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    SCIENTIFIC REPORT 2016 cruk.org


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    COVER IMAGE SCIENTIFIC Live small cell lung cancer cells, stained for actin, invading into a layer of matrigel. Colours represent depth at which the cells were imaged. REPORT 2016 Image supplied by Andrew Porter & Sophie Adlard (Cell Signalling) MANCHESTER INSTITUTE


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    CONTENTS DIRECTOR’S INTRODUCTION 04 Richard Marais 28 RESEARCH SERVICES RESEARCH PUBLICATIONS 58 RESEARCH HIGHLIGHTS 2016 07 Molecular Oncology Steve Bagley 48 THESES 69 Esther Baena 30 Advanced Imaging and Flow Cytometry CANCER RESEARCH UK Prostate Oncobiology Duncan Smith 50 SEMINAR SERIES 2016 70 MANCHESTER INSTITUTE Biological Mass Spectrometry Facility POSTGRADUATE EDUCATION 72 Crispin Miller 32 RNA Biology Kim Acton and Lisa Doar 50 OPERATIONS 76 RESEARCH GROUPS John Brognard 34 Biological Resources Unit CANCER RESEARCH UK’S LOCAL 82 Santiago Zelenay 12 Cancer Inflammation and Immunity Signalling Networks in Cancer Natalia Moncaut 51 ENGAGEMENT AND DEVELOPMENT Amaya Viros 36 Transgenic Production Facility ACKNOWLEDGEMENT FOR FUNDING OF 86 Iain Hagan 14 Skin Cancer and Ageing Garry Ashton 52 THE CANCER RESEARCH UK Cell Division Histology MANCHESTER INSTITUTE Nic Jones 16 Georges Lacaud 38 Stem Cell Biology Mark Craven 54 CAREER OPPORTUNITIES AT THE CANCER 87 Cell Regulation Laboratory Services RESEARCH UK MANCHESTER INSTITUTE Angeliki Malliri 18 Valerie Kouskoff 40 Stem Cell and Haematopoiesis Wolfgang Breitwieser 54 CONTACT DETAILS 88 Cell Signalling Claus Jørgensen 42 Molecular Biology Core Facility Caroline Dive 20 Clinical and Experimental Pharmacology Systems Oncology Marek Dynowsk 55 Michela Garofalo 44 Scientific Computing Donald Ogilvie 24 Drug Discovery Transcriptional Networks in Lung Cancer Hui Sun Leong 55 Computational Biology Support Tim Somervaille 26 Leukaemia Biology The Cancer Research UK Manchester Institute is located across The Paterson Building (shown here) and The Manchester Cancer Research The Manchester Cancer Research Centre Building Centre Building 2 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE 3


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    DIRECTOR’S INTRODUCTION An organoid labelled for SiR-actin. Image supplied by Renaud Mevel (Stem Cell Biology) It has been a busy and successful year for the Institute. Highlights from 2016 include the demonstration by our Clinical and Experimental Pharmacology (CEP) group, in collaboration with AstraZeneca, of impressive pre-clinical activity of a Wee1 inhibitor in combination with olaparib in small cell lung cancer. Director of the Cancer Research This has led to a clinical trial in this disease that will begin early UK Manchester Institute this year, at The Christie NHS Foundation Trust and several Professor US sites. Richard Marais occurs in older patients. Also impressive was International PhD Student Cancer Conference Caroline Dive’s successful bid, together with in Cambridge, and Romina Girotti won the We are also pleased that, building on the oncogenic KRAS also regulates tumour cell Professor Peter Kuhn at the University of inaugural Christopher J. Marshall award for impressive work from our Leukaemia Biology signalling through the stromal cells. Also, Southern California, for funding from former US studies on signal transduction and melanoma, group, the first Phase 1 clinical trial data with my group reported the development of a Vice President Joe Biden’s Cancer Moonshot which was presented by the Society for ORY-1001 – a first in class inhibitor of the histone comprehensive precision medicine platform for initiative. The aim of this programme is to Melanoma Research at their annual meeting in demethylase LSD1 – was presented at the melanoma in Cancer Discovery that makes use develop and refine technologies to analyse tiny Boston in November. Such external recognition American Society of Hematology meeting in of liquid biopsies and circulating-free DNA to numbers or residual circulating cancer cells in is highly rewarding and in total members of the San Diego in December 2016. This revealed that monitor responses to therapy and identify the blood to identify the patients who are likely Institute received 26 prizes and awards during ORY-1001 promotes differentiation of acute mechanisms of resistance. to relapse. We are also participating in several the year. Each year, the Institute also bestows its myeloid leukemia blast cells in patients, an exciting collaborations with our local partners. own award, the Dexter Prize for Young exciting outcome that is now being taken Extending the breadth and depth of our During 2016, CRUK MI played major roles in Scientists. The Prize is named after Mike Dexter, forward in collaboration with Oryzon Genomics research portfolio is critical to achieving our Manchester’s successful bid for CRUK Major a former Director of the Paterson Institute for and Roche. We congratulate the Drug aims and to this end, it is important that we Centre status and Manchester’s successful bid Cancer Research (the former name of the Discovery Unit (DDU) on declaring its first attract additional funding to support our work. for a NIHR Biomedical Research Centre (BRC). Institute) and in 2016 it was awarded to Dan pre-clinical candidate drug, which targets the To increase our success in winning external Both of these initiatives bring significant Wiseman. Dan is a Clinical Fellow who tyrosine kinase RET. This important milestone grants, we therefore established a Grants additional support to our research and will allow completed his PhD in the Leukaemia Biology was the result of a collaboration with the Committee in 2016 to oversee the preparation us to make progress on our major objective of group in 2016. His project focused on IDH venture capital company 6th Element Capital of applications and ensure a robust system of delivering personalised medicine for cancer mutations in Acute Myeloid Leukaemia and who partially funded this work. The drug internal peer review prior to submission. I am patients in the North-West. Our Deputy resulted in a significant body of work candidate will now progress through to clinical delighted that Iain Hagan agreed to chair this Director, Caroline Dive, is working closely with encompassing both the basic biology of this trials in non-small cell lung cancer patients committee, because he brings considerable clinical colleagues and leading the cancer disease as well as more translational aspects. I whose tumours harbour RET-fusion experience, having been a member of several precision medicine theme in the BRC. am delighted that he is continuing his interest in oncogenes. Finally, during the summer, my grant funding committees and review panels. IDH in his post-doctoral studies in the own group, Molecular Oncology, underwent a This committee is already having an impact, and It is always pleasing to see the success of our Leukaemia Biology group having secured a very successful quinquennial review of our I am also delighted at the success we enjoyed in scientists recognised with prestigious prizes and highly competitive Bloodwise Clinician Scientist research programme. our grant applications in 2016. Notably awards. Caroline Dive was the recipient of the Fellowship. successes were Iain Hagan’s Investigator Award AstraZeneca British Pharmaceutical Society Highlights from our publications include from the Wellcome Trust, which will enable him Prize for Women in Pharmacology, while Amaya It was immensely rewarding to showcase Caroline Dive’s study in Nature Medicine to expand his work on the spatial and temporal Viros received the Leo Pharma Research Manchester and its success to the world’s showing how to distinguish chemosensitive control of mitotic commitment. Similarly, Foundation Award, and I was delighted to cancer research community by hosting the from chemorefractory small cell lung cancer Amaya Viros, a former post-doctoral fellow in receive the European Society for Pigment Cell European Association for Cancer Research patients using the genetic features of circulating the Molecular Oncology group, secured a Research Fritz Anders Medal. At The University (EACR) meeting in the city during the summer. It tumour cells. Another notable publication was Wellcome Trust-funded Clinical Scientist of Manchester annual awards ceremony, Tim was an honour to chair this meeting in my a study published in Cell from one of our Junior Fellowship to enable her to establish an Somerville was named one of four Postgraduate capacity as EACR President (2014-16). Moreover, Group Leaders, Claus Jørgensen. Claus’ independent group at CRUK MI. Her Skin Students of the Year and I was honoured to Caroline Dive chaired a vibrant and stimulating group reported that in pancreatic ductal Cancer and Ageing group will investigate how receive one of four Researcher of the Year National Cancer Research Institute meeting in adenocarcinoma, in addition to its conventional the ageing microenvironment of the skin Awards. Emma Williams was judged to have Liverpool in November, which included an role in directly driving tumour cell proliferation, contributes to the more aggressive disease that given the best oral presentation at the excellent plenary lecture from Iain Hagan and a 4 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE DIRECTOR’S INTRODUCTION 5


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    DIRECTOR’S INTRODUCTION (CONTINUED) RESEARCH An in vitro co-culture of pancreatic cancer cells and cancer-associated fibroblasts. HIGHLIGHTS Image supplied by Colin Hutton (Systems Oncology) In this section we highlight some research publications from 2016 which report significant advances in specific areas. The selected papers demonstrate the breadth and the quality of the research being undertaken by the groups at the Cancer Research UK Manchester Institute. Tape CJ, Ling S, Dimitriadi M, McMahon KM, isoforms regulates the DNA damage response. Worboys JD, Leong HS, Norrie IC, Miller CJ, npj Genomic Medicine, 2016; 1, Article number: Poulogiannis G, Lauffenburger DA, 16020. Jørgensen C. Oncogenic KRAS Regulates Tumor Cell Blood vessels within a tumour are disorganised, Signaling via Stromal Reciprocation. leading to oxygen starvation (hypoxia). High Cell, 2016; 165(4):910-20. levels of hypoxia are associated with poor patient outcome and resistance to therapy, Pancreatic ductal adenocarcinoma (PDAC) is a making it a crucial aspect of tumour biology. dismal disease with five-year survival levels at When a gene is expressed to make a protein, the Women in Science workshop that was Kouskoff is continuing her research at the less than 5%. This is due to late diagnosis, cell makes an RNA copy of the gene’s DNA. This organised and chaired by Caroline. Division of Musculoskeletal and Dermatological aggressive disease progression and limited is then translated into a protein sequence that Sciences at The University of Manchester. We treatment options. PDAC is characterised by folds up to make the final protein. For most Engaging with the supporters who fund our thank both them and their research teams for activating mutations in the oncogene KRAS, genes, some additional processing called research is incredibly important and the past their hard work and contributions to the Institute with co-occurring inactivation of CDKN2A, ‘alternative splicing’ is performed, in which year offered some unique opportunities to over the last few years and wish them all every TP53 and SMAD4. A hallmark of PDAC is an parts of the RNA are chopped out and the engage with the general public and to success in the future. excessive expansion of genetically normal remaining bits spliced together. This lets the communicate the progress that we are making. stromal cells, such as fibroblasts, which cell make a set of different, but related, proteins In particular, 2016 marked Manchester’s tenure Finally, looking forward, during 2017 we shall generate a pro-tumorigenic microenvironment from the same gene. as European City of Science. Our staff and continue with further Group Leader recruitment that decreases the sensitivity of tumour cells to students took part in a “science lates” evening at in our areas of priority while continuing to treatments. However, the mechanisms By using RNA-sequencing to monitor changes Manchester Museum and joined in with a develop our other activities. This includes whereby tumour cells recruit and co-opt to the RNA within cell lines starved of oxygen, soapbox science event at Piccadilly Gardens in continuing to work with our partners at ‘normal’ cells, and how these cells in turn the group identified specific genes that changed the centre of Manchester. We held two open University College London and in Belfast to influence tumour cells, are poorly understood. their splicing patterns to make RNA molecules days where we invited our supporters into the develop our Lung and Prostate Centres of Interrogating how tumour cells communicate that could not be translated into a valid protein. Institute as well as hosting a regular series of Excellence respectively. Similarly, we shall with resident fibroblasts, we observed that They then saw the same pattern occurring in laboratory tours. During these events, we continue to collaborate with colleagues in the tumour cells co-opt neighbouring fibroblasts patient samples from The Cancer Genome Atlas explained to the general public how we use CRUK Manchester Centre and the MCRC over to produce a reciprocal signal, ultimately (TCGA), and showed that it affected a significant animals in our research, because although most plans to develop the MCRC Phase 2 Building altering tumour cell function. Specifically, the number of genes associated with repairing DNA of our work does not involve animals, some which will culminate in a new research facility tumour cell response to expression of damage. This is exciting because it reveals a new animal research is essential if we are to that will house the Manchester Centre for oncogenic KRAS was greatly influenced by the way that cells can switch on and off specific understand, prevent and cure cancer. As part of Cancer Biomarker Sciences. This initiative is led presence of fibroblasts. As such, we observed genes important in cancer. our drive to be open about how, when and why by Caroline Dive and will support a range of that specific kinases, for example AKT, were we use animals, we put on displays at various translational and clinical studies to accelerate only activated in tumour cells when these were Draper JE, Sroczynska P, Tsoulaki O, Leong HS, events including one at the Manchester Science biomarker research into patient benefit. During allowed to communicate with fibroblasts. Fadlullah MZ, Miller C, Kouskoff V, Lacaud G. Festival, and I am delighted that the efforts of the the summer, the Institute will undergo a Importantly, tumour cells were not sensitive to RUNX1B expression is highly heterogeneous team that took part were acknowledged by a Quinquennial Review by an international panel inhibitors of AKT when cultivated alone, but and distinguishes megakaryocytic and erythroid national award in public engagement from the of experts to assess our achievements over the only when co-cultured with fibroblasts. This lineage fate in adult mouse hematopoiesis. organisation Understanding Animal Research. last five years and consider our strategy for the suggests that future efforts to evaluate drug PLoS Genetics, 2016; 12(1):e1005814. years ahead. We look forward to sharing our targets should include stromal components. During the summer, we said goodbye to two of success with the panel and receiving their input The transcription factor RUNX1 is a master our Group Leaders. John Brognard returned to to help us progress our scientific agenda over Memon D, Dawson K, Smowton CSF, regulator of blood cell production. It controls the US to establish a group at the National the next five years. Xing W, Dive C, Miller CJ. the accurate production of the various types of Cancer Institute in Maryland while Valerie Hypoxia-driven splicing into noncoding blood cells throughout adult life. Mutations in 6 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH HIGHLIGHTS 7


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    RESEARCH HIGHLIGHTS (CONTINUED) Antonello J, Morrow CJ, Hodgkinson CL, a microarray from SCLC patient tissues we Morris K, Priest L, Carter M, Miller C, Hughes A, showed that high VM levels correlated with Blackhall F, Dive C, Brady G. worse prognosis. Importantly, using copy RUNX1 cause defects in diverse blood lineages levels of mutant DNA predicted clinical Molecular analysis of circulating tumor cells number alterations, we used our CTC-derived in human patients, including different types of responses and allowed detection of relapse due identifies distinct copy-number profiles in patient explant models to confirm that VM leukaemia as well as blood clotting defects to common resistance mechanisms weeks patients with chemosensitive and vessels were tumour derived and that VE-Cad due to a shortage of platelet-producing ahead of clinical imaging. Patient-derived chemorefractory small-cell lung cancer. expressing CTCs were of SCLC origin. By megakaryocytes. RUNX1 is expressed from two xenografts (PDXs) were used to test hypothesis- Nature Medicine, 2016 Epub 21 November knocking down VE-Cad in a VM proficient SCLC promoters, which produce several distinct RNA driven, personalised therapies and to validate cell line, we showed in the xenograft model that transcripts and protein isoforms. To investigate combination therapies. As an alternative, in Small cell lung cancer (SCLC), the most VE-Cad was required for VM which increased the timing and localisation of the expression of cases where tumours were not accessible, we aggressive form of lung cancer, kills around initial tumour growth dynamics. Despite these two promoters (termed distal and generated circulating tumour cell derived 220, 000 patients per year globally, with no increased intratumoural cisplatin in VM proximal), the Stem Cell Biology group created xenografts (CDXs) from blood biopsies. CDXs significant change in survival or treatment proficient versus deficient tumours, VM a mouse model with reporter genes expressed recapitulated the genetic and histologic features options beyond platinum-based chemotherapy proficient tumours were less cisplatin sensitive. under the control of the Runx1 promoters. In a of patient tumours and could predict response in 30 years. Most patients respond to The functional significance of VM in SCLC previous study, they determined the activities to therapy. Our findings demonstrate the power chemotherapy well initially, but relapse rapidly suggests that understanding its regulation may and requirements for the Runx1 promoters at of a personalised medicine approach in which with acquired drug resistance. Approximately present opportunity for therapeutic the initiation of blood production in the alterations identified by NGS can be used to 20% of patients present with chemorefractory interventions. The impact of VM on developing embryo. In this study, the group monitor treatment responses, reveal resistance disease progressing within 90 days of treatment. metastases is now under investigation. investigated the output from the two promoters mechanisms, and may represent actionable We generated the first DNA-based biomarker in adult organs, including bone marrow, spleen targets that can be tested using xenograft for SCLC in a 31 patient study which was based James DI, Smith KM, Jordan AM, Fairweather and thymus. They demonstrated that the distal models. on copy number alterations (CNA) measured EE, Griffiths LA, Hamilton NS, Hitchin JR, Runx1 promoter is highly expressed but the pre-treatment in single circulating tumour cells Hutton CP, Jones S, Kelly P, McGonagle AE, proximal promoter activity is more restricted Marei H, Carpy A, Woroniuk A, Vennin C, (CTCs). This CTC CNA classifier anticipated Small H, Stowell AI, Tucker J, Waddell ID, and in particular marks the point in adult blood White G, Timpson P, Macek B, Malliri A. whether patients would be chemosensitive or Waszkowycz B, Ogilvie DJ. production where the red blood cell and Differential Rac1 signalling by guanine chemorefractory. When examined in CTCs First-in-Class Chemical Probes against megakaryocyte pathways separate. The nucleotide exchange factors implicates FLII in from initially chemosensitive patients who then Poly(ADP-ribose) Glycohydrolase (PARG) different proteins produced by these two Runx1 regulating Rac1-driven cell migration. progressed, the classifier did not correlate with Inhibit DNA Repair with Differential promoters may therefore have different roles in Nature Communications, 2016; 7:10664. that of chemorefractory patients’ CTCs at Pharmacology to Olaparib. driving the production of these two cell types. baseline, implying distinct mechanisms of ACS Chemical Biology, 2016; 11(11):3179-3190. They also determined that expression of the The protein Rac1 has been implicated in the inherent and acquired chemotherapy leukaemic fusion protein AML1-ETO9a resulted formation of many tumour types and the resistance. Larger studies are now required to DNA repair pathways are often compromised in in preferential proximal upregulation suggesting dissemination of metastatic cells. Upon qualify this classifier. Currently patients receive cancer cells and this presents a double-edged that its expression may be important to activation by guanine nucleotide exchange chemotherapy upon a SCLC diagnosis. Until sword for tumours. Whilst the compromised establish a pre-leukaemic environment. factors (GEFs), Rac1 associates with a variety of improved targeted treatments are forthcoming, repair mechanisms allow cells to rapidly gain proteins in the cell thereby regulating various it is very unlikely that the classifier will be used to mutations conferring benefits such as drug Girotti MR, Gremel G, Lee R, Galvani E, functions, including cell migration and invasion. alter treatment. However, the classifier could be resistance, these cells become highly Rothwell D, Viros A, Mandal AK, Lim KH, However, activation of Rac1 is also required for helpful in designing early clinical trials of dependent on the remaining pathways for Saturno G, Furney SJ, Baenke F, Pedersen M, epithelial cells to adhere strongly one to the emerging therapies where anticipation of the survival. Blockade of these remaining processes Rogan J, Swan J, Smith M, Fusi A, Oudit D, other, raising the possibility of exacerbating duration of first line chemotherapy response with a small molecule can lead to therapeutic Dhomen N, Brady G, Lorigan P, Dive C, tumour progression when targeting Rac1. could be beneficial. benefit. This approach is exemplified by Marais R. Avoiding this complication calls for the inhibitors of the DNA repair protein poly(ADP- Application of sequencing, liquid biopsies, identification of factors that control the Williamson SC, Metcalf RL, Trapani F, Mohan S, ribose) polymerase (PARP). However, many and patient-derived xenografts for selection of Rac1-driven cellular responses. Antonello J, Abbott B, Leong HS, Chester CPE, components of these pathways are poorly personalized medicine in melanoma. Interestingly, there are at least 20 GEFs involved Simms N, Polanski R, Nonaka D, Priest L, Fusi A, understood, partly because of a lack of Cancer Discovery, 2016; 6(3):286-99. in Rac1 activation, suggesting a more complex Carlsson F, Carlsson A, Hendrix MJC, Seftor chemical tools to probe their role in disease. In role of GEFs in regulating Rac1 signalling besides REB, Seftor EA, Rothwell DG, Hughes A, this study, the Drug Discovery Unit (DDU) reveal Melanoma is the most aggressive form of skin promoting guanine nucleotide exchange. In this Hicks J, Miller C, Kuhn P, Brady G, Simpson KL, a unique compound which specifically targets cancer. Recent breakthroughs in the study, we highlighted the role of two Rac- Blackhall FH, Dive C. poly(ADP-ribose) glycohydrolase (PARG), a vital development of immune checkpoint inhibitors specific GEFs, Tiam1 and P-Rex1, in dictating Vasculogenic mimicry in small cell lung cancer. component in the repair of DNA single strand and targeted therapies have transformed the contrasting biological outcomes downstream Nature Communications, 2016; 7:13322. breaks. PARG was previously considered by management of melanoma and extended of Rac1: Tiam1 inhibits, whereas P-Rex1 some to be undruggable, given the nature of the survival. Nonetheless, most patients still promotes migration. Importantly, proteomic The majority of small cell lung cancer patients active site but this study (arising from a succumb to metastatic disease. Biomarkers to analysis uncovered a role for both GEFs in present with widespread metastases and collaboration with AstraZeneca) revealed a determine which patients will benefit from a modulating the Rac1 interactome, which results consistently circulating tumour cells (CTCs) are novel start point that induced its own unique specific treatment, to detect disease in the stimulation of GEF-specific signalling relatively prevalent in this tumour type. We binding site. The DDU used computational, progression early and to select effective cascades. In particular, we demonstrate that discovered a rare CTC subset which co- medicinal chemistry and assay development second-line therapies are needed. We have P-Rex1 stimulates migration through enhancing expressed the endothelial marker vascular- expertise to discover the first cell-permeable, developed a personalised medicine platform the interaction between Rac1 and the actin- endothelial cadherin (VE-Cad) with epithelial selective inhibitors of PARG. The team utilising tumour tissue and liquid biopsies in remodelling protein flightless-1 homologue, to cytokeratins, a phenotype consistent with anticipate that this compound will help unravel combination with next generation sequencing modulate cell contraction. vasculogenic mimicry (VM). VM is a hypoxia the role of PARG in DNA repair and other (NGS) and xenograft models. Analysis of driven process whereby tumour cells form biological processes, eventually leading to the circulating tumour DNA (ctDNA) revealed that Carter L, Rothwell DG, Mesquita B, Smowton C, ‘endothelial-like’ vessels. We hypothesise that discovery of novel therapeutics. Leong HS, Fernandez-Gutierrez F, Li Y, Burt DJ, VM may support tumour dissemination. Using 8 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH HIGHLIGHTS 9


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    CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH GROUPS 10 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE 11


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    www.cruk.manchester.ac.uk/Research/CRUK-MI-Groups/Cancer-Inflammation-and-Immunity/Home Building on from a recent study where we use of in vitro assays with the analysis of uncovered a fundamental role for genetically engineered cancer mouse models prostaglandin E2 (PGE2) in cancer immune that very closely reflect the genetics and CANCER INFLAMMATION evasion (Zelenay et al. Cell 2015), our group investigates the basis for how different types of pathology of human cancer. Furthermore, our current analyses of publically available cancer AND IMMUNITY inflammation are established or manipulated by a growing tumour and to what extent manipulation of the local inflammatory patient datasets underscored a notable conservation of the COX-driven mouse inflammatory signature and extended our response constitutes an opportunity for original early findings in human cutaneous therapeutic intervention. In this proof of primary melanoma to advanced metastatic principle study we demonstrated that melanoma and to other cancer types, The clinical success of therapies that harness the ability of melanoma, colorectal or breast cancer cells suggesting that COX-activity might be a driver the immune system to recognise and eliminate cancer cells is rendered unable to produce PGE2 by genetic of immune suppression across different means are greatly impaired in their ability to malignancies. currently extending to numerous malignancies, including cancer form tumours and frequently completely types previously thought to be refractory to immunotherapy. regressed. Cancer cells, impaired in their Manipulating inflammation to raise ability to produce PGE2, failed to grow in cancer immunogenicity However, complete and durable tumour regressions are observed An additional key finding from this study on immunocompetent animals while grew only in a reduced minority of patients highlighting the need for progressively, as their parental counterparts, which most of our initial research questions are Group Leader mechanistic studies to uncover the basis for absent or partial in immunodeficient hosts highlighting an based was that reduction of PGE2 levels using essential role for PGE2 in immune escape. COX inhibitors synergised with immune Santiago Zelenay responses. The Cancer Inflammation and Immunity group Moreover, tumours formed by cancer cells checkpoint blockade in promoting immune- Scientific Officer investigates the underlying mechanisms that allow cancer cells to deficient in cyclooxygenase (COX) 1 and 2, the dependent tumour growth control in pre- Chikkanna-Gowda CP evade natural and therapy-induced tumour immunity, combining rate limiting enzymes for PGE2 production, clinical models. Indeed, combining the COX- showed a drastic shift in the local inflammatory inhibitor aspirin, one of the most widely used Postdoctoral Fellows the use of genetically engineered pre-clinical cancer models with drugs, with anti-PD-1 blockade, promoted signature characterised by lower expression of Eduardo Bonavita1 Victoria Pelly1 the analysis of samples from cancer patients. Our ultimate goal cancer-promoting factors and a concomitant much more rapid adaptive immune-mediated is to develop novel targeted interventions to disrupt immune increase in mediators normally associated with tumour eradication than either treatment Graduate Student T cell-dependent tumour control (Figure 1). alone. Thus, in a project of great translational Eimear Flanagan1 suppression, promote tumour immunity and enhance the and clinical significance, we are further Thus, our findings suggest that manipulation of 1 efficacy of cancer therapy. the type of inflammation and subversion of characterising the role of COX-inhibitors in joined in 2016 immune surveillance by a growing tumour is potentiating anti-tumour immune responses The remarkable success of immunotherapy, therapy are still poorly defined. This knowledge essential for its ability for progressive growth. following conventional and immune-based mainly from strategies based on immune is essential to the design of novel interventions to Figure 1 Our current working model postulates that cancer therapies. These activities included the checkpoint blockade, has fully reinvigorated enhance the efficacy of cancer therapy. In this Cyclooxygenase (COX)-driven tumour-derived PGE2 allows cancer immune design of a clinical trial to test these production of the inflammatory interest in its application to the cancer context, our group actively investigates the escape by shifting the intra-tumoural immune combinations in cancer patients in lipid PGE2 by cancer cells fuels immunology field. These therapies have shown signals that drive innate and adaptive immunity tumour-promoting inflammation environment from one favourable to collaboration with oncologists from The great promise for the treatment of advanced against malignant cells aiming to distinguish and allows progressive tumour anti-tumour immunity to one that fuels tumour Christie NHS Foundation Trust and a research cancers and also, more recently, as an adjuvant factors that accelerate cancer progression from growth. Cancer cell-specific growth (Figure 1; Zelenay & Reis e Sousa agreement with a pharmaceutical company to therapy following tumour resection. Arguably those that mediate tumour elimination. COX-deficiency alters the OncoImmunology 2016). evaluate the efficacy of improved newly the most important open question in the field is inflammatory profile at the developed COX-2 inhibitors as potential tumour site increasing why only some patients benefit from treatment Tumour-protective versus We are currently further investigating the additions to immunotherapies based on anti-tumour mediators and and particularly why full and long-lasting clinical tumour-promoting inflammation underlying basis for this dominant and essential immune checkpoint blockade. Similarly, we are enabling immune-dependent outcomes are observed only in a much reduced Inflammation is a common feature of most, if tumour control and eradication. role of PGE2 in immune evasion, combining the investigating whether combination with COX subset of patients. Intense recent research on not all, clinically apparent tumours. This type of inhibitors may be useful to a broader range of this topic points to the presence of pre-existing inflammatory response, however, commonly cancer therapies, where their efficacy rely, at tumour-infiltrating T cells, particularly CD8+ T associated with the presence of myeloid cells least in part, on the stimulation of anti-tumour cells, as one of the best predictive markers for such as macrophages or specific subsets of immunity, such as chemotherapy and clinical benefit following immune checkpoint regulatory T cells, typically fuels tumour growth, PGE2-­‐driven  cancer-­‐promo7ng   radiotherapy. blockade. This is consistent with the notion that aggressiveness and can drive resistance to inflamma7on   these drugs work, at least in part by blocking cancer treatment, including therapies aimed at Finally, using an approach that combines once inhibitor receptors upregulated in activated boosting the anti-tumour immune response. IL-­‐6,  CXCL1,  G-­‐CSF   again in vitro assays with the analysis of in vivo endogenous T cells. Thus, rather than tumours being inflamed or not COX-­‐expressing   inflamed, they exhibit quantitative and cancer  cells   cancer models and human samples and These observations have prompted the qualitatively different cellular and molecular Poorly  immunogenic   datasets, we embarked on a project specifically classification of tumours into ‘hot’ T cell- inflammatory profiles. Tumour-infiltrating progressive  tumour   designed to identify other potential inflamed tumours, and ‘cold’ non-inflamed inflammatory cells, such as macrophages, T immunomodulatory and immunosuppressive X   tumours lacking T cell infiltration, and stimulated cells, neutrophils, dendritic cells and other white factors that, similarly to PGE2, act in a dominant PGE2   the study of the principles and rules that account cells can be found in various numbers and Chemoprotec7ve   manner. These factors, unlike recessive for the establishment and maintenance of these displaying a wide spectrum of differentiation inflamma7on   mechanisms of immune escape, constitute two tumour phenotypes. Indeed, the signals, status. All these cells can have a dual role CD103+   ideal therapeutic targets to unleash anti-cancer factors and pathways controlling the priming of, impacting favourably or negatively on cancer DCs   IL-­‐12,  type  I  IFN,  IFNγ,  CXCL10   immunity and to enhance the efficacy of recruitment to and function of, T cells within progression depending on their number, relative Immune-­‐mediated   standard and immune-based therapy. COX-­‐deficient   tumours in natural conditions or following composition and activation phenotype. tumour  rejec7on   cancer  cells   Publications listed on page 58 12 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE CANCER INFLAMMATION AND IMMUNITY 13


