx consortium that will map intra-tumour Cassandra Hodgkinson chemosensitivity of the patient from which they Caroline Dive Clare Hodgson heterogeneity and the evolution of Non Small Cell Lung Hana Jelassi were derived. Importantly, we have developed Paul Kelly sequential CDX models from individual patients’ Cancer (NSCLC). This year our annual report outlines the Matthew Lancashire blood samples collected at baseline, prior to Daniel Morris development of unique patient derived CTC based mouse Karen Morris patient responses to first cycle of chemotherapy, and again as patients relapse models, to study the biology of SCLC and to test novel agents, Jackie Pierce Tony Price with drug resistant disease. These models are as well as the progress made by our circulating nucleic acids Lynsey Priest Robert Sloane now being interrogated to facilitate an understanding of acquired drug resistance and biomarkers team. Nigel Smith to identify potential targets that could be Nahida SultanaMiah1 evaluated by our Drug Discovery Unit. Graduate Students B Danielle Potter The Nucleic Acids Biomarkers Team (NAB) Deputy Group Leader Shaun Villa2 Cancers arise and develop through the multistep accumulation of somatic mutations Ged Brady Small Cell Lung Cancer – the need for chemotherapy resistance. However, a major Laboratory Manager responsible for tumour growth, metastasis and improved preclinical models to explore barrier to more comprehensive understanding Martin Greaves Disease Focus biology and test novel therapies of human SCLC biology is the paucity of fresh, the development of treatment resistance. Team Leaders Scientific Assistant Circulating biomarkers provide one of the most Small cell lung cancer (SCLC) represents sufficient and sequential tumour biopsies for Fiona Blackhall Aileen Jardine promising means of serially monitoring a ~15-20% of all lung cancer cases and is research as surgery is rarely performed. There Andrew Renehan characterised by a high proliferative rate are few useful preclinical models of SCLC with patient’s cancer and raises the possibility of Malcolm Ranson Admin Assistant resulting in rapid tumour growth, early which to progress translational research and Lisa Waters more effective personalised therapies. A patient Staff Scientists metastatic dissemination and an aggressive drug development. blood sample may contain 0-1000s CTCs in Jeff Cummings clinical course. Most cases are initially chemo 1 joined in 2013 amongst 108 normal white blood cells (WBCs) Dominic Rothwell along with nanogram quantities of tumour and radiosensitive, but disease relapse invariably CTC derived patient explant mouse models 2 left in 2013 Jonathan Tugwood occurs with emergence of treatment resistance (CDX), a unique approach to study Small derived circulating free DNA (cfDNA). The NAB Clinical Lecturers such that overall survival rarely exceeds two Cell Lung Cancer team led by CEP Deputy Group Leader, Ged Alastair Greystoke2 years. The major genetic aberrations in SCLC Having demonstrated the prevalence of SCLC Brady, was established in 2011 to develop Cliona Kirwan involve p53 (75-90%) and Rb (78-90%), along CTCs, we sought to develop patient derived in circulating biomarkers in a clinic ready format. In Matthew Krebs 2013, the NAB team has established the with mutually-exclusive amplification of MYC vivo models of SCLC, reasoning that tumour Associate Scientists family genes in 18-31% of patients. Whilst many initiating cells must be present within this C following methodological protocols: Christopher Morrow scientific hypotheses have been generated invasive tumour cell subpopulation. Blood Kathryn Simpson using long established SCLC cell lines, they have samples were obtained from patients with • Novel stable whole blood processing not been upheld in the clinic where trials with chemonaïve, extensive stage SCLC. To establish Figure 1 delivering CTCs and cfDNA up to 4 days Service Manager Development of SCLC Patient CTC derived mouse post collection targeted therapies in SCLC have proved whether patients’ CTCs could form tumours in David Moore models: A, schematic of protocol for enrichment and universally disappointing. Readily accessible immune-compromised mice, 10ml of blood enumeration of CTCs from SCLC patients’ blood • NGS based mutation calling and copy Postdoctoral Fellows patient derived preclinical models are required. from each patient was enriched for CTCs and samples; B, example of subcutaneous CDX tumour; C, Alison Backen The frequent, rapid and marked biological the enriched fraction injected subcutaneously comparison of the pathology and number variation (CNV) of tumour Becky Bola transition from chemotherapy sensitive to into the flanks of NOD.scid IL2γ (NSG) mice. immunohistochemistry of example CDX tumour with samples Radoslaw Polanski2 matched patient diagnostic cytology specimen. resistant disease suggests that SCLC has much The number of epithelial CTCs (EpCam+/CK+) to reveal regarding novel drivers of acquired implanted was estimated in a paired 7.5ml blood 24 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE CLINICAL AND EXPERIMENTAL PHARMACOLOGY 25

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    CLINICAL AND EXPERIMENTAL PHARMACOLOGY (CONTINUED) • Routine single CTC isolation, Sanger whole blood for four days at room temperature. sequencing and NGS based CNV analysis This simplification will greatly improve sample analysis for multi-site clinical trials and allows • Platforms for multiplex NGS analysis of the analysis of cfDNA as well as CTC mRNA and single CTCs DNA from the same blood sample. To establish molecular profiles of individual CTCs we have • Single cell mRNA profiling by array, established a three step protocol (Figure 2), a) RNA-Seq and qPCR CTC enrichment removing >99% of WBCs; b) single cell identification/isolation and c) • Routine plasma cfDNA qPCR analysis Genomic DNA (gDNA) and mRNA analysis. For each patient sample the appropriate CTC • Initial NGS of cfDNA including detection enrichment protocol was chosen based on the of a potential novel resistance biomarker type of cancer and the downstream analysis required. For routine gDNA analysis of single • Plasma miRNA profiling for clinical and CTCs we established a simple and pre-clinical monitoring representative whole genome amplification (WGA) approach. Over the last year we have • Bioinformatic pipeline for sequencing successfully developed and applied next and CNV analysis in collaboration with generation sequencing (NGS) approaches to Crispin Miller’s group determine gDNA copy number variation (CNV) and mRNA profiles from individual cells. Fundamental to progress with nucleic acids Bioinformatic analysis of CTC CNV data (in based biomarker development, we improved collaboration with Crispin Miller) has established blood collection and processing by ensuring similarities between SCLC CTCs and matching minimal loss or change in cellular and CDX tumours as well as heterogeneity extracellular nucleic acids following storage of accompanying treatment and relapse. CTCs from a patient with non small cell lung cancer enriched DAPI Cytokeratin using ISET microfiltration. Figure 2 In collaboration with Nitzan Rosenfeld (Cancer sequencing of plasma and tumour DNA from Tumour cells are identified Schematic for the enrichment Research UK Cambridge Institute) we have also patients with advanced colorectal cancer through their expression of the and isolation of CTCs prior to established a focussed NGS approach (Tagged (collaboration with Marilyn M. Li Baylor Genetics epithelial marker cytokeratin molecular analysis. WBC are (green) and leukocytes through used as negative controls and amplicon sequencing or TAm-seq) for single Dept) showed mutation profiling of cfDNA can expression of CD45 (red). The the protocol was initially CTCs analysis. complement tumour profiling by identifying co-expression of cytokeratins validated using LS174T epithelial mutations missed by tumour sequencing alone and VE-cadherin (blue) by cells spiked in healthy volunteer For mRNA analysis we can now carry out and was also able to detect the emergence of a tumour cells is under blood: (CK= cytokeratins). accurate and reproducible transcriptional cfDNA mutation that may be a potential investigation by our laboratory in association with a study of profiling at the single cell (CTC) level and have chemotherapy resistance mechanism. We have tumour cell vasculogenic devised approaches that allow profiling of single developed a highly sensitive protocol for plasma mimicry. cells stored at room temperature for four days. based miRNA analysis that can now be utilised Image supplied by Robert CD45 VE-Cadherin We have successfully used the mRNA profiling methods to identify stem cell linked signatures for longitudinal monitoring of tumours in mice and patients. Metcalf. in enriched primary non-small cell lung cancer (NSCLC) cancer initiating cells, and this work Publications listed on page 57 was judged as the best poster (from a total of over 100) at the 9th International Symposium on Minimal Residual Cancer (Paris, Sept 2013). We have developed our cfDNA program by establishing both whole genome and focused cfDNA NGS analysis. Initial longitudinal 26 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE CLINICAL AND EXPERIMENTAL PHARMACOLOGY 27

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    screening for proteins that have the ability to modulate chromatin structure and facilitate DNA DAMAGE RESPONSE respond to DNA damage in a manner that is DNA repair reactions in response to poly(ADP- blocked by treatment with clinically relevant ribose) signalling (Mehrotra et al., Mol Cell 2011). PARP inhibitors, such as olaparib. Our goal is to characterise some of the obtained candidate Another class of DNA damage response proteins and elucidate their exact biochemical proteins that is a focus of our research is the functions in DNA repair, as well as their mode of macrodomain proteins. The macrodomain is regulation in response to DNA damage. another evolutionary widespread module with Previously, in screening for proteins with the the capacity to bind poly(ADP-ribose) and we ability to bind poly(ADP-ribose), we discovered a recently identified several human macrodomain poly(ADP-ribose)-binding zinc finger motif protein factors that are recruited to broken (PBZ). PBZ is a structurally distinctive, atypical DNA ends in a poly(ADP-ribose)-dependent Many cancer therapy procedures, such as radiotherapy and type of zinc finger that is associated with several manner. These include a histone H2A variant proteins involved in response to DNA damage called MacroH2A and several other some types of chemotherapy, work by overwhelming the (Ahel at al., Nature 2008). uncharacterised macrodomain proteins, capacity of the cell to repair DNA damage, resulting in cell such as TARG1 and MACROD1. One of the human proteins containing a PBZ death. Most rapidly dividing cells - cancer cells - are motif is a protein called Checkpoint protein with Structural and functional analysis of the preferentially affected by such treatments, providing the FHA and RING domains (CHFR). CHFR is an poly(ADP-ribose)-degrading enzymes and ubiquitin ligase frequently inactivated in human their validation as a targets for cell- opportunity to use DNA damaging agents to selectively kill epithelial tumours, which acts as a key regulator permeable inhibitor design cancer cells. In addition, the genomic instability is the driving of the poorly understood early mitotic Available data indicates that inhibiting PARG Group Leader checkpoint that transiently delays chromosome might offer a promising and beneficial approach force of cancer development, which requires multiple DNA condensation and nuclear envelope breakdown in the treatment of cancer and cardiovascular Ivan Ahel1 mutations resulting in loss of cellular growth control. In order in response to variety of stresses. The conditions. However, unlike the case of PARP Postdoctoral Fellows elucidation of the function of the PBZ motif inhibitors, progress in developing permeable, Dragana Ahel 1 to accelerate the accumulation of genetic changes, cancers gave us a vital clue to discover that the CHFR- small-molecule PARG inhibitors has been Benito Banos1 Rosa Morra1 often sacrifice specific DNA repair pathways. This can make dependent checkpoint is regulated by PARPs limited, partly due to the lack of functional and and that the PBZ motif in CHFR protein is critical structural data for the human PARG protein. Roko Zaja1 cancer cells additionally susceptible to DNA damaging agents for checkpoint activation. Another PBZ- Recently, we solved the crystal structures of Senior Scientific Officer and/or to inhibitors that block alternative repair pathways. For regulated protein we are studying is a protein several PARG enzymes from bacteria and lower Ria Weston1 called Aprataxin-PNK-like factor (APLF). APLF eukaryotes, which gave the first insight into the these reasons, studying the protein components involved in uses tandem PBZ repeats for direct interaction basic principles of PARG structure and its Graduate Students Eva Barkauskaite1 repair of damaged DNA has been proven a valuable strategy in with poly(ADP-ribosyl)ated PARP1, which allows mechanism of catalysis (Slade et al., Nature APLF’s timely localisation to the sites of DNA 2011; Barkauskaite et al., Nat Commun. 2013). Michael Tallis1 searching for novel approaches and targets in cancer therapy. Figure 1 Active site of human TARG1 damage. We previously discovered that the role These structures revealed that the PARG 1 left in 2013 protein with bound ADP-ribose. of APLF is to act as a histone chaperone to catalytic centre is a diverged type of Poly(ADP-ribosyl)ation in regulation specific recruitment and scaffolding of DNA macrodomain and demonstrated that they are of DNA repair repair complexes. In addition, the damage- likely to prove useful in guiding structure-based Poly(ADP-ribosyl)ation is a post-translational induced poly(ADP-ribosyl)ation has a role in discovery of new classes of PARG inhibitors. protein modification that controls several relaxation of the chromatin structure and in Despite these advances, detailed structural nuclear processes known to be important for apoptotic signalling. The recent development of information on the human PARG is still lacking. genome stability, including DNA repair, potent PARP inhibitors has provided powerful Our goal is to solve the structures of human of regulation of chromatin structure, cell cycle tools to study pathways regulated by poly(ADP- PARG in complex with the substrate analogues checkpoint, transcription, apoptosis and mitosis. ribose), as well as providing a promising novel and inhibitors which, in combination with Poly(ADP-ribose) is a highly negatively charged class of drugs for cancer treatment. Specifically, solution and cell biology studies, should address polymer that is formed from repeating selective inhibition of the DNA break repair the mechanism, structure and regulation of ADP-ribose units linked via glycosidic ribose- pathway using permeable PARP inhibitors has human PARG, as well as providing a foundation ribose bonds, and is synthesised by the been proven highly effective against breast and for the development of small, cell-permeable poly(ADP-ribose) polymerase (PARP) family of ovarian cancers (Bryant et al., Nature 2005). PARG inhibitors. 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 The PARG enzyme is unable to cleave the -ation is performed by the hydrolytic action of processes is likely of vital importance for mono(ADP-ribose) unit directly linked to the an enzyme called poly(ADP-ribose) selecting and developing efficient therapies. protein targets; accordingly, we recently glycohydrolase (PARG), which specifically identified TARG1 (C6orf130) protein as an targets ribose-ribose bonds and cleaves Identification and characterisation of novel enzyme responsible for this last step of poly(ADP-ribose) into ADP-ribose monomers. poly(ADP-ribose)-regulated factors poly(ADP-ribosyl)ation reversal in human cells The role of poly(ADP-ribosyl)ation is best Our laboratory is particularly interested in the (Sharifi et al., EMBO J. 2013) (Figure 1). We are understood in the regulation of DNA repair, identification of novel DNA repair pathways and studying further this protein to understand its which is controlled by the three PARPs protein functions regulated by poly(ADP-ribosyl) mechanism of action and the exact cellular responsive to DNA strand breaks (PARP1, PARP2 -ation, in order to identify components of these pathways it regulates. and PARP3). Poly(ADP-ribosyl)ation arising at the pathways that can be exploited as targets for sites of damaged DNA serves as a platform for cancer therapy. For this, we have been Publications listed on page 58 28 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE DNA DAMAGE RESPONSE 29

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    Finally, for our more advanced lead optimisation DRUG DISCOVERY Target Hit Lead Lead Clinical projects, in collaboration with colleagues in Selection Identification Identification Optimisation Development www.cruk.manchester.ac.uk/drugdiscovery CEP, we have developed pharmacokinetic and biomarker technologies. These are very Project 1 important capabilities, enabling the selection Project 2 of the best compound(s) and models for Project 3 demonstration of preclinical proof of concept Project 4 and ultimately the identification of cancer patients most likely to benefit from the novel Project 5 target approach in clinical trials. Cancer Research UK During 2013, we have advanced our drug discovery We remain actively involved in the broader Cancer Research UK drug discovery portfolio significantly and now have two projects in the programme. During the summer of 2013 we lead optimisation phase. We also currently have three earlier underwent a successful five year (quinquennial) review with an international expert panel. hit-to-lead projects tackling a variety of cancer targets. These The group was complimented on many activities are underpinned by multiple collaborations, both aspects of its progress, including our closely integrated multidisciplinary team, clinical within and beyond Manchester. In particular, during 2013, alignment of our projects, rapid decision we have signed four collaboration agreements with making and project portfolio progression and Group Leader we have recently been notified that our funding pharmaceutical and biotech companies to enhance our has been extended for a further five years (until Donald Ogilvie hit-finding capabilities. March 2019). Head of Chemistry Allan Jordan Figure 1 project, we have worked closely with James People and Publications Project Portfolio required extensive mutation and expression Drug Discovery Unit active During the last year as our project portfolio Lynch in Tim Somervaille’s Leukaemia Biology Head of Bioscience Our current portfolio consists of two lead data are available, is underway and this has portfolio. has moved forward we recruited Ben Acton to Ian Waddell group in the development and use of a novel, optimisation projects, directed against led to new collaborations with target facilitate more advanced biological and Figure 2 proximal cell biomarker for LSD1 activity. The oncogene and DNA repair targets respectively, validation partners. pharmacokinetic testing of compounds. We Chemists CD86 expression in THP1 cells, DDU provided an irreversible LSD1-selective Ali Raoof and three hit identification projects against two as determined by ELISA, have also taken on our first PhD student, Daniel inhibitor (compound 3) and this was used to Alison McGonagle epigenetic and oncogene signalling targets Capabilities following 24hr incubation with Mould, who previously completed an industrial treat THP1 AML cells for 24 hours. Amanda Lyons2 (Figure 1). For the more advanced lead Once a target has been selected for drug various concentrations of LSD1 Transcriptome analysis revealed CD86, a placement year in our laboratory and was Bohdan Waszkowycz optimisation projects, the pharmacology of discovery, the next stage is to try and identify inhibitors 3, 22 & 23 Colin Hutton ligand for the co-inhibitory immune response awarded North West student of the year 2013 by our novel compounds is being explored in prototype small molecules, or “hits”, that interact the Society of the Chemical Industry for his James Hitchin receptors CTLA4 and CD28, as one of the five Kate Smith cells, both internally and with key expert with the target molecule. Using our fragment- project report. most upregulated genes. Inhibitor dose- Kristen Goldberg collaborators. In support of all of our projects, based ligandability assessment platform, we dependent upregulation of the corresponding Niall Hamilton we have initiated a range of target biology and have concluded that some of our novel targets Although wider reporting of our most Rebecca Newton CD86 protein was then demonstrated using technology-related collaborations, both within carry a low/medium likelihood of finding new advanced work is usually delayed by the need Stuart Jones both flow cytometry and ELISA methods. The and beyond Manchester. hit matter. In order to maximise our chance of to file patents first, four papers directly relating negative regulation of CD86 expression by Bioscientists success with these types of target, we have to Drug Discovery activities were published in LSD1 was confirmed using lentiviral shRNAs Alex Boakes Deployment of bioinformatics has had a major negotiated four screening collaborations with 2013. A further four publications, related to (Lynch et al., Anal Biochem. 2013). Ben Acton1 impact on our portfolio this year, both upfront in industrial partners giving us access to millions of work with collaborators in the Institute and Dominic James drug target selection, bringing forward new diverse proprietary compounds. In two cases, Emma Fairweather While this work was progressing, two putative beyond, also appeared in 2013. Moreover, we hypotheses for evaluation, and also at later with AstraZeneca and GlaxoSmithKline, this has are now progressing our first patent filings, Gemma Hopkins reversible inhibitors of LSD1 were disclosed. Graeme Thomson stages. In particular, we have built and deployed already yielded novel chemical startpoints for providing legal protection for our discoveries These were quickly synthesised in the DDU Helen Small an innovative collateral vulnerability our projects. In another of these deals, with the in the laboratory. and rapidly tested in the CD86 cell biomarker Mandy Watson bioinformatics pipeline to identify novel biotech company HitGen, we are accessing Mark Cockerill assay (see Figure 2 and Hitchin et al., Med Chem candidate drug discovery targets. In this >400 million DNA-encoded compounds. The Future Nicola Hamilton Commun. 2013). This experiment confirmed approach, we seek to identify functional Our local Cancer Research Technology During 2013, we have advanced our project Nikki March the highly potent, selective activity of the Phil Chapman homologues of genes that are selectively representative, Martyn Bottomley, has played portfolio into the lead optimisation stage and irreversible standard compound 3 (CD86 IC50 Samantha Fritzl deleted in cancer cells that are essential for their a key role in securing these important have enhanced our capabilities both internally 14nM; cytotoxicity IC50 >20uM). One of the Sarah Holt survival. Selective pharmacological targeting of collaborations. and with four industrial collaborations. In 2014, competitor reversible inhibitors (compound 23) these homologues is potentially lethal for we begin a new five year funding cycle and our Graduate Student also showed potent, selective activity (CD86 cancer cells while sparing normal cells. The After hit matter is identified, one of the ultimate aim for that period is to deliver our first Daniel Mould1 (PhD) IC50 1.2uM; cytotoxicity IC50 >20uM) in cells, output from the collateral vulnerability analysis challenges in a drug discovery project is to candidate drug. In the short term, we look while the other (compound 22) showed little Undergraduate Students in lung cancer is now being followed up by assess rapidly and quantitatively the activity and forward to progressing rapidly our lead CD86 activity and a negative margin to Kate Bowler1 functional target validation studies in Caroline selectivity of novel inhibitor compounds in cytotoxicity (CD86 IC50 >20uM; cytotoxicity optimisation projects while simultaneously Eleanor French2 Dive’s Clinical and Experimental Pharmacology intact cells. These cell assays are not only IC50 <2uM). These data demonstrate the bringing forward new target opportunities. group (SCLC) and John Brognard’s Signalling important to direct our own drug discovery 1 joined in 2013 power of this novel assay to assess and Networks in Cancer team (NSCLC). Exploration efforts but also serve to benchmark competitor Publications listed on page 59 2 left in 2013 discriminate between these competitor of additional cancer types, for which the compounds as they emerge. In the LSD1 reversible LSD1 inhibitors. 30 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE DRUG DISCOVERY 31

