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    SCIENTIFIC REPORT 2018 cruk.org


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    SCIENTIFIC REPORT COVER IMAGE STEF-depleted cell showing nucleus (blue) with active Rac1 (green) targeted to the nuclear membrane where it has restored the cables of 2018 the actin cap (red). Outline of the cell shown in magenta. Image supplied by Andrew Porter (Cell Signalling) MANCHESTER INSTITUTE


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    CONTENTS DIRECTOR’S INTRODUCTION 04 Tim Somervaille 30 RESEARCH HIGHLIGHTS 2018 07 Leukaemia Biology RESEARCH SERVICES RESEARCH PUBLICATIONS 62 Richard Marais 32 Steve Bagley 52 CANCER RESEARCH UK Molecular Oncology Advanced Imaging and Flow Cytometry Facilities SEMINAR SERIES 2018 70 MANCHESTER INSTITUTE POSTGRADUATE EDUCATION 72 Esther Baena 34 Duncan Smith 53 Prostate Oncobiology Biological Mass Spectrometry Facility OPERATIONS 75 RESEARCH GROUPS Santiago Zelenay 14 Crispin Miller 36 Jen Hughes and Lisa Doar 53 CANCER RESEARCH UK’S LOCAL 82 Cancer Inflammation and Immunity RNA Biology Biological Resources Unit ENGAGEMENT AND DEVELOPMENT Iain Hagan 16 Amaya Virós 38 Natalia Moncaut 55 ACKNOWLEDGEMENT FOR FUNDING OF 86 Skin Cancer and Ageing Transgenic Production Facility THE CANCER RESEARCH UK Cell Division MANCHESTER INSTITUTE Maximiliano Portal 18 Georges Lacaud 40 Garry Ashton 55 Stem Cell Biology Histology CAREER OPPORTUNITIES AT THE CANCER 87 Cell Plasticity & Epigenetics RESEARCH UK MANCHESTER INSTITUTE Angeliki Malliri 20 Claus Jørgensen 42 Mark Craven 56 Systems Oncology Laboratory Services CONTACT DETAILS 88 Cell Signalling Caroline Dive 22 Michela Garofalo 44 Wolfgang Breitwieser 56 Clinical and Experimental Pharmacology Transcriptional Networks in Lung Cancer Molecular Biology Core Facility Caroline Springer 26 Robert Bristow 46 Marek Dynowski 57 Drug Discovery Translational Oncogenomics Scientific Computing Robert Metcalf 28 Patricia Muller 48 Hui Sun Leong 58 Head and Neck Cancer Biology Tumour Suppressors Computational Biology Support The Cancer Research UK Manchester Institute is temporarily located at Alderley Park in Cheshire until we return to our original site in The Oglesby Cancer Research Building Withington. Some research groups and staff remain in the Oglesby Cancer Research Building. 2 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE 3


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    DIRECTOR’S INTRODUCTION Novel luminal progenitor marker of castration-resistant prostate cells – LY6D (red) – allows isolation of these rare cells and by expansion ex-vivo produce a new range of treatments for prostate cancer. Image supplied by Ivana Steiner (Prostate Oncobiology) Over the past year, we have continued to recover from the fire in April 2017 that caused so much damage to our main research facility, the Paterson Building, and by the spring of 2018, we had completed our relocation to our interim home at Alderley Park in Cheshire. Professor To mark the one-year anniversary of the fire, we that our staff and students have attended over Richard Marais held an event at Alderley Park to reflect on the the last year, and we look forward to many more Director of the Cancer Research year and to acknowledge the heroic collective occasions over the coming year where we will UK Manchester Institute effort that was required to complete our move share our progress with those who make our My group has also shown that DNA isolated tumours that are deficient in homologous so quickly. It was also an opportunity to research possible. from plasma can be used as an indicator of recombination. A partnership agreement to celebrate what we have achieved scientifically tumour burden and as a prognostic biomarker develop these PARG inhibitors has been signed despite the extensive disruption we endured. We are proud of our scientific achievements of for overall survival of melanoma patients. The with IDEAYA Biosciences, an oncology-focused On that day, we were delighted to be joined by the year and the progress that we have CEP group extended their work on the biotechnology company, in early 2018. many of our colleagues from The University of continued to make despite the disruption. generation of xenograft models derived from Manchester and the other various external Scientific highlights from the year include a tumour cells isolated from the blood of small Two of our Senior Group Leaders, Iain Hagan organisations who together played a key role in study in Nature Communications from the cell lung cancer patients. Using this approach, and Tim Somervaille underwent quinquennial our recovery. It was an upbeat occasion that Tumour Suppressors’ group who found that a they have identified the utility of Wee1 and PARP reviews in September and despite the disruption demonstrated the resilience and spirit of the combination of host p53 mutation status and inhibition in certain genetic backgrounds that following the fire, their programmes were rated Institute. Although temporary, we are making tumour cell engulfment may promote genomic mimic loss of BRCA function; this approach has very highly. Santiago Zelenay underwent a the most of our new home and seeking new instability. The Leukaemia Biology group now entered clinical trials. In another study, they successful mid-term review having developed opportunities that our environment offers, while uncovered the surprising mechanism by which used short-term cultures from these models ex an exciting portfolio of projects. Earlier this year, working hard to maintain our vital relationships LSD1 inhibition leads to differentiation of acute vivo to expedite the testing of novel therapeutic The University of Manchester formed a new with our many academic and clinical colleagues myeloid leukaemic blast cells. Contrary to the approaches and reduce the number of mice research institute – The Lydia Becker Institute of on both the University main campus and the assumption that the mechanism of action of the required to unravel new lung cancer biology. Immunology and Inflammation; Santiago is the Christie site. I am pleased that our new location inhibitors would be via the abrogation of LSD1 co-lead for the Cancer Immunology section has not prevented us from inviting our histone demethylase activity, the group found The Drug Discovery Unit (DDU) is continuing to together with Rob Bristow. There is further supporters to visit the Institute, to tour our that they disrupt an interaction with the GFI1 develop well under the Directorship of Caroline representation from the Institute including facilities and talk to our scientists. It has been a transcription factor suggesting the potential of Springer who joined from The Institute of Amaya Viros and Rob Metcalf and from Caroline pleasure to meet so many supporters at our further therapeutic options for this type of Cancer Research in autumn 2017. Several Dive who supervises a PhD student together laboratories but also at the various other events leukaemia. The Transcriptional Networks in members of her team moved with her from with Lydia Becker Director Tracy Hussell. Other Lung Cancer group identified micro-RNAs that London and, together with existing members of developments in immune-oncology include modulate gene expression changes that the group, they moved into specialised drug the tumour inflammation and immunology Institute Deputy Director discovery laboratories in Alderley Park in early monitoring laboratory (TIIML) which the CEP promote lung tumorigenesis in a KRAS – Professor Caroline Dive proudly 2018. They are working with an exciting group have set up, led by Elaine Kilgour, to displays her CBE medal dependent manner and determined that they act by turning off key tumour suppressor genes. portfolio of projects, many of which have arisen support biomarker sciences for immune-based The Prostate Oncobiology group found a way from projects within the Institute. The DDU therapeutics. CEP have also played a major part to identify prostate cancer cells that are resistant have also been part of two collaborative studies in the success of the first phase of TARGET to hormone deprivation therapy. In a relating to their PARG inhibitor programme. The which is a ctDNA-driven selection phase I trial. collaborative study with colleagues in Clinical first with the Mitosis and Cancer Pharmacology The molecular profiling of patients in TARGET is and Experimental Pharmacology (CEP) and my group led by Stephen Taylor at The University of underpinned by ctDNA-based liquid biopsies Molecular Oncology group, and with clinicians Manchester, demonstrating the potential for performed by the CEP team and supported by at the Christie, we analysed the genomic PARG inhibition as a therapeutic option for the scientific computing infrastructure at CRUK characteristics of prostate lesions that can be some ovarian cancers that do not display PARP MI. The overall project is a team approach with identified by a commonly used MRI technique. inhibitor sensitivity. The second study with the Christie and The University of Manchester, Our results suggest that complex analysis is colleagues at the Netherlands Cancer Institute including the clinical lead Matt Krebs who needed in order to not underestimate the shows that loss of PARG is a potential completed his PhD with CEP in 2011. The complexity of a tumour and thus the risk of mechanism of resistance to PARP inhibition in project is also supported by the work of the progression. 4 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE DIRECTOR’S INTRODUCTION 5


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    DIRECTOR’S INTRODUCTION (CONTINUED) digital Experimental Cancer Medicine Team CEP with an American Association for Cancer RESEARCH who created eTARGET, a platform that allows researchers, scientists and clinicians to meet virtually and review patient details to support Research (AACR) Women in Cancer Research Scholar Award for Francesca Chemi and a poster prize awarded to Sam Humphrey at the HIGHLIGHTS assessment and clinical decision-making. The CRUK Lung Cancer Centre of Excellence ctDNA activity within CEP is also playing a key workshop. role in supporting the recently initiated PrecisionPanc project aimed at delivering Our Grants Committee continues to provide an personalised treatments for pancreatic cancer invaluable service to the Institute’s scientists in patients based on molecular profiling. Both conducting a peer review of applications prior In this section we highlight some research publications from TARGET and PrecisionPanc are exemplars of to submission. There were some notable 2018 which report significant advances in specific areas. The the personalised medicine agenda that is key to successes, including Isabel Romero-Camarero the mission of the Manchester Institute. from the Leukamia Biology group who was selected papers demonstrate the breadth and the quality of the awarded a Kay Kendall Leukaemia Fund Junior research being undertaken by the groups at the Cancer Certain indicators of our success such as Research Fellowship to understand the funding applications and other awards have regulation of FOXC1 in Acute Myeloid Research UK Manchester Institute. been affected this year as a result of the Leukaemia. Eduardo Bonavita from the Cancer disruption caused by the fire and relocation. Inflammation and Immunity group was Mackay HL, Moore D, Hall C, Birkbak NJ, Enhancer activation by pharmacologic There have, however, been some notable awarded an Advanced Fellowship from EMBO Jamal-Hanjani M, Karim SA, Phatak VM, displacement of LSD1 from GFI1 induces achievements to celebrate. In May I was both and Maria Roel from Prostate Oncobiology Piñon L, Morton JP, Swanton C, Le Quesne J, differentiation in acute myeloid leukemia. delighted and honoured to join the ranks of so received an I2C Postdoctoral award from the Muller PAJ. Cell Reports 2018; 22(13):3641-3659. many of my scientific heroes by receiving the Spanish government. Genomic instability in mutant p53 cancer cells fellowship of the Royal Society. In June, upon entotic engulfment. Lysine-specific Demethylase 1A (LSD1) has Caroline Dive, Deputy Director of the Institute I am delighted to be part of a Grand Challenge Nature Communications 2018; 9(1):3070. recently emerged as a candidate therapeutic and Head of the CEP group visited Buckingham consortium funded by CRUK and led by target in cancer, at least in part due to its high Palace to receive the CBE that she was awarded Stephen Elledge at Harvard Medical School that Cell-in-cell (CIC) structures in histopathological expression in poor prognostic sub-groups of in the New Years’ Honours list for services to shall investigate why specific oncogenes are sections of tumours are defined by the patients with the disease. Initially identified as a cancer research. Rob Bristow who moved to associated with particular cancers but not with presence of one whole cell enclosed in a core component of an RCOR1 and histone Manchester in 2017 was awarded the Research others. Caroline Dive is part of two CRUK- tumour cell. How CIC are formed and how they deacetylase transcription corepressor complex Leadership Award from Prostate Cancer funded consortia; the first is an Accelerator contribute to cancer remains poorly called CoREST, LSD1 was later found to Canada. Senior Group Leaders Georges Lacaud Award to find new personalised approached to understood. The Tumour Suppressor group demethylate histone tails. A first-in-man phase 1 and Angeliki Malliri were awarded treat bowel cancer; the second is an Early investigated CIC structures in lung cancers and trial of the LSD1 inhibitor ORY1001 (from Oryzon Professorships by The University of Manchester Detection Award to determine whether identified an association between the presence Genomics) recently demonstrated that LSD1 and gave enjoyable inaugural addresses monitoring of patients’ liquid biopsies collected of mutant p53 proteins and CIC structures. In inhibitors promote blast cell differentiation in describing their careers to date at an event held in the community from patients with resected cancer cell lines these structures were formed patients with Acute Myeloid Leukaemia (AML) at Alderley Park. Angeliki was also the recipient non-small cell lung cancer can predict early by a process of entotic engulfment. Engulfed associated with translocations targeting the of the Philip Godfrey Fund Memorial Award relapse. Sara Valpione from my group was tumour cells either died or escaped regardless Mixed Lineage Leukaemia gene. The from the Biochemical Society, while Zoi awarded an EORTC Translational Research of their p53 status, but exerted a profound assumption had been that differentiation is Diamantopoulou from her group received the Grant to study the tumour and effect on the host cell. In p53 null host cells the induced through blockade of LSD1’s histone BACR Chris Marshall Prize for Cell Signalling and microenvironment transcriptomic engulfed cells most often caused replication demethylase activity. However, members of the her PhD student Joe Maltas won a poster prize landscape of melanoma. stress and subsequent death of the host cell. In Leukaemia Biology group observed that rapid, at the International PhD Student Cancer contrast, host mutant p53 cells were much extensive drug-induced changes in transcription Conference held at The Francis Crick Institute. There is much to look forward to in the coming more likely to survive this stress, but at the cost occurred without genome-wide accumulation year. We continue to make progress in the of aberrant cell divisions, multinucleation and of the histone modifications targeted for Stuart Williamson from CEP was awarded a planning of the replacement for the Paterson genomic rearrangements. Xenograft injections demethylation by LSD1 at sites of LSD1 binding. prestigious CRUK-Fulbright Scholarship to Building. The ambition is to build a larger facility of high-engulfing cells showed enhanced They also found that a demethylase-defective spend six months at Stanford University to allow for greater interaction and synergy with growth over less frequent engulfers. These mutant rescued LSD1 knockdown AML cells as developing his research into vasculogenic our clinical colleagues and to facilitate a findings suggest that pro-tumourigenic cell efficiently as the wild-type construct. Rather, mimicry in the laboratory of Julien Sage. multidisciplinary approach to our research. engulfment activity is associated with mutant LSD1 inhibitors disrupt the physical interaction Rebecca Lee from my group was awarded the There are several new clinical trials starting in p53 expression, and that the combination of of LSD1/CoREST with the SNAG-domain Association of Cancer Physicians McElwain 2019 that are combined efforts between the cell engulfment and p53 mutant status may be transcription factor GFI1, which is bound to a Prize as well as the Institute’s Dexter Prize for Institute’s scientists and our clinical colleagues. a key factor in chromosomal aberrations in discrete set of enhancers located near to critical Young Scientists in recognition of her research In July, many of our scientists will be human tumours. genes which regulate myeloid differentiation. demonstrating the utility of ctDNA analysis for participating in a conference hosted in The consequent inactivation of GFI1 leads to predicting both disease-free and long term Manchester which will highlight some of these Maiques-Diaz A, Spencer GJ, Lynch JT, increased enhancer histone acetylation within survival of stage II/III melanoma patients interactions and their role in the development of Ciceri F, Williams EL, Amaral FMR, Wiseman hours which directly correlates with up following resection. Melanie Galvin won the phase 1 clinical trials. This promises to be an DH, Harris WJ, Li Y, Sahoo S, Hitchin JR, Mould regulation of nearby, subordinate genes. Andrew Blake Tribute Award for her work in CEP excellent showcase for the exciting DP, Fairweather EE, Waszkowycz B, Jordan refining methods of tumour passage resulting developments taking place in our city and the AM, Smith DL, Somervaille TCP. in the use of fewer mice and eliminating the collaborations that underpin them. need for surgery. There was further success in 6 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH HIGHLIGHTS 7


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    RESEARCH HIGHLIGHTS (CONTINUED) This mitotic cell (DNA stained in blue) has been left at 4°C Somerville TDD, Simeoni F, Chadwick JA, and cellular mechanisms underlying this critical for 30 minutes, which causes depolymerisation of Williams EL, Spencer GJ, Boros K, Wirth C, endothelial-to-haematopoietic transition (EHT) microtubules (magenta), then Tholouli E, Byers RJ, Somervaille TCP. remain poorly understood. In this study, briefly returned to 37°C, causing Derepression of the iroquois homeodomain members of the Stem Cell Biology group a burst of microtubule growth, transcription factor gene IRX3 confers investigated the role of histone deacetylases marked by EB1 staining (green) differentiation block in acute leukemia. during EHT. They first pharmacologically at the growing tips. Cell Reports 2018; 22(3):638-652. inhibited HDAC activity with the pan HDAC Image supplied by Andrew Porter inhibitor Trichostatin A and observed a (Cell Signalling) The acute myeloid leukaemias (AML) are a significant impairment in haematopoietic cell heterogeneous group of proliferative generation from embryonic stem cell-derived malignancies characterised by a hierarchically- HE cells in vitro and from embryo HE cells ex organised cellular structure and accumulation vivo. They then focused on HDAC1 and HDAC2 of poorly differentiated myeloid blasts in bone that are prevalent members of the HDAC family. marrow and blood. Through in silico and Loss of either of these epigenetic silencers functional experiments, the Leukaemia Biology through conditional genetic deletion reduced group identified a significant role for the haematopoietic transition from HE. Combined iroquois homeodomain transcription factor deletion of HDAC1 and HDAC2 was totally gene IRX3 in the differentiation block of AML, incompatible with blood generation. In order to which is the cardinal pathologic feature of the define the molecular changes occurring in disease. IRX3 is normally expressed in the Hdac1 and Hdac2 knockout HE cells, they developing nervous system, limb buds and performed global transcriptomic analysis and heart, whereas transcript levels are very low in determined the genome wide DNA binding normal human bone marrow cells. They patterns of HDAC1 and HDAC2. These analyses observed high IRX3 expression in ~30% of identified TGF-β signalling as one of the patients with AML and ~50% and ~20% pathways controlled by HDAC1 and HDAC2. respectively of those with T-or B-acute They experimentally demonstrated that lymphoblastic leukaemia. Forced expression of activation of this pathway in HE cells reinforces IRX3 alone was sufficient to immortalise normal haematopoietic development. Altogether these were significantly elevated in plasma and in ‘strap’ the nucleus in place. In this study, bone marrow stem and progenitor cells in vitro results establish that HDAC1 and HDAC2 are tumours from patients that underwent surgical scientists from the Cell Signalling group showed and induce lymphoid leukaemias in vivo. critical during EHT, modulate TGF-β signalling resection of early stage NSCLC compared to that the protein STEF, an activator of RAC, a Moreover, IRX3 knockdown induced terminal and that stimulation of this pathway in HE cells normal lung. Mechanistically, we showed that protein which in turn controls actin fibres, is differentiation of IRX3 high AML cells. might therefore be beneficial for producing ELK1 is responsible for miR-30c and miR-21 required for cell migration. Moreover, they Combined expression of IRX3 and Hoxa9 in blood cells for regenerative therapies. transcriptional activation through direct binding showed that STEF localises to the outer nuclear murine bone marrow stem and progenitor cells to the miRNA proximal promoter regions. Taken membrane where it regulates perinuclear RAC1 impeded normal T-progenitor differentiation in together, this study proves that miR-30c and activity. STEF depletion reduced apical lymphoid culture and substantially enhanced Shi L, Middleton J, Jeon YJ, Magee P, Veneziano miR-21 are important mediators of KRAS-driven perinuclear actin cables (a phenotype rescued the morphologic and phenotypic D, Laganà A, Leong HS, Sahoo S, Fassan M, tumorigenesis and could be valid biomarkers by targeting active Rac1 to the nuclear differentiation block of AML in myeloid Booton R, Shah R, Crosbie PAJ, Garofalo M. in NSCLC. envelope), increased nuclear height, consistent leukaemia transplantation experiments, KRAS induces lung tumorigenesis through with the impaired constraint of the nucleus, and through suppression of a myelomonocytic microRNAs modulation. Woroniuk A, Porter A, White G, Newman impeded nuclear orientation during migration. differentiation program. Likewise, in cases of Cell Death and Disease 2018;9(2):219. DT, Diamantopoulou Z, Waring T, Rooney STEF down-regulation also decreased nuclear primary human AML, high IRX3 expression is C, Strathdee D, Marston DJ, Hahn KM, stiffness and reduced expression of TAZ- strongly associated with reduced KRAS is one of the most mutated oncogenes in Sansom OJ, Zech T, Malliri A. regulated genes, indicating an alteration in myelomonocytic differentiation. Overall, our human cancers. MicroRNAs (miRNAs) are small STEF/TIAM2-mediated Rac1 activity at the mechanosensing pathways as a consequence results demonstrate that tissue-inappropriate non-coding RNAs of ~22 nucleotides in length nuclear envelope regulates the perinuclear of disruption of the actin cap. This study derepression of IRX3 contributes to the block of which play a main role in cancer development actin cap. therefore sheds light on the mechanisms myelomonocytic differentiation in AML. and spread. By using inducible human and Nature Communications 2018; 9(1):2124. regulating the perinuclear actin cap, a key mouse cell lines and by overexpressing either regulator of nuclear morphology and hence cell Thambyrajah R, Fadlullah MZH, Proffitt M, Patel wild-type or mutant KRAS (KRASG12D) the Cell migration is important for many migration. R, Cowley SM, Kouskoff V, Lacaud G. Transcriptional Networks in Lung Cancer group physiological processes, such as embryo HDAC1 and HDAC2 modulate TGF-β signaling identified KRAS-modulated microRNAs in development, wound healing and immune during endothelial-to-haematopoietic non-small cell lung cancer (NSCLC). They responses, but is also required for the Barros-Silva JD, Linn DE, Steiner I, Guo G, Ali A, transition. showed that miR-30c and miR-21 were dissemination of cancer cells. When cancer Pakula H, Ashton G, Peset I, Brown M, Clarke Stem Cell Reports 2018; 10(4):1369-1383. significantly upregulated by both KRAS isoforms cells invade, they often have to squeeze through NW, Bronson RT, Yuan GC, Orkin SH, Li Z, and induced drug resistance by silencing NF1, tight spaces, which requires correct orientation Baena E. The first haematopoietic stem and progenitor RASA1, BID and RASSF8. Systemic delivery of of the nucleus, the largest organelle of the cell. Single-cell analysis identifies LY6D as a marker cells are generated during development from a LNA-anti-miR-21 in combination with cisplatin An important cytoskeletal structure that linking castration-resistant prostate luminal cells specific type of endothelium called in vivo suppressed the development of lung regulates nuclear morphology and orientation to prostate progenitors and cancer. haemogenic endothelium (HE) through a tumours in a mouse model of lung cancer during migration is the perinuclear actin cap, an Cell Reports 2018; 25(12):3504-3518. trans-differentiation process. The molecular (KRASLSLG12D). Furthermore, miR-30c and miR-21 array of actin cables above the nucleus that 8 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH HIGHLIGHTS 9


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    RESEARCH HIGHLIGHTS (CONTINUED) and during treatment. The team found that cultures have advantages over long established baseline cfDNA concentration correlated with cell lines that have undergone irreversible drift in pre-treatment tumour burden and the culture and add a new tool to understand the Prostate cancer is one of the leading causes of remain the gold-standard therapeutic correlation was maintained during treatment. biology of SCLC and test targeted therapies. morbidity and mortality in men. While early procedure. However, more recently, there is a Additionally, higher levels of baseline cfDNA stage PCa patients can be treated and cured trend towards the use of multiparametric (mp) levels were associated with worse prognosis. In with local therapies (surgery/radiotherapy), MRI to trigger biopsies in PCa patients, particular, a cut-off value of baseline Lallo A, Frese KK, Morrow CJ, Sloane R, Gulati S, treatment options for metastasised patients combined with genetic testing to refine risk cfDNA=89pg/μL identified two distinct Schenk MW, Trapani F, Simms N, Galvin M, remains palliative. Antiandrogen therapy is the stratification. mpMRI is supposed to provide prognostic groups (HR=2.22 for high cfDNA, Brown S, Hodgkinson CL, Priest L, Hughes A, mainstay therapy for these patients; however, superior images of higher resolution than P=0.004). Patients with cfDNA ≥89pg/μL had Lai Z, Cadogan E, Khandelwal G, Simpson KL, patients will inevitably develop resistance to ultrasound. Importantly, the study led by shorter survival (10.0 versus 22.7 months, Miller C, Blackhall F, O'Connor MJ, Dive C. these therapies. Notably, the exact identity of researchers in Prostate Oncobiology and P=0.009; HR=2.22 for high cfDNA, P=0.004) The combination of the PARP inhibitor Olaparib castrate resistant (CR) cells and their relation to Molecular Oncology, identified that even with and the significance was maintained when and the WEE1 inhibitor AZD1775 as a new CR prostate cancer (CRPC) is unresolved. In this this superior technique >10% potentially compared to the standard melanoma therapeutic option for small cell lung cancer. paper, the Prostate Oncobiology group use significant PCas are missed because they are prognostic biomarker (LDH) in a multivariate Clinical Cancer Research 2018; 24(20):5153- single-cell gene profiling to analyse the not detected by mpMRI. They hypothesised that analysis. Thus, the group have demonstrated 5164. molecular heterogeneity in basal and luminal- the genomic makeup of these “invisible” lesions that cfDNA is a surrogate biomarker of tumour compartments. Within the luminal could provide important insights into their burden in metastatic melanoma patients, and Small cell lung cancer (SCLC) is a highly compartment, we identify a subset of cells metastatic capacity and help to assess their moreover it is prognostic for overall survival. aggressive lung cancer that metastasises early intrinsically resistant to castration with a potential lethality. To address this question, the with poor prognosis. For the past three decades, bi-lineage gene expression pattern. We team have recently completed a study standard of care chemotherapy has resulted in discover LY6D as a marker of CR prostate correlating genomics and mpMRI in men Lallo A, Gulati S, Schenk MW, Khandelwal G, temporary tumour responses followed by progenitors with multipotent differentiation and undergoing radical prostatectomy in order to Berglund UW, Pateras IS, Chester CPE, Pham disease relapse in the majority of patients and enriched organoid-forming capacity. Lineage elucidate the genomic characteristics of mpMRI TM, Kalderen C, Frese KK, Gorgoulis VG, Miller novel therapies are urgently required. In this tracing further reveals that LY6D+ CR luminal visible and non-visible tumours and to assess C, Blackhall F, Helleday T, Dive C. study and in collaboration with AstraZeneca, cells can produce LY6D- luminal cells. In the inter-relationship. They found that the Ex vivo culture of cells derived from circulating members of the Clinical and Experimental contrast, in luminal cells lacking PTEN, LY6D+ intra-tumour heterogeneity within visible tumour cell xenograft to support small cell lung Pharmacology group exploited their SCLC cells predominantly give rise to LY6D+ tumour mpMRI lesions bears the risk of misclassifying cancer research and experimental therapeutics. circulating tumour cell patient derived explant cells, contributing to high-grade PIN lesions. patients when using genomic biomarkers from British Journal of Pharmacology [Epub 14 models (CDX) to test the new drug combination The team used gene expression analyses from a single biopsy. These findings have practice November 2018] of a DNA damage repair inhibitor (PARP) and a patients’ biopsies and identified that LY6D changing implications as they indicate that cell cycle regulator inhibitor (WEE1 kinase). The expression correlates with early disease restricting biopsies to mpMRI visible lesions In 2014, the Clinical and Experimental rationale for this combination in SCLC is centred progression, including progression to CRPC. underestimates the complexity of PCa, which Pharmacology group pioneered a new on high levels of endogenous DNA damage, Their work thus identifies a subpopulation of bears the risk of misclassifying the patients’ risk approach to generate clinically relevant models rapid proliferation rate, absence of G1 luminal progenitors characterised by LY6D for progression. of small cell lung cancer (SCLC) using a patient’s checkpoint function due to mutant TP53 and expression and intrinsic castration resistance. 10ml blood sample from which they enriched oncogene driven replication stress. The These studies suggest that LY6D may serve as a circulating tumour cells and explanted them combination efficacy was variable across CDX prognostic marker for advanced prostate Valpione S, Gremel G, Mundra P, Middlehurst P, into immune compromised mice (Hodgkinson models derived from patients with differing cancer, which will allow researchers to further Galvani E, Girotti MR, Lee RJ, Garner G, et al, Nature Medicine). The resultant so called degrees of initial chemosensitivity, but superior stratify risk profiles for PCa patients and to help Dhomen N, Lorigan PC, Marais R. ‘CDX’ models were a landmark for SCLC to standard of care chemotherapy (cisplatin/ tailor more specific therapies. Plasma total cell-free DNA (cfDNA) is a surrogate research. However, these in vivo models take etoposide). In one CDX model (with a PALB2 biomarker for tumour burden and a prognostic several months to develop and need to be mutation driving ‘BRCAness’), the combination biomarker for survival in metastatic melanoma passaged up to three times before experiments resulted in cures. Of importance, they tested the Marina A. Parry, Shambhavi Srivastava, Adnan patients. can commence. To both reduce the number of novel combination in a pair of CDX models Ali, Alessio Cannistraci, Jenny Antonello, João European Journal of Cancer 2018; 88:1-9. animals required (in line with the 3Rs’ principles) made from the same patient prior to treatment Diogo Barros-Silva, Valentina Ubertini, Vijay and to accelerate their studies testing novel and again post treatment at disease relapse. Ramani, Maurice Lau, Jonathan Shanks, The total metastatic tumour volume or burden therapeutics for this aggressive lung cancer, the Here the combination showed promise in the Daisuke Nonaka, Pedro Oliveira, Thomas is prognostic in melanoma. However, group developed ex vivo short-term cultures pre-treatment model but was ineffective at Hambrock, Hui Sun Leong, Nathalie Dhomen, determining the tumour burden is extremely from established CDX tumours. They defined disease progression. These data have informed Crispin Miller, Gerard Brady, Caroline Dive, time-consuming and not feasible in clinical the reversible and irreversible molecular early clinical trials of Olaparib and AZD1775 that Noel W. Clarke, Richard Marais, Esther Baena. practice. The aim of the study undertaken by changes that occur during short term culture are currently underway. Genomic evaluation of multiparametric members of the Molecular Oncology group and showed that they can genetically magnetic resonance imaging-visible and was to assess the potential of plasma total cell manipulate CDX cultures and return CDX cells to -nonvisible lesions in clinically localised free DNA as surrogate biomarker of tumour mice for in vivo function testing and cell tracking prostate cancer. burden and prognosis in metastatic melanoma experiments without distorting their tumour European Urology Oncology [Epub 18 patients. With this purpose, they measured growth dynamics. They reported studies using September 2018] tumour burden in melanoma patients by CDX cultures to screen novel therapeutics calculating the total volumes (the sum of single before selecting promising candidates to The diagnosis of prostate cancer is based on metastases) visualised by standard scans and validate in vivo, providing data to support early imaging studies, followed by ultrasound- correlated that to total plasma circulating clinical trials. In summary, short-term CDX guided biopsies of suspicious lesions, which cell-free DNA (cfDNA) concentration, before 10 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH HIGHLIGHTS 11