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    www.cruk.manchester.ac.uk/Research/CRUK-MI-Groups/Cell-Division/Home further load on the SAC by anti-mitotics is Figure 1: The Spindle pole body Polo sufficient to trigger apoptosis in the tumour cells, acts as a signalling hub to trigger while simply delaying exit in non-transformed CELL DIVISION Cdk1-Cyclin B activation Cdk1-Cyclin B activity is held in check in interphase as a P Cdk1 P Cdc25 Cdk1 cells that have a normal karyotype. However, anti-mitotics only work in certain tumour settings, as cells can “slip” out of mitosis from the consequence of phosphorylation of Cdk1 by Wee1. Cdc25 removes Cyclin B Cyclin B mitotic arrest without completing either cell the inhibitory phosphate to trigger Wee1 division or triggering death. Slippage occurs mitosis. This trigger level of when Cdk1-Cyclin B activity falls below a Cdk1-Cyclin B then activates polo threshold level that is required to maintain the kinase to further boost Cdc25 Polo mitotic state. There is therefore great interest in activity and inhibit Wee1 to make Cut12 finding therapeutic routes to block slippage and The inappropriate proliferation of cancer cells can arise from this transition a bi-stable switch so reinforce the impact of anti-mitotics to trap between two distinct fates. The unchecked cell division, a failure to engage cell death pathways, affinity of PP1 for Cut12 sets the PP1 slippage-prone cells in mitosis. A simple threshold for the activation of Polo approach would be to block the phosphatase or simultaneous changes in both. Understanding how the diverse kinase. When PP1 is tightly bound activities that counteract Cdk1-Cyclin B activity cues are integrated to co-ordinate cell division and death therefore to Cut12, Polo activity cannot rise to set the Cdk1-Cyclin B slippage threshold. to threshold levels that trigger the sits at the heart of our need to understand the biology of cancer. feedback controls that drive PP1 and protein phosphatases 2A (PP2A) Furthermore, DNA damaging and anti-mitotic therapies owe division. If PP1 is absent, Polo account for over 90% of the serine/threonine activity rises inappropriately to Group Leader much of their success to the checkpoint pathways that ensure inhibit Wee1 and remove the need directed phosphatase activity in human cells. upon anchorage to the SPB. This network is Iain Hagan that transition through the cell division cycle only occurs when for Cdc25. anchored by a second scaffolding molecule; PP1 is recruited to docking sites from where it dephosphorylates targets. Hetero-trimeric PP2A Associate Scientist genome integrity is guaranteed. Figure 2: The mitotic PP1-PP2A Sid4. We recently found that signalling from Sid4 enzymes comprise single scaffolding and phosphatase relay can compensate for mitotic commitment Agnes Grallert catalytic subunits, alongside one of four different PP1 and PP2A activities are all deficiencies when Cut12 function is repressed upon entry into mitosis. types of regulatory subunit. Multiple, alternatively Postdoctoral Fellow We therefore study the targets of two of these propagating throughout the cell has been compromised. Thus, dialogue between the The mode of PP2A repression is spliced, genes give the potential to generate Lenka Halova therapeutically important checkpoint pathways: consolidated by other data to suggest that mitotic exit and mitotic commitment scaffolds unclear, however, it is well hundreds of variants of each type of PP2A the commitment to, and the exit from, the the centrosome provides a specific determines when division will be initiated. This established that Cdk1-Cyclin B complex in humans, whereas fission yeast can Scientific Officers physical process of genome segregation, microenvironment for the activation of phosphorylation represses PP1 dialogue provides a plausible rationale for the Ben Hodgson live on one of each, or, in the case of PP2A-B55, mitosis. Because the regulatory networks that Cdk1-Cyclin B to trigger the G2/M transition. activity. Cyclin B destruction then use of the centrosome as a signalling hub: Kuan Yoow Chan none. PP2A-B55, PP2A-B56 and PP1 have each control cell division are highly conserved, we Our studies of the fission yeast centrosome allows PP1 to auto-catalytically convergence of signals from multiple pathways remove this inhibitory phosphate been proposed to drive mitotic exit. PP1, study the simple, unicellular, fission yeast to equivalent, the spindle pole body (SPB), provide to a limited number of neighbouring scaffolds, from itself. As PP1 is bound to the PP2A-B55 and PP2A-B56 activities decline identify the key questions to ask of the molecular insight into how this switch may clustered together on the SPB, can integrate the B55 regulatory subunit of upon mitotic commitment. analogous controls in the much more complex operate. We have been able to exploit the inputs from the different pathways to generate a PP2A-B55 at this time, PP1 context of human cell division. malleability of yeast to show that release of reactivation immediately restores coherent signal that sets the flux through We found that direct recruitment of PP1 to Cdk1-Cyclin B or Polo kinase activity at the SPB, PP2A-B55 activity. In contrast, outgoing signalling cascades. We are pursuing PP2A-B55 and PP2A-B56 re-activates these In a typical cell division cycle the G1 gap phase but at no other location in the cell, triggers the PP2A-B56 is unable to recruit PP1 the molecular basis for this dialogue and the PP2A phosphatases to support appropriate precedes DNA replication in Synthesis (S phase), G2/M transition. This mechanistic insight because Polo kinase function of the equivalent kinases and similar phosphorylates a residue within mitotic progression/exit in fission yeast. before a second gap phase, G2, separates S from suggests that the correlative observations of centrosomal scaffolds in human cells. the PP1 docking site on the Mitotic inhibition of PP1 arises from direct genome segregation in Mitosis (M phase). Cdk1-Cyclin B activation in metazoa reflect a regulatory B56 subunit. Once Polo phosphorylation by Cdk1-Cyclin B. The Growth, developmental and environmental cues true functional relationship between the Anti-mitotic drugs, such as the microtubule activity declines at the end of destruction of Cyclin B subsequently allows regulate the G1/S and G2/M transitions to control centrosome and the activation of Cdk1-Cyclin B mitosis, PP2A-B55 can overcome stabilising derivatives of Taxol, arrest mitotic PP1 to auto-dephosphorylate and restore its the rate of proliferation. Passage through these to initiate mitosis. Furthermore, our studies of the Polo activity towards this site and progression because the lack of chromosomal own phosphatase activity. Reactivated PP1 then key transitions is driven by the activation of SPB scaffold Cut12 provide insight into just how remove the inhibitory phosphate alignment on the mitotic spindle stimulates the reactivates PP2A-B55. Polo phosphorylation distinct CDK-Cyclin complexes. The G2/M great an impact centrosomal control can have. from the PP1 docking site of B56. spindle assembly checkpoint (SAC). SAC Consequently PP1 can be recruited of the PP1 docking site of PP2A-B56 initially transition is a critical safeguard of genome Simply blocking the recruitment of protein activation blocks the destruction of Cyclin B that to PP2A-B56 and this second PP2A blocks PP1 binding to PP2A-B56. When Polo integrity; incomplete DNA replication or DNA phosphatase 1 (PP1) to the SPB enables us to is required for mitotic exit. Prolonged SAC activity is reactivated at the end of activity declines in mitotic exit, PP2A-B55 damage triggers checkpoint pathways that block delete the cdc25+ gene without impacting upon dependent arrest triggers apoptotic cell death. mitosis. Reprinted by permission dephosphorylates the Polo phosphorylation the G2/M transition to ensure that chromosomes viability. The basis for this bypass of the from Macmillan Publishers Ltd: As the genomic instability of tumour cells places site on B56 to allow PP1 to reactivate PP2A-B56 are not segregated when incomplete or requirement for this essential mitotic inducer Nature 517:94-98, copyright 2015. a disproportionate demand upon SAC function, (Figure 2). Dr Anja Hagting in Professor Jonathan damaged. The G2/M transition is driven by appears to lie in the influence that the Cut12/PP1 Pines’ lab (Institute of Cancer Research, London) activation of the Cdk1-Cyclin B protein kinase. axis holds over Polo kinase activity. Polo activity found a relationship between PP1 and PP2A-B56 Wee1-related kinases phosphorylate the shows a direct, inverse, correlation with PP1 PP2A-B56 PP2A-B55 that mirrors our findings in yeast. We are now catalytic subunit, Cdk1, to inhibit the complex recruitment to the SPB indicating that the SPB extending these studies in human cells and during interphase. This phosphate is removed by appears to act as a catalyst to release Polo using yeast to identify the PP1 target in the Cdc25 phosphatases to promote mitotic entry. activity throughout the cell (Figure 1). PP1 PP2A complexes and will characterise the Cdk1-Cyclin B activation promotes a positive Inappropriate elevation of Polo activity probably fundamental properties of PP2A phosphatases. feedback loop that boosts Cdc25 and inhibits removes the requirement for Cdc25 because it Our findings will impact upon cancer research Wee1 activities to ensure that mitotic inappropriately represses Wee1 activity, as it beyond cell cycle control as PP2A components commitment is a rapid and irreversible bi-stable would ordinarily do during a normal mitotic switch (Figure 1). In humans and fission yeast, commitment. Polo and regulators are frequently mutated in cancer. this feedback control exploits polo kinase to Cdk1-Cyclin B kinase determine the timing of mitotic entry. The importance of signalling from the SPB is Publications listed on page 58 re-enforced by the counterintuitive observation Interphase Mitosis The observation that active Cdk1-Cyclin B that the signalling network that controls the appears on human centrosomes before timing and execution of cytokinesis also relies Maximal PP1 Association with PP2A holoenzyme 14 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE CELL DIVISION 15


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    www.cruk.manchester.ac.uk/Research/CRUK-MI-Groups/Cell-Regulation/Home A A) A) A) CELL REGULATION MAPK - High MAPK - High MAPK - High MAPK - Low MAPK - Low MAPK - Low B) B) C) C) B) C) B C The stress-activated kinase pathways play an essential role in the cellular response to many different extracellular and intracellular signals and as a result regulate many key biological processes including cell proliferation and cell death. Cancer cells are exposed to various stress conditions such as oncogenic stress and oxidative stress and it is not surprising therefore that these pathways have been Group Leader implicated in cancer development and progression. Their Nic Jones involvement appears to be complex and can either promote or Scientific Officer inhibit tumour growth in a context-dependent manner. Our goal is Steve Lyons to better understand the role and molecular function of these utilises expression information from 18 ATF2 the outgrowth of highly proliferative tumours Graduate Student pathways in particular human cancer types. Figure 1: The status of stress- targets, is significantly associated with risk of that possess increased metastatic potential. Alekh Thapa response pathways in primary disease recurrence in independent patient prostate cancers. cohorts – supporting the notion that progressive To elucidate the function of ATF2 in prostate A) Analysis of reverse-phase disease is associated with impaired expression of cells we used CRISPR-based gene editing to We have predominantly focused on the JNK determine whether a tumour remains indolent protein array data from TCGA was stress-responsive genes. ablate ATF2 expression in the cell line PNT2. stress-activated kinase pathway and one of its or progresses are only poorly understood, and used to quantify phosphorylation of the three major MAP kinases When cultured in low serum concentrations, downstream targets, namely the transcription this impedes the development of better tools for (JNK, ERK and p38). Hierarchical Since ATF2 drives transcription in response to ATF2 KO cells continue to incorporate BrdU factor ATF2. The role of JNK in tumourigenesis the management and treatment of prostate clustering reveals two groups of its phosphorylation by MAPKs, we asked more readily than their WT counterparts, has been studied in a variety of mouse tumour cancer patients. tumours possessing either high whether the expression of ATF2 targets was revealing a function for ATF2 in the regulation of models and in most cases shown to be tumour- or low MAPK activation. B & C) correlated with the activation status of these proliferation. Furthermore, we find that the suppressive. We have shown that JNK We have analysed the expression of ATF2 target Gene set enrichment analysis kinases in prostate tumours. Focusing on JNK, expression of ATF2 targets is negatively suppresses Ras-dependent tumour formation in genes in prostate tumours at different stages of reveals distinct patterns of gene p38 and ERK, we performed hierarchical correlated with markers of cellular proliferation an orthotopic mouse model of liver cancer and development using several publically available expression associated with MAPK activation status; whereas clustering on RPPA (Reverse-phase protein array) in several datasets. Thus, tumours displaying that this effect is mediated through its regulation datasets. The results indicate that many ATF2 expression of ATF2 targets data available in the TCGA PRAD dataset. This high levels of MAPK activation (and ATF2 target of ATF2 activity. Tumour suppression by JNK is targets are strikingly under-expressed in correlates with elevated MAPK approach reveals that the activation status of all expression), are characterised by low expression lost in the absence of ATF2 or by mutation of the metastatic versus primary tumours. By this activation in prostate tumours (B), three kinases is strikingly similar, and that primary of cell-cycle genes. Taken together, these data key residues in ATF2 that are phosphorylated by criterion, impairment of ATF2 function occurs in genes associated with cellular tumours are readily organised into two classes suggest a role for ATF2 in the negative regulation JNK. We identified a novel ATF2-driven all metastatic prostate tumours and is not proliferation are highly expressed having either high or low activation of MAPKs. of proliferation in prostate tumours. transcriptional programme activated in response restricted to tumours with specific genetic in tumours with relatively low Furthermore, gene set enrichment analysis of to stress stimuli in a JNK-dependent manner and lesions. Furthermore, we find that in primary MAPK phosphorylation (C). these two clusters reveals that expression of Characterisation of tumour suppression by showed a striking down-regulation of this tumours, reduced expression of ATF2 targets is ATF2 targets is significantly associated with MEKK1-MKK4 signalling programme in several human tumour types related to Gleason score. Analysis of two MAPK activation. Stratifying the RPPA dataset by Characterisation of various mouse models (including breast, lung and ovarian) compared to independent patient cohorts reveals a clear Gleason score reveals a clear trend towards provides compelling evidence for a tumour normal tisues. These studies strongly indicate trend, with low grade tumours having the reduced MAPK activation in high grade tumours. suppressive role of JNK signalling in human that ATF2 and its upstream regulator JNK play an highest, and high grade tumours the lowest, Therefore our data indicate that high risk lesions cancer. Whilst JNK is not recurrently mutated in important tumour supressive role in human expression of ATF2 targets. This suggests that are characterised by a loss of MAPK signalling tumours it is clear that genes encoding several cancer development. Furthermore, these ATF2-dependent gene expression is lost initially which results in impaired expression of ATF2 upstream kinases of the pathway do harbour observations are supported by the presence of during development of the primary tumour, and targets. missense mutations at a reasonably high inactivating mutations in several kinases lying that impaired ATF2 function is associated with frequency. Of particular interest are the MAP3 upstream of JNK in a number of human cancers. increased risk of metastasis. 3-4 % of prostate tumours harbour homozygous kinase, MEKK1 and the MAP2 kinase, MKK4. In loss of the chromosomal locus 2q31, with the breast and ovarian tumours, these genes are The role of stress-dependent transcription Given that following radical prostatectomy, peak of deletion occurring in the vicinity of the subject to inactivation by either deletion or in prostate cancer disease recurrence is caused by the presence of ATF2 gene. Although the number of tumours missense mutation, and intriguingly, the Prostate cancer is often a stable disease with small metastatic lesions, we hypothesised that lacking ATF2 is relatively low, our analysis presence of an inactivating mutation in one of modest symptoms that can be managed the expression of ATF2 targets should correlate indicates that they possess an aggressive these kinases is almost always exclusive of a conservatively. In fact, the great majority of men with clinical outcome. Accordingly, we find that phenotype. The presence of these deletions mutation in the other suggesting that they are diagnosed with relatively benign, indolent low expression of several ATF2 target genes correlates with a high risk of disease recurrence function together to suppress tumour formation. tumours that remain localised to the prostate strongly correlates with a high risk of recurrence. following radical prostatectomy, and these To understand their function in suppression we gland. On the other hand, a minority of patients Based on these findings, we used a tumours are characterised by high expression of have established and are characterising will develop aggressive tumours that have the bioinformatics approach to derive a prognostic markers of cellular proliferation. These appropriate mouse models in which floxed potential to metastasize, and endanger life. gene signature based solely on ATF2 target observations suggest that genetic loss of ATF2 alleles of either MEKK1 or MKK4 are deleted in a However, the molecular mechanisms that genes. The resulting prognostic index, which may alter the course of the disease, promoting tissue-specific manner. 16 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE CELL REGULATION 17


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    www.cruk.manchester.ac.uk/Research/CRUK-MI-Groups/Cell-Signalling/Home samples, including Dukes stages A-C, from a A well-characterised patient cohort using a TIAM1 A antibody. Intriguingly, we detected TIAM1 not CELL SIGNALLING only in the cytoplasm, but also in cell nuclei and showed that nuclear and cytoplasmic TIAM1 staining intensity decreased with advancing Dukes stage. Thus, TIAM1 expression is negatively associated with colon cancer progression, consistent with our previous finding that TIAM1 antagonised progression of intestinal tumours in ApcMIN/+ mice. Further analysis of these data also revealed that patients with high Recurrent mutations and overexpression in tumours and cell lines nuclear TIAM1 had significantly better survival implicate the small GTPase RAC and its activators, the guanine B B than those with low nuclear TIAM1. Subsequent nucleotide exchange factors (GEFs), in the development and experiments into the mechanism by which TIAM1 antagonises CRC progression showed spreading of a range of human cancers. Furthermore, the effects of that TIAM1 localises in the nucleus of CRC cells deleting genes encoding RAC proteins or RAC GEFs in mouse or of via a functional nuclear localisation signal. These experiments also identified TIAM1 as a critical inhibiting RAC-GEF/RAC interactions with drugs strongly suggest antagonist of CRC migration and invasion Group Leader that targeting RAC signalling could constitute a cancer treatment. through inhibition of TAZ and YAP transcriptional Angeliki Malliri However, to be effective, only RAC-dependent effects that promote co-activators. Thus, our findings have identified a critical role for TIAM1 in colon cancer Postdoctoral Fellows tumour growth and progression should be targeted, while those progression through regulating TAZ/YAP activity Zoi Diamantopoulou functions that antagonise tumour progression or are essential in and showed that high levels of nuclear TIAM1 Matteo Menotti1 could serve as a good prognostic factor for Aishwarya Payapilly healthy cells should be spared. CRC patients. Andrew Porter phosphorylation of TIAM1 by SRC suppressed Scientific Officers RAC, a member of the family of RHO-like TIAM1 antagonises malignant progression Figure 1: (A) RAC GEFs regulate AJ disassembly (Woodcock et al., Mol Cell 2009). GEFs determine differential RAC signalling Gavin White GTPases, cycles between a GDP- and a via regulating cell‒cell adhesion RAC signalling via serving as RAC In another study from our laboratory (Vaughan Activation of RAC can lead to contradictory Anna Woroniuk 1 GTP-bound state. When GTP-bound, it binds Despite their slower growth, skin tumours activators as well as scaffolding et al., Cell Reports 2015), we found that TIAM1 is migratory phenotypes— cell‒cell adhesion proteins. The RAC GEFs TIAM1 ubiquitylated and degraded upon treatment versus motility— raising the possibility that Graduate Students to various effector molecules that stimulate arising in Tiam1-deficient mice progressed and P-REX-1 lead to different downstream responses including, notably, more frequently to malignancy, suggesting of cells with hepatocyte growth factor (HGF), targeting RAC in a clinical setting could worsen Sophie Adlard1 effects on cell migration via actin cytoskeletal reorganisation. Multiple that retention of TIAM1 impedes malignant a cytokine that is abundant in cancer and tumour progression. This calls for the Joe Maltas regulating the RAC interactome. Anna Woroniuk2 mechanisms control RAC activity including progression, consistent with down-regulation (B) Schematic representation of promotes invasion of cancer cells. We mapped identification of factors that both influence the nucleotide binding and hydrolysis regulated by of TIAM1 in skin malignancies in the mouse the P-REX1-RAC-FLII signalling the ubiquitylation site on TIAM1 and also selection of RAC-driven cellular processes as Summer Student GEFs and GTPase Activating Proteins (GAPs) chemical carcinogenesis model (Malliri et al., cascade. Activation of RAC by identified the responsible E3 ligase as being well as mediate RAC’s effects. In a recent study Daniel Blears1,2 respectively, subcellular localisation, modulation Nature 2002). One mechanism by which TIAM1 P-REX1 results in RAC binding to the HECT family member HUWE1. Moreover, (Marei et al., Nat Commun. 2016), we FLII. P-REX1, via its scaffolding we showed that interfering with TIAM1 demonstrated that two RAC GEFs TIAM1 and 1 joined in 2016 of RAC protein levels, and post-translational and RAC suppress malignant progression is ability, binds to FLII through its 2 modification including isoprenylation and, as we through stimulating cell–cell adhesion. ubiquitylation by depleting HUWE1 or mutating P-REX1 promote RAC-driven cell‒cell adhesion left in 2016 GEL domain and brings it in close and others have demonstrated, ubiquitylation Previously, we identified β2-syntrophin, a proximity to active RAC further the ubiquitylation site retards the scattering and and RAC-driven cell migration and invasion and SUMOylation (Castillo-Lluva et al. component of the dystroglycan adhesion stimulating the RAC-FLII invasion of cells through delaying AJ respectively, through regulating the RAC Oncogene 2013; Castillo-Lluva et al. complex, as a TIAM1 interacting partner. Our interaction via the LRR domain of disassembly. HGF and HUWE1 are plentiful in interactome. While TIAM1 promotes the Nat Cell Biol. 2010). study (Mack et al. Nat Cell Biol. 2012) uncovered FLII. Through this interaction, lung cancer. We showed that HUWE1 and association of RAC with proteins that stimulate P-REX1 induces phosphorylation TIAM1 expression are inversely correlated in the formation and maintenance of cell‒cell a novel role for this complex in promoting tight of MLC (pMLC) thereby lung cancer specimens and significantly that adhesions and consequently inhibits migration, RAC GEFs are typically large proteins containing junction formation and the development of enhancing cell contraction in a multiple protein‒protein interaction domains. apicobasal polarity through generating a RAC HUWE1 promotes lung cancer invasion by we established that P-REX1 stimulates migration FLII-dependent manner. This Besides stimulating guanine nucleotide activity gradient in the membrane region cascade accounts, in part, for degrading TIAM1. Potentially, drugs capable of through augmenting the interaction between exchange, GEFs function as molecular scaffolds encompassing these junctions. P-REX1-RAC-driven cell disrupting the HUWE1-TIAM1 interaction could RAC and the actin-remodelling protein targeting active RAC to particular subcellular migration. antagonise invasion of lung and other cancer Flightless-1 homolog (FLII), to modulate cell locations and potentially increasing the local Malignant progression can entail the loss of cells, reducing the risk of metastasis. contraction in a RHOA-ROCK independent concentration of selective effector molecules, cell‒cell adhesion. Over-expression of activated manner. Thus, we provided direct evidence that thereby influencing downstream processes. RAC or TIAM1 promotes the formation of TIAM1 localises in the nucleus of colorectal RAC GEFs are determinants of selectivity in Through influencing selectivity in RAC adherens junctions (AJs) and the associated cancer cells and inhibits their migration signalling events downstream of RAC and also signalling, GEFs could therefore perform unique generation of an epithelial-like phenotype in and invasion identified FLII as a novel mediator of RAC-driven signalling roles that could be important for mesenchymal cell lines (Malliri & Collard, Curr Previously, using recombinant mouse models, migration and invasion, which now needs tumourigenesis. Indeed, mice deficient for the Opin Cell Biol 2003). Moreover, TIAM1 is required we showed that TIAM1 cooperates with WNT evaluation as a factor promoting tumour RAC GEF TIAM1 are resistant to the formation of for both the formation as well as the signalling during the initiation of colorectal metastasis. skin tumours induced by chemical carcinogens maintenance of cadherin-based adhesions cancer (CRC) but then antagonises CRC tumour that target H-Ras and the few resulting tumours (Malliri et al., J Biol Chem 2004). The progression (Malliri et al., J Biol Chem. 2006). Publications listed on page 58 grow very slowly (Malliri et al., Nature 2002). Thus oncoprotein SRC, a non-receptor tyrosine However, how TIAM1 influences CRC initiation TIAM1, we infer, plays a unique role in mediating kinase, targets AJs for disassembly. Previously, and progression remained obscure. To further RAS transformation that the Cell Signalling group we revealed that SRC phosphorylates TIAM1 address the influence of TIAM1 on CRC and is elucidating, focusing currently on RAS-driven inducing its cleavage by Calpain and its increase our understanding of its clinical role, we lung cancer. depletion from adherens junctions. Blocking probed a tissue microarray comprising 650 18 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE CELL SIGNALLING 19


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    www.cruk.manchester.ac.uk/Research/CRUK-MI-Groups/CEP/Home Sarah Pearsall2 first line chemotherapy. These exciting data standards using CellSearch (EpCam based CTC Max Schenk2 contributed to AstraZeneca’s decision to take this capture) in 1,615 blood samples within 13 clinical drug combination to clinical trial in SCLC in 2017 trials. Cognisant of the need to evaluate EpCAm CLINICAL AND EXPERIMENTAL Scientific Officers Mahmood Ayub Kieran Bradbury2 with The Christie NHS Foundation Trust as one of the clinical trial sites. positive and negative CTCs, we evaluated three marker independent CTC platforms (Parsortix, PHARMACOLOGY Nicholas Brittain2 Stewart Brown Debbie Burt Vasculogenic mimicry in SCLC Our research on vasculogenic mimicry (VM), Clearbridge Spiral Chip and RareCyte AccuCyte/ CyteFinder) where CTCs are enriched on the basis of cell size, buoyancy and/or deformability and Fouziah Butt (Williamson et al. Nature Communications 2016; biobanked resultant CTC enriched samples. A Mathew Carter see Research Highlights) describes the ability of versatile ‘liquid staining’ method for CTC John Castle Brian Chan1 tumour cells to adopt endothelial characteristics characterisation is in the final stages of validation and form fluid conducting channel-like structures and will be deployed to enumerate and Clinical and Experimental Pharmacology has two major Christopher Chester1 Samson Chinien1 independent of host vasculature. We showed that characterise our biobank of CTC enriched research goals: firstly to discover, develop, validate, and implement Jakub Chudziak 1 VM occurs in SCLC and correlates with worse samples in order to better capture CTC Lucy Coverley2 patient overall survival. VM was observed in CDX heterogeneity. During 2016, we have initiated biomarkers to support personalised medicine for cancer patients Suzanne Dalby1 thus enabling further study. The endothelial several exciting new collaborations with key as we expand the Manchester Centre for Cancer Biomarker Sarah Evans marker VE-Cadherin co-localised with VM external partners including Amgen (SCLC CTC Lynsey Franklin Sciences; and secondly to develop a patient relevant platform in Melanie Galvin channels in CDX and via copy number analysis of based biomarkers), Merck (NSCLC CTC based laser capture micro-dissected CDX tissue and biomarkers), Carrick Therapeutics (biomarkers to order to discover and test novel therapies for small cell lung cancer Keal Gracey2 Hannah Gregson disaggregated single cells, we confirmed that VM support development of their CDK7 inhibitor in Group Leader that can be translated to the clinic. Highlights this year included: (i) Grace Hampson cells are of tumour origin. We identified sub- breast cancer), and AstraZeneca (biomarkers to Rebekah Pringle Caroline Dive the initiation of the first international collaboration within the Sarah Hilton populations of VE-Cadherin expressing CTCs and support therapeutics targeting DNA damage in collaboration with Prof. Peter Kuhn at The repair in lung cancer). Deputy Group Leader ‘Cancer Moonshot’ programme between CRUK and the USA NCI Michael Hoffs2 University of Southern California, we isolated Nadia Iqbal Ged Brady to develop early detection biomarkers based on single circulating Aileen Jardine single VE-Cadherin expressing CTCs and New academic collaborations have been set up confirmed their tumour origin using their High with Professor Charles Swanton at University Staff Scientists tumour cell analysis; (ii) development of the first DNA based liquid Hana Jelassi Noel Kelso Definition Single Cell Assay (HD-SCA) (the College London, for CTC based biomarker Dominic Rothwell Jonathan Tugwood biopsy for SCLC that anticipates chemoresponse; (iii) studies that Scarlett Martindale ‘Cancer Moonshot’ technology) (Figure 2). analysis on DARWIN trials as sub-studies of the Simrah Mohammad revealed the functional significance of vasculogenic mimicry for Karen Morris Knock down of VE-Cadherin in a xenograft model CRUK Lung Cancer Centre of Excellence flagship Associate Scientists reduced the intratumoural delivery of cisplatin TRACERx (TRAcking non-small cell lung Cancer Kathryn Simpson SCLC spread and drug resistance; (iv) the translation of a novel Joanne Norris consistent with a VM-mediated improved blood Evolution through therapy [Rx]) programme, and Dawar Pasha2 Kris Frese drug combination for SCLC to the clinic based on data from our Jackie Pierce supply. However, subsequent drug response with Alastair Greystoke (CEP alumnus) within the was reduced, implicating VM signalling in Newcastle ECMC to assess CTCs in thyroid Service Manager CTC patient derived explant models; and (vi) leadership of the Alan Redfern2 Caroline Roberts2 chemoresistance. The molecular regulation cancer. We installed and qualified the Aushon Tony Price personalised medicine theme in the newly awarded Manchester Karishma Satia2 and role of VM in metastasis is under investigation. CiraPlex system for multiplex ELISA analysis, and Nicole Simms Postdoctoral Fellows Biomedical Research Centre. Nigel Smith this platform has been used to analyse proof of Jenny Antonello Use of CDX models to develop biomarkers concept biomarkers in several trials of anti- Sarah Taylor Becky Bola1 Pharmacodynamic biomarker development in angiogenic therapies. Simon Topham2 Francesca Chemi2 CDX runs in parallel with testing of novel targeted Highlights from the Preclinical In collaboration with Pharma partners, we are Louise Walkin1 Fabiola Fernandez-Gutierrez2 Pharmacology Team testing new therapies in CDX with parallel Sally Wood1 therapies with a view to conversion to CTC Highlights from the Nucleic Acids Sakshi Gulati Barbara Mesquita biomarker development in CDX and in CTCs in Paul Wright 1 based assays for the clinic. We have focused Biomarkers Team (NAB) Francesca Trapani SCLC CTC and drug development order to rapidly translate promising treatments to on the DNA-damage response pathway, The Nucleic Acids Biomarkers (NAB) team, led by Laboratory Manager Stuart Williamson This team, led by Kris Frese and Kathryn Simpson, early phase trials at The Christie NHS Foundation frequently aberrant in SCLC, using a Ged Brady, continued their development and Matthew Lancashire Sumitra Mohan expanded our panel of SCLC CDX from 17 to 35 Trust. p53 aberrations render the G1 checkpoint combination of immunohistochemical and application of molecular profiling methods models, including seven matched pairs compromised in all SCLC patients thus placing Laboratory Support Technician immunofluorescence-based single and dual suitable for monitoring tumour status from a Bioinformaticians increased reliance on the G2 checkpoint to stall staining methods. Of considerable interest is our simple blood sample. representing disease at chemonaive baseline Andrew Stevens Antony Chui and then again at progression after treatment the cell cycle and allow DNA damage repair. In recent finding that CDX cells are capable of Christopher Smowton1 with platinum/etoposide standard of care collaboration with AstraZeneca, we completed ECMC/Lung Cancer Centre metastasis in the mouse with the same tropism Molecular Analysis of CTCs Bedirhan Kilerci2 testing of the combination of their Wee1 G2 Project Manager seen in the donor patient (Figure 3). A major focus is the genomic analysis of individual chemotherapy (SOC). Our panel now Charlotte Minter1 Clinical Informaticians recapitulates the spectrum of patient responses checkpoint kinase inhibitor AZD1775 and their CTCs with bioinformatic analysis of CTC copy Jenny Bradford2 to SOC. The panel is being used to explore DNA damage repair inhibitor olaparib (a PARP Executive Assistant to Biomarkers to support clinical trials number alterations (CNA) to establish if patterns Fouziah Butt 2 mechanisms of inherent and acquired inhibitor) in several SCLC CDX models. This In the past 12 months, our biomarkers’ portfolio of CNA are linked to response to therapy. We Professor Dive Laura Hutchinson2 combination promoted durable tumour supported 36 clinical trials (26 academic established that CNA patterns identified in SCLC chemoresistance using both CRISPR-based Ekram Aidaros Lisa Kennedy2 forward genetics as well as computational regression in a chemosensitive CDX model with sponsored, 10 pharmaceutical company CTCs obtained prior to patient treatment Donal Landers2 bioinformatics, and putative mediators of complete tumour regression for up to a year Administrative Coordinators sponsored and 12 NIHR badged), and 23 correlated with response to standard of care Paul O’Regan2 after final drug combination treatment. Efficacy Maria McGloin1,2 experimental medicine studies. Our focus has chemotherapy (Carter et al. Nat Med. 2016; see Jenny Royle2 chemoresistance are undergoing validation. A Lisa Waters Laura Stephenson2 key part of our strategy has been the was maintained if the novel combination was remained the development and application of Research Highlights). Examination of CNA profiles Ashley West 2 Julie Stevenson2 development of CDX ex vivo cultures that can administered after standard of care but was less liquid biopsies - specifically CTCs and circulating obtained from patients’ CTCs following treatment Jason Swift 2 durable. The response to olaparib/AZD1775 was DNA (ctDNA) - that reduce reliance on invasive - and relapse indicated that the genetic basis for be genetically modified, and re-implanted back 1 left in 2016 into mice (Figure 1). Importantly, CDX cells less impressive in baseline chemorefractory 2 joined in 2016 and not always feasible or repeatable - tumour failure to respond to initial treatment differs from Clinical Fellows Victoria Foy2 maintain their transcriptional profiles and CDX, but still outperformed cisplatin/etoposide. biopsies. that seen at relapse after an initial response. Robert Metcalf1 chemosensitivity throughout this process, By testing in a baseline/progression CDX These observations are the first to establish a link Danielle Shaw1 making this a suitable platform for functionally matched pair, we learned that the new Highlights from the Cells and between genetic changes in CTCs and clinical validating candidate mediators of combination was most effective at baseline Proteins Biomarkers Team outcomes. Graduate Students chemoresistance. with efficacy lost at progression, arguing for This team, led by Jonathan Tugwood, developed Jakub Chudziak2 rapid introduction of the combination after and validated several new CTC based assays and The NAB team also examined the genetic status Alice Lallo enumerated CTCs to good clinical practice (GCP) of single CTCs from patients with primary 20 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE CLINICAL AND EXPERIMENTAL PHARMACOLOGY 21