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    patients with low/normal levels of base line IL-6 IMMUNOLOGY and low anti-superantigen antibody levels, a statistically significant treatment advantage for overall survival was seen. In North America and Western Europe, this subgroup accounts for 40-50% of the total number of advanced renal cell cancer patients (J Clin Oncol 31, 2013 (suppl; abstr 3073)). Additional analyses of the ANYARA Phase II/III study data are on-going with future development strategies aiming at a phase II/III study with ANYARA in combination with a tyrosine kinase inhibitor in the favourable 5T4 oncofoetal glycoprotein expression by many different RCC subgroup. Figure 1 relapse whilst asymptomatic with low volume cancer types with only low level detection in some normal 5T4 antibody inhibition of radiological disease, but there is no survival 5T4 antibody drug conjugates (ADC) (Pfizer) leukaemia spread tissues, as well as its mechanistic involvement in cancer spread, 100µg mAb 5T4, but not normal benefit from early chemotherapy treatment ADCs chemically combine the specificity of the (www.ctc.ucl.ac.uk/TrialDetails. antibody with a cytotoxic drug. The challenge is has driven the development and clinical testing of several 5T4 mouse serum (NMS), (both given day one and then every other aspx?TrialID=75&TrialName=TRIOC). to produce an efficacious and safe agent and directed immunotherapies. Since 1989, our translational day for 10 days) or AMD3100 this demands optimising the properties of a (CXCR4 inhibitor- plerixafor at 5T4 antibody targeted superantigen suitable TAA specific antibody in combination research has been powered by pivotal academic/clinical 1.25mg/kg, given daily for 10 therapy (with Active Biotech) with the linkage chemistry and the payload days) blocks spread of collaborations, most recently with the Groups of Professors intravenous Sup5T4 leukaemia Bacterial superantigens, such as Staphylococcal characteristics. A recent development of Group Leader (5x10e6). Significant reduction in Enterotoxin A (SEA), can activate T cells by this approach by Pfizer has used a 5T4 Robert Hawkins, Vaskar Saha and Henry Kitchener within the humanised monoclonal antibody (A1) linked by Peter L. Stern1 total tumour load and for spread linking the latter through binding to a particular University of Manchester’s Institute of Cancer Sciences (ICS), to the ovaries at day 40 for mAb5T4 compared to either family of V-beta chain containing T cell sulphydryl-based conjugation to deliver a Postdoctoral Fellows receptors (TCRs) to MHC class II molecules on tubulin inhibitor, monomethyllauristan F (MMAF) Owen McGinn1 interfaced with commercial partnerships, many of which were NMS or AMD3100 treated antigen presenting cells. With an antibody- via a maleimidocaproyl linker (Sapra et al. Mol. animals (Mann-Whitney). Julie Brazzatti1 Darren Roberts1 initiated through Cancer Research Technology. This final superantigen fusion protein, large amounts of Cancer Ther., 2013). This conjugate, cytotoxic and cytokine producing T cells can be (A1mcMMAF), showed potent in vivo activity in a report aims to update progress of this body of work. targeted by the antibody specificity for a TAA for variety of tumour models, with induction of Clinical Fellow Saladin Sawan1 in vivo tumour treatment. A succession of long term regression after the last dose. 5T4 vaccine (TroVax® with Oxford BioMedica) TroVax® or placebo, along with either 5T4-Fab-superantigen fusions designed to Evidence of the selective accumulation of the Scientific Officer 5T4 (but not control) conjugates with release of Cheryl Petit 1 Vaccine immunotherapy aims to overcome the interferon-α, IL-2 or sunitinib as first line overcome the toxicity associated with MHC relative poor immunogenicity of tumour treatment (Amato et al. Clin. Cancer Res., 2010). class II binding, and avoiding any pre-existing the payload and consequent mitotic arrest in 1 associated antigens (TAA) by presentation in a While TroVax® was safe and well tolerated in all immunity to the bacterial protein, have been the tumour tissue was demonstrated. left in 2013 foreign viral vector with the principle goal of these patients, it failed to meet its primary developed and tested in preclinical and clinical generating effector T cells able to kill 5T4 endpoint as there was no significant difference studies. The latest version (ANYARA or Depending on the particular tumour, 3-10mg/ positive tumours. The highly attenuated and in survival for the TroVax® and placebo treated naptumomab estafenatox) incorporates a kg doses given every 4 days were sufficient to modified vaccinia virus ankara (MVA), expressing groups. However, in the subset of patients with a hybrid SEA/E-120 superantigen sequence produce a complete pathogenic response; this either human or mouse 5T4, was used for good prognosis and receiving IL-2, there was a with additional point mutations, reducing was independent of the degree of successful evaluation of immunogenicity and significantly improved survival with TroVax® MHC class II binding and antigenicity heterogeneity in 5T4 expression. This effect was anti-tumour activity in preclinical studies. compared to the placebo group. Interestingly, a (Forsberg et al. J. Immunother., 2010). shown to be consistent with the targeting of Following this, a succession of phase I or II high antibody response was associated with tumour initiating cells (TICs) within the tumours. clinical trials in colorectal, prostate and renal cell longer survival within the TroVax® treated group A multinational (UK, Russia, Ukraine, Bulgaria, The overall therapeutic value is enhanced by the carcinoma (RCC) patients (including with (Harrop et al., Cancer Immunol Immunother. Romania), randomised Phase II/III study of targeting of the most aggressive and chemotherapy or cytokine treatments) 2011). It is tempting to speculate that such ANYARA, in combination with interferon-alpha tumourigenic populations within tumours established the optimal dose and route of antibodies might be mechanistically involved in versus interferon-alpha alone, in 533 advanced (TICs), and its testing in a clinical setting is vaccination as well as safety, tolerability, and reducing cancer spread. This is supported by the RCC patients has been completed. The eagerly awaited. vaccine immunogenicity. Two or three TroVax® observation that monoclonal antibody (mAb) to treatment was safe but the primary endpoint of immunisations were needed to generate 5T4 5T4 can prevent the spread of 5T4 positive survival advantage in the intention to treat Building on the above, funding from Leukaemia specific cellular immunity and this was Sup-B15 B-ALL (acute lymphoblastic leukaemia) population was not reached. This appears to be and Lymphoma Research (Stern, McGinn, Saha) independent of the vector specific response, cells in a xenograft model (Figure 1). It is possible a consequence of higher levels of pre-formed and Wellcome Trust for a Clinical Research leading to a protocol of multiple booster that the observed influence of spread might, in antibodies against the superantigen component Fellow (Louise Wan) on a collaborative project vaccinations; there was an association of 5T4 part, derive from inhibition of 5T4 glycoprotein of ANYARA in the patients recruited from East (with Stern, Gilham & Kitchener) are allowing immune responses with better clinical outcome function in regulating chemokine or Wnt European countries. Thus a subgroup analysis, further evaluation of the role of chemokine and in some cases. signalling pathways (Castro et al., Leukemia excluding patients with high levels of Wnt pathways as well as 5T4 targeted 2012; Kagermeier-Schenk et al., Developmental pre-formed antibodies, resulted in a trend for immunotherapies in leukaemia and ovarian A phase III trial in RCC patients was designed to Cell 2011). TroVax® is now being tested in ovarian survival benefit with ANYARA treatment. cancer respectively within ICS. determine if the addition of TroVax® to available cancer patients to see if there is benefit from an Interestingly, high baseline levels of IL-6 were standard of care therapy could improve survival immunological response with an extended time associated with a poorer outcome, and a Publications listed on page 59 for patients with metastatic RCC. 733 patients to progression. A significant number of patients hypothesis generating analysis of the 25 % of were randomised to receive 7/8 injections of with advanced ovarian cancer develop CA-125 32 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE IMMUNOLOGY 33

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    Figure 2 previous studies showed that nuclear PtdIns5P INOSITIDE LABORATORY regulates the accumulation of toxic reactive oxygen species (ROS), possibly through regulating the NRF2 transcription pathway. Our recent studies show that oxidative stress induced PtdIns5P increases PKB activation and cell survival. Isoform-specific RNAi studies show that the PIP2K2A isoform regulates stress induced PtdIns5P accumulation and PKB activation, while paradoxically loss of PIP4K2α also inhibits cell growth in a PKB independent manner. How exactly PtdIns5P regulates PKB is Phosphoinositides are a family of lipid second messengers Figure 3 under study, the importance of which is underlined by the intensity with which the that are regulated in response to environmental changes pharmaceutical industry has pursued inhibition by a network of kinases and phosphatases. Alterations in of the PI3K/PKB pathway as a means to halt tumour growth. Thus the inhibition of PIP4K2α phosphoinositide levels can regulate many different cancer- might compromise tumour cell growth, relevant pathways including cell survival, proliferation, however the paradoxical activation of PKB might eventually stimulate tumour growth migration, cell substratum interactions and transcription. if tumour cells can bypass the growth inhibition pathway. Group Leader In cancer cells, PtdIns(4,5)P2 is at the heart of gene can be amplified as part of the ERBB2 1 In order to unravel how PtdIns5P regulates Nullin Divecha phosphoinositide signalling and can be amplicon. Not surprisingly, increased PIP4K2β cellular processes, we have searched for synthesised by two different kinase pathways. expression was associated with poor patient Associate Scientist PtdIns5P interacting proteins and have identified De-regulation of the PIP5K or PIP4K pathway survival, probably as a consequence of its David Jones1 the PHD (Plant Homeo-Domain) finger as a leads to different outcomes in cancer signalling, co-amplification with ERBB2 (Figure 2). Figure 2 leukaemic human cell lines, murine cell lines receptor that is regulated by nuclear Postdoctoral Fellow suggesting that each pathway controls specific Interestingly, some tumours also have marked Left panel: Distribution of phosphoinositides. PHD fingers are cross- Maria Carla Motta1 downstream targets. increases in PIP4K2β expression in the absence PIP4K2B gene expression was and of primary human tumour-derived AML of ERBB2 amplification, although low patient cells. Strikingly, decreased PIP42α expression braced Zinc fingers, some of which can interact assessed across 2999 primary Scientific Officer did not inhibit normal human haematopoietic with and decode changes in histone tail PIP4K and PtdIns5P numbers prevented an analysis of its correlation breast cancer tumours Yvette Bultsma1 integrated from 17 studies. stem cell growth or differentiation, suggesting modifications to regulate chromatin structure PtdIns(4,5)P2 is present in the plasma membrane with patient outcome. and in the nucleus and can be synthesised by The data are presented as a that inhibitors of PIP4K2α might be used as and gene transcription. We found that the Graduate Student frequency distribution of therapeutics in AML. Other studies in muscle PHD finger of TAF3, a component of the Rebecca Foulger two different families of kinases utilising two Surprisingly, the immunohistochemistry study PIP4K2B expression. The basal transcription complex that regulates different substrates (Figure 1). There are three also showed that low PIP4K2β expression (the samples were amalgamated into cells showed that reducing the expression of 1 left in 2013 isoforms of PIP4Ks of which the α is enzyme is expressed in normal breast tissue) three groups based on PIP4K2B PIP4K2β, which is normally highly expressed in transcription and cell differentiation, interacts with phosphoinositides. We characterised predominantly cytosolic, β is cytosolic and also correlated with worse patient survival. expression for further analysis: muscle, stimulated the ability of stem like mutants that no longer interact with PtdIns5P nuclear, and γ localises to internal membrane This is strikingly illustrated in the analysis of the Top 10% (red) middle 80% (green) myoblast cells to differentiate into myotubes. compartments. We developed a specific mRNA profiles from 3000 breast tumours and the lowest 10% (blue). High These studies suggest that PIP4K2β expression and used these to demonstrate that changes in expression of PIP4K2B (Red) nuclear PtdIns5P are required for the antibody to PIP4Kβ and interrogated tissue (Figure 2). To begin to define if decreased was clearly associated with limits differentiation, perhaps preventing stem cell depletion during successive rounds of transcription of a subset of TAF3-regulated micro arrays of 500 advanced human breast PIP4K2B expression might be causal in patient high ERBB2 expression (inset). Right panel: Cumulative muscle injury, a process that is disrupted in genes through its interaction with the PHD tumour samples associated with patient survival, we analysed its expression and the recurrence-free outcome diseases such as muscle dystrophies. The effect finger. Mutants that do not interact with outcome data. These data demonstrated that consequences of manipulating its expression in was plotted for low, middle and PtdIns5P still interact with components of the Figure 1 increased PIP4K2β expression in tumours was human breast tumour cell lines. Our studies high PIP4K2B expression with of manipulating the expression levels of PIP4K basal transcription machinery and can interact The phosphatidylinositol-5- highly correlated with ERBB2 expression. show that decreased PIP4K2β expression colours defined above. isoforms on cell fate in different model systems phosphate kinase (PIP4K) is outline in Figure 3. with H3K4me3 modified histone tails. and phosphatidylinositol-4- Analysis of mRNA profiles derived from 3000 decreases the expression of the tumour tumours associated with patient outcome suppressor protein E-cadherin. E-cadherin Figure 3 phosphate kinase PIP5K An illustration of how isoforms of How PIP4Ks control cell fate is far from clear. We are using TAF3 as a model to understand pathway contribute to supported the above conclusion (Figure 2) and regulates cell-cell adhesion, and loss of PIP4K control cell fate in different We have previously hypothesised that PtdIns5P how changes in nuclear PtdIns5P directly phosphatidylinositol(4,5) analysis of genome amplifications in breast E-cadherin expression increases the cells systems. OX= overexpression bisphosphate (PtdIns(4,5) P2) is a key signalling intermediate and its regulate gene transcription, which might tumour samples demonstrated that the PIP4K2B propensity to undergo an epithelial to and Ex= expression. synthesis, however the major sub-cellular compartmental specific regulation underlie our observation of the impact of synthetic pathway is likely mesenchymal transition (EMT), an acquired Characterised changes in downstream pathways after by PIP4K will regulate specific downstream PIP4K2B expression on muscle cell through PIP5K. PIP4K characteristic important in the development of manipulation of PIP4K are pathways. For example, oxidative signalling differentiation. Our studies highlight the regulates the levels of metastatic tumour cells and the dissemination illustrated with red arrows. For plays a key role in aging and cancer; in response importance of the expression of different phosphatidylinositol-5- of tumour cells to distant organs. p21, p27, e-cadherin and ROS phosphate (PtdIns5P). to oxidative stress, the induction of adaptive isoforms of PIP4Ks in controlling cell fate and, arrows reflect changes in their Diacylglycerol (DAG) activates responses ultimately controls cell fate. We together with novel studies (Cantley laboratory) In other cell types, targeted RNAi studies and levels while for PKB and TAF3 protein kinase C (PKC) which they represent change in activity. discovered that oxidative stress leads to a rapid showing synthetic lethality of PIP4K2α and regulates the phosphorylation RNAi screening strategies demonstrated that decreasing the expression of PIP4Kα specifically and reversible increase in the levels of both β loss in p53 null tumours (Emerling et al., Cell and intracellular activity of PIP5K. We expect that an inhibitor of cytoplasmic and nuclear PtdIns5P and that 2013), suggest that isoform specific inhibitors reduced tumour cell growth in colorectal, PIP5K will inhibit both the PI3K PtdIns5P signalling impacts on cell survival. of PIP4Ks might be useful to target cancer cell breast, pancreatic and leukaemic human and the Phospholipase C (PLC) The PKB, FOXO and NRF2 pathways are growth and other disease states. pathway, while specific inhibitors tumour cell lines. Together with Dr. T. Somervaille, we showed that limiting the evolutionarily highly conserved regulators of the PIP4K will increase cellular Publications listed on page 60 and nuclear PtdIns5P levels. expression of PIP4K2α reduces cell growth of that control responses to oxidative insults. Our 34 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE INOSITIDE LABORATORY 35