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    CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH GROUPS Section of healthy skin highlighting the different cell types that develop into melanoma (pink) or squamous cell carcinoma (green and red). Image taken on the Olympus VS120 Virtual Slide Microscope. Image supplied by Candelaria Bracalente (Molecular Oncology) 12 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE 13


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    www.cruk.manchester.ac.uk/Our-Research/Cancer-Inflammation-and-Immunity treatment induced cancer cell death is associated with the induction of immunological tolerance against dead-cell associated antigens and/or pro- CANCER INFLAMMATION tumourigenic inflammatory responses that instead fuel tumour progression and spread. The AND IMMUNITY underlying basis for these opposing outcomes is largely unknown. We have thus embarked upon a new project intended to shed light onto this issue by exploiting our expertise on the analysis of the immune and inflammatory landscape of tumours. Immunotherapies have led to remarkable clinical responses across a Overall our recent findings support a model in wide range of cancer types. The development of these cancer which the tumour inflammatory profile at treatments builds on more than one hundred years of basic research baseline and early on-treatment largely dictates cancer progression and treatment outcome. in immunology. Under the premise that further fundamental Therefore, to fully characterise the inflammatory research will provide insights into how to make immunotherapies and immune landscape of tumours, over the last year we have significantly increased our use of more efficient and safer, our group studies the principles that multiplex immunoassays and next generation Group Leader determine the susceptibility of tumours to spontaneous or therapy- sequencing techniques. The use of bioinformatics Santiago Zelenay induced cancer immunity. Combining the use of preclinical cancer has thus become an essential and regular component of our research toolkit. Postdoctoral Fellows models with bioinformatic analysis of cancer patient samples, we NHS Foundation Trust specialising in breast Eduardo Bonavita have uncovered key cellular and molecular regulators of cancer Figure 1 cancer, we will soon start testing in the clinic Finally, our on-going research projects have Agrin Moeini1 Our working model postulates some of our more exciting preclinical findings. emphasised how much there is still to learn about Victoria Pelly immunity. Targeting these offers promising therapeutic avenues to that the tumour inflammatory key aspects of the immune response to cancer Scientific Officer enhance the efficacy of current cancer therapies. profile at baseline and on- treatment influences tumour In another project, we continue our efforts to and have triggered new fundamental questions. identify dominant mediators of immune evasion. To start answering them our group has expanded Shih-Chieh Chiang1 outgrowth and spread. Within this framework, the current basic We argue that these mechanisms, unlike and embarked upon further collaborations with Graduate Students Immunotherapy shares a podium of oncology have uncovered an essential role for natural killer research questions of the Cancer recessive mechanisms of immune escape, such industry and prominent cancer biologists and Charlotte Bell treatments available alongside more cells in spontaneous and immune-checkpoint Inflammation and Immunity as loss of tumour antigens or downregulation of renowned immunologists, including Alberto Christian Bromley conventional options such as surgery, blockade-induced cancer immunity. Eduardo group are centred on identifying the antigen-presentation machinery by cancer Mantovani (Humanitas University), Carlos Caldas Eimear Flanagan cellular and molecular mediators cells, constitute attractive therapeutic targets to chemotherapy and radiotherapy. Checkpoint Bonavita gave a talk on this work during the 5th (CRUK Cambridge Institute), Daniel Davies that regulate the type of 1 inhibitors in particular constitute the most European Congress of Immunology held in boost the efficacy of current immunotherapies. (University of Manchester) and Nadia Guerra Joined in 2018 inflammatory response at the promising pan-cancer drugs promoting Amsterdam in September 2018. Through in silico tumour bed. Screening a large and heterogeneous panel of (Imperial College London). Some of these significant patient benefit across multiple analysis of cancer patient datasets, we have also murine cancer lines of diverse tissue origins and collaborations have already resulted in high- malignancies. Numerous recent clinical trials found a remarkable conservation in the genetic makeup, we have uncovered conserved profile collaborative research articles published in have shown that these drugs can induce inflammatory profile of our cancer mouse cancer cell intrinsic features that selectively Nature and Cell (Molgora et al. Nature 2017; responses that frequently outperform the models and multiple human malignancies. define cancer lines with unique and profound Bottcher et al Cell 2018). mainstream treatments. Nonetheless, the Moreover, we have derived an inflammatory ability to regulate multiple immune fraction of patients experiencing profound and gene signature that predicts overall survival in a cell types. Publications listed on page 62 durable responses is still limited and restricted to wide range of cancer types. This signature also certain tumour types. Likewise, these treatments associates with the outcome from immune Despite recent breakthroughs with many of these are associated with adverse, and often life checkpoint blockade in various patient datasets, treatment modalities aimed at harnessing the threatening, effects. Critically, we currently have outperforming previously published immune anti-tumour function of the immune system, no reliable means to predict which patients will signatures. Encouraged by these findings and in cytotoxic therapy remains the standard of care derive benefit from treatment and whether a collaboration with Cancer Research for most unresectable malignancies. Tumour patient will develop toxicities. In the Cancer Commercial Partnerships, we have filed an shrinkage following chemotherapy or Inflammation and Immunity group we believe application to patent our method for predicting radiotherapy is largely attributed to the damaging that increasing our understanding of the outcome from these immunotherapies. effects of these treatments on rapidly principles and rules that define the proliferating cells. In addition to their direct killing immunogenicity of cancer will help answer In parallel, we have been evaluating the effect on cancer cells, growing evidence also these major open questions. We carry out basic therapeutical significance of our findings and points to a major role for the immune system in and translational research mining cancer patient found that the efficacy of immune checkpoint regulating the mid and long-term efficacy of datasets and simultaneously study genetically inhibitors can be greatly enhanced by co- these mainstream clinical practices. These engineered cancer models to establish administering anti-inflammatory drugs in various observations are consistent with the notion that cause-and-effect relationships in relevant preclinical models. Victoria Pelly gave a talk at certain cell death modalities, by virtue of the in vivo settings. the ISREC-SCC Symposium 2018: Horizons of release of the so-called damage-associated Cancer Biology and Therapy in Lausanne molecular patterns, can stimulate innate immune Relying on murine cancer models, we have describing our progress in this highly clinically antigen-presenting cells, drive T cell responses made great progress characterising and relevant line of research. This work has also been against antigens contained within the dying cells comparing the cellular and molecular vital for the design of a soon to open clinical trial. and promote tumour growth control. However, composition of tumours with unequivocal In this study, led by Anne Armstrong, a this concept is highly controversial, with an progressive or regressive fates. In doing so, we Consultant Medical Oncologist at the Christie equally vast amount of literature suggesting that 14 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE CANCER INFLAMMATION AND IMMUNITY 15


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    www.cruk.manchester.ac.uk/Our-Research/Cell-Division Cdk1-Cyclin B activation promotes the activities of an array of mitotic protein kinases that phosphorylate a broad spectrum of targets to CELL DIVISION drive chromosome condensation and the formation and function of the division apparatus. Once each phase of mitosis is complete, each phosphorylation event is reversed to support transition to the next phase of division. As a protracted mitotic arrest can trigger apoptosis, there is great interest in learning more about the biology of protein phosphatases to identify routes that could enhance the mitotic delay that The inappropriate proliferation of cancer cells can arise from naturally arises as a consequence of the unchecked cell division, a failure to engage cell death pathways, or abnormal karyotype of tumour cells. simultaneous changes in both. Understanding how the diverse cues PP1 and the protein phosphatase 2A isoforms are integrated to co-ordinate cell division and death is therefore PP2A-B55 and PP2A-B56 play key roles in mitotic critical to understanding the biology of cancer. signalling network that controls the timing and control. PP1 is recruited to docking sites from Figure 1: Feedback control by execution of mitotic exit events also relies upon where it dephosphorylates targets. Hetero- Polo kinase in Cdk1-Cyclin B the anchorage of a signalling network to an SPB trimeric PP2A enzymes comprise single Group Leader Indeed, DNA damaging and anti-mitotic We study three core aspects of cell cycle activation at the G2/M transition scaffolding and catalytic subunits, alongside one associated scaffold. In this case it is the Iain Hagan therapies owe much of their success to the control: (1) the role played by the centrosome Cdk1-Cyclin B activity is held in of four different types of regulatory subunit. anchorage of the Septum Initiation Network (SIN) checkpoint pathways that ensure transition in determining when cells commit to genome check in interphase as a Multiple, alternatively spliced, genes give the consequence of phosphorylation to the scaffold Sid4 that is essential for the correct Associate Scientist through the cell division cycle only occurs when segregation; (2) the biology of the Wee1 family potential for hundreds of variants of PP2A-B55 of Cdk1 by Wee1. Cdc25 removes control of mitotic exit under normal conditions Agnes Grallert genome integrity is guaranteed. We therefore of kinases that restrain entrance into division in and PP2A-B56 in humans, whereas fission yeast the inhibitory phosphate to and for the ability to restrain division when mitotic study the targets of two of these therapeutically response to genome stress; and (3) the can live on one of each, or in the case of Honorary Consultant Oncologist trigger mitosis. This trigger level spindle function or cytokinesis is perturbed. important checkpoint pathways: (1) the regulation of the mitotic exit protein of Cdk1-Cyclin B then activates PP2A-B55, none. Andrew Hudson Unexpectedly, we identified a second signal commitment to; and (2) the exit from, mitosis – phosphatases whose activities counteract polo kinase to further boost transduction network on Sid4 that determines Postdoctoral Fellows the physical process of genome segregation. those of the pro-mitotic kinases in order Cdc25 activity and inhibit Wee1 to After confirming that PP1, PP2A-B55 and the level of Cut12/PP1/Polo signalling required to Because the regulatory networks that control to drive cells out of division. make this transition a bi-stable Daniela Eckert promote mitotic commitment. Such confluence PP2A-B56 activities decline upon mitotic Zoe Edwards cell division are highly conserved, we switch between two distinct states. Our work shows that of signalling networks at the centrosome commitment in fission yeast, we found that direct Lenka Halova complement our studies of cell division in The initial appearance of active Cdk1-Cyclin recruitment of PP1 to PP2A-B55 and PP2A-B56 Wei Ma signalling events on the fission provides a plausible rationale for the use of the human cell lines with manipulation of the B on human centrosomes, before propagating reactivates these PP2A phosphatases to support Pawan Singh yeast equivalent of the centrosome as a signalling hub: signals from simple, unicellular, fission yeast in order to throughout the cell, suggests that the centrosome, the spindle pole appropriate mitotic progression/exit. Mitotic Neslihan Zohrap1 multiple pathways, to a limited number of identify the key questions to ask of the centrosome provides a specific body, determine when this switch inhibition of PP1 arises from direct neighbouring scaffolds on the SPB, can set the Scientific Officers analogous controls in the complex context of microenvironment to trigger the G2/M transition. is flipped to trigger division. phosphorylation by Cdk1-Cyclin B. The flux through the outgoing signalling cascades Kuan Yoow Chan2 human cell division cycle control. Our studies of the fission yeast centrosome destruction of Cyclin B subsequently allows PP1 Figure 2: The mitotic PP1-PP2A that determine the timing of mitotic progression. Eleanor Wendy Trotter equivalent, the spindle pole body (SPB), provide to auto-dephosphorylate and restore its own phosphatase relay In a typical cell division cycle the G1 gap phase molecular insight into how and why this switch phosphatase activity. Reactivated PP1 then Graduate Students PP1 and PP2A activities are all We are applying the philosophies acquired during precedes DNA replication in S phase, before a may operate. repressed upon entry into mitosis. reactivates PP2A-B55. Polo phosphorylation of Zoe Lee these studies of fission yeast cell cycle control to second gap phase, G2, separates S from The mode of PP2A repression is the PP1 docking site of PP2A-B56 initially blocks Millie Jones1 address the role/s played by Wee1 family kinases genome segregation in mitosis (M phase). We were able to drive fission yeast cells into unclear however, it is well PP1 binding to PP2A-B56. When Polo activity in human cell cycle progression. While the 1 Joined in 2018 Growth, developmental and environmental cues division by releasing Cdk1-Cyclin B or Polo established that Cdk1-Cyclin B declines in mitotic exit, PP2A-B55 phosphorylation represses PP1 development of a Wee1 inhibitor has rapidly 2 Left in 2018 control the rate of proliferation by determining kinase activity at the SPB. In contrast, release at dephosphorylates the Polo phosphorylation site activity. Cyclin B destruction then progressed through to clinical trials, we still know the timing of progression through both the point any other location around the cell had no impact on B56 to allow PP1 to reactivate PP2A-B56 allows PP1 itself to auto- remarkably little about the basic biology and use of commitment to the cell cycle in G1 phase, upon division timing. Our attempts to define the (Figure 2). We are now assessing the impact of catalytically remove this of the three Wee1 family kinases in the cancer cell known as the “restriction point”, and the molecular basis for such a striking impact have inhibitory phosphate from itself. phosphorylation on PP2A-B55 function and cycle. As a full understanding of Wee1 biology is transition from G2 into M. Passage through these been guided by lessons from the SPB scaffold As PP1 is bound to the B55 seeking the phosphorylation events on PP2A key to identifying the optimal context and full key transitions is driven by the activation of Cut12. Simply blocking the recruitment of regulatory subunit of PP2A-B55 at complexes that are reversed by PP1 recruitment spectrum of opportunity for Wee1 inhibition in distinct CDK-Cyclin protein kinase complexes. protein phosphatase 1 (PP1) to Cut12 enabled this time, PP1 reactivation the clinic, we are addressing when, where and to restore PP2A-B55 and PP2A-B56 activities. The G2/M transition is a critical safeguard of us to delete the cdc25+ gene without immediately restores PP2A-B55 activity. In contrast, PP2A-B56 is how, these kinases are used in human cancer genome integrity; incomplete DNA replication compromising viability. This bypass of the unable to recruit PP1 because cells. or DNA damage triggers checkpoint pathways requirement for an otherwise essential mitotic Polo kinase phosphorylates a that block the G2/M transition, to ensure that inducer arose from the impact of the Cut12/PP1 residue within the PP1 docking chromosomes are not segregated when axis on Polo kinase activity. Polo activity was site on the regulatory, B56 incomplete or damaged. The G2/M transition is inappropriately elevated by the abolition of PP1 subunit. Once Polo activity driven by activation of the Cdk1-Cyclin B protein recruitment to Cut12. Enhanced Polo activity declines at the end of mitosis, kinase. Wee1 related kinases phosphorylate the probably overcomes the need for Cdc25 PP2A-B55 can overcome Polo activity towards this site and catalytic subunit, Cdk1, to inhibit the complex because it boosts Polo’s ability to inhibit Wee1 to remove the inhibitory phosphate during interphase. This phosphate is removed by such a degree that it completely silences Wee1. from the PP1 docking site of B56. Cdc25 phosphatases to promote mitotic entry. Then, without the kinase putting the phosphate Consequently PP1 can be Cdk1-Cyclin B activation promotes a positive onto Cdk1, there is no need for the phosphatase recruited to PP2A-B56 and this feedback loop that boosts Cdc25 and inhibits that normally reverses the missing second PP2A activity is Wee1 activities to ensure that mitotic phosphorylation. reactivated at the end of mitosis. Reprinted by permission from commitment is a rapid and irreversible bi-stable Macmillan Publishers Ltd: Nature switch (Figure 1). SPB/centrosomal control of cell cycle 517:94-98, copyright 2015. progression extends beyond Cut12. The 16 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE CELL DIVISION 17


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    www.cruk.manchester.ac.uk/Our-Research/Cell-Plasticity-and-Epigenetics CELL PLASTICITY & EPIGENETICS During neoplastic development cancer cells are constantly exposed to ever-changing conditions, first within their niche of origin and further by the ecosystems encountered whilst colonising other tissues. The fast-paced nature of the environment drives micro- evolutionary processes that give rise to different sub-clones of cancer cells within a tumour leading to increased heterogeneity Institute Fellow during the course of the disease. This heterogeneity can arise either Maximiliano Portal as a result of somatic mutations acquired through the development Postdoctoral Fellow of the tumour or due to the ability of the same genotype to produce Yelizaveta Shlyakhtina1 many discrete, sometimes dramatically different, phenotypes. The Graduate Student latter phenomenon is called phenotypic plasticity and provides a Katherine L Moran1 dynamic source of diversity that may support rapid adaptation 1 Joined in 2018 during various environmental perturbations. Our group is interested in unravelling the molecular details underlying cellular plasticity in order to understand the molecular mechanisms that cancer cells use to escape from therapeutic paradigms. An outstanding question in the cancer field establishing experimental validation pipelines The past year was one of settlement and growth activating apoptotic cascades that will only lead Figure 1: Our model system. relates to whether the observed resistance to a that will enable us to identify in a robust manner for our group. We successfully completed a first to DNA damage at the cellular dismantling stage. a) Line graph representing the given treatment is an inherent feature of a which pathways are indeed fundamental to round of recruitments and rapidly established a This, together with the reversible nature of TRAIL response of hRAS(G12V) subpopulation of cells contained within the naïve maintain the resistant state and to describe their transformed clonal cell working environment within CRUK MI. We tolerance observed in clonal cell populations, population or whether this feature arises “de role in the acquisition and/or propagation of the populations when challenged initiated successful pilot experiments and strongly suggests that the phenomenon of with TRAIL for a given period of novo” in response to treatment. In an effort to TRAIL-tolerant phenotype. established many of the bioinformatic pipelines fractional killing is mainly epigenetically time and then released from the discriminate between these possibilities, we required to analyse our output data. Particularly, encoded rather than based on the acquisition of selective pressure. Apoptosis was found that hRAS(G12V)-transformed clonal Following our initial observations, the main goal we focused our attention onto setting up permanent genetic alterations. This particularity daily induced by TRAIL (maximum populations harbour up to 8 discrete and of our work is to unravel the key molecular appropriate model systems to answer our singles out the TRAIL system as an ideal model dose) and determined by transcriptionally divergent subpopulations that players and mechanisms that orchestrate the PARP-cleavage 4hs after queries and got everything up and running to study the inheritance of epigenetically co-exist within treatment naïve cells. Strikingly, establishment, maintenance and temporal induction (maximum PARP swiftly. encoded traits as the TRAIL-tolerant phenotype activity). The main biological each individual subpopulation responds to TRAIL propagation of stable phenotypic states within is propagated throughout several cell divisions phases observed are depicted. treatment by re-adjusting their gene expression isogenic populations of cancer cells that are The model system after TRAIL withdrawal and fades away gradually b) tSNE plot representing the profile generating once again up to 8 discrete exposed to a cytotoxic challenge. In those lines, Our group is interested in exploring the until full reversion is reinstated. identified 8 subpopulations divergent subpopulations. This result indicates we hypothesise that differential RNA expression mechanistic details underlying cellular plasticity observed in hRAS(G12V) that cellular plasticity may play a major role in and/or epigenetic landscapes within isogenic and in particular its relevance in drug tolerance. The data transformed clonal cell lines as determining the outcome of the apoptotic populations determine phenotypic lineages that determined by single-cell RNA To do so, we take advantage of the exquisite Our initial experiments demonstrate that a response and suggests that resistance to can significantly vary in their response to the sequencing. response exhibited by cancer cells when variety of hRAS(G12V) in vitro transformed cell treatment can be sustained concomitantly by cytotoxic agent; yet all may eventually lead to the challenged with TRAIL (Tumor-necrosis factor lines display fractional killing and reversible several discrete transcriptional and/or epigenetic establishment of resistance. Moreover, our data Related Apoptosis Inducing Ligand). Briefly, resistance to TRAIL. We were able to map the states. supports the notion that the ability of a cell to TRAIL triggers apoptosis by a clearly defined precise moment where the resistant state is change its molecular properties and convert to a molecular route and, though breakthrough acquired within the clonal population, the Moreover, our results show that resistant cells are different stable phenotype – known as lineage evidence supports the use of TRAIL as a duration of the molecular “memory” and its dramatically different when compared to naïve switching – lies at the basis of non-genetic therapeutic agent for many cancer types, propagation in the absence of the challenging populations at the signal transduction level. adaptation. Overall, our research will begin to fractional killing and reversible resistance have agent. Interestingly, we were able to recapitulate Indeed, a handful of tyrosine phosphorylation shed light onto the molecular mechanisms been observed in many cases. Notably, the the acquisition, propagation and reversion of the modulated pathways are solely activated in the orchestrating acquisition of “temporal molecular primary mode of action of many currently used resistant phenotype sequentially, thus not only resistant state and we are now exploring the memory” and will provide a first insight into the therapeutic agents is to directly induce DNA suggesting that the process is reversible and immediate consequences of such events in our machineries that grant the inheritance of complex damage, thereby being mutagenic by default. In epigenetically encoded but also demonstrating system. Thus, we are currently in the process of traits during adaptation to a new environment. net contrast, TRAIL promotes cell death by first the robustness of the model system. 18 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE CELL PLASTICITY & EPIGENETICS 19


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    www.cruk.manchester.ac.uk/Our-Research/Cell-Signalling Figure 1. Model describing the role of perinuclear STEF. CELL SIGNALLING STEF at the nuclear envelope generates a localised pool of active Rac1 which is required for the formation and/or stabilisation of perinuclear actin cables and for perinuclear actomyosin contractility. Disruption of the perinuclear actin cap in STEF depleted cells alters the structural and morphological properties of Recurrent mutations and overexpression in tumours implicate the nucleus, with consequences for directed cell migration and the small GTPase RAC and its activators, the guanine nucleotide mechanotransduction. exchange factors (GEFs), in the formation and progression of cancers. Furthermore, the effects of deleting genes encoding RAC proteins or RAC GEFs in mouse, or of inhibiting RAC-GEF/RAC interactions with drugs, strongly suggest that targeting RAC Group Leader signalling could constitute a cancer treatment. disassembly and scattering of epithelial cells by regulator of the perinuclear actin cap, a Angeliki Malliri the oncoprotein SRC requires phosphorylation contractile structure composed of thick, aligned and consequent cleavage and depletion of actomyosin filaments that interact with the Postdoctoral Fellows However, owing to multiple physiological roles GEFs might determine whether it has a pro- TIAM1 from cell-cell adhesions (Woodcock et al., apical surface of the interphase nucleus via the Zoi Diamantopoulou2 of RAC and RAC functions that antagonise or anti-migratory phenotype. GEFs are Mol Cell 2009; 33: 639). Moreover, in another Linker of the Nucleus and Cytoskeleton (LINC) Pauline Jeannot study from our laboratory (Vaughan et al., Cell complex. The actin cap functions to constrain Matteo Menotti tumour dissemination, sustained suppression of typically large proteins harbouring multiple RAC signalling could be detrimental. Given this protein-protein interaction domains and, Rep 2015; 10: 88), we found that TIAM1 is the nucleus and is required for efficient re- Aishwarya Payapilly challenge, the research of the Cell Signalling besides stimulating guanine nucleotide ubiquitylated and degraded specifically from the orientation of the nucleus in polarising cells. The Andrew Porter2 group aims to distinguish RAC-dependent exchange, they act as molecular scaffolds cell-cell adhesions upon treatment of cells with tension force required to constrain and anchor Scientific Officers effects that promote tumour growth and targeting active RAC to particular subcellular hepatocyte growth factor (HGF), a cytokine that the nucleus is dependent on actomyosin Gavin White progression from those that antagonise tumour locations and potentially increasing the local is abundant in cancer and promotes invasion of contractility generated by the actin motor Andrew Porter1 cancer cells. Non-muscle myosin IIB (NMMIIB); depletion of progression so that RAC signalling might be concentration of selective effector molecules targeted more effectively. influencing downstream processes. In a recent NMMIIB causes nuclear expansion and an Graduate Students study (Marei et al., Nat Commun. 2016; 7: 10664), Apart from these studies showing that TIAM1 over-rotation phenotype. There is mounting Ryan Guilbert1 Joe Maltas RAC, a member of the RHO-like family of we provided evidence that such a hypothesis is inhibits migration by promoting cell-cell evidence that the actin cap works to bridge the Hannah Reed GTPases, cycles between a GDP- and a correct. We demonstrated that two RAC GEFs, adhesion we recently identified another extracellular environment and the nucleus via its GTP-bound state. When GTP-bound, it interacts TIAM1 and P-REX1, promote RAC-driven mechanism by which TIAM1 hinders migration. connection with actin cap associated focal 1 Joined in 2018 with various effector molecules that regulate inhibition and RAC-driven promotion of cell We demonstrated that TIAM1 localises in the adhesions and the LINC complex, providing a 2 Left in 2018 nucleus of several colorectal cancer cell lines direct, rapid pathway for mechanotransduction. several cellular processes including proliferation migration respectively, through regulating the and migration. Multiple mechanisms control RAC interactome. We showed that TIAM1 and that nuclear TIAM1 inhibits their migration We showed that STEF localises to the nuclear RAC activity, including control of nucleotide promotes the association of RAC with proteins via suppressing the interaction of the envelope, co-localising with the key perinuclear binding and hydrolysis by GEFs and GTPase known to be important for the formation and transcriptional co-activator TAZ with its cognate proteins Nesprin-2G and NMMIIB, where it Activating Proteins (GAPs) respectively, maintenance of cell-cell adhesions, while transcription factor TEAD. Suppression of this regulates the activity of perinuclear Rac1. We regulation of subcellular localisation and P-REX1 enhances the interaction between RAC interaction by TIAM1 inhibited expression of TAZ/ also showed that STEF depletion reduces apical modulation of RAC protein levels (reviewed in and proteins promoting cell migration, such as YAP target genes implicated in epithelial- perinuclear actin cables (a phenotype rescued Porter et al., Small GTPases 2017; 7: 123). As the actin-remodelling protein Flightless-1 mesenchymal transition, cell migration, and by targeting active Rac1 to the nuclear envelope), mentioned above, several studies using homolog. Thus, in this study, we provided direct invasion. Consistent with these in vitro data, we increases nuclear height and impairs nuclear recombinant RAC and RAC GEF mice have evidence that RAC GEFs are critical determinants showed that TIAM1 localised not only in the re-orientation during front-rear polarisation. shown that RAC is required for the formation and of selectivity in signalling events downstream cytoplasm, but also in nuclei of tumours of a Moreover, we observed a decrease in pMLC and growth of tumours. However, the role of RAC in of RAC. colorectal cancer microarray. We also showed myosin-generated tension at the nuclear the malignant progression of tumours is not that nuclear staining intensity significantly envelope in STEF-depleted cells, indicating that always positive. There are cases where deletion Role of RAC and its regulators in inhibiting decreased with advancing Dukes stage and that localised STEF-mediated Rac1 activity might of RAC GEFs leads to tumours with increased migration and antagonising malignant patients with high nuclear TIAM1 had regulate NMMIIB activity to promote stabilisation malignancy and there are reports suggesting progression significantly better survival than those with low of the actin cap. Consistent with these data, STEF that reduced RAC levels correlate with more Mice deficient for the RAC activator TIAM1 are nuclear TIAM1 (Diamantopoulou et al., Cancer depletion resulted in a decrease in nuclear aggressive tumours (Porter et al., Small GTPases resistant in the formation and growth of Cell 2017; 31:621). stiffness and reduced expression of TAZ- 2017; 7: 123). In line with these studies, in vitro RAS-induced skin tumours. However, tumours regulated genes, indicating that data have shown that activation of RAC may lead arising in Tiam1-deficient mice progressed more Role of RAC and its regulators in promoting cell mechanosensing pathways are altered as a to opposing migratory phenotypes raising the frequently to malignancy (Malliri et al., Nature migration consequence of disruption of the actin cap possibility that targeting RAC in a clinical setting 2002; 417: 867). This can be explained by the role As mentioned above, the RAC activator P-REX1 (Figure 1 and Woroniuk et al., Nat Commun. could exacerbate tumour progression. For these of TIAM1 and RAC in the regulation of cell-cell promotes cell migration and invasion. Apart 2018; 9: 2124). We therefore suggest that the reasons it is important to identify the factors that adhesion; TIAM1 is required for both the from P-REX1, we have previously shown that functional role of STEF at the nuclear envelope influence the selection of RAC-driven cellular formation as well as the maintenance of activation of RAC by another RAC-specific GEF, impacts on directed cell migration, which we processes. We hypothesised that RAC GEFs may cadherin-based adhesions (Malliri et al., J Biol STEF/TIAM2, promotes cell migration (Rooney et show is compromised in STEF depleted cells. be critical determinants of downstream RAC Chem 2004; 279: 30092). Consistent with these al., EMBO Rep. 2010; 11: 292). In a recent study, signalling and that activation of RAC by different early findings, optimal cell-cell adhesion we established that this RAC activator is a critical Publications listed on page 62 20 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE CELL SIGNALLING 21


