SCIENTIFIC REPORT 2019 cruk.org SCIENTIFIC COVER IMAGE Primary cutaneous (nests of purple cells) invading the dermis. The specimen is REPORT stained with Masson’s trichrome stain. Cyan blue reveals collagen fibre architecture.

Image supplied by Amaya Viros (Skin 2019 and Ageing) MANCHESTER INSTITUTE CONTENTS

DIRECTOR’S INTRODUCTION 04 Tim Somervaille 32 RESEARCH SERVICES RESEARCH PUBLICATIONS 64 RESEARCH HIGHLIGHTS 2019 07 Leukaemia Duncan Smith 52 THESES 69 Richard Marais 34 Biological Mass Spectrometry Facility SEMINAR SERIES 2019 70 CANCER RESEARCH UK Molecular Jen Hughes and Lisa Doar 53 MANCHESTER INSTITUTE OPERATIONS 72 Esther Baena 36 Biological Resources Unit Prostate Oncobiology Wolfgang Breitwieser 55 POSTGRADUATE EDUCATION 80 RESEARCH GROUPS Molecular Biology Core and CANCER RESEARCH UK’S LOCAL 84 Caroline Dive 14 Amaya Virós 38 Computational Biology Support Facility ENGAGEMENT AND DEVELOPMENT Cancer Centre Skin Cancer and Ageing Georges Lacaud 40 Garry Ashton 56 ACKNOWLEDGEMENT FOR FUNDING OF 86 Santiago Zelenay 20 THE CANCER RESEARCH UK Stem Biology Histology Cancer Inflammation and Immunity MANCHESTER INSTITUTE Claus Jørgensen 42 Natalia Moncaut 56 Iain Hagan 22 CAREER OPPORTUNITIES AT THE CANCER 87 Systems Oncology Transgenic Production Facility Cell Division RESEARCH UK MANCHESTER INSTITUTE Marek Dynowski 58 Maximiliano Portal 24 Michela Garofalo 44 Scientific Computing CONTACT DETAILS 88 Cell Plasticity & Epigenetics Transcriptional Networks in Lung Cancer Jeff Barry 59 Angeliki Malliri 26 Robert Bristow 46 Flow Cytometry Cell Signalling Translational Oncogenomics Steve Bagley 60 Caroline Springer 28 Patricia Muller 48 Visualisation, Irradiation & Analysis Drug Discovery Tumour Suppressors Robert Metcalf 30 Head and Neck Cancer Biology

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 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE 3 Sciences at The , prostate cancer progression and treatment together with colleagues in the Leukaemia response. Biology group, uncovered an intriguing DIRECTOR’S relationship between splicing and epigenetic Our early career researchers’ efforts were regulation underpinning a new molecular recognised at several meetings during the INTRODUCTION subtype of Acute Myeloid Leukaemia. summer. Postdoctoral Research Fellow, Lucas Alongside, the Leukaemia Biology group Trucco, from the Molecular Oncology team continue to develop their programme on the received the AACR Scholar-in-Training Award to potential AML target Lsd1 and have identified attend the AACR Annual Meeting, while Brian mTORC1 inhibition as a synergistic therapeutic Lee from Systems Oncology was awarded a approach. poster prize at the BACR Tumour Microenvironment meeting. Several of our PhD During 2019 we continued our recovery from the 2017 Paterson I was delighted that the DETECTION clinical trial students attended the International PhD Student was funded by CRUK and that the CACTUS trial Cancer Conference in Amsterdam, with two of Building fire by consolidating our interim base at Alderley Park. is now up and running. Both applications for our students, Max Schenk and Chris Bromley, The Paterson Building has been demolished and good progress these melanoma trials have been driven by winning prizes for their presentations. Sakis Rebecca Lee, a former clinical fellow in the Paliouras received a University of Manchester is being made on the plans for its replacement. Molecular Oncology group, and rely on the Doctoral Academy Graduate Society ctDNA expertise developed in the CBC. Also, conference award and a BACR-CRUK student many of our Group Leaders contributed to the travel award to attend the AACR annual meeting. The project received a significant boost in the patients following lung cancer surgery with the Professor successful application for an Academic In July, the MCRC and Experimental Cancer summer through a £25m award from the aim of using these “liquid biopsy” approaches to Researcher Clinical Training Innovation in Medicine Centre organised an exciting meeting Richard Marais Research England UK Research Partnership detect relapse at an early stage. The TARGET Cancer programme (ARCTIC) as part of the in Manchester “Phase 1: Where Science Investment Fund and towards the end of 2019, trial has been another exciting development for CRUK Manchester Centre’s Clinical Academic Becomes Medicine” at which Julie Stevenson Director of the Cancer Research the Rewrite Cancer campaign was launched by the CBC in recent years, together with clinical UK Manchester Institute Training (CAT) initiative and we look forward to and Paul O’Regan from the digital Experimental the project's