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    Figure 1 Figure 1 of the EP400 complex (which physically LEUKAEMIA BIOLOGY In vitro and in vivo effects interacts with MYC) in murine and human AML of EPC knockdown. www.cruk.manchester.ac.uk/leukaemia (A) Representative images show cells. Together, these data suggest a role for EPC1 and EPC2 knockdown EP400 complex members in regulating MYC abolishes the clonogenic turnover in AML cells, and in preventing potential of primary human AML leukaemia cell apoptosis. cells from a patient with a t(9;11) translocation. Primary AML cells We further demonstrated that the acute from a patient with a t(9;11) were infected with lentiviruses accumulation of MYC following EPC1 targeting EPC for knockdown, or knockdown contributed to apoptosis. This was a non-targeting control (NTC), suggested by four lines of evidence. First, both with GFP as the selectable the accumulation of MYC and onset of marker. (B) Graph shows Epigenetic dysfunction plays a critical but incompletely percentage human AML cell apoptosis after EPC1 knockdown in THP1 cells were found to be MAP kinase dependent, understood role in the pathology of myeloid cancers. In the engraftment in mouse bone marrow (BM) 150 days following because both were significantly reduced by past year, the Leukaemia Biology Laboratory has reported transplant of 1.25 x105 FACS- purified GFP+ EPC knockdown or treatment of cells with MEK inhibitors U0126 or PD184352. Second, treatment of cells with the results of a genetic knockdown screen designed to reveal control cells into sub-lethally 10058-F4, a small molecule inhibitor of irradiated neonatal immune novel dependencies of myeloid leukaemia cells on key deficient mice (n=4 per cohort). MYC:MAX dimerisation (which did not prevent (C) Representative FACS plot accumulation of MYC), also significantly regulators of the structure and function of chromatin. shows engraftment in BM of reduced EPC1 knockdown-induced apoptosis. primary human AML cells in a Third, concomitant knockdown of MYC and control mouse from (B). (D) Group Leader We discovered that Enhancer of Polycomb potential in vitro (Figure 1A) through induction of Representative images show EPC1 delayed apoptosis by comparison with Figure 2 EPC1 knockdown alone. Finally, acute induction Tim (EPC) 1 and 2, components of the EP400 apoptosis, and abolished leukaemia-initiating Epc1 or Epc2 knockdown spares the clonogenic potential of of MYC levels in THP1 cells using a doxycycline- chromatin regulatory complex, are required to potential in vivo as demonstrated by syngeneic Somervaille prevent AML cells (but not normal cells) and xenogeneic transplantation (Figures 1B and murine KIT+ HSPCs. inducible system led to a significant and persistent increase in apoptosis and a modest Postdoctoral Fellows undergoing apoptosis. Identification of genes 1C). Forced expression of human EPC1 or EPC2 Figure 2 but significant reduction in proliferation. These Xu Huang selectively required for the function of rescued the clonogenic potential of knockdown Accumulation of MYC after EPC knockdown. data were published as an article in Leukemia in James Lynch leukaemia cells, but not normal bone marrow AML cells. Similar results were obtained Murine MLL-AF9 leukaemic October, with Xu Huang as the first author. cells, is an important strategy to pinpoint new following EPC knockdown in cell lines Clinical Research Fellows splenocytes (A) or KIT+ HSPCs (B) therapeutic targets for drug development. representative of other disease subtypes (e.g. were infected with lentiviruses Brigit Greystoke2 Inhibition of LSD1 as a potential therapy Dan Wiseman Kasumi1, NB4, U937, HL60), suggesting a wide targeting Epc1 or Epc2 for for AML To identify novel chromatin genes that regulate dependency of AML cells on EPC. By contrast, knockdown, or a non-targeting control (NTC), with GFP as the The last 12 months have also seen progress Scientific Officers leukaemogenic potential, we adopted a Epc1 or Epc2 knockdown did not significantly Gary Spencer selectable marker. Cells were towards a first-into-man trial of a first-in-class targeted shRNA knockdown screening affect the clonogenic (Figure 1D) or Filippo Ciceri1 FACS-purified 48 hours inhibitor of the histone demethylase, LSD1. Our approach. The knockdown library consisted of reconstitution potential of normal murine following lentiviral infection. publication of data demonstrating that Graduate Students 1040 individual lentiviral vectors targeting 272 haematopoietic stem and progenitor cells Representative western blots pharmacological inhibition of LSD1 promoted Julian Blaser2 3 genes with Gene Ontology chromatin (HSPC). This was even though levels of Epc1 and show expression of the indicated proteins in FACS-purified cells. differentiation of human AML cells (Harris et al., Filippo Ciceri2 annotations, and the screen was performed in Epc2 expression and efficiencies of knockdown Tim Somerville Cancer Cell 2012) has led to a fruitful human THP1 cells as a representative myeloid of both transcript and protein were similar to Emma Williams1 transcriptional changes previously associated collaboration with Oryzon Genomics based in leukaemia line. Lentiviral supernatants were those observed in AML cells. Experiments with with a MYC oncogenic signature. These Barcelona, Spain. Their advanced lead 1 joined in 2013 prepared individually in 96-well plates and the normal human CD34+ HSPC gave similar results: changes were not seen following Epc1 or molecule, ORY1001, which is a derivative of the 2 left in 2013 consequences for THP1 cell proliferation or the clonogenic and multilineage differentiation Epc2 knockdown in normal granulocyte- monoamine oxidase inhibitor tranylcypromine, 3 apoptosis following infection by each were potentials of EPC1 and EPC2 knockdown CD34+ co-supervised with macrophage progenitor cells; indeed the is ready for the clinic. Contingent upon ethical Nullin Divecha assessed using an alamarBlue cell biomass cells were maintained with respect to myeloid changes in gene expression observed were approval, we plan to initiate, in early-mid 2014, a until February 2013 assay. In addition to MLL itself and MEN1, which lineage colonies, although a reduction in quite distinct from those observed in AML clinical trial of this molecule in patients with is essential for the oncogenic potential of MLL erythroid burst-forming units was noted. Thus, cells, as expected given the different functional relapsed AML at The Christie NHS Foundation fusion oncogenes, this approach identified the there is a selective dependency of AML cells, but outcomes. Our data raised a question as to Trust, Manchester. In parallel, we have homologous Enhancer of Polycomb genes not normal HSPC, on EPC1 and EPC2. whether Epc knockdown activated MYC in continued to work on understanding the EPC1 and EPC2 as required for AML cell leukaemia cells. mechanisms by which tranylcypromine derivate proliferation and/or survival. EPC is conserved Using exon arrays, we next investigated the LSD1 inhibitors, such as ORY1001, promote and essential in yeast, fly and mouse and its transcriptional consequences of Epc1 and Epc2 Western blotting demonstrated that in AML differentiation of AML cells; we plan to report gene product forms part of the EP400 knockdown in murine normal and leukaemic cells there was a marked accumulation of MYC our findings in this area in 2014. More generally, chromatin regulatory complex, variants of granulocyte-macrophage progenitor cells. protein, but not transcripts, following Epc1 or our future plans will focus on the identification which include the TIP60 histone Changes in gene expression following Epc1 Epc2 knockdown (Figure 2A) which was not and validation of additional candidate acetyltransferase complex and a MYC knockdown were highly correlated with those observed in normal HSPCs (Figure 2B). therapeutic targets in AML, as well as the binding complex. To date there is no following Epc2 knockdown, suggesting their Cycloheximide chase assays demonstrated an evaluation of candidate compounds using in information as to any role for EPC1/2 in gene products have similar but non-redundant increased half-life of MYC in Epc1 knockdown vitro and in vivo model systems that take normal or malignant haematopoiesis. functions. The most remarkable observation AML cells by comparison with control cells. advantage of patient AML cells as a prelude to was that prior to apoptosis of AML cells, there Similar accumulations of MYC protein (and early phase clinical trials. Knockdown of EPC1 or EPC2 in mouse was relative up-regulation of transcriptional induction of apoptosis) were observed following MLL-AF9 AML cells, human THP1 cells and programs associated with the oncogenic knockdown of genes coding for other members Publications listed on page 61 primary patient cells, abrogated clonogenic potential of MLL leukaemia stem cells, including 36 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE LEUKAEMIA BIOLOGY 37

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    complexity of mechanisms of resistance to MOLECULAR ONCOLOGY these drugs (Figure 1). Our studies suggest that www.cruk.manchester.ac.uk/molecularoncology drugs that target both the BRAF and EGFR pathways may delay the onset of resistance in some patients and may even provide second-line treatments for patients who have failed treatment. Finally, we have continued to use next generation sequencing to characterise the genomes of human melanomas. We performed whole genome, or whole exome Our group focuses on cancer cell biology, with particular sequencing on ten mucosal melanomas. The mutation signature did not implicate UVR emphasis on melanoma, a form of skin cancer that affects over exposure in the genesis of this disease and we 12,000, and kills over 2,000 people in the UK each year. The observed a significantly lower number of single nucleotide variants (SNVs), but a significantly NRAS gene is mutated in 20% of melanomas and BRAF is higher number of copy number variations and mutated in a further 40% of cases and the proteins these genes structural changes than are seen in mucosal melanoma (Furney et al, 2013a). These data express are components of the RAS/RAF/MEK/ERK pathway, a show that mucosal and common cutaneous conserved signalling module that regulates cell growth and melanomas are different diseases with a distinct Group Leader genesis. The data suggest that structural survival. Importantly, drugs that inhibit this pathway can variations in the chromosomes play a more Richard Marais achieve impressive responses in melanoma patients whose significant role in mucosal than in common Senior Clinical Scientist cutaneous melanomagenesis, but the Amaya Viros tumours carry mutations in BRAF, but most patients develop consequences of these differences to disease Postdoctoral Fellows resistance to these drugs after a relatively short period of Figure 1 progression and treatment are currently unclear. genes, whose growth is then accelerated by the Franziska Baenke disease control. An important concept to emerge from these The EGF receptor confers BRAF drugs. Single lesions can be easily removed by We have also performed whole genome Kelly Brooks inhibitor resistance in BRAF- Simon Furney observations is the need to understand the genetics and mutant melanoma cells. LHS surgical approaches, but removal of multiple sequencing on 12 uveal melanoma samples Romina Girotti Upper panels: phospho-protein lesions can prove problematic. We (Furney et al, 2013b). Uveal melanoma is the Gabriela Gremel 1 biology of individual patients’ tumours so that their treatment arrays for A375 and A375/R cell demonstrated that topical application of most common eye malignancy and it has a very Koorosh Korfi Matthew Martin2 can be tailored for their particular disease. Thus, a key aim of lines. Lower panels: The SFKs confer BRAF inhibitor resistance 5-fluorouracil elicits regression in paradox- poor prognosis, being fatal in about half of induced lesions, providing a relatively simple patients. Surprisingly, uveal melanoma had a Berta Lopez Sanchez-Laorden our group is to develop “personalised medicine” protocols for in BRAF-mutant melanoma cells: phospho-protein arrays. RHS: and inexpensive treatment for patients with very low tumour burden, with very small Grazia Saturno melanoma patients. Drugs that target both the BRAF large fields of tumours for whom surgery is not number of SNVs (only ~2,000 per genome) and HaoRan Tang (BRAFi) and EGFR (EGFRi) desirable (Viros et al, 2013). a small number of structural chromosome pathways may provide a variations. Again we did not observe a UVR Bioinformatician treatment strategy for patients A key approach we take is to develop melanoma Drugs that inhibit BRAF can drive a curious Although BRAF drugs are effective in the mutation signature, suggesting that UVR does Mike Gavrielides who develop resistance to BRAF models driven by expression of oncogenes in paradox in cells. When BRAF is mutated they majority of melanoma patients with a BRAF not play a role in uveal melanomagenesis. In inhibitors. Scientific Officers melanocytes. In humans, these specialised inhibit MEK/ERK signalling, but when NRAS is mutation, approximately 20% of melanoma addition to recurrent mutations in GNAQ or Sarah Ejiama1 pigment cells largely reside in the skin, where mutated, they hyper-activate MEK/ERK patients do not respond to these drugs despite GNA11 (11/12 samples) and BAP1 (7/12 samples), Lesley-Anne Foster1 the presence of a BRAF mutation (primary one of their major functions is to provide signalling. This is because in the presence of previously described as the most common Clare McManus protection from the harmful effects of the active RAS, BRAF inhibitors drive BRAF into a resistance), and the majority of patients who do oncogenes and tumour suppressors in uveal Eamonn Morrison ultraviolet radiation (UVR) that is present in complex with a closely related protein called respond, relapse on treatment due to the melanoma, we also identified mutations in Graduate Student sunlight. However, melanocytes also reside in CRAF, activating CRAF and thereby MEK/ERK development of secondary resistance. In 2013, SF3B1 in ~15% of the tumours. The tumours Kate Hogan organs such as the brain, eyes, ears and heart signalling. In 2013, we demonstrated that a third we reported that hyper-activation of epidermal with SF3B1 mutations had a better prognosis where they are presumed or known to perform closely related protein called ARAF is not growth factor receptors (EGFR) can drive than those with loss of chromosome 3 and MSc Student other specialist functions. We previously functionally redundant with CRAF in this process resistance in some patients (Girotti et al. 2013). these events generally appear to be mutually Courtney Thwaites1 demonstrated that oncogenic BRAF drives (Rebocho and Marais, 2013). It appears also to We developed cell lines resistant to BRAF exclusive. SF3B1 encodes for a component of 1 joined in 2013 melanomagenesis, but although it can initiate be activated by BRAF, but rather than binding to inhibitors and discovered that they had elevated the splicosome machinery and accordingly, the 2 left in 2013 this process, it does so by cooperating with BRAF, it appears to stabilise the BRAF:CRAF EGFR, and SRC family kinase (SFK) signalling. mutations in SF3B1 were associated with additional genetic abnormalities. In 2013, we complex, at least in some cells. These data add Importantly, the combination of EGFR and BRAF differential splicing of specific coding and reported developmental melanoma models another layer of complexity to the paradox, but drugs was able to inhibit the growth of the non-coding RNA. Again, these data suggest driven by oncogenic NRAS (Pedersen et al, the consequences of this are currently unclear. resistant lines both in vitro and in vivo. that uveal melanoma is a distinct disease that is 2013). We found that somatic mutations in NRAS Importantly, we observed increased EGFR and driven by distinct genetics, highlighting the in embryonic melanocytes appear to be a risk In 2012, we demonstrated that a consequence SFK activity in tumours from patients who had need to improve our understanding of factor for leptomeningeal melanosis, a rare, but of the BRAF inhibitor paradox is induction of developed resistance to BRAF drugs and melanoma biology to allow us to develop new inevitably fatal form of childhood melanoma of secondary non-melanoma skin cancers in showed that tumours from resistant patients treatment approaches for melanoma patients. the CNS. These studies have improved our ~30% of patients (keratoacanthomas and were susceptible to the combination of BRAF understanding of the genetics underlying this squamous cell carcinomas). This is because the and EGFR drugs. These data established that Publications listed on page 61 particular disease and provided a relevant and paradox accelerates growth of pre-existing increased EGFR signalling can mediate tractable model system for its further study. tumours, bearing mutations in one of the RAS resistance to BRAF drugs, adding to the 38 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE MOLECULAR ONCOLOGY 39

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    forms of the kinase. Next we determine SIGNALLING NETWORKS IN CANCER phenotypic effects of expressing the WT, KD www.cruk.manchester.ac.uk/signallingnetworks and mutant forms of the target kinase on proliferation, survival and transformed properties of appropriate tumour and normal cell lines. We then verify the function of the kinase using si/shRNA and evaluate the role of the endogenous kinase in regulating cellular phenotypes associated with tumourigenesis. We also investigate the molecular mechanisms utilised by the cancer mutants to promote tumourigenesis. For example, if the mutation is Signalling pathways dictate a range of important cellular an activating mutation, we will identify cancer relevant substrates that are phosphorylated by outcomes, ranging from cell death to replication and cellular the cancer mutants to promote tumourigenesis. migration. Genetic lesions that skew the balance of these Finally, we will assess the consequences of somatic mutations utilising cell lines that pathways towards abnormal growth, proliferation, and cell harbour endogenous mutations in the target survival, are the fundamental mechanisms that cause normal kinase. The overall goal of these studies will be to identify common and convergent pathways cells to become premalignant. Kinases are the key regulators utilised by cancer cells to promote lung of signalling pathways (similar to transistors in a circuit) and Figure 1 tumours of any origin. Specifically, we identified tumourigenesis and identify convergent and Group Leader Targeted genetic dependency FGFR4, PAK5, and MLK1 as kinases that harbour essential targets that could be exploited for the dictate the activation or amplification of a given signal that screen to identify novel development of novel therapeutics for the John Brognard actionable mutations; mutated novel GOF mutations in lung cancer patients ultimately leads to cellular fate decisions. When these FGFR4, MAP3K9 and PAK5 are and this results in hyperactivation of the MEK/ treatment of lung cancer patients. Postdoctoral Fellows ERK pathway (Figure 1). The mutation frequency Shameem Fawdar1 kinases are hyperactivated, or inactivated by genetic illustrated examples. Patient tumours are exome for the genes we identified ranged from 2-10% The next generation of personalised medicine is Anna Marusiak mutations, they become the main drivers of tumourigenesis sequenced and treatment is stratified based upon the of lung cancers; given the frequency of lung becoming a reality in non-small cell lung cancer cancer in the population, these targets could be (NSCLC). Normal cellular growth relies on the Clinical Fellow and thus serve as primary targets for the development of mutational profile of each exploited by pharmaceutical companies for interaction of networks of kinases (enzymes) Andrew Hudson individual. Treatment options small molecule inhibitors. include novel pharmacological drug discovery development. These types of that turn cellular processes ‘on’ and ‘off’. Some Scientific Officers compounds to specifically inhibit screening approaches have the potential to NSCLC patients have a specific genetic change Natalie Stephenson driver oncogenes, and/or that generates an “always on” version of a kinase identify both therapeutic targets and Eleanor Wendy Trotter Cancer genomic sequencing studies and functional impact of somatic mutations in novel targeting downstream the main biomarkers. (EML4-ALK is the name of the genetically genome-wide siRNA screens are highlighting or understudied kinases identified in lung hyper-activated pro-proliferative Graduate Students activated kinase). Cells with this mutated kinase signalling pathway, for example Zoe Edwards the amazing diversity in the kinases required to cancer genomic screens. We use the following Our final strategy involves an unbiased are unable to turn ‘off’ certain cellular processes inhibition of MEK. Ewelina Testoni maintain tumourigenic phenotypes or permit bioinformatics applications to assess the kinome-wide evaluation of all kinases focusing and therefore grow out of control to form a drug resistance and emphasise the importance functional impact of somatic mutations: PMUT, on amplification and somatic mutations in tumour. Patients with this defective kinase 1 left in 2013 that neglected or understudied kinases play in polyphen2, mutationtaster, snpeffect, SIFT, and squamous cell lung cancer. Utilising online data benefit significantly from treatment with a small the development and maintenance of a tumour. SNPs and go. Kinases where a majority of portals such as cBio, the lab concentrates on molecule inhibitor (Crizotinib) that targets the Thus a major goal of our lab is to identify and mutations are predicted to be “likely cancer”, kinases that are both amplified and somatically activated kinase to turn ‘off’ the pathway. The elucidate novel kinase drivers that are essential “pathological” or likely to have a “high” mutated in an effort to identify kinases where major aim of our research is to identify new for lung tumour development, required for functional impact are further evaluated in the increased expression or mutational activation druggable targets by screening lung cancer maintenance of lung tumorigenic phenotypes lab. Additionally, we model many of the drives lung tumourigenic phenotypes. We start cells to find the next set of hyperactivated genes or play a role in lung cancer therapeutic mutations that score highly in our analysis to with the kinome wide approach; a candidate that are required for lung cancer cell survival. resistance. determine the structural consequences of the kinase is identified that is frequently amplified or putative driver mutations concomitant with mutated in lung cancer, and regions of Kinase mutations in other cancers Genetic Drivers of Lung Cancer experiments in the lab. We have found that this mutations are highlighted. This kinase is then In addition, the lab is focused on a novel family Lung cancer patients receive significant benefit approach works extremely well in identifying studied in depth in the laboratory to determine if of kinases capable of promoting resistance to from targeted therapies aimed at mutationally LOF mutations and candidate tumour it is required to maintain lung cancer cell survival targeted therapies for melanoma patients. activated kinases, such as EGFR or EML4-ALK, suppressing kinases. and proliferation and what downstream Guided by cancer genomic studies the lab has where inhibition of the activated kinase mechanisms are utilised to promote elucidated a novel family of MEK kinases that suppresses proliferation and promotes cell In a second approach we use genetic these phenotypes. can promote resistance to RAF inhibitors in death. However, despite the significant dependency screens to identify mutationally melanoma. Future work will be aimed at advances in targeted therapies for lung cancer activated drivers of lung cancer. Utilising cancer Characterisation of mutant kinases understanding the molecular mechanisms that patients, only approximately 20% of lung cancer genomic sequencing data from the Sanger To characterise the mutant kinases, our general dictate pathway activation downstream of these patients can be stratified for treatment with Institute we depleted six lung cancer cell lines of strategy is to first assess the functional kinases. Lastly, we have identified a novel targeted therapies. Therefore, it is necessary to all somatically mutated proteins to discover consequences of somatic mutations on overall tumour suppressing kinase in colon cancer and elucidate novel signalling pathways that are novel genetic dependencies and identify low kinase activity utilising in vivo and in vitro kinase we aim to elucidate why LOF mutations in this essential to maintain lung cancer cell survival. frequency driver mutations. Targeted genetic activity assays. We compare the activity of the kinase are essential for the development of The lab utilises three strategies to identify such dependency screens are an effective way to kinases harbouring cancer mutations colon cancer. pathways in lung cancer. In the first approach uncover low frequency oncogenes that can (engineered through site-directed mutagenesis) we use bioinformatic tools to evaluate the serve as targets for therapeutic intervention for to WT, kinase dead (KD) and hyperactivated Publications listed on page 62 40 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE SIGNALLING NETWORKS IN CANCER 41