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    www.cruk.manchester.ac.uk/Our-Research/CEP/CEP-Home Laura Stephenson Julie Stevenson METASTATIC CELLS ARE DETECTED IN MULTIPLE ORGANS OF CDX17P, Siobhan Southam INCLUDING BRAIN CLINICAL AND EXPERIMENTAL Jason Swift Jenny Ward Clinical Fellow Micrometastases Tumour Lungs Liver Brain Ovary Bone PHARMACOLOGY Alicia Conway and DTCs were CDX17 Victoria Foy Human Matthew Howell3 detected in lungs, mitochondria liver, brain, ovary, Graduate Students and bone of Alessia Catozzi CDX17P animals, Jakub Chudziak whereas no In 2018 the Clinical and Experimental Pharmacology group had Sam Humphrey3 micrometastases Human Alice Lallo three major goals: 1) to discover, develop, validate and implement Sarah Pearsall were observed in mitochondria the analysed organs biomarkers to support personalised medicine for cancer patients; Max Schenk CDX17P of CDX17 animals. 2) to characterise and exploit our expanding panel of circulating Scientific Officers Eleanor Anderson2 NCAM tumour cell derived explant models of small cell lung cancer (SCLC) Mahmood Ayub * for discovery of new targets and to test novel therapies for this Samantha Barlow2 Dominic Birch2 Group Leader aggressive cancer of unmet clinical need; and 3) to develop new Kieran Bradbury Caroline Dive teams with broadened scope as we continue to develop the Nicholas Brittain Laura Booth2 Figure 1. Immunohistochemistry showing examples of brain and liver metastases from a subcutaneous CDX tumour Deputy Group Leader Manchester Centre for Cancer Biomarker Sciences. New initiatives Henry Brown1 stained with Haematoxylin and Eosin (H&E). NCAM (CD56) staining identifies neuroendocrine cells and human cells Stewart Brown stain with a human specific antibody to a mitochondrial protein. Ged Brady include: (i) development of a community-based project for frequent Debbie Burt Staff Scientists monitoring of minimal residual disease using liquid biopsies from Holly Butterworth Mathew Carter Elaine Kilgour Dominic Rothwell patients whose lung cancer was resected with curative intent; (ii) Alex Clipson member POU2F3, have been shown to promote patients to establish if these approaches can be Jonathan Tugwood following our feasibility study, the roll out of the second phase of Joanne Felce Alice Fickling neuroendocrine and non-neuroendocrine used as a routine patient monitoring tool for Associate Scientists our TARGET trial where circulating tumour DNA profiling assists Rachael Fortune-Grant 1 differentiation states, respectively. These assessing tumour load, treatment response and Lynsey Franklin transcription factors define three of the four patient outcomes. Initial data indicates that CNA Kris Frese selection of the most appropriate trial for patients entering The Melanie Galvin transcriptional groups, and we are currently readouts can be used to measure both treatment Kathryn Simpson Christie NHS Foundation Trust Phase I Trials Unit; (iii) biomarker Weronika Golinska investigating both their role in tumour response and disease relapse including in limited Keal Gracey1 Service Manager analysis on immunotherapy trials in our tumour immunology and maintenance and progression and seeking stage SCLC. The NAB team is also building upon Eleanor Gregory2 Tony Price druggable downstream targets. our observation reported last year that CNA inflammation monitoring laboratory; (iv) initiation of studies that Hannah Gregson Grace Hampson patterns identified in SCLC circulating tumour Postdoctoral Fellows Katarzyna Bloch2 exploit nanoparticles aimed to increase liquid biopsy sensitivity; Thomas Helps2 The majority of SCLC patients are diagnosed cells obtained prior to patient treatment Rebekah Higgins with aggressive metastatic disease. We have correlated with response to standard of care Francesca Chemi and (v) establishment of our Bioinformatics and Biostatistics Team. Sarah Hilton developed SCLC CDX models that metastasise to chemotherapy (Carter et al. Nature Medicine Adam Fletcher Michael Hoffs Sumitra Mohan clinically relevant sites including the liver and 2017). Ongoing studies are now focusing on Nadia Iqbal 1 Barbara Mesquita1 brain that we can now use to interrogate the extending both the number of patients The CEP group is subdivided into nine teams; care for small cell lung cancer remained Aileen Jardine Maria Peiris-Pages underlying mechanisms (Figure 1). examined through national and international Hana Jelassi Ruth Stoney Preclinical Pharmacology (PP), Nucleic Acids unchanged for over three decades with only the collaborations, as well as streamlining single Noel Kelso Stuart Williamson Biomarkers (NAB), Cells and Proteins (CAP) recent introduction of immunotherapy in third Simrah Mohammad Genetically related models from the same CTC analysis. incorporating the Tumour Immunology and line therapy benefitting a minority of patients. Derrick Morgan2 patient can demonstrate different metastatic Bioinformaticians Inflammation Laboratory (TIIML), Tissue Although SCLC phenotypic heterogeneity was Karen Morris capabilities, thus facilitating our ability to discover The utility of non-small cell lung cancer CTCs Anthony Chiu1 Tine Descamps2 Biomarkers (TB), Biomarker Bioinformatics and reported 30 years ago, the current World Health Cristien Natal 1 genomic regulators of dissemination. Within the CRUK Lung Cancer Centre of Statistics (BBS), Quality Assurance (QA), the Organization classification of lung tumours Kamrun Nessa Saba Ferdous2 Furthermore, our new brain metastasis models Excellence-led TRACERx (TRAcking non-small digital Experimental Cancer Medicine Team recognises a single morphological classification. Anthony Oojageer Sakshi Gulati1 offer a unique opportunity to interrogate this cell lung Cancer Evolution through therapy [Rx]) Christina Parkes1 Cheryl Jones2 (digital ECMT), CEP operations (OPS) and CEP Due in part to the failure to identify novel biology and investigate potential therapeutic consortium (PI, Professor Charles Swanton, Safwan Patel2 Chang Kim1 administration (AD). In the past 12 months, our treatments for SCLC, there have been recent Bethanie Neale2 Jackie Pierce opportunities. UCL), we have continued to examine the genetic biomarkers’ portfolio supported 22 clinical trials attempts to develop a molecular classification Alan Redfern status of single CTCs compared to tumour Simon Pearce2 (16 academic sponsored, 6 pharmaceutical system that can aid in the implementation of Mitchell Revill2 Liquid biopsies for SCLC profiles from patients with resectable NSCLC. Matthew Roberts2 William Rowe2 company sponsored and 6 NIHR badged), and precision medicine. To this end, the PP team Caroline Roberts Targeted therapies are now entering clinical trials We have applied both CNA and whole exome 20 experimental medicine studies. During 2018, applied RNAseq to our panel of 45 patient Jordan Roebuck2 Cong Zhou2 for SCLC but serial tissue biopsies are sequencing (WES) to single pulmonary vein we initiated several exciting new collaborations circulating tumour cell derived explant (CDX) Karishma Satia Nicole Simms1 challenging. With this in mind, the NAB team circulating tumour cells (PV-CTCs) detected in Clinical Informaticians with AstraZeneca, Bristol Myers Squibb, Merck, models and the data are being used to Daniel Slane-Tan continue to develop and test liquid biopsies for early stage NSCLC at surgical resection with Jenny Bradford Novartis and PsiOxus Therapeutics. Our PP team hierarchically cluster tumours into subgroups Fouziah Butt Nigel Smith this disease. We have developed a novel genome curative intent. An examination of 103 patients has also set up collaborative studies on SCLC that may provide insight to SCLC biology. Victoria Stevenson1 wide ctDNA copy number aberration (CNA) from TRACERx confirmed findings from our Katherine Dempsey2 Paul Fitzpatrick with Amgen, Epigene Therapeutics, FLXBio and Principal component analysis using the 1000 Sarah Taylor1 approach and targeted NGS of ctDNA on 110 previous pilot study that PV-CTC detection is AstraZeneca. most variable transcripts reveals at least four George Thompson2 Andrew Hughes genes relevant to the SCLC genomic landscape associated with lung cancer relapse, groups, suggesting that there may be Simon Topham Laura Hutchinson and novel therapeutics. Having completed a independently of tumour stage. In a patient Erich van Hechnova2 Donal Landers Highlights functionally distinct subtypes. Several pilot study of 69 extensive and limited stage where we compared primary tumour, PV-CTCs Amelie Viratham Leanne Ogden Understanding SCLC biology and the search transcriptional drivers including the proneural Paul O’Regan Hannah Walsh2 SCLC patients, we are extending analysis to and a pleural metastasis detected 10 months for new therapies basic helix-loop-helix transcription factors Daniel White include longitudinal profiles of over 130 SCLC after surgery, 50% of PV-CTC private mutations Jenny Royle Despite multiple clinical trials, the standard of ASCL1 and NEUROD1, as well as the POU family 22 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE CLINICAL AND EXPERIMENTAL PHARMACOLOGY 23


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    expression analysis from Formalin Fixed Paraffin CLINICAL AND EXPERIMENTAL PHARMACOLOGY (CONTINUED) Embedded tissue using the nanostring platform. Implementation of these assays within clinical studies is beginning in blood samples from the Laboratory Manager undetected in the primary tumour or circulating will give blood samples on a monthly basis in TRIBE study (Tyrosine Kinase Inhibitor Therapy In Matthew Lancashire tumour DNA at surgery were detected in the their community pharmacy, for collection and Renal Cell Carcinoma Immune Biomarker metastasis. The data from this patient formally transport to CEP with an aim to develop a liquid Evaluation) undergoing analysis for activated, Laboratory Support Technician Andrew Stevens link CTCs to metastasis and demonstrate a case biopsy that picks up relapsing disease earlier. exhausted and memory T-cell subsets. The where PV-CTCs represented a minor subclone PD-L1 CTC assay is being developed as an BRC Project Manager disseminating early that was responsible for ctDNA applicable to clinical trials exploratory endpoint in a SCLC clinical trial. As Jonathan Wake disease relapse. The TARGET Trial (Tumour chARacterisation to part of the iMATCH (Innovate Manchester Guide Experimental Targeted therapy), was Advanced Therapies Centre Hub) project, EA to Caroline Dive Liquid biopsies for earlier detection and minimal initiated in 2015 and is led from the clinic by CEP validation of a multiplex ELISA assay for Ekram Aidaros-Talbot residual disease monitoring in NSCLC alumnus Dr Matthew Krebs (The Christie NHS monitoring cytokine storms in patients receiving Administrative Coordinators We continue to collaborate with Dr Phil Crosbie Foundation Trust). We have regular Molecular advanced T-cell therapies is nearing completion. Suzanne Bickley2 and his clinical colleagues at Manchester Tumour Board meetings to integrate clinical Immunotherapy associated markers are also Fiona Mckenzie-Wilde1 University NHS Foundation Trust (MFT) having data, tumour mutation profiling (archival and/or being developed for the TARGET study, including Lisa Waters1 processed and banked blood samples from 748 fresh biopsies) and contemporaneous CEP analysis of infiltrating lymphocytes and PD-L1 Megan Wright 1 “high risk, hard to reach” subjects who have ctDNA data utilising our visualisation tool expression in tumour tissue alongside 1 taken part in a community-based lung health eTARGET, developed by the digital Experimental measurement of tumour mutation burden by Left in 2018 2 check study which offered low dose CT Cancer Medicine Team (digital ECMT). We analysis of ctDNA. Joined in 2018 3 Joined from RNA scanning. We are now evaluating blood completed the feasibility stage A for 100 patients Biology in 2018 biomarkers that may identify those with early where ctDNA analysis by NGS of 641 cancer- Digital clinical trials stage lung cancer using both plasma based associated genes and a comparison to matched The digital Experimental Cancer Medicine Team assays aimed at identifying DNA (ctDNA), tumour data revealed a 79% concordance (www.digitalecmt.org) has the capability to test microRNA and proteins that are linked to cancer, (Rothwell et al, in press Nature Medicine). We Figure 3. Lorigan (The Christie NHS Foundation Trust), we new technology, devices, data-science as well as cell based assays, including the “no cell have initiated TARGET stage B and ctDNA The handheld device that allows developed a GCP validated droplet digital PCR techniques and new ways of working under analysis has now been successfully applied to patients to input their own data is left behind” High Definition Single Cell Analysis (ddPCR) ctDNA assay which is ready for clinical trial conditions that brings researchers, whilst on a clinical trial. (HDSCA) platform for detecting CTCs. Evaluation over 300 patients with improved sample deployment in the upcoming advanced clinicians, technology and patients together to of HDSCA will be carried out in a continued processing and ctDNA CNA analysis. A ctDNA cutaneous melanoma phase II trial (CACTUS), to innovate in early clinical trials. One example of collaboration with the system developer based measure of tumour mutational burden define a switch in treatment from targeted to this approach is eTARGET, a decision support tool Professor Peter Kuhn (University South (TMB) is currently being developed. Ongoing immunotherapy. that integrates clinical and genomic NGS data to California), and plasma assays will include studies are expanding the TARGET biomarkers to facilitate decision-making for matching patients established published assays and novel assays in include tissue based immune markers, which In order to increase assay sensitivity, the NAB with clinical trials and has transformed the way development within CEP. We were recently along with ctDNA TMB will be assessed as a team are examining approaches to specifically that the Manchester Molecular Tumour Board for awarded additional CRUK funding and with this means of identifying patients who may benefit enrich plasma tumour DNA using established the TARGET trial works. we are now setting up a minimal residual disease from immune checkpoint therapies. ctDNA methylation assays and, in collaboration monitoring study in partnership with Lloyds In collaboration with Professor Richard Marais with Professor Kostas Kostarelos (The National As another example of an on-going digital Pharmacies where, after their surgery, patients (Molecular Oncology group) and Professor Paul Graphene Centre), asking whether nanoparticles clinical trial, PROACT (Patient Reported Opinions (lipid- and graphene-based) or ‘nanonets’ can be About Clinical Trial Tolerability) that was used to scavenge nucleic acids in the blood and developed with patients, is a mobile phone app Figure 2. be ‘tuned’ to preferentially enrich different or website tool that allows patients to say how The TIMML biomarker 'toolkit'. tumour derived particles and nucleic acids. they are feeling on an early clinical trial (Figure 3). Figure 3. The handheld device that allows Biomarkers to inform immunotherapy trials patients to input their own data whilst on a The Tissue Biomarker and Nucleic Acid clinical trial. Biomarker teams are working closely with the TIIML to build and expand our immune- Video, voice or text messages are sent between oncology ‘biomarker toolkit’ for analysis of patients and their medical teams. Initial feedback tumour tissue and blood samples to support has been very positive with over 90% of patients immune-oncology trials (Figure 2). offered PROACT taking part. The digital ECMT has also developed a digital clinical trial for Liquid biopsy assays are now available including patients to be able to monitor their kidney lymphoid and myeloid flow cytometry panels, function at home, which is planned to start in multiplex ELISAs for cytokine analysis and an 2019. assay for assessment of PD-L1 expression on CTCs enriched by CellSearch. For analysis of the Publications listed on page 62 immunogenic status of the tumour microenvironment, we developed immunohistochemistry (IHC) assays for a range of immune markers and are currently developing IHC assays for the Mismatch Repair Deficiency proteins MSH2, MSH6, MLH1 and PMS1. We have also established a robust and reproducible workflow for RNA extraction and immune gene 24 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE CLINICAL AND EXPERIMENTAL PHARMACOLOGY 25


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    www.cruk.manchester.ac.uk/Our-Research/Drug-Discovery Alex Stowell Our gene-directed enzyme prodrug therapy In the longer term, our portfolio is being Adnana Tudose2 (GDEPT) programme uses engineered vaccinia repopulated from the impressive number of Mandy Watson2 viral vectors to target tumours selectively and early targets which have been sourced from our DRUG DISCOVERY Graduate Students Elizabeth Hogg3 produce a unique bacterial enzyme locally, which is able to convert subsequently Senior Group Leader collaborators, such as Professors Richard Marais, Caroline Dive, Iain James Stratford1 administered prodrugs to cytotoxic drugs thus Hagan, Dr Claus Jorgensen and beyond, with killing the tumour cells. In collaboration with leveraged additional expertise. We are Undergraduate Students Professor Marais we have demonstrated particularly excited by the opportunity for Felix Rummel2 selective tumour targeting with our viral vectors discovery of inhibitors for lung cancer, given the Hannah Fortune1 and long-term tumour xenograft regressions in academic and clinical expertise present in the EA to Caroline Springer mice, and are currently working to generate Manchester Institute, the Christie NHS improved vaccinia vectors. In addition, we will be Foundation Trust and the CRUK Lung Cancer The last year has continued to be a time of advancement for the Jayne Fowler1 looking to assess the potentially beneficial Centre of Excellence. Drug Discovery Unit, under new leadership and direction with the 1 Joined in 2018 immune responses to our GDEPT system, in 2 Left in 2018 appointment of Caroline Springer as Director, and moving to 3 Joint with Systems Oncology collaboration with Institute colleague Dr Across all our projects, we will ensure that our Santiago Zelenay. DDU projects are integrated with Professor temporary laboratories at Alderley Park in early 2018. Caroline also Caroline Dive’s biomarker discovery programme brought with her a number of new cancer drug target projects Originally initiated somewhat earlier in our so that all nominated targets have selection and which are being progressed in tandem with ongoing DDU projects portfolio, and in collaboration with AstraZeneca, predictive biomarkers and with the excellent we have recently initiated a new project aimed committed clinicians in the Christie NHS Director by the integrated team. at reversing hormone therapy resistance in Foundation Trust. Caroline Springer prostate cancer. Drawing upon the disease expertise of the Manchester Institute in this area, Following our move in 2018, we have greatly Head of Chemistry Our pan-RAF drug has now completed its Phase progressed our LOX drug discovery programme with experts such as Professor Rob Bristow, we valued the opportunity to work together in the Allan Jordan I clinical trial at The Christie and Royal Marsden to late lead optimisation. Our advanced believe this approach has the potential to offer new laboratories at Alderley Park as a fully NHS Foundation Trusts, and we look forward to inhibitors have demonstrated significant significant benefit to men with castrate-resistant integrated DDU. We are extremely fortunate to Deputy Head of Chemistry Dan Niculescu-Duvaz1 the commencement of Phase 2 trials. This orally therapeutic activity against a number of primary prostate cancer. This emerging project also have a portfolio of numerous projects with bioavailable, well-tolerated panRAF/SRC drug tumour models and pronounced anti-metastatic integrates our new Structural Biology alliance excellent CRUK MI collaborators, ranging from Head of Biochemistry was discovered in collaboration with Professor efficacy in a metastatic tumour model. Professor with Professor Richard Bayliss at the University target validation to Phase I clinical trials, on many Graeme Thomson Richard Marais and is designed to treat mutant Marais’ group has discovered new biology of Leeds, and will allow us to understand, on a different targets involved in a range of cancer BRAF or mutant RAS melanoma and other concerning LOX interaction with signalling molecular level, how our compounds interact indications. Head of Cellular Pharmacology tumour types driven by these mutant oncogenes. pathways, which provides good biological with their target at the atomic level. We will Graeme Walker rationale for combining targeted agents with our exploit this information to design improved Publications listed on page 63 Chemists Our DNMT-1 and RET pre-clinical candidate LOX inhibitors. We will develop our inhibitors compounds and, in doing so, increase the rate Mohammed Aljarah development programmes also continue to further and select drug candidates to progress to of progression of this project. These efforts will Michael Brown make good progress and we anticipate these toxicology studies in 2019 before moving into be greatly enhanced through the work of Dr Rae Vikki Clayton compounds will enter first-in-human trials in the early clinical trials in patients, as monotherapy Lawrence, our new Principal Computational Mark Dodsworth near future. and in combinations. Chemist, who joined the team earlier this year. Laura Johnson Chris Kershaw Our partnership with IDEAYA Bioscience on Cancer stem cells (CSCs) are a subset of tumour Rae Lawrence1 Leo Leung our Poly(ADP-ribose) glycohydrolase (PARG) cells with the ability to perpetuate cancer growth Rebecca Newton programme continues to develop. Following indefinitely. CSCs are involved in tumour Ali Raoof on from an out-licencing agreement, IDEAYA progression, resistance to treatment and Kate Smith gained access to multiple proprietary series of recurrence in many cancers. Current therapies Figure 1. Deborah Smithen our small molecule inhibitors of PARG. In this target bulk tumour cells, but CSCs escape A potent and selective CRUK MI collaboration we are now assessing in vivo resulting in tumour regrowth and treatment designed ligand bound to a target Bioscientists involved in inhibiting CSC growth. Alex Baker activity. The goal is to nominate a pre-clinical failure. Thus there is a need for drugs to target Habiba Begum candidate shortly. the CSC subpopulation within tumours, to use in Elizabeth Blaikley combination with the standard of care. We are Aimee Blair1 Metastasis is the main cause of death in cancer, pursuing a drug discovery programme to target Charlotte Burt2 and we are excited to assess the potential of our cancer stem cells that has progressed rapidly to Mairi Challinor1 lysyl oxidase (LOX) inhibitors to treat primary lead optimisation. We have discovered potent Andrew Clifton1 tumours and metastases in patients. LOX is an and selective inhibitors of our CSC target, which Emily Crowley1 Zoi Diamantopoulou1 enzyme that regulates cross-linking of structural have promising pharmacokinetic profiles (Figure Cath Eberlein2 proteins in the extracellular matrix and plays a 1). Our medicinal chemistry has been greatly Joanna Grabarek critical role in metastasis and in tumour growth supported by internal computational chemistry Louise Griffiths in many cancers. LOX is a validated therapeutic and by crystallography in collaboration with Nicola Hamilton target, and our aim, in collaboration with Leicester University (funded by a CRUK Ben Hodgson Professor Marais, is to discover first-in-class, Accelerator Award). We are collaborating with Dominic James2 orally bioavailable small molecule LOX inhibitors. Professor Richard Marais and The University of Paul Kelly Filipa Lopes Despite being a protein that cannot be purified Manchester based expert Dr Robert Clarke, in Nikki March2 or crystallised, with funding from Wellcome and elucidating the biology of our target. Robert McLeary CRUK we have discovered LOX inhibitors with Alice Pilborough1 good pharmacokinetics properties and have 26 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE DRUG DISCOVERY 27


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    www.cruk.manchester.ac.uk/Our-Research/Head-and-Neck-Cancer-Biology HEAD AND NECK CANCER BIOLOGY The particular focus of the Head and Neck Cancer Biology group is on salivary gland cancers because there is a significant unmet need in this patient group. Genomic profiles in head and neck tumour samples will be examined to identify alterations that can be targeted by drug therapies. Our primary aim is to develop a new biological understanding of how changes at the gene and protein level result in Institute Fellow tumour growth and metastasis. Novel insights provided by this Robert Metcalf 1 approach will allow drugs to be screened using cell lines and mouse 1 Joined in 2018 models. Those drugs which are found to be effective in laboratory studies will be taken forwards to clinical trials in patients with the goal of improving patient survival. Initial research will focus on adenoid cystic models using shRNA and CRISPR/Cas9 or carcinoma (ACC), working in collaboration with dCas9-KRAB followed by lentiviral mediated Professor Caroline Dive and the Clinical and MYB rescue. To validate candidates identified Experimental Pharmacology group. ACC is a through this approach as therapeutic targets, in salivary gland cancer, which metastasises in over vitro and in vivo drug screening will be 50% of patients leading to a median survival of 3 performed in models of ACC. As metastasis is a Figure 1. Approach to identify and validate candidate therapeutic targets in adenoid cystic carcinoma. years, with no effective drug therapies. Due to defining feature of ACC, in vivo and ex vivo Step 1: ChIP-Seq data using ACC tumours to determine MYB binding (downloaded from ENCODE 1 Drier et al., Nature Genetics 2016) was analysed to identify chromosomal rearrangements, 90% of ACC fluorescence/luminescence imaging studies in MYB binding peaks coinciding with promoter and enhancer histone marks (histone 3 lysine 4 mono-methylation (H3K4me1) and tri-methylation (H3K4me3) and lysine 27 acetylation (H3K27ac)) in the regions of transcriptional start sites of biologically plausible genes. Example shown is in the region of CDK6. Step 2: tumours are driven by overexpression of the isogenic models will be used to identify ACC patient samples are analysed and to determine the prevalence of candidates in tumour compared with normal salivary gland. Step 3: ACC cells are myeloblastosis (MYB) transcription factor gene functional predictors of metastasis. Fundamental screened in vitro and in vivo for evidence of sensitivity/resistance. In the example shown, ACC patient derived tumour xenografts show tumour growth family members: a-MYB and c-MYB. The overall discoveries in ACC biology will be translated to inhibition with pharmacological inhibition of CDK4/6. In parallel, in Step 4: CRISPR-Cas9 and shRNA approaches are undertaken to knock-down/knock-out goal is to develop new therapies for ACC by patients using tumour/liquid biopsies to identify MYB and MYL1 to determine the functional significance of MYB/MYBL1 bound genes on tumour growth and metastasis. identifying mechanisms through which MYB and validate prognostic and predictive drives tumour growth and metastasis, and to biomarkers. develop predictive and prognostic biomarkers to optimise patient treatment. Findings can be rapdly translated from this research to develop biomarker led clinical trials Chromatin immunoprecipitation sequencing for this patient group. As there is a high risk of has identified MYB downstream targets that are metastasis and no standard drug therapies, if this enriched for genes controlling cell cycle research is successful it would have immediate regulation. As the cell cycle is dysregulated global impact in this patient population. The aim through multiple mechanisms in most human is to develop a platform to broaden research tumours and offers avenues for therapeutic focus across other rare sub-types of head and targeting, our initial focus is to determine neck cancers. In addition, understanding of MYB whether and how MYB overexpression impacts biology in ACC provided by this study may on cell cycle control in adenoid cystic provide insight into other solid tumours where carcinoma. MYB is frequently overexpressed including sub-groups of patients with breast and For functional studies to identify critical effector colorectal cancers. genes downstream of MYB, we are developing isogenic MYB knock-down/knock-out ACC Publications listed on page 64 28 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE HEAD AND NECK CANCER BIOLOGY 29


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    www.cruk.manchester.ac.uk/Our-Research/Leukaemia-Biology gene GFI1. Given that physical association of LSD1 with the N-terminal SNAG domain of GFI1 is essential for the function of GFI1 as a transcription LEUKAEMIA BIOLOGY repressor, we evaluated whether OG86 disrupts this interaction and found in immunoprecipitation experiments that it did. In fact, using ChIP sequencing we discovered that GFI1, LSD1 and its CoREST complex binding partner RCOR1 strongly associate one with another on chromatin at enhancer regions genome-wide. Treatment of AML cells with an LSD1 inhibitor targets GFI1/CoREST chromatin- A core focus of the team is to identify new therapeutic targets for raising the possibility that other mechanisms may bound complexes for disruption and release to development through to the clinic, for longer term patient benefit. Figure 1: be important. To begin to address this question the nucleoplasm. Through generation and Along these lines, over the last seven years we have had a significant Heat maps show ChIP signal for some years back James Lynch, former expression of conditional expression constructs, the indicated proteins or histone Postdoctoral Fellow in the lab, treated AML cells where LSD1 was fused directly to the DNA investment in the story of LSD1. Lysine Specific Demethylase 1 is one marks at 1,560 GFI1-bound with a potent LSD1 inhibitor and performed ChIP binding domain of GFI1, Gary Spencer was able of a number of epigenetic regulators which have recently emerged enhancers, ranked top to bottom according to GFI1 peak strength, sequencing and RNA sequencing analyses. Our to show that inhibitor-induced myeloid prediction based on the literature was that we differentiation in AML cells is entirely dependent as candidate therapeutic targets in cancer. in THP1 AML cells treated with would see accumulation of histone methylation upon the physical separation of LSD1 from GFI1. DMSO control or OG86, a Group Leader marks genome wide at sites of LSD1 binding To determine the consequences of LSD1:GFI1 tranylcypromine derivative Tim Somervaille It was initially identified as a core component of called EUROSTARS to run this first-in-class, inhibitor of LSD1, for 24 hours. close to genes whose expression changed physical separation and concomitant inactivation significantly. To our surprise we did not find this at of GFI1 at GFI1-bound enhancers, Alba Maiques- Postdoctoral Fellows an RCOR1 (CoREST) histone deacetylase (HDAC) first-in-man trial which opened in 2014 in the UK all, even though drug treatment very strongly Diaz performed additional ChIP sequencing Alba Maiques-Diaz transcription corepressor complex and later at The Christie NHS Foundation Trust and at sites promoted significant changes in gene experiments. She found that most GFI1-bound Isabel Romero-Camarero found to have lysine-specific demethylase across Spain and France. The trial completed its expression, with concomitant cellular enhancers were pre-loaded with two myeloid Bettina Wingelhofer1 activity. With regard to its enzymatic function, recruitment in late 2016 with evaluation of the differentiation. This critical observation led to a lineage transcription factors called SPI1 and Niall Gilding1 LSD1 is a flavin adenine dinucleotide (FAD) results currently being finalised for publication in series of experiments led jointly by Gary Spencer, CEBPA, and that following treatment of cells with dependent homologue of the amine oxidase early 2019. This study in patients with relapsed or James Lynch and Alba Maiques-Diaz over several LSD1 inhibitor, there was loss of CoREST complex Clinician Scientist family with an ability to demethylate refractory acute leukaemia evaluated the safety, years, which have revealed the actual mechanism histone deacetylases HDAC1 and HDAC2 and Dan Wiseman2 monomethyl or dimethyl lysine 4 (K4) of histone pharmacokinetics, and preliminary activity of by which LSD1 inhibition promotes differentiation gain of the histone acetyltransferase CBP and the Clinical Research Fellows H3 as well as a number of other targets such as iadademstat and determined the recommended in AML. We reported our findings in Cell Reports methyltransferase MLL4. In keeping with this we Mark Williams DNMT1 and TP53. dose as a single agent. We found iadademstat to in 2018. observed increased histone acetylation at John Chadwick be well tolerated with most adverse events GFI1-bound enhancers within hours of drug The interest in LSD1 as a therapeutic target in consistent with transient myelosuppression. Figure 2: Bioinformatician Summary of mechanism by We first noted using comparative transcriptomic treatment (Figure 1) and a slower latency cancer arose from the observation of its high Encouragingly, in terms of responses we techniques that pharmacologic inhibition of LSD1 accumulation of histone methylation by 48 Fabio Amaral which LSD1 inhibitors promote level expression in poor prognosis sub-groups of observed one complete remission and a number mimics knockdown of the transcription factor hours. Interestingly, in contrast to histone differentiation in AML. Senior Scientific Officer prostate, lung, brain and breast cancer, as well as of haematological improvements with induction acetylation, the accumulation of histone Gary Spencer in certain haematologic malignancies. In 2010 of blast cell morphological and molecular methylation at GFI1-bound enhancers did not William Harris, then a graduate student in the lab, differentiation, in keeping with expectations correlate well with the increased expression of Scientific Officer noticed that expression of LSD1 was very high in from our prior pre-clinical data. These data have Priyanka Bhattacharya the subordinate network of genes controlled by murine leukaemia stem cells driven by an led on to a Phase 2a study called ALICE, which is these enhancers. MLL-AF9 fusion oncogene, but was down a pilot study to assess the safety, tolerability, dose Graduate Students Fabrizio Simeoni regulated as those cells underwent finding and efficacy of ORY-1001 (iadademstat) Thus using a tractable experimental system and Francesco Camera differentiation. This prompted him to perform a in combination with azacitidine as first line confirmatory analyses in patient cells, we have knockdown experiment and he discovered that therapy in older patients with acute myeloid shown that both irreversible and reversible 1 Joined in 2018 LSD1 transcript depletion resulted in leukaemia leukaemia; the first patient was recruited to this inhibitors of LSD1 promote differentiation 2 Left in 2018 cell differentiation. The first drug found to inhibit protocol just before Christmas 2018. More through disruption of the protein:protein LSD1 was tranylcypromine, a monoamine generally, interest in LSD1 as a therapeutic target interaction of GFI1 with LSD1 (Figure 2), rather oxidase inhibitor previously used in the treatment continues to increase worldwide with multiple than impairment of histone demethylation. Our of depression. We found that this drug, as well as pharmaceutical companies currently studies reveal a critical role for both GFI1 and some derivative compounds synthesised for us conducting early phase clinical trials with related LSD1 as key contributors to the cardinal by James Hitchin, Donald Ogilvie and Allan compounds in diverse clinical settings such as pathologic feature of MLL-translocated AML, the Jordan in the Drug Discovery Unit, induced myelofibrosis, Ewing sarcoma and small cell lung differentiation block of immature blast cells. An murine and primary human leukaemia cells to cancer. intriguing final point is that drug discovery differentiate whether tested in vitro or in vivo. programs focusing on inhibition of demethylase These data were published in Cancer Cell in With LSD1 inhibitors in early phase clinical trials, activity as a target have generated compounds 2012 and led to our establishing a collaboration an appreciation of their mechanism of action is that function through an unexpected with Tamara Maes and Carlos Buesa at Oryzon essential. Given the well-established activity of mechanism. The important possibility is raised Genomics in Barcelona to set up a Phase 1 trial of LSD1 as a histone demethylase, the assumption that a search for compounds that maximally their highly potent and selective orally-active has been that LSD1 contributes to gene disrupt SNAG domain:LSD1 interactions might LSD1 inhibitor ORY-1001, now called repression by removing monomethyl and yield molecules with higher potential therapeutic iadademstat. dimethyl histone marks from lysine 4 of histone efficacy. H3, and that this is the key activity targeted for We won funding through a commercial- potential therapeutic effect. However, LSD1 also Publications listed on page 64 academic European Union funding scheme interacts with multiple transcription factors 30 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE LEUKAEMIA BIOLOGY 31