partners to raise the final £20m colleagues at the Christie Hospital. This ctDNA- welcoming some of the trainees to CRUK MI in Cancer Medicine Team were jointly awarded required for the build. As 2020 proceeds, we driven selection phase I clinical trial is being run due course. Our scientists also played a major prizes for their posters on eTARGET - a digital look forward to finalising the design and across Experimental Cancer Medicine Centres role in the success of several other CRUK solution for the Manchester Molecular Tumour watching the new facility start to emerge and in the North and was also published in a Manchester Centre initiatives such as the ACED Board. PhD students Denys Holovanchuk, Amy take shape. Medicine publication, and the trial will now be early detection alliance and the radiation McCarthy and Jakub Chudziak all received expanded to assess patient survival. It is very research network RADNET, while the Belfast- awards to attend conferences, Amin Ali received Despite the disruption of the move, we have pleasing to see how well the CBC has recovered Manchester Movember Centre of Excellence for The University of Manchester’s Presidential celebrated many scientific highlights that from the fire. Prostate Cancer, which involves three of our Award and Joe Maltas was awarded The illustrate the depth and breadth of research at research groups, was renewed in 2019. Humane Research Trust Margaret Pritchard the Institute. It was a particularly successful year The Institute’s Drug Discovery Unit (DDU) has Moreover, together with partners in Italy and Memorial Prize. Postdoctoral Research Fellow for the Institute’s Deputy Director Caroline Dive also made an excellent recovery, since Spain, the CBC’s digital Experimental Cancer Victoria Pelly received a CRUK Research Travel and her Clinical and Experimental establishing their facilities at Alderley Park in Medicine Team were awarded a CRUK Award, which facilitated a visit to the group of Pharmacology group who continue to be at the early 2018. Their programme of greater Accelerator award to produce digital tools for Ton Schumacher at the NKI. We have continued forefront of the development of to integration with the Institute’s scientists is clinical teams across the CRUK Experimental to celebrate our achievements in pursuing aid clinical decision making. In April, they developing well in addition to fostering exciting Cancer Medicine Centres’ network providing developments in the principles of the 3Rs – received an “Outstanding” rating at their new collaborations with the wider CRUK real-time access to patient data to facilitate Replacement, Reduction and Refinement – quinquenniual review, which took place over community including a joint position with faster clinical decision-making. Georges during in vivo procedures and to promote two days at Alderley Park. Later in the year, they Stephen Taylor at The University of Manchester, Lacaud, who leads the Stem Cell Biology group, sharing and dissemination of ideas and best were part of the successful renewal of the CRUK and a second joint position with Richard Bayliss received funding from the charity Bloodwise practice, we held a joint 3Rs’ Poster event with Lung Cancer Centre of Excellence, and towards at The University of Leeds. They published two (now Blood Cancer UK) to investigate MOZ AstraZeneca and Agenda Life Sciences. I am the end of the year, we renamed the group as studies in 2019 that are the first to describe orally histone acetyl transferase activity in leukaemia. delighted that CRUK MI picked up awards in all the Cancer Research UK Manchester Institute available drug-like compounds against lysyl three categories at this event, through the work Cancer Biomarker Centre (CBC) to recognise oxidase (LOX). This forms cross-links A number of our staff and students received of Lisa Doar, Chris Below, Callum Hall and the scope and extent of their exciting scientific between collagens and elastin in the awards and prizes during the year. Caroline Dive Yannick von Grabowiecki. activities. We also welcomed Dr Alistair Kerr and extracellular matrix and plays a critical role in was one of The University of Manchester’s Dr Carlos Garcia Lopez to the CBC as the new regulating tumour growth and metastasis. Their Researcher of the Year, and she received the Junior Group Leader Santiago Zelenay received Head of the Bioinformatics and Biostatistics work has added to the growing interest in LOX 2019 Heine H. Hansen Lectureship Award for the inaugural BIAL Prize in Biomedicine, along team, and as an Institute Fellow respectively. as a potential therapeutic target, and the lead Small Cell