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    Figure 1 development of these two lineages in close STEM CELL BIOLOGY The Histone Acetyl Transferase association provided the basis for the hypothesis (HAT) activity of MOZ regulates www.cruk.manchester.ac.uk/stemcellbiology proliferation of haematopoietic that they arise from a common precursor, a cell and neuronal stem cells called the haemangioblast. A conflicting theory MOZ histone acetyl transferase however, associates the first haematopoietic activity prevents entry into early cells to a phenotypically differentiated replicative senescence by endothelial cell with haematopoietic potential, regulating the expression of the i.e. a haemogenic endothelium. Support for the tumour suppressor p16INK4a . In the absence of MOZ HAT activity, haemangioblast concept was initially provided the levels of p16INK4a are by the identification during embryonic stem T: Telencephalon FL: Fetal Liver significantly increased in both cells differentiation of a clonal precursor, the BM: Bone Marrow haematopoietic and neural stem blast colony-forming cell (BL-CFC), which gives NSC: Neural Stem Cell HSC: Hematopoietic Stem Cell cell compartments. Upon Transcription factors bind to specific sequences in DNA p16INK4a expression upregulation, rise after four days of culture to blast colonies with both endothelial, smooth muscle and and control how genes are transcribed into RNA and, as a these cells leave the cell cycle to become senescent, haematopoietic potential. Recent studies have H2B-VENUS was strongly correlated with α-Sma consequence, indirectly control the translation of RNA resulting in severely impaired haematopoietic and neural now provided further evidence for the presence expression during the in vitro differentiation of of this tri-potential precursor in vivo. We have this reporter ES cell line (Figure 2A). In addition, into functional proteins. stem cell self-renewal. recently established a new model of the enrichment for expression of a panel of haematopoietic development based on the in smooth muscle markers indicated that Genes encoding the AML1/RUNX1 transcription progenitor and stem cells, inducing an early vitro differentiation of embryonic stem cells. We H2B-VENUS+ cells accurately represent a factor, and its cofactor CBFβ, are frequently entrance into replicative senescence and demonstrated that haematopoietic cells are smooth muscle cell lineage. Furthermore, we rearranged or mutated in human leukaemias, loss of self-renewal. Silencing of p16Ink4a by generated from the haemangioblast through detected, mostly in the H2B-VENUS sorted cells, Group Leader such as acute myeloid leukaemia (AML) and genetic deletion reverses the proliferative the formation of a haemogenic endothelium transcripts of the long isoform of Smoothelin-B, Figure 2 intermediate. During this process, the suggesting that our ES differentiation conditions Georges acute lymphoblastic leukaemia (ALL). Consistent defect in both MozHAT-/- haematopoietic and Smooth Muscle cells develop independently from haemogenic endothelial cells lose their preferentially generate vascular rather than with its implication in leukaemia, RUNX1 neural progenitors. Lacaud has also been shown to be critical for haemogenic endothelium endothelial identity, altering their flat, adherent visceral smooth muscle cells. Altogether, these A. Immmuno-staining of smooth appearance into the characteristic round shape results indicate that H2B-VENUS detection Postdoctoral Fellows haematopoietic development. Similarly, the Altogether, these results provide new insights muscle cells generated upon ES transcriptional co-activator MOZ is involved in into the control of stem and progenitor cell of mobile haematopoietic precursor cells. allows the direct quantification or isolation of Julia Draper cell differentiation (red: SMA Kiran Batta three independent myeloid chromosomal proliferation and identify an unexpected role of Smooth muscle actin, green; smooth muscle cells. With this reporter ES cell Roshana Thambyrajah translocations fusing MOZ to the partner MOZ-mediated acetylation in the regulation of H 2 B:Venus). B. Model of The haemangioblast generates blast colonies line, we then confirmed that clonal BL-CFCs Michael Lie-A-Ling genes CBP, P300 or TIF2 in human leukaemia. p16Ink4a expression (Figure 1). We propose that generation of smooth muscle containing both haematopoietic, endothelial generate smooth muscle cells. To determine if Guilherme Costa2 cells by Flk 1+ cells and and smooth muscle cells. These vascular the generation of smooth muscle cell is Anne Largeot 1 Our group studies the function of RUNX1 and this mechanism could also be critical for the haemangioblast. MOZ in haematopoietic development and self-renewal of other types of stem cells. These smooth muscle cells represent a major associated with, or independent from, the Scientific Officer maintenance, in order to better understand findings also suggest that, in the context of component of the cardiovascular system. emergence of the haemogenic endothelium Rahima Patel how alterations of these functions lead to leukaemia, the repressive activity mediated by During vasculogenesis, newly formed intermediate cell population, we examined the leukaemogenesis. MOZ acetylation on the expression of the endothelial vessels become rapidly associated presence of H2B-VENUS+ cells in Graduate Students with mural cells of the smooth muscle cell subpopulations containing this precursor. We Milena Mazan2 tumour suppressor p16Ink4a might be further MOZ regulates the expression of the exacerbated in fusion proteins created upon lineage. These cells are either referred to as indeed observed the presence of some Elli Marinopoulou Magdalena Florkowska tumour suppressor p16Ink4a and entry into translocations at the human MOZ locus. As vascular smooth muscle cells if they encircle H2B-VENUS+ cells in these haemogenic replicative senescence of neuronal and such, these MOZ leukaemic fusion proteins larger vessels, or as pericytes if they reside within endothelium populations. However, these cells 1 joined in 2013 haematopoietic stem cells would inhibit the triggering of senescence and the wall of small vessels, such as capillaries and lacked haematopoietic potential and therefore 2 left in 2013 The histone acetyl-transferase MOZ (Monocytic favour the development of leukaemia. post-capillary venules. These cells regulate did not correspond to functional haemogenic Leukaemia Zinc Finger protein; MYST3, or blood flow through contraction and have been endothelium, indicating that smooth muscle KAT6A) is a key regulator of haematopoiesis, Generation of smooth muscle cells by proposed to control endothelial cell cells are largely generated independently from frequently found translocated in acute haemangioblast proliferation and differentiation. Although the haemogenic endothelium. Altogether, myeloid leukaemia. Previous studies have There is a worldwide shortage of matched the role of smooth muscle cells in the these findings are consistent with an early revealed a crucial role of MOZ in controlling donors for blood stem cell transfer of leukaemia pathophysiology of cancer is still unclear, separation between haemogenic endothelium haematopoietic stem cells and progenitors’ or lymphoma patients. The generation of evidence suggests that aberrations in and smooth muscle lineages during (HSC/Ps) proliferation. blood cells upon the in vitro differentiation pericyte-endothelial cell signalling networks development (Figure 2B). Whether signalling by of embryonic stem (ES) cells or induced could contribute to tumour angiogenesis smooth muscle contributes to some extent to We recently established that this effect is not pluripotent stem (iPS) cells could represent a and metastasis. the generation of blood cells from haemogenic limited to the haematopoietic compartment, powerful approach to generate the autologous endothelium is still unclear. This study provides but extends to neuronal stem cells and cell populations required for these To further investigate the development of new and important insights into haematopoietic progenitors (NSC/Ps), suggesting that these two transplantations. In this context, it is important smooth muscle cells, we generated a mouse and vascular development, which may help types of cells, HSCs and NSCs, use the same to further understand the development of reporter ES cell line in which the expression of drive further progress towards the development mechanism involving MOZ-driven acetylation in blood cells. the fluorescent protein, H2B-VENUS, is driven of bioengineered vascular grafts, or help order to maintain their capacity to proliferate. from the α-SMA (Smooth Muscle Actin) discover new opportunities to control tumour We then demonstrated that the HAT activity of The earliest site of blood cell development in regulatory sequences. We demonstrated that angiogenesis and metastasis. MOZ is critical for silencing expression of the the mouse embryo is the yolk sac where blood this reporter cell line allows us to efficiently track tumour suppressor p16Ink4a . In the absence of islands, consisting of haematopoietic cells smooth muscle development during murine ES Publications listed on page 62 MOZ HAT activity, expression of p16Ink4a is surrounded by a layer of angioblasts, develop at cell differentiation. The expression of up-regulated in haematopoietic and neuronal approximately day 7.5 of gestation. The parallel 42 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE STEM CELL BIOLOGY 43

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    STEM CELL HAEMATOPOIESIS mesoderm, we generated embryonic stem (ES) cells and transgenic mouse lines carrying www.cruk.manchester.ac.uk/sch a foxf1-venus knock-in allele (Figure 2A). When FLK1+ cells, isolated from in vitro differentiated Foxf1venus/+ ES cells, were cultured to generate blood progenitors, we observed the formation of a distinct FOXF1::VENUS+ subpopulation. Interestingly, CD41+ blood progenitors were not VENUS+ (Figure 2B). This observation was further confirmed in gastrulating embryos where FOXF1::VENUS expression was exclusively detected within the yolk sac and did not Early events of embryonic development can be recapitulated co-segregate with CD41 expression. Further analysis of E7.5 embryo tissue sections revealed in vitro upon the differentiation of pluripotent stem cells, such that while FOXF1::VENUS expression was seen in as embryonic stem cells or induced pluripotent stem cells. the allantois, the amnion and the mesothelium lining the exocoelomic cavity, it was not However, many pluripotent cell lines are notoriously refractory detected in the prospective blood islands. to differentiation toward some of these specific lineages. To further assess the relationship between FOXF1 expression and blood specification, we Given the therapeutic potential of cell (Figure 1). The molecular mechanisms Figure 2 or skeletal muscle lineages. It remains to be generated an ES cell line in which the expression Group Leader populations generated upon in vitro controlling the specification of mesoderm are A) Schematic representation of determined, however, what this cell context of Foxf1 can be induced by doxycycline. Upon differentiation, it has become essential to just starting to be unravelled, and the overall the foxf1 knock-in construct in culture of in vitro differentiated FLK1+ cells, we Valerie determine how pluripotent stem cells can be transcriptional network orchestrating this which the Venus cassette specificity implies and how it impinges on observed that the induction of FOXF1 expression replaces the first exon of foxf1. A MESP1 activity. Little is known about the Kouskoff directed to specific fates for the generation of process is still poorly understood. In particular, PGK-neomycin resistance specification of mesoderm to the mesothelium dramatically compromised the production of lineages such as blood, endothelial or cardiac how alternate cell fates are regulated in nascent cassette was initially present in that forms an epithelial monolayer lining all blood progenitors. Taken together, our data Postdoctoral Fellows cells to be used for therapeutic purposes. To mesoderm is mostly unknown. the construct for selection body cavities and organs. Apart from its lateral suggest that FOXF1 expression is incompatible Alexia Eliades purpose but was removed via date, the molecular mechanisms controlling the plate mesoderm origin, nothing is known with blood specification and actively represses Maud Fleury Flp/Frt deletion. B) Wild-type Eva Garcia-Alegria specification of mesodermal precursors to each The specification of blood and endothelial about the molecular control underlying the the specification of mesoderm precursors to (foxf1+/+) and Foxf1::Venus Josh Lilly of these fates remain poorly understood. lineages is tightly associated, depending on knock-in (foxf1venus/+) ES lines specification of this lineage that shares a haematopoiesis. similar signalling pathways and transcription were differentiated for 3 days common mesodermal origin with the Scientific Officers Specification of mesoderm derivatives factors for their emergence. We and others through embryoid body cardiovascular system. The potential role of FOXF1 in cardiac Stella Pearson formation; FLK1+ cells were At the early stage of embryonic development, have shown that the FLK1-VEGF signalling axis specification was assessed using an ES cell Kirstie Wallace isolated and further the multipotent epiblast gives rise to the three and the ETS transcription factor ETV2 are FOXF1, a novel regulator of mesoderm line in which both Foxf1 alleles were deleted. differentiated in haemangioblast Graduate Students germ layers which are subsequently specified to essential for the generation of both lineages. culture conditions promoting specification Upon in vitro differentiation, the absence of Sara Cuvertino form all tissues of the developing organism. However, both FLK1 and ETV2 are widely the formation of CD41 Forkhead box (FOX) transcription factors belong FOXF1 expression led to a dramatic increase Genny Filiciotto1 Initiation of germ layer formation occurs expressed in nascent mesoderm and are haematopoietic progenitors. to an evolutionary conserved family of proteins in cardiogenesis potential, suggesting an Andrzej Mazan Flow cytometry analysis for through a process known as gastrulation, in unlikely to be responsible for restricting named after the Drosophila forkhead gene active repression of this programme by FOXF1 Sharmin Naaz2 FOXF1::VENUS and CD41 which cells from the epiblast undergo an mesoderm specification to blood and/or whose mutation results in a spiked head expression. In line with this observation, expression was performed at day 1 joined in 2013 epithelial to mesenchymal transition and ingress endothelium fate. Directly regulated by ETV2, 1, 2 and 3. phenotype. There are 44 FOX proteins in the explant culture of yolk-sac derived from Foxf1- 2 joint with ACBB group through the primitive streak to generate the bHLH transcription factor SCL is required mouse genome, all characterised by a forkhead deficient embryos were capable of generating mesoderm and endoderm. Cells exiting the for both haematopoiesis through its control DNA-binding motif, and assembled into 19 cardiomyocytes, further delineating a role posterior part of the primitive streak form the of haemogenic endothelium formation. subgroups based on sequence homologies for FOXF1 in the repression of the cardiac extra-embryonic and lateral plate mesoderms, Interestingly, SCL was recently shown to inside and outside the fork-head domain. programme in mesoderm precursors. Genome which later are specified to become blood repress cardiogenesis in prospective FOXF1, one of the two members of the FOXF wide experiments are currently underway to precursors, endothelial and smooth muscle, haemogenic endothelium, suggesting that subgroup, is expressed widely in extra- further understand how, at the molecular forming the vasculature, heart and mesothelium even after specification the fate of mesoderm embryonic and lateral plate mesoderm, and its level, FOXF1 represses both haematopoiesis lineages remains plastic and requires active deletion leads to early embryonic lethality with a and cardiogenesis. Mesothelium appeared to Figure 1 repression to alternative fates. complete absence of vasculogenesis and be the only mesoderm derivative in which high Schematic representation of the mis-expression of haematopoietic and vascular expression of FOXF1 is observed. Interestingly, cellular steps leading to the Specification of the cardiac tissues depends markers. At a later stage of embryonic in the adult organism, the mesothelium retains formation of mesoderm derivatives. on the transcription factor MESP1, a master development, heterozygosity or mutation in remarkable progenitor characteristics with the regulator of cardiac development expressed in FOXF1 is linked to lung malformation and ability to differentiate into myofibroblast, the primitive streak. Upon enforced expression, alveolar capillary dysplasia involving the smooth muscle and endothelium upon injury MESP1 promotes the generation of all cardiac abnormal development of the vascular system or specific signals. It will be important in the lineages, controlling the downstream cascade in the lungs. Given its expression pattern and future to explore the possible role of FOXF1 in of transcriptional regulators. A recent study, null phenotype, we became interested in the the maintenance of this progenitor potential. however, has challenged this view, implicating potential role of this transcription factor during MESP1 more widely in the patterning of mesoderm specification. Publications listed on page 62 mesoderm. Depending on specific cell contexts, MESP1 was shown to specify In order to characterise in detail its expression, mesoderm toward haematopoietic, cardiac to track and isolate FOXF1-expressing 44 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE STEM CELL HAEMATOPOIESIS 45

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    CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES Imaging with gated stimulated emission depletion (gSTED) microscopy. Actin has been labelled with Alexa488-Phalloidin. Each pixel of the image is 19nm in size and the whole image took twelve seconds to capture. After image capture, deconvolution has been applied with the Huygens software resulting in an overall resolution of around 28-30nm. The spotted nature of the image is due to the size of the labelling complex which blocks additional binding sites. Image supplied by Steve Bagley. 46 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES 47

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    modelling of the variation that exists in a allows the high resolution study of cells under RESEARCH SERVICES population of cells, which can be exploited for laser illumination. The system produces a www.cruk.manchester.ac.uk/Research/ the analysis of potential biomarkers. high-resolution image where the relationship between two labelled proteins can be The introduction of the Seahorse XFe allows characterised in such a way that each pixel of researchers to measure the extracellular flux of the image equates to 19nm. This level of the two major energy pathways of the cell, resolution is far greater than achieved through mitochondrial respiration and glycolysis, in a use of more traditional systems, for example a multi-well plate and in real-time. With few cells, confocal microscope, which typically has a this technology analyses the effects of up to resolution of around 200nm. The antibody- four compounds on cellular metabolism over label complex used to visualise and provide minutes, hours or days. contrast has an approximate size of 35-45nm, Towards the end of 2012, HEFCE awarded £8.7 million to the hence the gSTED system produces data that Within the histology imaging section of the cannot be resolved by the confocal microscope. University of Manchester from the UK Research Partnership facility, a new scanner has been installed which The gSTED resolution improvement is made Investment Fund, to be used for the provision of equipment to permits the sampling of tissue by searching for possible by side stepping some of the barriers cells that exhibit user-defined parameters based that are inherent in traditional microscope support cutting edge cancer research. Most of this equipment on their spectral qualities. By utilising a illumination and design. will be housed in the existing core facilities within the Institute spectrophotometer, immuno-histochemical and fluorescently labelled tissues are imaged, As demands upon the facility will increase in the and, as the sections below indicate, this opportunity is already modelled and analysed. This allows for the coming year with the appointment of new providing a boost to the range of services available. removal of background auto-fluorescence research groups within the Institute and the Chief Laboratory Officer (which is inherent in all cells and tissues) and the completion of the Manchester Cancer Research application of multiple labels so that complex Centre building, a major external review of the Stuart Pepper Chief Laboratory Officer installing systems that provide new techniques questions can be asked of primary tissue. The facility was undertaken to assess the established Stuart Pepper to ensure the Institute maintains its cutting edge technique is beneficial when detecting rare cells structures, workflows, efficiency, management data collection capabilities. New developments within a tissue section which exhibit and staffing levels. The review incorporated the Most of the purchasing during 2013 has been to have taken place across all parts of the facility: morphological or protein expression differences comments of Group Leaders at the Institute. In support genomic sequencing, histological flow cytometry; whole slide histology scanning; when compared to the main population. light of the review, several new structures are to analysis and FACS analysis and details are given high content scanning; and microscopy. be put in place to enable the facility to meet below of the equipment that has been A new member of staff has been appointed future demands. procured. Extra funding was also provided by Within flow cytometry, a BD Aria III and BD during this year to manage histological imaging, the MCRC to support purchase of a gated STED Fortessa were purchased to increase sample to train and provide assistance to the users, and microscope, a truly state of the art imaging throughput. The BD Aria has more lasers than to develop techniques for the image analysis of Biological Mass Spectrometry Facility system, discussed further under ‘Advanced the standard off-the-shelf system, allowing tissues. The development of scanning, analysis Duncan Smith, Yvonne Connolly, John Griffiths Imaging and Flow Cytometry’. greater versatility for future projects, such as and modelling is on-going as researchers are sorting Confetti cell populations. The Confetti asking more involved questions of tissue that The role of the facility is to enable research Given the recent major investment into the technique produces a population of cells that requires assessment at the molecular level. groups to access cutting edge proteomic service units, this seemed like a good stochastically express several fluorescent workflows. The lab is active in routine service opportunity to undertake a full review of the proteins, which in turn are passed down to the High content screening (HCS) has been provision, project design and data interpretation, core facilities to ensure that the services are daughter cells, a technique that is heavily utilised undertaken within the facility since 2004 with a through to the bespoke collaborative being developed in a way that closely matches in stem cell studies. As the amount of primary variety of microscope systems, however in 2011 development of novel workflows designed to the needs of the researchers. An external panel samples has increased, the system has been the purchase of a HCS solution allowed for a answer previously intractable biological of experts visited the Institute in November and installed in a class II cabinet to ensure purity of greater level of automation of cellular analysis. questions. The foundations of the facility are met with service managers and key users. The sort and operator safety. The BD Fortessa was Within the Institute, HCS is predominantly used based on maximising CRUK MI’s research outcome was very positive and will help guide installed at the same time as the Aria, providing by two large research groups; for the output by balancing high quality service the development of the services during 2014. an additional platform for cytometry analysis. assessment of drug screens and the analysis of provision with active research and development The system has automation for high throughput circulating tumour cells. This year, an to evolve new workflows and technology key to multi-well plate analysis, thus increasing the rate automated loader arm was installed which delivering a world-class facility. Advanced Imaging and Flow Cytometry of data collection for the exploration of protein permits batch processing of up to 80 multi-well Steve Bagley, Jeff Barry, Helen Bradley1, expression within large cell populations. plates, consequently allowing a higher We have pushed our current Orbitrap Mike Hughes, Abi Johnson, Kang Zeng throughput of data. technology further than ever with the 1 joined in 2013 An Amnis ImageStream X II has been installed in development of ion trap centric data the CEP laboratory, which allows all samples to In the microscopy part of the facility, the independent acquisition methods that facilitate The facility has experienced substantial be run to GCP(L) standardisation. The installation of the two-photon confocal more comprehensive analysis of every analyte growth during 2013, both in data complexity equipment is a combination of cytometer, microscope and the gated stimulated emission within a given sample. This year has seen a and with an increase in equipment provision, microscope and high content system which depletion (gSTED) nano-scope has allowed massive improvement in our ability to both consequently allowing the researcher to study allows visualisation of every non-adherent cell researchers to visualise cells with a greater clarity detect and map sites of SUMO modification by the molecule, the cell, the population and the in a sample, containing up to twelve different than was previously possible. The two-photon bespoke enzymatic digestion, chemical tissue. In response to the research requirements labels per cell, at high capture rates (up to 4,000 microscope images deep into tissue, enabling derivitisation and subsequent diagnostic ion of the Institute, there was a real need to increase cells per second) in ten minutes. The nature of the visualisation of cell invasion, tumour generation (Chicooree et al). We have further capacity and accelerate workflows, as well as the equipment allows for mathematical formation and the interaction of cells with enhanced this approach towards fully connective tissues. The gSTED nano-scope quantitative analysis by utilising mTRAQ labelling 48 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES 49