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    provides an improved biomarker readout arena was also maintained by attendance at CLINICAL AND EXPERIMENTAL PHARMACOLOGY (CONTINUED) (Figure 4). NIHR refresher GCP training, a North of England Regional Forum in October 2016 and the Annual ctDNA analysis - the TARGET initiative and SCLC The Quality Assurance Team Research Quality Association (RQA) Conference In collaboration with Richard Marais and with In addition to supporting on-going Good in November 2016. Andrew Hughes, Matthew Krebs, Emma Dean Clinical Practice (GCP) related activities within and Natalie Cook at The Christie Early Clinical CEP, much of the activity for the QA team in 2016 The new Clinical Informatics team and Trials Unit, and Andrew Wallace and William centred on the re-implementation of internal the iDECIDE Programme Newman at the Manchester Centre for Genomic auditing processes, the development and The Clinical Informatics team of the Manchester Medicine, the NAB team is conducting extensive implementation of a non-conformance process, Centre for Cancer Biomarker Sciences is being analysis of ctDNA within the TARGET precision the roll-out of refresher GCP training & Quality established. Its first research programme, medicine initiative that seeks to identify the Management System (QMS) training to longer iDECIDE is a five year collaboration between four optimal allocation of a patient to an available serving members of staff and maintaining up to strategic partners/leaders in oncology research: Phase I trial. We have processed ctDNA from the date knowledge in an increasingly changing the Manchester Centre for Cancer Biomarker first 100 TARGET patients using a workflow regulatory environment. Implementation of Sciences; The University of Manchester Faculty based on a sensitive next generation sequencing harmonised risk-based internal audit and of Biology Medicine and Health; The Christie (NGS) approach that enables parallel sequencing non-conformance processes were delivered to NHS Foundation Trust Clinical Trials Unit; and non-small cell lung cancer (NSCLC) and, as part of >600 cancer associated genes selected to provide evidence of on-going assurance of AstraZeneca. The iDECIDE programme Figure 1: CTCs enriched from of the TRACERx (TRAcking non-small cell lung cover a wide range of actionable drug targets. A compliance with GCP and a framework for commenced in January 2016 supported patient blood are implanted into Cancer Evolution through therapy [Rx]) study dedicated QA scientist is currently working with continual improvement to both local CEP by AstraZeneca (£11.5M). mice to generate CDX models. Figure 3: CDX cells are capable of led by Charles Swanton, compared CTC and us to ensure our ctDNA NGS assays become management and The University of Manchester Cells from these CDX can metastasis with the same tropism tumour profiles. Initial results identified both GCP compliant to allow clinical decision making. as observed in the donor patient. Research Compliance Committee. Four internal The overarching iDECIDE vision is to enable subsequently be cultured ex vivo CNA patterns and mutations that are shared The extended feasibility phase of the project IHC for the neuroendocrine audits of laboratory processes and 14 audits of better, early clinical trial decision making that and genetically modified using lentiviruses before re-implanting between CTCs and the matched tumour as well demonstrated that blood samples could be biomarker CD56 (left panel) and laboratory notebooks were conducted directly benefits the patient. Specifically, into mice. These culture-derived as changes that differ. The data indicate that the processed and NGS data generated with a anti-human mitochondria (right alongside 28 internal data audits to assure the iDECIDE will: CDX maintain the original combined single cell CTC and tumour analysis panel) positive cells in liver of a CDX turnaround time suitable for routine feedback accuracy of reported data and compliance with characteristics of the donor identifies additional layers of heterogeneity that bearing mouse where the model to the TARGET Molecular Tumour Board. A was derived from a patient with GCP, sponsor requirements and local • enable all experts, including patients, to work tumour. would not be seen by either approach alone and comparison of the cfDNA NGS data to the liver metastases. procedures, allowing us to address issues arising together as a team, striving to evolve the that this will further our understanding of how patient’s archival tumour biopsies profiled and prevent reoccurrence. Refresher GCP science of cancer medicine tumour clonal heterogeneity impacts upon using a standard Oncocarta gene panel showed training and QMS training were also developed • transform clinical trials through precision therapeutic outcomes. impressive overlap in detected mutations. and implemented to ensure awareness of science: delivering treatments that work for Ongoing development aims to increase ctDNA regulatory requirements in addition to the people, healthcare and society The established ability within NAB to genetically assay sensitivity and improve classification of underlying principles and rationale for GCP • empower patients to become active profile single CTCs as well as micro-dissected ctDNA mutations not covered or identified by Laboratory Quality Management Systems. The collaborators and co-scientists in our clinical cell subpopulations was exploited to compare tumour analysis. QA team also maintained external links and trials to make personalised medicine a reality split sample matched CDX tumours and CTCs. In collaborated with The Christie QA team, ECMC a case study of a NSCLC patient, a CDX was The NAB team is also examining ctDNA obtained Quality Assurance in Translational Science The newly established team (currently 11 staff) generated despite lack of detectable CellSearch from plasma of patients with SCLC to establish network and the newly formed Manchester brings a broad range of skills sets including CTCs. Molecular analysis of this CDX and NGS mutation profiles using the same process Corridor QA/GxP Network, sharing knowledge, clinical, business analysis, clinical informatics, matched size enriched CTCs revealed developed for the TARGET initiative as well experience and practices. Awareness of current patient centricity, software/enterprise systems predominantly mesenchymal phenotypes in establishing ctDNA CNA profiles. This will allow regulatory issues and hot topics within the GCP and biomarker development. The team has both with common somatic mutations us to compare ctDNA and CTC CNA patterns now formal collaborations in place with the Figure 2: Single Cell CNA analysis confirming the tumour origin of these CTCs and establish the utility using either readout Blood Draw Manchester Royal Infirmary (Nephrology) of VE-Cadherin negative and (Morrow et al. Ann Oncol. 2016). alone or whether the combination of both Day 0 and AstraZeneca Research & Development VE-Cadherin positive CTCs from a SCLC patient indicate two Informatics with further collaborations planned Pa#ent 1 Plasma genetically distinct sub- Day 28 in 2017. The Joint Steering Committee was also populations of cells. A CTCs initiated and formally agreed to provide robust Extrac,on of were identified using the HD-SCA Circula,ng Free DNA governance. (cfDNA) assay whereby cells were stained Day 175 for DAPI (blue), VE-Cadherin Whole Genome NGS The key goals for 2017 are the delivery of: (white), CKs (red), CD45 (green) Library and identified DAPI+/VE- Cadherin-/CK+/CD45- CTCs (i) • new independent iDECIDE Microsoft Azure Low Pass Whole Day 0 Pa#ent 2 and (ii) and DAPI+/VE-Cadherin+/ Genome Sequencing environment supporting the deployment of CK+/CD45- CTCs (iii) and (iv). A our principal systems – Real-time Analytics for reference white blood cell (wbc) Bioinforma,c Analysis Day 45 Clinical Trials (REACT) and Patient Reported To Iden,fy CNA is included which is negative for Outcomes About Clinical Tolerability all markers except CD45 (v). B CNA analysis of cells from A (PROACT) shows the two populations of Figure 4. Workflow and Examples of Whole Genome Copy Number Alterations (CNA) • the REACT Patient Tracker component CTCs based on VE-Cadherin seen in Plasma cfDNA from two patients with Small Cell Lung Cancer (SCLC): (patient summaries) expression also have different The workflow to the left summarises the entire process from blood collection to the generation • the PROACT Protocol in studies at The Christie genomic architecture, VE- of CNA patterns. CNA data is presented by chromosome order (1-22); regions with copy - this protocol will be sponsored by The Cadherin- CTCs are clonal (i) and number gains coloured red/yellow (yellow indicating a higher degree of copy number gain) Christie and paid for using the iDECIDE grant (ii) whereas VE-Cadherin+ CTCs and regions with copy number loss coloured blue. In the three sequential time-points analysed • new integrated REACT exposure, biomarker show non-clonal arrangements for Patient 1 the Day 0 and Day 28 samples show little or no Copy Number Changes, whereas (iii) and (iv). Representative images prominent changes are seen in the Day 175 sample indicating low levels of circulating tumour and efficacy visualisations PoC in H1 2017 and profiles are shown. DNA (ctDNA) within the 1st month, followed by an increase in ctDNA reflecting disease progression. In contrast, for Patient 2 both time-points show similar pronounced CNA Publications listed on page 60 patterns indicating the presence abundance of ctDNA at both time points. 22 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE CLINICAL AND EXPERIMENTAL PHARMACOLOGY 23


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    www.cruk.manchester.ac.uk/Research/CRUK-MI-Groups/Drug-Discovery Undergraduate Students Shaun Johns2 Klaudia Milc1 DRUG DISCOVERY 1 joined in 2016 2 left in 2016 points. There will be significant capability build quinquennium was to deliver our first over the coming months as the group candidate drug into pre-clinical development, 2016 has been a year of considerable success for the Drug Discovery undertakes full scale phenotypic cell based through our own efforts or in partnership with Unit (DDU) and has seen the achievement of several significant screening for the first time. a collaborator. Having achieved this highly significant milestone, our primary focus is now milestones. In April, we disclosed for the first time our novel tool Further strengthening our portfolio of projects to ensure the long term sustainability of our compounds against the DNA repair target PARG at the AACR Annual have been several advanced discussions which project portfolio. We are investing significant we anticipate will bring new drug discovery effort in the identification and validation of the Meeting in New Orleans. In October, we announced that the lead targets and new partnerships with the next generation of drug discovery targets for the series of compounds developed for our collaborative programme pharmaceutical industry into the DDU in the Unit to work on, to ensure continued delivery of Group Leader with GlaxoSmithKline (GSK) had been returned to them for coming months. further candidate drug molecules. Donald Ogilvie accelerated progression toward pre-clinical candidate selection. The Future Publications listed on page 62 Head of Chemistry Most notable, however, was the declaration of our first pre-clinical The primary aim of the DDU in this Allan Jordan candidate compound, arising from our RET inhibitor project. Head of Bioscience Ian Waddell Figure 1: The aim of this latter project, which is partially to CRUK. Again, this direct appreciation of the funded by the venture capital company 6th value created in a Manchester-based project Novel chemical startpoints, Chemists bound to the kinase domain of Jonathan Ahmet Element Capital (6EC), is to develop new represents a significant acknowledgement of RET, are being developed toward Roger Butlin2 treatments for lung cancer patients whose our success and capabilities. This collaborative alternate lead compounds. Niall Hamilton2 tumours harbour activating gene fusions in RET, project remains highly active within GSK, who Ligand-protein structures were James Hitchin2 a receptor tyrosine kinase. This particular are now driving the lead series towards the clinic obtained through collaboration Stuart Jones2 genetic alteration is found in approximately one in no less than three disease areas, including with the Cancer Research UK Chris Kershaw Structural Biology Accelerator percent of all lung cancers but has recently been oncology. The DDU team here in Manchester Alan Lee1,2 Award. Alison McGonagle associated with sixteen percent of non-small cell are now focusing upon the delivery of a Rebecca Newton lung cancer cases in non-smokers. The chemically distinct back-up inhibitor compound Ali Raoof selection of a pre-clinical development series for this challenging target. Kate Smith candidate is a first for the team as a whole and Roger Taylor1 represents a major achievement for a group of Whilst the past year has seen significant focus on Bohdan Waszkowycz our size. Importantly, this nomination has also these two major projects, other projects within triggered the availability of extra funds from 6EC, the team continue to make good progress: Bioscientists Figure 2: Ben Acton supporting both the additional work required to Docked model of the PARG Mentxu Aiertza Otxoterna2 take the candidate drug through safety testing Our PARG programme recently disclosed the chemical probe compound Habiba Begum and into clinical trials and the development of a first ever cell-active tool compounds against this PDD00017273 bound to the Elizabeth Blaikley back-up series. Work to deliver this back-up challenging target, both as published research human PARG protein. Charlotte Burt series is primarily being undertaken in the labs articles and at the American Association for Phil Chapman here at the CRUK Manchester Institute. We are Cancer Research meeting in New Orleans in Mark Cockerill Cath Eberlein1 also working closely with our local experts in April. This disclosure was the subject of two Emma Fairweather The Christie NHS Foundation Trust Clinical Trials podium presentations and two very busy Samantha Fritzl Unit to develop efficient ways of identifying posters. These tool compounds are now Joanna Grabarek 1 suitable patients, in order to help facilitate the available to our collaborators and the wider Louise Griffiths design of future clinical trials for these community, as we seek to expand upon the Nicola Hamilton compounds. understanding of this critical but poorly Gemma Hopkins2 understood component of DNA repair. We hope Dominic James Paul Kelly Our second success stemmed from our that these investigations will suggest new Valentina Lukinovic1 ongoing epigenetics collaboration with GSK in therapeutic applications for PARG inhibitors and Nikki March Philadelphia (US). Following a very successful will catalyse further efforts in the area. The Helen Small2 drug discovery collaboration running in parallel project is also the subject of late-stage Alex Stowell at both sites, the Drug Discovery Unit has now partnering discussions. Graeme Thomson transferred the lead chemical series (which was Adnana Tudnose designed, synthesised, tested and developed Alongside this, our SMARCA2 project continues Graeme Walker Mandy Watson here in Manchester) to GSK. This is the first time to progress. This biological target has been the we have transferred a late-stage programme to a subject of intense interest over the past two Graduate Students pharmaceutical partner and in doing so, the years or so from many organisations but it has, to Elizabeth Hogg team triggered a success payment being made date, proved difficult to find good chemical start Daniel Mould 24 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE DRUG DISCOVERY 25


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    www.cruk.manchester.ac.uk/Research/CRUK-MI-Groups/Leukaemia-Biology/Home It is also well established that LSD1 physically interacts with SNAG domain transcription factors, and that this interaction is absolutely LEUKAEMIA BIOLOGY required for the function of these factors as transcription repressors. Following analysis of the transcriptome of drug treated leukaemia cells, we found that LSD1 inhibition mimicked knockdown of the SNAG domain transcription factor GFI1. This led us to discover that LSD1 inhibitors target the protein:protein interaction of LSD1 with GFI1, promoting the physical separation of one from another, and also more A key focus of the laboratory is the translation of our basic science generally the physical separation of LSD1 from discoveries into the clinic aiming for future patient benefit. In chromatin (Figure 2). Using an elegant inducible 2012 we reported in Cancer Cell that LSD1 (for lysine-specific fusion transcription factor system developed by Gary Spencer in the lab we went on to demethylase 1) contributes to the differentiation block that is the had argued that histone tail methylation was demonstrate that the differentiation resulting an irreversible phenomenon. LSD1 is a flavin core pathologic feature of acute myeloid leukaemia (AML). This Figure 1: adenine dinucleotide (FAD) dependent from treatment of AML cells with LSD1 inhibitors Effect of treatment of THP1 AML depended upon physical separation of LSD1 led to our collaboration with Oryzon Genomics, a Spanish cells with the tranylcypromine- homologue of the amine oxidase family from GFI1. The consequence of disruption of the Group Leader pharmaceutical company with a first-in-class inhibitor of LSD1. In derivative LSD1 inhibitor OG86. with an ability to demethylate monomethyl GFI1:LSD1 interaction following LSD1 inhibition THP1 AML cells were treated with or dimethyl lysine 4 (K4) of histone H3. Tim Somervaille turn, this led to a first-in-man Phase 1 trial of Oryzon’s advanced 250nM OG86 or DMSO vehicle was a localised increase in histone acetylation at the surrounding chromatin, consistent with Postdoctoral Fellows lead compound ORY-1001 in the clinical setting of AML, with The for 24 hours in semi-solid culture. Exemplar flow cytometry plot In view of the high level of expression of LSD1 a model whereby GFI1/LSD1-repressed (or Gauri Deb Christie NHS Foundation Trust a major recruiting centre. Data from in a range of poor prognosis sub-groups of (left panel) and bar chart (right primed) enhancers become activated Alba Maiques-Diaz panel) indicate change in prostate, lung, brain and breast cancer, as well as this trial were presented for the first time at the American Society following drug treatment. Thus, unexpectedly, Isabel Romero-Camarero expression of the monocyte/ in certain haematological malignancies, there pharmacological inhibitors of LSD1 are effective Clinician Scientist of Hematology in December 2016, and show that, as in our pre- macrophage differentiation has been much interest in the potential of LSD1 through disrupting a protein:protein interaction marker CD86 and the stem cell inhibitors as novel therapies in cancer. Given the Dan Wiseman clinical models, treatment of patients with ORY-1001 promotes marker KIT. Error bars refer to known enzymatic activity of LSD1, the rather than, as expected, through disrupting catalytic activity. Clinical Research Fellows differentiation of AML blast cells in blood and bone marrow. These SEM; n=4. assumption in the field has been that LSD1 Mark Williams exciting translational findings serve as a platform for further clinical contributes to gene repression by removing Our studies highlight GFI1 and its interaction with John Chadwick 1 monomethyl and dimethyl histone marks from development of a novel differentiation therapy for patients with AML. lysine 4 of histone H3 and that this is the key LSD1 as critical targets for differentiation therapy Bioinformatician in human AML with MLL translocations, a class activity targeted for potential therapeutic effect. Fabio Amaral 1 of leukaemia typically distinguished by A major milestone in 2016 for the group has predicted by our previously published pre- However, LSD1 also interacts with multiple monoblastic/monocytic morphology. Our been the completion, in collaboration with clinical studies, in the extension phase of the transcription factors raising the possibility that Scientific Officer findings emphasise that compounds developed Gary Spencer Oryzon Genomics, of a first-in-man, first-in- trial (which was focused in particular on patients other mechanisms may be significant. through drug discovery programs focused on class Phase 1 clinical trial of ORY-1001, a with MLL leukaemias), patients showed optimising inhibition of the demethylase activity Graduate Students tranylcypromine-derivative inhibitor of lysine- morphological and pharmacodynamic features In in vitro experiments we observed that rapid, of LSD1 unexpectedly block both structural and Emma Williams extensive drug-induced changes in transcription specific demethylase 1 (LSD1, also known as of blast cell differentiation following drug catalytic activities. Our data illustrate a paradigm Fabrizio Simeoni occurred without accumulation of the histone KDM1A). The data arising from the study were treatment. This indicates that, at least for certain for epigenetic therapy whereby, through presented at the American Society for patients with AML, inhibition of LSD1 represents modifications targeted for demethylation by 1 joined in 2016 disruption of the protein:protein interaction Hematology meeting in San Diego in December an entirely new approach to differentiation LSD1, and that a demethylase-defective mutant between a transcription repressor and an 2016. This trial follows on from a pre-clinical therapy. These exciting findings are now being rescued LSD1 knockdown AML cells as efficiently Figure 2: epigenetic regulator, repression is released study from our lab which was originally taken forward in collaboration with Roche, Mechanism of action of LSD1 as wild-type protein. These experimental and dynamic enhancer acetylation and gene published in Cancer Cell in 2012. In that report which has formed a partnership with Oryzon inhibitors. findings called into question the hypothesis expression ensue. Further, they refine the role we demonstrated that LSD1 contributes to the Genomics. Further clinical trials with ORY-1001 that it was the enzymatic activity of LSD1 that is of LSD1 in leukaemic haematopoiesis as an differentiation block in acute myeloid leukaemia are underway in the clinical setting of small cell targeted by inhibitors of LSD1. epigenetic reader of transcription factor binding (AML) (in particular in the Mixed Lineage lung cancer (SCLC), where novel therapies are sites, in addition to its role elsewhere as an eraser Leukaemia (MLL) molecular subtype of the desperately needed. In addition to AML, a strong of histone methylation marks. disease) and is targeted by novel derivatives of pre-clinical signal for LSD1 inhibition has been tranylcypromine to promote differentiation of observed in SCLC. These functional studies, which have revealed leukaemic blast cells. A block in myeloid an unexpected mechanism of action of LSD1 differentiation is one of the core pathologic Understanding the mechanism by which inhibitors, are being taken forward in the lab by features of AML. pharmacological inhibition of LSD1 promotes Gauri Deb and Alba Maiques-Diaz, who are differentiation in AML (Figure 1) is key to trying to uncover novel genes and cellular The Phase 1 trial was funded by a co-operative optimising future use of ORY-1001 and related pathways which, when inhibited or blocked, grant from the European Union EUROSTARS drugs in patients. LSD1 was initially identified as a collaborate with LSD1 inhibition to promote scheme awarded in late 2013 to Oryzon core component of an RCOR1 (CoREST) histone myeloid differentiation. Such cellular co- Genomics and The University of Manchester. deacetylase (HDAC) transcription corepressor targeting strategies may hold out a prospect The trial, which in its first phase was a standard complex and later found to have histone tail for future patient benefit in clinical trials. dose escalation design, demonstrated that lysine-specific demethylase activity. Indeed ORY-1001 was well tolerated by patients without LSD1 was the first protein described to have Publications listed on page 63 unexpected side effects. Importantly, and as histone demethylase activity. Prior to this some 26 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE LEUKAEMIA BIOLOGY 27


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    www.cruk.manchester.ac.uk/Research/CRUK-MI-Groups/Molecular-Oncology/Home patient’s treatment cycles with targeted therapies, immunotherapies and chemotherapies. We discovered that the MOLECULAR ONCOLOGY patient had two tumours that responded differently to each of these treatment modalities (Figure 2), and this study demonstrates the power of ctDNA. We are now elaborating those studies by developing clinical trials that will use this approach to determine the optimal time for switching between targeted and immuno- therapies in individual patients. My group studies cancer biology and we use the new knowledge Although our studies are largely focused on we generate to improve cancer patient care. We are developing new melanoma, we are using the approaches we anti-cancer drugs, and are developing precision medicine protocols DAPI! have developed for melanoma to investigate the CD3! biology of other cancers. In 2016, we reported to tailor treatments to individual patients. Our aim is to develop multi- the use of whole genome sequencing in a CD4! disciplinary teams of scientists and clinicians to develop new CD8! chronic myeloid leukaemia (CML) patient whose diagnostic tools and new treatments for cancer patients. tumour had developed resistance to the 3rd PD-1! generation ABL inhibitor ponatinib. Our analysis Group Leader revealed that by combining the BCL2 inhibitor Richard Marais During the last decade, the treatment of we demonstrated that acquired resistance to navitoclax (ABT-263) with ponatinib, we could Erasmus student combinations of targeted and immuno- inhibit growth of the patient’s tumour cells. Last Associate Scientist melanoma has advanced rapidly and led to BRAF inhibitors is associated with a switch in therapies, and investigate how best to combine Monika Wensing1 year we elaborated this study in B-cell acute Nathalie Dhomen significant improvements in patient survival. The metabolism from glucose to glutamine (Baenke and schedule these treatments for the best lymphoblastic leukemia (B-ALL), an aggressive discovery that the BRAF gene is mutated in about et al, 2016). Despite this, there is increased 1 joined in 2016 outcomes for patients. disease that is lethal in ~50% of adult patients. Senior Clinical Scientists half of melanomas, has led to the development oxidative metabolism and the cells show a 2 left in 2016 Martin Cook We observed that inhibition of the protein kinase of drugs that inhibit the mutant protein and slow greater dependence on glutamine and One of the other major clinical challenges with Amaya Viros2 MEK synergised with inhibition of the BCL-2/ the growth of the tumours to provide significant mitochondrial function for proliferation and the new treatments is to know precisely how BCL-XL anti-apoptotic proteins to induce death extensions of patients’ lives. In parallel, drugs survival. Consequently, the resistant cells are patients are responding and when they are Senior Research Scientist of B-ALL cells are driven by various genetic Adele Green have been developed to induce the immune more sensitive to mitochondrial poisons and about to relapse. In this setting, an early warning Figure 1: abnormalities, suggesting the potential of this system to attack the tumours, and these also inhibition of glutaminolysis, providing exciting of impending treatment failure will allow better Multiplex staining of cells of the combination in this disease. Postdoctoral Fellows prolong patient survival and in some cases even therapeutic opportunities to explore. immune system in the spleen of adjustment of the various treatment modalities, Franziska Baenke lead to cures. Despite these remarkable an immuno-competent mouse. Candelaria Bracalente1 and also allow second and third-line treatments We have developed several powerful advances, many patients’ tumours develop The sort of complexity described above as The image shows the presence to be applied earlier and with more precision. Kelly Brooks2 approaches that can be used to test hypothesis- resistance to targeted therapies, and many melanoma cells adapt to the presence of the of CD3+ (purple), CD4+ (green) Alessio Cannistraci and CD8+ (red) T cells, and also Last year we reported that circulating tumour driven treatments with the aim of translating patients do not respond to immunotherapies, various drugs creates enormous clinical DNA (ctDNA) can be used to meet this challenge Elena Galvani shown is staining for PD-1. DAPI is these into the clinic to improve patient care. Our Romina Girotti2 and as a consequence, most patients with challenges for patient care, and last year we (Girotti et al, 2016). Several studies have used as a counter-stain for the biomarker studies in particular have given us the Gabriela Gremel metastatic melanoma still die of their disease. described a platform of approaches that can be nucleus. established that ctDNA is released from tumours confidence to use our approaches to examine Koorosh Korfi2 A major challenge for the field is therefore to used to allow patient treatment adaptations as into patient blood and although it is technically other cancers, and in the coming years we will Katharina Mahal 1 develop approaches that can get the best out this occurs (Girotti et al, 2016). We used next Amit Mandal2 challenging to detect, it can be used to monitor be reporting on how we are using these of these remarkable new drugs. We need to generation sequencing to reveal the landscape how tumours are responding to treatment. In a Piyushkumar Mundra1 approaches to examine the genomic landscape learn how to combine and schedule the drugs of genomic changes in patients’ tumours and further elaboration of this approach, we Marina Parry of prostate cancer in order to determine how Grazia Saturno2 to achieve the best responses for patients as a to follow how these change over time. We performed whole exome sequencing on the we can use our approaches to impact the care HaoRan Tang whole and also how to personalise the developed patient-derived xenografts (PDX) by ctDNA from a patient from whom we were of other types of cancer. Lucas Trucco1 treatment to get the best for individual patients. growing individual patient tumours in immuno- unable to obtain tumour biopsies (Gremel et al, For this we require prognostic and predictive compromised mice, and used these to test novel 2016). We discovered a small number of Clinical Fellows Publications listed on page 63 biomarkers so that each patient receives the best treatment strategies and provide proof-of- mutations, and analysed those across the Pablo Garcia Martinez1 first line treatment, and that their treatment is principle of effectiveness so that we can begin to Lauren Harries1 Rebecca Lee adapted as the tumours evolve to try to escape develop new hypothesis-driven clinical trials. the first and each subsequent line of treatment. While PDX technology is a powerful approach to Figure 2: DTIC Imat. Ipi. Pembr. Pacli/Carbo. Pacli. Scientific Officers test targeted and chemotherapies, it cannot be A patient with two tumours with 30 Variant allele frequency (%) Luke Chisholm There is great complexity in the mechanisms used to test immunotherapies because these distinct responses to therapy is Tumour 1 Grace Garner2 revealed by ctDNA. The image 25 Tumour 2 that underpin the development of resistance experiments are conducted in immuno- Megan Grant shows longitudinal analysis of to targeted therapy, and in the last year we compromised mice. We have therefore refined 20 Jonathan Greenall 1 ctDNA from a melanoma patient described two studies that focused on the BRAF/UVR driven mouse melanoma who was treated sequentially with Clare McManus 15 Philippa Middlehurst 1 metabolism. Through a collaboration with Dr model that we reported in 2014 to test decarbazine (DTIC), imatinib Paul Montgomery1 Beloueche-Babari at the ICR in London, we immunotherapies, because the tumours (imat.), ipilimumab (ipi.), 10 reported that BRAF inhibition caused a decrease develop in fully immuno-competent pembrolizomab (Pembr.), and Matthew Smith paclitaxol (Pacli) with or without 5 Tumour 2. Joshua Tweedy1 in glycolytic activity that resulted in reduced genetically-engineered mice and, as in humans, Sally Wood2 extracellular lactate and a build-up of are driven by ultraviolet light. To understand carboplatin (Carbo). The data 0 show distinct responses by the 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 intracellular glucose and glutamate, consistent how these tumours respond to specific Weeks on follow-up Graduate Students tumours to imatinib, with a reduction in glucose consumption when treatments, we have established techniques to pembrolizomab, and paclitaxol, Kate Hogan2 Denys Holovanchuk BRAF is inhibited in BRAF mutant melanoma monitor how different drugs affect the infiltration revealing the complexity of cells (Delgado-Goni et al, 2016). This data may of specific cells of the immune system responses that can occur with allow us to image cells to measure their (illustrated in Figure 1), and over the next few targeted, immuno and chemo- response to these drugs. In a related study, years we will use our models to test various therapies. 28 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE MOLECULAR ONCOLOGY 29