Lung Cancer in recognition of her with his former mentor at The Francis Crick inhibitors developed by the DDU display pioneering discovery research in this field. The Institute, Caetano Reis e Sousa. This new award One of the CBC’s key studies was published in promising effects in delaying tumour growth in Deputy Group Leader of the CBC, Ged Brady, recognises significant achievements in Nature Medicine and it describes the prediction a mouse model of breast cancer. was awarded a Professorship by The University biomedicine published over the past decade. of non-small cell lung cancer (NSCLC) relapse of Manchester in recognition of his During his time at the Crick, Santiago discovered through pulmonary venous circulating tumour The Cell Signalling group uncovered a new contributions to nucleic acid biomarker a mechanism used by to escape cells. These cells are isolated from stage I-IIIa regulator of mitotic spindle orientation through research. Manchester Cancer Research Centre detection by the immune system, which has the NSCLC patients during surgery, and through the interaction of the calcium/calmodulin- Director and CRUK MI Senior Group Leader Rob potential to be exploited therapeutically. This their enumeration, risk of recurrence can be dependent serine protein kinase (CASK) with the Bristow was elected Fellow of the Academy of work has formed the basis of several clinical predicted. Prof Dive’s team is partnering with tumour suppressor Dlg1. Former Clinical Fellow, Medical Sciences for advancing medical trials exploring the combination of anti- Lloyds Pharmacies in Greater Manchester where Dan Wiseman, now Oglesby Senior Leukaemia science through his outstanding contributions inflammatory drugs with immunotherapy in a phlebotomists take blood samples from Research Fellow in the Division of Cancer towards understanding the genomics of range of cancers and is underpinning the

4 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE DIRECTOR’S INTRODUCTION 5 DIRECTOR’S INTRODUCTION (CONTINUED)

research programme of Santiago's Cancer across the CRUK Institutes. I look forward to RESEARCH Inflammation and Immunity group. seeing the outcomes from this initiative in the coming years. HIGHLIGHTS Each year the Institute awards its own prize to recognise the most outstanding achievements Our Science Take Away (STAy) group continues by a young scientist. Named after a former to drive a programme of events for our cohort Institute Director, the 2019 Dexter award was of early career researchers and to organise presented jointly to Mark Williams and Alice opportunities to engage with the public. In 2019, Lallo. During his time as a Clinical Research they invited and hosted several of our external In this section we highlight some research publications from Fellow in the Leukaemia Biology group, Mark seminar speakers, organised media training and uncovered the molecular mechanism grant writing workshops and put together a 2019 which report significant advances in specific areas. The underpinning up-regulation of the drug efflux highly successful World Cancer Day “Behind the selected papers demonstrate the breadth and the quality of the pump ABCB1, which is a key driver of Labels” public engagement evening - a large chemoresistance in acute myeloid leukaemia. event incorporating clinician, researcher, and research being undertaken by the groups at the Cancer His work, published in the Journal of Clinical patient talks, lab tours and table-top activities. Research UK Manchester Institute. Investigations, suggested a potential The group put forward their views as part of therapeutic approach to overcome this form of Cancer Research UK’s consultation process with resistance and enhance the activity of ABCB1 researchers over the PlanS coalition on open Chemi F, Rothwell DG, McGranahan N, Gulati detected 10 months later (91%) than with the inhibitors. Mark is set to pursue a career as a access publishing. Together with Steve Bagley S, Abbosh C, Pearce SP, Zhou C, Wilson GA, primary tumour (79%), suggesting that early- clinician scientist in the field of stem-cell (Head of Visualisation, Irradiation and Analysis) Jamal-Hanjani M, Birkbak N, Pierce J, Kim CS, disseminating PV-CTCs were responsible for transplantation, and in August 2020 we look and Gill Campbell (Grants Advisor), the group Ferdous S, Burt DJ, Slane-Tan D, Gomes F, disease relapse. The evolutionary origin of the forward to welcoming him back to CRUK MI, submitted a successful application to the Royal Moore D, Shah R, Al Bakir M, Hiley C, Veeriah S, PV-CTCs and metastasis was confirmed by through a University of Manchester Presidential Society