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    number of staff and provide new platforms for expression of different proteins within the same in vivo imaging. tumour tissue/cells, and the interactions between proteins, and for the first time on a A significant change to the law took place at the large-scale to understand how these markers start of the year with the implementation of interact. Ultimately, this will allow us to study Directive 2010/63EU across the UK. This has led tumour heterogeneity from a different angle to some local changes for the management of that complements efforts in understanding the the Certificate of Designation, now known as genetic/molecular heterogeneity of tumours. the Licence 2C for the establishment. Two new We have also facilitated the adoption of other roles have also been created: Training and sophisticated multiplex labelling techniques, Competency Officer and Named Information including multiplex quantum-dot based Officer. These two roles will help to ensure that immunofluorescence and proximity ligation all Personal Licence Holders have access to the assay for in situ detection of molecular most recent information on relevant procedures interactions. and will be able to carry out experiments to the highest possible standards. As the expansion of tissue biomarkers research continues to gather pace, the construction of tissue microarrays (TMAs), using both the ATA27 Histology and MTA1 platforms, has also increased. High Garry Ashton, Caron Abbey, Michelle throughput, accurate TMA construction of Greenhalgh (MCRC, Tissue Biobank) tumour specific and custom arrays has resulted David Millard, Deepti Wilks (Haematological in the production of extremely high quality Malignancy Biobank) TMAs giving true representation. The service is now embedded within the facility with TMAs The past year has once again been extremely from disease groups including breast, chemistries that facilitate quantitative analysis in required the rederivation of over 100 lines and it busy as the Histology unit continues to develop melanoma, prostate (cores and chips), bladder, both MS and MSMS modes. In the sample has been a significant challenge to manage this and expand. The range and complexity of the lymphoid, small cell and non-small cell lung preparation area, we have utilised a range of rederivation programme whilst maintaining a services offered continues to grow, all of which cancer having all been constructed. In addition, immobilised proteases with enhanced activities fully functional service. As 2013 closes, the have seen exceptional demand over the period. mouse model and cell pellet control in an attempt to enhance proteolysis in rederivation is nearing completion and should The continued professional development and microarrays have been constructed. workflows where this step is currently be finished in the early stages of 2014. The next retention of staff, with particular focus on cross performance limiting, such as the analysis of step will be an expansion of stock in the new training, has ensured that the unit continues to Today, the nature of oncology research requires complex protein mixtures. We have also been facility, followed by a winding down of the offer a comprehensive and flexible service at all more downstream analysis on smaller clinical very active in developing the next generation of breeding program in the existing facility. Once times. The unit specialises in the histological samples and as a result we are continually data analysis tools, including de novo this move is accomplished, there will be the preparation and analysis of a wide range of looking at developing workflows requiring less sequencing and intelligent integration of capacity for a significant expansion from the samples, including tissues from mouse models, material from both laser capture multiple database search algorithms into a current level of approximately 2000 cages up to whole mouse embryo preps, cell preps, and microdissection and macrodissection. Recent unifying results report. a maximum of 5,500 cages. human biopsies. The samples are in either work has allowed us to extract both RNA and paraffin or frozen format. DNA, sufficient in quantity and quality for Next In 2013, staff members from the facility were Experimental Services Generation Sequencing from relatively small authors of six peer reviewed publications. A The experimental area has been supported by The laboratory was refurbished earlier in the amounts of material, whilst still leaving material collaboration with the Cell Division group licensed technicians who have delivered a year allowing better use of existing space and for future subsequent analysis. Workflows for showcased the power of our comprehensive varied range of non-surgical and surgical thus creating a second clinical sample both nucleic acid and proteomic analysis from phosphomapping workflow by helping uncover procedures for 11 research groups. As always, preparation area. In addition, more space has the same sample, taking into account tissue fundamental aspects of mitotic commitment we have ensured that the highest quality of care since been secured and it is hoped that the new heterogeneity, have also been developed and (Grallert et al). has been provided, and that Home Office immunohistochemistry platform will be housed used successfully. legislation has been adhered to, by performing in this area. The existing automated daily health and welfare checks along with immunohistochemistry (IHC) platforms The unit continues to process FFPE and frozen Biological Resources Unit extensive monitoring for animals under continue to offer a high throughput, routine, samples for the Manchester Cancer Research procedure. troubleshooting and antibody validation service. Centre Biobank. To date samples from over The animal facility has been fully occupied Demand on this service has once again seen a 5200 patients have been collected. Blood, bone throughout 2013, both for Transgenic A range of techniques have been supported by sharp rise with many antibodies being validated. marrow and plasma (at various disease status Production and Experimental Services, with a the facility, including subcutaneous The new joint post between the Histology and time points) from over 330 haematological total of 15 Project Licences in place with primary implantation of slow-releasing hormone pellets, Advanced Imaging and Flow Cytometry malignancy patients has also been collected. So and secondary availability. The renewal of the parenteral injections, gavage, blood sampling Facilities, focussing on the numerical analysis of far, over 64 research projects have been Transgenic Service Licence over the summer with and without recovery, whole body histological data in tissue biomarker expression approved. Samples have also been released for has ensured production of progeny for the next irradiation, local irradiation to subcutaneous studies, is proving very successful. method validation studies. Histology and five years for the scientific programmes. tumour and in vivo imaging. For most of the year molecular pathology, with the adoption of the experimental facility has run close to Multispectral imaging has allowed the Histology sophisticated labelling techniques and the Transgenic Services capacity. As the move of the transgenic facility facility to evaluate the methodologies of truly availability of new platforms/technologies, is at To facilitate expansion of the transgenic service described above progresses, there will be a multiplex immunohistochemistry. This allows very exciting point, placing the Histology facility the entire facility is relocating to space in the great opportunity to expand the experimental researchers to study both the levels of at the forefront of biomarker analysis in University Incubator Building. This move has service. Funds are in place to increase the 50 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES 51

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    oncology. The addition of a new platform and a The Laboratory Services team also provides a lines in use in the building can now be regularly new member of staff in early 2014 will allow the critical safety function for the Institute by authenticated by Short Tandem Repeat (STR) unit to focus primarily on its development within carrying out Legionella monitoring. Once a profiling. This technique is the gold standard these areas. month all 400 taps in the building are run to method for checking cell line identity but ensure there is no build-up of Legionella in requires some care when interpreting results. pipes. Alongside these principle tasks the team The MBCF team has considerable experience in Laboratory Services also maintains the Institute’s film processor, and this area to help interpret any unexpected Mark Craven, Tony Dawson, Corinne Hand, organises the provision of clean lab coats and results. Alongside the regular mycoplasma John Higgins, Frances Hockin, Amy Moloney, first aid items throughout the building. screening, this service gives confidence that all Christine Whitehurst cell lines in use are appropriate for the experiments being carried out. During 2012, work had been carried out to Molecular Biology Core Facility and Cancer improve the laboratory facilities available for Research UK Microarray Service Demand for the daily Sanger sequencing Laboratory Services to make media and other Stuart Pepper, Chris Clark, Yvonne Hey, Nelson service has continued to be high over the last solutions, and to provide a new autoclave and Iley1, Gill Newton, Leanne Wardleworth1, John year, whereas demand for the automated other equipment. This work has allowed the Weightman, Jodie Whittaker2 plasmid DNA extraction has fluctuated. At the team to operate very efficiently during 2013 and 1 joined in 2013 2 left in 2013 start of 2013, we had two new Next Generation provide a highly reliable service. Aside from the Sequencing (NGS) platforms delivered – a HiSeq main role of providing sterile glass and The Molecular Biology Core Facility (MBCF) 2500 and a MiSeq. The team has worked hard to plasticware, the facility also generates provides a range of services to support the implement the various workflows needed on approximately 1200L of sterile water, 600L of research groups on site. These include the new platform and start delivering results. PBS, 400L of liquid and up to 3000 agar plates. diagnostic services, such as cell line The facility now offers full genome sequencing authentication and mycoplasma screening, and and exome sequencing for DNA, and has The team make daily collections to remove dirty research services for expression profiling and protocols for total RNA sequencing or polyA glassware from laboratories and make daily DNA sequence analysis. selected RNA sequencing. The team has also deliveries to the labs and research areas of new run sequencing on ChIP samples and PCR sterile items, such as filter tips and microtubes, The core routine services have continued to run products. In total, forty data sets have been Scientific Computing supplying approximately 1700 non-filter pipette smoothly and had constant demand delivered in the first year of operation, though Wei Xing, Zhi Cheng Wang tip boxes, 400 filtered pipette tip boxes and 600 throughout the year. Cell line authentication is this is likely to increase considerably next year as packs of sterile microtubes to laboratories every now well established in the Institute and all cell demand for the service has increased towards Dr Wei Xing joined us during the summer, from month. the end of 2013. To help maintain throughput of the Institute of Cancer Research in London, to the service, an automation platform has been become the Head of Scientific Computing. The purchased to help process larger sample newly created department has been established Co-culture of breast cancer batches. Once fully established this will allow to provide the computational support needed cells (green), and normal the service to offer a good turnaround time for within the Institute to handle the high volumes human fibroblasts (non-green). larger experiments. of genomics and imaging data arising from our Cancer cells interact with deep sequencing, proteomics and microscopy stromal cells, in this case For expression profiling of individual genes, platforms. To do this we are building a High fibroblasts, to influence their gene expression profile. A rather than entire transcriptomes, the facility has Performance Computing (HPC) facility and data transcription factor (red) is found two ABI7900 machines with a choice of 96 or centre, significantly extending our current to accumulate in the fibroblast 384 well format. These systems, in conjunction capabilities. Wei’s team will provide the nuclei upon co-culturing with with our Probe Library, continue to be heavily infrastructure and expertise required to provide cancer cells. used and provide a very cost effective route for this essential resource to the Institute. HPC has Image provided by Haoran Tang small experiments on small numbers of become a pre-requisite for the proper from Molecular Oncology. samples. This year a new platform has been exploitation of cancer genomics data, in part, a added to support larger qPCR projects. The consequence of the high volumes of data Fluidigm Biomark HD allows the analysis of up produced by recent advances in deep to 96 genes on samples in a single run. Initial sequencing, imaging, and mass spectrometry. data looks very good and this platform will be These have the potential to revolutionise valuable both for large expression studies, and translational cancer research and to offer new for analysis of expression variability in single therapeutic strategies for personalised and cells. stratified medicine. The CRUK Affymetrix microarray service has Wei’s primary focus will be to provide the also continued to experience significant underlying infrastructure needed to support demand throughout the year. Although many integrated analysis of cancer data sets and groups have switched to NGS for expression intelligent bio-computing platforms, and will be work, there is still demand for microarray working closely with the Computational Biology analysis to allow comparison of new Support team to help provide the platforms that experiments to legacy data. To date, demand for support our pipelines for proteomics and this service has not significantly dropped and genomics data analysis. Zhi Cheng Wang has there are already projects ready for processing joined the Scientific Computing department in 2014. from the Institute’s IT team. 52 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES 53

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    CANCER RESEARCH UK MANCHESTER INSTITUTE PUBLICATIONS AND ADMINISTRATION MDCK cells forming cysts and other 3D structures in collagen, stained with β-Catenin (a basal marker, green), GP135 (an apical marker, red) and actin (blue). Image supplied by Andrew Porter, Cell Signalling. 54 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE CANCER RESEARCH UK MANCHESTER INSTITUTE 55

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    RESEARCH PUBLICATIONS Crispin Miller (page 14) S, Miller CJ, Buffa F, Harris AL, and West CM. (2013) Tay YD, Patel A, Kaemena DF, and Hagan IM. (2013) Dalton LE, Kamarashev J, Barinaga-Rementeria Applied Computational Biology and An in vivo hypoxia metagene identifies the novel Mutation of a conserved residue enhances the Ramirez I, White G, Malliri A, Hurlstone A. (2013) Bioinformatics hypoxia inducible factor target gene SLCO1B3. sensitivity of analogue-sensitised kinases to Constitutive Rac Activation Is Not Sufficient to Eur J Cancer 49, 1741-1751. generate a novel approach to the study of mitosis Initiate Melanocyte Neoplasia but Accelerates Refereed Research Papers in fission yeast. J Cell Sci 126, 5052-5061. Malignant Progression. J Invest Dermatol 133(6), Betts GN, Eustace A, Patiar S, Valentine HR, 1572-81. Irlam J, Ramachandran A, Merve A, Homer JJ, Karim Labib (page 16) Other Publications Moller-Levet C, Buffa FM, Hall G, Miller CJ, Cell Cycle Hagan IM and Grallert A. (2013) Daugaard M, Nitsch R, Razaghi B, McDonald L, Harris AL, and West CM. (2013) Spatial control of mitotic commitment in fission Jarrar A, Torrino S, Castillo-Lluva S, Rotblat B, Li L, Prospective technical validation and assessment of Refereed Research Papers yeast. Biochem Soc Trans 41, 1766-1771. Malliri A, Lemichez E, Mettouchi A, Berman JN, intra-tumour heterogeneity of a low density array Foltman M, Evrin C, De Piccoli G, Jones RC, Penninger JM, Sorensen PH. (2013) hypoxia gene profile in head and neck squamous Edmondson RD, Katou Y, Nakato R, Shirahige K, Hace1 controls ROS generation of vertebrate cell carcinoma. Eur J Cancer 49, 156-165. and Labib K. (2013) Nic Jones (page 20) Rac1-dependent NADPH oxidase complexes. Eukaryotic replisome components cooperate to Cell Regulation Nat Commun 4, 2180. Dhani DK, Goult BT, George GM, Rogerson process histones during chromosome replication. DT, Bitton DA, Miller CJ, Schwabe JW, and Cell Rep 3, 892-904. Refereed Research Papers Tanaka K. (2013) Walczynski J, Lyons S, Jones N, and Caroline Dive (page 24) Mzt1/Tam4, a fission yeast MOZART1 homologue, Nkosi PJ, Targosz BS, Labib K, and Breitwieser W. (2013) Clinical and Experimental Pharmacology is an essential component of the gamma-tubulin Sanchez-Diaz A. (2013) Sensitisation of c-MYC-induced B-lymphoma cells complex and directly interacts with GCP3Alp6. Mol Hof1 and Rvs167 have redundant roles in to apoptosis by ATF2. Oncogene doi: 10.1038/ Refereed Research Papers Biol Cell 24, 3337-3349. actomyosin ring function during cytokinesis in onc.2013.28. Adamski J, Price A, Dive C, and Makin G. (2013) budding yeast. PLoS One 8, e57846. Hypoxia-induced cytotoxic drug resistance in Eustace A, Mani N, Span PN, Irlam JJ, Taylor J, osteosarcoma is independent of HIF-1Alpha. Betts GN, Denley H, Miller CJ, Homer JJ, Rojas AM, Sengupta S, van Deursen F, de Piccoli G, and Angeliki Malliri (page 22) PLoS One 8, e65304. Hoskin PJ, Buffa FM, Harris AL, Kaanders JH, and Labib K. (2013) Cell Signalling West CM. (2013) Dpb2 integrates the leading-strand DNA Bouranis L, Sperrin M, Greystoke A, Dive C, A 26-gene hypoxia signature predicts benefit from polymerase into the eukaryotic replisome. Refereed Research Papers and Renehan AG. (2013) hypoxia-modifying therapy in laryngeal cancer but Curr Biol 23, 543-552. Castillo-Lluva S, Tan CT, Daugaard M, The interaction between prognostic and not bladder cancer. Clin Cancer Res 19, 4879-4888. Sorensen PH and Malliri A. (2013) pharmacodynamic biomarkers. Br J Cancer The tumour suppressor HACE1 controls cell 109, 1782-1785. Fawdar S, Trotter EW, Li Y, Stephenson NL, Hanke F, Iain Hagan (page 18) migration by regulating Rac1 degradation. Marusiak AA, Edwards ZC, Ientile S, Waszkowycz B, Cell Division Oncogene 32, 1735-1742. Cawthorne C, Burrows N, Gieling RG, Morrow Miller CJ, Brognard J. (2013) CJ, Forster D, Gregory J, Radigois M, Smigova Targeted genetic dependency screen facilitates Refereed Research Papers Chicooree N, Connolly Y, Tan CT, Malliri A, A, Babur M, Simpson K, Hodgkinson C, Brown identification of actionable mutations in FGFR4, Grallert A, Chan KY, Alonso-Nunez ML, Madrid M, Li Y, Smith DL, Griffiths JR. (2013) G, McMahon A, Dive C, Hiscock D, Wilson I, MAP3K9, and PAK5 in lung cancer. Proc Natl Acad Biswas A, Alvarez-Tabares I, Connolly Y, Tanaka K, Enhanced detection of ubiquitin isopeptides using and Williams KJ. (2013) Sci USA 110(30), 12426-31. Robertson A, Ortiz JM, Smith DL, and Hagan reductive methylation. J Am Soc Mass Spectrom [18F]-FLT positron emission tomography can be IM. (2013) 24(3), 421-30. used to image the response of sensitive tumors to Hall JS, Iype R, Armenoult LS, Taylor J, Miller CJ, Removal of centrosomal PP1 by NIMA kinase PI3-kinase inhibition with the novel agent GDC- Davidson S, de Sanjose S, Bosch X, Stern PL, and unlocks the MPF feedback loop to promote mitotic Chicooree N, Griffiths JR, Connolly Y, Tan CT, 0941. Mol Cancer Ther 12, 819-828. West CM. (2013) commitment in S. pombe. Curr Biol 23, 213-222. Malliri A, Eyers CE, Smith DL. (2013) Poor prognosis associated with human A novel approach to the analysis of SUMOylation Cummings J, Morris K, Zhou C, Sloane R, papillomavirus alpha7 genotypes in cervical Grallert A, Patel A, Tallada VA, Chan KY, Bagley S, with the independent use of trypsin and elastase Lancashire M, Morris D, Bramley S, Krebs M, carcinoma cannot be explained by intrinsic Krapp A, Simanis V, and Hagan IM. (2013) digestion followed by database searching utilising Khoja L, and Dive C. (2013) radiosensitivity. Int J Radiat Oncol Biol Phys 85, Centrosomal MPF triggers the mitotic and consecutive residue addition to lysine. Rapid Method validation of circulating tumour cell e223-229. morphogenetic switches of fission yeast. Commun Mass Spectrom 27(1), 127-34. enumeration at low cell counts. BMC Cancer Nat Cell Biol 15, 88-95. 13, 415. Ramachandran A, Betts G, Bhana S, Helme G, Blick C, Moller-Levet C, Saunders E, Valentine H, Pepper 56 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH PUBLICATIONS 57