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    www.cruk.manchester.ac.uk/Our-Research/Molecular-Oncology Rebecca Lee2 Our studies reveal a complex interaction laboratory and clinic, representing a Zena Salih1 between UVR and melanoma and have shown collaborative effort between ourselves and the Sara Valpione that even a few incidences of UVR exposure Clinical and Experimental Pharmacology group MOLECULAR ONCOLOGY Scientific Officers Rebecca Atkinson-Dell2 increase the risk of melanoma. Further, we provide insight into the correlation between the at CRUK MI and the clinicians of the Melanoma Group at the Christie NHS Foundation Trust. Christopher Chester tumour’s genomic landscape and disease Darryl Coles progression. Going forward, we will study the In the past year, we also collaborated with and Megan Grant clinical features of patients in the two groups to supported leading researchers in the fields of Clare McManus understand the relationship between melanoma epidemiology and histopathology to Philippa Middlehurst2 UVR-induced genomic alterations and survival. characterise further and understand better Paul Montgomery2 Joshua Tweedy distinct melanoma subtypes. In one study, we Better understanding of melanoma biology that provide new insights into naevoid melanomas, We study cancer biology and work with clinicians so that the new emerges from the type of study described above which we propose can be further classified into Translational Research knowledge we generate can be used to improve cancer patient care, Project Manager underpins our efforts to translate our findings two types, with distinct clinical and primarily in melanoma but also in other diseases including breast Jackie Mitchell into the clinic. To that end, we recently initiated a histopathological features. In another study, clinical trial based on laboratory discoveries, we presented data that suggests nodular and pancreatic cancers. Our multi-disciplinary approach allows Graduate Students following our recent reports that circulating melanoma (NM), a histopathologically distinct Luke Chisholm scientists and clinicians to work closely together. Our aim is to Denys Holovanchuk tumour DNA (ctDNA) in the blood of high-risk subtype of cutaneous melanoma, is a distinct improve our understanding of cancer biology and develop new Matthew Wilson (stage III) melanoma patients (so-called “liquid fast-growing entity from the outset. These two biopsies”) can be a surrogate of patient tumour studies have implications for the prognosis of Group Leader drugs and diagnostic tools that can be used to optimise cancer 1 Joined in 2018 burden. We hypothesised that ctDNA could these rare melanoma subtypes. Finally, in Richard Marais treatment and improve patient care. 2 Left in 2018 reveal how patients are responding to treatment addition to our work in melanoma, in the past and so developed CAcTUS (CirculAting Tumour year we also harnessed our expertise in genomic Associate Scientists DNA gUided therapy Switch), a biomarker-driven and transcriptomic tumour analysis in a Nathalie Dhomen Developing more effective prevention, diagnosis accelerate melanoma development (Figure 1A). phase II trial to investigate if ctDNA can guide a collaborative study with the Prostate Valeria Pavet and treatment of cancer, begins with an We found that the type of genomic changes in switch between targeted and immune therapy in Oncobiology group at CRUK MI. We increased understanding of cancer biology, and short-wavelength UVR-exposed tumours had a patients with advanced cutaneous melanoma. characterised the genomic, epigenomic and Senior Clinical Scientist in particular the mechanisms that drive cancer predominance of a pattern of mutations known CAcTUS is due to open in the next few months transcriptomic features of clinically localised Martin Cook initiation. To this end, we established a as signature 7, which has previously been with the aim of maximising response in prostate cancer, and in particular assessed these Senior Research Scientist programme to determine how ultraviolet associated with UVR-induced DNA damage; this melanoma by using ctDNA to determine when in the context of tumour visibility by Adele Green radiation (UVR) causes melanoma. It is widely pattern was absent in the tumours from the mice to switch between first and second line therapies multiparametric MRI. accepted that exposure to UVR, either naturally that did not receive short-wavelength UVR. (Figure 2). This trial is a testament to our Postdoctoral Fellows from sunlight or artificially from tanning beds, is Strikingly, we found a similar dichotomy in extraordinarily successful relationship between Publications listed on page 65 Franziska Baenke associated with increased melanoma incidence. human melanoma, with ~85% of cases showing Jing Bi1 However, the relevance of the genomic changes a predominance of signature 7 and ~15% where Candelaria Bracalente Alessio Cannistraci that result from different components and signature 7 was not predominant. Thus, Elena Galvani patterns of UVR exposure to the progression of although patients generally present with Pauline Hascoët this disease is poorly understood. In a recent clinically similar disease, our data suggests at Figure 2: Katharina Mahal2 study, we examined the genomic alterations in least two distinct mutation processes drive this Trial schema for CAcTUS. HaoRan Tang tumours from our mouse melanoma model, disease. Notably these two groups show Lucas Trucco following exposure to different wavelengths, differences in overall- and disease-free survival Bioinformaticians patterns and quantities of UVR. We found that (Figure 1B). Our data suggests that there are at Piyushkumar Mundra exposure to short wavelength UVR accelerates least two types of cutaneous melanoma with Shambhavi Srivastava2 melanoma development and increases distinct aetiologies leading to different disease mutation burden compared to long wavelength courses and outcomes. Clinical Fellows UVR. We also found that only a few exposures to Pablo Garcia Martinez short wavelength UVR were enough to Figure 1: UVR exposure accelerates melanomagenesis and can imprint specific mutation signatures on tumours (A) Rate at which melanomas develop in a physiologically relevant mouse model of melanoma either in the absence of UVR exposure, or after 4 or up to 26 UVR exposures. (B) Five-year overall survival of patients with cutaneous melanoma segregated by a predominance of the UVR mutation signature (signature 7) in the tumour genomes. 32 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE MOLECULAR ONCOLOGY 33


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    www.cruk.manchester.ac.uk/Our-Research/Prostate-Oncobiology Figure 1: HORMONE-NAÏVE CASTRATED Schematic diagram showing LY6D LY6D LY6D LY6D LY6D PROSTATE ONCOBIOLOGY HOMEOSTASIS LY6D+ luminal progenitors in CASTRATION homeostasis and PCa tumorigenesis (upon tamoxifen induced Pten-loss in K8- + Tamoxifen K8-CreER + Tamoxifen CreER;PtenL/L mice) derived from LY6D LY6D LY6D LY6D LY6D putative CR LY6D+ luminal cells LY6D+ (with red bars), or LY6D - luminal castration-resistant cells in hormone-naïve or luminal cell castrated mice. + Testosterone LY6D LY6D Personalised treatment for prostate cancer (PCa), the most LY6D- commonly diagnosed cancer in men in the UK, remains a Luminal cell challenge because there are yet no clearly defined molecular subtypes to predict patient response. In recent years, deep HORMONE-NAÏVE CASTRATED LY6D LY6D LY6D LY6D sequencing and single cell studies have significantly advanced our CASTRATION LY6D understanding of the cellular composition and gene expression Induction programs that shape tumour architecture, opening new routes to TUMORIGENESIS Group Leader + Tamoxifen Pten-loss + Tamoxifen K8-CreER Esther Baena tackle cancer. The goal of the Prostate Oncobiology group is to LY6D LY6D LY6D LY6D LY6D LY6D LY6D Postdoctoral Fellows apply these techniques to understand the prostate cellular landscape Valentina Ubertini and its evolution during neoplastic transformation to facilitate the Maria Roel Sánchez1 development of better therapies. + Testosterone Scientific Officer LY6D LY6D LY6D LY6D LY6D Pengbo Wang LY6D It has now become evident that tumours are heterogeneity of PCas and may therefore be Graduate Students Ivana Steiner composed of co-evolving heterogeneous unsuitable to assess the patients' risk based on Alexander Du Feu1 sub-clones, which have fundamental genomic analyses. On the contrary, limiting implications for the response to treatment. biopsies to mpMRI visible-only lesions may Clinical Fellow Ultrasound-guided biopsies of PCa-suspicious underestimate the patients’ individual risk for lineage and to a lesser degree also in the basal prostate cells from the SCA1high and SCA1low/- Amin Ali1 lesions remain the gold-standard therapeutic disease progression and metastasis. lineage. We found that a subset of prostate cells gates were enriched with organoid-forming procedure. However, there is a trend towards the in the luminal lineage co-express multiple basal multipotent luminal progenitors and formed Visiting ERASMUS Student use of multiparametric (mp)MRI to trigger Our study showed that PCa tumours undetected (e.g. Krt5, Krt14, Trp63) and luminal (e.g. Krt8, increasing numbers of multipotent organoids in Silvia D’Ambrosio1,2 biopsies in PCa patients combined with genetic by mpMRI can harbour genomic alterations, Krt18, and Ar) markers together with prostate the absence of androgen. Taken together, these 1 Joined in 2018 testing to refine risk stratification. Importantly, which are commonly seen in metastatic stem/progenitor marker genes (e.g., Ly6d, Trop2, findings suggest that LY6D expression correlates 2 Left in 2018 >10% potentially significant PCas are missed by castration resistant prostate cancer (mCRPC), Sca1, Cd133, Cd166). Many of these stem/ with PCa initiation and progression to castration- this approach because they are not detected by including RB1 and TP53 loss, and MYC progenitor genes are found within a bi-lineage resistant growth from the luminal lineage (Figure mpMRI; the genomic makeup of these “invisible” amplification. In our recently published work gene set expressed in both basal cells and a 1). Importantly, in support of this hypothesis, lesions could provide important insights into mpMRI non-visible tumours could be regarded portion of luminal cells. An important analysis of human PCa cohorts revealed that their metastatic capacity and help to assess their as genomically aggressive and could potentially observation from our study is that many higher LY6D expression levels are associated potential lethality. give rise to lethal clones. Thus, our study further castration-resistant (CR) prostate cells in the with more aggressive disease and worse emphasises the complexities of diagnosis in luminal lineage exhibit a bi-lineage expression outcome, suggesting that LY6D may serve as a To address this question, we have recently clinically localised PCa. Importantly, it highlights signature similar to that of intermediate cells, prognostic biomarker for advanced PCa. completed a study correlating genomics and the shortcomings of this new diagnostic raising a possibility that such intermediate cells mpMRI in men undergoing radical method, using mpMRI on its own as a triage test, may be intrinsically CR. Further studies are warranted to determine the prostatectomy in order to elucidate the genomic and the need of additional biomarkers to inform cellular composition of tumours during characteristics of mpMRI visible and non-visible patient diagnosis and prediction of treatment Our studies identified LY6D as a novel marker progression and their association with mpMRI tumours and to assess the inter-relationship. We response. Thus, our study has the potential of linking intermediate cells to prostate stem/ visibility, as well as the precise role of LY6D in found that the intra-tumour heterogeneity being practice changing by refining diagnostic progenitor cells and CR prostate cells. LY6D is a prostate epithelial heterogeneity, PCa initiation within visible mpMRI lesions bears the risk of testing in PCa. gene with yet no established role in prostate and progression to adenocarcinoma, to validate misclassifying patients when using genomic development or cancer. It is a member of the its utility as a novel biomarker for patient biomarkers from a single biopsy. For instance, In a complimentary study focused on the Ly6/uPAR family, characterised by their roles in stratification, and to assess the impact of CR considering a previously validated threshold for heterogeneity of PCa, we assessed the cellular cell proliferation, cell-cell interaction, immune LY6D+ cells as novel therapeutic target for the percentage of genomic aberrations composition within the prostate epithelium in cell maturation, and cytokine production, which patients with lethal PCa. (PGA≥7.49%), of the two cores obtained from the the context of response to therapy (i.e. are all essential components of tumour initiation same visible lesion, one core can classify the androgen-deprivation) by combining single cell and progression. However, in PCa, the functional Publications listed on page 65 patient as low risk, while the other core can profiling and functional characterisation. For this, role of LY6D for tumorigenesis and tumour classify the patient as high risk. Similarly, intra- we compared the mouse prostate of intact mice maintenance remains unknown. Our in vitro and tumour transcriptomic heterogeneity within with those that underwent androgen- in vivo data showed that LY6D+ cells in the visible mpMRI lesions can also lead to deprivation upon surgical castration. Our single luminal lineage represent luminal progenitors misclassification. As a result, single mpMRI cell expression analysis revealed an unexpected inherently resistant to androgen deprivation and targeted biopsy poorly reflects the genomic molecular heterogeneity in the prostate luminal with regenerative capacity. We found that LY6D+ 34 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE PROSTATE ONCOBIOLOGY 35


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    RNA BIOLOGY The RNA Biology group is interested in how changes in transcription drive oncogenic transformation, how sets of genes work together to regulate the behaviour of normal cells, and how these patterns of gene activity change in tumours. We are particularly interested in long noncoding RNAs (lncRNAs). These are transcripts that are never translated into proteins, but are increasingly being shown to act as Group Leader part of the regulatory systems within a cell. Crispin Miller1 Advances in nucleotide sequencing have led to core mechanisms that underpin these processes Senior Scientific Officer Keren Dawson1,3 the discovery of thousands of human genes that are evolutionarily conserved within humans, the express RNA molecules that do not encode implication is that there will be similar types of projects have made it possible to identify predictive of disease-specific survival. They thus Postdoctoral Fellows proteins. Despite their lack of coding potential, lncRNAs acting in human cells. We are seeking Jing Bi1,3 Figure 1: Identifying polygenic specific genes that are mutated more often provide a potential explanation of how the same these noncoding RNAs typically feature similar these lncRNAs. Garima Khandelwal 1 mini-driver signatures than would be expected by chance, and are thus hallmark phenotypes can emerge in different patterns of histone marks to protein coding Laura Bennett 1 (a) A coexpression network built likely to be ‘driver’ genes that perform a critical tumours with a high degree of inter-tumour genes, produce RNA molecules that are often To do this we have been exploiting the from RNA seq data derived from Chang Sik Kim1,2 role in the processes that govern oncogenic heterogeneity. processed to incorporate 5' caps and poly(a) availability of large cohorts of RNA sequencing matched adjacent normal tissue transformation. This has rapidly advanced Graduate Students tails, are often alternatively spliced, and are data derived from patient tumours; using these in LUAD patients. (b) Mutations from independent tumours are the field. In silico error correction improves cfDNA Mairah Khan1,4 consistently differentially expressed between to search for noncoding RNAs that are Sam Humphrey1,3 different conditions. These observations differentially expressed in tumour cells, and then mapped onto the network, and mutation calling Matt Howell1,3 modules with a disproportionate One of the most striking results from these The possibility of characterising a tumour by the together suggest that noncoding genes are applying computational approaches to infer Ronnie Rodriguez Pereira1,3 number of mutations are sequencing projects has been the degree of mutations detected in the circulating free DNA under regulatory control, and are thus unlikely to potential functions for these transcripts. Our identified. (c) While a significant heterogeneity in mutation patterns – to the (cfDNA) found in peripheral blood has the 1 be transcriptional artefacts. A key question goal therefore, is to identify previously number of genes are mutated in a Left in 2018 extent that there is often little or no overlap potential to revolutionise cancer genomics 2 Joint with CEP therefore, is the extent to which noncoding uncharacterised lncRNAs expected to have an module, different genes in the between the sets of mutations identified in through the development of novel ‘liquid 3 Joined other CRUK MI RNAs exert an important and previously important role in driving tumour growth and module are mutated in different patients. Figure derived from data different tumours. A consequence of this is that biopsies’ that support tumour profiling. This is group/facility in 2018 underappreciated role within a tumour cell. maintenance. These data are revealing many used in Bennett et al., 2018. even the most commonly occurring driver challenging because circulating tumour DNA 4 Joined Division of Cancer lncRNAs that interact directly with DNA Sciences group at University of mutations do not occur in every patient. For (ctDNA) accounts for only ~0.1% of the total The RNA Biology group is particularly interested sequences, and we are therefore particularly Manchester in 2018 example, the most frequently mutated gene in cfDNA. Reliable profiling therefore requires high in noncoding RNAs greater than 200 focused on those that directly associate with lung adenocarcinomas (LUAD), TP53, is altered sequencing depth and multiple rounds of PCR nucleotides in length. These long noncoding chromatin. in only half of all LUAD tumours (Imielinski et al., amplification. This leads to elevated PCR error RNAs (lncRNAs) are now known to outnumber 2012; Bennett et al., 2018). This is surprising, rates, and loss of mutation calling accuracy. protein coding genes, but their relatively recent High performance computing and big data given that all cancers share a set of hallmark discovery means that only a small number have analysis phenotypes (Hanahan and Weinberg, 2000), In collaboration with the CEP group we have been assigned a function. Those that have been Much of the work in the group makes use of irrespective of their particular mutational developed a novel algorithm for PCR error studied however, have been shown to perform a large volumes of DNA and RNA sequencing data profiles. It thus raises a fundamental question: correction that exploits the redundancy that diversity of roles, driven by their ability to derived from human tumours. We have how do two tumours share the same canonical occurs when sequencing many PCR amplicons hybridise to specific nucleotide sequences developed our own de novo annotation hallmark phenotypes when there is no overlap arising from the same initial cfDNA fragment through complementary base-pairing. This strategies, making it possible to discover between mutational profiles? (Kim et al., 2018). Our purely in silico approach allows them to target specific DNA and RNA previously unannotated lncRNAs, and then to molecules, and to fold into secondary structures ask how their patterns of expression change in achieves similar levels of performance to more We set out to address this apparent paradox by complex strategies that use additional protocol that define their interactions with particular tumours. This requires substantial computing building models that integrate somatic mutation steps and calibration samples. Our approach is proteins. As a consequence, lncRNAs perform a power. The RNA Biology group is highly data with gene expression profiles derived from useful because these alternative strategies add wide range of targeting and scaffolding roles interdisciplinary, and makes use of techniques LUAD patients (Figure 1, Bennett et al., 2018). cost and complexity to the protocol, and may throughout the cell. from computer science and Artificial One possibility is that in tumours that lack a also reduce the efficiency of library preparation. Intelligence. The CRUK MI has a large High canonical driver mutation, oncogenesis might This is particularly undesirable when dealing In previous work we made substantial use Performance Computing (HPC) cluster allowing instead be driven by the accumulation of with limited amounts of ctDNA material. of the model system fission yeast us to employ big data techniques to cluster (Schizosaccharomyces pombe) to study the multiple weak-effect mutations congregating datasets, to seek patterns in these data, and to basic mechanisms by which noncoding RNAs around the same cellular subsystems. Our ask how these patterns change in tumour cells. function. These revealed an extensive network analyses provided significant support for this Publications listed on page 66 of regulatory noncoding RNAs that act upon the ‘mini-driver model’ of oncogenesis, and led to Polygenic mini-drivers genome to control gene expression. Since the the identification of a novel mutation signature Large-scale tumour genome sequencing 36 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE RNA BIOLOGY 37


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    www.cruk.manchester.ac.uk/Our-Research/Skin-Cancer-and-Ageing SKIN CANCER AND AGEING Figure 1: The primary focus of our studies is melanoma and skin cancer in the The proportion of primary elderly population. Melanoma affects over 12,000 people and cutaneous melanomas arising at specific anatomic sites varies causes over 2,000 premature deaths each year in the UK, and 85% of depending on age. Old patients have more melanomas in heavily deaths occur in patients older than 60. Age is the most powerful sun-damaged skin. predictor of outcome together with primary tumour thickness. Institute Fellow One factor underpinning the increased aged patients better than the current guidelines Amaya Virós incidence of melanoma in the elderly is that as of staging. Because checkpoint inhibitors are we age we acquire more ultraviolet (UV)-driven showing promising results in the adjuvant setting Postdoctoral Fellows damage. However, although more melanomas to treat early stage melanoma patients, and the Tim Budden1 appear in the elderly population and more cost burden to health care limits access to these Katharina Roeck2 frequently occur at anatomic sites that have drugs, we are additionally investigating if certain Clinical Fellow accumulated sun damage over the years, they genetic markers linked to high risk of progression Sarah Craig can also arise at rarely exposed sites (Figure 1). can be used to identify patients with a higher rate The aged skin anatomy and function varies of response to immunotherapy. Graduate Student greatly depending on the amount of sun Shilpa Gurung1 damage that has accumulated over the years, In addition to the age-specific tumour biology, with chronically damaged sites presenting we are studying the role of the aged skin Pre-Doctoral Clinical Academic Fellow greater tissue decay, more wrinkles and more microenvironment in melanoma progression in Charles Earnshaw1 UV-driven mutations in the aged cells. Our lab the elderly patient. Previous work has shown that aims to understand how differences to the multiple cells composing the melanoma tumour 1 Joined in 2018 pattern of skin ageing (or accumulated sun stroma contribute to disease severity. For 2 Left in 2018 damage) and differences in the tumour are example, certain macrophage subtypes and first examining how skin degrades and how this that form the connective extracellular matrix. We linked to greater incidence and mortality in the fibroblasts alter the immune and metabolic affects the main physiological functions. One are investigating how UV damage versus sun- aged subset of the population. We also aim to milieu at the site of melanoma metastases and important feature of human skin is that structural protected, chronological cutaneous ageing identify new strategies to improve the outcome promote disease spread. With ageing there is an and functional decay depend greatly on the affects homeostasis. Human skin affords the of old patients with early stage, primary high-risk increase in melanoma incidence at all anatomic environmental pressure. Thus, skin from sun- opportunity to study the cellular and acellular disease. sites, but there is a specific increase in incidence protected sites becomes paucicellular across the differences in an organ subject to different levels particularly at anatomic sites with chronic sun three layers, and there is less connective tissue in of extrinsic or environmental pressure. Finally, this An important focus of our research is the tumour damage. Some melanoma subtypes that arise at the dermis, leading to thinning across all the year has given us a significant opportunity to biology of melanoma in the elderly patient. The sites of chronic sun exposure, over severely layers of the skin. In contrast, skin from establish clinical collaborations that will allow us genomic characteristics driving melanoma sun-aged skin, have a better outcome; but sun-exposed sites present a predominant pattern to explore these questions in human tissue. specifically in the aged population have not paradoxically, overall melanoma deaths increase of dermal connective tissue degradation, where been investigated. This year we have made in parallel to melanoma incidence in the elderly. collagen fibre networks disappear to form Publications listed on page 66 progress towards describing the molecular Our lab is addressing the role of stroma in globules of degraded collagen and other fibres aspects that define melanoma in the elderly promoting or inhibiting melanoma progression patient. For this major aim of our lab, we have at the earliest stages of disease. We are looked at the mutation rates, mutation types and particularly interested in the earliest primary patterns of DNA damage accumulated in stage, when melanomas that arise melanoma cells during ageing. Additionally, we predominantly from intra-epidermal are performing a comprehensive genomic melanocytes breach the dermo-epidermal basal analysis of age-specific melanoma molecular membrane to invade the deeper cutaneous characteristics linked to poor outcome in the tissue and prepare for lymphatic, local and elderly population. We are identifying the unique neurovascular spread. features that define melanoma in elderly patients and are investigating the specific changes that One intriguing aspect of melanoma progression can reliably identify old patients at high risk of in the elderly is the higher likelihood of spreading melanoma-specific death. We aim to identify the via the vascular system, and one possibility could DNA and/or RNA changes that can be used as a be that structural and functional changes in the predictor tool in clinical practice, and we are aged cutaneous microenvironment are working to test genetic markers that can stratify responsible for this phenomenon. Thus, we are 38 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE SKIN CANCER AND AGEING 39


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    www.cruk.manchester.ac.uk/Our-Research/Stem-Cell-Biology Figure 1: RUNX1 Dosage Plays an Essential Role at the Initial Stages RUNX1 levels control both the of Blood Formation STEM CELL BIOLOGY initiation and completion of blood formation from haemogenic endothelium (HE). Blood Cell Development In naive HE, RUNX1 transcription and protein levels are low. Low Maturation HE EHT RUNX1 activity is required to mature the naive HE population and initiate a productive EHT. Once EHT is initiated a higher Blood Mature RUNX1 dose is subsequently Naive HE Mature HE Progenitors Blood Cells The genes encoding for core binding factors AML1/RUNX1 and required in order to produce mature haematopoietic cells. CBFβ are frequently rearranged or mutated in human leukaemias, This shift in RUNX1 dosage and/ RUNX1 Expression and Activity such as acute myeloid leukaemia (AML) and acute lymphoblastic or activity may rely on an increase in Runx1 transcription, the switch Runx1 transcription leukaemia (ALL). Consistent with its implication in leukaemia, the from the Runx1b to Runx1c isoform and potentially also by a Haematopoietic RUNX1 activity transcription factor RUNX1 has also been shown to be critical for change in RUNX1 binding Low Low RUNX1 Required High RUNX1 Required High haematopoietic development. Our group studies the function of partners. SOXF transcription factor family members like SOX7 CBFβ Group Leader RUNX1 in haematopoietic development, maintenance and may play a role in the latter SOX7 Georges Lacaud malignancies in order to better understand how alterations of these process. SOX7 can interact with RUNX1 in HE and has previously RUNX1 RUNX1 Postdoctoral Fellows functions might lead to cancer. been shown to compete with RUNX1 co-activator CBFβ. Neo Wen Hao1 Divya Malik differentiation. To evaluate the effects of different 20% of all the AML cases. In mice models, full Muhammad Maqbool 1 RUNX1 dosage in haematopoietic development development during EHT in committed CD41+ RUNX1B expression levels on HE and EHT, we length AML-ETO on its own, expressed from a Alterations in RUNX1 dosage or activity in the haematopoietic progenitors and is the main utilised a mESC line that we previously generated viral vector or a targeted integration, is not able to Scientific Officers Michael Lie-a-Ling blood system have been associated with several active promoter in the adult haematopoietic in which Runx1b transcription is under control of induce leukaemia. However, AML1-ETO9a (AE9a), Rahima Patel2 blood disorders. Both reduction system. At a functional level, various proteins a doxycycline inducible tet-on system and the an alternatively spliced form of AML1-ETO, which (thrombocytopenia, myelodysplastic syndrome) have been shown to be able to modulate RUNX1 endogenous alleles have been deleted. Analyses is thought to act as a dominant inhibitor of AML1/ Graduate Students and gain (Down syndrome haematopoietic activity including CBFβ. CBFβ is a critical RUNX1 of this line in the presence of either a wildtype or RUNX1, has been shown to cause rapid Renaud Mevel disorders) of functional Runx1 alleles lead to cofactor that heterodimerises with RUNX1, a disrupted Cbfb locus allowed us to evaluate the development of leukaemia in mice upon Ewan Selkirk 1 haematological abnormalities and imbalances. enhances its DNA-binding affinity and protects it Muhammad Fadlullah Wilmot effects of a wide range of RUNX1B levels on retroviral transfer. Seeking to establish an In addition, RUNX1 dosage plays a crucial role in from degradation. RUNX1 protein is barely blood cell development from HE. We found that inducible AE9a-based mouse leukaemia model 1 Joined in 2018 the maintenance of leukaemias harbouring detectable in Cbfb-/- cells and the mouse Cbfb at the earliest stages, low levels of RUNX1B in HE in our lab, we first established a mESC line 2 Left in 2018 core-binding factor related translocations. knockout model is embryonic lethal and shows are crucial for the maturation of the HE and the containing a doxycycline AE9a IRES GFP Several studies have clearly established that an almost identical phenotype to the Runx1 initiation of EHT. Circumventing HE maturation inducible cassette. Doxycycline induced during the ontogeny of the blood system Runx1 knockout model, suggesting that the phenotype by increasing RUNX1 levels resulted in abortive expression of AE9a in this mESC line was able to haploinsufficiency or mutations result in a is mainly caused by a reduction in RUNX1 activity differentiation, highlighting the need for a phase block the generation of haematopoietic cells decreased generation of haematopoietic stem levels. More recently, SOXF transcription factor of low level RUNX1 expression in the HE to initiate during in vitro differentiation. Based on these and/or progenitor cells. At this early stage in family members SOX7 and SOX17 have emerged EHT successfully. However once EHT was results, we subsequently generated a mouse line development, the first blood cells arise from a as novel important regulators of RUNX1 activity. initiated, an increase in RUNX1 activity was from these inducible AE9a mESC. The mice specialised endothelium (haemogenic These transcription factors have established required to successfully exit EHT and complete expressing AML1-ETO9a developed extra endothelium or HE), via a process termed roles in vasculogenesis and angiogenesis, the blood formation process. We further medullary haematopoiesis followed by the endothelial-to-haematopoietic transition (EHT). suggesting that they are potentially able to demonstrated that SOX7 and RUNX1 interact in development of acute myeloid leukaemia with a Although we and others have shown that RUNX1 interact with RUNX1 in the context of HE HE and generated data, suggesting that SOX7 latency of around 6 months. The disease latency is critical for EHT and consequently the development. Indeed both SOX7 and SOX17 are might modulate RUNX1 activity by protecting it could be significantly shortened to 3 months emergence of blood cells from HE, little is co-expressed with RUNX1 during a narrow from degradation and sequestering it from its when AML1-ETO9a was induced on a P53-/- known about the precise role of RUNX1 dosage temporal window of haematopoietic activator CBFβ. Altogether, our study indicates background. We collaborated with the groups of in this process. We therefore sought to define development that encompasses the HE stage. that RUNX1B is essential not only for the initiation Contanze Bonifer (Birmingham University) and the role and importance of RUNX1 dosage in HE Furthermore, both factors have been proposed of the EHT but also for its completion and that Olaf Heidenreich (Newcastle University) to use and EHT. to be negative regulators of RUNX1 these two events require different levels of this model to evaluate new therapeutic strategies transcriptional activity and their overexpression RUNX1B. for AML. We demonstrated that AML1-ETO drives At the transcriptional level Runx1 is controlled by blocks haematopoietic development. leukaemia by directly promoting cell cycle two alternative promoters that generate the The CDK4/6 inhibitor palbociclib inhibits progression and that palbociclib, a clinically transcripts encoding for the two main RUNX1 Studying the role of RUNX1 dosage in detail expansion of AML1/ETO leukaemia in vivo approved inhibitor of CCND-CDK4/6 complexes, isoforms. The P1, or distal promoter, controls the during the initiation of the haematopoietic Human acute leukaemias are characterised by hampers leukaemic growth in vivo. These results expression of the distal RUNX1 isoform RUNX1C, system from HE is particularly challenging in vivo the presence of recurrent chromosomal establish that inhibition of G1 CCND-CDK and the P2, or proximal promoter, controls the as temporal and stage specific modulation of abnormalities, which encode the formation of complexes are a promising therapeutic strategy proximal isoform RUNX1B. In mice, Runx1 P2 gene dosage in embryos is difficult to achieve. chimeric transcription factors. The core binding for the treatment of AML1-ETO AML. promoter activity starts early during To circumvent this limitation, we took advantage factors AML1/RUNX1 and CBFβ are the most haematopoietic development and is detected in of the in vitro mouse embryonic stem cell frequent targets of these genetic alterations. The Publications listed on page 66 HE in which it is the sole active Runx1 promoter, (mESC) differentiation system. Indeed, mouse t(8;21) translocation, resulting in AML1-ETO indicating that the RUNX1B isoform is ESCs have been shown to recapitulate key fusion and the inv(16) generating the responsible for the initiation of EHT. In contrast events of early embryonic yolk sac SHMMC-CBFβ fusion, account for more than to P2, the Runx1 P1 promoter is activated later in haematopoiesis including HE formation and 40 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE STEM CELL BIOLOGY 41