Summer Science Exhibition 2020. Summers Y, Crosbie P, Ward S, Mesquita B, phylogenetic analysis that revealed both the Fellowship. Unfortunately, this event was postponed due to Dynowski M, Biswas D, Tugwood J, Blackhall F, PV-CTCs and metastasis were part of the same the SARS-CoV2 pandemic, but we look forward Miller C, Hackshaw A, Brady G, Swanton C, specific branch, which was distinct from all Alice Lallo’s work in the CBC focused on to them displaying their exhibit on the theme of Dive C; TRACERx Consortium. other subclones of the primary tumour. Finally, characterising DNA damage response the Institute’s work on the tumour Pulmonary venous circulating tumor cell they examined the shared by deficiencies (DDR) in small cell lung cancer microenvironment at some point in the future. dissemination before tumor resection and PV-CTCs and the metastatic biopsy yet absent using cell lines and circulating tumour cell disease relapse. from the primary tumour. In this patient, the derived explant (CDX) models. Her research has It is a great privilege to engage with CRUK’s Nature Medicine 2019; 25(10):1534-1539. PV-CTC and metastatic-associated mutations supported translation of the combination of supporters, facilitated through the excellent included a putative inactivating driver two DDR inhibitors to the clinic and resulted in a work of Tim Hudson, CRUK’s Research In early stage non-small cell lung cancer in the tumour-suppressor gene LZTS1, which first author publication in Clinical Cancer Engagement Manager based in Manchester. (NSCLC), tumour recurrence post-surgery has been shown to inhibit tumour migration and Research. A critical part of her study involved During 2019, we welcomed supporters to our occurs in approximately 50% of cases and most whose lower expression has been linked to poor the use of ex vivo CDX cultures to screen for Laboratories at both Alderley Park and the commonly at distant sites. In this study, overall survival in NSCLC. In summary, they drug efficacy as well as investigating molecular Oglesby Cancer Research Building while also members of the Cancer Biomarker Centre showed the potential utility of PV-CTCs as early mechanisms of drug response. In 2019, Alice attending events such as CRUK’s Relay for Life addressed whether pulmonary vein circulating predictors of NSCLC recurrence after surgery. published her second first author paper in the Stockport. We hosted a visit by three members tumour cells (PV-CTCs) detected in early stage British Journal of Pharmacology, in which she of the Science Museum’s exhibition curation NSCLC at surgical resection with curative intent Rothwell DG, Ayub M, Cook N, Thistlethwaite F, described the development and team to help them prepare for the CRUK- represent lethal subclone(s) responsible for Carter L, Dean E, Smith N, Villa S, Dransfield J, characterisation of her CDX ex vivo culture sponsored cancer exhibition, which is due to subsequent disease relapse. They enriched and Clipson A, White D, Nessa K, Ferdous S, Howell system. take place in 2021. enumerated blood samples from 100 patients M, Gupta A, Kilerci B, Mohan S, Frese K, Gulati S, enrolled into the TRACERx study using Miller C, Jordan A, Eaton H, Hickson N, O'Brien Our core facilities continue to evolve and At the time of writing, we are in the midst of the CellSearch platform. PV-CTCs were detected in C, Graham D, Kelly C, Aruketty S, Metcalf R, support our research through the provision of SARS-CoV2 pandemic. Like many other 48% of patients but was not significantly Chiramel J, Tinsley N, Vickers AJ, Kurup R, cutting-edge technological platforms. The research establishments, we halted our associated with clinicopathologic factors such Frost H, Stevenson J, Southam S, Landers D, Mass Spectrometry facility was severely laboratory work during the lockdown and as age, sex, histologic subtype, pathologic Wallace A, Marais R, Hughes AM, Brady G, Dive affected by the Paterson Building fire but after a sought other ways to continue our research stage, smoking status, and treatment received. C, Krebs MG. sustained effort to replace equipment and efforts by working from home. I was very proud An exploratory analysis showed that PV-CTC- Utility of ctDNA to support patient selection for optimise the environment in which to house it, that so many of our researchers volunteered to high status (≥7 PV-CTCs per 7.5 ml blood) was early phase clinical trials: the TARGET study. the service returned to being fully operational assist the national effort on COVID-19 testing. significantly associated with lung cancer Nature Medicine 2019; 25(5):738-743. and I congratulate Duncan Smith and Yvonne