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    Gibb A, Greystoke A, Ranson M, Linton K, Neeson A caspase-3 ‘death-switch’ in colorectal cancer Donald Ogilvie (Page 30) Fawdar S, Trotter EW, Li Y, Stephenson NL, Hanke F, S, Hampson G, Illidge T, Smith E, Dive C, Pettitt A, cells for induced and synchronous tumor apoptosis Drug Discovery Marusiak AA, Edwards ZC, Ientile S, Waszkowycz B, Lister A, Johnson P, and Radford J. (2013) in vitro and in vivo facilitates the development of Miller CJ, Brognard J. (2013) A study to investigate dose escalation of minimally invasive cell death biomarkers. Cell Death Refereed Research Papers Targeted genetic dependency screen facilitates doxorubicin in ABVD chemotherapy for Hodgkin Dis 4, e613. Raoof A, Depledge P, Hamilton NM, Hamilton NS, identification of actionable mutations in FGFR4, lymphoma incorporating biomarkers of response Hitchin JR, Hopkins GV, Jordan AM, Maguire LA, MAP3K9, and PAK5 in lung cancer. Proc Natl Acad and toxicity. Br J Cancer 109, 2560-2565. Stovold R, Meredith SL, Bryant JL, Babur M, McGonagle AE, Mould DP, Rushbrooke M, Small Sci USA 110, 12426-12431. Williams KJ, Dean EJ, Dive C, Blackhall FH, White A. HF, Smith KM, Thomson GJ, Turlais F, Waddell ID, Greystoke A, Harris G, Jenkins M, Goonetilleke D, (2013) Waszkowycz B, Watson AJ, Ogilvie DJ. (2013) Hitchin JR, Blagg J, Burke R, Burns S, Cockerill MJ, Moore D, Lancashire M, Ranson M, Hughes A, Neuroendocrine and epithelial phenotypes in Toxoflavins and deazaflavins as the first reported Fairweather E E, Hutton C, Jordan AM, McAndrew Clack G, and Dive C. (2013) small-cell lung cancer: implications for metastasis selective small molecule inhibitors of tyrosyl-DNA C, Mirza A, Mould D, Thomson GJ, Waddell I, Assessment of diurnal changes and confounding and survival in patients. Br J Cancer 108, 1704-1711. phosphodiesterase II. J Med Chem 56, 6352-6370. Ogilvie DJ. (2013) factors that affect circulating cell death biomarker Development and evaluation of selective, reversible levels: a short communication. J Pharm Biomed Other publications Thomson G, Watson A, Caldecott K, Denneny O, LSD1 inhibitors derived from fragments. Med Chem Anal 84, 184-188. Fusi A, Metcalf R, Krebs M, Dive C, Depledge P, Hamilton N, Hopkins G, Jordan A, Commun 4(11), 1513-1522. Blackhall F. (2013) Morrow C, Raoof A, Waddell I, Ogilvie D. (2013) Honeychurch J, Dive C, and Illidge TM. (2013) Clinical utility of circulating tumour cell detection Generation of assays and antibodies to facilitate the Lynch JT, Cockerill MJ, Hitchin JR, Wiseman DH, Synchronous apoptosis in established tumors leads in non-small-cell lung cancer. Curr Treat Options study of human 5’-tyrosyl DNA phosphodiesterase. Somervaille TC. (2013) to the induction of adaptive immunity. Oncol 14, 610-622. Anal Biochem 436, 145-150. CD86 expression as a surrogate cellular biomarker Oncoimmunology 2, e24501. for pharmacological inhibition of the histone Thomson GJ, Hamilton NS, Hopkins GV, Waddell demethylase lysine-specific demethylase 1. Anal Horsley L, Cummings J, Middleton M, Ward T, Ivan Ahel (page 28) ID, Watson AJ, Ogilvie DJ. (2013) Biochem 442, 104-106. Backen A, Clamp A, Dawson M, Farmer H, Fisher N, DNA Damage Response A fluorescence-based assay for the apurinic/ Halbert G, Halford S, Harris A, Hasan J, Hogg P, apyrimidinic-site cleavage activity of human Morley AD, Pugliese A, Birchall K, Bower J, Brennan Kumaran G, Little R, Parker GJ, Potter P, Saunders Refereed Research Papers tyrosyl-DNA phosphodiesterase 1. Anal Biochem P, Brown N, Chapman T, Drysdale M, Gilbert IH, M, Roberts C, Shaw D, Smith N, Smythe J, Taylor A, Barkauskaite E, Brassington A, Tan ES, Warwicker 440, 1-5. Hoelder S, Jordan A, Ley SV, Merritt A, Miller D, Turner H, Watson Y, Dive C and Jayson GC. (2013) J, Dunstan MS, Banos B, Lafite P, Ahel M, Mitchison Swarbrick ME, Wyatt PG. (2013) A phase 1 trial of intravenous 4-(N-(S- TJ, Ahel I, Leys D. (2013) Belot A, Kasher PR, Trotter EW, Foray AP, Debaud Fragment-based hit identification: thinking in 3D. glutathionylacetyl)amino) phenylarsenoxide Visualization of poly(ADP-ribose) bound to PARG AL, Rice GI, Szynkiewicz M, Zabot MT, Rouvet I, Drug Discov Today 8(23-24), 1221-7. (GSAO) in patients with advanced solid tumours. reveals inherent balance between exo- and Bhaskar SS, Daly SB, Dickerson JE, Mayer J, Cancer Chemother Pharmacol 72, 1343-1352. endo-glycohydrolase activities. Nat Commun O’Sullivan J, Juillard L, Urquhart JE, Fawdar S, 4, 2164. Marusiak AA, Stephenson N, Waszkowycz B, W Peter Stern (page 32) Linnik IV, Scott ML, Holliday KF, Woodhouse N, Beresford M, Biesecker LG, C M Black G, René C, Immunology Waterton JC, O’Connor JP, Barjat H, Liess C, Ulloa Sharifi R, Morra R, Appel CD, Tallis M, Chioza B, Eliaou JF, Fabien N, Ranchin B, Cochat P, Gaffney J, Young H, Dive C, Hodgkinson CL, Ward T, Jankevicius G, Simpson MA, Matic I, Ozkan E, Golia PM, Rozenberg F, Lebon P, Malcus C, Crow YJ, Refereed Research Papers Roberts D, Mills SJ, Thompson G, Buonaccorsi GA, B, Schellenberg MJ, Weston R, Williams JG, Rossi Brognard J, Bonnefoy N. (2013) Bosch FX, Broker TR, Forman D, Moscicki AB, Cheung S, Jackson A, Naish JH, Parker GJ. (2013) MN, Galehdari H, Krahn J, Wan A, Trembath RC, Protein kinase cdelta deficiency causes mendelian Gillison ML, Doorbar J, Stern PL, Stanley M, Arbyn Noninvasive tumor hypoxia measurement using Crosby AH, Ahel D, Hay R, Ladurner AG, Timinszky systemic lupus erythematosus with B cell-defective M, Poljak M, Cuzick J, Castle PE, Schiller JT, magnetic resonance imaging in murine U87 glioma G, Williams RS, Ahel I. (2013) apoptosis and hyperproliferation. Arthritis Rheum Markowitz LE, Fisher WA, Canfell K, Denny LA, xenografts and in patients with glioblastoma. Magn Deficiency of terminal ADP-ribose protein 65, 2161-2171. Reson Med doi: 10.1002/mrm.24826. glycohydrolase TARG1/C6orf130 in neurodegenerative disease. EMBO Journal 32, Melis MH, Simpson KL, Dovedi SJ, Welman A, 1225–1237. MacFarlane M, Dive C, Honeychurch J, Illidge TM. (2013) Other Publications Sustained tumour eradication after induced Barkauskaite E, Jankevicius G, Ladurner AG, Ahel I, caspase-3 activation and synchronous tumour Timinszky G. (2013) apoptosis requires an intact host immune response. The recognition and removal of cellular poly(ADP- Cell Death Differ 20, 765-773. ribose) signals. FEBS J 280, 3491-3507. Polanski R, Hodgkinson C, Fusi A, Nonaka D, Priest Tallis M, Morra R, Barkauskaite E, Ahel I. (2013) L, Kelly P, Trapani F, Bishop P, White A, Critchlow SE, Poly(ADP-ribosyl)ation in regulation of chromatin Smith PD, Blackhall FH, Dive C, Morrow CJ. (2013) structure and the DNA damage response. Activity of the Monocarboxylate Transporter 1 Chromosoma Oct 27. [Epub ahead of print] inhibitor AZD3965 in Small Cell Lung Cancer. Clin Cancer Res Nov 25. [Epub ahead of print] Zaja R, Mikoc A, Barkauskaite E, and Ahel I. (2013) Molecular Insights into Poly(ADP-ribose) Simpson KL, Cawthorne C, Zhou C, Hodgkinson Recognition and Processing. Biomolecules 3, 1-17. CL, Walker MJ, Trapani F, Kadirvel M, Brown G, Dawson MJ, MacFarlane M, Williams KJ, Whetton AD, Dive C. (2013) 58 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH PUBLICATIONS 59

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    Franco EL, Steben M, Kane MA, Schiffman M, Meijer Jones DR, Ramirez IB, Lowe M, Divecha N. (2013) leukaemia may be obscured by cryopreservation. Falck Miniotis M, Arunan V, Eykyn TR, Marais R, CJ, Sankaranarayanan R, Castellsagué X, Kim JJ, Measurement of phosphoinositides in the zebrafish Br J Haematol 163, 538-541. Workman P, Leach MO, Beloueche-Babari M. Brotons M, Alemany L, Albero G, Diaz M, Sanjosé S. Danio rerio. Nat Protoc 8, 1058-1072. (2013) (2013) Huang X, Spencer GJ, Lynch JT, Ciceri F, MEK1/2 inhibition decreases lactate in BRAF-driven Comprehensive control of human papillomavirus van den Bout I, Jones DR, Shah ZH, Halstead Somerville TD, Somervaille TC. (2013) human cancer cells. Cancer Res 73(13), 4039-49. infections and related diseases. Vaccine 31 Suppl, JR, Keune WJ, Mohammed S, D’Santos CS, Enhancers of Polycomb EPC1 and EPC2 sustain the 7:H1-H31. Divecha N. (2013) oncogenic potential of MLL leukemia stem cells. Furney SJ, Turajlic S, Stamp G, Nohadani M, Carlisle Collaboration of AMPK and PKC to induce Leukemia Oct 29. doi: 10.1038/leu.2013.316. A, Thomas JM, Hayes A, Strauss D, Gore M, van den phosphorylation of Ser413 on PIP5K1B resulting in Oord J, Larkin J, Marais R. (2013) Nullin Divecha (page 34) decreased kinase activity and reduced PtdIns(4,5)P2 Lynch JT, Cockerill MJ, Hitchin JR, Wiseman Genome sequencing of mucosal melanomas Inositide Laboratory synthesis in response to oxidative stress and energy DH, Somervaille TC. (2013) reveals that they are driven by distinct mechanisms restriction. Biochem J 455, 347-358. CD86 expression as a surrogate cellular biomarker from cutaneous melanoma. J Pathol 230(3), 261-9. Refereed Research Papers for pharmacological inhibition of the histone Elouarrat D, van der Velden YU, Jones DR, Other Publications demethylase lysine-specific demethylase 1. Anal Niculescu-Duvaz D, Niculescu-Duvaz I, Moolenaar WH, Divecha N, Haramis AP. (2013) Keune WJ, Jones DR, Divecha N. (2013) Biochem 442, 104-106. Suijkerbuijk BM, Ménard D, Zambon A, Davies L, Role of phosphatidylinositol 5-phosphate 4-kinase PtdIns5P and Pin1 in oxidative stress signaling. Adv Pons JF, Whittaker S, Marais R, Springer CJ. (2013) alpha in zebrafish development. Int J Biochem Cell Biol Regul 53, 179-189. Lynch JT, Somerville TD, Spencer GJ, Huang X, Potent BRAF kinase inhibitors based on Biol 45, 1293-1301. Somervaille TC. (2013) 2,4,5-trisubstituted imidazole with naphthyl and Shah ZH, Jones DR, Sommer L, Foulger R, TTC5 is required to prevent apoptosis of acute benzothiophene 4-substituents. Bioorg Med Chem Gupta A, Toscano S, Trivedi D, Jones DR, Mathre S, Bultsma Y, D’Santos C, Divecha N. (2013) myeloid leukemia stem cells. Cell Death Dis 4, e573. 21(5), 1284-304. Clarke JH, Divecha N, Raghu P. (2013) Nuclear phosphoinositides and their impact on Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) nuclear functions. FEBS J 280(24), 6295-310. White DJ, Unwin RD, Bindels E, Pierce A, Teng HY, Pedersen M, Küsters-Vandevelde HV, Viros A, regulates TOR signaling and cell growth during Muter J, Greystoke B, Somerville TD, Griffiths J, Groenen PJ, Sanchez-Laorden B, Gilhuis JH, van Drosophila development. Proc Natl Acad Sci USA Lovell S, Somervaille TC, Delwel R, Whetton AD, Engen-van Grunsven IA, Renier W, Schieving J, 110, 5963-5968. Tim Somervaille (page 36) Meyer S. (2013) Niculescu-Duvaz I, Springer CJ, Küsters B, Leukaemia Biology Phosphorylation of the leukemic oncoprotein EVI1 Wesseling P, Blokx WA, Marais R. (2013) Keune WJ, Sims AH, Jones DR, Bultsma Y, Lynch on serine 196 modulates DNA binding, Primary melanoma of the CNS in children is driven JT, Jirström K, Landberg G, Divecha N. (2013) Refereed Research Papers transcriptional repression and transforming ability. by congenital expression of oncogenic NRAS in Low PIP4K2B expression in human breast tumors Alimam SM, Mamat MK, Kulkarni S, PLoS One 8, e66510. melanocytes. Cancer Discov 3(4), 458-69. correlates with reduced patient survival: A role for Somervaille TC. (2013) PIP4K2B in the regulation of E-cadherin expression. Sudden onset bilateral deafness as a Williams MS, Ali N, Nonaka D, Bloor AJ, Somervaille Smith MP, Ferguson J, Arozarena I, Hayward R, Cancer Res 73(23), 6913-25. presentation of chronic myeloid leukaemia. TC. (2013) Marais R, Chapman A, Hurlstone A, Wellbrock C. Br J Haematol 160, 3. Fatal invasive aspergillosis of the larynx. Eur J (2013) Jones DR, Foulger R, Keune WJ, Bultsma Y, Haematol 90, 354. Effect of SMURF2 targeting on susceptibility to MEK Divecha N. (2013) Greystoke BF, Huang X, Wilks DP, Atkinson S, inhibitors in melanoma. J Natl Cancer Inst 105(1), PtdIns5P is an oxidative stress-induced second Somervaille TC. (2013) Other Publications 33-46. messenger that regulates PKB activation. FASEB J Very high frequencies of leukaemia-initiating Wiseman DH, Greystoke BF, Somervaille TC. (2013) 27, 1644-1656. cells in precursor T-acute lymphoblastic The variety of leukemic stem cells in myeloid Girotti MR, Pedersen M, Sanchez-Laorden B, Viros malignancy. Oncogene doi: 10.1038/onc.2013.269. A, Turajlic S, Niculescu-Duvaz D, Zambon A, Sinclair J, Hayes A, Gore M, Lorigan P, Springer C, Larkin J, Jorgensen C, Marais R. (2013) Richard Marais (page 38) Inhibiting EGF receptor or SRC family kinase Molecular Oncology signaling overcomes BRAF inhibitor resistance in melanoma. Cancer Discov 3(2), 158-67. Refereed Research Papers Escuin-Ordinas H, Atefi M, Fu Y, Cass A, Ng C, Maertens O, Johnson B, Hollstein P, Frederick DT, Huang RR, Yashar S, Comin-Anduix B, Avramis E, Cooper ZA, Messiaen L, Bronson RT, McMahon M, Cochran AJ, Marais R, Lo RS, Graeber TG, Granter S, Flaherty K, Wargo JA, Marais R, Herschman HR, Ribas A. (2013) Cichowski K. (2013) COX-2 inhibition prevents the appearance of Elucidating distinct roles for NF1 in cutaneous squamous cell carcinomas accelerated melanomagenesis. Cancer Discov 3(3), 338-49. by BRAF inhibitors. Mol Oncol S1574- 7891(13)00163-4 doi: 10.1016/j. Viros A, Hayward R, Martin M, Yashar S, Yu CC, molonc.2013.11.005. Sanchez-Laorden B, Zambon A, Niculescu-Duvaz D, Springer C, Lo RS, Marais R. (2013) Furney SJ, Pedersen M, Gentien D, Dumont AG, Topical 5-fluorouracil elicits regressions of BRAF Rapinat A, Desjardins L, Turajlic S, Piperno- inhibitor-induced cutaneous squamous cell Neumann S, de la Grange P, Roman-Roman S, carcinoma. J Invest Dermatol 133(1), 274-6. Stern MH, Marais R. (2013) SF3B1 mutations are associated with alternative Other Publications splicing in uveal melanoma. Cancer Discov Martin M, Marais R. (2013) 3(10), 1122-9. Braking BRAF: AMPK leaves ERK stranded in the desert. Mol Cell 52(2), 155-6. 60 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH PUBLICATIONS 61