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    cruk.manchester.ac.uk/Research/CRUK-MI-Groups/Prostate-Oncobiology/Home Castration-resistant! Regeneration! PROSTATE ONCOBIOLOGY +/- Testosterone! pten, etv1! pten, etv1! Acquired! Prostate cancer (PCa) is a very heterogeneous disease both clinically resistance ! Basal/Luminal-origin ! and biologically. Amongst men in the UK, PCa is the most commonly diagnosed cancer, and the second leading cause of cancer-related death. Although androgen deprivation therapy (ADT) is initially effective, most patients inevitably progress to castration- Castration! - Testosterone! resistant prostate cancer (CRPC). In the last decade considerable resistant PCa Group Leader progress has been accomplished in understanding the molecular (CRPC)! Esther Baena events that lead to PCa, although identification of the cell type(s) Postdoctoral Fellows involved in neoplastic transformation is still not clear with Intrinsic resistance ! João D. Barros-Silva contradictory reports published in the literature. Understanding Valentina Ubertini the multistep process of prostate neoplastic transformation genes. We have identified expression of a novel RNA levels of the inherently-CR markers at Clinical Fellow Adnan Syed Ali1 would undoubtedly facilitate the development of new Figure 1: potential marker for prostate luminal progenitors diagnosis correlates with aggressive disease and N has not been implicated in prostate biology. diagnostic and prognostic markers as well better therapies. Schematic prostate duct in the that disease recurrence, and more importantly, with Scientific Officer hormone-naïve, castrated and To test this hypothesis, we have evaluated those development of CRPC. In parallel, these novel Pengbo Wang regenerated states. Most luminal prostate epithelial subpopulations from HN mice cell-surface markers are under evaluation in The prostate gland is comprised of basal cells these studies support the existence of CR cells undergo apoptosis during for organoid culture, recently established in the human prostate cells and patient tissue Graduate Students castration, whereas most basal (which separate luminal cells from basement multipotent and unipotent luminal progenitor laboratory for in vitro characterisation of stem/ microarrays, and we will move towards the Ivana Steiner cells survive; hence, the process membrane), luminal secretory epithelial cells, (LP) cells that repopulate the luminal lineage of regeneration primarily progenitor cells, particularly luminal progenitors. isolation of human tumour subpopulations to Tristan Henser-Brownhill 1 and scattered rare neuroendocrine cells. upon androgen-induced regeneration. produces luminal cells. Different We found that this newly defined prostate assay directly for tumour-initiation/propagation 1 joined in 2016 Luminal cells produce most of the components Furthermore, luminal cells have been shown cell types of origin in the lineage subpopulation has a higher organoid-forming capacity. of the seminal plasma, such as the Prostate as the preferred cell-of-origin of PCa. Lineage- hierarchy might then generate capacity, and are multipotent in vitro forming Specific Antigen (PSA) that have revolutionised tracing experiments also reveal that PCa may distinct tumour subtypes organoids from distinct morphologies and To assess how mutations in the CR stem/ the diagnosis and follow-up of PCa. The have a basal origin; however, basal cell-derived following oncogenic cellular content, including solid mass organoids, progenitor subpopulations might promote transformation. function of neuroendocrine cells in prostate PCa exhibits a longer latency, due to initial acinar organoids composed of a lumen tumour initiation/progression to CRPC, we are biology is unknown although it has been linked differentiation of basal cancer cells to luminal surrounded by multiple layers of cells, and currently characterising mouse and human to aggressive prostate cancer. The role of the cancer cells. The recently developed organoid translucent organoids with a large and hollow models with several genetic alterations such as basal cells is also poorly understood in culture system has allowed detection of lumen surrounded by a thin layer of cells. Of Pten deficiency and ETS factors overexpression homeostasis and disease. Indeed, PCa multipotent or unipotent LPs in vitro from both note, we found that they formed organoids in an (i.e ETV1). We will test the requirements for progression is characterised by a reduction of human and mouse origins. Despite these efforts, androgen-independent manner. selected ETV1/PTEN inflammation-associated basal cells to undetectable levels. the identity of CR prostate cells in vivo, Conditional genetic marking permits both targets for tumour growth and immune cell particularly CR luminal cells, and their lineage and temporal control of genetic recruitment using mouse tissue and human PCa Understanding the cell distribution in a given contribution to CRPC remain largely unresolved. alterations, thereby allowing initiation of PCa primary cell organoid co-culture. We anticipate population under homeostasis, androgen- specifically in CR or HN luminal cells. By that understanding the role of CR stem/ deprived (castration), and regeneration To address this gap in knowledge, we utilised a comparing the phenotypes of PCa arising from progenitor cells in tumourigenesis and the conditions may facilitate further understanding novel Fluidigm multiplex quantitative PCR different luminal cells of origin, we obtained interactions with their niches may lead to of tumour initiation, and tumour evolution (qPCR)-based single cell expression analysis evidence to support that tumour lesions identification of novel targets to tackle towards the castration-resistant stage. Unlike platform to interrogate expression profiles of Figure 2: originating from CR luminal cells are more propagation of prostate cancer. basal cells, which are largely insensitive to individual prostate cells sorted from hormone- Regeneration capacity ex vivo advanced. Indeed, PCa patients expressing high androgen deprivation, the majority of luminal naïve (HN) and castrated mice, and coupled the of prostate epithelial organoids cells undergo apoptosis during castration, analysis with organoid culture and in situ from lineage-marked luminal although a small proportion remain castration- lineage-tracing. By unsupervised clustering cells in response to AR. Mouse Hormone-naive Castration (DHT-) Regenerated (DHT- > DHT+) prostate organoids in hormone- resistant (CR). Prostate basal cells possess analysis, we observed that prostate epithelial naïve conditions showing nuclear multipotent stem cell activity and can cells within the luminal lineage appeared more AR staining, after castration/ differentiate into luminal cells and serve as a heterogeneous than those in the basal lineage. androgen deprivation (-DHT, cell-of-origin of PCa, as revealed by We found that cells of the luminal lineage from without dihidrotestosterone transplantation and injury-repair assays. HN mice could be separated into at least five stimulation) showing However, lineage-tracing studies using luminal- subsets. Krt8high-subsets are characterised by cytoplasmic AR staining. Ex-vivo regeneration capacity was specific mouse models demonstrate that the differential mRNA expression patterns of induced by stimulation with DHT prostate luminal lineage in adults is largely differentiation genes, prostate stem/progenitor (DHT- >DHT+) and evaluated by 20µm 20µm 20µm self-sustained by luminal cells. In particular, marker genes; and some subsets express basal nuclear AR staining. AR 30 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE PROSTATE ONCOBIOLOGY 31


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    www.cruk.manchester.ac.uk/Research/CRUK-MI-Groups/RNA-Biology-Computational-Biology/Home Figure 1: Using Complex Networks, to RNA BIOLOGY interrogate expression data from patient-derived lung adenocarcinoma samples (left) provides a principled mathematical framework with which to identify modular regulatory systems and pathways (right) that are perturbed in tumours. When long noncoding RNAs are included in these The RNA Biology group is interested in why less than 2% of the models, these approaches help predict biological function for human genome encodes amino acids, and what this noncoding un-annotated transcripts. sequence does differently in cancer. By studying these parts of the genetic code, we aim to uncover new processes regulating how tumours grow and develop. A major focus of our work is directed at the study of noncoding RNAs. These are transcripts that are Group Leader expressed, but never translated into proteins. The group is highly Crispin Miller interdisciplinary, and uses computer science, mathematics, and noncoding transcripts’ behaviour is the way Hypoxia has a major influence on the cell, and Scientific Officer computational biology to generate new hypotheses that we they fold in three dimensions. We are affects numerous aspects of cancer biology Keren Dawson explore experimentally at the bench. combining these with techniques from including down regulation of DNA repair machine learning and using these to infer pathways, promotion of pro-survival Postdoctoral Fellows the molecules with which lincRNAs interact. phenotypes and increased proclivity for invasion Jing Bi Although less than 2% of the human genome highly collaborative, with members of the group and metastasis. In a study published this year we Laura Bennett Garima Khandelwal encodes amino acids, about 70% of it is embedded in with collaborators in other parts High Performance Computing showed using cell lines that tumour hypoxia is Chang Kim transcribed into RNA, even though these of the Institute. As the group has grown, it Many of the analyses we perform are associated with genome-wide changes in transcripts are never translated into proteins. became appropriate to bring everyone together, computationally demanding, and we make alternative splicing (Memon et al. 2016). Graduate Students This pervasive transcription is a relatively recent and this year we were able to move into our own substantial use of the Institute’s ~2,000 core Interestingly, many of the genes that changed Sam Humphrey discovery, enabled by rapid advances in lab space. High Performance Computing (HPC) facility to were associated with the DNA damage Mairah Khan microarray and deep sequencing technologies. Andrew Jenkins1 perform these intense calculations. We are response, and switched from expressing a As a result, the vast majority of these transcripts The majority of our work is focused on lung developing novel software that makes use of protein coding mRNA to expressing a noncoding Danish Memon1 Ronnie Rodrigues Pereira2 have yet to be assigned a function. Those that cancer, through our involvement in the CRUK MapReduce to parallelise calculations across the splice variant instead. In many cases, this have been studied, however, have been shown Lung Cancer Centre of Excellence, and through HPC system, while other computational happened through a process of intron retention, Clinical Fellow to perform a wide variety of roles, acting in all our computational work, we also have biologists in the group are exploiting access to in which an intron that is usually removed from Matt Howell2 compartments in the cell and functioning significant collaborations with other groups in the large amounts of RAM (> 2TB) provided on the transcript is no longer spliced out. We then through a broad range of interactions with the Institute, including an extensive collaboration some of our compute nodes to build enormous showed that this allows cells to shut down 1 left in 2016 proteins, with DNA, and with other RNA with the Clinical and Experimental 2 joined in 2016 data structures and explore them efficiently. protein expression, revealing for the first time molecules. Pharmacology group (page 20). Recently Ron that alternative splicing into a noncoding isoform Rodrigues Pereira joined us as a PCUK funded Alternative splicing in hypoxia can act as a novel way of regulating gene A major challenge when studying noncoding graduate student to study the role of noncoding When a gene is transcribed, the nascent RNA is expression. This has the potential to increase the RNAs (ncRNAs) experimentally is that the wide RNAs in prostate cancer, in collaboration with often processed to systematically remove number of regulatory pathways that impinge on variety of functions that they can perform is the Prostate Oncobiology group (page 30). certain portions of the transcript, called introns, a gene, and to change the kinds of changes we accompanied by an increasingly diverse set of and to splice together the remaining sections, look for when we are studying the effects of possible experiments that might be used to Computational approaches to long known as exons. This process allows genes to cancer on the expression patterns within a characterise them. We are developing novel noncoding RNAs produce more than one transcript, depending tumour. computational approaches to help us make A major class of ncRNAs are long intergenic on which introns are spliced out of the predictions about how a noncoding RNA might noncoding RNAs (lincRNAs). These are >200 sequence, and to express different splice- We then looked at RNA-sequencing data from function, and to predict which biological nucleotides in length and expressed from genes variants at different times, and in different patient samples from The Cancer Genome Atlas, processes it might be involved in. This allows us that are spatially distinct from protein coding conditions. The majority of human protein- by building transcript models from scratch and to narrow down the set of experiments we might genes. Many of these transcripts are 5' capped, coding genes are alternatively spliced, potentially using these to ask whether we saw the same then perform to study an interesting ncRNA in spliced, 3' poly(A)-tailed and differentially adding considerable functional diversity to the patterns in real tumours as we had seen in the more depth. By combining these approaches expressed. We are using mathematical set of proteins in a cell’s repertoire. cell lines. These data revealed widespread with the analysis of RNA-sequencing data from techniques from graph theory to build complex changes from coding to noncoding patient samples, we are able to prioritise our networks, and to provide a principled way in We are interested in how splicing patterns might transcription, and the same characteristic analyses towards ncRNAs of most relevance which to analyse changes in lincRNA expression. change in tumours and, in particular, how they pattern, in which many DNA damage response in cancer. These techniques from Big Data analysis, more are altered as cells respond to a lack of oxygen (a genes switch to expressing a noncoding RNA as commonly applied to social networks, have condition known as hypoxia). This is important their major isoform. This was associated with Having identified candidate ncRNAs through allowed us to start to predict lincRNA function, because the majority of solid tumours are poor patient outcome. our in silico work, we then pursue them helping when we select them for detailed hypoxic; a condition that is associated with poor experimentally at the bench. Approximately mechanistic study at the bench. We are also patient outcome and resistance to radiotherapy. Publications listed on page 64 50% of the group is now bench-based. The developing novel approaches to investigate RNA bench work within the group has always been structure, since a major determiner of 32 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE RNA BIOLOGY 33


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    Figure 1: Diagram showing JNK signalling SIGNALLING NETWORKS pathway in colon cancer cells expressing MLK4-WT versus cells expressing MLK4-LOF mutant. IN CANCER MLK4-WT activates JNK signalling and downstream targets including p21 and p15, which might lead to inhibition of proliferation or OIS (oncogene- induced senescence). LOF mutations in MLK4 block Cancer genomic sequencing has significantly impacted our MLK4-WT activation and JNK pathway signalling, which results understanding of the temporal and spatial genetic alterations that in overcoming OIS and increased proliferation of colon cancer lead to tumorigenesis. This information enables the development of cells. targeted therapies that result in durable and less toxic responses in patients. In regard to kinases, the biomedical community has focused research efforts on approximately 200 kinases among the Group Leader 538 kinases present in the human kinome, yet siRNA screens and John Brognard1 cancer genomic studies indicate that the vast majority of these Postdoctoral Fellows unexplored kinases (approximately 300) are implicated in cancer Natalie Stephenson1 and harbour putative driver mutations. required to maintain tumorigenic phenotypes, summary, our results suggest Pedro Torres-Aryuso1 we reconstituted its signalling axis in colon that NSCLC patients with ABL1 mutations cancer cells harboring MLK4-inactivating could be stratified for treatment with imatinib Clinical Fellow The major focus of our research is to elucidate novel tumour suppressing kinases in colon and mutations. We found that restoring MLK4 activity in combination with other therapies. Andrew Hudson1 novel cancer-associated kinases in the lung cancer that include MLK4 and DAPK3. In a reduced cell viability, proliferation, and colony Senior Scientific Officer unexplored kinome, guided by bioinformatics second approach we use genetic dependency formation in vitro and delayed tumour growth in We are continuing to focus on novel druggable Eleanor Wendy Trotter1 and functional genomic approaches, with screens to identify mutationally activated drivers vivo. Mechanistic investigations established that drivers of lung cancer with a focus on resident an overarching aim of understanding the of lung cancer. Targeted genetic dependency restoring the function of MLK4 selectively genes on the 3q amplicon in lung squamous cell Scientific Officer induced the JNK pathway and its downstream carcinoma and novel genetic dependencies in Zoe Edwards1 molecular mechanisms utilised by these kinases screens are an effective way to uncover low to promote tumorigenesis. Through use of in frequency oncogenes that can serve as targets targets, cJUN, ATF3, and the cyclin-dependent KRAS positive lung adenocarcinomas. vivo patient derived xenograft mouse models, for therapeutic intervention for tumours of any kinase inhibitors CDKN1A and CDKN2B. Our Graduate Students Ewelina Testoni1 we will translate these findings to the clinic and origin. Specifically we identified FGFR4, PAK5, work indicates that MLK4 is a novel tumour- Publications listed on page 65 encourage drug development programs and MLK1 as kinases that harbour novel gain of suppressing kinase harbouring frequent LOF 1 left in 2016 focused on these novel targets. The overall goal function (GOF) mutations in lung cancer patients mutations that lead to diminished signalling in Figure 2: Structural analysis of of our research is to provide a platform for and these mutations result in hyperactivation of the effects of movements in the JNK pathway and enhanced proliferation in transformational research to identify novel the MEK/ERK pathway. The mutation frequency the glycine rich motif. colon cancer. druggable drivers so that the vast majority of for the genes we identified ranged from 2-10% of The likely position of ATP (dark blue sticks) and Magnesium Somatically mutated ABL1 is an actionable and cancer patients can begin to benefit from lung cancers; given the frequency of lung (green sphere) within MLK4 as essential NSCLC survival gene precision medicine based targeted therapies. cancer in the population, these targets could be determined by aligning the Collectively this research should identify new exploited by pharmaceutical companies for The lack of actionable mutations in patients with structures to human CDK2 genetic drivers, targets for therapeutic drug discovery development. non-small cell lung cancer (NSCLC) presents a crystalised with ATP present (pdb intervention, and novel mechanisms of accession code: 1HCK). Images significant hurdle in the design of targeted tumorigenesis. show the change in position of therapies for this disease. We identified Recurrent MLK4 loss-of-function mutations mutants (A) E314K (shown in red), somatically mutated ABL1 as a genetic (B) H261Q (shown in pale blue), dependency that is required to maintain NSCLC The lab utilises a multitude of strategies to suppress JNK signalling to promote colon (C) H261Y (shown in blue) and (D) identify critical pathways required to promote tumorigenesis cell survival. We demonstrated that NSCLC Y330H (shown in orange) tumorigeneis. These include high-throughput The lab is also interested in MLK4, which is a cells with ABL1 mutations are sensitive to ABL compared to the WT structure bioinformatics and structural modelling, siRNA member of the mixed-lineage family of kinases (shown in black). Facing view (i) is inhibitors and we verified that the drug-induced screening, and precision genome editing to that regulate the JNK, p38, and ERK kinase rotated 90° counter clockwise to effects on cell viability are specific to establish various functional genomic signalling pathways. MLK4 mutations have been give side view (ii). Images pharmacological inhibition of the ABL1 kinase. approaches to identify novel drivers. Utilising identified in various human cancers, including produced using PyMol. Furthermore, we confirmed that imatinib bioinformatics we identify novel kinases frequently in colorectal cancer, where their suppresses lung tumour growth in vivo, enriched for functional mutations to hone in on function and pathobiological importance have specifically in lung cancer cells harboring a activated enzymes that can serve as drug targets. been uncertain. We assessed the functional gain-of-function mutation in ABL1. Consistent We then assess the structural consequences of a consequences of MLK4 mutations in colon with structural modelling, we demonstrated that subset of mutations in the respective kinases, tumorigenesis. Biochemical data indicated that a mutations in ABL1 identified in primary NSCLC where crystal structures are available, to majority of MLK4 mutations are loss-of-function tumors and a lung cancer cell line increase determine if the mutations likely increase or (LOF) mutations that can exert dominant- downstream pathway activation compared to decrease catalytic activity. These approaches negative effects. In seeking to understand the wild-type ABL1. Finally, we observe that the ABL1 have been successful in identifying kinases with abrogated activity of these mutants, we cancer mutants display an increased cytosolic activating mutations in lung cancer (ABL1 – elucidated a new MLK4 catalytic domain localisation, which is associated with the Testoni et al EMBO Mol Med. 2016), as well as structure. To determine whether MLK4 is oncogenic properties of the ABL1 kinase. In 34 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE SIGNALLING NETWORKS IN CANCER 35


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    www.cruk.manchester.ac.uk/Research/CRUK-MI-Groups/Skin-Cancer-and-Ageing/Home SKIN CANCER AND AGEING Skin cancer predominantly affects the ageing population and the prognostic factors into account there is a survival Biology of tumours and adjuvant care of discrepancy between elderly and young elderly patients majority of skin cancer deaths affect people who are older than 50. patients, and age is the strongest independent The salient features that define a poor outcome Although most deaths are due to melanoma, skin cancers of other adverse prognostic factor together with tumour in tumours from aged patients are currently thickness. Importantly, older patients are more poorly understood. We are defining the cell origins also affect overwhelmingly the aged population. This likely to suffer from multiple melanocytic and epidemiological, clinical, microenvironmental subgroup of patients has a particularly poor outcome and the non-melanocytic skin cancers, which and genetic features that characterise high-risk reasons underlying this adverse prognosis are poorly understood. underscore the need for tailored, specialist tumours to better stratify disease, guide care in this high-risk group. prevention and follow-up care. Old patients Group Leader who have had a primary melanoma removed Amaya Virós1 The Skin Cancer and Ageing group studies the patients who are older than 50 years of age, and Susceptibility to skin cancer in the elderly host and are non-metastatic are at high risk of disease mechanisms that drive poor outcome in the mortality is specifically increasing in the elderly. The environmental risk factor for skin cancer is progression, but there are currently no approved Postdoctoral Fellow ultraviolet radiation (UVR), which increases risk to approaches of adjuvant therapy for this cohort. elderly population. Skin cancer morbidity and Melanomas in this patient group more frequently Katharina Röck 1 mortality represents a critical current problem present as aggressive primary lesions - thick neoplasia of keratinocytic lineage in a linear We are developing approaches of secondary Graduate Student in health care. We focus our research on primary tumours that predominantly comprise association to lifetime UVR exposure. By prevention to delay or inhibit skin cancer Matthew Winder1 understanding the changes in aged skin that the high-risk primary melanoma stages (Stage contrast, the relationship between UVR and recurrence to improve the care of elderly promote skin cancer progression and the IIB-IIC). The overall survival for stage IIB-IIC melanoma is multifaceted, with a less clear-cut patients, who are at highest risk of disease 1 joined in 2016 biology of aggressive disease that affect the patients of all ages at 5 years is 60% and 45%, correlation between total UVR exposure and progression. elderly to identify new strategies of adjuvant respectively, despite being localised to the skin higher incidence of disease. Importantly, UVR therapy. and non-metastatic at diagnosis. There is a confers higher risk of melanoma progression gradual decline in 5-year survival with increasing once the disease is established. As elderly Unique features of skin cancer in the aged decades of life, with an almost 20% decrease patients are the least likely to survive skin cancer, population from ages 60 to 69, up to ages greater than 80 we are addressing how the interaction between Cancer is more common in elderly patients years. Additional characteristics of poor aged skin and the environment modulates skin and melanoma incidence continues to rise prognosis, such as ulceration and elevated cancer initiation and progression. predominantly in this population. Approximately mitotic rate, are also more common in the 80% of melanoma deaths in the UK occur in elderly. However, even after taking the main Figure 1: Age-specific melanoma mortality rates. Data prepared by CRUK 36 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE SKIN CANCER AND AGEING 37


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    $RUWLF /XPHQ www.cruk.manchester.ac.uk/Research/CRUK-MI-Groups/Stem-Cell-Biology/Home $RUWLF /XPHQ bound by GFI1 and/or GFI1B. Notably, the &'581;*),*),% (QGRWKHOLDO &HOOV resulting list of candidates contained genes &'581; involved in cardiovascular development, blood STEM CELL BIOLOGY &'581;*),*),% (QGRWKHOLDO &HOOV &'581; vessel maintenance and remodelling. Altogether our findings suggest that during the emergence of HSCs, GFI1 and GFI1B epigenetically silence &'581;*),F.,7 the endothelial programme to allow the &'581;*),F.,7 acquisition of a round non-adherent cellular morphology and the release of newly formed Gfi1ko/Gfi1Bko Gfi1het/Gfi1Bko Gfi1het/Gfi1Bhet Gfi1ko/Gfi1Bhet blood cells into the circulation. Gfi1ko/Gfi1Bko Gfi1het/Gfi1Bko Gfi1het/Gfi1Bhet Gfi1ko/Gfi1Bhet RUNX1 isoforms in normal and malignant Genes encoding the AML1/RUNX1 transcription factor and its haematopoiesis cofactor CBFβ are frequently rearranged or mutated in human As with other mammalian Runx genes, Runx1 leukaemias such as acute myeloid leukaemia (AML) and acute has two promoters, P1 (distal) and P2 (proximal), which generate the distinct protein isoforms lymphoblastic leukaemia (ALL). Consistent with its implication embryonic lethality observed in the absence of RUNX1C and RUNX1B respectively. The activities in leukaemia, RUNX1 has also been shown to be critical for Figure 1: GFI1 and GFI1B are RUNX1. We hypothesised that the lack of an and specific relevance of these two promoters critical for the EHT giving rise to early phenotype might be due to a functional in adult haematopoiesis remain to be fully haematopoietic development. HSCS. Top. Schematic compensation for the loss of one gene by the elucidated. Utilising a dual reporter model we representation of the expression other. Indeed, the two GFI1 and GFI1B proteins Group Leader demonstrated that the distal P1 promoter is of RUNX1, GFI1, GFI1B and c-KIT exhibit very high levels of homology in their Georges Lacaud Similarly, the transcriptional co-activator MOZ the gain of haematopoietic cell surface marker during the formation of functional domains and were previously shown broadly active in adult haematopoietic stem and is involved in recurrent independent myeloid expression. In the AGM, the EHT process results haematopoietic clusters in the progenitor cell (HSPC) populations. By contrast Postdoctoral Fellows dorsal aorta. Bottom. to be functionally interchangeable in the adult the activity of the proximal P2 promoter, and chromosomal translocations in human in the generation of haematopoietic clusters in Julia Draper Immunohistochemistry on haematopoietic system. We therefore examined RUNX1B expression, is more restricted and its leukaemia. Our group studies the function the dorsal aorta containing cells with a HSC Kiran Batta Gfi1ko/Gfi1bko,Gfi1het /Gfi1bko, the consequences of deleting both proteins upregulation in both the immature Lineage- of RUNX1 and MOZ in haematopoietic phenotype. More recently, HE cells have been Roshana Thambyrajah Gfi1het /Gfi1bhet and Gfi1ko/ using Gfi1 and Gfi1b GFP knock-in mice. In the development and maintenance in order to shown to also give rise to blood generated in the Sca1high cKithigh (LSK) and bipotential Pre- Michael Lie-A-Ling Gfi1bhet E10.5 AGMs for CD31 double knockout embryos, strong defects in the Anne Largeot better understand how alterations of these yolk sac, a process that is recapitulated in vitro (red), GFI1 and GFI1B (green) and Megakaryocytic/Erythroid Progenitor (PreMegE) EHT were observed; yolk sac derived blood cells populations coincides with a loss of erythroid functions might lead to leukaemogenesis. during ESCs culture system. Although HE has c-KIT (yellow). Scientific Officer were absent from the circulation and specification. Accordingly the PreMegE now been clearly established as the cellular Rahima Patel haematopoietic clusters were not observed population can be prospectively separated Clinical needs for blood cell populations source of the first HSCs in vivo, the molecular in the AGM (Figure 1 bottom). Instead, we into “pro-erythroid” and “pro-megakaryocyte” Bone marrow transplantations are well- and cellular mechanisms orchestrating this Graduate Students found GFP+ cells accumulating in the yolk sac populations based on P2 activity. Prospective Renaud Mevel established cellular therapies for the treatment intriguing trans-differentiation remain largely vasculature or embedded within the endothelial isolation of these two populations provides the Zaki Fadlullah1 of a variety of haematopoietic malignancies, unknown. One important clue in understanding lining of the dorsal aorta, demonstrating opportunity to further investigate and define leukaemia or genetic haematopoietic disorders. this process was provided by the observation the critical requirement for GFI1 and GFI1B in the molecular mechanisms involved in 1 joined in 2016 The success of these transplantations relies on a that the transcription factor RUNX1 is critical for the EHT. megakaryocytic/erythroid (Mk/Ery) cell fate rare population of haematopoietic stem cells the generation of blood cells by EHT. In the (HSCs), which can reconstitute the entire blood absence of this transcription factor, HE cells do decisions. GFI1 and GFI1B have been shown to repress and immune system cells. However, a major not lose their endothelial identity nor do they transcription in a MEL (murine erythroleukemia) Recent studies have revealed that in addition to a restriction to the wider application of these acquire a haematopoietic fate. Figure 2: RUNX1B might promote cell line, by recruiting the chromatin regulatory tumour suppressor role, WT AML1/RUNX1 is also curative treatments is the difficulty, or even the pre-leukaemia. A. AML mouse model expressing a doxycycline- CoREST complex containing the histone required for the promotion of leukaemogenesis possibility, of finding a compatible source of Taking advantage of this critical role of inducible AML1-ETO9a linked demethylase LSD1 (KDM1A). To investigate if this in certain leukaemia subtypes. Notably, AML1- donor tissue. The scarcity in matched donors RUNX1 in the EHT process, we identified the to a GFP. B. Induction of complex was involved in EHT, we examined the ETO-driven AML appears to be dependent on could potentially be overcome in the future by transcriptional repressors GFI1 and GFI1B as AML1-ETO9a for 8 days and consequences of LSD1 inactivation on this the provision of unlimited and renewable direct transcriptional targets of RUNX1 during maintaining some WT RUNX1 activity to protect FACs sort of cell population. C. transition. LSD1 genetic deletion or inhibition sources of HSCs from pluripotent stem cells the EHT. To investigate the relevance of these Upregulation of RUNX1 P2 the leukaemic cells from apoptosis. However, it impaired the emergence of round non-adherent is unclear whether AML1-ETO oncogene such as embryonic stem cells (ESCs) or patient- two proteins in the EHT, we evaluated their ability expression in AML1-ETO9a haematopoietic cells. Finally to identify direct expression promotes the expression of one derived induced pluripotent stem cells (iPSCs). to rescue this transition in Runx1-/- HE cells. (GFP+) expressing cells. D. RUNX1B might be involved in the transcriptional targets of GFI1 and GFI1B, we Runx1 promoter over another. To address this Furthermore, ESCs- or iPSC-derived HSCs We observed that ectopic expression of Gfi1, persistence and amplification of compared changes in global gene expression question, we utilised a novel AML mouse model could represent a cellular source to generate or Gfi1b, restored many features of the EHT pre-leukaemic cells. profiles upon LSD1 inhibition with lists of genes expressing a doxycycline-inducible various specific immune cells for adoptive process. The cells acquired a round non- cancer immunotherapy. The fulfillment of these adherent morphology and silenced the A C AML1-ETO9a linked to GFP (Figure 2) and studied A C the impact of AML1-ETO9a on Runx1 isoform promises relies on a better understanding of the expression of endothelial markers. To confirm molecular and cellular mechanisms underlying the association of GFI1 and GFI1B with the EHT in expression in vivo by isolating AML1-ETO- the development of the haematopoietic system. vivo, we analysed in detail their expression in the expressing (GFP+) and non-expressing (GFP-) mouse AGM region. We observed that Gfi1 and bone marrow. We analysed LSK, PreGM and New insights into the birth of haematopoietic Gfi1b were successfully expressed during the GMP cell populations as they contain the stem cells EHT process giving rise to haematopoietic leukaemia propagating cell fraction in Recent studies have established that the first clusters (Figure 1 top). Furthermore, we numerous AML patient samples. In all haematopoietic stem cells originate in the observed that transplantation of the endothelial populations, expression of AML1-ETO was intra-embryonic aorta-gonad-mesonephros cells expressing Gfi1 resulted in long-term correlated with a specific upregulation of Runx1 (AGM) region from a specialised endothelium, repopulation of irradiated recipient mice directly B D P2 expression. Our results suggest that RUNX1B i.e. a haemogenic endothelium (HE) through an demonstrating their HSC potential. Although B D activity generated by RUNX1 P2 promoter might Leukaemia specifically participate and support a pre- endothelial to haematopoietic transition (EHT). these findings suggest the importance of GFI1 Leukaemia leukaemic phenotype in emerging AML cells. The EHT process is characterised by the loss of and GFI1B in the EHT, none of the respective endothelial identity concomitant with the knockouts recapitulated the early block in acquisition of a round cellular morphology and haematopoietic development and early Publications listed on page 65 38 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE STEM CELL BIOLOGY 39