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    www.cruk.manchester.ac.uk/Our-Research/Systems-Oncology Figure 1: Pancreatic Ductal SYSTEMS ONCOLOGY Adenocarcinoma (PDA) is characterised by extensive stromal reaction and desmoplasia. Immunohistochemistry for epithelia (pan-cytokeratine), activated fibroblasts (alpha smooth muscle actin, αSMA) and collagen (Massons Trichrome) shown on pancreatic tissue Pancreatic cancer, and specifically Pancreatic Ductal isolated from Genetic Engineered Mouse model of Pancreatic Adenocarcinoma (PDA), is a dismal disease with a median survival Cancer. Shown is normal wild below six months and an average five-year survival rate below 5%. type (WT), KRas expressing early stages pancreatic ductal This is due to the aggressive nature of the cancer, a lack of effective neoplasia (KC) or KRas/P53R172H therapy, as well as late diagnosis. Consequently, while PDA is only the expressing PDA. Notably, the epithelia lose its structure 11th most common occurring cancer in the UK, it is currently the 4th progressively as disease develops alongside an extensive fibroblast Group Leader largest contributor to cancer related deaths. activation and collagen Claus Jørgensen deposition. Postdoctoral Fellows The most frequent occurring genetic mutations therapies co-targeting tumour cell intrinsic Brian Lee have been identified with activating mutations in dependencies together with tumour cell Jingshu Xu 1 the oncogene KRAS and inactivation of the extrinsic dependencies on stromal reciprocal Giulia Veluscek tumour suppressor CDKN2A in more than 90% signals. of all cases, and loss of SMAD4 and TP53 Scientific Officer Xiaohong Zhang function occurring in 55% and 85% of all cases To address how pancreatic cancer cells (PDA respectively. Treatments targeting tumour cell cells) co-opt resident fibroblasts, we recently Graduate Students dependencies on these mutations are not used a co-culture system where PDA cells with Amy McCarthy currently available in the clinic. Less frequent an inducible mutant KRAS (G12D) were directly Colin Hutton mutations can be grouped according to the co-cultured with naïve fibroblasts (Tape et al, Elizabeth Hogg2 deregulated pathways, where DNA repair Cell 2016). Using our recently implemented and Christopher Below mechanisms are inactive in ~20% of all PDA. This optimised system for long term cell-specific importantly, what these data highlight is that the ablate their pro-tumorigenic effect on the offers novel ways to treat PDA in the clinic, some labelling (Tape et al, Mol Cell Proteomics 2014) tumour cell function is drastically regulated by tumour cells. We are currently working closely Clinical Fellow Konstatinos Georgiadis of which will be tested through the PRECISION- we analysed cell-specific changes in tumour cell stromal elements (such as the fibroblasts) and with the Drug Discovery Unit at CRUK Panc framework (see below). A hallmark of PDA signalling as a consequence of fibroblast that these elements should be included in our Manchester Institute, directed by Professor 1 Left in 2018 is an extensive stromal infiltrate that makes up co-option. Specifically, we observed that model systems to gain a better understanding of Caroline Springer, to develop this project further. 2 Joint with DDU 80% of the tumour volume. This desmoplastic activated KRAS in the tumour cells lead to putative therapeutic targets. reaction consists of a pathological remodelled increased activation of the MEK-MAPK pathway, Delivering personalised medicine in PDA – extracellular matrix and influx of fibroblasts and but not of the PI3K-AKT pathway. In contrast, Defining and targeting the tumour PRECISION-Panc immune cells (Figure 1). The microenvironment inclusion of fibroblasts enabled tumour cells to microenvironment in PDA Personalised therapy, the subscription of a has been shown to contribute to therapeutic engage additional pathways, which included In order to define interdependencies between therapy that is matched to specific resistance, immune tolerance and tumour activation of the PI3K-AKT pathway. Tumour tumour and stromal cells it is critical to map the characteristics of individual tumours, has progression. Importantly, very little is still known cells expressing KRAS secrete abundant levels of cellular and extracellular component in the benefitted cancer patients enormously, but is still about the mechanisms whereby the tumour the morphogen sonic hedgehog (SHH), but are microenvironment. We have therefore started to not available to patients with PDA. In an effort to cells co-opt host cells to establish this hostile themselves insensitive to the ligand. However, catalogue, isolate and characterise individual improve treatment options and patient selection environment. Delineating these mechanisms is fibroblasts are highly responsive to SHH and elicit stromal elements (including both cellular and in PDA, we are involved in establishing a national therefore important and may lead to the a response that includes increased production extracellular components). The critical aim of infrastructure where individual tumours are identification of novel therapeutic targets in both and secretion of ECM proteins and the growth these analyses is to determine whether individual subjected to molecular profiling, enabling the tumour and stromal cells. factors IGF-1 and GAS6. These ligands then stromal cell populations (or extracellular matrix patients to be matched with selected treatments. engage cognate receptors on the tumour cells components) differentially alter the tumour cell These clinical trials are underpinned by the Tumour-stroma signalling in Pancreatic and activate the PI3K-AKT pathway. Critically, phenotype and whether this results in a development of biomarkers and pre-clinical Ductal Adenocarcinoma PDA cells grown in the presence of fibroblasts differential sensitivity to therapy. Using a research to further refine treatment strategies The central aim of the Systems Oncology group also deregulate their metabolic pathways, where combination of proteomics and transcriptomics targeting specific dependencies. Understanding is to determine how tumour cells exchange the proteomic composition of the mitochondria analyses we are defining the key pathways the role of the microenvironment in shaping the information with host cells to support tumour and ensuing function shifts dramatically. In regulating tumour cell resistance. In parallel we therapeutic response across selected patient growth and resistance to therapies. Specifically, addition, tumour cells gain the ability to grow are identifying targetable pathways in the populations is critical to define whether we aim to describe the key mechanisms under anchorage independent conditions and tumour stroma and optimising their use for approaches targeting the tumour stroma should whereby tumour cells co-opt stromal cells and display decreased level of apoptosis. Blocking combination therapy. We have recently be delivered in a personalised manner, or conversely how the contextual impact of the the signals exchanged between tumour cells completed a screen to identify candidate targets whether a broader non-selected approach can microenvironment steer specific cancer cell and fibroblasts (SHH, GAS6 and IGF-1) normalise in the pancreatic stroma and have in initial be taken. phenotypes. Understanding these rules will the tumour cell function, suggesting that these experiments described how targeting these enable development of synergistic combination pathways are content-dependent targets. More stromal cells results in their ‘normalisation’ to 42 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE SYSTEMS ONCOLOGY 43


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    www.cruk.manchester.ac.uk/Our-Research/Transcriptional-Networks-in-Lung-Cancer TRANSCRIPTIONAL NETWORKS IN LUNG CANCER Lung cancer causes the most cancer-related deaths in the world and the main obstacles to a cure are late diagnosis and chemoresistance. The major interest in our group is to identify the causes behind lung cancer spread and resistance to chemotherapy. Over the last decade, a growing number of non-coding transcripts (ncRNAs) have been found to have a pivotal role in gene regulation Group Leader and cell biology. MicroRNAs (miRNAs) are single stranded RNAs of Michela Garofalo 19-25nt in length, that negatively regulate gene expression by Postdoctoral Fellows translational inhibition or degradation of the mRNA targets. MiRNAs Lei Shi are differentially expressed in almost all types of human cancers Tiziana Monteverde versus the normal tissue counterpart and are key players in cancer Scientific Officer Peter Magee onset and progression. Graduate Students Athanasios Paliouras To investigate whether KRAS, one of the most hydrolysis of GTP to GDP to switch off the KRAS Manuela La Montagna mutated oncogenes in lung adenocarcinoma, signalling. Mutations in KRAS are very frequent in was able to modulate miRNAs expression, we NSCLC (~30%) (Timar J, 2014) and in lung overexpressed wild type and mutant KRAS adenocarcinoma harbouring K-Ras mutations, (KRASG12D) in non-small cell lung cancer (NSCLC) although so far no targeted drug has cells. We identified two miRNAs, miR-30c and demonstrated efficacy, at least not in the clinical ALK-EML4 lung tumours specificity towards EML4-ALK-mutant cells. We Figure 1. Working Model. In NSCLC small molecule inhibitors for mutant are further investigating whether these CDK miR-21, significantly upregulated in wild type and setting. One of our goals was to identify K-RAS- KRAS, through the transcription kinases have offered unprecedented success in inhibitors are well tolerated in vivo in mouse mutant forms and showed that miR-30c and modulated miRNAs that, by targeting molecules factor ELK1, activates miR-30c miR-21 induce cell proliferation and enhance involved in the RAS pathway, can be employed the management of disease. One of the most models. and miR-21, which in turn, by migration/invasion by inhibiting crucial tumour as therapeutic tools in lung cancer. By downregulating NF1, RASA1, successful examples is Echinoderm Microtubule suppressor genes. Systemic delivery of overexpressing wild-type or mutant KRAS RASSF8 and BID, regulates KRAS, Like-4-Anaplastic Lymphoma Kinase (EML4- Publications listed on page 68 anti-miR-21 in combination with cisplatin in vivo (KRASG12D) and using inducible human and NF-kB and the intrinsic apoptotic ALK)-mutant NSCLC, which affects 4-5% of lung suppressed the initiation of lung tumours in a mouse cell lines, we analysed KRAS-regulated pathway, inducing lung cancer patients (Gainor et al., 2013). Several tumorigenesis and inhibiting EML4-ALK inhibitors have already been approved mouse model of lung cancer. miRNAs in NSCLC. We showed that miR-30c apoptosis in NSCLC. MiR-30c and and miR-21 are significantly upregulated by both by the FDA, namely crizotinib, ceritinib, alectinib, miR-21 are released into the A subset of lung adenocarcinomas is driven by KRAS isoforms and induce drug resistance and brigatinib and lorlatinib. Even though the bloodstream and could be the EML4-ALK translocation. While ALK inhibitors enhance cell migration/invasion through the potential biomarkers for early objective response rate for the ALK inhibitors in the clinic lead to excellent initial responses, inhibition of important tumour suppressor NSCLC detection. crizotinib and alectinib in the clinic surpasses acquired resistance to these inhibitors due to genes, such as RASA1 and RASSF8. MiR-30c and 60%, patients typically develop resistance to on-target mutations, or parallel pathway miR-21 levels were elevated in tumours from these inhibitors and relapse soon thereafter alterations, represents a major clinical challenge. patients that underwent surgical resection of (Hida et al., 2017). We discovered that EML4-ALK cells with early stage NSCLC compared to normal lung acquired resistance to crizotinib, ceritinib or and in plasma from the same patients. Systemic In order to mimic the context of acquired alectinib overexpress specific Cyclin Dependent delivery of LNA-anti-miR-21 in combination with resistance to ALK inhibitors in vitro, we utilised Kinases (CDKs) and CDK inhibitors halt tumour cisplatin in vivo suppressed the development of cell lines with acquired resistance to crizotinib growth and robustly induce apoptosis in this lung tumours in a KRASG12D-driven genetic (CrizR), ceritinib (CeritR) and alectinib (AlecR) by setting. mouse model of lung cancer. Mechanistically, long-term exposure to these drugs. RNA-seq we demonstrated that ELK1 is responsible for identified a cell cycle dysregulation in crizotinib- KRAS and non-coding RNAs miR-30c and miR-21 transcriptional activation by resistant cells, evidenced by an upregulation of The proto-oncongene RAS encodes three direct binding to the miRNA proximal promoter CDKs and their partner Cyclins. Following this different RAS proteins: HRAS, NRAS and KRAS, regions (Figure 1). In summary, our study defines observation, we treated EML4-ALK drug- regulated by guanine nucleotide exchange that miR-30c and miR-21 may be valid resistant cells with different CDK inhibitors which factors (GEFs), which stimulate RAS activation biomarkers for early NSCLC detection and their led to a rapid induction of apoptosis, while through GDP for GTP exchange, and by GTPase- silencing could be beneficial for therapeutic sparing normal epithelial cells, and exhibiting activating proteins (GAPs), which catalyse the applications (Shi L. et al., 2018). 44 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE TRANSCRIPTIONAL NETWORKS IN LUNG CANCER 45


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    http://www.cruk.manchester.ac.uk/Our-Research/Translational-Oncogenomics thirds of patients and this specific subclonal PNT2 Parent PNT2 –BRCA2+/GT #16 architecture is associated with adverse clinicopathological features (Espiritu et al.Cell, TRANSLATIONAL 2018). Early tumour development is characterised by point mutations and deletions followed by ONCOGENOMICS later subclonal amplifications and changes in trinucleotide mutational signatures. Patients with low-risk monoclonal tumours rarely relapse after primary therapy (l7%), while those with high-risk polyclonal tumours frequently do (61%). In further PNT2-BRCA2+/GT #17 PNT2 –BRCA2+/GT #18 bioinformatics analyses, we quantified hypoxia in Defining aggressive features of localised prostate cancer 8,006 tumours across 19 tumour types using TCGA and ICGC datasets and observed that in ten Control of genome stability requires careful coordination between tumour types, hypoxia was associated with cell cycle checkpoint control and DNA repair mechanisms. Defects elevated genomic instability. In aggressive localised PCa, hypoxia was associated with in the repair of DNA double-stranded breaks have been associated elevated rates of chromothripsis and genetic with acquiring genetic instability, particularly in genes responsible for instability, particularly in polyclonal tumours. homologous recombination such as BRCA1 and BRCA2. Although models of BRCA2 heterozygosity with isogenic Further work using pan-cancer approaches showed that elevated hypoxia is associated with Group Leader germline mutations in DNA repair genes are rare in localised prostate Figure 2: Accumulation of DNA damage in controls. Since men with germline BRCA2 increased mutational load across cancers, mutations are at high risk of developing a lethal Robert Bristow cancer (PCa); mutations in the Breast Cancer susceptibility-2 cells heterozygous for BRCA2. disease we want to understand the role of BRCA2 irrespective of the underlying mutational class. CRISPR/Cas9 gene editing Importantly, we observed an association Scientific Officer (BRCA2) gene is the most frequent DNA repair defect observed and technology was used to target a loss in driving genetic instability and between hypoxia and signatures associated with tumourigenesis. At present, there are very few Steve Lyons this confers an 8-9 fold increased risk of developing lethal prostate gene-trapping cassette to intron cell lines that lack functional BRCA2, a fact that defective homologous recombination, DNA 7 of the BRCA2 gene in the Postdoctoral Fellows cancer and subsequent failure of standard of care treatment, prostate epithelial cell line, PNT2. has hampered research into BRCA2 generally. mismatch repair and base excision repair. This provides evidence that aggression is associated Richard Rebello1 Christoph Oing1 shortening the overall cancer-specific survival to 5-8 years post Clones with integration of the gene trap were isolated by This probably stems from essential roles of with a constellation of features that we term BRCA2 in DNA replication and mitosis. Our aim is diagnosis for 50% of patients, compared with >90% for stage- selection on Puromycin- to develop a panel of BRCA2-null prostate cell tumour nimbosus– an aggressive cellular Graduate Students containing medium. Inactivation phenotype in which co-incident hypoxia, genetic Alex Suvacs1 matched at diagnosis non-carriers (Castro et al. JCO, 2013). of one BRCA2 allele by gene lines, using a strategy based on CRISPR-Cas9 technology to reversibly knockout the BRCA2 instability and aggressive sub-pathologies Ronnie Rodrigues trapping (BRCA2+/GT ) was gene by means of a gene trap. We will build on co-occur (Chua et al. Eur Urol, 2017). We are Jack Ashton1 associated with spontaneous Germline mutations in BRCA2: A model for WNT signalling, defective mitotic control and accumulation of DNA damage this by generating murine and human prostate currently exploring whether nimbosus is 1 Joined in 2018 localised aggression DNA repair and altered androgen signalling. during culture, as revealed by epithelium which are BRCA2 function- operational in high-risk, locally advanced ad Recently, our group, in collaboration with These findings suggest that in untreated BRCA2- increased levels of gamma-H2AX compromised and/or co-express secondary and oligometastatic prostate cancers using both scientists in Melbourne, used whole genome associated prostate cancers, pathways are (green signal), in three tertiary mutations of interest to understand the pre-clinical models (e.g. isogenic prostate cancer independent clones compared to cells lines with DNA repair defects) and by sequencing to explore the genetic defects in already upregulated that herald resistance to the parental PNT2 cells. molecular pathology of this disease and identify tissues derived from untreated prostate cancers hormone therapy and genetic instability. key vulnerabilities. characterising the genomes in clinical specimens Magnification x400, bar 75 arising in men with familial BRCA2 mutations derived from patients with aggressive PCa. microns. Blue signal, DAPI. (Fraser et al. 2017, Taylor et al. 2017). We We now aim to determine the cancer cell Aggression in sporadic prostate cancer: compared germline BRCA2-associated signalling program of BRCA2-deficient PCa clonal evolution and outcome mutations to those found in sporadic prostate using non-malignant prostate epithelium and In a cohort of close to 300 localised prostate cancers and found a number of genetic changes understand the cellular pathogenesis which tumours, we detected multiple subclones in two associated with germline BRCA2 mutation. gives rise to this disease. During the genesis Surprisingly, these changes are generally not of our lab this year we have employed observed in untreated localised prostate cancer, CRISPR-Cas9 targeting of BRCA2 in human but do occur in to metastatic castrate-resistant immortalised non-tumorigenic and prostate Figure 3: disease These included altered beta catenin- cancer cell lines and to this end have generated Features of localised, aggressive prostate cancers. A prostate cancer nimbosus, is associated with intraductal (IDC) and cribriform subpathologies, Figure 1: hypoxia and elevated SChLAP1. Features of germline mutant These patients are at high risk of BRCA2-associated prostate disease recurrence and cancer. metastases compared to those Early in the development of without these features. CA = BRCA2-Mutant PCa, the disease cribriform architecture; IDC = is characterised by amplifications intraductal carcinoma; PSA = in 3q, MED12, MED12L and MYC, prostate-specific antigen; global genomic instability, and a RNA-ISH = RNA in situ global hypomethylation profile. hybridisation. Chua et al, Eur In comparison, these features are Urology, 2017. restricted to relatively advanced sporadic prostate cancers. Taylor et al., Nat Commun, 2017. Taylor et al; Nature Comm, 2017 Figure 4: Evolutionary Trajectory of BRCA2- and Sporadic PCa 46 Harbouring SCIENTIFIC REPORT 2018 CANCERIDC RESEARCH UK MANCHESTER INSTITUTE TRANSLATIONAL ONCOGENOMICS 47


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    www.cruk.manchester.ac.uk/Our-Research/Tumour-Suppressors a a b c TUMOUR SUPPRESSORS Nuclei / EV mCherry / 237H GFP The most frequently mutated gene in all cancers is the tumour suppressor p53 (TP53). In our lab, we are interested in the different stress. Mutant p53 cells were able to survive this While analysing CIC, we found that in the same mutations in TP53 and the functional consequences of these Figure 2. stress, albeit at the cost of aberrant divisions and cell type, different GOF mutations of p53 have a) Z-stack image of a cell-in-cell multinucleation. When implanted as co-cultures different potencies of cell engulfment. Similarly, mutations in lung cancers. The mutation frequency for p53 in all structure in H1299 cells; green: in recipient immune-compromised mice, the when we provided our cell lines with these cancers is about 60%, but can be variable in individual cancers. In H1299 cells stably expressing mutant p53 and GFP, red: H1299 heterogeneous populations of cells, in which the different mutants to the lab of H. Vakifametoglu- non-small cell lung cancer (NSCLC) the frequency is about 30%, but cells stably expressing an empty highest numbers of engulfing cells were Norberg, Eriksson et al discovered differences in (EV) control plasmid and detected, grew faster. This suggests a tumour metabolism between p53 mutants regulating Institute Fellow in small cell lung cancer (SCLC) p53 is mutated in nearly all cases mCherry, blue: dapi. Top and left promoting role for cell engulfment. We glycolysis (Eriksson, Mol Cell Biol, 2017). These Patricia Muller (90% or more). The vast majority of p53 mutations occur in the DNA panels are showing 3D vertical investigated cell engulfment in tumours of about data suggest that while many consider GOF and horizontal cross sections of 300 lung cancer patients. In histology, mutants as similar, they can actually have Postdoctoral Fellow binding domain of p53 (Figure 1). When looking at the mutation the Z-stack. engulfment can be seen as CIC (cell-in-cell) dramatically different functional consequences Yannick von Grabowiecki1 frequency in SCLC compared to all cancers, there are clear b) Cell-in-cell structure in non-small cell lung cancer. structures that resemble the appearance of to a cell. In our lab we are interested in these Graduate Student differences (Figure 1) that cannot be solely attributed to the ‘smokers’ c) Survival of lung cancer patients engulfed cells in tissue culture (Figure 2b): an differences and are focussing on the different p53 based on the presence of CIC outside larger cell with a half-moon shaped mutations seen in SCLC. We have generated a Callum Hall mutation signature. In particular, mutations in the N-terminus, the structures. nucleus that contains a ‘rounded up inside cell’ in library of frequent and less frequent SCLC p53 1 Joined in 2018 C-terminus and at certain regions in the DNA binding domain of p53 a vacuole. As mutant p53 is often overexpressed mutations and are testing these in functional in cancers, we stained the tissues for p53 and assays in lung cancer cell lines. Pilot data revealed are more prone to mutate in SCLC compared to all cancers (Figure 1). identified an association between mutant p53 a number of differences that comprise loss of These data suggest selective advantages for these mutations to staining and the presence of CIC, which was certain wildtype p53 functions as well as gain of occur in SCLC. particularly apparent in tumours that had other functions. We therefore aim to heterogeneous p53 staining. In this patient systematically characterise and categorise these cohort, we could also identify a correlation mutations and compare these to SCLC patient Mutations in p53 can result in loss of p53 a role for these proteins in mediating mutant between the presence of CIC and decreased characteristics. Several of the mutations we expression, or expression of mutant p53 p53-dependent chemoresistance. survival (Figure 2c). In patients, we also noted that selected are present in CDXs (circulating tumour proteins. Previous data from our lab has CIC was associated with multinucleation and cell derived explants) of SCLC patients, which we indicated that although the mutant p53 proteins Most recently we noted that mutant p53 proteins aberrant mitotic structures, which generally are plan to use to validate our results. These data often lose wildtype function, these are more also gain the capacity to promote engulfment of indicative of chromosomal changes. In could therefore provide evidence of functional harmful than a simple loss of p53 function/ neighbouring tumour cells (Mackay et al 2018) collaboration with TracerX, we determined that differences in p53 mutations in SCLC. activity. Mutant p53 proteins gain the ability to (Figure 2a). This was most often observed in CIC was indeed associated with genomic inhibit the p53 family members p63 and p73 and heterogeneous populations of cells comprising instability. Together these data demonstrate a Finally, we identified that specific metals can the microRNA machine protein Dicer to p53 null and mutant p53 cells. Mechanistically, it role for CIC in promoting tumourigenesis. Future change the function of wildtype p53 and make it promote invasion and metastasis. This inhibition mostly resembled the process of entosis, research will focus on further elucidating this role. behave like a mutant p53 by promoting invasion leads to an enhanced recycling of integrins and requiring ROCK activity and cell-cell adhesion and metastasis or interacting with mutant- growth factor receptors to the cell membrane, molecules such as E-cadherin and beta-catenin. Figure 3. specific interactant proteins. Smokers’ lungs are mediated by RCP (Rab Coupling Protein). We are Upon engulfment of neighbours, p53 null cells Infra-red fluorescent image of exposed to a variety of metals and in many following up on these data and are investigating were more likely to die as a result of replication micrometastases in the lung of cancers increased metal levels can be detected. iRFP H1299 cells injected We characterised the mechanistical changes in intravenously in NSG mice p53 that occur upon metal exposure and are percent in all cancers currently validating our findings in patients and Figure 1. p53 mutation spectrum in SCLC versus all cancers percent in sclc mice. For the mouse studies, we will use iRFP p53 mutations frequency in SCLC 7 (infra-red fluorescent protein)-expressing stable compared to the frequency of all 6 cell lines for heterotopic xenografts, with which cancers (based on TCGA data) 5 we can monitor and count micrometastases to frequency (%) 4 the lung (Figure 3). These data will provide 3 important information between p53 functions 2 and changes in extracellular environments, 1 influencing/promoting tumorigenesis. 0 p53 protein Publications listed on page 68 N-terminus DNA binding domain C-terminus 48 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE TUMOUR SUPPRESSORS 49


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    CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES 50 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE 51