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    Marais R, Sellers W, Livingston D, Mihich E. (2013) Newton AC, GaoT, Brognard J: Compositions and Vasculogenic Mimicry (VM) Twenty-fourth annual Pezcoller symposium: Methods for Treating Diseases Associated with describes the ability of aggressive tumour cells to mimic Molecular basis for resistance to targeted agents. PHLPP. US Patent Pending 60/667,709, filed: March properties of endothelial cells Cancer Res 73, 1046-1049. 31, 2006. (WO/2006/105490) that enable de novo generation of tumour-derived vascular Sanchez-Laorden B, Viros A, Marais R. (2013) networks and provide microcirculation independently Mind the IQGAP. Cancer Cell 23(6), 715-7. Georges Lacaud (page 42) of the host. CD31 (right most Stem Cell Biology panel) staining is combined with Girotti MR, Marais R. (2013) PAS staining (second from right), Deja Vu: EGF receptors drive resistance to BRAF Refereed Research Papers to identify endothelial cells and inhibitors. Cancer Discov 3(5), 487-90. Stefanska M, Costa G, Lie-A-Ling M, Kouskoff V, determine the basement membranes of blood vessels and Lacaud G. (2013) tumour cells in which any Zambon A, Niculescu-Duvaz D, Smooth muscle cells largely develop independently structure containing CD31- Niculescu-Duvaz I, Marais R, Springer CJ. (2013) of functional hemogenic endothelium. Stem Cell positive immunoreactivity will be BRAF as a therapeutic target: a patent review (2006 defined as a blood vessel, while – 2012). Expert Opin Ther Pat 23(2), 155-64. Res 12, 222-232. VM structures will be strictly AC, Santibanez-Koref M, Walters K. (2013) Akira Orimo Bayesian inference supports a location defined as CD31-negative Perez-Campo FM, Costa G, Lie-A-Ling M, PAS-positive structures (the and neighbour-dependent model of DNA Refereed Research Papers Rebocho AP, Marais R. (2013) Stifani S, Kouskoff V, Lacaud G. (2013) methylation propagation at the MGMT images are from small cell lung Polanska UM, Orimo A. (2013) ARAF acts as a scaffold to stabilize BRAF:CRAF MOZ-mediated repression of p16INK4a is critical cancer and are supplied by gene promoter in lung tumours. Carcinoma-associated fibroblasts: non- heterodimers. Oncogene 32(26), 3207-12. for the self-renewal of neural and hematopoietic Francesca Trapani, Clinical and J Theor Biol 336, 87-95. Experimental Pharmacology). neoplastic tumour-promoting mesenchymal stem cells. Stem Cells. 2013 Dec 4. doi: 10.1002/ cells. J Cell Physiol 228, 1651-1657. stem.1606. Bosch FX, Broker TR, Forman D, Moscicki AB, John Brognard (page 40) Gillison ML, Doorbar J, Stern PL, Stanley M, Other Publications Signalling Networks in Cancer Other Publications Arbyn M, Poljak M, Cuzick J, Castle PE, Schiller Togo S, Polanska UM, Horimoto Y, Orimo A. Perez-Campo FM, Costa G, Lie-a-Ling M, JT, Markowitz LE, Fisher WA, Canfell K, Denny (2013) Refereed Research Papers Kouskoff V, Lacaud G. (2013) LA, Franco EL, Steben M, Kane MA, Schiffman Carcinoma-associated fibroblasts are a Belot A, Kasher PR, Trotter EW, Foray AP, Debaud The MYSTerious MOZ, a histone acetyltransferase M, Meijer CJ, Sankaranarayanan R, Castellsagué promising therapeutic target. Cancers (Basel) 5, AL, Rice GI, Szynkiewicz M, Zabot MT, Rouvet I, with a key role in haematopoiesis. Immunology X, Kim JJ, Brotons M, Alemany L, Albero G, Diaz 149-169. Bhaskar SS, Daly SB, Dickerson JE, Mayer J, 139, 161-165. M, Sanjosé S. (2013) O’Sullivan J, Juillard L, Urquhart JE, Fawdar S, Comprehensive control of human Marusiak AA, Stephenson N, Waszkowycz B, W papillomavirus infections and related diseases. Vaccine 31 Suppl, 7:H1-H31. Duncan Smith Beresford M, Biesecker LG, C M Black G, René C, Valerie Kouskoff (page 44) Eliaou JF, Fabien N, Ranchin B, Cochat P, Gaffney Stem Cell Haematopoiesis Crosbie PA, Harrison K, Shah R, Watson AJ, Refereed Research Papers PM, Rozenberg F, Lebon P, Malcus C, Crow YJ, Agius R, Barber PV, Margison GP, Povey AC. Hálová L, Du W, Kirkham S, Smith DL, Brognard J, Bonnefoy N. (2013) Refereed Research Papers (2013) Petersen J. (2013) Protein kinase cdelta deficiency causes Stefanska M, Costa G, Lie-A-Ling M, Kouskoff V, Topographical study of O(6)-alkylguanine DNA Phosphorylation of the TOR ATP binding mendelian systemic lupus erythematosus with B Lacaud G. (2013) alkyltransferase repair activity and N7- domain by AGC kinase constitutes a novel cell-defective apoptosis and hyperproliferation. Smooth muscle cells largely develop independently mode of TOR inhibition. J Cell Biol 203(4), Arthritis Rheum 65, 2161-2171. of functional hemogenic endothelium. Stem Cell methylguanine levels in resected lung tissue. Chem Biol Interact 204, 98-104. 595-604. Res 12, 222-232. Fawdar S, Trotter EW, Li Y, Stephenson NL, Senthong P, Millington CL, Wilkinson OJ, Chicooree N, Griffiths JR, Connolly Y, Hanke F, Marusiak AA, Edwards ZC, Ientile S, Perez-Campo FM, Costa G, Lie-A-Ling M, Marriott AS, Watson AJ, Reamtong O, Eyers CE, Smith DL. (2013) Waszkowycz B, Miller CJ, Brognard J. (2013) Stifani S, Kouskoff V, Lacaud G. (2013) Williams DM, Margison GP, Povey AC. (2013) Chemically facilitating the generation of Targeted genetic dependency screen facilitates MOZ-mediated repression of p16INK4a is critical The nitrosated bile acid DNA lesion O6- diagnostic ions from SUMO(2/3) remnant identification of actionable mutations in FGFR4, for the self-renewal of neural and hematopoietic carboxymethylguanine is a substrate for the isopeptides. Rapid Commun Mass Spectrom 27, MAP3K9, and PAK5 in lung cancer. Proc Natl Acad stem cells. Stem Cells. 2013 Dec 4. doi: 10.1002/ human DNA repair protein O6-methylguanine- 2108-2114. Sci USA 110, 12426-12431. stem.1606. DNA methyltransferase. Nucleic Acids Res 41, 3047-3055. Weekes MP, Tan SY, Poole E, Talbot S, Antrobus Other Publications Other Publications R, Smith DL, Montag C, Gygi SP, Sinclair JH, Belot A, Brognard J, Crow YJ, Bonnefoy N. (2013). Perez-Campo FM, Costa G, Lie-a-Ling M, Lehner PJ. (2013) Author reply. Arthritis Rheum doi: 10.1002/ Kouskoff V, Lacaud G. (2013) Zhang F, Tsunoda M, Suzuki K, Kikuchi Y, Wilkinson O, Millington CL, Margison GP, Latency-associated degradation of the MRP1 art.38234. The MYSTerious MOZ, a histone acetyltransferase Williams DM, Czarina Morishita E, Takénaka A. drug transporter during latent human with a key role in haematopoiesis. Immunology 139, cytomegalovirus infection. Science 340(6129), Fawdar S, Edwards ZC, Brognard J. (2013) (2013) 161-165. 199-202. Druggable drivers of lung cancer. Oncotarget 4, Structures of DNA duplexes containing O6- 1334-1335. carboxymethylguanine, a lesion associated with gastrointestinal cancer, reveal a mechanism for Active Patents Geoff Margison inducing pyrimidine transition mutations. Kozikowski AP, Dennis PA, Brognard J, and Sun H: Nucleic Acids Res 41, 5524-5532. Akt Inhibitors, Pharmaceutical Compositions, and Refereed Research Papers uses Thereof. US Patent: 7,378,403, 2008. Bonello N, Sampson J, Burn J, Wilson IJ, McGrown (WO/2004/022569) G, Margison GP, Thorncroft M, Crossbie P, Povey 62 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH PUBLICATIONS 63

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    THESES Filippo Ciceri Leukaemia Biology Group Studies on the proliferation of AML blasts Emily Holmes Cell Regulation The identification of novel targets of the Filippo Ciceri fission yeast Sty1 MAPK Leila Khoja CEP Clinical Significance and Utility of Circulating Tumour Cells in Pancreatic Carcinoma and Melanoma Emily Holmes Georgi Marinov Immunology Trafficking and functional interactions of oncofoetal trophoblast glycoprotein 5T4 Andrzej Mazan Stem Cell Haematopoiesis Elucidating the role of Sox7 in the generation of haemogenic endothelium Andrzej Mazan Milena Mazan Stem Cell Biology Exploring the role of GFI1 and GFI 1B in early haematopoiesis Tumour-stroma interface. GFP expressing tumour cells (green) re-arrange collagen bundles (grey) Sharmin Naaz perpendicularly to the tumour edge to facilitate tumour ACBB growth and invasion. In contrast, collagen bundles (grey) The role of non-coding RNAs at the onset in the adjacent normal tissue run parallel to the tumour Milena Mazan of haematopoietic commitment edge acting as a physical barrier to limit tumour progression. Image provided by Haoran Tang (Molecular Oncology) Monika Stefanska (nee Antkiewicz) Stem Cell Biology Investigating the origin of primitive erythrocytes and vascular smooth muscle cells Sharmin Naaz 64 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE THESES 65

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    EXTERNAL SEMINAR SPEAKERS 2013 The seminar series that we run is vital for the Institute, Luca Pellegrini Department of Biochemistry, connecting world-class researchers across the broad spectrum Cambridge University of cancer research. 2013 was another successful year for Rickard Sandberg scientific interaction with an excellent set of internationally Karolinska Institutet, Stockholm renowned speakers visiting the Institute. In its fifth year, The Owen Sansom Breakthrough Breast Cancer Research Unit seminar series The Beatson Institute for Cancer Research, continues to produce an outstanding range of speakers. The Glasgow postdoctoral researchers at the Institute also give weekly Stephen Taylor seminars which are very well attended and help to integrate the Institut Gustave Roussy, Paris entire cancer research efforts of the Institute. Johannes Zuber Institute of Molecular Pathology, Vienna Chris Bakal Tony Hunter Institute of Cancer Research, London The Netherlands Cancer Institute, Amsterdam Breakthrough Breast Cancer Research Unit Seminar Series 2013 Nick Cross Claus Jorgensen National Genetics Reference Laboratory, Institute of Cancer Research, London Alan Ashworth Wessex Institute of Cancer Research, London Stefan Knapp Bill Earnshaw Nuffield Department of Medicine, Oxford Cathrin Brisken Wellcome Trust Centre for Cell Biology, École Polytechnique Fédérale de Lausanne, University of Edinburgh Richard Lamb Lausanne Liverpool Cancer Research UK Centre Oskar Fernandez-Capetillo Ferruccio Galbiati Spanish National Cancer Research Center, Oliver Maddocks University of Pittsburgh School of Medicine, Madrid The Beatson Institute for Cancer Research, Pittsburgh Glasgow Greg Findley Beatrice Howard Samuel Lunenfeld Research Institute, Ontario Chris Maher Breakthrough Research Centre, London London Research Institute, London Margaret Frame Rama Khokha Edinburgh Cancer Research Centre Matthias Merkenschlager Ontario Cancer Institute, Ontario MRC Clinical Sciences Centre, Imperial David Glover College, London Jeffrey Rosen University of Cambridge Baylor College of Medicine, Texas Sebastian Nijman Mary J.C. Hendrix CeMM Research Center for Molecular John J. Wysolmerski Lurie Children’s Research Center, Northwestern Medicine, Vienna Yale School of Medicine, Connecticut University, Illinois Christian Ottensmeier Martin Humphries University of Southampton Wellcome Trust Centre for Cell-Matrix Research, University of Manchester 66 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE EXTERNAL SEMINAR SPEAKERS 2013 67

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    The annual CRUK MI Colloquium, held in CEP (in collaboration with MCRC colleagues); POSTGRADUATE EDUCATION September, is an excellent opportunity for our at AstraZeneca they receive training in clinical www.cruk.manchester.ac.uk/education new intake of students to meet other trials management, regulatory interaction, established PhD students, members of the translational research through project Institute, including Group Leaders, Postdoctoral management, and attend investigator meetings. Fellows, and Scientific Officers. This forum Clinical training includes one research clinic communicates up to date science in the form of per week, training in clinical trial design and oral presentations given by Group Leaders and methodology, ICH-GCP, EU Directives and second year PhD students as well as poster research governance. Biomarker method presentations from a range of scientists across development and application take place on the Institute. Poster prizes are awarded, both sites in all projects, with mutual benefit as including the Lizzy Hitchman Prize for the best each Fellow brings newly acquired knowledge The Cancer Research UK Manchester Institute (CRUK MI) offers poster presented by a PhD student or Clinical to the other site. Regular meetings take place Fellow. In 2013, Daniel Wiseman from the between the Fellows, their supervisors, as well a range of graduate degrees for students interested in a career Leukaemia Biology was the recipient of the as other staff members involved in the project, involving cancer research. The Institute considers education of Lizzy Hitchman Prize for his work describing a ensuring effective collaboration and an potential test to predict relapse in Acute Myeloid integrated approach. both research and clinical scientists to be a major investment in Leukaemia. the future of cancer research, and has an excellent track record Education Committee 2013 PhD studentships The Education Committee acts for of launching careers in basic, translation and clinical research. All of our CRUK core funded studentships are of postgraduate students based within CRUK Julie Edwards four years duration, and consist of an approved core funded research groups and consists of Postgraduate Education Manager As part of this commitment, we have an active designed not only to provide formal points at research project in one of our research groups. Senior Group Leaders, the Chief Operating postgraduate programme that provides which progress (of both the student and the Some students have joint supervisors in different Officer and the Postgraduate Education excellent students and clinical research fellows project) can be monitored, but also to help groups, fostering important collaborations and Manager of CRUK MI. the opportunity to study for cancer-related PhD develop the presentation skills that are so providing exposure to different disciplines. or MD degrees. This is achieved through a fundamental to the majority of careers in Recruitment is highly competitive, with Our goal is for every student to have a project training programme that aims to improve science and elsewhere. Graduate training is hundreds of applicants competing for around that is both achievable and intellectually effectiveness in research, provide professional monitored by an Education Committee, which four to eight places each year. Interviews are stimulating and demanding. Projects and and management skills and enhance career features CRUK core-funded Group Leaders, typically conducted over a two-day period in students are monitored by the Education development. Ninety-nine percent (99%) of our scientists and student representatives (see early January. Committee which makes sure that the students in the past eight years have found below). A main supervisor and a second or proposed plan of research is suitable, and that employment after graduation; half of these are co-supervisor is nominated for each student, All of our students benefit from access to progress is made consistently throughout the Ian Waddell Postgraduate Tutor in American or European laboratories, while who is able to provide additional advice and advanced state-of-the-art facilities, including course of the studentship. Various assessments 20% continue to progress in their clinical careers consultation on both academic and non- advanced imaging, biological mass throughout the studentship, including regular in the NHS. Students leave the CRUK MI with academic matters. Each student is also assigned spectrometry, microarrays, flow cytometry, talks, progress meetings and written reports, are excellent career prospects across the world. an advisor (similar to a personal tutor on an histology and next generation sequencing. Our key to ensuring successful completion of the undergraduate programme) whose role is to research groups offer PhD studentships and PhD programme. Such assessments not only In 2013, we welcomed three graduate students provide impartial support and advice, while projects covering the entire breadth of research monitor progress, but also help to develop to our PhD programme, working in a variety of further support is also available individually from within the Institute. performance and presentations skills. fields from drug discovery, to stem cells and the Education Committee chair, Postgraduate leukaemia biology. It was also particularly Tutor, Postgraduate Manager, or collectively as Fellowships in Clinical Valerie Kouskoff gratifying to see that, over the past twelve the Education Committee Administration Pharmacology Research (Chair, Education Committee) months, eleven of the Institute’s publications Group. In order to help train the next generation of Julie Edwards had students as first authors in journals as clinical pharmacologists with expertise in Karim Labib diverse as Melanoma Research, Nature The CRUK MI runs an external seminar series oncology, the CRUK MI, in collaboration with (until July) Communications and Stem Cell Research. featuring talks from many of the key players in the MCRC and AstraZeneca, established in 2007 a fellowship scheme in Clinical Pharmacology Angeliki Malliri During the course of the year, a total of five PhD cancer research, and students are expected to students and one Clinical Fellow were awarded attend all of these external seminars. The Research. The fellowships are open to Donald Ogilvie their PhDs. speakers are internationally renowned scientists applicants who have obtained, or are close to Tim Somervaille and we consider it essential that our students obtaining, their Completed Certificate of Ian Waddell The Cancer Research UK Manchester are exposed to outstanding work from the Specialist training (CCST) in Medical Oncology. (Postgraduate Tutor from October) Graduate Programme leaders in different disciplines, which will give Caroline Wilkinson We aim for each student to receive high quality them a broad understanding of many aspects of Each Clinical Pharmacology Research Fellow undertakes a three-year PhD project, which Richard Marais - Ex-Officio Member training in scientific research through an cancer research and basic biology. In addition, (from February) intellectually demanding but achievable we hold a series of weekly postdoctoral provides training in biomarker discovery, research programme. Each project is peer- research seminars that the students attend. method development/validation, and clinical trial methodology. During tenure at the Christie/ Student Representatives reviewed in advance and monitored throughout While students themselves are asked to give Hadir Marei (until December) the course of their studies via a mixture of oral talks at key points during their PhD, they also CRUK MI, the post holders receive clinical supervision from Malcolm Ranson, and Danish Memon (from November) presentations, written reports, and progress have opportunities to present their work at lab Alekh Thapa (from December) meetings. These modes of assessment are meetings and during student forums within the laboratory-based training from Caroline Dive in Institute. 68 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE POSTGRADUATE EDUCATION 69

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    OPERATIONS The Operations Department provides the necessary During 2013, the Estates team have carried out a team supports the research groups by providing number of small works’ projects to adapt effective and efficient professional advice when infrastructure and services to facilitate the running of the laboratories and offices for the arrival of new costing new research proposals and Institute. During 2013, Caroline Wilkinson and Stuart Pepper groups and the relocation of others. The Estates administering existing grants. team endeavour to identify sustainable solutions were appointed Chief Operating Officer and Chief Laboratory when putting new schemes together to help Towards the end of 2012, we were advised that Officer respectively. Finance and purchasing, as well as Estates reduce the Institute’s carbon footprint whenever an application to UK Research Partnership possible. Lighting is one of the main users of Investment Fund (UKRPIF) had been successful. and Logistics, fall under the leadership of Margaret Lowe while energy in a building and the team is hoping to This has resulted in the purchase of several Caroline Wilkinson Chief Operating Officer Stuart Pepper oversees IT as well as Health and Safety; Rachel reduce our energy usage by replacing the significant pieces of equipment during 2013 existing luminaires with LED lighting. with further purchases planned throughout Powell is head of HR and Caroline Wilkinson is responsible for all 2014. aspects of Scientific Administration and acts as the primary The team has been proactive throughout the year, attending to many legislative requirements; Health & Safety point of operational contact within the Institute for both The examples include Legionella best practices and Colin Gleeson University of Manchester and Cancer Research UK. fire alarm testing. Team members have attended relevant courses to improve skills and keep their During the year, an increased emphasis on the knowledge up to date with current working importance of reporting accidents and near This year, the Operations team welcomed The department has been incredibly busy this practices and changing legislation; the team misses led to an increase in the number of Ekram Aidaros and David Stanier to the year with arrangements for the Institute name attended an asbestos awareness training course reported incidents that more accurately reflect Stuart Pepper Administration Department; Lewis Parkinson change that took effect on the 1st October. It was in December. the situation in the Institute. We achieved a Chief Laboratory Officer joined Estates and Neepa Begum was recruited an exciting time for us and we launched a twitter doubling in the number of reported near misses, to the Finance team. Hong Mach joined IT to feed @CRUK_MI and Facebook page under our Finance & Purchasing which, it is suspected, have been historically provide specialist support for Apple devices new name. The department has assisted with Margaret Lowe, Neepa Begum1, David Jenkins, under-reported. There was only one RIDDOR while Tom Bolton was recruited to the position the organisation of several events over the Denise Owen, Muhammad Raja, Debbie reportable incident in 2013; this involved an of Web Developer. course of the year, including the Institute Trunkfield escape of liquid nitrogen from a brand new Colloquium and quinquennial/mid-term 1 joined in 2013 vessel. As a result, we reviewed our A major project for all operational staff during reviews. Administrative support is provided for arrangements for liquid nitrogen safety resulting the year involved the Institute’s change of name the external seminar series, which has been a The Institute Finance Team supports the in the provision of personal low-oxygen which came into effect at the beginning of great success in 2013, and the 2014 programme Director with the management of the Institute’s monitors and the repeat of a cryogenic training October. This involved a great deal of work is already in place. We aim to provide a varied £21m budget, which is devolved to the various session. Margaret Lowe leading up to the event including replacing programme of national and international groups and service units. The team provides a Head of Finance signage and the design and development of a speakers, serving to foster collaboration and comprehensive service to the Institute, which Other health and safety management system new website. encourage positive interaction within the wider covers all areas of Procurement and Finance, performance indicators were encouraging. A scientific community. Details can be found at ensuring we comply with the University financial survey to assess the effectiveness of the Administration Department www.cruk.manchester.ac.uk/seminars regulations and procedures. processes for capturing new starters for Ruth Perkins, Ekram Aidaros, Steven Morgan, induction training indicated that it was very David Stanier Estates The University has upgraded the Oracle system effective, as all new starters in the previous eight Steve Alcock, Graham Hooley, Lewis Parkinson, in 2013. This has involved close liaison with the months had been inducted. Furthermore, The Administration department has expanded Tony Woollam Project Team to ensure as little disruption as surveys showed that statutory required this year with the addition of David Stanier as possible within the Institute. There have performance testing of all fume cupboard and Administration Assistant who provides support The past year has seen our team expand with inevitably been a few minor issues but the effect safety cabinets had been completed. Other Rachel Powell to Caroline Wilkinson, Chief Operating Officer; the arrival of Lewis Parkinson who has been on the scientists has been minimal. site-wide initiatives included compliance checks Head of Human Resources to the HR department; and cover for Steve appointed as an Apprentice Building Services on stocks held of chemical weapons, Morgan on Reception. Ruth Perkins took up the Engineer. He will gain great benefit from over a The Institute has been successful in securing desensitised explosives, dangerous pathogens position of Executive Assistant to the Director hundred years of combined experience in the several new external grant awards that will be and toxins, Euratom materials and radioactive when Siana Peters left the Institute in May and Estates team. activated in 2014 and we are also awaiting the waste disposal records. she is now supported by Ekram Aidaros, outcome of several other applications. The Administrative Services Coordinator. 70 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE OPERATIONS 71