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    75% of adult ALL. Genes involved in B cell A B development such as EBF1, IKZF1, PAX5 or PBX1 are frequently found mutated in BCP-ALL. STEM CELL Additional cytogenetic abnormalities are frequently detected and often define specific HAEMATOPOIESIS subtypes of the disease with unique prognostic features. While childhood BCP-ALL is curable in most cases with a survival rate approaching 80%, the rate of cure for adult patients presenting with BCP-ALL is only 40%. Despite progress in the treatment of BCP-ALL, intensive chemotherapy regimens cause life-threatening complications During vertebrate ontogeny, blood, endothelium, smooth muscle and the outcome of those who relapse is poor. and cardiac tissue develop from the mesoderm germ layer to form There is a great need for the development of less the cardiovascular system. Whilst we are starting to understand short-term in vivo engraftment. Genome-wide toxic compounds and novel therapies for the analysis revealed that the enforced expression of treatment of relapsed and specific subgroups of better the molecular mechanisms underlying this developmental Figure 1: Sox7 in adult bone marrow cells impairs B cell BCP-ALL patients with poor prognosis. process, the overall network of transcription factors and signalling SOX7-expressing bone marrow cells retain an immature maturation while promoting a stem/progenitor signature. Given the impact of Sox7 expression in The deregulation of SOX gene expression has pathways governing cell fate decisions during cardiovascular morphology been widely documented in cancer, and a few (A) Cells isolated from wild type ex vivo cultures, we investigated whether Group Leader development remains largely unknown. The research in our bone marrow present the Sox7-enforced expression in vivo affects the studies have identified a direct involvement of Valerie Kouskoff1 laboratory aims at further understanding the regulatory network expected heterogeneous homeostasis of the haematopoietic system. Our SOX factors in tumorigenesis. The expression of morphologies of myeloid and findings demonstrated that in vivo the ectopic SOX7 is frequently down-regulated in solid Postdoctoral Fellows that orchestrates this developmental process. Many transcriptional erythroid cells. (B) Most bone expression of SOX7 led to B lymphopoiesis tumours such as prostate, colon and Alexia Eliades1 regulators implicated in haematopoietic specification during marrow cells expressing SOX7 present a homogenous impairment and expansion of haematopoietic endometrial cancers, but the functional Eva Garcia-Alegria1 relevance of these findings remains unknown. A Josh Lilly1 embryogenesis are also linked to leukaemogenesis events, often as immature and blast-like progenitors in the bone marrow associated with recent study has also revealed a tumour morphology. fibrosis, splenomegaly and extra-medullary Bioinformatician a result of aberrant expression. Through a better understanding of hematopoiesis. suppressor role for SOX7 in acute myeloid Zaki Fadlullah1 the function of these transcriptional regulators at the onset of leukaemia. In our study, we established that When ectopically expressed in blood SOX7 is specifically and frequently expressed in Graduate Students haematopoietic specification, we hope to gain insights into their progenitors, all SOXF factors were shown to alter human BCP-ALL, without an obvious association Genny Filiciotto potential role in the initiation and maintenance of haematological the balance between proliferation and with specific chromosomal abnormalities or Sara Menegatti cytogenetic subtypes. Furthermore, our data malignancies. Our work on the transcription factor SOX7 revealed differentiation. Given their known degree of demonstrated that the down-regulation of SOX7 redundancy, it is very likely that all three SOXF 1 left in 2016 that this factor is a critical regulator of embryonic development and factors activate a similar transcriptional program in BCP-ALL human cell lines induced a that its aberrant expression in B cell acute lymphoblastic leukaemia that promotes proliferation at the expense of significant decrease in proliferation, even though lineage differentiation. Based on the known SOX7 transcript levels were only decreased by is linked to enhanced proliferation and dissemination of the functions of the SOXF factors, two possible 50%. Finally, we showed that in vivo the down- leukaemic cells. explanations can be suggested for these regulation of SOX7 delays the onset of BCP-ALL observations: i) the transcriptional program and decreases the leukaemic burden, The SOX family of transcription factors is highly embryo-derived cells, or in in vitro differentiated activated by all SOXF in blood cells is similar to establishing the critical contribution of SOX7 conserved throughout evolution and in mouse embryonic stem cells, was shown to promote the one activated by SOX17 in foetal expression in maintaining the high proliferative comprises 20 members divided into subgroups the self-renewal of early blood progenitors haematopoietic stem cells which promotes the status of the leukaemic cells. based on sequence similarities. During harbouring endothelial-like features and to block active proliferation of these cells; SOX7 and embryogenesis, SOX factors are essential for further differentiation to committed lineages. SOX18 are able to activate this programme when Together, our study suggests that SOX7 the regulation of many developmental ectopically expressed in a blood-specific cell expression in BCP-ALL is an important factor processes. In addition to their critical roles during SOX7 expression promotes the expansion of context; ii) All three SOXF factors are expressed in contributing to leukaemia. It will be important in embryogenesis, SOX factors are also often adult blood progenitors and blocks B cell haemogenic endothelium and might in this future work to define the downstream implicated in the maintenance and identity of development cellular context induce a transcriptional programme regulated by SOX7 in BCP-ALL and stem cell populations. SOX7 and its two close Given the potential of SOXF factors in programme promoting proliferation, which can to identify the key pathways regulating the homologues, SOX17 and SOX18, belong to the maintaining the self-renewal properties of be induced in a blood-specific cell context upon proliferative potential controlled by SOX7. They SOXF subgroup and play important roles in embryonic blood progenitors, we hypothesised ectopic expression. The transcriptional targets might represent potential novel druggable cardiovascular development. The SOXF factors that the ectopic expression of SOX7 may also specifically activated by the SOXF factors to targets for the treatment of poor prognosis and have also been implicated in the development of confer a proliferative or survival advantage to regulate self-renewal are still currently unknown; relapsed BCP-ALL. Alternatively, the the haematopoietic system, the embryonic adult haematopoietic cells. Using a transgenic this will be an important avenue of investigation identification of small molecules that can origin of which is closely linked to endothelium inducible mouse model, we explored the for future studies. interfere with SOX7 transcriptional activity either development. We and others have consequences of SOX7 ectopic expression on through SOX7-DNA interaction or through demonstrated that the ectopic expression of adult haematopoiesis. First, we showed that the SOX7 promotes the maintenance and protein complex formation will represent SOXF factors in embryonic haematopoietic cells enforced expression of Sox7 ex vivo in adult proliferation of B cell precursor acute interesting avenues of investigation with leads to dramatic alterations in the balance bone marrow cells dramatically increases lymphoblastic cells potential therapeutic benefits. between proliferation and differentiation. Our proliferation and impairs the differentiation of B Acute lymphoblastic leukaemia (ALL) is the most group showed that Sox7 expression was up- lymphocytes. The enforced expression of Sox7 common cancer in children and comprises Publications listed on page 66 regulated in mesoderm precursors at the onset in bone marrow cells led to the ex vivo neoplastic precursor cells committed to the B of blood specification and down-regulated as maintenance and expansion of a progenitor cell (BCP-ALL) or the T cell (T-ALL) lineages. differentiation progresses to committed blood population able to give rise to myeloid, erythroid BCP-ALL represents the majority of ALL, lineages. The enforced expression of SOX7 in and lymphoid lineages and to provide accounting for up to 85% of childhood ALL and 40 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE STEM CELL HAEMATOPOIESIS 41


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    www.cruk.manchester.ac.uk/Research/CRUK-MI-Groups/Systems-Oncology/Home Nucleus S632 S414 S300 PTRF Y53 FUNCTIONAL LINK LMNA Nucleoskeletal S167 T302 Figure 1: VIME ER regulation PREDICTED S22 S78 S170 S176 KINASE ACTIVITY Contact-initiated signalling S5830 HSPB1 F125A TPD52 SYSTEMS ONCOLOGY Mitochondria between tumour cells (blue, top) ITPR3 AHNK S511 PROTEINS: Membrane S916 S3426 trafficking Involved in and endothelial cells (red, S184 S438 S104 cytoskeletal FMNL STK10 S519 Calcium signaling PEA15 Macrotubule remodeling bottom) regulate a number of STIM1 S226 KPCA S221 destabilization signalling pathways controlling S257 SVIL S63 Involved in S509 cell adhesion S1176 Rho pathway CAMKII S25 STMN1 processes such as cell adhesion, MYPT1 S1179 RHG35 S16 S1444 KTN1 Y187 PATHWAYS: cell migration and cell shape. The T153 TLN1 ARHG2 ARAP3 S2040 S425 MK01 S141 Signaling INCREASED receptor tyrosine kinase (EPHA2) S172 S151 Cell-ECM S85 PAK2 MAPK pathway Signaling DECREASED is specifically regulated in the interaction PAXI RAF signaling CLIP1/2 tumour cells upon endothelial LM2 S348 INTEGRINS PHOSPHOSITES: S1081 S1459 cell interaction, where the S831 S706 S1443 T1463 S734 S1166 S259 RAF1 FLNA Y772 S466 Core S2152 EGFR S43 The main aim of the Systems Oncology group is to understand activation loop phosphorylation CD97 CD44 IQGA1 S897 EPHA2 LIPB1 ITB4 PECA1 S160 phosphorylation is decreased (Locard-Paulet et al, EPHA2 S230 CTNB1 ITB4 ACVL1 HUVECs how signal transduction mechanisms are deregulated in tumour Sci Signal 2016). S2397 S135 S897 ZYX CTND1 S552 S1474 S1069 Core phosphorylation AHNK S583 S51 MAP4 cells, with a specific focus on delineating the role of the S5448 S1073 S143 FLNA BNIP2 PKN2 RHG01 S1179 S768 S2152 Rho pathway Membrane trafficking microenvironment. It is widely appreciated that solid tumours S462 Calcium signaling NOS3 NEST S114 Macrotubule P4K2A S2670 Y187 contain a multitude of infiltrating ‘normal’ host cells in addition to ER ITPR3 CAMKII S116 MK01 stabilization STMN1 Caveolae MAPK pathway KPCA S25 the malignant cancer cells. Such host cells form the cellular basis formation/regulation PTRF PEA15 S104 MK03 Y204 S82 Group Leader of the tumour stroma and consist of immune cells, endothelial S167 SUN2 Nucleoskeletal HSPB1 S12 regulation Mitochondria Nucleus Claus Jørgensen cells as well as fibroblasts. Postdoctoral Fellows perverse stromal infiltrate that makes up to 80% a consequence of PSC co-option. Initially we Kelly Broster1 Interactions between tumour cells and stromal This approach enabled the identification of of the tumour volume. This desmoplastic observed that activated KRAS in the tumour cells Brian Lee cells have been shown to modulate a number numerous receptors and signalling pathways reaction consists of a pathological remodelled leads to increased activation of the MEK-MAPK Giulia Veluscek2 Jingshu Xu2 of key processes such as tumour progression, that were specifically regulated in endothelial extracellular matrix and influx of fibroblasts and pathway, but not of the PI3K-AKT pathway. In metastasis, metabolism and response to therapy. and tumour cells as they interact (Figure 1). immune cells. The microenvironment has been contrast, inclusion of PSCs permitted tumour Scientific Officers However, while the functional importance of shown to contribute to therapeutic resistance, cells to activate additional pathways, where the Emma Newsham1 the tumour stroma is reasonably clear, the We observed that the receptor tyrosine kinase immune tolerance and tumour progression. number of regulated phosphorylation sites Xiaohong Zhang2 mechanisms whereby tumour cells co-opt EPHA2 displayed a decrease in the level of Importantly, very little is still known about the doubled. This included the activation of the stromal cells and the means by which coerced phosphorylation of the activation loop mechanisms whereby the tumour cells co-opt PI3K-AKT pathway. Interestingly, tumour cells Graduate Students stromal cells support tumour cells to facilitate (suggesting a decrease in the activity) specifically hosts cells to establish this hostile environment. expressing KRAS secrete abundant levels of the Amy McCarthy Colin Hutton malignant progression are not well understood. in tumour cells engaging with endothelial cells. Delineating these mechanisms is therefore morphogen sonic hedgehog (SHH), but are Delineating these mechanisms is essential to Interestingly, ablating EPHA2 in tumour cells, important and may lead to the identification of themselves insensitive to the ligand. However, 1 left in 2016 our understanding of the basic biology of or blocking the interaction between EPHA2 and Figure 2: novel therapeutic targets in both the tumour and PSCs are highly responsive to SHH and elicit a 2 joined in 2016 tumour progression, but also for the its cognate ligand Ephrin-A1, increased the Establishing a reciprocal stromal cells. response that includes increased production development of therapeutic strategies ability of tumour cells to adhere to and migrate signalling axis between and secretion of ECM proteins and the growth co-targeting the tumour stroma. across an endothelial monolayer. This was pancreatic cancer and stromal How do co-opted stromal fibroblasts regulate factors IGF-1 and GAS6. These ligands then confirmed in vivo, where cells deficient in EPHA2 cells (fibroblasts). Expression of tumour cells? engage cognate receptors on the tumour cells oncogenic KRAS in tumour Understanding how tumour cells escape the displayed increased retention in the lung To address how pancreatic cancer cells (PDAC and activate the PI3K-AKT pathway. Critically, cells leads to a cell autonomous vascular system during metastasis parenchyma, a typical site of metastasis. activation of MAPK signalling cells) co-opt resident fibroblasts, the pancreatic PDAC cells grown in the presence of PSCs also Tumour cell metastasis accounts for 90% of Together, these and other results led us to and secretion of SHH, which stellate cells (PSC), we used a co-culture system deregulate their metabolic pathways, where the cancer related deaths. As tumour cells escape propose a model for EPHA2-dependent engage fibroblasts in a non-cell where PDAC cells with an inducible mutant proteomic composition of the mitochondria and the primary tumour, enter the vascular system regulation of transendothelial cell migration: autonomous manner. Coerced KRAS (G12D) were directly co-cultured with ensuing function shifts dramatically. In addition, and exit to proliferate at the metastatic site, they contact between tumour and endothelial cells fibroblasts in turn elicit a naïve PSCs (Tape et al, Cell 2016). Using our tumour cells gain the ability to grow under interact with a number of host cells. In particular, leads to EPHA2 activation and a repulsion of reciprocal signal (GAS6 and recently implemented and optimised system for anchorage independent conditions and display IGF-1) to engage additional very little is known of the mechanisms whereby tumour cells. However, metastatic tumour cells long-term cell-specific labelling (Tape et al, Mol decreased levels of apoptosis. Blocking the signalling cascades in the tumour cells in the vascular system adhere to have developed mechanisms where EPHA2 is PDAC tumour cells (Tape et al, Cell Proteomics 2014) we then discerned signals exchanged between tumour cells and and cross the endothelial monolayer at the rapidly inactivated and switches repulsion into Cell 2016). cell-specific changes in tumour cell signalling as fibroblasts (SHH, GAS6 and IGF-1) normalises metastatic site. To address this, we developed a adhesion thus facilitating extravasation. the tumour cell function, suggesting that these co-culture system where tumour cells are plated pathways are context-dependent targets. More on top of an endothelial cell monolayer. As cells Tumour-stroma signalling in Pancreatic Ductal Tumour Cell Stromal Cell importantly, what these data highlight is that the interact, receptors between the two cell-types Adenocarcinoma Oncogenic Signalling Flux tumour cell function is drastically regulated by engage to regulate signalling pathways in a Pancreatic Ductal Adenocarcinoma (PDAC) has Reciprocal stromal elements (such as the fibroblasts) and cell-specific manner. Building on our previous a dismal prognosis with a median survival below that these elements should be included in our experience with cell-specific labelling six months and an average five-year survival model systems to gain a better understanding (Jorgensen et al, Science 2009), we applied below 5%. This is due to the aggressive nature of Non-Cell of putative therapeutic targets. methods to differentially label the proteome of the cancer, a lack of effective therapy and late Autonomous tumour and endothelial cells with isotopomeric diagnosis. The most frequently occurring Publications listed on page 67 versions of amino acids (SILAC labelling, Ong et genetic mutations have been identified with al, Mol Cell Proteomics 2002). We then activating mutations in the oncogene KRAS and Cell-Autonomous conducted an analysis of differentially regulated inactivation of the tumour suppressor CDKN2A G12D Cell-Autonomous KRAS Threshold signalling pathways in tumour cells that were in more than 90% of all cases and loss of SMAD4 either co-cultured with endothelial cells or left in and TP53 function occurring in 55% and 85% of Oncogenic Signalling Space solution (Locard-Paulet et al, Sci Signal 2016). all cases respectively. A hallmark of PDAC is a 42 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE SYSTEMS ONCOLOGY 43


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    www.cruk.manchester.ac.uk/Research/CRUK-MI-Groups/Transcriptional-Networks-in-Lung-Cancer/Home TRANSCRIPTIONAL NETWORKS IN LUNG CANCER Figure 1: KRAS-modulated RAS Lung cancer causes the most cancer-related deaths in the world and microRNAs. KRAS activation or GDP the main obstacle to a cure is chemoresistance. The main interest of overexpression induces the upregulation of microRNAs with our group is to identify the causes behind lung cancer development oncogenic function. Modulating GAP GEF and resistance to chemotherapy. Over the last decade, a growing KRAS-regulated microRNAs can stop KRAS-driven lung number of non-coding transcripts (ncRNAs) have been found to tumourigenesis. RAS have a pivotal role in gene regulation and cell biology. The most GTP Group Leader well- known ncRNAs are microRNAs (miRNAs), single stranded RNAs Michela Garofalo of 19–25 nucleotides in length, which negatively regulate gene Postdoctoral Fellows expression by translational inhibition or degradation of the mRNA microRNAs?? Srivatsava Naidu1 targets. miRNAs are differentially expressed in almost all types of Lei Shi human cancers versus the normal tissue counterpart and are key Scientific Officer Peter Magee players in cancer onset and progression, functioning as tumour promoters (TP) or tumour suppressors (TS). The application of Graduate Students RAF PI3K Ral Tiam1 Athanasios Paliouras miRNAs to cancer therapeutics and diagnostics is emerging as an GEF Manuela La Montagna2 important field of gene therapy. Thus far both miRNA replacement MEK AKT Ral RAC 1 left in 2016 and miRNA inhibition strategies have been successfully used to 2 joined in 2016 restore normal gene networks in vitro and in vivo, evidencing the ERKs mTOR NFKB PAK1 huge potential of microRNAs in the fight against cancer. PDGFRs axis cellular proliferation, migration and survival. Lung cancer ranks first in cancer morbidity and There are five different isoforms of PDGF that group is focusing on PDGFR-modulated tools in lung cancer (Figure 1). Enforced mortality rates globally. The most frequently activate cellular response through receptors microRNAs to better define this pathway and expression of either KRASWT or KRAS harbouring diagnosed histological sub-type, NSCLC, alpha (PDGFR-α) and beta (PDGFR-β). Both identify new targets for drug development and the G12D mutation (with an amino acid accounts for 80–85 % of cases, with a PDGF ligands and the receptors have been targeted therapy. substitution at position 12 leading to constitutive disappointing five-year survival rate of 15.9%. detected in lung cancer cells but not in normal activation of KRAS signalling) in NSCLC cells During the last decade, several effective targeted cells and are markers of worse prognosis. KRAS induced the expression of several microRNAs. therapies have been developed; Erlotinib, Targeting the PDGFR-α/β axis has proven useful The proto-oncongene RAS encodes three We are defining the molecular mechanisms Gefitinib and most recently Osimertinib and for the therapy of those cancers with alterations different GTPases: HRAS, NRAS and KRAS. RAS through which these KRAS-modulated crizotinib/ceritinib for patients with EGFR in such genes including point mutations and proteins switch between 'on' and 'off' microRNAs might be involved in lung activating mutations and ALK/EML4 gene fusions that result from chromosomal conformations that are mediated by the binding tumorigenesis using in vitro experiments translocations, respectively. However, rearrangements. In both cases, these alterations of GTP and GDP, respectively. The transition and in vivo mouse models. EGFR-mutant and ALK-rearranged cancers activate the kinase domain in the absence of the between these two states is regulated by constitute less than one-fifth of all NSCLC cases ligand, giving rise to a permanent signal for cell guanine nucleotide exchange factors (GEFs), and patients that initially respond well to therapy proliferation. Most gastrointestinal tumours which stimulate KRAS activation through GDP inevitably relapse a few months later. Thus, (GISTs) have activating mutations in the KIT for GTP exchange, and by GTPase-activating identification of other potential molecular receptor tyrosine kinase or PDGFR-α and proteins (GAPs), which catalyse the hydrolysis of targets and novel therapeutic approaches is of respond well to imatinib. However, in NSCLC, GTP to GDP to switch off KRAS signalling (Figure primary importance. It is now accepted that PDGFRs lack the genetic change that activates 1). Mutations in KRAS are very frequent in NSCLC NSCLC is not a singular entity but a the kinases, and as a consequence they do not (~30%) and are markers of worse prognosis in heterogeneous disease and therefore requires respond to treatment with imatinib. Therefore, lung adenocarcinoma harbouring K-Ras targeted therapies. In this regard, a pathway that further understanding of the molecular mutations, but so far no specific drug has holds significant promise involves platelet- mechanisms involved in the PDGFR axis may demonstrated efficacy. One of our current derived growth factors (PDGFs) which bind to provide the crucial information necessary to interests is to identify K-RAS-regulated PDGF receptors resulting in the activation of a improve its targeting strategies and to develop microRNAs that, by targeting molecules involved downstream signalling cascade that promotes new drug families with synergistic effects. Our in the RAS pathway, can be used as therapeutic 44 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE TRANSCRIPTIONAL NETWORKS IN LUNG CANCER 45


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    CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES 46 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE 47


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    www.cruk.manchester.ac.uk/Research modalities and to train scientists to apply these approaching a thickness of 1mm have become techniques to their research. The technologies commonplace for in vitro assay functions such available cover: flow cytometry cell sorting and as phenotypic screens for drug discovery. RESEARCH SERVICES analysis; widefield and confocal microscopy; two-photon microscopy; high content screening; super resolution imaging; X-ray Working in collaboration with the Histology facility, tissue screening and its analysis has been sources; image processing and analysis; and a huge undertaking over the year as automated support for in vivo imaging. histology imaging via cameras and spectrophotometry techniques has resulted in Over the year, new equipment has been the processing of over 12,000 slides. As a introduced in response to the scientific demand consequence, the Imaging and Cytometry of our research groups for increasingly more facility has responded by introducing a Maintenance of cutting edge core facilities requires constant novel imaging processes and to be able to consultancy service for the analysis and acquisition of new equipment as technologies continually develop extend or advance pre-existing methods, informatics of tissue to enable researchers to new capabilities. The following section describes some of the new including an additional automated scanning apply suitable analysis routines and to collate the system for high resolution histology (Leica subsequent data using informatics software. equipment that has been bought over the last year, but also Versa), a supplementary high content screening highlights a focus on continual development of how services are system (Perkin Elmer Operetta) for 3D organoid- Training has been another main feature of this based studies, and an automated flow cytometry year’s efforts; included in the training regime provided to meet the needs of the Institute. analysis system (BD Canto). has been: Chief Laboratory Officer Stuart Pepper Method development has been a major a) Over thirty staff trained in the image Chief Laboratory Officer from the imaging systems available to requirement this year to support new processing and analysis of histology data Stuart Pepper researchers on site. This in turn allows us to technologies including single cell sorting for using contrast based methods fully leverage the benefit of development molecular biology applications, serial section b) Implementation of flow cytometry sorting Over the last three years the Biological work described below that has been carried reconstruction for 3D histology, establishing advanced users’ course for ‘after hours’ usage Resources Unit (BRU) has seen a shift towards a out in the Histology facility. major screens of up to 70 plate assays, assistance c) Over forty researchers trained in flow more collaborative environment where work is in the application of CYTOF, high multiplexing cytometry analysis using traditional and carried out by BRU and research staff side by Towards the end of 2015 a new facility was for automated quantitative histology analysis, imaging methods side. This brings benefits both in an enhanced created to allow the development of novel development of in vivo analysis routines d) Over thirty users trained in image processing work environment and also in the efficient use of transgenic mouse lines. This facility is now fully (photons, acoustics and CT), screening tools for and analysis to aid high content screening the facility. As reported below we have seen a operational with several clones in development. the analysis of 3D cultures and confetti imaging. applications significant increase in the volume of work that Alongside the two other animal facilities e) Twenty-five researchers trained in the the facility can support. A similar approach has (breeding and experimental), the Institute is As researchers ask more complex phenotypic application of cell irradiation. now been implemented in the FACS facility, with now able to carry out all aspects of in vivo (cell behaviour, gene and protein expression, training offered to scientists so that they can run research from generation and breeding of interaction, morphology and function) questions As more complex techniques are being applied complex FACS experiments alongside facility novel transgenic lines through to dosing, of clinical samples, the requirement for multiple to the equipment, the facility members have also staff. Again this brings the joint benefit of surgery and imaging protocols as required by labels in flow cytometry analysis increases. In undergone several training courses and enhancing communications, and increasing the research programs. response, a critical part of the facility’s role is to meetings including screening, flow cytometry volume of work that can be accommodated in advise on the appropriate use of combinations panel design, imaging cytometry, management the facility. A final mention goes to the continual of markers. and good laboratory practice. development of Next Generation Sequencing Elsewhere we have seen changes that reflect the (NGS). NGS is established as a supremely The team has been complemented by the This year our work on the development of high increasing importance of support for data powerful tool in many areas of biology and appointment of Heather Woodhouse who content screening techniques was presented at analysis. The laboratory based facilities are protocols for routine sequencing of DNA and manages the day to day running of high content the American Association for Cancer Research benefitting from closer integration with the RNA are well established. More recently robust screening. These techniques have expanded Annual Meeting and two chapters have been Scientific Computing (Sci Com) and protocols for sequencing of single cells have considerably over the last five years as eighteen published discussing phenotypic screening and Computational Biology Support teams become available and this is expanding the research groups now utilise three systems. The microscopy. The facility also took part in over managed by Crispin Miller. In particular the Mass possibilities for this technology to map resulting complex data sets require sophisticated thirty lab tours for Cancer Research UK Spectrometry team are working closely with genomic and transcriptomic differences mathematical modelling of single cells and supporters and five talks were given to the bioinformaticians in the development of between individual cells. sub-populations for drug screening, 3D cultures general public. Data from the facility was also automated analysis pipelines for data sets and biomarker discovery applications. Working featured in many fundraising and research generated by the facility. For users of the facilities Advanced Imaging and Flow Cytometry in collaboration with Scientific Computing we engagement projects. this collaborative approach allows seamless Steve Bagley, Jeff Barry (Deputy Manager, Flow now have software systems in place for the integration between generation and analysis of Cytometry), Toni Banyard, Helen Carlin, Abi statistical analysis of phenotype across many As the facility both introduces and develops new data sets. In the Imaging facility one post is now Johnson, Isabel Peset Martin, Kang Zeng, millions of cells. technologies, its success is reflected in how the fully dedicated to support image analysis; with Heather Woodhouse1, Bogdan Potereas1 equipment has been employed over the year. the increased use of automated imaging systems 1 joined in 2016 Within the facility, there is an increasing demand Analysis shows that we have engaged with over some of the major challenges are now for in vitro applications that better mimic in vivo 270 users, in 240 different projects, where the connected to the collection, storage and The Imaging and Cytometry facility’s remit is to conditions hence the escalated requirement for equipment was in operation for over 32,000 processing of images. By providing more provide state of the art tools for both basic and confocal and multi-photon microscopy. As hours, which in turn generated over 120 support for image analysis, and working with Sci translational cancer studies (from molecular research projects move from 2D cellular studies Terabytes of data, leading to more than twenty Com on aspects of data management, the interactions in primary cells through to to 3D, and over time to 4D, there is a necessity to five papers featuring data generated in the facility is able to obtain the maximum benefit tissue-wide responses) to develop new develop tools for imaging thick samples in an facility. environmental context. Tissue sections 48 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES 49


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    Pancreatic tumour stained RESEARCH SERVICES (CONTINUED) with a stromal cell marker. Image supplied by Colin Hutton (Systems Oncology) Biological Mass Spectrometry Facility imported lines are now held at the Paterson Duncan Smith, Yvonne Connolly, John Griffiths1 Building, until re-derivation (via embryos) to the 1 left in 2016 Incubator Building is complete. Live mice are not brought into the Incubator Building directly to Our remit is to facilitate the access of research maintain the high health status of the facility and groups to protein characterisation by mass avoid possible contamination. spectrometry (MS). The use of MS, and affiliated technologies, has become widespread in Approximately 37 new lines were set up at the biological research in recent years due to its Incubator Building during 2016, mostly by analytical strengths when analysing protein crossing existing lines, but included nine newly biochemistry. MS approaches can be employed imported lines and seven lines created by to ask questions about many aspects of a embryo microinjections. Cryopreservation of protein’s life in the cell, including modulation of embryos and sperm (twice weekly) has resulted protein levels and post-translational in the archiving (closing of live colonies) for 47 modification (such as phosphorylation, lines during 2016. A further 22 live colonies were acetylation and ubiquitylation), site assignment closed in 2016 (cryopreservation not required). and quantification. The dynamic nature of the proteome makes it an ideal candidate for study Currently, the Transgenic Breeding facility has Biological Resources Unit our most commonly used strain of mouse in cancer progression as all molecular events around 145 breeding lines of genetically altered Experimental Services interacted with the swing 40% of the time during leading to disease biology are executed at the (GA) mice, cared for by 10 staff, for the Institute’s Team Leader: Lisa Doar the monitoring sessions, which represents a protein level. scientists. Although the numbers of lines have significant refinement and development for the decreased, the number of cages is increasing The last year has been an exciting one for the welfare of our mice. The facility has been used by the majority of (currently at approximately 2,300). This is due to experimental team with a steady increase in the groups in 2016 with the identification of protein smaller (maintenance) lines having been closed, number of people working in the facility and the Our plan for the coming year is to ascertain binding partners, phosphorylation site mapping with increased breeding for the newer lines, to number of projects completed. We have seen a whether we can refurbish our quarantine facility and label free protein profiling being particularly establish stock of appropriate genotypes for ~20% increase in the number of cages in the unit. to improve collaboration with other institutes popular. We benefit from having access to two experimental use. Additional services include One of the biggest challenges for this year has that have a different health status for working world-class LCMS platforms (a Thermo Orbitrap timed mating set up and the import and export been coping with and processing the number of with PDX models. These improvements will aim Fusion and Sciex 6600) that complement each of strains (exports in 2016 included Australia, training requests we have received from new to provide access opportunities to alternative other and allow us to perform proteomics Greece, Portugal and Argentina). licensees joining the Institute. We have imaging modalities based at other institutes in experiments in a wide applications area. established a new system and database to help the local area. The facility manages the genotyping service, manage the requests more efficiently and so far During the year, we implemented the migration where all samples are sent to an external this process is handling the increased training Transgenic Production Facility of data analysis pipelines onto high performance company (Transnetyx, USA) and genotype requirements well. Natalia Moncaut, Mark Willington, virtual machines in collaboration with the results are downloaded into the stock records. Athina Papaemmanouil1 Scientific Computing and Computational We have had another piece of imaging 1 joined in 2016 Biology Support teams. This will facilitate an Prompted by an increase in numbers of GA equipment installed into the unit this year – a enhanced level of data processing in addition to animals produced and used in recent years micro-CT scanner. This enables us to visualise The Transgenic Production Facility generates significant amounts of automation. The pipeline across the UK, the Home Office Animals in tumour growth in the lungs and bones and is new genetically modified mice working closely migration will continue into 2017. Science Regulation Unit (ASRU), in consultation now used on a weekly basis by two of the lung with the research groups and according to their with breeding experts and establishments cancer groups. As with all the other imaging specific needs. The service is responsible for the The facility has spent a significant amount of (including the BRU Transgenic Team), drafted an equipment in the facility, staff in the entire process of generating a transgenic mouse time investigating the utility of automating our assessment framework. As a guide for experimental team have been trained so that we model: from its initial design by employing the gel based sample preparation workflow this year. establishments to self-assess their GAA breeding can offer a quality service to the researchers. most appropriate targeting strategy, to the The process was successfully concluded at the practices and identify strengths and areas for generation of all required components: end of 2016 with the purchase of a new robotic improvement, the team have been included in a As part of our on-going commitment to keep microinjection, mouse surgery, screening of liquid handling system. The new system should ‘pilot’ (with five other establishments) with ASRU. the service up to date, members of the targeted events and breeding of founder mice. be installed in Q1 2017 and will significantly Within an initial meeting and facility tour in May experimental team have attended several Depending on the project type and its improve throughput and facilitate the and outcome review in October, of 51 points conferences and symposia again this year. We complexity, we offer two approaches to target a development of new protein preparation raised across 10 focus areas, including colony are also proud that one of our technicians won locus of interest. The first involves the workflows of the future. management, genotyping, team working etc., the Institute’s Annual 3Rs’ Prize (aimed at the co-delivery of CRISPR-Cas reagents into 28 were rated as satisfactory, 20 rated as replacement, reduction and refinement of embryonic stem (ES) cells and the second Biological Resources Unit ‘examples of excellent practice that can be animal work) with her poster on how she has approach employs the direct microinjection of Transgenic Breeding shared’ and three ‘relatively minor areas’ where improved practices for monitoring of ovarian CRISPR-Cas components into one-cell stage Team Leader: Kim Acton some improvement could be made – for which tumours, which is a fantastic achievement for mouse embryos. plans are being put in place. This excellent the team. Another important project that has The Biological Resources Unit (BRU) transgenic outcome is a great reflection on the organisation been carried out by one of our other technicians It has been an exciting and challenging first year mouse breeding facility is at The University of and functioning of the unit. is determining whether cable-tie ‘swings’ hung for the team. Since its inception we have Manchester’s Incubator Building. Only newly from the cage bars are a good form of established a new molecular biology laboratory enrichment for mice. The results showed that and an ES cell culture room in the Paterson 50 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE 51