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    www.cruk.manchester.ac.uk/Research RESEARCH SERVICES The introduction of Mass Cytometry (CyTOF) has Biological Mass Spectrometry Facility been a major initiative this year; the technology Duncan Smith, Yvonne Connolly is based on both flow cytometry coupled with time of flight mass spectrometry, which permits 2018 has been a year of change for the Mass the inquiry of protein expression on a single cell Spectrometry facility. In Q1 2018, we moved into basis. Antibodies are labelled with rare earth Alderley Park tasked with relocation of the three heavy metals rather than light emitting tandem Mass Spectrometers previously situated During the last year there have been several significant investments fluorochromes in traditional flow cytometry in the Paterson Building. This has involved in the core facilities and it has been great to see the continued – consequently crossover of signals and re-location, installation, testing, repair and development of new services as the core facilities have settled into detection of discrete labels becomes easier to instrument performance testing to ensure the achieve. Working alongside the Systems hardware could perform at their previous levels. new laboratory spaces at Alderley Park. Oncology group, panels have been formed for After extensive efforts, one system was found to human and murine to assist researchers in be beyond repair and another is in the final adopting this technology. The introduction of stages of testing. It has been necessary to Chief Laboratory Officer teams have adapted to new working this technology has been quite an undertaking, outsource MS provision for the vast majority of Chief Laboratory Officer Stuart Pepper arrangements with the breeding facility however the advantage of being to be able 2018. The Institute submitted a tender for two delivering mice on a twice weekly basis to the Stuart Pepper The recent purchase of a CyTOF system in the Alderley Park site. With all in vivo work the detect over 50 distinct events per cell leads to new Mass Spectrometers in Q3 2018 and functional and phenotypic assays of the tumour successfully purchased an Orbitrap Lumos and Advanced Imaging and FACS facility has been a Institute is highly committed to the principles of niche. Q-Exactive HFX, two state-of-the-art great example of close partnership between the 3Rs, and we were very pleased when one of our proteomics platforms with superior Systems Oncology group and the core facility, staff was invited to speak at the NC3Rs oncology Histological whole slide scanning and analysis performance capabilities to anything CRUK MI leading to the establishment of this exciting workshop this year. was a process first introduced into the facility in has had access to previously. Excitingly, both technology as a service available to all groups. 2008. Over this time an array of instruments and systems were installed at AP in November 2018. Two other new platforms have also been Laboratory services have adapted to working software have been developed to provide data at The new systems represent the very best procured to support our Biological Mass across two sites, with the main function based at the single cell or across the whole tissue. These platforms available for proteomics. The first Spectrometry facility. Our older Orbitrap the Oglesby Cancer Research Building and efforts have been made in conjunction with the service provision samples were run on the new instrument did not survive the fire in the Paterson some reagents transferred to Alderley Park by Histology facility – both facilities working systems in late December 2018. We are now Building and so needed to be replaced, and a our Logistics team. together can identify the correct tools for the establishing our portfolio of workflows on the strategic decision was made to also upgrade the whole histological workflow. In the last year the new hardware that will bring an end to the need Q-Exactive. The addition of two new Looking back at 2018 it is good to see how software HALO has been introduced for to outsource MS provision over the course of Q1 instruments to our Mass Spec facility means that quickly the various specialist facilities have histological image analysis; this permits a range 2019. we now have state of the art equipment to settled into the new space at Alderley Park, and of topographical and single cell analysis support a broad range of protein analysis have maintained momentum for continual techniques such as those enumerating RNA and applications. A final new technology to mention enhancement of the technology platforms gene expression, cell and object-based analysis, Biological Resources Unit is the 10X Genomics Chromium platform which available to support the research programs of along with bioinformatics for histology data. Transgenic Breeding was installed in our Molecular Biology Core the Institute. Research potential is to be extended in the Team Leader: Jen Hughes¹, Kim Acton² Facility this year. This was bought primarily to coming year with the purchase of a replacement 1 Joined in 2018 ² Left in 2018 support single cell RNA sequencing, but will also whole slide imaging system, which will permit be used for single cell CNV mapping and ATAC Advanced Imaging and Flow Cytometry brightfield, darkfield, polarised and fluorescence The BRU Transgenic Breeding Team breeds sequencing. Steve Bagley, Jeff Barry, Michele Fresneda imaging. mice to meet the requirements of CRUK MI Alarcon¹, Antonia Banyard, Helen Carlin, Jack researchers. Ten staff members currently provide The ever increasing range of equipment Eastham¹, Isabel Peset Martin, Heather The facility data load year on year is on the day-to-day care for 133 different transgenic generating large and complex data sets puts Woodhouse, Kang Zeng increase and so with the laboratory’s relocation mouse lines spread across approximately 2,400 greater demands on our Scientific Computing 1 Joined in 2018 this has provided an opportunity to investigate all cages in a facility located within the main Facility. Following a successful relocation of our of our workflows. Consequently, we are working University campus. Services offered include HPC infrastructure, much of the focus over the The Imaging and Cytometry facility’s remit is to closer with the Scientific Computing team who rederivations using fresh and frozen sperm and last year has been on increasing the stability of all provide state-of-the-art tools for both the handle all of our live data. embryos, pairing mice for breeding, monitoring aspects of the system not just to benefit all the fundamental and translational study of cancer timed matings, recording and weaning litters, ear users of the service, but such that a growing – from molecular interactions in primary cells In the next year we will also see a major change snipping for identification and genotyping, number of clinical pipelines can be hosted and through to tissue-wide responses. Taking our to the facility as Imaging and Flow Cytometry managing the genotyping service (using an operated to the standard required for GCP lead from the requirements of the group leaders become separate facilities. Jeff Barry will be external service provider), translating and compliance. As part of this work an extra and their research, the facility is able to adapt as heading the Flow Cytometry facility whilst the transferring genotyping results, monitoring 1.4PByte of storage to enhance our backup the team responds by introducing new Imaging facility will develop new modalities. tumour prone lines for onset of symptoms and capacity has been added to the system. equipment, methods and workflows at both Initiatives are planned for analysis and cryopreservation of lines that can be archived. In Alderley Park and at the Oglesby Cancer distribution of data along with increased support accordance with Home Office requirements the Across the Institute there are three teams Research Building (formerly the MCRC Building). of assisting in preclinical image analysis. mice are closely monitored in order to ensure supporting in vivo research that include Technologies include microscopy (confocal and high welfare standards. The 3Rs (Replacement, production of novel transgenic mice, breeding super resolution), high content screening, of a wide range of transgenic strains and a facility histological imaging and flow cytometry sorting to support experimental procedures. All three and analysis. 52 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES 53


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    RESEARCH SERVICES (CONTINUED) Reduction and Refinement) initiatives The cages at Alderley Park are smaller than the The Zeiss AiryScan microscope undertaken in the last year include adapting and ones we previously used, meaning we have can capture many more cells at high resolution simultaneously. standardising the ear snipping process and to clean the mice out every week rather than Here, HCT116 colon cancer cells grouping male mice at time of weaning where fortnightly as we were doing in the past. We have are stained for their microtubule possible, so that the numbers of singly housed therefore had to recruit an extra two temporary cytoskeleton. Cells are males are reduced. posts to match the increasing workload. undergoing division, migration and clustering, allowing The last year has been a busy one with 39 new Our ultrasound imaging machine was water cell-by-cell examination of the breeding lines being started and 19 lines being damaged during the fire so we have purchased a effects of individual proteins or drug targets. closed. The breeding facility is housed in a clean newer model which is more user friendly than unit with a high health status and is kept free the original one and with better image quality Image supplied by Andrew from common mouse pathogens, which means and the ability to produce 3D images much Porter (Cell Signalling) that new transgenic mouse lines from external faster. Consequently demand for ultrasound sources cannot be brought in directly as live work has increased greatly and is now used by a mice. New lines coming from external sources range of research groups to monitor pancreas, instead have to be transferred in as either bladder and prostate tumours, as well as looking embryos or sperm and thoroughly health at spleen inflammation and liver metastasis. We screened in order to ensure that the resulting have also used the kit to successfully carry out offspring are pathogen free. When live mice are image guided injections into tumours in the sent they are housed in quarantine facilities bladder for the first time ever. during the rederivation process. Another piece of equipment we had on trial in Five of the 39 new lines have been rederived, December 2018 is the ‘Pearl’ by LiCor – this is an using embryos and live mice sent from optical imaging modality which uses near-infra- Singapore, USA, London, Birmingham and red fluorescence to monitor tumour Manchester. Another 8 of the new lines have development. The issue with general been transferred from the Transgenic fluorescence imaging is that many of the tissues Production Facility and the remainder have been in the mice auto-fluoresce at the same generated by crossing mice from existing lines. wavelength we are trying to look at, generating more background noise. This system uses a Mice are transferred on request in twice weekly slightly different wavelength so we should get a shipments to the BRU Experimental Team at much better signal. We are comparing the three Alderley Park. After transfer a minimum of 1 week systems we have available so the results should acclimatisation is required before mice can be be really interesting. enrolled in experiments. As well as shipping to Alderley Park, this year we have also shipped Surgical work has been a big focus this year and mice to USA, Spain and the University BSF facility. we have spent a lot of time implementing Transgenic Production Facility transgenic approaches like targeting mouse improvements and training licensees in these Natalia Moncaut, Mark Willington, Athina embryonic stem cells or random transgenesis. One of our main aims for 2018 was to source techniques. One key area has been looking at Papaemmanouil The TPF is also responsible for the design of specialist mouse management software in order alternative forms of wound closure which are targeting strategies using the CRISPR technology to improve work flow, reduce errors and allow more comfortable for the mouse. We have been The Transgenic Production Facility (TPF) offers a for in vitro applications, such as targeting information to be accessed more easily. The moving towards using glue or sutures rather than comprehensive service in the generation of different cell lines. One interesting project we are procurement process has now been completed wound clips as they can very easily be put in too genetically modified mouse lines using the involved in is in vivo targeting to produce cancer and we are beginning to implement actions tight and cause skin-healing issues. We have set CRISPR technology. The Facility is responsible mouse models. This strategy will bypass the necessary for the changeover which will be up several new surgical models across the for the design of the best targeting strategy and generation of new lines along with their completed in 2019. research groups this year, including an for the production of all the reagents required for associated time and breeding costs. orthotopic eye model and an orthotopic bladder the process. model. We have also introduced refinements to The field of animal transgenesis is evolving in an Biological Resources Unit our orthotopic mammary fat pad model so we Generating mouse models remains instrumental unprecedented manner with more efficient and Experimental Services can now carry this out non-surgically. In in revealing the complexities of human cancer precise tools to make the technology more Team Leader: Lisa Doar September, the NC3Rs invited us to talk at a biology. Working together with different research versatile and available to any application. TPF is symposium they were running in collaboration groups at the Institute, this last year we have regularly participating in annual meetings and Moving to Alderley Park and having everyone with CRUK where the focus was on setting up successfully produced new mouse lines courses in order to keep up with the latest back together was a great morale boost for the new cancer models and skill-sharing between including the conditional and constitutive advances in this technology. Together with other team. Throughout the year our workload has institutes. Joanne Roberts and Janet Watson knock-outs of specific genes involved in transgenic facilities within the UK, we are increased steadily and by Q3 was back to pre-fire presented the talk and discussed these particular different aspects of the human disease. Also we organising a series of technical workshops to levels – this is quite an achievement and three models, generating much interest and generated new strains carrying patient-specific build an active community of transgenic testament to how hard the team have worked. discussion points. point mutations and larger knock-ins. New technologists. projects have been started involving different 54 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES 55


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    RESEARCH SERVICES (CONTINUED) Histology protein immunohistochemistry on single tissue Institute’s scientific infrastructure. For a number process hundreds to many thousands of single Garry Ashton, Caron Abbey, Keren Dawson¹, sections is also now in routine use. of years the NGS service has been supported cells for transcriptome and genome Janice Kerrigan², Katherine Lally¹, Marta mainly by three platforms; Illumina’s HiSeq2500, interrogations. These types of single cell Madureira da Graca, Usman Mahmood, Emma Laser capture microdissection followed by the NextSeq500 and MiSeq. Methodologies include analyses are rapidly gaining traction as methods Watson, Deepti Wilks (Haematological downstream extraction of both RNA and DNA, sequencing of whole genomes, whole exomes, of choice in immunological and cancer biology Malignancy Biobank) giving sufficient quantity and quality for NGS transcriptomes, PCR amplicons, as well as investigations and consequently, over the last 1 Joined in 2018 2 Left in 2018 from relatively small amounts of material, is now interrogations of protein-DNA interactions (e.g. year we have processed an ever increasing routine and has seen a large increase in demand. ChIP-Seq), and chromosomal architecture (4C-, number of single cell NGS projects from a The Histology facility continues to underpin the The evaluation of several commercial extraction HiC-Seq). The increasing demand for the diverse set of tissues, including blood, bone research activities of a large number of both kits has been undertaken whilst the technology is paralleled by a continuously marrow, xenografts, and tumour biopsy material. basic and translational research groups within development of laser capture microdissection growing range of methods and the service has the CRUK MI. It allows the adoption of tissue- together with immunohistochemistry is been striving to develop, validate, and improve Another exciting development in the NGS field is based experimental approaches to all research ongoing. NGS workflows, including novel methods for presented by long read sequencing. MBCF has programmes. The unit’s remit is to offer a full ChIPSeq (NEBNext UltraII), analysis of immune been an early adopter of this technology and we range of both routine and advanced histological The use of our existing tissue microarray archive repertoire (Immunoseq), and improved genome have validated a number of workflows using services for oncology research. As the range and and the construction of new arrays have again and transcriptome analysis of formalin fixed Oxford Nanopore’s MinION platform for genome, complexity of the services offered continues to proved extremely popular. All TMAs give true samples (SureselectXT, RNADirect). transcriptome, and targeted sequencing. In grow, the training and continued professional sample representation and are of the highest combination with the high throughput capability development of staff has ensured the unit quality. TMAs from disease groups including A rapidly developing area in the NGS field is and accuracy of Illumina’s short read technology, continues to offer a comprehensive and flexible breast, melanoma, prostate (cores and chips), single cell (sc) analysis. Supported by a set of we have been exploiting long read information service at all times. bladder, lymphoid, small cell and non-small cell state-of-the-art automation platforms (Echo, using Nanopore sequencing for, e.g. the lung cancer plus mouse model and cell pellet Mantis, Bravo), MBCF has developed workflows interrogation of chromosomal rearrangements, The core facility is now housed at Alderley Park controls are all available. for high throughput methodologies for sc-RNA or improved accuracy in determining transcript with a small satellite lab offering routine sequencing (Smartseq2, Celseq2). In addition we splice variants. histology services and immunohistochemistry One interest of the Stem Cell Biology group is to have installed a 10XGenomics Chromium also based within the Oglesby Cancer Research study the development of the haematopoietic platform for automated single cell encapsulation A recent addition to MBCF’s services has been Building (formerly the MCRC Building). Two new system and identify new regulators of this to support sc-RNA sequencing and genomic the provision of bespoke chemical compound scientific officers have also been recruited within process. Histology has been instrumental in analysis including sc-CNV and sc-ATAC Seq. As a libraries for drug screening purposes. This the year. characterising some of these newly identified result, MBCF now has the capabilities to rapidly service is supported by an automation set up for regulators by performing section and Histology routinely processes, embeds and whole-mount staining of mouse embryos. The Some proteins undergo major sections both human and mouse tissues in facility has also optimised the combination of changes in localisation during the addition to organotypic assays, spheroids, agar in-situ hybridisation (ISH) staining and cell cycle. NuMA (magenta), plugs and cell pellets. Vibratome sections of multiplexed IHC using fluorescent labelling for contained in the cell nucleus until fresh tissue (50–250μm) have also been this project. In addition we have provided the start of cell division, will move extensive support in the development of to the mitotic spindle (cyan) prepared for the set up of ex vivo cultures of multiplexed IHC antibody panels (up to 5 poles. NuMA concentration tumours and to evaluate and develop three increases to anchor microtubules dimensional studies. Several special stains have fluorescent labels). These panels have been for correct orientation of the been used by various groups including Masson applied to a variety of tissue samples, including mitotic spindle and separation of Trichrome, PAS and reticulin stains. In addition, mouse prostate organoids and urogenital chromosomes. Imaged on the the unit continues to process FFPE and frozen systems, and human patient tissue microarrays Zeiss AiryScan super-resolution samples for the MCRC Biobank. To date samples in order to characterise the expression of RUNX1 microscope. from over 10,000 patients have been collected. in the context of other markers. The unit continues to be used heavily by the CEP Image supplied by Andrew In addition blood, bone marrow and plasma Porter (Cell Signalling) from haematological malignancy patients have Preclinical and IHC Biomarkers teams. CDX also been collected and processed. The samples models are phenotyped routinely on our are of the highest quality ensuring maximum automated IHC platforms ensuring consistency, value to any research program. reproducibility and standardisation. The high throughput routine immunohistochemistry service, troubleshooting Molecular Biology Core Facility and antibody validation services have once again Wolfgang Breitwieser, Andzhela Abu Rashed, seen exceptional demand. In addition the unit Chris Clark, Gillian Williams¹, Amy Priestman, has incorporated sophisticated labelling Rachel Horner, and John Weightman techniques into routine practice. mRNA 1 Left in 2018 in situ hybridisation and multiplex immunohistochemistry are labelling techniques The Next Generation Sequencing (NGS) services used by several research groups. Multiplexing offered by the Molecular Biology Core Facility using both mRNA in situ hybridisation and (MBCF) provide a critical component to the 56 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES 57


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    RESEARCH SERVICES (CONTINUED) high throughput compound dispensing (Access, finishing their work on a new web framework cBioPortal and REACT (REal-time Analytics for Generation Sequencing, Mass Spectrometry, Echo). Using these automation platforms we also called Octopus. Clinical Trials). The results of this work are also and Advanced Bio-imaging. Computational support high throughput compound dispensing, beneficial for other projects including those that Biology Support is responsible for the initial and combinatorial dosing experiments. As more Octopus is an easy to use web frontend that can inform regarding the genomic status of a pre-processing and analysis of data sets researchers recognise the benefits of automated contains several modules for the pre-processing patient. generated by the Institute’s Core Facilities, and compound dispensing, the service is of sequencing data. Currently, modules for also for providing bio-statistical support to experiencing an increasing number of complex pre-processing Illumina and Nanopore A novel platform for analysing, visualising and research groups. For this the Service draws on projects. The recent installation of an instrument sequencing data and a 10x Genomics module managing data from in vitro screening assay the most advanced methodologies for analysis specific combination screening software thus are based on the Cell Ranger software for single technologies based on the Genedata Screener® of DNA-, RNA-, ChIP-seq, proteomics and allows us to program work flows for complex cell DNA sequencing. Pre-processing includes software has been implemented for DDU by bio-molecular imaging. The team utilise plate layouts. steps for demultiplexing as well as automated SciCom. The software and its components were open-source as well as custom-built software quality and contamination checks. The modules installed on our virtual server infrastructure in tools running on the on-site high performance Scientific Computing and were implemented by SciCom in close close cooperation with Genedata, Advanced computing facility and we also contribute to the Computational Biology Support cooperation with the Molecular Biology Core Imaging and IT core facilities. The installation building of automated workflows in data Facility and the CEP NAB team. Great progress serves as a prototype to further data integration processing. For example, we have also been These two services provide high performance has been made regarding the implementation of projects. A bioinformatic analysis pipeline for involved in developing models and algorithms computing and data analysis solutions for the the bioinformatic mutation calling pipelines for high content single cell analysis was successfully using R code to study the survival of patients Institute’s scientists. the TARGET trial. The focus in 2018 was on implemented with CEP and the University of across a number of cancer types. The team’s increasing the stability, the reproducibility and Southern California’s Convergent Science other contributions include the development of portability of the pipelines, using technologies Initiative in Cancer. The standardised gene signatures to predict outcomes of patients, Scientific Computing like Linux containers. These containers allow the components of the compute intensive pipeline e.g. in lung carcinoma. In addition, we help to Marek Dynowski, Kevin Doyle¹, Jack Heal², Rishi creation of defined software stacks, so that the are created by Peter Kuhn’s lab at USC and run interrogate publically available data resources, Ramgolam¹, Neil Venables², ZhiCheng Wang pipelines can easily be ported on cloud or on the SciCom virtualisation infrastructure. e.g. The Cancer Genome Atlas (TCGA) or the 1 Joined in 2018 2Left in 2018 standby High-Performance Computing systems Database for Genotypes and Phenotypes during a Disaster Recovery Process. The different In 2018 the re-installation of the SciCom High (dbGaP). To aid this process we have drafted The Scientific Computing core facility (SciCom) versions of the pipelines for TARGET are Performance Computing (HPC), virtualisation guidelines to help facilitate access for the faced both challenges and opportunities in automated, so that they can be integrated as a and storage infrastructure were fully completed. Institute’s investigators to open as well as 2018. It started well with Rishi Ramgolam joining module into the Octopus framework. This will We used this opportunity to optimise hardware restricted information, e.g. gene expression the team in January. He is a software developer greatly simplify their usage and improve designs and software configurations as well as information, and de-identified clinical and with a focus on implementing bioinformatic reproducibility by reducing the chance for the introduction of new cluster utilities to demographic data. analysis pipelines for clinical trials. Kevin Doyle human errors. New precision medicine solutions improve the stability, manageability and started in November as Linux Systems are being developed in cooperation with the efficiency of the systems. For instance, the In 2018 the CBS team contributed to a number administrator and will work on the infrastructure TARGET team and digital ECMT that allow the introduction of the new workspace concept has of high profile publications. In one study we for running virtual servers. Sadly, the software loading of sequence data into cBioPortal, which led already to a more efficient use of cluster’s supported the bio-statistical analysis of histone architects Neil Venables and Jack Heal left enable the uploading of patient’s tumour biopsy storage resources, since unnecessary data is methylation changes upon pharmacological SciCom in September 2018, but not before and circulating tumour DNA sequence data into automatically cleaned up. The security and intervention in AML cells (Maiques et al., Cell stability of the cluster could be further improved Reports 2018). Our contribution included through a comprehensive Linux operating evaluation of ChIPSeq data as well as Gene Set Super-resolution microscopy system and batch system upgrade on the Enrichment Analysis (GSEA). In a separate probes the structure of cells in new ways. Here, Pericentrin Phoenix HPC cluster, which was carried out at investigation on tumours that lacked known (orange) forms an almost perfect the end of the year. Finally, additional 1.4 PByte driver mutations, we interrogated these tumours ring around the centrosome, a storage and backup capacity for the SciCom for the presence of chromosomal abnormalities complex structure that helps research storage were purchased in December. and copy number gains using Whole Genome organise the microtubule This storage upgrade and the purchase of new Sequencing, and using Whole Exome cytoskeleton of the cell. The hardware for running compute intensive virtual Sequencing for the identification of actionable centrosome is in close proximity to the cell nucleus (blue), where servers in 2019 allows the implementation of mutations (Torres Ayuso et al., NPJ Genomic the mottled pattern gives a hint at new integrated data analysis methods, fully Medicine 2018). In a study of microRNA the structure of the exploiting the potential of the high-speed access mediated tumour suppression, we undertook chromosomes inside. to a shared central storage system miRNA expression analysis of TCGA datasets from lung adenocarcinoma (LUAD) samples as Image supplied by Andrew well as tumour cells (Shi et al., Cell Death & Porter (Cell Signalling) Computational Biology Support Disease 2018). Current projects involve for Hui Sun Leong2, Sudhakar Sahoo, Samuel example the analysis of p53 transcriptional Taylor, Pieta Schofield, Nitin Sharma1 network in non-small cell lung cancer, lncRNA- 1 Joined in 2018, 2Left in 2018 mediated transcriptional and post- transcriptional regulation of metastasis in lung The Computational Biology Support Team cancer, and KRAS copy number association with provides expertise in the analysis of large worsened prognosis in pancreatic cancer. biological data sets that originate from high throughput technologies such as Next 58 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES 59


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    CANCER RESEARCH UK MANCHESTER INSTITUTE PUBLICATIONS AND ADMINISTRATION Multiplexed immunofluorescent staining of an ex-vivo model of prostate development. Image scanned on the Leica Aperio Versa. Image supplied by Renaud Mevel (Stem Cell Biology) 60 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE 61


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    Cancers: Correlative Results from AZD9496 Molecular characterisation and liquid Oral SERD Phase I Trial. Clinical Cancer biomarkers in Carcinoma of Unknown Primary RESEARCH Research [Epub 6 August 2018] (CUP): taking the 'U' out of 'CUP'. British Journal of Cancer [Epub 23 December 2018] PUBLICATIONS Torres-Ayuso P, Sahoo S, Ashton G, An E, Simms N, Galvin M, Leong HS, Frese KK, Simpson K, Cook N, Hughes A, Miller CJ, Blackhall F, Frese KK, Simpson K, Kilgour E, Brady G, Dive C. (2018) Marais R, Dive C, Krebs MG, Brognard J. (2018) Will liquid biopsies improve outcomes for Signaling pathway screening platforms are an patients with small-cell lung cancer? efficient approach to identify therapeutic Lancet Oncology, 19(9):e470-e481. targets in cancers that lack known driver mutations: a case report for a cancer of unknown primary origin. NPJ Genomic Drug Discovery (page 26) In silico error correction improves cfDNA Medicine, 3:15. Caroline Springer/Donald Ogilvie Cancer Inflammation mutation calling. Bioinformatics [Epub 6 and Immunity (page 14) December 2018] Lallo A, Frese KK, Morrow CJ, Sloane R, Gulati Refereed research publications Santiago Zelenay S, Schenk MW, Trapani F, Simms N, Galvin Waszkowycz B, Smith KM, McGonagle AE, Lallo A, Gulati S, Schenk MW, Khandelwal G, M, Brown S, Hodgkinson CL, Priest L, Hughes Jordan AM, Acton B, Fairweather EE, Griffiths Refereed research publications Berglund UW, Pateras IS, Chester CPE, Pham A, Lai Z, Cadogan E, Khandelwal G, Simpson LA, Hamilton NM, Hamilton NS, Hitchin JR, Böttcher JP, Bonavita E, Chakravarty P, TM, Kalderen C, Frese KK, Gorgoulis VG, KL, Miller C, Blackhall F, O'Connor MJ, Hutton CP, James DI, Jones CD, Jones S, Blees H, Cabeza-Cabrerizo M, Sammicheli S, Miller C, Blackhall F, Helleday T, Dive C. Dive C. (2018) Mould DP, Small HF, Stowell AIJ, Tucker JA, Rogers NC, Sahai E, Zelenay S, Reis e Ex vivo culture of cells derived from circulating The combination of the PARP inhibitor Waddell ID, Ogilvie DJ. Sousa C. (2018) tumour cell xenograft to support small cell olaparib and the WEE1 inhibitor AZD1775 as a Cell-Active Small Molecule Inhibitors of the NK Cells Stimulate Recruitment of cDC1 lung cancer research and experimental new therapeutic option for small cell lung DNA-Damage Repair Enzyme Poly(ADP- into the Tumor Microenvironment therapeutics. British Journal of Pharmacology cancer. Clinical Cancer Research, 24(20): ribose) Glycohydrolase (PARG): Discovery Promoting Cancer Immune Control. [Epub 14 November 2018] 5153-5164. and Optimization of Orally Bioavailable Cell, 172(5):1022-1037. Quinazolinedione Sulfonamides. Journal Parry MA, Srivastava S, Ali A, Antonello J, Backen AC, Lopes A, Wasan H, Palmer DH, of Medicinal Chemistry [Epub 19 November Other publications Cannistraci A, Leong HS, Barros-Silva J, Duggan M, Cunningham D, Anthoney A, 2018] Bonavita E, Pelly VS, Zelenay S. (2018) Ubertini V, Ramani V, Lau M, Shank J, Nonak Corrie PG, Madhusudan S, Maraveyas A, Ross Resolving the dark side of therapy-driven D, Oliveira P, Hambrock T, Dhomen N, Miller PJ, Waters JS, Steward WP, Rees C, McNamara Tumbale P, Schellenberg MJ, Mueller GA, cancer cell death. Journal of Experimental C, Brady G, Dive C, Clarke N, Marais R, MG, Beare S, Bridgewater JA, Dive C, Valle Fairweather E, Watson M, Little JN, Krahn J, Medicine, 215(1):9-11. Baena E. JW. (2018) Waddell I, London RE, Williams RS. (2018) Genomic evaluation of multiparametric Circulating biomarkers during treatment in Mechanism of APTX nicked DNA sensing and magnetic resonance imaging-visible and patients with advanced biliary tract cancer pleiotropic inactivation in neurodegenerative receiving cediranib in the UK ABC-03 trial. Cell Signalling (page 20) -nonvisible lesions in clinically localised British Journal of Cancer, 119(1):27-35. disease. EMBO J, 37(14). pii: e98875. Angeliki Malliri prostate cancer. European Urology Oncology [Epub 4 September 2018] Maiques-Diaz A, Spencer GJ, Lynch JT, Ciceri Salem A, Mistry H, Backen A, Hodgson C, F, Williams EL, Amaral FMR, Wiseman DH, Refereed research publications Jayson GC, Zhou C, Backen A, Horsley L, Koh P, Dean E, Priest L, Haslett K, Trigonis Harris WJ, Li Y, Sahoo S, Hitchin JR, Mould DP, Woroniuk A, Porter A, White G, Newman DT, Marti-Marti K, Shaw D, Mescallado N, Clamp I, Jackson A, Asselin MC, Dive C, Renehan Fairweather EE, Waszkowycz B, Jordan AM, Diamantopoulou Z, Waring T, Rooney C, A, Saunders MP, Valle JW, Mullamitha S, Braun A, Faivre-Finn C, Blackhall F. (2018) Smith DL, Somervaille TCP. (2018) Strathdee D, Marston DJ, Hahn KM, Sansom M, Hasan J, McEntee D, Simpson K, Little RA, Cell death, inflammation, tumor burden, and Enhancer Activation by Pharmacologic OJ, Zech T, Malliri A. (2018) Watson Y, Cheung S, Roberts C, Ashcroft L, proliferation blood biomarkers predict lung Displacement of LSD1 from GFI1 Induces STEF/TIAM2-mediated Rac1 activity at the Manoharan P, Scherer SJ, Del Puerto O, cancer radiotherapy response and correlate Differentiation in Acute Myeloid Leukemia. Cell nuclear envelope regulates the perinuclear with tumor volume and proliferation imaging. Reports, 22(13):3641-3659. actin cap. Nature Communications,9(1):2124. Jackson A, O'Connor JPB, Parker GJM, Dive C. (2018) Clinical Lung Cancer, 19(3):239-248.e7. Other publications Plasma Tie2 is a tumor vascular response Gogola E, Duarte AA, de Ruiter JR, Wiegant biomarker for VEGF inhibitors in metastatic Germano G, Mauri G, Siravegna G, Dive C, WW, Schmid JA, de Bruijn R, James DI, Payapilly A, Malliri A. (2018) colorectal cancer. Nature Communications, Pierce J, Di Nicolantonio F, D'Incalci M, Guerrero Llobet S, Vis DJ, Annunziato S, van Compartmentalisation of RAC1 signalling. 9(1):4672. Bardelli A, Siena S, Sartore-Bianchi A. (2018) den Broek B, Barazas M, Kersbergen A, van de Current Opinion in Cell Biology, 54:50-56. Parallel Evaluation of Circulating Tumor DNA Ven M, Tarsounas M, Ogilvie DJ, van Vugt M, and Circulating Tumor Cells in Metastatic Wessels LFA, Bartkova J, Gromova I, Andújar- Paoletti C, Schiavon G, Dolce EM, Darga EP, Colorectal Cancer. Clinical Colorectal Cancer, Sánchez M, Bartek J, Lopes M, van Attikum H, Clinical and Experimental Carr TH, Geradts J, Hoch M, Klinowska T, 17(1):80-83. Borst P, Jonkers J, Rottenberg S. (2018) Lindemann J, Marshall G, Morgan S, Patel P, Pharmacology (page 22) Rowlands V, Sathiyayogan N, Aung K, Selective Loss of PARG Restores PARylation Caroline Dive Hamilton E, Patel M, Armstrong A, Jhaveri K, Other publications and Counteracts PARP Inhibitor-Mediated Im SA, Iqbal N, Butt F, Dive C, Harrington EA, Conway AM, Mitchell C, Kilgour E, Brady G, Synthetic Lethality. Cancer Cell, 33(6):1078- Refereed research publications Barrett JC, Baird R, Hayes DF. Dive C, Cook N. 1093. Kim CS, Mohan S, Ayub M, Rothwell DG, Circulating Biomarkers and Resistance to Dive C, Brady G, Miller C. Endocrine Therapy in Metastatic Breast 62 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH PUBLICATIONS 63