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    providing increased functionality where have also been able to make significant savings required. The team support and maintain of just over £4,000 this year by setting up orders more than 500 PC and Apple based desktops that cover multiple deliveries from a single as well as over 100 mobile devices including company with a number of suppliers. Blackberry, iPads and mobile tablets, therefore it is vital that front-end and back-end systems Researchers can order central stores stock items are continuously updated and upgraded to via the intranet which are then distributed by the ensure system security and stability. Logistics team who also ensure that adequate stock levels are maintained at all times. Included During 2013, the on-going need for additional in this system are the enzymes and media data storage has meant we have increased our stored in the Institute freezers (Sigma, Invitrogen, storage capability to over 200TB to meet the Roche, Promega, New England Biolabs, Fisher requirements of our staff and accommodate the kits and Qiagen). The department works closely scientific needs for the coming year. A review of with all the research groups and helps out the storage strategy will be undertaken in the where necessary, for example by tracing and New Year to take into account the research confirming delivery of goods with suppliers. undertaken at the CRUK MI as well as the new The team also manages the moving of heavy MCRC building. The IT team are looking forward equipment or furniture, and the reconfiguration to the challenge of supporting the new MCRC of meeting rooms for numerous events which building which will have a diverse customer this year has included various fundraising base resulting in complex interactions in terms events, QQRs and events for the Manchester of IT systems. The team will be looking to devise Science Festival. creative solutions to ensure that the Institute Some of the Health and Safety initiatives managers and staff on all employment-related extends the high standards of IT, that current Scientific Operations undertaken in 2013 involved the Institute’s core matters such as recruitment, policy guidance, users have become accustomed to, across to Caroline Wilkinson, Tom Bolton1 2, Gillian facilities. To increase protection when sorting legislation and best practice. the new MCRC building. Campbell live cells, bespoke microbiological safety 1 cabinets were selected and installed in the Flow During 2013, 44 individuals were successfully joined in 2013 The recent separation of the high performance Cytometry facility. Accordingly, all live cell appointed who will endeavour to complement 2 joint with MCRC cluster into a dedicated new department has sorting can be contained within safety cabinets, and enhance the work of the Institute. The led the IT department to focus their attention whose performance can be validated by KI department has continued its drive for efficiency Scientific administration is overseen by the Chief on the core IT challenges of the Institute’s containment testing. In the Biological by evaluating where we advertise our vacancies. Operating Officer, Caroline Wilkinson, who infrastructure. As a consequence of the rise in Resources Unit, monitoring was undertaken to This is to ensure that we are attracting the best provides support to the Director in order to the number of Apple desktop systems, IT assess the levels of airborne concentrations of candidates to the deliver the aims and objectives facilitate the day-to-day running of the Institute. support for Apple devices has been doubled. animal allergens. The results indicated that in of the Institute and to ensure that we remain a The team is also responsible for producing a The new name for the Institute necessitated most of the facility, allergen levels were leading cancer research establishment. variety of scientific communications for the the rebranding of all IT systems which was undetectable or very low indeed. Allergen was Institute including publications such as the achieved in a seamless manner. detected in the cage cleaning area and action is We have continued our joint partnership Annual Scientific Report, the Institute’s being undertaken to improve matters and so working with the unions which has resulted in Newsletter, writing material for the intranet and Logistics reduce the possibility of exposure to as low as the revision of many policies such as the Career external website and for the Institute’s social Maurice Cowell, Edward Fitzroy, Sedia Fofana, reasonably practicable. Respiratory protective Path for Scientists, Special Leave Policy and the media presence. Talks and tours are also Stephen Keane1, Andrew Lloyd, Jonathan Lloyd equipment testing was also carried out. Career Break Policy. We are committed to provided for a packed programme of 1 developing an environment where staff are able joined in 2013 fundraisers’ events throughout the year. During Health and Safety training has been delivered to fully contribute to the service while feeling 2013, Tom Bolton was recruited to the position including Induction training as well as during valued and respected. These values are The Logistics facility plays a vital role in of Web Developer and took on the re-design specialist sessions for working with biological reinforced by the Institute with the revision and supporting the research carried out at the and development of the external website to agents and clinical material, working with re-launch of the Respect at Work Policy. Institute. This includes the receipting, checking, coincide with the rebranding of the Institute. hazardous chemicals and completing risk booking in and distribution of goods ordered by assessments. These sessions were well Over the next year, the main focus for the staff as well as the collection and removal of In addition to receiving core funding from attended. As the new MCRC building nears HR department will be the successful waste. The team also provides assistance with CRUK, our researchers also apply for external completion, equipment acquisition and recruitment of new groups to the Institute moving equipment and supplies and therefore awards to extend the portfolio of research that formalising relationships with stakeholders and the implementation of the new online helps facilitate internal rearrangements and the we can undertake. All grant applications has begun. probationary system. arrival of new groups. Some larger liquid submitted by our researchers are screened for nitrogen vessels are now in operation, the appropriate ethical approvals, as well as the Human Resources Information Technology increasing the usage of nitrogen and its ability of the Institute to accommodate the Rachel Powell, Laura Jones, Julie Jarratt, Lisa Malik Pervez, Hong Mach1, Brian Poole, Steve transportation to the labs. This is performed proposed programme of work. We also have a Rumsey1 Royle, Zhi Cheng Wang2, Matthew Young three times a week with dry ice deliveries taking rigorous internal peer review process for grant 1 1 place twice a week. During 2013, the Institute applications which is organised by Gill joined in 2013 joined in 2013 used approximately 90 litres of liquid nitrogen in Campbell, the Institute’s Grants Advisor. Gill also 2 Moved to Scientific Computing team storage vessels. Gas cylinders are also Over the past year, the HR Department has provides support to the Institute’s researchers monitored and replaced as necessary. We have through the grant preparation and submission continued to successfully provide a professional The CRUKMI IT team have continued to been able to make savings by assessing our gas process. This year, saw an increase in the and proactive HR service to the Institute. This maintain IT services to the high standards cylinders and to cutting down on those in less number of submissions including a successful includes providing advice and guidance to that are expected by service users as well as demand thus decreasing rental charges. We 72 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE OPERATIONS 73

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    application to Prostate Cancer UK, for the award gap between cancer drug discovery and early of a Movember Prostate Cancer Centre of development. It will take potential cancer drugs, Excellence (in collaboration with Queen’s primarily discovered by Cancer Research UK, University Belfast). from discovery through to entry to Phase II clinical trials before partnering with In September, the team organised a one day pharmaceutical and biotechnology companies. meeting between the Institute’s scientists and those working on cancer-related aspects at The By arrangement with The University of University of Manchester’s Faculty of Life Manchester, CRT owns and is responsible for Sciences. This was highly successful and we are the development and commercialisation of planning to make this a regular event. intellectual property arising from Cancer Research UK funded research at The University During the year, we modified the Institute’s new of Manchester (including the Cancer Research staff induction process to include information UK Manchester Institute). Our relationship with regarding research integrity. In addition, Gill the Cancer Research UK Manchester Institute Campbell was appointed as the Institute’s reflects the specific requirements of the Research Integrity Officer to help promote and scientist, Cancer Research UK Manchester maintain the highest standards of rigour and Institute, Cancer Research UK and the individual integrity in all aspects of our research. project. To effectively facilitate these requirements and interactions, Martyn Cancer Research Technology Bottomley, a CRT Business Manager, is based Martyn Bottomley on-site at the Cancer Research UK Manchester Institute and is dedicated to working closely with Cancer Research Technology (CRT) is a the staff at the Institute and also The University specialist oncology focused development and of Manchester. Martyn is here to offer access to commercialisation company wholly owned by oncology-focused expertise in technology Cancer Research UK. CRT aims to maximise evaluations, patent applications and patient benefit from publicly funded research management, funding for development, worldwide by advancing research discoveries commercialisation, drug discovery, market into development with pharmaceutical and intelligence, and project management. He also biotechnology parties. works closely with UMIP, The University of Manchester technology transfer organisation. At CRT, we bridge the fundamental gap CRT continues to work very closely with the between cutting edge academic research and Drug Discovery Laboratories based at the industrial development of cancer therapeutics Cancer Research UK Manchester Institute to and diagnostics. We achieve this by working facilitate the development of small molecules closely with prestigious international research drug therapies to satisfy the unmet clinical institutes, such as the Cancer Research UK needs of cancer patients. In the year to date, we Manchester Institute and funding bodies to have negotiated four collaboration agreements develop, protect and commercialise oncology with commercial partners for the Drug related discoveries. Core activities of business Discovery Laboratories, including AstraZeneca, development and drug discovery are supported GSK and HitGen. CRT is also currently actively by specialists, integrated in the business with managing a broad portfolio of development expertise in patents, legal, finance and programmes and exciting licensing marketing. Our exclusive focus in oncology opportunities originating from the Cancer provides an unrivalled depth of knowledge and Research UK Manchester Institute that continue experience in cancer-specific translational to attract commercial partners. We look forward Normal human colon stained with an antibody to the development and commercialisation. We now to building on our success and continuing to marker MUC2 (brown). MUC2 is used as a marker of also have access to the CRT Pioneer Fund; this work closely with the Cancer Research UK specialised Goblet cells and is deregulated in several £50m fund has been established with Cancer Manchester Institute to advance discoveries to tumour types. The tissue has been counterstained with haematoxylin to show the nuclei of cells (blue). Research Technology (CRT) and the European beat cancer in the years ahead. Investment Fund (EIF) to bridge the investment Image provided by Darren Roberts, Immunology. 74 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE OPERATIONS 75

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    picture to its place in the research journey. partnership projects with schools involved with CANCER RESEARCH UK’S Visitors were invited to add their messages of the University of Manchester’s SUPI (School and RESEARCH ENGAGEMENT support to the gallery walls in return for a charity University Partnership) initiative. The Institute’s donation. Buy Art Fair’s founder Thom scientists have many fundraising plans, such as Hetherington said ‘Cancer Research UK’s running the Manchester Marathon, the Great involvement really brought something to the fair Manchester Run and climbing Scafell Pike. this year, and hopefully it generated profile and opportunity for you’. It is unlikely that our 40,000 supporters in Greater Manchester would make the In 2014, we hope to build on the success of contribution to our work that they do without 2013, including the Manchester Science Festival, the passion and enthusiasm of the Institute’s which takes a look at the past, present and scientists, who are always keen and willing to The Cancer Research UK Manchester Institute has had a future of cancer research as part of Manchester share their work in new and creative ways. Histories Festival and cross-disciplinary fantastic year of supporting fundraisers, thanking them and joining them in a range of activities. Diane Hughes shares ideas of how patients could better inform Cancer Research UK’s research More than 1000 charity supporters, twice as Institute scientists themselves have also taken policy and progress during many as in 2012, have visited the Institute this part in a series of fundraising activities, raising September’s Patient year and taken part in a range of activities, from tens of thousands of pounds for research. Engagement event at the Cancer Research UK Manchester a film screening to lab tours and interactive These range from the most challenging 40 mile Institute Cancer Research UK’s hands-on science taster sessions. Many more Keswick to Barrow walk and 24 hour Relay for Research Engagement have joined us outside the Institute for events Life, to running through clouds of paint in the Manager and activities, including science festivals and Colour Run, practising yoga poses for Stand up projects with local schools, and in total there to Cancer, growing a series of stylish Eve Hart1 have been more than 260 events in which moustaches during Movember, and even Institute scientists interacted face-to-face with representing scientific research through the the public who fund our work. medium of cake during the charity’s inaugural Science Cakes competition. The range of visitors to the Institute this year has Nelson Iley talks about the work been wider than ever before. In September, we of the Molecular Biology Core piloted an open afternoon for local businesses Facility before one of our visitors to encourage them to support our work, an get hands on with a pipette event to be repeated twice in the first half of 2014. In the same month, more than 30 people affected by cancer visited us as part of a network Cancer Research UK’s of patient engagement events taking place Research Engagement around the UK at the same time. The events, Manager which included a live webcast by CRUK Chief James Dunphy2 Executive Harpal Kumar, will help the charity to develop a new strategy to better involve people 1 joined in 2013 affected by cancer in research. Colette Hughes 2 left in 2013 attended with her sister Diane. She said ‘We are a family who carry the BRCA2 gene and my sister is on personalised treatment [an early The Cancer Research UK Gallery at Buy Art Fair 2013, phase clinical trial at the Christie NHS Spinningfields, Manchester Foundation Trust]. Getting more survivors to talk to the scientists and seeing what is being done from both viewpoints would be just brilliant’. Outside the Institute, a new audience were exposed to our work and encouraged to support us when we established a surprising ‘Gallery of Research’ as part of the Buy Art Fair in Before and after. The Cancer central Manchester. Thousands of visitors saw Research UK Manchester Institute’s Drug Discovery team Steve Bagley’s images hung alongside original tackle Movember. In the first paintings and artworks, telling the story of photo, Stuart is the control cancer research from DNA to drug discovery. subject. The team raised a The gallery even had its own catalogue in the brilliant total of £1366. form of image flashcards that related each 76 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE CANCER RESEARCH UK’S RESEARCH ENGAGEMENT 77

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    ACKNOWLEDGEMENT FOR FUNDING OF THE CAREER OPPORTUNITIES AT THE CANCER CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH UK MANCHESTER INSTITUTE The total funding of the CRUK Manchester Institute for 2013 was The Cancer Research UK Manchester Institute is located alongside £20.8m. The major source of this funding was awarded by Cancer The Christie NHS Foundation Trust, and has a strong programme of Research UK (CRUK) via a core grant of £10.1m, plus additional strategic basic and translational research. There are very close links with clinical funding of £3.7m. This funding enables the various scientific groups and translational research groups throughout the Christie Hospital site. and service units within the Institute to carry out their research. The Institute offers excellent laboratory facilities In addition to postgraduate and postdoctoral and outstanding core facilities, including molecular opportunities, the Institute is still seeking to recruit CRUK MANCHESTER INSTITUTE FUNDING 2013 services, next generation sequencing, real-time outstanding candidates to the positions of Junior PCR, a microarray platform, proteomics, flow and Senior Group Leaders. The packages provided 13.6% cytometry, histology, advanced imaging, and the are extremely attractive and commensurate with production of knock-in/knock-out animal models. the experience of the applicant, with significant 21.5% Details of all groups and facilities are given in this funding for personnel, recurrent expenditure and report, and can guide interested parties to the equipment. Junior Group Leaders are appointed KEY appropriate contacts. for an initial six-year period with a review between five and six years for consideration of promotion to CRUK Core Grant 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. CRUK Strategic Funding of degrees by research which is described in the section on Postgraduate Education. We encourage Specific vacancies can be found on our web pages HEFCE 46.7% applications from suitably qualified graduates to (www.cruk.manchester.ac.uk/Jobs/), but suitably 14.7% apply to join either the PhD or MD programmes. qualified and enthusiastic individuals should Other Sources Graduates with a first or 2.1 honours degree in a contact the Institute at any time to enquire about biological science can apply each year to train for a career possibilities. four-year PhD in one of our research laboratories. The University of Manchester offers a wide range of training for new and existing students which provides opportunities to acquire skills that will complement the research programme and help The infrastructure of the CRUK Manchester Institute • BBSRC achieve personal and career development goals. At is funded by HEFCE generated income at a cost of • Leukaemia & Lymphoma Research Fund the Institute, we also ensure that postgraduate £2.4m. This was further enhanced by £2.3m from • Abbott Laboratories students are provided with high quality, relevant and the UK Research Partnership Investment Fund • Christie Hospital NHS Foundation Trust appropriate training alongside development (UKRPIF). • Association for International Cancer Research opportunities. The Institute also has a well- • Medical Research Council developed process for ensuring excellent pastoral The balance of the Institute’s funding is received • Kay Kendal Leukaemia Fund care and mentoring for all students. from a number of additional sources. The research • Wellcome Trust carried out through these additional projects • Parsortix Postdoctoral applicants of high calibre are regularly enhances and supports the research undertaken by • Pancreatic Cancer Research Fund sought. Although Postdoctoral Fellows will be the core funding. • Academy of Medical Sciences encouraged to apply for their own fellowships, • Immunogen Inc funded positions are available for outstanding These sources are as follows: candidates. Interested applicants should contact We are immensely grateful to all our sponsors. the Group Leaders directly, with details of their • AstraZeneca research interests and recent experience. • European Commission • European Research Council 78 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE CAREER OPPORTUNITIES AT THE MANCHESTER INSTITUTE 79

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    CONTACT DETAILS Cancer Research UK Manchester Institute ISSN 1479-0378 Director: Professor Richard Marais Copyright © 2013 Cancer Research UK Tel: +44(0) 161 446 3156 Fax: +44(0) 161 446 3109 Edited by: Caroline Wilkinson Gillian Campbell Address Cancer Research UK Manchester Institute OUT-PATIENT ENTRANCE Wilmslow Road NATHAN HOUSE PRIVATE Manchester PATIENTS UNIT ENTRANCE M20 4BX United Kingdom MANCHESTER INSTITUTE ENTRANCE e-mail: enquiries@cruk.manchester.ac.uk website: www.cruk.manchester.ac.uk MAIN HOSPITAL ENTRANCE Tel +44(0) 161 446 3156 Electronic version of this report can be found at: www.cruk.manchester.ac.uk/About/ 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 80 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK MANCHESTER INSTITUTE

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    Cancer Research UK Manchester Institute Wilmslow Road Manchester M20 4BX United Kingdom Telephone +44(0) 161 446 3156 www.cruk.manchester.ac.uk www.cruk.manchester.ac.uk

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