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    to evaluate, develop and automate both mRNA evaluation DNA/RNA was extracted from 282 RESEARCH SERVICES (CONTINUED) in situ hybridisation and multiplex individual prostate samples from 36 patients. immunohistochemistry. We have offered these The quality was high, with median DIN 6.4 platforms as services and incorporated (2.4-8.7) and median RIN 7.2 (0-9) and the sophisticated labelling techniques into several amounts obtained were more than sufficient for Building. The animal work, based at The full range of both routine and advanced groups’ research programs. These techniques downstream analysis; median DNA recovered University of Manchester’s Incubator Building, histological services for oncology research. In allow researchers to gain more information 1.25ug (0.13-6ug) and RNA recovered 0.37ug has also been reorganised to ensure regular 2016, Histology continued to develop and about the relationships between cells from (0-4). These results led to very high quality provision of mouse embryos. During 2016 we expand. As the range and complexity of services different lineages on a single tissue sample or to sequencing data, which has in turn led to novel initiated seven fully customised projects offered continues to grow, the training and obtain gene expression information in the findings in prostate cancer genetics which are incorporating the different approaches. With the continued professional development of staff has context of tissue or cell morphology. currently being prepared for publication. increase in workload, a new member of staff was ensured the facility continues to offer a recruited to the team in September with comprehensive and flexible service at all times. In a collaborative project, a 6plex In a separate project somatic molecular profiling responsibilities in the molecular biology and cell Space has been acquired and equipment immunohistochemistry panel was evaluated, is being performed by NGS on approximately 110 culture area. purchased that has allowed us to begin whereby percentage staining in single-multiple resected thymomas. Thymoma is a rare establishing a laboratory that can operate to protocols was compared. Cell counts from five malignant tumour of thymic gland that New targeting technologies are changing GCP standards. A basic sample barcoding and areas on each slide were used to form constitutes 5 histological subtypes (type A, AB, completely the field of mouse production, tracking system (for nucleic acid extraction comparisons using linear regression for each B1, B2, and B3). Due to the rarity, molecular and consequently the field is evolving rapidly. across research groups and core facilities) was single-multiple signal. The results indicate that findings have been scarcely reported in the In order to keep on top of these new also implemented to complement our existing antibody order may have an effect on staining literature. A further complication is that this developments the Transgenic Production tissue labelling and barcoding system. quality in multiplexed immunohistochemistry group of tumours often contain heavy Facility team attended the annual International and, therefore, while multiplexed inflammatory cellular components, which are Society of Transgenic Technologies meeting as The facility routinely processes, embeds and immunohistochemistry is a promising concept, non-neoplastic in nature, particularly type B1 well as several national conferences, including a sections both human and mouse tissue in this effect needs to be borne in mind in group in which the vast majority of cellular CRISPR Conference at the Wellcome Trust addition to organotypic assays, spheroids, agar experimental design. components are non-neoplastic inflammatory Sanger Institute. plugs and cell pellets. Vibratome sections from cells. In order to extract optimal tumour DNA for fresh tissue (50–250μm) have also been One interest of the Stem Cell Biology group is to NGS, sections of type B1 thymomas were Histology prepared for the set up of ex vivo cultures of study the development of the blood system in immunohistochemically stained with pan- Garry Ashton, Caron Abbey, Adam Burke, tumours and to evaluate and develop three the mouse and to identify new regulators of this cytokeratin using clone AE1/AE3, and positive Emma Watson, Usman Mahmood1, Deepti dimensional studies. A number of research process. mRNA in situ hybridisation is currently tumour cells were dissected using laser-capture Wilks (Haematological Malignancy Biobank) groups have used several special stains, being used to study the expression of these microdissection. 1 joined in 2016 including Masson Trichrome, PAS and reticulin. genes. The group are also investigating the role of RUNX1 in prostate cancer. The Histology The use of our existing tissue microarray archive The Histology facility underpins the research The high throughput routine facility has provided extensive support in the and the construction of new arrays have again activities of a large number of both basic and immunohistochemistry service, troubleshooting development of multiplexed proved extremely popular. All TMAs give true translational research groups within CRUK MI. and antibody validation services have immunohistochemistry panels to study the sample representation and are of the highest It allows the adoption of tissue-based experienced exceptional demand this year. In expression of RUNX1, in the context of other quality. TMAs from disease groups including experimental approaches to all research addition, the availability of the new Leica and established markers, on patient tissue micro- breast, melanoma, prostate (cores and chips), programmes. The facility’s remit is to offer a Roche platforms has also allowed us to continue arrays constructed in the facility. Multiplex bladder, lymphoid, small cell and non-small cell immunohistochemistry is also being used for lung cancer plus mouse model and cell pellet the Molecular Oncology group to define controls are all available. Pancreatic stellate cells. subpopulations of lymphocytes infiltrating Image supplied by Colin Hutton frozen mouse melanoma tissue. The facility continues to be used heavily by the (Systems Oncology) CEP preclinical team and IHC biomarkers teams. The Prostate Oncobiology group have used CDX models are phenotyped routinely on our immunohistochemistry initially to characterise automated IHC platforms ensuring consistency, the lymphocyte antigen 6 complex, locus D reproducibility and standardisation. The Leica RX progenitor subpopulation in the mouse prostate. platform is also used to stain slides that are Following this, multiplex IHC is being used to scanned on the RareCyte platform. These slides characterise both FFPE and frozen organoid are stained with a cocktail of antibodies to samples (basal lineage, luminal lineage, identify circulating tumour cells (CTCs) in patient proliferation marker and functional response to blood samples. CEP has a collaborative stimuli by androgen receptor). Evaluation of the relationship with the Histology facility which acts potential role of this novel marker in prostate as an excellent platform for scientific discussion cancer patients’ outcome, using multiplex IF on and knowledge exchange. prostate cancer patients’ tissue-microarrays is ongoing in order to stratify patients and predict Alongside the work described above, the their clinical outcome. Histology facility also has a critical role supporting the MCRC Biobank. The two teams The microdissection/macrodissection of tissue have a close working relationship which means and downstream extraction of both RNA and that there is a seamless connection between the DNA, sufficient in quantity and quality for NGS collection, storage, quality control and from relatively small amounts of material is now processing of samples held by the Biobank. routine. In a recent project with the Molecular Oncology group, following pathological 52 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE 53


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    Pancreatic cancer-associated RESEARCH SERVICES (CONTINUED) fibroblast and cancer cell co-culture. Image supplied by Colin Hutton (Systems Oncology) Laboratory Services and after almost a decade of service, the existing Mark Craven, Tony Dawson, Corinne Hand, qPCR setup has been replaced by 2 ABI Petra Kubinova1, Jolanta Organisciak2, Quantstudio 5 (384-well) and 1 Quantstudio 3 Adriana Tudelo, Christine Whitehurst (96-well) instruments. Their impact has been 1 joined in 2016 2left in 2016 immediately felt by their ease of access and use, and fast and reliable performance. During 2016, Laboratory Services continued to Introduced in the previous year, the Compound support the research activities within the Library Service has seen a steady increase in Paterson and MCRC Buildings along with the demand in 2016. Supported by currently four Wolfson Molecular Imaging Centre (WMIC). We small to medium sized compound libraries, we staff dedicated departments within the Paterson assist drug screens by high throughput and MCRC Buildings, both containing dispensing of compounds using the Labcyte equipment for glass washing and autoclaving Access/Echo platform. The service is also items for the research groups to use. increasingly used in compound dosing experiments and combinatorial assays. Our primary role is to supply and deliver sterile The last year has also seen a continued rise in glassware, plastics, water and simple buffers to demand for Next Generation Sequencing (NGS), labs. We collect any dirty glassware and plastic which is demonstrated by the fact that over this ware twice a day and refill labs with new stock period all but one of the research groups used each day. this service. To reflect the versatility of the technology we have validated and adapted a Via our purpose-built clean room within the number of novel NGS applications, especially Paterson Building, we also offer a range of RNASeq methods, where we now offer solutions Scientific Computing data sources, such as sequencers or bacteriological media and agar plates across the for a wide range of inputs and sample qualities. Marek Dynowski1 , ZhiCheng Wang, microscopes, into the SciCom storage site. We manufacture these items using For example, due to the nature of the fixation Christopher Wirth, Christopher Smowton2 infrastructure – a prerequisite for establishing standardised recipes to maintain a consistent process the investigation of nucleic acids from 1 effective and highly-automated data lifecycle joined in 2016 2left in 2016 product but also prepare and adapt bespoke FFPE samples has been difficult. However, management. material if required by the research groups. This improved extraction methods and applications This year Marek Dynowski joined the Institute to year, we manufactured a total of 3500 litres of for library preparation have made it possible to A virtualisation infrastructure was introduced lead the Scientific Computing Team. The year liquid media, 3200 agar plates and sterilised exploit FFPE DNA and RNA for NGS. We have to optimise hardware usage, which led to a has been very productive, with the team 9500 litres of PBS. therefore validated appropriate methods and reduction in administrative effort and expense, introducing a number of new services, and added them to routine services for genome and while still providing new services such as a making continued improvements to the stability In other roles, we maintain and clean the two transcriptome analysis. central versioning repository for source code and efficiency of the high performance dark rooms operating film developers. We also computing (HPC) and storage systems. New job Gitlab. It allows programmers to keep track of organise the supply of clean lab coats for general Another focus of assay development has been their code while ensuring that code developed management software MOAB has been and tissue culture work as well as for visitors. The on workflows for high throughput RNASeq of by CRUK MI is centrally documented as well as installed, increasing both the reliability of the department oversees the monthly pipette clinics FACS sorted single cells. This application allows backed up and archived. system and the efficiency of job scheduling. In and the annual servicing of lab kit such as us to process hundreds of cells in one cooperation with the Estates team, several anemometers and personal oxygen monitors. experiment and is currently used, for example, Several pipelines developed by the changes were made to improve the stability of Additionally within the Paterson Building we for cell lineage tracing in haematopoiesis. Of Computational Biology Support team, the the system and drive it towards high availability. monitor and replace first aid and eye wash particular importance is the automated plate RNA Biology group and CEP were improved and These measures allow us to fully exploit the supplies and ensure all taps are opened as part of handling and liquid dispensing at very low accelerated through close cooperation with potential of our water-cooled machine room the Legionella testing programme each month. volumes. To achieve this we have exploited our Christopher Wirth and Christopher Smowton. and uninterruptible power supply. To improve assortment automation platforms, e.g. by using services when problems arise, or when new Molecular Biology Core Facility the Formulatrix Mantis for cDNA preparation, In the autumn, Snakemake was introduced as software needs to be installed, an IT Support Wolfgang Breitwieser, Chris Clark, Toni Grady, the Agilent Bravo for magnetic bead clean up our new workflow management system on the Ticket system has been set up and put into Emily Hulme1, Gillian Newton, Leanne steps, and the Labcyte Echo for cDNA HPC cluster Troodon, which, in combination production. This new system decreases Wardleworth, John Weightman quantitation and sequencing library preparation. with our new job management system, leads to response times for users of SciCom services and 1 Consequently the assay miniaturisation provided substantially increased throughput in our data joined in 2016 hardware and helps to optimise the SciCom by the automation also resulted in a significant analysis pipelines. support. reduction in the cost of processing per single While in the last year there remained a steady Two NVIDIA K40 graphic accelerators, which are cells. Computational Biology Support demand for a number of Molecular Biology Core proving especially useful for molecular dynamics Facility (MBCF) core applications, including simulations, were integrated into the job Hui Sun Leong, Sudhakar Sahoo, Sanger sequencing and microbe testing, Scientific Computing and Samuel Taylor1, Pieta Schofield1, management system and are now accessible to requests for other services such as cell line Computational Biology Support Shambhavi Srivastava1 all our users. authentication and sample QC (Bioanalyzer and 1 joined in 2016 These two services provide high performance Tapestation) have experienced a significant The first step in creating a fast 10GbE network computing and data analysis solutions for the increase. High throughput instrumentation for connection between SciCom and the CRUK MI The continual increase in the amount of Institute’s scientists and are overseen by quantitative PCR is a backbone of MBCF support Core Facilities was taken in cooperation with the experimental data derived from high-throughput Crispin Miller. IT department. It allows tighter integration of technologies has caused a paradigm shift in 54 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE 55


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    RESEARCH SERVICES (CONTINUED) biological research, with a shift from a We have also been actively expanding into new traditionally hypothesis-driven to a data-driven areas to help support scientific advances in other approach. Given the size and complexity of research technologies. For example, we are these big data, scientists are in need of rigorous working closely with the Molecular Biology Core and scalable methods to unearth the exciting Facility (MBCF) to develop approaches for knowledge captured within the data. The performing NGS on archival materials derived Computational Biology Support (CBS) team from formalin-fixed paraffin embedded (FFPE) helps researchers in the CRUK Manchester tumour samples, and to evaluate the potential Institute meet this challenge by providing use of FFPE samples in cancer genomics studies. expertise in data analysis of next generation sequencing (NGS) experiments, and other In 2017, CBS will be working with the Biological large-scale biological experiments that require Mass Spectrometry Facility and Systems bioinformatics support. Oncology group to develop dedicated workflows for analysing label-free quantitative CBS underwent a major expansion in 2016. With the recruitment of two new computational biologists, the group was able to extend its service provision beyond genomics data analysis proteomics, SILAC/iTRAQ and SWATH datasets generated by research programmes within the Institute. We will also be collaborating with the Scientific Computing team to develop CANCER RESEARCH UK to the processing of high-throughput automated NGS pipelines with improved proteomics datasets, as well as supporting the performance and throughput. bioinformatics needs of members from the Movember Prostate Cancer Centre of Excellence through the recruitment of an additional MANCHESTER Prostate Cancer UK funded member of staff. During the course of 2016, CBS collaborated with experimental researchers within the Institute on various NGS projects involving RNA-seq, small RNA-seq, ChIP-seq, genome-wide CRISPR/Cas9 knockout screens, whole-exome and whole-genome sequencing. For example, the team have been working closely with members from the Clinical and INSTITUTE Experimental Pharmacology group to develop dedicated data analysis workflows for detecting somatic mutations and copy number aberration PUBLICATIONS events in circulating tumour DNA samples and liquid biopsies. Some of the collaborative efforts AND ADMINISTRATION have contributed to high-impact publications (Carter L et al. Nat Med. 2016; Williamson SC et al. Nat Commun. 2016; Tape CJ et al. Cell. 2016; Draper JE et al. PLoS Genet. 2016). Staining of secretory luminal cells (CK8+) in glands of normal murine prostate. Image supplied by João Diogo Barros Silva (Prostate Oncobiology) 56 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE 57


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    RESEARCH PUBLICATIONS Santiago Zelenay (page 12) Chemical Fixation. Fission Yeast: a HUWE1 is a critical colonic tumour suppressor Other publications Cancer Inflammation and Immunity Laboratory Manual CSHLP 208-212. gene that prevents MYC signalling, DNA Marei H, Malliri A. (2016) damage accumulation and tumour initiation. Rac1 in human diseases: The therapeutic Other publications Hagan IM. (2016) EMBO Molecular Medicine, Epub 2016 potential of targeting Rac1 signaling regulatory Zelenay S, Reis E Sousa C. (2016) Staining Fission yeast Filamentous actin with December 21. mechanisms. Small GTPases, Epub 2016 Reducing prostaglandin E2 production to raise fluorescent phalloidin conjugates. Fission July 21. cancer immunogenicity. Oncoimmunology, Yeast: a Laboratory Manual CSHLP 213-216. Marei H, Carpy A, Macek B, Malliri A. (2016) Proteomic analysis of Rac1 signaling regulation Marei H, Malliri A. (2016) 5(5):e1123370. Hagan IM, Grallert A, Simanis V. (2016) by guanine nucleotide exchange factors. Cell GEFs: Dual regulation of Rac1 signaling. Small Analysis of the Schizosaccharomyces pombe Cycle, 15(15):1961-74. GTPases, Epub 2016 June 17. cell cycle. Fission Yeast: a Laboratory Iain Hagan (page 14) Manual CSHLP 271-282. Marei H, Carpy A, Woroniuk A, Vennin C, Porter AP, Papaioannou A, Malliri A. (2016) Cell Division White G, Timpson P, Macek B, Malliri A. (2016) Deregulation of Rho GTPases in cancer. Small Hagan IM, Grallert A, Simanis V. (2016) Differential Rac1 signalling by guanine GTPases, 7(3):123-38. Other publications Cell Cycle Synchronization of nucleotide exchange factors implicates FLII in Grallert A., Hagan I.M. (2016) Schizosaccharomyces pombe by Centrifugal regulating Rac1-driven cell migration. Nature Elementary protein analysis in Elutriation of Small Cells. Fission Yeast: a Communications, 7:10664. Schizosaccharomyces pombe. Fission Laboratory Manual CSHLP 283-290. Yeast: a Laboratory Manual CSHLP 119-121. Hagan IM, Grallert A, Simanis V. (2016) Mouse pancreatic intraepithelial Grallert A, Hagan IM. (2016) neoplasms stained with SMA Cell Cycle Synchronization of Preparation of Protein Extracts from (Red), LOX (Green), PDFR Beta Schizosaccharomyces pombe by Lactose (Grey) and DAPI (Blue). Schizosaccharomyces pombe Using Gradient Centrifugation to Isolate Small Cells. Image supplied by Trichloroacetic Acid Precipitation. Fission Fission Yeast: a Laboratory Manual CSHLP Haoran Tang (Molecular Yeast: a Laboratory Manual CSHLP 122-126. 291-294. Oncology) Grallert A, Hagan IM. (2016) Hagan IM, Grallert A, Simanis V. (2016) Small-Scale Immunoprecipitation from Synchronizing Progression of Fission Yeast Cell Extracts. Fission Yeast: a Schizosaccharomyces pombe Cells Laboratory Manual CSHLP 127-133. from Prophase through Mitosis and into S Phase with nda3-KM311 Arrest Release. Fission Grallert A., Hagan I.M. (2016) Yeast: a Laboratory Manual CSHLP 295-299. Large-Scale Immunoprecipitation from Fission Yeast Cell Extracts. Fission Yeast: a Hagan IM, Grallert A, Simanis V. (2016) Laboratory Manual CSHLP 134-140. Synchronizing Progression of Schizosaccharomyces pombe Cells from G2 Hagan IM, Bagley S. (2016) through Repeated Rounds of Mitosis and Fixed-Cell Imaging of Schizosaccharomyces S Phase with cdc25-22 Arrest Release. Fission pombe. Fission Yeast: a Laboratory Yeast: a Laboratory Manual CSHLP 300-304. Manual CSHLP 193-198. Hagan I.M. (2016) Immunofluorescence Microscopy of Angeliki Malliri (page 18) Schizosaccharomyces pombe Using Cell Signalling Chemical Fixation. Fission Yeast: a Laboratory Manual CSHLP 199-207. Refereed Research Papers Myant KB, Cammareri P, Hodder MC, Wills J, Hagan IM. (2016) Von Kriegsheim A, Győrffy B, Rashid M, Polo S, Chromatin and Cell Wall staining of Maspero E, Vaughan L, Gurung B, Barry E, Schizosaccharomyces pombe Using Malliri A, Camargo F, Adams DJ, Iavarone A, Lasorella A, Sansom OJ. (2016) 58 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH PUBLICATIONS 59


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    Inhibition of PI3K/BMX Cell Survival Pathway Gandara DR, Slotman BJ, Blackhall F, Goss G, RESEARCH PUBLICATIONS (CONTINUED) Sensitizes to BH3 Mimetics in SCLC. Molecular Thomas R, Rudin CM, Hirsch FR. (2016) Cancer Therapeutics, 15(6):1248-60. Small Cell Lung Cancer: Can Recent Advances in Biology and Molecular Biology Be Morrow CJ, Trapani F, Metcalf RL, Bertolini G, Translated into Improved Outcomes? Journal Caroline Dive (page 20) Carlsson F, Carlsson A, Hendrix MJ, Seftor RE, Hodgkinson CL, Khandelwal G, Kelly P, Galvin ofThoracic Oncology, 11(4):453-74. Clinical and Experimental Pharmacology Seftor EA, Rothwell DG, Hughes A, Hicks J, M, Carter L, Simpson KL, Williamson S, Wirth Miller C, Kuhn P, Brady G, Simpson KL, C, Simms N, Frankliln L, Frese KK, Rothwell Russell MR, Walker MJ, Williamson AJ, Refereed Research Papers Blackhall FH, Dive C. (2016) DG, Nonaka D, Miller CJ, Brady G, Blackhall Gentry-Maharaj A, Ryan A, Kalsi J, Skates S, Russell MR, D'Amato A, Graham C, Crosbie EJ, Vasculogenic mimicry in small cell lung cancer. FH, Dive C. (2016) D'Amato A, Dive C, Pernemalm M, Humphryes Gentry-Maharaj A, Ryan A, Kalsi JK, Fourkala Nature Communications, 7:13322. Tumourigenic non-small-cell lung cancer PC, Fourkala EO, Whetton AD, Menon U, EO, Dive C, Walker M, Whetton AD, Menon U, mesenchymal circulating tumour cells: a Jacobs I, Graham RL. (2016) Jacobs I, Graham RL. (2016) Gremel G, Lee RJ, Girotti MR, Mandal AK, clinical case study. Annals of Oncology, Protein Z: A putative novel biomarker for early Novel risk models for early detection and Valpione S, Garner G, Ayub M, Wood S, 27(6):1155-60. detection of ovarian cancer. International screening of ovarian cancer. Oncotarget. Epub Rothwell DG, Fusi A, Wallace A, Brady G, Dive Journal of Cancer, 138(12):2984-92. 2016 November 26 C, Dhomen N, Lorigan P, Marais R. (2016) Illidge TM, McKenzie HS, Mayes S, Bates A, Distinct subclonal tumour responses to therapy Davies AJ, Pettengell R, Stanton L, Cozens K, Testoni E, Stephenson NL, Torres-Ayuso P, Carter L, Rothwell DG, Mesquita B, Smowton revealed by circulating cell-free DNA. Annals of Hampson G, Dive C, Zivanovic M, Tipping J, Marusiak AA, Trotter EW, Hudson A, C, Leong HS, Fernandez-Gutierrez F, Li Y, Burt Oncology, 27(10):1959-65. Gallop-Evans E, Radford JA, Johnson PW. Hodgkinson CL, Morrow CJ, Dive C, Brognard DJ, Antonello J, Morrow CJ, Hodgkinson CL, (2016) J. (2016) Morris K, Priest L, Carter M, Miller C, Hughes A, Crosbie PA, Shah R, Krysiak P, Zhou C, Morris K, Short duration immunochemotherapy Somatically mutated ABL1 is an actionable and Blackhall F, Dive C, Brady G. (2016) Tugwood J, Booton R, Blackhall F, Dive C. followed by radioimmunotherapy essential NSCLC survival gene. EMBO Molecular analysis of circulating tumor cells (2016) consolidation is effective and well tolerated in Molecular Medicine, 8(2):105-16. identifies distinct copy-number profiles in Circulating Tumor Cells Detected in the relapsed follicular lymphoma: 5-year results patients with chemosensitive and Tumor-Draining Pulmonary Vein Are from a UK National Cancer Research Institute Girotti MR, Gremel G, Lee R, Galvani E, chemorefractory small-cell lung cancer. Associated with Disease Recurrence after Lymphoma Group study. British Journal of Rothwell D, Viros A, Mandal AK, Lim KH, Nature Medicine, Epub 2016 November 21 Surgical Resection of NSCLC. Journal of Haematology, 173(2):274-82. Saturno G, Furney SJ, Baenke F, Pedersen M, Thoracic Oncology, 11(10):1793-7. Rogan J, Swan J, Smith M, Fusi A, Oudit D, Zhou C, Clamp A, Backen A, Berzuini C, Weiskopf K, Jahchan NS, Schnorr PJ, Cristea Dhomen N, Brady G, Lorigan P, Dive C, Marais Renehan A, Banks RE, Kaplan R, Scherer SJ, Jahchan NS, Lim JS, Bola B, Morris K, Seitz G, S, Ring AM, Maute RL, Volkmer AK, Volkmer R. (2016) Kristensen GB, Pujade-Lauraine E, Dive C, Tran KQ, Xu L, Trapani F, Morrow CJ, Cristea S, JP, Liu J, Lim JS, Yang D, Seitz G, Nguyen T, Application of Sequencing, Liquid Biopsies, Jayson GC. (2016) Coles GL, Yang D, Vaka D, Kareta MS, George J, Wu D, Jude K, Guerston H, Barkal A, Trapani F, and Patient-Derived Xenografts for Systematic analysis of circulating soluble Mazur PK, Nguyen T, Anderson WC, Dylla SJ, George J, Poirier JT, Gardner EE, Miles LA, de Personalized Medicine in Melanoma. Cancer angiogenesis-associated proteins in ICON7 Blackhall F, Peifer M, Dive C, Sage J. (2016) Stanchina E, Lofgren SM, Vogel H, Winslow Discovery, 6(3):286-99. identifies Tie2 as a biomarker of vascular Identification and Targeting of Long-Term MM, Dive C, Thomas RK, Rudin CM, van de progression on bevacizumab. British Journal of Tumor-Propagating Cells in Small Cell Lung Rijn M, Majeti R, Garcia KC, Weissman IL, Sage Other publications Cancer, 115(2):228-35. Cancer. Cell Reports, 16(3):644-56. J. (2016) O'Connor JP, Aboagye EO, Adams JE, Aerts CD47-blocking immunotherapies stimulate HJ, Barrington SF, Beer AJ, Boellaard R, Williamson SC, Metcalf RL, Trapani F, Mohan S, Potter DS, Galvin M, Brown S, Lallo A, macrophage-mediated destruction of Bohndiek SE, Brady M, Brown G, Buckley DL, Antonello J, Abbott B, Leong HS, Chester CP, Hodgkinson CL, Blackhall F, Morrow CJ, Dive small-cell lung cancer. Journal of Clinical Chenevert TL, Clarke LP, Collette S, Cook GJ, Simms N, Polanski R, Nonaka D, Priest L, Fusi A, C. (2016) Investigation, 126(7):2610-20 deSouza NM, Dickson JC, Dive C, Evelhoch JL, Faivre-Finn C, Gallagher FA, Gilbert FJ, Kirwan CC, Bundred NJ, Castle J, Clarke R, Gillies RJ, Goh V, Griffiths JR, Groves AM, Dive C, Morris J, Holcombe C, Harvey JR. Halligan S, Harris AL, Hawkes DJ, Hoekstra Crystal structure of a potent (2016) OS, Huang EP, Hutton BF, Jackson EF, Jayson and cell-active inhibitor bound to the human PARG protein. PO-36 - Thrombin Inhibition Preoperatively GC, Jones A, Koh DM, Lacombe D, Lambin P, Image supplied by Bohdan (TIP) in early breast cancer, the first clinical trial Lassau N, Leach MO, Lee TY, Leen EL, Lewis Waszkowycz (Drug Discovery) of NOACs as an anti-cancer agent: trial JS, Liu Y, Lythgoe MF, Manoharan P, Maxwell methodology. Thrombosis Research, RJ, Miles KA, Morgan B, Morris S, Ng T, 140 Suppl 1:S189-90. Padhani AR, Parker GJ, Partridge M, Pathak AP, Peet AC, Punwani S, Reynolds AR, Robinson Bunn PA Jr, Minna JD, Augustyn A, Gazdar AF, SP, Shankar LK, Sharma RA, Soloviev D, Ouadah Y, Krasnow MA, Berns A, Brambilla E, Stroobants S, Sullivan DC, Taylor SA, Tofts PS, Rekhtman N, Massion PP, Niederst M, Peifer Tozer GM, van Herk M, Walker-Samuel S, M, Yokota J, Govindan R, Poirier JT, Byers LA, Wason J, Williams KJ, Workman P, Yankeelov Wynes MW, McFadden DG, MacPherson D, TE, Brindle KM, McShane LM, Jackson A, Hann CL, Farago AF, Dive C, Teicher BA, Waterton JC. (2016) Peacock CD, Johnson JE, Cobb MH, Wendel Imaging biomarker roadmap for cancer HG, Spigel D, Sage J, Yang P, Pietanza MC, studies. Nature Reviews Clinical Oncology, Krug LM, Heymach J, Ujhazy P, Zhou C, Goto Epub 2016 Oct 11 K, Dowlati A, Christensen CL, Park K, Einhorn LH, Edelman MJ, Giaccone G, Gerber DE, Swanton C, Soria JC, Bardelli A, Biankin A, Salgia R, Owonikoko T, Malik S, Karachaliou N, Caldas C, Chandarlapaty S, de Koning L, Dive 60 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH PUBLICATIONS 61