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    RESEARCH PUBLICATIONS (CONTINUED) Image of focal expression of Other publications Torres-Ayuso P, Sahoo S, Ashton G, An E, the progenitor marker LY6D Simeoni F, Somervaille TCP. (2018) Simms N, Galvin M, Leong HS, Frese KK, (magenta) in the human Revert the SIRT: Normalizing SIRT1 Activity in Simpson K, Cook N, Hughes A, Miller CJ, prostate. Myelodysplastic Stem Cells. Cell Stem Cell, Marais R, Dive C, Krebs MG, Brognard J. (2018) Image supplied by Ivana Steiner 23(3):315-317. Signaling pathway screening platforms are an (Prostate Oncobiology) efficient approach to identify therapeutic targets in cancers that lack known driver Molecular Oncology (page 32) mutations: a case report for a cancer of Richard Marais unknown primary origin. NPJ Genomic Medicine, 3:15. Refereed research publications Trucco LD, Mundra PA, Hogan K, Garcia- Valpione S, Gremel G, Mundra P, Middlehurst Martinez P, Viros A, Mandal AK, Macagno N, P, Galvani E, Girotti MR, Lee RJ, Garner G, Gaudy-Marqueste C, Allan D, Baenke F, Cook Dhomen N, Lorigan PC, Marais R. (2018) M, McManus C, Sanchez-Laorden B, Dhomen Plasma total cell-free DNA (cfDNA) is a N, Marais R. surrogate biomarker for tumour burden and a Ultraviolet radiation-induced DNA damage is prognostic biomarker for survival in metastatic prognostic for outcome in melanoma. Nature melanoma patients. European Journal of Medicine [Epub 3 December 2018] Cancer, 88:1-9. Smith MP, Rana S, Ferguson J, Rowling EJ, Other publications Flaherty KT, Wargo JA, Marais R, Wellbrock C. Yates LR, Seoane J, Le Tourneau C, Siu LL, A PAX3/BRN2 rheostat controls the dynamics Marais R, Michiels S, Soria JC, Campbell P, of BRAF mediated MITF regulation in MITFhigh Normanno N, Scarpa A, Reis-Filho JS, Rodon /AXLlow melanoma. Pigment Cell and J, Swanton C, Andre F. (2018) Melanoma Research [Epub 1 October 2018] The European Society for Medical Oncology (ESMO) Precision Medicine Glossary. Annals Head and Neck Maes T, Mascaró C, Tirapu I, Estiarte A, of Oncology, 29(1):30-35. Ciceri F, Lunardi S, Guibourt N, Perdones A, Parry MA, Srivastava S, Ali A, Antonello J, Cancer Biology (page 28) Lufino MMP, Somervaille TCP, Wiseman DH, Cannistraci A, Leong HS, Barros-Silva J, Robert Metcalf Duy C, Melnick A, Willekens C, Ortega A, Ubertini V, Ramani V, Lau M, Shank J, Nonak Refereed research publications Martinell M, Valls N, Kurz G, Fyfe M, Castro- D, Oliveira P, Hambrock T, Dhomen N, Miller Prostate Oncobiology (page 34) Palomino JC, Buesa C. (2018) C, Brady G, Dive C, Clarke N, Marais R, Esther Baena Rack S, Rahman R, Carter L, McKay C, ORY-1001, a Potent and Selective Covalent Baena E. Metcalf R. KDM1A Inhibitor, for the Treatment of Acute Genomic evaluation of multiparametric Refereed research publications Impact of tumour profiling on clinical trials in Leukemia. Cancer Cell, 33(3):495-511.e12 magnetic resonance imaging-visible and Parry MA, Srivastava S, Ali A, Antonello J, salivary gland cancer. Clinical Otolaryngology -nonvisible lesions in clinically localised Cannistraci A, Leong HS, Barros-Silva J, [Epub 13 August 2018] Somerville TDD, Simeoni F, Chadwick JA, prostate cancer. European Urology Oncology Ubertini V, Ramani V, Lau M, Shank J, Nonak Williams EL, Spencer GJ, Boros K, Wirth C, [Epub 4 September 2018] D, Oliveira P, Hambrock T, Dhomen N, Tholouli E, Byers RJ, Somervaille TCP. (2018) Miller C, Brady G, Dive C, Clarke N, Marais Leukaemia Biology (page 30) Derepression of the Iroquois Homeodomain Kugel CH 3rd, Douglass SM, Webster MR, Kaur R, Baena E. Tim Somervaille Transcription Factor Gene IRX3 Confers A, Liu Q, Yin X, Weiss SA, Darvishian F, Al-Rohil Genomic evaluation of multiparametric Differentiation Block in Acute Leukemia. Cell RN, Ndoye A, Behera R, Alicea GM, Ecker BL, magnetic resonance imaging-visible and Refereed research publications Reports, 22(3):638-652. Fane M, Allegrezza MJ, Svoronos N, Kumar V, -nonvisible lesions in clinically localised Maiques-Diaz A, Lynch JT, Spencer GJ, Wang DY, Somasundaram R, Hu-Lieskovan S, prostate cancer. European Urology Oncology Somervaille TCP. (2018) Paredes R, Schneider M, Stevens A, White DJ, Ozgun A, Herlyn M, Conejo-Garcia JR, [Epub 4 September 2018] LSD1 inhibitors disrupt the GFI1 transcription Williamson AJK, Muter J, Pearson S, Kelly JR, Gabrilovich D, Stone EL, Nowicki TS, Sosman repressor complex. Molecular and Cellular Connors K, Wiseman DH, Chadwick JA, Löffler J, Rai R, Carlino MS, Long GV, Marais R, Ribas Barros-Silva JD, Linn DE, Steiner I, Guo G, Ali Oncology, 5(4):e1481813. H, Teng HY, Lovell S, Unwin R, van de Vrugt HJ, A, Eroglu Z, Davies MA, Schilling B, A, Pakula H, Ashton G, Peset I, Brown M, Smith H, Kustikova O, Schambach A, Schadendorf D, Xu W, Amaravadi RK, Menzies Clarke NW, Bronson RT, Yuan GC, Orkin SH, Li Maiques-Diaz A, Spencer GJ, Lynch JT, Ciceri Somervaille TCP, Pierce A, Whetton AD, Meyer AM, McQuade JL, Johnson DB, Osman I, Z, Baena E. (2018) F, Williams EL, Amaral FMR, Wiseman DH, S. (2018) Weeraratna AT. (2018) Single-cell analysis identifies LY6D as a marker Harris WJ, Li Y, Sahoo S, Hitchin JR, Mould DP, EVI1 carboxy-terminal phosphorylation is Age Correlates with Response to Anti-PD1, linking castration-resistant prostate luminal Fairweather EE, Waszkowycz B, Jordan AM, ATM-mediated and sustains transcriptional Reflecting Age-Related Differences in cells to prostate progenitors and cancer. Cell Smith DL, Somervaille TCP. (2018) modulation and self-renewal via enhanced Intratumoral Effector and Regulatory T-Cell Reports, 25(12):3504-3518 Enhancer Activation by Pharmacologic CtBP1 association. Nucleic Acids Research, Populations. Clinical Cancer Research, Displacement of LSD1 from GFI1 Induces 46(15):7662-7674. 24(21):5347-5356. Wang P, Dreger M, Madrazo E, Williams CJ, Differentiation in Acute Myeloid Leukemia. Samaniego R, Hodson NW, Monroy F, Baena Cell Reports, 22(13):3641-3659. 64 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH PUBLICATIONS 65


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    RESEARCH PUBLICATIONS (CONTINUED) E, Sánchez-Mateos P, Hurlstone A, Redondo- mini-drivers associated with relapse after Microtubules (orange) give Muñoz J. (2018) surgery in lung adenocarcinoma. Science structure to cells and form the basis of the mitotic spindle WDR5 modulates cell motility and morphology Reports, 8(1):14830. in dividing cells (centre cell), and controls nuclear changes induced by a 3D which help segregate environment. Proc Natl Acad Sci U S A, Torres-Ayuso P, Sahoo S, Ashton G, An E, chromosomes (cyan) into 115(34):8581-8586. Simms N, Galvin M, Leong HS, Frese KK, two new daughter cells. Simpson K, Cook N, Hughes A, Miller CJ, Protein EB1 (magenta) is seen at the growing tips of the Marais R, Dive C, Krebs MG, Brognard J. (2018) microtubules. In the bottom RNA Biology (page 36) Signaling pathway screening platforms are an right, a dense patch of Crispin Miller efficient approach to identify therapeutic microtubules marks the targets in cancers that lack known driver centrosome location in a cell mutations: a case report for a cancer of at an earlier phase of division. Refereed research publications Kim CS, Mohan S, Ayub M, Rothwell DG, Dive unknown primary origin. NPJ Genomic Image supplied by Andrew C, Brady G, Miller C. Medicine, 3:15. Porter (Cell Signalling) In silico error correction improves cfDNA mutation calling. Bioinformatics [Epub 6 Lallo A, Frese KK, Morrow CJ, Sloane R, Gulati December 2018] S, Schenk MW, Trapani F, Simms N, Galvin M, Brown S, Hodgkinson CL, Priest L, Hughes A, Lallo A, Gulati S, Schenk MW, Khandelwal G, Lai Z, Cadogan E, Khandelwal G, Simpson KL, Berglund UW, Pateras IS, Chester CPE, Pham Miller C, Blackhall F, O'Connor MJ, Dive C. TM, Kalderen C, Frese KK, Gorgoulis VG, Miller (2018) C, Blackhall F, Helleday T, Dive C. The combination of the PARP inhibitor olaparib Ex vivo culture of cells derived from circulating and the WEE1 inhibitor AZD1775 as a new tumour cell xenograft to support small cell lung therapeutic option for small cell lung cancer. cancer research and experimental Clinical Cancer Research, 24(20):5153-5164. therapeutics. British Journal of Pharmacology [Epub 14 November 2018] Skin Cancer Parry MA, Srivastava S, Ali A, Antonello J, and Ageing (page 38) Cannistraci A, Leong HS, Barros-Silva J, Amaya Virós Ubertini V, Ramani V, Lau M, Shank J, Nonak D, Berry P, Mueller D, Elder A, Bomken SN, Pal D, HOXB4 promotes hemogenic endothelium Oliveira P, Hambrock T, Dhomen N, Refereed research publications Allan JM, Veal GJ, Cockerill PN, Wichmann C, formation without perturbing endothelial cell Miller C, Brady G, Dive C, Clarke N, Marais Trucco LD, Mundra PA, Hogan K, Garcia- Vormoor J, Lacaud G, Bonifer C, Heidenreich development. Stem Cell Reports, 10(3):875- R, Baena E. Martinez P, Viros A, Mandal AK, Macagno N, O. (2018) 889. Genomic evaluation of multiparametric Gaudy-Marqueste C, Allan D, Baenke F, Cook The oncogenic transcription factor RUNX1/ magnetic resonance imaging-visible and M, McManus C, Sanchez-Laorden B, Dhomen ETO corrupts cell cycle regulation to drive Draper JE, Sroczynska P, Fadlullah MZH, Patel -nonvisible lesions in clinically localised N, Marais R. leukemic transformation. Cancer Cell, R, Newton G, Breitwieser W, Kouskoff V, prostate cancer. European Urology Oncology Ultraviolet radiation-induced DNA damage is 34(4):626-642. Lacaud G. (2018) [Epub 4 September 2018] prognostic for outcome in melanoma. Nature A novel prospective isolation of murine fetal Medicine [Epub 3 December 2018] Baron CS, Kester L, Klaus A, Boisset JC, liver progenitors to study in utero Khandelwal G, Miller C. (2018) Thambyrajah R, Yvernogeau L, Kouskoff V, hematopoietic defects. PLoS Genetics, Improved PDX and CDX Data Processing- Other publications Lacaud G, van Oudenaarden A, Robin C. 14(1):e1007127. Response. Molecular Cancer Research, Craig S, Earnshaw CH, Virós A. (2018) (2018) 16(11):1814. Ultraviolet light and melanoma. Journal of Single-cell transcriptomics reveal the dynamic Garcia-Alegria E, Menegatti S, Fadlullah MZH, Pathology, 244(5):578-585. of haematopoietic stem cell production in the Menendez P, Lacaud G, Kouskoff V. (2018) Hudson AM, Stephenson NL, Li C, Trotter E, aorta. Nature Communications, 9(1):2517. Early human hemogenic endothelium Fletcher AJ, Katona G, Bieniasz-Krzywiec P, generates primitive and definitive Howell M, Wirth C, Furney S, Miller CJ, Stem Cell Biology (page 40) Lie-A-Ling M, Marinopoulou E, Lilly AJ, hematopoiesis in vitro. Stem Cell Reports, Brognard J. (2018) Georges Lacaud Challinor M, Patel R, Lancrin C, Kouskoff V, 11(5):1061-1074. Truncation- and motif-based pan-cancer Lacaud G. (2018) analysis reveals tumor-suppressing kinases. Refereed research publications Regulation of RUNX1 dosage is crucial for Thambyrajah R, Fadlullah MZH, Proffitt M, Science Signaling, 11(526). Martinez-Soria N, McKenzie L, Draper J, efficient blood formation from hemogenic Patel R, Cowley SM, Kouskoff V, Lacaud G. Ptasinska A, Issa H, Potluri S, Blair HJ, Pickin A, endothelium. Development, 145(5). (2018) Bennett L, Howell M, Memon D, Smowton C, Isa A, Chin PS, Tirtakusuma R, Coleman D, HDAC1 and HDAC2 modulate TGF-β Signaling Zhou C, Miller CJ. (2018) Nakjang S, Assi S, Forster V, Reza M, Law E, Teichweyde N, Kasperidus L, Carotta S, during endothelial-to-hematopoietic Mutation pattern analysis reveals polygenic Kouskoff V, Lacaud G, Horn PA, Heinrichs S, transition. Stem Cell Reports, 10(4):1369-1383. Klump H. 66 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH PUBLICATIONS 67


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    RESEARCH PUBLICATIONS (CONTINUED) Transcriptional Networks in Select additional publications Gravells P, Neale J, Grant E, Nathubhai A, Olsen CM, Pandeya N, Thompson BS, Smith KM, James DI, Bryant HE. (2018) Dusingize JC, Subramaniam P, Nagle CM, Lung Cancer (page 44) Radiosensitization with an inhibitor of Green AC, Neale RE, Webb PM, Whiteman DC; Michela Garofalo Hadjidemetriou M, McAdam S, Garner G, Thackeray C, Knight D, Smith D, Al-Ahmady Z, poly(ADP-ribose) glycohydrolase: A QSkin Study. (2018) Mazza M, Rogan J, Clamp A, Kostarelos K. comparison with the PARP1/2/3 inhibitor Hormonal and reproductive factors and Refereed research publications olaparib. DNA Repair, 61:25-36. incidence of basal cell carcinoma and Shi L, Middleton J, Jeon YJ, Magee P, The human in vivo biomolecule corona onto PEGylated Liposomes: A proof-of-concept squamous cell carcinoma in a large, Veneziano D, Laganà A, Leong HS, Sahoo S, Little RA, Jamin Y, Boult JKR, Naish JH, prospective cohort. Journal of the American Fassan M, Booton R, Shah R, Crosbie PAJ, clinical study. Advanced Materials, [Epub 28 November 2018] Watson Y, Cheung S, Holliday KF, Lu H, Academy for Dermatology, 78(3):615-618. Garofalo M. (2018) McHugh DJ, Irlam J, West CML, Betts GN, KRAS induces lung tumorigenesis through Ashton G, Reynolds AR, Maddineni S, Clarke Olsen CM, Green AC. (2018) microRNAs modulation. Cell Death & Disease, Jordan AM. (2018) Artificial Intelligence in drug design-the storm NW, Parker GJM, Waterton JC, Robinson SP, Risk of invasive melanoma in patients with 9(2):219. O'Connor JPB. (2018) rheumatoid arthritis treated with biologics: an before the calm? ACS Med Chem Lett. 9(12):1150-1152 Mapping hypoxia in renal carcinoma with updated meta-analysis. Annals of the Other publications oxygen-enhanced MRI: comparison with Rheumatic Diseases, 77(8):e49. Paliouras AR, Monteverde T, Garofalo M. (2018) intrinsic susceptibility MRI and pathology. Oncogene-induced regulation of microRNA Lamarca A, Nonaka D, Breitwieser W, Ashton G, Barriuso J, McNamara MG, Moghadam S, Radiology, 288(3):739-747. expression: Implications for cancer initiation, progression and therapy. Cancer Letters, Rogan J, Mansoor W, Hubner RA, Clark C, Chakrabarty B, Valle JW. (2018) Olsen CM, Green AC, Pandeya N, 421:152-160. Whiteman DC. PD-L1 expression and presence of TILs in small intestinal neuroendocrine tumours. Trends in melanoma incidence rates in eight Oncotarget, 9(19):14922-14938. susceptible populations to 2015. Journal of Tumour Supressors (page 48) Investigative Dermatology [Epub 19 December Patricia Muller Callender JA, Yang Y, Lordén G, Stephenson 2018] NL, Jones AC, Brognard J, Newton AC. (2018) Refereed research publications Protein kinase Cα gain-of-function variant in Rueegg CS, Stenehjem JS, Egger M, Mackay HL, Moore D, Hall C, Birkbak NJ, Alzheimer's disease displays enhanced catalysis Ghiasvand R, Cho E, Lund E, Weiderpass E, Jamal-Hanjani M, Karim SA, Phatak VM, Piñon by a mechanism that evades down-regulation. Green AC, Veierød MB. L, Morton JP, Swanton C, Le Quesne J, Muller Proc Natl Acad Sci U S A, 115(24):E5497-E5505. Challenges in Assessing the Sunscreen- PAJ. (2018) Melanoma Association. International Journal Genomic instability in mutant p53 cancer cells of Cancer [Epub 16 November 2018] upon entotic engulfment. Nature Communications, 9(1):3070. Duffy DL, Zhu G, Li X, Sanna M, Iles MM, Jacobs LC, Evans DM, Yazar S, Beesley J, Law MH, Kraft P, Visconti A, Taylor JC, Lui F, Wright STEF-depleted cell showing MJ, Henders AK, Bowdler L, Glass D, Ikram nucleus (blue) with active Rac1 AM, Uitterlinden AG, Madden PA, Heath AC, (green) targeted to the nuclear membrane where it has Nelson EC, Green AC, Chanock S, Barrett JH, restored the cables of the actin Brown MA, Hayward NK, MacGregor S, Sturm cap (red). Other actin RA, Hewitt AW; Melanoma GWAS structures are shown in white. Consortium, Kayser M, Hunter DJ, Newton Bishop JA, Spector TD, Montgomery GW, Image supplied by Andrew Porter (Cell Signalling) Mackey DA, Smith GD, Nijsten TE, Bishop DT, Bataille V, Falchi M, Han J, Martin NG. (2018) Novel pleiotropic risk loci for melanoma and nevus density implicate multiple biological pathways. Nature Communications, 9(1):4774. Koh U, Janda M, Aitken JF, Duffy DL, Menzies S, Sturm RA, Schaider H, Betz-Stablein B, Prow T, Soyer HP, Green AC. 'Mind your Moles' study: protocol of a prospective cohort study of melanocytic naevi. BMJ Open, 8(9):e025857. 68 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH PUBLICATIONS 69


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    EXTERNAL SEMINAR Confocal microscopy image of a brain metastatic lesion in a murine model of SPEAKERS 2018 melanoma brain metastases. Melanoma cells (red) invading from leptomeninges into the brain parenchyma, surrounded by resident astrocytes (green). Cell nuclei are represented in blue. Image supplied by Denys The seminar series that we run is vital for the Institute, connecting Holovanchuk (Molecular Oncology) world-class researchers across the broad spectrum of cancer research. We have enjoyed another successful year for scientific interaction 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 speakers. Postdoctoral researchers and other scientists at the Institute also give weekly seminars which are very well attended and help to integrate the entire cancer research efforts of the Institute. Julia Newton-Bishop Violeta Serra Martine Jager Breast Cancer Now Seminars University of Leeds Vall d'Hebron Institute of Oncology Leiden University Daniel Rea Holger Bastians Jakob Nilsson Antony M. Carr Institute of Cancer and Genomic Sciences Goettingen Center for Molecular The Novo Nordisk Foundation Center for Genone Damage and Stability Centre, - University of Birmingham Biosciences (GZMB) Protein Research, University of Copenhagen University of Sussex Mohamed Bentires-Alj Dominique Bonnet Ian Collins Marianna Kruithof-de Julio University of Basel The Francis Crick Institute Cancer Research UK Cancer Therapeutics Unit University of Bern at The Institute of Cancer Research Christina Scheel Ingo Ringshausen Bertie Göttgens German Research Center for Environmental University of Cambridge Paul Huang University of Cambridge Health - Institute of Stem Cell Research The Institute of Cancer Research Stefan Fröhling Vladimir Kirkin Antonis Antoniou German Cancer Research Center Stefano Piccolo The Institute of Cancer Research University of Cambridge University of Padova Balca Mardin Serena Nik-Zainal BioMed X Innovation Center Eleonora Leucci University of Cambridge KU Leuven John Le Quesne Maggie Cheang University of Leicester Ken Lau The Institute of Cancer Research Vanderbilt University Ravid Straussman Phil Jones Weizmann Institute of Science Trudy G. Oliver University of Cambridge - Wellcome Trust Huntsman Cancer Institute Sanger Institute Dr Nicholas McGranahan UCL Dieter Saur Katherina Stankova Technical University of Munich (TUM) Maastricht University Andreas Trumpp German Cancer Research Center (DKFZ) Jonathan Brody John Poirier Thomas Jefferson University Memorial Sloan Kettering Cancer Centre Catrin Pritchard University of Leicester 70 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE EXTERNAL SEMINAR SPEAKERS 2018 71


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    www.cruk.manchester.ac.uk/education POSTGRADUATE EDUCATION The Cancer Research UK Manchester Institute offers a postgraduate co-located with us. The aim is to provide a work describing a novel approach in identifying forum for discussions and training related to two fibroblast populations in pancreatic degree (PhD) for students interested in a career involving cancer research, communication of scientific tumours. research. The Institute considers education of both research and engagement and development of social and clinician scientists to be a major investment in the future of cancer networking opportunities. Topics in 2018 have Cancer Research UK contributes towards an included their annual Science Showdown, exclusive annual International PhD Student research, and has an excellent track record of launching careers in patient-focused engagement event “STAy Cancer Conference (IPSCC) allowing high basic, translational and clinical research. Patient With Us” and various science calibre students (typically in their 2nd and 3rd Postgraduate communications. years) from top cancer research institutes Education Manager across Europe to organise and present at their As part of this commitment, we have an active fundamental to the majority of careers in The CRUK Manchester Institute Colloquium own scientific conference. Core participating Julie Edwards postgraduate programme that provides science and elsewhere. Graduate training is takes place annually in September, and is an institutes include London Research Institute students and clinical research fellows of monitored by the Education Committee, excellent opportunity for our new intake of (LRI), Cambridge Institute (CI), Beatson Institute outstanding potential the opportunity to study staffed by the Institute’s group leaders and students to meet other established PhD (BICR), Netherlands Cancer Institute (NKI), for a cancer-related PhD degree. This is student representatives (see below). A main students, members of the Institute, including European School of Molecular Medicine, Milan achieved through a training programme that supervisor and a second or co-supervisor are group leaders, postdoctoral fellows, and (SEMM, IFOM & IFEO), and the German Cancer aims to improve effectiveness in research, nominated for each student, who are able to scientific officers. This forum communicates up Research Centre (DKFZ). provide professional and management skills provide additional advice and consultation on to date science in the form of oral presentations and enhance career development. Our PhD both academic and non-academic matters. given by group leaders and second year PhD In 2018, the 12th IPSCC was organised by PhD students have exceptional employment Each student is also assigned an advisor (similar students, as well as poster presentations from a students from The Francis Crick Institute, prospects following graduation, with the great to a personal tutor on an undergraduate range of scientists across the Institute covering London and held in June. The Institute was Postgraduate Tutor majority (>95%) continuing in academia, programme) whose role is to provide impartial all aspects of cancer research. Poster prizes are represented by 12 of our PhD students in their industry or healthcare, and securing positions support and advice in a pastoral capacity. Angeliki Malliri in destinations across the UK, Europe and the Further support is also available individually awarded, including the Lizzy Hitchman Prize for 2nd and 3rd years, and the forum provided a the best poster presented by a PhD student or unique opportunity for the students to present USA. from the Director of Postgraduate Education, clinical fellow. In 2018, The Lizzy Hitchman their work and network with some of Europe’s Postgraduate Tutor, Postgraduate Manager, or student prize went to PhD Student Colin Hutton best cancer research institutes. The students In 2018, we welcomed nine graduate students collectively as the Education Committee from the Systems Oncology group for his PhD enjoyed a welcome talk from Professor Sir Paul and one clinical research fellow to our PhD Administration Group. programme, working in a variety of fields from translational oncogenomics, cell plasticity and The CRUK MI runs an external seminar series epigenetics, molecular oncology, skin cancer featuring talks from many of the key players in and ageing, prostate oncobiology, through to cancer research. The speakers are cell biology. It was also particularly gratifying to internationally renowned scientists and we Postgraduate Director see that, over the past twelve months, one of consider it essential that our students are and Chair of the Education our students has published two first author exposed to outstanding research from leaders Committee papers in Clinical Cancer Research and British in different disciplines, which will give them a Tim Somervaille Journal of Pharmacology. broad understanding of many aspects of cancer research and basic biology. In addition, The Cancer Research UK Manchester we hold a series of weekly postdoctoral Graduate Programme research seminars and attendance from PhD We aim for each student to receive high quality students is an integral part of the seminar training in scientific research through an programme. While students themselves are intellectually demanding but achievable asked to give talks at key points during their research programme. Each project is peer- PhD, they also have opportunities to present reviewed in advance and monitored their work at lab meetings and during student throughout its course through a mixture of oral forums within the Institute. STAy (short for presentations, written reports, and progress Science TakeAway) is a group run by junior meetings. These modes of assessment are scientists in the CRUK Manchester Institute. designed not only to provide formal points at Meetings are open to all early career scientists which progress (of both the student and the - PhD students, postdocs and scientific officers project) can be monitored, but also to help from the Institute and The University of develop the presentation skills which are so Manchester Division of Cancer Sciences 72 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE POSTGRADUATE EDUCATION 73


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    POSTGRADUATE EDUCATION (CONTINUED) OPERATIONS Nurse, and keynote lectures from Dr Patrick Our goal is for every student to have a project Vallance, Professor Fiona Watt, Professor Ester that is both achievable and intellectually Hammond and Professor Steve Jackson. stimulating and demanding. Projects and students are monitored by the Education The conference was attended by over 90 Committee which makes sure that the students in total with 18 talks and 74 posters proposed plan of research is suitable, and that scheduled over the two and a half days. It is progress is made consistently throughout the mandatory for participating student delegates course of the studentship. Various assessments The Operations’ team provides the necessary services that to submit a poster to showcase their research throughout the studentship, including regular either through a poster or oral presentation. talks, progress meetings and written reports, facilitate the running of the Institute. It is overseen by four Posters are scored and ranked by the student are vital to ensuring successful completion and operational managers. Caroline Wilkinson is the Chief Operating delegates during the poster sessions, with the graduation for the PhD degree. Such Officer with responsibility for scientific administration and top three posters receiving prizes. CRUK assessments help not only to monitor progress, Manchester Institute student Joe Maltas, from but also help to develop performance and communications. She also acts as the primary point of operational the Cell Signalling group, was awarded joint first presentations skills. contact within the Institute for both The University of Manchester poster prize for showcasing his work on “The Chief Operating Officer and Cancer Research UK. Rachel Powell is Head of the Human nuclear roles of the Rac activator tiam1 in Education Committee Members NSCLC”. Caroline Wilkinson Resources team which this year welcomed Laura Bayliff; Stuart Tim Somervaille Pepper is the Chief Laboratory Officer overseeing all aspects of PhD studentships Postgraduate Director and Chair of general laboratory management and also oversees IT and Health All of our CRUK core funded studentships are of Education Committee four years’ duration, and consist of an approved Angeliki Malliri and Safety. The fourth operational manager is Mike Berne who research project in one of our core funded Postgraduate Tutor started at the Institute in 2018 to head our Finance team following research groups. Some students have joint Richard Marais Margaret Lowe entering a phased retirement. Margaret has served supervisors in different groups, fostering Ex-Officio Member important collaborations and providing Julie Edwards the Institute with distinction for over 25 years and we wish her all the exposure to different disciplines. Recruitment is Postgraduate Manager best for the future when she leaves us in early 2019. Mike brings a highly competitive, with 300-500 applicants Chief Laboratory Officer wealth of experience from the finance team at The University of competing for around four to eight places each Claus Jørgensen Georges Lacaud Stuart Pepper Manchester’s Faculty of Biology, Medicine and Health and has year. Interviews are typically conducted annually over a two-day period in early January. Jonathan Tugwood settled well into Institute life. Caroline Wilkinson Our students benefit from access to advanced Wolfgang Breitwieser This year has seen some restructuring of the Team. The team grew to four members this state-of-the-art facilities, including advanced Operations’ team with Logistics reporting to year; we welcomed Jayne Fowler as Executive imaging, biological mass spectrometry, flow Student Representatives Colin Gleeson, and Neil Carne and Tony Assistant to the new Director of the Drug cytometry, histology and next generation Woollam from the Estates team seconded to Discovery Unit and Belen Conti was promoted sequencing. Our research groups offer PhD Denys Holovanchuk² Jakub Chudziak The University of Manchester and our Logistics to a new position as Executive Assistant to the studentships and projects covering the entire team respectively. Belen Conti was promoted Senior Management Team, while Delydd Jones breadth of research within the Institute Callum Hall¹ to the position of Executive Assistant to Stuart joined us as Admin Services Coordinator. The currently based over two sites at Alderley Park, Head of Finance 1 Joined in 2018 Pepper and Caroline Wilkinson, and Jayne extra support has been invaluable during this Cheshire and the Olgesby Cancer Research 2 Left in 2018 Mike Berne Fowler was recruited as Executive Assistant to extremely busy year. Building, Manchester (formerly the Manchester Caroline Springer (Director of the DDU). Delydd Cancer Research Centre Building). Jones joined the Institute to replace Belen in The team provides administrative support to the Director’s Office team. The year has been the Director and the Institute Faculty in addition Education Committee 2018 dominated by moving the Institute to Alderley to assisting with the Institute’s move to Alderley The Education Committee acts for Park and working with our operational Park and the organisation of several events over postgraduate students and consists of group counterparts at the site to ensure a smooth the course of the year, including the Paterson leaders, the Chief Operating Officer, the transition to our interim premises. The team has Fire Anniversary Event and Institute Colloquium. Postgraduate Tutor and the Postgraduate also been working hard on arrangements for the Belen also manages the Institute’s social media Education Manager from the CRUK Manchester redevelopment of the Paterson Building site. accounts. Institute. Head of Human Resources Delydd has taken over from Belen in organising Rachel Powell Institute Administration Team the external seminar series, which has Ruth Cox, Maria Belen Conti, Jayne Fowler1 , continued to be a great success in 2018. The Delydd Jones1 seminars serve to foster collaboration and 1 encourage interaction with the wider scientific Joined in 2018 community. We ensure that staff at both Alderley Park and OCRB are able to participate Ruth Cox is Executive Assistant to the Institute in the visits and view the seminars using a Director and manager of the Institute Admin 74 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE OPERATIONS 75