Pleasingly, we have reopened our laboratories, relapse and remained an independent predictor Connelly for all their sterling work during this albeit with the restrictions imposed by reduced in multivariate analysis when adjusting for Researchers in the Cancer Biomarker Centre challenging time. An evolving theme has been occupancy and social distancing, but I am again tumour stage. The team molecularly profiled have analysed the genetic mutations in a blood increased collaboration between the facilities proud that our staff are back at work and PV-CTCs to establish genetic links between sample of cancer patients, which could help to great effect together with better integration pursuing their projects with so much renewed PV-CTCs, the primary tumour and the relapse match those patients with no other treatment with our Computational Biology and Scientific enthusiasm. tumour. In a case study, genomic profiling of options to clinical trials with experimental Computing activities. During the year, plans single PV-CTCs collected at surgery revealed medicines. Together with colleagues at The developed to hold a core facilities retreat to higher mutation overlap with metastasis provide a platform for enhanced discussion

6 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH HIGHLIGHTS 7 RESEARCH HIGHLIGHTS (CONTINUED) (62.5%) and 27/31 (87%) of patients with locally 10-fold reduction of CNA and no notable CNA advanced and metastatic disease respectively. change in a sixth patient. At relapse, CNAs were Copy number changes were detected in cfDNA detectable in four of the five patients and the Christie NHS Foundation Trust, AstraZeneca blood test is successful at matching patients to of 10 patients of whom seven exhibited gain of sixth patient with undetectable CNA relapsed and the NIHR Manchester Biomedical Research early phase clinical trials and the impact this has chromosome 12p harbouring KRAS as well as a with only brain metastasis and stable lung Centre (BRC), in this promising study they on their overall survival. There is also an option canonical KRAS codon 12 mutation. In disease. The liquid biopsy approaches reported demonstrated that a blood test can be carried of referring patients to other clinical trial sites multivariable Cox Regression analysis, they here have been shown to provide effective out and analysed in a timeframe that can help within the Experimental Cancer Medicine show for the first time that patients withKRAS baseline analysis and longitudinal monitoring clinicians select a matched, targeted treatment. Centre (ECMC) network, if suitable matched copy number gain and KRAS mutation have of both LS and ES disease and are now being Currently, enrolment to trials depends on a trials are available in other parts of the country. significantly worse outcomes, suggesting that incorporated into extended SCLC studies patient’s type of cancer or genetic data this may be linked to PDAC progression. The and trials. obtained from an invasive tumour biopsy, Mohan S, Ayub M, Rothwell DG, Gulati S, Kilerci simple cfDNA assay the team describe will which is often months or years old and may not B, Hollebecque A, Sun Leong H, Smith NK, enable determination of the presence of KRAS Porter AP, White GRM, Mack NA, Malliri A. represent a patient’s current disease due to their Sahoo S, Descamps T, Zhou C, Hubner RA, copy number gain and KRAS mutations in larger The interaction between CASK and the tumour tumours’ evolutionary changes over time. McNamara MG, Lamarca A, Valle JW, Dive C, studies and clinical trials. suppressor Dlg1 regulates mitotic spindle Brady G. orientation in mammalian epithelia. The team from CBC showed that a small Analysis of circulating cell-free DNA identifies Mohan S, Foy V, Ayub M, Leong HS, Schofield P, Journal of Cell Science 2019; 132(14) jcs230086. volume of blood can contain up-to-date KRAS copy number gain and mutation as a Sahoo S, Descamps T, Kilerci B, Smith NK, genetic information about a patient’s cancer to novel prognostic marker in pancreatic cancer. Carter M, Priest L, Zhou C, Carr TH, Miller C, Epithelial cells normally align their divisions with inform treatment choices. In this feasibility Scientific Reports 2019; 9(1):11610. Faivre-Finn C, Blackhall F, Rothwell DG, Dive C, the plane of the epithelium, ensuring that study of the first 100 patients, 11 were enrolled Brady G. daughter cells are produced side-by-side. In 3D onto an available and molecularly matched Using serial biopsy of pancreatic ductal Profiling of circulating free DNA using targeted this leads to single layers of epithelial cells clinical trial. adenocarcinoma (PDAC) to chart tumour