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    Mouse mammary tumour Active Patents/Filings RESEARCH PUBLICATIONS (CONTINUED) stained with FGF1 (Red), McGonagle EA, Jordan AM, Waszkowycz B, FGFR1 (Green), SMA (Grey) Hutton CP, Waddell I, Hitchin JR, Smith KM, and DAPI (Blue). Image supplied by Haoran Hamilton NM. Tang (Molecular Oncology) “2,4-Dioxo-Quinazoline-6-Sulphonamide C, Feunteun J, Leung SY, Marais R, Mardis ER, Identification of selective inhibitors of RET and Derivatives as Inhibitors of PARG” PCT Int. Appl. McGranahan N, Middleton G, Quezada SA, comparison with current clinical candidates WO2016092326, 16 June 2016. Rodón J, Rosenfeld N, Sotiriou C, André F. through development and validation of a (2016) robust screening cascade. F1000Research, McGonagle EA, Jordan A, Waszkowycz B, Consensus on precision medicine for 5:1005. Hutton C, Waddell I, Hitchin JR, Smith KM, metastatic cancers: a report from the MAP Hamilton NM. conference. Annals of Oncology, 27(8):1443-8. Hornyak P, Askwith T, Walker S, Komulainen E, “PARG Inhibitory Compounds” PCT Int. Appl. Paradowski M, Pennicott LE, Bartlett EJ, WO2016097749, 23 June 2016. Brissett NC, Raoof A, Watson M, Jordan AM, Donald Ogilvie (Page 24) Ogilvie DJ, Ward SE, Atack JR, Pearl LH, Stockley ML, Macdonald EC, Shah P, Jordan Drug Discovery Caldecott KW, Oliver AW. (2016) AM, Hitchin JR, Hamilton NM. Mode of action of DNA-competitive small “Autotaxin inhibitory compounds” PCT Int. Appl. Refereed Research Papers molecule inhibitors of tyrosyl DNA WO2016124939, 11 August 2016. Jones S, Ahmet J, Ayton K, Ball M, Cockerill M, phosphodiesterase 2. The Biochemical Fairweather E, Hamilton N, Harper P, Hitchin J, Journal, 473(13):1869-79. Jordan A, Levy C, Lopez R, McKenzie E, Packer Tim Somervaille (page 26) M, Plant D, Simpson I, Simpson P, Sinclair I, Jordan AM, Begum H, Fairweather E, Fritzl S, Leukaemia Biology Other publications Somervaille TC, Small H, Spencer GJ, Goldberg K, Hopkins GV, Hamilton NM, Lyons Maiques-Diaz A, Somervaille TC. (2016) Thomson G, Tonge M, Waddell I, Walsh J, AJ, March HN, Newton R, Small HF, Refereed Research Papers LSD1: biologic roles and therapeutic targeting. Waszkowycz B, Wigglesworth M, Wiseman Vishwanath S, Waddell ID, Waszkowycz B, Jones S, Ahmet J, Ayton K, Ball M, Cockerill M, Epigenomics, 8(8):1103-16. DH, Ogilvie D. (2016) Watson AJ, Ogilvie DJ. (2016) Fairweather E, Hamilton N, Harper P, Hitchin J, Discovery and Optimization of Allosteric Anilinoquinazoline inhibitors of the RET kinase Jordan A, Levy C, Lopez R, McKenzie E, Packer Deb G, Somervaille TC. (2016) Inhibitors of Mutant Isocitrate Dehydrogenase 1 domain-Elaboration of the 7-position. M, Plant D, Simpson I, Simpson P, Sinclair I, Antagonizing Self-Renewal in Acute Myeloid (R132H IDH1) Displaying Activity in Human Bioorganic & Medicinal Chemistry Letters, Somervaille TC, Small H, Spencer GJ, Leukemia: ID2 Takes the Stage. Cancer Cell, Acute Myeloid Leukemia Cells. Journal of 26(11):2724-9. Thomson G, Tonge M, Waddell I, Walsh J, 30(1):5-7. Medicinal Chemistry, 59(24):11120-11137. Waszkowycz B, Wigglesworth M, Wiseman Stowell AI, James DI, Waddell ID, Bennett N, DH, Ogilvie D. (2016) Truman C, Hardern IM, Ogilvie DJ. (2016) Discovery and Optimization of Allosteric James DI, Durant S, Eckersley K, Fairweather E, A high-throughput screening-compatible Inhibitors of Mutant Isocitrate Dehydrogenase Richard Marais (page 28) Griffiths LA, Hamilton N, Kelly P, O'Connor M, Molecular Oncology Shea K, Waddell ID, Ogilvie DJ. (2016) homogeneous time-resolved fluorescence 1 (R132H IDH1) Displaying Activity in Human An assay to measure poly(ADP ribose) assay measuring the glycohydrolase activity of Acute Myeloid Leukemia Cells. Journal of Refereed Research Papers glycohydrolase (PARG) activity in cells. human poly(ADP-ribose) glycohydrolase. Medicinal Chemistry, 59(24):11120-11137. Spender LC, Ferguson GJ, Liu S, Cui C, Girotti F1000Research, 5:736. Analytical Biochemistry, 503:58-64. MR, Sibbet G, Higgs EB, Shuttleworth MK, Somerville TD, Somervaille TC. (2016) Hamilton T, Lorigan P, Weller M, Vincent DF, Chapman PJ, James DI, Watson AJ, Hopkins Newton R, Bowler KA, Burns EM, Chapman Tissue-inappropriate derepression of FOXC1 is Sansom OJ, Frame M, Dijke PT, Marais R, GV, Waddell ID, Ogilvie DJ. (2016) PJ, Fairweather EE, Fritzl SJ, Goldberg KM, frequent and functional in human acute Inman GJ. (2016) IncucyteDRC: An R package for the dose Hamilton NM, Holt SV, Hopkins GV, Jones myeloid leukemia. Molecular & Cellular Mutational activation of BRAF confers response analysis of live cell imaging data. SD, Jordan AM, Lyons AJ, Nikki March H, Oncology, 3(2):e1131355. sensitivity to transforming growth factor beta F1000Research, 5:962. McDonald NQ, Maguire LA, Mould DP, Purkiss inhibitors in human cancer cells. Oncotarget, AG, Small HF, Stowell AI, Thomson GJ, Wiseman DH, Williams EL, Wilks DP, Sun 7(50):81995-82012. James DI, Smith KM, Jordan AM, Fairweather Waddell ID, Waszkowycz B, Watson AJ, Leong H, Somerville TD, Dennis MW, Struys EE, Griffiths LA, Hamilton NS, Hitchin JR, Ogilvie DJ. (2016) EA, Bakkali A, Salomons GS, Somervaille TC. Delgado-Goni T, Miniotis MF, Wantuch S, Hutton CP, Jones S, Kelly P, McGonagle AE, The discovery of 2-substituted phenol (2016) Parkes HG, Marais R, Workman P, Leach MO, Small H, Stowell AI, Tucker J, Waddell ID, quinazolines as potent RET kinase inhibitors Frequent reconstitution of IDH2(R140Q) Beloueche-Babari M. (2016) Waszkowycz B, Ogilvie DJ. (2016) with improved KDR selectivity. European mutant clonal multilineage hematopoiesis The BRAF Inhibitor Vemurafenib Activates First-in-Class Chemical Probes against Journal of Medicinal Chemistry, 112:20-32. following chemotherapy for acute myeloid Mitochondrial Metabolism and Inhibits Poly(ADP-ribose) Glycohydrolase (PARG) Inhibit leukemia. Leukemia, 30(9):1946-50. Hyperpolarized Pyruvate-Lactate Exchange in DNA Repair with Differential Pharmacology to Other publications BRAF-Mutant Human Melanoma Cells. Olaparib. ACS Chemical Biology, 11(11):3179- Jordan AM, Grant RP. (2016) Korfi K, Smith M, Swan J, Somervaille TC, Molecular Cancer Therapeutics, 15(12):2987- 3190. Communicating Our Science to Our Dhomen N, Marais R. (2016) 2999. Customers: The Vital Role of Passionate Public BIM mediates synergistic killing of B-cell acute Watson AJ, Hopkins GV, Hitchin S, Begum H, Advocacy. ACS Medicinal Chemistry Letters, lymphoblastic leukemia cells by BCL-2 and Cammareri P, Rose AM, Vincent DF, Wang J, Jones S, Jordan A, Holt S, March HN, Newton 7(12):1010-1011. MEK inhibitors. Cell Death & Disease, 7:e2177 Nagano A, Libertini S, Ridgway RA, Athineos R, Small H, Stowell A, Waddell ID, Waszkowycz D, Coates PJ, McHugh A, Pourreyron C, Dayal B, Ogilvie DJ. (2016) JH, Larsson J, Weidlich S, Spender LC, 62 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH PUBLICATIONS 63


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    Draper JE, Sroczynska P, Tsoulaki O, Leong LA, Read JA, Brassington C, Pollard HK, RESEARCH PUBLICATIONS (CONTINUED) HS, Fadlullah MZ, Miller C, Kouskoff V, Lacaud Phillips C, Green I, Overman R, Collier M, G. (2016) Testoni E, Miller CJ, Hunter T, Sansom OJ, RUNX1B Expression Is Highly Heterogeneous Brognard J. (2016) and Distinguishes Megakaryocytic and Recurrent MLK4 Loss-of-Function Mutations Sapkota GP, Purdie KJ, Proby CM, Harwood C, Feunteun J, Leung SY, Marais R, Mardis ER, Erythroid Lineage Fate in Adult Mouse Suppress JNK Signaling to Promote Colon CA, Leigh IM, Clevers H, Barker N, Karlsson S, McGranahan N, Middleton G, Quezada SA, Hematopoiesis. PLoS Genetics, 12(1):e1005814. Tumorigenesis. Cancer Research, 76(3):724-35. Pritchard C, Marais R, Chelala C, South AP, Rodón J, Rosenfeld N, Sotiriou C, André F. Marusiak AA, Stephenson NL, Baik H, Trotter Sansom OJ, Inman GJ. (2016) (2016) EW, Li Y, Blyth K, Mason S, Chapman P, Puto Inactivation of TGFβ receptors in stem cells Consensus on precision medicine for drives cutaneous squamous cell carcinoma. metastatic cancers: a report from the MAP LA, Read JA, Brassington C, Pollard HK, Georges Lacaud (page 38) Phillips C, Green I, Overman R, Collier M, Stem Cell Biology Nature Communications, 7:12493. conference. Annals of Oncology, 27(8):1443-8. Testoni E, Miller CJ, Hunter T, Sansom OJ, Brognard J. (2016) Refereed Research Papers Gremel G, Lee RJ, Girotti MR, Mandal AK, Recurrent MLK4 Loss-of-Function Mutations Lilly AJ, Costa G, Largeot A, Fadlullah MZ, Valpione S, Garner G, Ayub M, Wood S, Crispin Miller (page 32) Suppress JNK Signaling to Promote Colon Lie-A-Ling M, Lacaud G, Kouskoff V. (2016) Rothwell DG, Fusi A, Wallace A, Brady G, Dive RNA Biology Tumorigenesis. Cancer Research, 76(3):724-35. Interplay between SOX7 and RUNX1 regulates C, Dhomen N, Lorigan P, Marais R. (2016) hemogenic endothelial fate in the yolk sac. Distinct subclonal tumour responses to therapy Refereed Research Papers revealed by circulating cell-free DNA. Annals of Development, 143(23):4341-4351. Oncology, 27(10):1959-65. Carter L, Rothwell DG, Mesquita B, Smowton John Brognard (page 34) C, Leong HS, Fernandez-Gutierrez F, Li Y, Burt Signalling Networks in Cancer Obier N, Cauchy P, Assi SA, Gilmour J, DJ, Antonello J, Morrow CJ, Hodgkinson CL, Lie-A-Ling M, Lichtinger M, Hoogenkamp M, Korfi K, Smith M, Swan J, Somervaille TC, Morris K, Priest L, Carter M, Miller C, Hughes A, Refereed Research Papers Noailles L, Cockerill PN, Lacaud G, Kouskoff V, Dhomen N, Marais R. (2016) Blackhall F, Dive C, Brady G. (2016) Testoni E, Stephenson NL, Torres-Ayuso P, Bonifer C. (2016) BIM mediates synergistic killing of B-cell acute Molecular analysis of circulating tumor cells Marusiak AA, Trotter EW, Hudson A, Cooperative binding of AP-1 and TEAD4 lymphoblastic leukemia cells by BCL-2 and MEK identifies distinct copy-number profiles in Hodgkinson CL, Morrow CJ, Dive C, Brognard modulates the balance between vascular inhibitors. Cell Death & Disease, 7:e2177. patients with chemosensitive and J. (2016) smooth muscle and hemogenic cell fate. Smith MP, Brunton H, Rowling EJ, Ferguson J, chemorefractory small-cell lung cancer. Somatically mutated ABL1 is an actionable and Development, 143(23):4324-4340. Nature Medicine, Epub 2016 November 21 essential NSCLC survival gene. EMBO Arozarena I, Miskolczi Z, Lee JL, Girotti MR, Molecular Medicine, 8(2):105-16. Nasrallah R, Fast EM, Solaimani P, Knezevic K, Marais R, Levesque MP, Dummer R, Frederick Williamson SC, Metcalf RL, Trapani F, Mohan S, Eliades A, Patel R, Thambyrajah R, DT, Flaherty KT, Cooper ZA, Wargo JA, Antonello J, Abbott B, Leong HS, Chester CP, Marusiak AA, Stephenson NL, Baik H, Trotter Unnikrishnan A, Thoms J, Beck D, Vink CS, Wellbrock C. (2016) Simms N, Polanski R, Nonaka D, Priest L, Fusi A, EW, Li Y, Blyth K, Mason S, Chapman P, Puto Smith A, Wong J, Shepherd M, Kent D, Inhibiting Drivers of Non-mutational Drug Carlsson F, Carlsson A, Hendrix MJ, Seftor RE, Tolerance Is a Salvage Strategy for Targeted Seftor EA, Rothwell DG, Hughes A, Hicks J, Melanoma Therapy. Cancer Cell, 29(3):270-84. Miller C, Kuhn P, Brady G, Simpson KL, Organoids isolated and Blackhall FH, Dive C. (2016) expanded from a genetically Girotti MR, Gremel G, Lee R, Galvani E, Vasculogenic mimicry in small cell lung cancer. engineered mouse model of Rothwell D, Viros A, Mandal AK, Lim KH, pancreatic ductal Nature Communications, 7:13322. adenocarcinoma. Saturno G, Furney SJ, Baenke F, Pedersen M, Image supplied by Xiaohong Rogan J, Swan J, Smith M, Fusi A, Oudit D, Tape CJ, Ling S, Dimitriadi M, McMahon KM, Zhang (Systems Oncology) Dhomen N, Brady G, Lorigan P, Dive C, Marais Worboys JD, Leong HS, Norrie IC, Miller CJ, R. (2016) Poulogiannis G, Lauffenburger DA, Jørgensen Application of Sequencing, Liquid Biopsies, and C. (2016) Patient-Derived Xenografts for Personalized Oncogenic KRAS Regulates Tumor Cell Medicine in Melanoma. Cancer Discovery, Signaling via Stromal Reciprocation. Cell, 6(3):286-99. 165(4):910-20. Baenke F, Chaneton B, Smith M, Van Den Memon D, Dawson K, Smowton CSF, Xing W, Broek N, Hogan K, Tang H, Viros A, Martin M, Dive C, Miller CJ. (2016) Galbraith L, Girotti MR, Dhomen N, Gottlieb E, Hypoxia-driven splicing into noncoding Marais R. (2016) isoforms regulates the DNA damage response. Resistance to BRAF inhibitors induces Npj Genomic Medicine, 1:16020. glutamine dependency in melanoma cells. Molecular Oncology, 10(1):73-84. Morrow CJ, Trapani F, Metcalf RL, Bertolini G, Hodgkinson CL, Khandelwal G, Kelly P, Galvin M, Carter L, Simpson KL, Williamson S, Wirth C, Other publications Simms N, Frankliln L, Frese KK, Rothwell DG, Virós A, Girotti MR, Marais R. (2016) Nonaka D, Miller CJ, Brady G, Blackhall FH, So You Can Teach Old Fibroblasts New Tricks. Dive C. (2016) Cancer Discovery, 6(6):581-3. Tumourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: a Swanton C, Soria JC, Bardelli A, Biankin A, clinical case study. Annals of Oncology, Caldas C, Chandarlapaty S, de Koning L, Dive 27(6):1155-60. 64 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH PUBLICATIONS 65


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    developmental hematopoiesis using Endoglin RUNX1B Expression Is Highly Heterogeneous RESEARCH PUBLICATIONS (CONTINUED) regulatory elements as molecular probes. and Distinguishes Megakaryocytic and Blood, 128(15):1928-1939. Erythroid Lineage Fate in Adult Mouse Hematopoiesis. PLoS Genetics, 12(1):e1005814. Cuvertino S, Lacaud G, Kouskoff V. (2016) Roychoudhuri R, Paul F, Klippert J, Hammes A, Largeot A, Perez-Campo FM, Marinopoulou E, SOX7-enforced expression promotes the Other Publications Willnow T, Göttgens B, Dzierzak E, Zon LI, Lie-a-Ling M, Kouskoff V, Lacaud G. (2016) expansion of adult blood progenitors and Lilly AJ, Lacaud G, Kouskoff V. (2016) Lacaud G, Kouskoff V, Pimanda JE. (2016) Expression of the MOZ-TIF2 oncoprotein in blocks B-cell development. Open Biology, 6(7). SOXF transcription factors in cardiovascular Identification of novel regulators of mice represses senescence. Experimental pii: 160070. development. Seminars in Cell & developmental hematopoiesis using Endoglin Hematology, 44(4):231-7.e4. Developmental Biology, S1084-9521(16) regulatory elements as molecular probes. Cuvertino S, Filiciotto G, Masurekar A, Saha V, 30216-6. Blood, 128(15):1928-1939. Draper JE, Sroczynska P, Tsoulaki O, Leong HS, Lacaud G, Kouskoff V. (2016) Fadlullah MZ, Miller C, Kouskoff V, Lacaud G. SOX7 promotes the maintenance and Batta K, Menegatti S, Garcia-Alegria E, Cuvertino S, Lacaud G, Kouskoff V. (2016) (2016) proliferation of B cell precursor acute Florkowska M, Lacaud G, Kouskoff V. (2016) SOX7-enforced expression promotes the RUNX1B Expression Is Highly Heterogeneous lymphoblastic cells. Oncotarget, Epub 2016 Concise Review: Recent Advances in the In expansion of adult blood progenitors and and Distinguishes Megakaryocytic and July 7. Vitro Derivation of Blood Cell Populations. blocks B-cell development. Open Biology, 6(7). Erythroid Lineage Fate in Adult Mouse Stem Cells Translational Medicine, 5(10):1330- pii: 160070. Hematopoiesis. PLoS Genetics, 12(1):e1005814. Thambyrajah R, Patel R, Mazan M, Lie-A-Ling 1337. M, Lilly A, Eliades A, Menegatti S, Garcia- Cuvertino S, Filiciotto G, Masurekar A, Saha V, Other Publications Alegria E, Florkowska M, Batta K, Kouskoff V, Garcia-Alegria E, Menegatti S, Batta K, Lacaud G, Kouskoff V. (2016) Lilly AJ, Lacaud G, Kouskoff V. (2016) Lacaud G. (2016) Cuvertino S, Florkowska M, Kouskoff V. (2016) SOX7 promotes the maintenance and SOXF transcription factors in cardiovascular New insights into the regulation by RUNX1 and Emerging concepts for the in vitro derivation proliferation of B cell precursor acute development. Seminars in Cell & GFI1(s) proteins of the endothelial to of murine haematopoietic stem and lymphoblastic cells. Oncotarget, Epub 2016 Developmental Biology, S1084-9521 hematopoietic transition generating progenitor cells. FEBS Letters, 590(22):4116- July 7. (16)30216-6. primordial hematopoietic cells. Cell Cycle, 4125. 15(16):2108-2114. Thambyrajah R, Patel R, Mazan M, Lie-A-Ling Batta K, Menegatti S, Garcia-Alegria E, M, Lilly A, Eliades A, Menegatti S, Garcia-Alegria Florkowska M, Lacaud G, Kouskoff V. (2016) E, Florkowska M, Batta K, Kouskoff V, Lacaud G. Concise Review: Recent Advances in the In Eliades A, Wareing S, Marinopoulou E, Claus Jørgensen (page 42) Fadlullah MZ, Patel R, Grabarek JB, Plusa B, Systems Oncology (2016) Vitro Derivation of Blood Cell Populations. Lacaud G, Kouskoff V. (2016) New insights into the regulation by RUNX1 and Stem Cells Translational Medicine, 5(10):1330- The Hemogenic Competence of Endothelial Refereed Research Papers GFI1(s) proteins of the endothelial to 1337. Progenitors Is Restricted by Runx1 Silencing Mardakheh FK, Sailem HZ, Kümper S, Tape CJ, hematopoietic transition generating during Embryonic Development. Cell Reports, McCully RR, Paul A, Anjomani-Virmouni S, primordial hematopoietic cells. Cell Cycle, 15(10):2185-99. Jørgensen C, Poulogiannis G, Marshall CJ, 15(16):2108-2114. Valerie Kouskoff (page 40) Bakal C. (2016) Stem Cell Haematopoiesis Garcia-Alegria E, Iluit M, Stefanska M, Silva C, Proteomics profiling of interactome dynamics Eliades A, Wareing S, Marinopoulou E, Heeg S, Kimber SJ, Kouskoff V, Lacaud G, by colocalisation analysis (COLA). Molecular Fadlullah MZ, Patel R, Grabarek JB, Plusa B, Refereed Research Papers Vijayaraghavan A, Batta K. (2016) Biosystems, 13(1):92-105. Lacaud G, Kouskoff V. (2016) Lilly AJ, Costa G, Largeot A, Fadlullah MZ, Graphene Oxide promotes embryonic stem The Hemogenic Competence of Endothelial Lie-A-Ling M, Lacaud G, Kouskoff V. (2016) cell differentiation to haematopoietic lineage. Tape CJ, Ling S, Dimitriadi M, McMahon KM, Progenitors Is Restricted by Runx1 Silencing Interplay between SOX7 and RUNX1 regulates Science Reports, 6:25917. Worboys JD, Leong HS, Norrie IC, Miller CJ, during Embryonic Development. Cell Reports, hemogenic endothelial fate in the yolk sac. Poulogiannis G, Lauffenburger DA, Jørgensen 15(10):2185-99. Development, 143(23):4341-4351. Goode DK, Obier N, Vijayabaskar MS, C. (2016) Lie-A-Ling M, Lilly AJ, Hannah R, Lichtinger M, Oncogenic KRAS regulates tumor cell Garcia-Alegria E, Iluit M, Stefanska M, Silva C, Obier N, Cauchy P, Assi SA, Gilmour J, Batta K, Florkowska M, Patel R, Challinor M, signaling via stromal reciprocation. Cell, 165(4) Heeg S, Kimber SJ, Kouskoff V, Lacaud G, Lie-A-Ling M, Lichtinger M, Hoogenkamp M, Wallace K, Gilmour J, Assi SA, Cauchy P, 910-920. Vijayaraghavan A, Batta K. (2016) Noailles L, Cockerill PN, Lacaud G, Kouskoff V, Hoogenkamp M, Westhead DR, Lacaud G, Graphene Oxide promotes embryonic stem Bonifer C. (2016) Kouskoff V, Göttgens B, Bonifer C. (2016) Locard-Paulet M, Lim L, Veluscek G, cell differentiation to haematopoietic lineage. Cooperative binding of AP-1 and TEAD4 Dynamic Gene Regulatory Networks Drive McMahon K, Sinclair J, van Weverwijk A, Science Reports, 6:25917. modulates the balance between vascular Hematopoietic Specification and Worboys JD, Yuan Y, Isacke CM, smooth muscle and hemogenic cell fate. Differentiation. Developmental Cell, Jørgensen C. (2016) Goode DK, Obier N, Vijayabaskar MS, Development, 143(23):4324-4340. 36(5):572-87. Phosphoproteomic analysis of tumour- Lie-A-Ling M, Lilly AJ, Hannah R, Lichtinger M, endothelial signaling identifies EPHA2 as a Batta K, Florkowska M, Patel R, Challinor M, Nasrallah R, Fast EM, Solaimani P, Knezevic K, Largeot A, Perez-Campo FM, Marinopoulou E, negative regulator of transendothelial Wallace K, Gilmour J, Assi SA, Cauchy P, Eliades A, Patel R, Thambyrajah R, Lie-a-Ling M, Kouskoff V, Lacaud G. (2016) migration. Science Signaling, 9(414):ra15. Hoogenkamp M, Westhead DR, Lacaud G, Unnikrishnan A, Thoms J, Beck D, Vink CS, Expression of the MOZ-TIF2 oncoprotein in Kouskoff V, Göttgens B, Bonifer C. (2016) Smith A, Wong J, Shepherd M, Kent D, mice represses senescence. Experimental Kümper S, Mardakheh FK, McCarthy A, Yeo M, Dynamic Gene Regulatory Networks Drive Roychoudhuri R, Paul F, Klippert J, Hammes A, Hematology, 44(4):231-7.e4. Stamp GW, Paul A, Worboys J, Sadok A, Hematopoietic Specification and Willnow T, Göttgens B, Dzierzak E, Zon LI, Jørgensen C, Guichard S, Marshall CJ. (2016) Differentiation. Developmental Cell, Lacaud G, Kouskoff V, Pimanda JE. (2016) Draper JE, Sroczynska P, Tsoulaki O, Leong Rho-associated kinase (ROCK) function is 36(5):572-87. Identification of novel regulators of HS, Fadlullah MZ, Miller C, Kouskoff V, Lacaud essential for cell cycle progression, G. (2016) senescence and tumorigenesis. Elife, 5:e12994 66 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH PUBLICATIONS 67


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    RESEARCH PUBLICATIONS (CONTINUED) THESES Live image of small cell lung cancer cells labelled for actin, grown as a three-dimensional spheroid, leaving the main body of cells (bottom of the image) and invading into a layer of matrigel. Cell colours correspond to depth at which they were imaged, showing the cells moving in three Kate Hogan Alekh Thapa dimensions. Molecular Oncology Cell Regulation Image supplied by Andrew Porter & Sophie Adlard (Cell Signalling) The gene-gene and gene- The role of MEKK1-MKK4 environment interactions in signalling in tumour biology melanoma Dan Wiseman Andrew Hudson Leukaemia Biology Signalling Networks in Cancer Isocitrate dehydrogenase Novel in-silico approaches to mutations as targets for identify driver mutations in monitoring and therapy in AML cancer genomics data Anna Woroniuk Ewelina Testoni Cell Signalling Signalling Networks in Cancer The role of the Rac1 activator Somatically mutated ABL1 is an Additional Publications Mihic-Probst D, Shea C, Duncan L, de la STEF/Tiam2 in front-rear actionable and essential lung Fouchardiere A, Landman G, Landsberg J, ven polarity and migration cancer survival gene Gaignaux A, Ashton G, Coppola D, De Souza Y, den Oord J, Lowe L, Cook MG, Yun SJ, Clarke De Wilde A, Eliason J, Grizzle W, Guadagni F, L, Messina J, Elder DE, Barnhill RL. (2016) Gunter E, Koppandi I, Shea K, Shi T, Stein JA, Update on Thin Melanoma: Outcome of an Rob Metcalf Danielle Shaw Sobel ME, Tybring G, Van den Eynden G, International Workshop. Advances in Anatomic Clinical and Experimental Clinical and Experimental Betsou F. (2016) Pathology, 23(1):24-9. Pharmacology Pharmacology A Biospecimen Proficiency Testing Program for Biobank Accreditation: Four Years of The clinical utility and Magnetic resonance imaging Experience. Biopreservation & Biobanking, characterisation of circulating biomarkers of angiogenesis and 14(5):429-439. tumour cells in lung cancer apoptosis in advanced cancer Long J, Basu Roy R, Zhang YJ, Antrobus R, Du Y, Smith DL, Weekes MP, Javid B. (2016) Plasma Membrane Profiling Reveals Upregulation of ABCA1 by Infected Macrophages Leading to Restriction of Mycobacterial Growth. Frontiers in Microbiology, 7:1086. Du W, Forte GM, Smith D, Petersen J. (2016) Phosphorylation of the amino-terminus of the AGC kinase Gad8 prevents its interaction with TORC2. Open Biology, 6(3). pii: 150189. 68 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE THESES 69


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    EXTERNAL SEMINAR SPEAKERS 2016 Multiplexed immunofluorescence for Runx1 (red) and pan-Cytokeratin (white) in a benign prostatic hyperplasia patient sample. Image supplied by Renaud Mevel (Stem Cell Biology) The seminar series that we run is vital for the Institute, connecting world-class researchers across the broad spectrum of cancer research. We have enjoyed another successful year for scientific interactions with an excellent set of internationally renowned speakers visiting the Institute. The Breast Cancer Now Research Unit seminar series also continues to produce an outstanding range of Kendle Maslowski Ewan Birney speakers. The postdoctoral researchers at the Institute also give Riken The European Bioinformatics Institute weekly seminars which are very well attended and help to integrate (EMBL-EBI) the entire cancer research efforts of the Institute. Lisa Belmont Ideaya Biosciences Karl Ekwall Karolinska Institutet Alberto Bardelli Stefanie Jeffrey Bernd Pulverer University of Torino Stanford University EMBO Press William G. Kaelin Dana-Farber Cancer Institute Rachael Natrajan Andrea Sottoriva Patricia Muller The Institute of Cancer Research Institute of Cancer Research MRC Toxicology Unit Geert Kops Hubrecht Institute Emmanuel Donnadieu Peter Campbell Gerard Evan Institut Cochin Wellcome Trust Sanger Institute University of Cambridge Leïla Perié Institut Curie Xosé Bustelo Juan Carlos Acosta Dirk Heerding University of Salamanca The University of Edinburgh GlaxoSmithKline Breast Cancer Now Seminars Norman Maitland Gabriel A. Rabinovich Nicola Valeri University of York Institute of Biology and Experimental The Institute of Cancer Research Renée van Amerongen Medicine (IBYME) University of Amsterdam Linda Bauld David Adams University of Stirling Mark O'Driscoll The Wellcome Trust Sanger Institute Simak Ali University of Sussex Imperial College London Cédric Blanpain Ton Schumacher Université Libre de Bruxelles Nizar Batada Netherlands Cancer Institute Jacco van Rheenen MRC Institute of Genetics and Molecular Hubrecht Institute Simon McDade Medicine (IGMM) Julio Saez-Rodriguez Queen's University Belfast RWTH-Aachen University Greg Hannon Arne Östman Cancer Research UK Cambridge Institute Richard J. Gilbertson Karolinska Institutet Carla Martins Cambridge Cancer Centre University of Cambridge Fran Balkwill Anne Ridley Barts Cancer Institute Sergio A. Quezada King's College London Jem Rashbass UCL Cancer Institute Public Health England Anne-Lise Børresen-Dale Roberto Buccione Oslo University Hospital Richard Bayliss EMBO Molecular Medicine David Carling University of Leeds Imperial College London Clare Davies Carlos Garcia Institute of Cancer and Genomic Sciences Dr Bissan Al-Lazikani The Francis Crick Institute Michael F. Olson The Institute of Cancer Research Cancer Research UK Beatson Institute Jack Cuzick Katie Ridd Wolfson Institute of Preventive Medicine Sean Knight Nature Communications Bruno Silva-Santos Guy’s and St Thomas’ NHS Foundation Trust Institute of Molecular Medicine (iMM Lisboa) 70 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE EXTERNAL SEMINAR SPEAKERS 2016 71