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    OPERATIONS (CONTINUED) video-link between sites. We aim to provide a Human Resources Information Technology varied programme of national and international Rachel Powell, Rachel Craven, Julie Jarratt, Steve Royle, Matthew Young, Hong Mach, Alongside rebuilding our IT infrastructure and speakers. Details can be found at www.cruk. Laura Jones, Emma Lloyd, Laura Bayliff¹, Brian Poole services afresh we are also now beginning to manchester.ac.uk/seminars. David Stanier³, Natalie Taylor² shape our return to our previous 'home' in The CRUK Manchester Institute IT team provides Withington based on The Christie NHS ¹ Joined in 2018 2 Left in 2018 ³ Joint with administration a full catalogue of IT services upon which our Foundation Trust site, in a new custom built Finance and Purchasing researchers and support staff alike now depend cancer research building to be shared by three Mike Berne¹, Margaret Lowe², David Jenkins, Over the past year, the HR department has for almost every aspect of their work. partners; CRUK MI, The University of Denise Owen, Muhammad Raja, Vikki Rosheski, continued to deliver a high quality proactive Manchester and The Christie. Debbie Trunkfield service to the Institute. The department Once again, 2018 was another year of ¹Joined in 2018 2 Phased retirement began in 2018 provides advice and guidance to managers and unprecedented change in IT. In readiness for Looking forward, the planning for our move staff on all employment-related matters such as the relocation and regrouping of the majority back will gather pace as the year progresses. It has very much been a year of transition for recruitment, policy guidance, employment of CRUK MI staff at Alderley Park (AP), Q1 2018 This will be an exciting time for all involved as the Institute Finance Team with the legislation and best practice. was primarily spent rebuilding our physical IT we start to design a cancer research facility for appointment of Mike Berne as the new Chief infrastructure on this new site. To this end, we the future. Finance Officer, replacing Margaret Lowe, who During 2018, we completed 126 recruitment have now implemented a dedicated CRUK MI began a phased retirement in July 2018. In rounds and successfully appointed 81 resilient wired and wireless network addition to this, there was also the transition individuals to enhance the work of the Institute. infrastructure across all CRUK MI research Safety and Facilities Management between working environments as the team This was compared to 105 recruitment rounds facilities at AP and re-established an enterprise- Colin Gleeson relocated to Alderley Park alongside a shift in in 2017; an increase of 20% in one year. Also, in class file storage facility for our research data. workloads as the team dealt with significant 2018 the department administered the This is based on a replicated design, hosted in Over the last year services related to a variety of increases in daily activity due to the successful promotion of 10 individuals. We two geographically separate datacentres, to functions across safety, logistics and laboratory management of fire-related expenditure. have continued our commitment to joint provide a resilient, high availability, redundant, support and these three elements have been The Institute Finance Team supports the partnership working with the Union, which has fit for purpose, storage facility. organised to sit together under a single Director with the management of the Institute's resulted in the revision of several HR policies maintenance facility that is managed by Colin £30m, which is devolved across the various and procedures and the renewal of the Q2 2018 and onwards was largely spent Gleeson. This puts safety at the heart of all Research Groups, Service Units and workforce agreement for scientists. There has supporting around 330 CRUK MI staff moving these vital support functions for the Institute, Operational Activities. This has been an area of also been a review of the salary scales for the into their new offices and labs at AP, whilst and also promotes efficient coordination of particular complexity this year, given the CRUK scientific and non-scientific pay and rebuilding our services to operate across activity between these teams. The relocation to expansive network as groups were originally grading framework. multiple sites, primarily Alderley Park and also Alderley Park placed many demands on these split across a number of distant sites, before the Oglesby Cancer Research Building. teams and it is a testament to the flexibility and finally managing to relocate, in the most part, to The Institute is committed to working towards Facilitating service provision across multiple hard work of everyone listed here that by the Alderley Park. the Athena Swan accreditation and this will be a sites has required careful planning and more end of summer 2018 the Institute was running priority over the next 12 months. We have widespread adoption of new services to efficiently. This section details some of the The team continues to support the research continued to provide support to our EU staff support remote working. For example, we have work that has taken place to allow the groups by providing effective and efficient during the uncertain time as the UK prepares to real-time multipoint network monitoring to relocation to be completed. professional advice when costing new research leave the European Union. Therefore, we were rapidly identify the source of any outages. proposals and contracts, while also providing pleased to announce to EU staff that The We also now make greater use of automated As we look ahead to the next couple of years guidance relating to any purchasing and University of Manchester was part of a new pilot deployment tools to deploy new client there will be an important role to play in logistical changes as a result of our new location. scheme from the Home Office which allows computers. In addition, our adoption of contributing to the new building, to ensure that staff to apply to continue to live and work in the 'self-service' application installation now the critical infrastructure necessary to run a While the fire created additional operations the UK after 2021. In addition, we have reimbursed enables research staff to resolve a significant research building can operate efficiently. Institute still receives funding from many staff the fees for the EU Settlement Scheme. number of IT Service Requests themselves. different sources and we have the on-going Health and Safety responsibility to ensure these funds are used in Next year, the focus will be on the Athena Swan The process of relocation and rebuilding has Colin Gleeson the intended manner following both the accreditation, recruitment of new research enabled us to work more closely with The financial regulations of the University as well as groups in line with the Institute’s strategy and University of Manchester IT Services and other The relocation of CRUK MI to Alderley Park was the Terms and Conditions of the funders. the implementation of a new candidate CRUK MI service groups and take advantage of completed in 2018. This demanded a wide We monitor all individual awards and provide management system to support the new synergies. In particular, we have formed range of activities, including laboratory the feedback required to the funders and the application process and enhance the closer working relationships with the Scientific re-design projects for some areas to guarantee Principle Investigators. candidate experience. Computing group and the Advanced Imaging the space was appropriate for our research group. This has enabled researchers to fast needs; operational meetings with our landlords track implementation of new technologies to to ensure the services they provide would be facilitate their research projects. sufficient for our needs; and major equipment rebuild and relocation to maintain a 76 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE OPERATIONS 77


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    OPERATIONS (CONTINUED) comprehensive range of research facilities and Logistics team, they continue to deliver office rearrangements. In April the team Scientific Operations and General available for our research. All of these activities items from OCRB to AP via the daily shuttle van successfully relocated all the Operations teams Administration required a considerable input from a health and service. from OCRB to AP. Caroline Wilkinson, Tom Bolton¹, Gillian safety point of view over a long and busy timeline. Campbell, Julie Edwards, Steve Morgan, In 2018 the team relocated the media service We now have a fully operational Logistic team Simon Poucher, David Stanier2 Concomitantly routine health and safety and now manufacture liquid media and agar at AP and they work closely with the various plates from the OCRB and deliver to the research ¹ Joint with MCRC ² Joint with HR activities continued, which included onsite Logistics and waste teams. inspections of facilities; reviewing risk groups across OCRB and AP. Scientific administration is overseen by assessments, including numerous new genetic The team has continued to deliver an efficient The Lab Services department also continues to Caroline Wilkinson who is the Chief Operating modification assessments; a review of our and effective service providing support for the support the research groups in other ways: Officer for the Institute and acts as the main current assessments for transgenics, creating research carried out at AP and OCRB. This • maintenance and servicing of the point of contact for both The University of new DSEAR assessments for our laboratories; includes the receipting, checking, booking in photographic dark rooms at each site; Manchester and Cancer Research UK. The arranging statutory inspections of equipment; and distribution of goods ordered by staff. We • providing a drop in monthly pipette clinic at team moved to Alderley Park in early 2018 and investigating incidents and non-compliance also facilitate the delivery of dry ice, liquid both sites; have helped with arrangements for operations issues and the development of working • organising the delivery of clean general and nitrogen and gas cylinders. at the Institute’s new location. The team is also relationships and liaising with health and safety tissue culture lab coats. responsible for communications and in 2018 colleagues at the AP site and University main Researchers can order central stores stock launched a new external website for the campus. We also greatly improved our Additionally, in consultation with the Health and items via the intranet, which can be collected Institute, which was produced by web first-aider provision with ten people gaining the Safety Manager, Mark Craven has maintained personally or distributed by the Logistics team. developer Tom Bolton with content overseen full 3-day first-aid at work qualification and appropriate first aid supplies across the sites. He Included in this system are the enzymes and by Gill Campbell. Gill is the Institute’s Grants another 6 gaining the 1-day emergency first-aid also supports the Chief Laboratory Officer and media stored in the Institute freezers at the Advisor who helps our scientists apply for qualification. coordinates servicing of shared equipment such OCRB (Sigma, Life tech, Promega, New external funding to extend the breadth of as microbiological safety cabinets and lab water England Bio labs, and Qiagen). A list of stocked research that we conduct. She works closely Further, in the latter half of the year, some final systems. Alongside the Facilities Manager, he items can be found on the intranet. We with the Institute’s Grants Committee, chaired close-down work for the old Paterson Building reports and resolves lab-based faults at AP. Mark continued to make savings by buying in bulk has coordinated the removal of CRUK MI by Iain Hagan, who undertake a review of all was completed. This involved the partial from our suppliers. laboratory waste ensuring compliance with prospective applications and also help prepare surrender of our authorisation for radioactive local rules. applicants for interviews associated with work within the Paterson Building, whilst at Over the past year the team has offered a fellowships or awards. Tom has adapted the the same time retaining a small laboratory for sample transfer service from OCRB to AP, with online PhD recruitment portal that he radioactive work within The Christie NHS daily transfers in the morning and afternoon; a produced in 2016 to create an online Foundation Trust site. We also embarked on Logistics daily afternoon service from AP to OCRB; and Andrew Lloyd, Michael Alcock, Edward Fitzroy, application system for staff recruitment, which the development of an application for depending on demand, a service to other Sedia Fofana, Stephen Keane, Jonathan Lloyd, will be launched in 2019. radioactive work at the Alderley Park site. In University of Manchester buildings. 2019, we look forward to contributing to the Robin Sherratt, William Glover, Nigel Fletcher, Tony Woollam Julie Edwards is the Postgraduate Education preparatory and planning work for the new Manager and has helped our students with the building on the old site. Electronics The past year has seen changes within the transition to Alderley Park and in particular with Yunis Al-hassan, Steve Powell, Tony Woollam team’s structure. At the beginning of the year, arranging extensions for those whose PhDs Michael Alcock was appointed Logistics were interrupted by the fire at the Paterson Laboratory Services As part of the Institute’s electrical and fire safety Supervisor and in the summer, Andy Lloyd was Building in 2017. Early on in 2018, we held our Mark Craven, Tony Dawson, Corinne Hand, strategies, this year the Electronics team have Petra Kubinova, Adriana Tudelo¹, Christine promoted to the role of Facilities Manager. PhD student recruitment day at Alderley Park PAT tested well over a thousand pieces of Whitehurst for the first time, which was organised by Julie electrical equipment across multiple sites. The It has been a challenging year for the Logistics and ably supported by David and Gill. David 1 Left in 2018 work included PAT testing our newly acquired team. With the additional support from Nigel Stanier is also responsible for general equipment. Additionally, the Institute electrical Fletcher (BRU) and Tony Woollam (Estates), we administration and oversees the Institute’s During 2018, Lab Services continued to support engineer has repaired numerous items of the various research buildings with a main base have managed to support both Olgesby transport arrangements between Alderley Park scientific research equipment. In some cases, of operations at the Oglesby Cancer Research Cancer Research Building and Alderley Park and the Oglesby Cancer Research Building. repairs are carried out at electronic component Building containing the glass washers and sites with our usual day to day service, and This latter site is home to two of our research level. This repair facility provides a significant autoclaves. From this base they supply clean, supported sample movement by introducing teams who have remained close to the Christie economic benefit to the Institute, in that sterile glass and plastics for the research groups a transport service. Hospital site for operational reasons. David is unnecessary expenditure on replacement based at OCRB and provide sterile plastics to the also the Institute’s Information Governance equipment can be avoided. The Institute groups at Alderley Park. At the start of the year the team worked Co-ordinator supporting Caroline Wilkinson as electrical engineer also tracks Institute extremely hard behind the scenes preparing for the Institute’s Information Governance equipment that are under warranty, service At AP the department continue to collaborate the relocation to AP. The team was tasked with Guardian. These responsibilities are required by contract or in-house repair. Again, this provides with the onsite support, facilities and catering setting up the office spaces, which involved The University of Manchester and interact with a significant economic benefit to the Institute. teams, and at OCRB deliver the service directly the University’s Information Governance Office large numbers of furniture deliveries and many to the lab groups. In partnership with the porters 78 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE OPERATIONS 79


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    OPERATIONS (CONTINUED) to help ensure that we operate to the Project Licences were reviewed by the AWERB. evening in a Manchester pub and at an event at We hope this will build deeper and more appropriate legislative requirements. Significant input is provided to the applicants by Manchester’s Science and Industry Museum, as strategic relationships with our funded centres, the Home Office Liaison Coordinator (HOLC), well as Institute Open Days; a workshop run by institutes and universities, as well as improving Caroline Wilkinson oversees the Research Named Veterinary Surgeon (NVS) and Named the Understanding Animal Research internal information flow and collaboration. Integrity Committee, which reviews all Animal Care and Welfare Officer (NACWO) at organisation in May helped our volunteers to manuscripts before final submission for the draft stage but review by the wider AWERB communicate comfortably with members of By arrangement with The University of publication to ensure that our research is membership contributes to further the public at these events. Manchester, CRUK owns and is responsible for conducted in accordance with the highest improvements. Licences have incorporated a the development and commercialisation of standards of integrity. This year, Simon Poucher number of techniques new to CRUK MI this intellectual property arising from CRUK-funded has taken on a role with the committee in order year (including a model of ocular melanoma, Cancer Research UK Commercial research at The University of Manchester. To to check that any in vivo research is reported in orthotopic brain tumours, and bladder Partnerships effectively facilitate this, Martyn Bottomley, a accordance with the NC3R’s ARRIVE catheterisation) and the AWERB has required Martyn Bottomley CRUK CP Translation Lead is based within guidelines. evidence of how licensees can develop Manchester and is currently hot-desking at the expertise. The NVS provides invaluable advice Cancer Research UK Commercial Partnerships Oglesby Cancer Research Building, UMIP and Steve Morgan continues in his role at the on all matters, including this year improved (CP) Team (formerly Cancer Research Alderley Park to work closely with the staff Oglesby Cancer Research Building where he surgical practice, scoring of skin ulceration Technology (CRT)) is a specialist oncology- funded by CRUK at The University of works closely with staff from the University’s effects and recommendations for best focused development and commercialisation Manchester. Martyn offers access to oncology- Faculty of Biology, Medicine and Health to anaesthetic equipment and stereotaxic rigs. A team which is part of Cancer Research UK's focused expertise in technology evaluations, ensure that reception runs smoothly and to total of 27,018 mice were used at the Institute in Research and Innovation Directorate. The CP patent applications and management, funding operate the Institute’s switchboard. regulated procedures under the Act in 2018. Team aims to maximise patient benefit from for development, commercialisation, drug CRUK-funded research worldwide by discovery, market intelligence, and project Licensees are required by law to report any advancing research discoveries into management. He also works closely with UMIP, Animal Welfare unforeseen adverse effects on animals or development with pharmaceutical and The University of Manchester technology Simon Poucher, Regulatory Liaison and breaches of the controls and limits in their biotechnology parties. We aim to bridge the transfer organisation. Training Officer, Janet Watson, Animal Welfare licence. Six incidents were self-reported to the gap between cutting-edge academic research and Ethical Review Body (AWERB) Chair, Home Office in the year, including unexpected and industrial development of cancer Martyn continues to work very closely with the Caroline Wilkinson, Establishment Licence reactions to treatments or unexpected tumour therapeutics and diagnostics. We achieve this Drug Discovery Unit (DDU) based at Alderley Holder, Stuart Pepper, (Deputy AWERB Chair) growth patterns; all of which were satisfactorily by working closely with prestigious Park to facilitate the development of drug resolved with the inspector. The AWERB international research institutes, such as the therapies to satisfy the unmet clinical needs of The Institute upholds the highest standards of reviews these reports and uses these to share Cancer Research UK Manchester Institute, and cancer patients. Martyn continues to be welfare for the laboratory mice used in our learning across licensees. funding bodies to develop, protect and involved with the management of research. All animal research activities are commercialise oncology-related discoveries. collaborations with Pharmaceutical partners conducted in full compliance with the Animals By the start of 2018, experimental work was such as Basilea and AstraZeneca, and also the (Scientific Procedures) Act 1986 (ASPA) and are fully established at Alderley Park under a mutual From April 2018, the CP Team has undergone a filing and management of a number of patent scrutinised by the Institute’s Animal Welfare and agreement with AstraZeneca to share their reorganisation from a “cradle to grave” model applications for the Drug Discovery Unit to Ethics Review Body (AWERB). This consists of facility and CRUK MI subsequently exited into functionally distinct sub-teams in order to protect novel compounds resulting from their experienced animal husbandry staff, a operations at the University’s Stopford building. provide greater strength, depth and research. During 2018, Martyn also completed veterinary surgeon, Institute scientists, a The CRUK MI AWERB has continued to interact accountability in our core activities supporting the license of the DNMT1 drug discovery statistician and lay members. The AWERB with other establishments through the NW translation and commercialisation, as well as program from the Manchester DDU to GSK. supports all staff involved with animal research, AWERB Hub; the Establishment Licence Holder providing clearer and more streamlined ensuring the provision of appropriate (ELH) and HOLC attend regular meetings with interfaces with other teams across Research Commercial Partnerships is also currently management structures and processes, staff the Home Office Animals in Science Regulation and Innovation with whom we collaborate to actively managing a broad portfolio of training, the facilities for the care and use of Unit (ASRU) and the ELH sits on the national achieve our joint goals of progressing CRUK development programmes and exciting mice, and encouraging implementation of the ELH forum as well as helping to train new science. licensing opportunities originating from the 3Rs’ principles (replacement, reduction and Establishment Licence Holders. Starting this Cancer Research UK Manchester Institute that refinement of animals). It also reviews the ethics year, the AWERBs of CRUK MI, AstraZeneca and The new structure comprises four core teams: continue to attract commercial partners. We of proposed collaborations and all grant Agenda Life Sciences, all based on the Alderley Opportunity Sourcing & Translation (OST); look forward to building on our successes and applications involving animal research. Park site, have held joint meetings to share Business Development & Transactions; continuing to work closely with the Cancer ideas, presentations and training opportunities Partnerships & Strategic Alliances; and Business Research UK funded researchers in Manchester In 2018, our AWERB met formally on seven - in October, the three held a joint 3Rs’ Operations. Notably, our interface with the under the new CP structure to advance occasions with two additional meetings poster event. academic community through OST will now discoveries to beat cancer in the years ahead. involving all licensees at the Institute. These two have a geographical focus, with four regions meetings were also attended by our Home Fulfilling our commitment under the each having an expanded team of Translation Office Inspector. In the year, six applications to Concordat on Openness on Animals Research, Managers and Executives under a Regional the Home Office for new Project Licences and CRUK MI staff have talked about our research Translational Lead and OST Associate Director. 11 applications for amendments to existing with mice to the public at a ‘Pint of Science’ 80 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE OPERATIONS 81


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    Staff from CRUK Corporate Partner, Flybe visit the MBCF labs in the Oglesby Cancer Research Building. CANCER RESEARCH UK’S RESEARCH ENGAGEMENT Cancer Researcher UK’s Research Engagement Team brings CRUK-funded research to life for its supporters, the public and its staff, working regionally with researchers to develop face-to-face engagement opportunities. The team creates compelling engagement content for local supporters and the public to drive activities to over 400 members of the public at researchers from the Manchester Institute interaction with life-saving research happening near them. Images top left to right. the Science and Industry Museum’s Pi: Platform captained by Steve Lyons. As well as organising a CRUK’s Events Interns meet MI for Investigation. Demonstrations included DNA host of pre-event fundraising and taking part in Research Engagement Manager PhDs in the Oglesby Cancer mutation and flow cytometry games, clinical the 24-hour walk event, the team hosted a Tim Hudson Almost 7,000 people interacted with the work of The Charity Champions enjoyed a talk by Claus Research Building to get trials activities and a special animal welfare research stall, talking to participants about their the Manchester Institute during 2018, at events in Jørgensen before meeting Lizzy Hogg in the hands-on with our research. station, hosted by the BRU team and others. work and getting hands-on with some our own labs or externally at fundraising events labs and learning about CyTOF with Steve Bagley. The University of Manchester’s strawberry DNA extraction. and science festivals. The Warburton family enjoyed speaking with Science Spectacular, part of the Further collaboration took place during the Caroline Dive, whose lab they have allocated Manchester Science Festival. Manchester Science Festival in September, when Our researchers engaged with national and During 16 visits to Institute labs at Alderley Park fundraising to for the coming year, and were MI staff teamed up with researchers from the international audiences too; PhD student, Denys and the Oglesby Cancer Research Building, 160 treated to a special visit to the CEP group’s Good Images bottom. Division of Cancer Sciences, the Biomedical Holovanchuk delivered a talk to school students donors, fundraisers, volunteers, corporate Clinical Practice lab. Institute scientists meet the public at the Science & Industry Research Council and The Christie NHS in Portugal, whilst Adele Green featured in a partners and CRUK staff had the opportunity to Museum’s Platform for Foundation Trust at The University of video exhibit for the Science Museum’s gain a close-up view of the research their Our scientists also took their work out to the Investigation. Manchester’s Science Spectacular, to engage acclaimed exhibition, The Sun: Living With Our support helps to fund. community at 25 external events. over 200 members of the public with a range of Star. research themes – prevention, early detection, A highlight came in the autumn when we In the spring, Institute scientists teamed-up with treatments and targeted therapies. Artistic collaborations resulted in works exhibited welcomed both the Warburton family and the Research Nurses from The Christie NHS in prestigious settings. Manchester Metropolitan company’s Charity Champions to Alderley Park, Foundation Trust, other University of Manchester The 13th Relay for Life Stockport took place in the University student, Alice Thickett visited Steve celebrating a four-year partnership which has research groups and others representing the height of the summer, featuring the team of Lyons and Steve Bagley in the Oglesby Cancer resulted in £1.5million towards research funding. CRUK Manchester Centre to deliver engagement 82 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE CANCER RESEARCH UK'S RESEARCH ENGAGEMENT 83


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    CANCER RESEARCH UK’S RESEARCH ENGAGEMENT Artist Alice Thickett with the piece she created following her work with our researchers. Alicia Marie-Conway was one of the MI scientists who joined with partners from The Christie and The University of Manchester to sign the Research Building and developed a piece titled joined signatures from over 80 physicists, STEM Bee. Cancer Research: Written in the Stars. The textile biologists, chemists, mathematicians and print features cell imagery from the labs, engineers in the city. After the trail ended the provided by Division of Cancer Sciences PhD bees were auctioned to raise funds for The Lord student Daiana Drehmer, and was exhibited as Mayor of Manchester’s charity – We Love MCR, part of the Pint of Science and Manchester to improve the lives and life chances of Science Festivals this year. Manchester people, with Kelly’s STEM Bee selling for an incredible £20,000. On a larger scale, Institute scientists and staff featured on one of 80 giant bee sculptures Huge thanks go to all the volunteer group which went on display in Manchester city centre, leaders, researchers, scientists and staff who as part of Bee In The City. Scientists were invited donate their time, energy and enthusiasm to to add their signatures to artist Kelly Stanford’s support our engagement activities. STEM Bee. The 12 cancer research autographs Images left to right. Relay for Life Stockport’s Researcher Team at this year’s event. Artist Kelly Stanford’s STEM Bee, displayed outside Manchester’s Oxford Road Rail Station as part of Bee in the City. Members of the Warburton family visit the CEP labs with Caroline Dive 84 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE CANCER RESEARCH UK’S RESEARCH ENGAGEMENT 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 2018 was The Cancer Research UK Manchester Institute has a strong £30m. The major source of this funding was awarded by Cancer programme of basic and translational research. There are close Research UK via a core grant of £13.3m plus additional strategic links with clinical and translational research groups throughout funding of £5.9m. This funding enables the various scientific groups the Christie Hospital site. and service units within the Institute to carry out their research. The Institute offers excellent laboratory facilities In addition to postgraduate and postdoctoral The infrastructure of the CRUK Manchester • European Organisation for Cancer Research and outstanding core facilities, including opportunities, the Institute is seeking to recruit Institute is funded by HEFCE generated income and Treatment of Cancer molecular biology services, next generation outstanding candidates to the positions of at a cost of £2.1m. • European Research Council sequencing, real-time PCR, mass spectrometry, Junior and Senior Group Leaders. The • Fondation ARC pour la Recherche flow cytometry, histology, advanced imaging, packages provided are extremely attractive The balance of the Institute’s funding is received sur le Cancer and a biological resources unit. Details of all and commensurate with the experience of from a number of additional sources. The • GlaxoSmithKline groups and facilities are given in this report, the applicant, with significant funding for research carried out through these additional • Harry J Lloyd Charitable Trust and can guide interested parties to the personnel, recurrent expenditure and projects enhances and supports the research • John Swallow Fellowship appropriate contacts. equipment. Junior Group Leaders are undertaken by the core funding. • Kay Kendall Leukaemia Fund appointed for an initial six-year period with • Leo Pharma Foundation Opportunities exist at a number of levels in the a review between five and six years for These sources are as follows: • Menarini Biomarkers Singapore Institute. We have a well-established consideration of promotion to Senior Group • Merck programme of degrees by research which is Leader, with Senior Group Leaders appointed • Amgen • Moulton Charitable Trust described in the section on Postgraduate to non-time limited positions. • Angle Inc • National Institute of Health Research Education. We encourage applications from • Astex Pharmaceuticals • Ono Pharmaceuticals suitably qualified graduates to apply to join Specific vacancies can be found on our web • AstraZeneca • Pancreatic Cancer Research Fund either the PhD or MD programmes. Graduates pages (http://www.cruk.manchester.ac.uk/ • Bioven • Pickering Leukaemia Research with a first or 2.1 honours degree in a biological Opportunities/Opportunities-Home) but • Bloodwise • Prostate Cancer UK science can apply each year to train for a suitably qualified and enthusiastic individuals • Carrick Therapeutics • Rosetrees Trust four-year PhD in one of our research should contact the Institute at any time to • CellCentric • Taiho Oncology Inc laboratories. The University of Manchester enquire about career possibilities. • Christie Hospital NHS Foundation Trust • The US Department of Health and offers a wide range of training for new and • Clearbridge Biomedicals Human Services existing students which provides opportunities • CRT Pioneer Fund • Wellcome Trust to acquire skills that will complement the • David & Ruth Lewis Trust • Worldwide Cancer Research research programme and help achieve personal • Euclises Pharmaceuticals Inc and career development goals. At the Institute, • European Commission We are immensely grateful to all our funders. we also ensure that postgraduate students are provided with high quality, relevant and appropriate training alongside development opportunities. The Institute also has a well- CRUK MANCHESTER INSTITUTE FUNDING 2018 developed process for ensuring excellent pastoral care and mentoring for all students. Postdoctoral applicants of high calibre are KEY regularly sought. Although Postdoctoral Fellows will be encouraged to apply for their 29.5% own fellowships, funded positions are available for outstanding candidates. Interested CRUK Core Grant applicants should contact the Group Leaders directly, with details of their research interests 43.5% CRUK Strategic Funding and recent experience. HEFCE Other Sources 7.1% 19.6% 86 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE CAREER OPPORTUNITIES AT THE CRUK MANCHESTER INSTITUTE 87


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    CONTACT DETAILS M6 to Preston, Carlisle, Scotland M62 to Leeds M6 M66 Rochdale Bury M62 Bolton M61 ISSN 1479-0378 Wigan Copyright © 2018 Cancer Research UK Oldham M60 Edited by: Caroline Wilkinson M602 Manchester Ashton-Under-Lyne Gillian Campbell City Centre M6 M67 M62 A57 to Sheffield M60 M62 Stockport M62 to Liverpool M6 M60 Warrington Altrincham M56 A34 Manchester Airport M56 M6 Wilmslow M6 to Birmingham, A537 London Knutsford Alderley Edge A34 Alderley Park Macclesfield Peak District A537 A34 Cancer Research UK Manchester Institute Cancer Research UK Director: Professor Richard Marais Cancer Research UK is a registered charity in England and Wales (1089464), Scotland Address (SC041666) and the Isle of Man (1103). Cancer Research UK Manchester Institute Registered address: Angel Building, 407 St The University of Manchester John Street, London, EC1V 4AD. Alderley Park SK10 4TG Tel 44(0) 20 1234 5678 United Kingdom www.cruk.org e-mail: enquiries@cruk.manchester.ac.uk website: www.cruk.manchester.ac.uk Electronic version of this report can be found at: Tel +44(0) 161 306 0871 www.cruk.manchester.ac.uk/About/ 88 SCIENTIFIC REPORT 2018 CANCER RESEARCH UK MANCHESTER INSTITUTE


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    CANCER RESEARCH UK MANCHESTER INSTITUTE Cancer Research UK Manchester Institute The University of Manchester Alderley Park SK10 4TG United Kingdom www.cruk.manchester.ac.uk www.cruk.manchester.ac.uk


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