and genome wide sequencing in patients with surrounding a hollow lumen or acini, such as evolution presents a significant challenge. The small cell lung cancer. those found in the kidney or breast. Spindle In the first of the two-part trial, called Cancer Biomarker Centre examined the utility of Journal of Thoracic Oncology [Epub 16 October misorientation can disrupt the integrity of the TARGET (Tumour chARacterisation to Guide circulating free DNA (cfDNA) as a minimally 2019] epithelial architecture, potentially promoting Experimental Targeted therapy), the researchers invasive approach across a cohort of 55 tumourigenesis. The Cell Signalling group were able to collect, process and analyse blood treatment-naïve patients with PDAC; 31 with Tissue biopsies and especially serial biopsies uncovered a new regulator of mitotic spindle samples from 100 patients in the Manchester metastatic and 24 with locally advanced disease. pose a significant challenge in SCLC as they are orientation, calcium/calmodulin-dependent area. The second part of TARGET, which is Somatic mutations in cfDNA were detected typically of poor quality and/or unavailable for serine protein kinase (CASK). CASK binds to and already underway, will show how often the using next generation sequencing in 15/24 research. Surgical resections of the tumour are recruits the tumour suppressor Dlg1 to the rare, yet by necessity most genomic studies so lateral cell membrane where Dlg1 in turn far have been performed on resected tumours recruits core components of the spindle and hence, under representative of the majority orientation complex. Cells depleted for CASK Multiplex IHC images of murine of the patients. Several targeted therapies have show reduced Dlg1 recruitment, misoriented prostate (pten-/-/ p53-/-) now entered clinical trials and analysis of cell divisions, and form 3D structures containing tumours stained for STING (green), CK8 (Red), and DAPI circulating free DNA (cfDNA) is a minimally multiple lumens. They showed the significance (Blue) following treatment with invasive approach for disease monitoring and of the CASK-Dlg1 interaction in two ways. First, radiotherapy. Radiotherapy leads stratification. To this end, members of the direct disruption of the CASK-Dlg1 interaction to an increase in STING staining. Cancer Biomarker Centre developed a novel by inducibly expressing an interfering peptide IHC staining was performed cfDNA workflow for disease monitoring using phenocopies the effect of depleting CASK. using the Bond (Rx) staining platform (CRUK MI Core facility) genome-wide copy number aberration (CNA) Second, depletion of Dlg1 results in spindle and imaged using Olympus analysis as well as targeted mutation analysis of misorientation, which can only be rescued by VS120 microscope (Advanced 110 SCLC associated genes. This workflow was expression of wild-type Dlg1 and not by Dlg1 imaging). applied to pre-treatment cfDNA from 69 lacking the CASK interaction domain. Together, patients with extensive-stage (ES) and limited- these results reveal the critical importance of Image supplied by Debayan Mukherjee (Targeted Therapy - stage (LS) SCLC to establish sensitivity and the CASK-Dlg1 interaction for spindle Division of Cancer Sciences, The feasibility of the approach. Their data show orientation, which has important implications University of Manchester) tumour-related changes (CNAs and/or somatic for the correct formation of epithelia. mutations) could be detected in cfDNA from 94% LS and 100% of ES patients. Among the 110 Leung L, Niculescu-Duvaz D, Smithen D, Lopes genes analysed, a total of 272 somatic F, Callens C, McLeary R, Saturno G, Davies L, mutations were identified, including driver Aljarah M, Brown M, Johnson L, Zambon A, alterations in 69% of samples of which 60% were Chambers T, Ménard D, Bayliss N, Knight R, Fish potentially targetable. In multivariate analysis, L, Lawrence R, Challinor M, Tang H, Marais R, both metrics from CNA analysis as well as the Springer C. number of mutations were significantly Anti-metastatic inhibitors of lysyl oxidase (LOX): associated with the stage of disease. Further, in a design and structure-activity relationships. subset of six patients the team explored the Journal of Medicinal Chemistry 2019; utility of cfDNA analysis for disease monitoring 62(12):5863-5884. using longitudinal samples. Four patients had baseline CNAs that were undetectable on Lysyl oxidase (LOX) is a secreted copper amine completion of treatment, a fifth patient had oxidase that serves as the master crosslinker of