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    www.cruk.manchester.ac.uk/education POSTGRADUATE EDUCATION The Cancer Research UK Manchester Institute (CRUK MI) offers a range of graduate degrees for students interested in a career involving cancer research. The Institute considers education of both research and clinician scientists to be a major investment in the future of cancer research, and has an excellent track record of launching careers in basic, translational and clinical research. Postgraduate Education Manager As part of this commitment, we have an The Cancer Research UK Manchester understanding of many aspects of cancer LSD1 inhibitors have the potential for treating Julie Edwards active postgraduate programme that provides Graduate Programme research and basic biology. In addition, we other haematological disorders such as students and clinical research fellows of We aim for each student to receive high hold a series of weekly postdoctoral research essential thrombocythemia. outstanding potential the opportunity to quality training in scientific research through seminars and attendance from PhD students is study for a cancer-related PhD degree. This is an intellectually demanding but achievable an integral part of the seminar programme. Cancer Research UK contributes towards an achieved through a training programme that research programme. Each project is peer- While students themselves are asked to give annual International PhD Student Cancer aims to improve effectiveness in research, reviewed in advance and monitored talks at key points during their PhD, they also Conference (IPSCC) allowing high calibre provide professional and management skills throughout its course through a mixture of oral have opportunities to present their work at lab students (typically in 2nd & 3rd years) from top and enhance career development. Our PhD presentations, written reports, and progress meetings and during student forums within the cancer research institutes across Europe to students have exceptional employment meetings. These modes of assessment are Institute. STAy (short for Science TakeAway) is a organise and present at their own scientific prospects following graduation, with the designed not only to provide formal points at group run by junior scientists in the CRUK conference. Core participating Institutes Postgraduate Tutor great majority (>95%) continuing in academia, which progress (of both the student and the Manchester Institute. Meetings are open to all include London Research Institute (LRI), industry or healthcare, and securing positions project) can be monitored, but also to help early career scientists - PhD students, Postdocs Cambridge Institute (CI), Beatson Institute Angeliki Malliri in destinations across the UK, Europe and develop the presentation skills which are so and Scientific Officers from the Paterson (BICR), Netherlands Cancer Institute (NKI), the USA. fundamental to the majority of careers in Building and the Manchester Cancer Research European School of Molecular Medicine, Milan science and elsewhere. Graduate training is Centre (MCRC) Building. The aim is to provide a (SEMM, IFOM & IFEO), and the German Cancer In 2016, we welcomed ten graduate students monitored by the Education Committee, forum for discussions and training related to Research Centre (DKFZ). and four clinical research fellows to our PhD staffed by the Institute’s Group Leaders and research, communication of scientific programme, working in a variety of fields from student representatives (see below). A main engagement and development of social and In 2016, the 10th IPSCC was organised by PhD RNA computational biology, clinical supervisor and a second or co-supervisor are networking opportunities. Topics in 2016 have students from the Cambridge Institute and 10 experimental pharmacology, leukaemia nominated for each student, who are able to included a careers panel and discussions students from the CRUK MI attended the biology, molecular oncology through to stem provide additional advice and consultation on around science communications. conference in June. It is mandatory for cell biology. It was also particularly gratifying to both academic and non-academic matters. participating student delegates to submit a Postgraduate Director see that, over the past twelve months, our Each student is also assigned an advisor (similar The CRUK Manchester Institute Colloquium poster to showcase their research either and Chair of the Education students had published first author papers in a to a personal tutor on an undergraduate takes place annually in September, and is an through a poster or oral presentation. Emma Committee variety of journals including Nature Medicine programme) whose role is to provide impartial excellent opportunity for our new intake of Williams from the Leukaemia Biology group, Tim Somervaille Leukemia, EMBO Molecular Medicine and support and advice in a pastoral role. Further students to meet other established PhD CRUK MI was selected to give an oral Nature Communications. During the course of support is also available individually from the students, members of the Institute, including presentation and she was voted as the recipient this year, a total of four PhD students and three Director of Postgraduate Education, Group Leaders, Postdoctoral Fellows, and for the prize of “best student oral presentation” Clinical Research Fellows [CRF] were awarded Postgraduate Tutor, Postgraduate Manager, or Scientific Officers. This forum communicates during the conference. their PhDs, a 100% success rate. From the collectively as the Education Committee up to date science in the form of oral seven graduates in 2016, two continued their Administration Group. presentations given by Group Leaders and PhD studentships careers in academia at the CRUK Manchester second year PhD students, as well as poster All of our CRUK core funded studentships are of Institute in post-doctoral positions with one The CRUK MI runs an external seminar series presentations from a range of scientists across four years’ duration, and consist of an approved student moving to a scientific position in featuring talks from many of the key players in the Institute covering all aspects of cancer research project in one of our core funded industry. Two CRFs returned to clinical training cancer research, and students are expected to research. Poster prizes are awarded, including research groups. Some students have joint at The Christie NHS Foundation Trust and one attend. The speakers are internationally the Lizzy Hitchman Prize for the best poster supervisors in different groups, fostering successfully attained a highly prestigious renowned scientists and we consider it presented by a PhD student or Clinical Fellow. important collaborations and providing Bloodwise Clinician Scientist award based at essential that our students are exposed to In 2016 PhD student Emma Williams from the exposure to different disciplines. Recruitment is the CRUK Manchester Institute and The Christie outstanding research from leaders in different Leukaemia Biology group was the recipient of highly competitive, with 300-500 applicants NHS Foundation Trust. disciplines, which will give them a broad this prize. Emma’s project has established that competing for around four-eight places each 72 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE POSTGRADUATE EDUCATION 73


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    POSTGRADUATE EDUCATION (CONTINUED) year. Interviews are typically conducted Education Committee 2016 annually over a two-day period in early January. The Education Committee (EC) acts for postgraduate students and consists of Group All of our students benefit from access to Leaders, the Chief Operating Officer, the advanced state-of-the-art facilities, including Postgraduate Tutor and the Postgraduate advanced imaging, biological mass Education Manager from the CRUK spectrometry, flow cytometry, histology and Manchester Institute. next generation sequencing. Our research groups offer PhD studentships and projects Our goal is for every student to have a project covering the entire breadth of research within that is both achievable and intellectually the Institute. stimulating and demanding. Projects and students are monitored by the Education Fellowships in Clinical Pharmacology Research Committee which makes sure that the In order to help train the next generation of proposed plan of research is suitable, and that clinical pharmacologists with expertise in progress is made consistently throughout the oncology, CRUK MI, in collaboration with the course of the studentship. Various assessments Manchester Cancer Research Centre (MCRC) throughout the studentship, including regular and AstraZeneca, established in 2007 a talks, progress meetings and written reports, fellowship scheme in clinical pharmacology are vital to ensuring successful completion and research. The fellowships are open to graduation for the PhD degree. Such applicants who have obtained, or are close to assessments help not only to monitor progress, obtaining, their Certificate of Completion of but also help to develop performance and Training (CCT) in Medical Oncology. presentations skills. Each research fellow undertakes a four year Education Committee Members PhD project, which provides training in biomarker discovery, method development/ Tim Somervaille validation, and clinical trial methodology. Postgraduate Director & Chair, During their tenure at The Christie NHS Education Committee Foundation Trust/CRUK MI, the post holders Angeliki Malliri receive support from their clinical supervisor, Postgraduate Tutor and laboratory-based training from Clinical and Richard Marais Experimental Pharmacology (CEP) Group Ex-Officio Member Leader Caroline Dive (in collaboration with Wolfgang Breitwieser MCRC colleagues). At AstraZeneca they receive John Brognard [until July] training in clinical trials management, Julie Edwards regulatory interaction, translational research Claus Jorgensen through project management, and attend Georges Lacaud [from November] investigator meetings. Clinical training includes Donald Ogilvie one research clinic per week, training in clinical Jonathan Tugwood [from January] trial design and methodology, ICH-GCP, EU Ian Waddell Directives and research governance. Biomarker Caroline Wilkinson method development and application take place on both sites in all projects, with mutual Student Representatives benefit as each fellow brings newly acquired knowledge to each site. Regular meetings take Emma Williams [until October] place between the fellows, their supervisors, as Amy McCarthy well as other staff members involved in the Denys Holovanchuk [from October] project, ensuring effective collaboration and an integrated approach. 74 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE POSTGRADUATE EDUCATION 75


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    OPERATIONS Mouse pancreatic ductal adenocarcinoma stained with SMA (Red), LOX (Green), PDFR The Operations Department provides the necessary infrastructure Beta (Grey) and DAPI (Blue). and services to facilitate the running of the Institute. Finance and Image supplied by Haoran Tang (Molecular Oncology) purchasing, as well as Logistics, fall under the leadership of Margaret Lowe while Stuart Pepper oversees IT, Estates and Health and Safety; Rachel Powell is head of HR and Caroline Wilkinson is responsible for all aspects of Scientific Administration and acts as the primary point Chief Operating Officer of operational contact within the Institute for both The University of Caroline Wilkinson Manchester and Cancer Research UK. This year, staff from across the Operations team have continued to help with arrangements to ensure that the Manchester Cancer Research Centre Building is fully operational and to assist with moving some of our research teams there. Other major projects included: the development of new online software tools for both our contribution review process and for PhD student applications, a major health and safety audit, and work towards our Athena SWAN application. Chief Laboratory Officer energy but give better illumination to provide a funding to allow us to increase the breadth of Stuart Pepper brighter work environment. research that we undertake. Director’s Office and within the team. There have been many Administration Services changes in the Paterson Building over the past Steve Alcock, the Estates Manager, will be The Institute receives funding from various Ruth Perkins, Maria Belen Conti1 year with groups moving around the Institute retiring in March 2017 after 20 years of service. different sources and we have the responsibility and some moving into the new MCRC Building Fortunately Steve’s replacement will be in to ensure that funds are used for the purpose 1 joined in 2016 across the road. These moves created a position prior to his departure to ensure a they are given. We monitor each individual substantial amount of small works in the smooth transition. award and provide financial information for the The office provides administrative support to Paterson Building in order to prepare the areas funders and principle investigators. the Director and to the Institute 's Group for the new incumbents. Finance and Purchasing Leaders. In addition, the department has Margaret Lowe, David Jenkins, Denise Health and Safety assisted with the organisation of several events The Estates team have been pro-active with Owen, Muhammad Raja, Vikki Rosheski, Colin Gleeson Head of Finance over the course of the year, including the refurbishment of the main ventilation plants Debbie Trunkfield Institute Colloquium, the quinquennial review which were installed in 1992. Air Handling Unit Margaret Lowe of the Director’s research group and the first 2 was refurbished earlier this year with AHUs 3, The Institute’s health and safety management The Institute Finance team supports the system was examined in 2015/2016 by means meeting of the Institute’s Scientific Advisory 4 and 6 being refurbished over the winter Director with the management of the Institute’s of an audit. The Health and Safety Management Board. Administrative support is provided for period. This will ensure their reliability for many £25m budget, which is devolved to the various Audit Profile (HASMAP) system was the external seminar series, which has been a years to come as well as being more groups and service units. The team provides a implemented as it is designed specifically for great success in 2016, hosting two seminars sustainable, utilising more energy efficient fan comprehensive service to the Institute, which the Higher Education sector. The audit was per week. The seminars serve to foster motors and cooling equipment. covers all areas of procurement and finance, undertaken by people external to, and collaboration and encourage positive ensuring we comply with the University independent of, the Institute and assessed: interaction within the wider scientific The reactive maintenance keeps the team busy financial regulations and procedures. community. We aim to provide a varied as does responding to any fault reports from • Five themes relating to leadership and programme of national and international around the building. The team also adhere to The Institute has been successful in securing integration (stage 1). Evidence was Head of Human Resources speakers. Details can be found at many legislative requirements which include several new external grant awards that were gathered by email www.cruk.manchester.ac.uk/seminars. the monitoring of Legionella and its associated activated in 2016 or will be activated early 2017. • Nine themes (stage 2), focusing on personal Rachel Powell work to comply with regulations. We are also awaiting the outcome of several protective equipment in the Drug Discovery Estates other applications. The team supports the Unit and the Cell Signalling group, and Steve Alcock, Graham Hooley, Steven Estates also play a role in efforts to continually research groups by providing effective and Manual Handling in the Logistics Department Powell, Tony Woollam reduce the energy footprint of the Institute. efficient professional advice when costing new There is a rolling program to replace traditional research proposals. Group Leaders are The audit took several weeks and involved Our newest member Steve Powell has settled fluorescent strip-lights with more energy encouraged to continue to apply for external collating and supplying evidence of our in well and is very enthusiastic about his role efficient LED lights. These newer lights use less 76 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE OPERATIONS 77


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    OPERATIONS (CONTINUED) the retirement of four members of staff. We connection via The University of Manchester have continued our commitment to joint campus network has also been upgraded partnership, working with the union which has to 10G. resulted in the revision of 17 HR policies and arrangements and staff attending interviews Human Resources procedures, including the Staff Training and The number of iOS devices used at the Institute with the auditor. The Executive Report 1 Rachel Powell, Rachel Craven , Natalie Development Policy, Recruitment and has continued to rise, reflecting a broad trend in summary showed that we had performed very Taylor1, Laura Jones, Julie Jarratt, Emma Selection Policy and the Career Path for many institutes and businesses. To enhance well in that we achieved nine “level 3” scores. Lloyd, David Stanier2 Scientists Policy. During 2016, we also our management and support provision for launched a new in house online performance these products new enterprise class software This was a pleasing outcome to the audit. A 1 joined in 2016 2joint with administration review system which was a great success. has been purchased that will provide greater remedial action plan was developed and control and flexibility. This will provide us with a implemented to address the minor action Over the past year, the HR department has Currently we are working towards the Athena management tool we can use to deploy and points raised by this audit. continued to deliver a high quality proactive Swan accreditation. A self- assessment team manage all Apple devices to ensure they are all service to the Institute and its staff. The has been established and is jointly chaired by optimally configured and running supported Compliance exercises including those of drug department provides advice and guidance to Professor Caroline Dive and Dr Caroline software versions. precursors, dangerous pathogens and toxins, managers and staff on all employment-related Wilkinson. A staff survey will be sent out to staff Euratom materials and DEFRA materials were matters such as recruitment, policy guidance, and students in 2017 and we look forward to Looking ahead to 2017, planning has carried out. The building-wide data were fed legislation and best practice. the outcomes that result from the work of this commenced for significant IT storage and back to the University Compliance Officer. Also, group and the benefits that it will bring to the security infrastructure upgrades to replace end statutory checks were made of local exhaust During 2016, 78 individuals were successfully Institute and its staff. of life equipment. This will include a refresh of ventilation and pressure vessels. appointed to enhance the work of the Institute. the IT storage along with two major initiatives Throughout the year we have developed new Next year, the focus will be on the recruitment to strengthen our defence against cyber Work to facilitate the smooth running of the advertising strategies for attracting candidates of new research Group Leaders and their threats, including installation of a new next newly opened MCRC Building continued to the Institute; such as advertising on social associated staff. generation firewall and enhanced protection together with The University of Manchester’s media with the creation of short video clips. against email borne spam, viruses and malware. Technical Operations Manager for the Building, The current headcount of staff at the Institute Information Technology Peter Reid. The first inspections of the lab areas has increased by 11.5% since 2015; from 280 to Steve Royle, Matthew Young, Hong Mach, Logistics were also undertaken and reports generated. 312 staff at the end of 2016. This recruitment Brian Poole Andrew Lloyd, Michael Alcock, Edward Fitzroy, Likewise inspections were carried out in the success highlights the continued growth and Sedia Fofana, Stephen Keane, Jonathan Lloyd, Paterson Building, monitoring health and safety development of CRUK MI, which includes the The CRUK Manchester Institute IT team Robin Sherratt1 and William Glover1 performance and taking remedial actions new iDECIDE Clinical Trials Bioinformatics provides a full catalogue of services including 1 where appropriate. In 2017 we shall continue team. email, desktop support, file storage and mobile joined in 2016 to embed our health and safety arrangements computing solutions. The CRUK MI network across both buildings. The department administered the successful supports over five hundred staff across the The past year has seen changes within the promotion of nine individuals and supported Paterson and MCRC Buildings. team’s structure with the arrival of Robin Sherratt and William Glover. The team has During 2016 the IT service underwent a expanded to cope with the increased demand detailed peer review process to look at how the on the service as it now also supports the service was performing. The report was very MCRC Building. Since October there has been positive, commenting that user feedback was a member of the logistics staff based there very good and that the services provided were offering goods in service and running of small appropriate for a modern research Institute. stores. 2016 has been a year of consolidation. We The team has continued to deliver an efficient have migrated and developed core services on and effective service providing support for the our new server farm and network infrastructure. research carried out in the Paterson Building We shall continue to build and provide new and MCRC Building. This includes the services on this platform going forward, to receipting, checking, booking in and provide our scientists with the high distribution of goods ordered by staff as well performance, high availability, fault tolerant as the collection and removal of waste. We IT services and resources needed to support currently recycle all the Institute’s waste their research. cardboard, plastic bottles, tin cans, wooden crates and pallets, ink toners, and scrap metal Throughout 2016, the MCRC Building has seen which reduces the amount of waste going a steady influx of new research groups. We have to landfill. completed the desktop deployment and network connectivity to accommodate these The Logistics team provides both buildings new groups and the MCRC Building, which is with liquid nitrogen collection and refill service now almost fully populated, is integrated into three days a week, and a dry ice service with our support activities. The network link deliveries taking place twice a week. Gas between the MCRC and the Paterson Buildings cylinders are also monitored and replaced has been upgraded to dual, redundant, 10G as necessary. fibre optic links. Also, the site internet 78 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE OPERATIONS 79


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    OPERATIONS (CONTINUED) in vitro studies. The remainder were used for Research UK Manchester Institute and is the maintenance and breeding of mice used in dedicated to working closely with the staff at procedures. We are proud of the quality of the Institute and also The University of animal welfare at the Institute and members Manchester. Martyn offers access to oncology- Researchers can order central stores stock organisations including the Wellcome regularly talk to the public about our use of focused expertise in technology evaluations, items via the intranet, which can be collected or Trust, the Rosetrees Trust and EMBO. mice in cancer research. In December 2016, patent applications and management, funding distributed by the Logistics team. We currently the Institute was recognised at a national event for development, commercialisation, drug stock over 100 stores items from tissue culture During the year, we produced our own organised by Understanding Animal Research discovery, market intelligence, and project essentials to cleaning products. Included in this software tools for our annual performance (UAR) on the Concordat on Openness with management. He also works closely with UMIP, system are the enzymes and media stored in appraisal system and for our PhD student the “Award for Public Engagement”. The University of Manchester technology the Institute freezers (Sigma, Invitrogen, Roche, recruitment. Both were introduced successfully transfer organisation. Promega, New England Bio labs, Bio-Rad, and have led to these processes being Cancer Research Technology Fisher kits and Qiagen). We now stock over 240 considerably streamlined. The team has Martyn Bottomley CRT continues to work very closely with the products, which are under continual review to worked on producing a new external website Drug Discovery Unit based at the Cancer meet the needs of users. We have been able to for the Institute which will be launched in 2017. Cancer Research Technology (CRT) is a Research UK Manchester Institute to facilitate make savings by buying in bulk from suppliers. specialist oncology-focused development the development of small molecule drug We have set up numerous “call off orders” for Animal Welfare and commercialisation company which has therapies to satisfy the unmet clinical needs of stores items, again making significant savings Simon Poucher, Regulatory Liaison and recently been integrated into Cancer Research cancer patients. This includes management of but also guaranteeing a stable stock. Training Officer, Janet Watson (AWERB Chair) UK’s Research and Innovation Directorate. collaborations with Pharmaceutical partners CRT aims to maximise patient benefit from such as Genentech, GSK, HitGen and The team also provides assistance with moving The welfare of mice that are used in CRUK-funded research worldwide by AstraZeneca and also the filing and heavy equipment or furniture helping facilitate experiments at the Institute is critical, not only advancing research discoveries into management of a number of patent internal rearrangements and the arrival of for the animals, but also because using mice development with pharmaceutical and applications for the Drug Discovery Unit to new groups. The last few months have been that are well cared for gives better quality of biotechnology parties. We aim to bridge the protect novel compounds resulting from their especially busy as the team has helped with data. One of the driving forces supporting all gap between cutting edge academic research research. This year has seen the first pre-clinical the relocation of multiple groups around the staff on animal welfare is the Animal Welfare & and industrial development of cancer candidate nomination from the DDU on the site. The team is also involved with the Ethics Review Body (AWERB). This consists of therapeutics and diagnostics. We achieve this RET project and also transfer of the lead series reconfiguration of meeting rooms for experienced animal husbandry staff, a by working closely with prestigious from the collaboration with GSK to them for numerous events being held at the Institute. veterinary surgeon, Institute scientists, a international research institutes, such as the further development. CRT is also currently In addition, the team looks after the Institute’s statistician and lay members. We have an Cancer Research UK Manchester Institute and actively managing a broad portfolio of “Shred It” service and water coolers. annual plan that covers areas of enhancement funding bodies to develop, protect and development programmes and exciting of animal facilities, care and husbandry of mice, commercialise oncology-related discoveries. licensing opportunities originating from the Scientific Operations and General encouraging implementation of the 3Rs Core activities of business development and Cancer Research UK Manchester Institute that Administration (Replacement, Reduction, Refinement of drug discovery are supported by specialists, continue to attract commercial partners. This Caroline Wilkinson, Tom Bolton1, Gillian animal use), reviewing animal ethics of integrated in the business with expertise in includes negotiation and completion of a Campbell, Julie Edwards, collaborations and grant applications from patents, legal, finance and marketing. significant number of agreements between the Steve Morgan, David Stanier2 Institute scientists, as well as management of Clinical Experimental Pharmacology group and compliance to the Animal Scientific Procedures Our exclusive focus on oncology provides an commercial collaborators. We look forward to 1 joint with MCRC, 2joint with HR Act and encouraging public engagement. unrivalled depth of knowledge and experience building on our successes and continuing to in cancer-specific translational development work closely with the Cancer Research UK Scientific administration is overseen by the In 2016, our AWERB met formally on six and commercialisation. We also have access to Manchester Institute to advance discoveries Chief Operating Officer, Caroline Wilkinson, occasions and reviewed four new project the CRT Pioneer Fund; this £70m fund has to beat cancer in the years ahead. who provides support to the Director in order licence applications and seven amendments been established with Cancer Research to facilitate the day-to-day running of the to the 21 project licences held at the Institute, Technology, the European Investment Fund Institute. The team is also responsible for before submission for review and granting by (EIF) and Battle Against Cancer Investment Trust producing a variety of scientific the Home Office. Additionally, we organise (BACIT) to bridge the investment gap between communications for the Institute including two annual meetings for project and personal cancer drug discovery and early development. publications such as the Annual Scientific licence holders where the success and best It will take potential cancer drugs, primarily Report, the Institute’s Newsletter, writing practice of working with mice was shared, discovered by Cancer Research UK, from material for the intranet and external website along with the latest news on animal welfare discovery through to entry to Phase II clinical and for the Institute’s social media presence. practices and legislation. These meetings are trials before partnering with pharmaceutical also attended by our Home Office Inspector. and biotechnology companies. Gill Campbell is the Institute’s Grants Advisor We hold an annual 3Rs’ Prize day and in 2016 who helps our researchers also apply for received thirteen entries describing ways in By arrangement with The University of external awards to extend the portfolio of which procedures undertaken on our mice Manchester, CRT owns and is responsible for research that we can undertake. This year, a can be replaced, reduced or refined. the development and commercialisation of Grants Committee has been established, intellectual property arising from Cancer chaired by Iain Hagan, to help our scientists A total of 25,252 mice were used and reported Research UK funded research at The University prepare the strongest possible applications on regulated procedures in 2016, which of Manchester (including the Cancer Research and ensure that there is a rigorous internal included 8,800 on experimental procedures. UK Manchester Institute). To effectively peer review process. This year we supported 1,788 mice were used for the creation of new facilitate this, Martyn Bottomley, a CRT Business successful applications to a variety of strains of mice and 905 were used for tissue in Manager is based on-site at the Cancer 80 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE OPERATIONS 81


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    Loose Change Buskers We celebrated International Clinical Trials Week celebrating their Queen’s in May with our Platform for Investigation day at Award for Voluntary Services the Museum of Science and Industry. This was our first solo event at the museum, taking over the ground floor with a range of hands-on activities that attracted just over 800 visitors. Highlights include new activities led by The Christie NHS Foundation Trust Radiotherapy Physics group, clinical trials activities with local research nurses and animal research activities which won the public engagement prize at the Understanding Animal Research Openness Awards in London towards the end of the year. The European City of Science Festival in July celebrated Manchester’s year as Europe’s science capital. We had a busy week of activities which included a science lates evening at In September, we took part in lightning talks and Manchester Museum, activities for the European science speed dating sessions at Manchester Commission’s “Science is a revolution” fair at the Museum’s European Research Night showcase, Museum of Science and Industry and taking part a fun evening filled with hands-on activities in soapbox science at Piccadilly Gardens. Our where our virtual reality lab tour proved to be a virtual reality lab tour also proved to be a hit. And we rounded off October with our highlight at The University of Manchester’s stand Manchester Science Festival events including at the festival’s flagship Euroscience Open Your Choice (an interactive board game about Forum conference. the challenges of research) and our “Cell Explorers” stand at The University of Manchester City Council leader Sir Richard Leese getting hands on in the MBCF lab Our main event for the festival was the Manchester’s Science Spectacular. week-long Fabric of Research Exhibition at Manchester Craft and Design Centre. Textile As well as engaging people with our research, designers from The University of Manchester staff at the Institute continue to be generous CANCER RESEARCH UK’S and Manchester Metropolitan University worked with researchers and patient volunteers to create fundraisers for the charity through a variety of events. We donned our unity bands in February RESEARCH ENGAGEMENT unique artworks that showcase our latest research and tell the personal stories of people affected by cancer. Particular thanks to Steve to celebrate World Cancer Day and enjoyed a sumptuous spread from the lovely people at Warburtons for Britain’s Biggest Breakfast in Bagley for providing a wealth of images and March. Our staff team for the Stockport Relay for Public engagement featured largely across the Institute again in expertise and to everyone who helped bring this Life held events throughout the year to raise a project to life. fantastic £2,757.86 to contribute to the 2016. We’ve had a busy year of welcoming visitors to CRUK MI, bringing our science to life at events across the city and getting involved with our own fundraising. Patient representative Jo Taylor sharing her experience of getting involved with research at our research café series Events at the Institute attracted over 1,500 on World Cancer Day in February, the talks have Cancer Research UK’s Research people interested in getting a ‘behind the scenes’ covered a wide range of topics including basic Engagement Manager insight into our work. Our regular programme of research, pharmacy’s role in clinical trials, lab tours (over 50 this year) gave our donors, insights into the role of a clinical scientist, patient Sive Finlay fundraisers, volunteers, ambassadors and involvement with research and an update on the corporate partners the opportunity to see the forthcoming proton beam therapy centre at work that their support helps to fund. Tour The Christie NHS Foundation Trust. highlights include the visit from the Sandbach and District Local Committee to celebrate their We held two successful open days in June and 60th year of fundraising for CRUK, engaging November which offered talks, tours and local councillors and MPs with our lung cancer activities to engage both CRUK supporters and research and a special visit from Loose Change local members of the public with our work. Buskers after they received the Queen’s Award Thanks to Café Vivo for their special opening for Voluntary Services having raised over arrangements and to all of our volunteers who £260,000 for the charity. helped to make the days so successful. Our new research café series gives staff, patients Outside the Institute, we took part in a range of and visitors an opportunity to find out more activities and events to raise awareness of about our work through short, informal talks in CRUK’s important role within Manchester’s the MCRC Building café (Café Vivo). Launched vibrant research community. 82 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE CANCER RESEARCH UK’S RESEARCH ENGAGEMENT 83


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    CANCER RESEARCH UK’S RESEARCH ENGAGEMENT Visitors learning about clinical impressive July event total of £84,617, all of trials at our June open day which will go directly to fund work at CRUK MI. In October, a team from Drug Discovery took on the Bear Grylls survival race at Tatton Park to raise £1,780 for Stand Up to Cancer while our fundraising fete at the MCRC Building offered plenty of tasty treats and unusual prizes in aid of the national campaign. We rounded off the year with Institute fundraising for Movember including ever popular bake sales and raffles from David Jenkins, special bootcamp yoga sessions led by Lisa Waters and a host of other activities that raised over £1,400 and got everyone into the Mo-spirit! It’s been a great year with many more exciting plans to come. Thank you to everyone across the Institute who is so generous with their time, energy and enthusiasm for supporting our many activities! Clockwise from top left: Elaine Mason and Nathalie Dhomen at the clinical trials and animal research activity stand for our June open day; The team behind our Understanding Animal Research public engagement prize; Research-inspired scarves at the Fabric of Research exhibition; Young visitors at Stockport Relay enjoying a virtual reality tour of our lab; Our inaugural pumpkin carving competition in aid of Stand Up to Cancer; David Jenkins – fundraiser extraordinaire! 84 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE 85


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    ACKNOWLEDGEMENT FOR FUNDING FOR THE CAREER OPPORTUNITIES AT THE CANCER CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH UK MANCHESTER INSTITUTE The total funding of the CRUK Manchester Institute for 2016 was The Cancer Research UK Manchester Institute is located alongside £25m. The major source of this funding was awarded by Cancer The Christie NHS Foundation Trust, and has a strong programme Research UK via a core grant of £12.2m plus additional strategic of basic and translational research. There are close links with funding of £4.8m. This funding enables the various scientific groups clinical and translational research groups throughout the Christie and service units within the Institute to carry out their research. Hospital site. The Institute offers excellent laboratory facilities In addition to postgraduate and postdoctoral The infrastructure of the CRUK Manchester • European Commission and outstanding core facilities, including opportunities, the Institute is still seeking to Institute is funded by HEFCE generated income • European Research Council molecular biology services, next generation recruit outstanding candidates to the positions at a cost of £2.1m. • GlaxoSmithKline sequencing, real-time PCR, mass spectrometry, of Junior and Senior Group Leaders. The • John Swallow Fellowship flow cytometry, histology, advanced imaging, packages provided are extremely attractive The balance of the Institute’s funding is received • Kay Kendall Leukaemia Fund and a biological resources unit. Details of all and commensurate with the experience of from a number of additional sources. The • Leo Pharma Foundation groups and facilities are given in this report, the applicant, with significant funding for research carried out through these additional • Lung Cancer Research Foundation and can guide interested parties to the personnel, recurrent expenditure and projects enhances and supports the research • Medical Research Council appropriate contacts. equipment. Junior Group Leaders are undertaken by the core funding. • Menarini Biomarkers Singapore appointed for an initial six-year period with • Moulton Charitable Trust Opportunities exist at a number of levels in the a review between five and six years for These sources are as follows: • Pancreatic Cancer Research Fund Institute. We have a well-established consideration of promotion to Senior Group • Angle Inc • Prostate Cancer UK programme of degrees by research which is Leader, with Senior Group Leaders appointed • Astex Pharmaceuticals • Rosetrees Trust described in the section on Postgraduate to non-time limited positions. • Astra Zeneca • Roy Castle Lung Cancer Foundation Education. We encourage applications from • BBSRC • The US Department of Health and suitably qualified graduates to apply to join Specific vacancies can be found on our web • Bloodwise Human Services either the PhD or MD programmes. Graduates pages (http://www.cruk.manchester.ac.uk/ • British Lung Foundation • Wellcome Trust with a first or 2.1 honours degree in a biological Jobs/), but suitably qualified and enthusiastic • Christie Hospital NHS Foundation Trust • Worldwide Cancer Research science can apply each year to train for a individuals should contact the Institute at any • Clearbridge Biomedicals four-year PhD in one of our research time to enquire about career possibilities. • CRT Pioneer Fund We are immensely grateful to all our sponsors. 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 CRUK MANCHESTER INSTITUTE FUNDING 2016 research programme and help achieve personal and career development goals. At the Institute, we also ensure that postgraduate students are provided with high quality, relevant and appropriate training alongside development 22.2% KEY opportunities. The Institute also has a well- developed process for ensuring excellent pastoral care and mentoring for all students. CRUK Core Grant Postdoctoral applicants of high calibre are regularly sought. Although Postdoctoral CRUK Strategic Funding Fellows will be encouraged to apply for their own fellowships, funded positions are available 8.6% for outstanding candidates. Interested 49.6% HEFCE applicants should contact the Group Leaders Other Sources directly, with details of their research interests and recent experience. 19.5% 86 SCIENTIFIC REPORT 2016 CANCER RESEARCH UK MANCHESTER INSTITUTE CAREER OPPORTUNITIES AT THE MANCHESTER INSTITUTE 87


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    CONTACT DETAILS ISSN 1479-0378 Copyright © 2016 Cancer Research UK Edited by: Caroline Wilkinson Gillian Campbell Cancer Research UK Manchester Institute Director: Professor Richard Marais Tel: +44(0) 161 446 3156 Fax: +44(0) 161 446 3109 Address Cancer Research UK Manchester Institute Wilmslow Road Manchester M20 4BX United Kingdom e-mail: enquiries@cruk.manchester.ac.uk website: www.cruk.manchester.ac.uk Tel +44(0) 161 446 3156 Electronic version of this report can be found at: Cancer Research UK www.cruk.manchester.ac.uk/About/ 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 88 SCIENTIFIC REPORT 2016 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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