8 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH HIGHLIGHTS 9 RESEARCH HIGHLIGHTS (CONTINUED) mTORC1 and LSD1 inhibition represent a mutations in the metabolic gene IDH2 and the candidate combination approach for enhanced spliceosome component SRSF2 in acute differentiation in MLL-translocated AML, which myeloid leukaemia (AML). In collaboration with collagens and elastin in the extracellular matrix. core leads to improved potency towards LOXL2 could be evaluated in early phase clinical trials. Memorial Sloan Kettering Cancer Center they LOX and its isoforms are critical mediators of via an irreversible mode of inhibition. Structure- confirmed this across several large publically tumour growth and metastatic spread, and activity relationship studies enabled the Williams MS, Amaral FM, Simeoni F, available sequencing datasets, characterising a have been firmly established as therapeutic development of a predictive LOXL2 3DQSAR Somervaille TC. new AML molecular subtype affecting 5-10% of targets in oncology and fibrosis. However, the model, and further selectivity and ADME A stress-responsive enhancer induces patients with distinctive clinical and DNA only effective LOX inhibitor available until assessment resulted in the identification of a dynamic drug resistance in acute myeloid methylation features. Through in vivo modelling recently is β-aminopropionitrile, a non drug-like lead compound (21b) that demonstrates leukemia. studies they demonstrated a functional molecule that is unsuitable for clinical excellent selectivity over common amine Journal of Clinical Investigation [Epub 26 collaboration between these mutations, which development. The Drug Discovery Unit oxidases and hERG, as well as improved Caco-2 November 2019] promoted acute leukaemogenesis when describe the discovery of a novel series of permeability and pharmacokinetic properties in present together as compared with either alone. aminomethylenethiophene (AMT) inhibitors in two rodent species. This inhibitor was also The drug efflux pump ABCB1 is a key driver of Comprehensive RNA sequencing of patient the first detailed study dedicated to inhibitors of evaluated in our transgenic LOX-driven breast chemoresistance, and high expression predicts samples, including a large cohort from the this LOX isoform. Significantly, they detailed cancer model where it displays excellent for treatment failure in acute myeloid leukaemia MCRC Biobank, identified substantially more structure-activity relationships around the AMT anti-tumour efficacy, with significantly reduced (AML). In this study, the Leukaemia Biology aberrant splicing in double mutant cases than pharmacophore leading to great improvement tumour growth, thus showing promise as a drug group identified and functionally validated the SRSF2 single-mutants. Amongst the genes most in potency over the HTS hit. This is followed by candidate for further study. network of enhancers that controls expression differentially spliced in this context was INTS3, further structural modifications to overcome in of ABCB1. They show that exposure of which encodes a component of the Integrator vivo stability issues while retaining LOX Deb G, Wingelhofer B, Amaral FMR, Maiques- leukaemia cells to daunorubicin activated an complex with multiple roles in transcriptional inhibition potency, leading to the orally Diaz A, Chadwick JA, Spencer GJ, Williams EL, integrated stress response-like transcriptional regulation. A double intron retention event available LOX inhibitor CCT365623. They Leong HS, Maes T, Somervaille TCP. program to induce ABCB1 through remodelling resulted in increased nonsense-mediated demonstrated that CCT365623 also inhibits Pre-clinical activity of combined LSD1 and and activation of an ATF4-bound, stress- decay, and was associated with broader LOXL2 at a similar level and has an excellent mTORC1 inhibition in MLL-translocated acute responsive enhancer. Protracted stress primed integrator dysfunction. Mechanistically they selectivity profile against four other non-LOX myeloid leukaemia. enhancers for rapid increases in activity showed that INTS3 missplicing was concordant amine oxidases. Above all, this inhibitor is Leukemia [Epub 28 November 2019] following re-exposure of cells to daunorubicin, with increased stalling of RNA polymerase II, effective in suppressing breast cancer providing an epigenetic memory of prior drug and dependent on mutant SRSF2 binding to cis metastasis to the lungs in their transgenic Acute myeloid leukaemia (AML) is a treatment. In primary human AML, exposure of elements in INTS3 mRNA. They further showed mouse model and thus demonstrating its heterogeneous group of proliferative fresh blast cells to daunorubicin activated the that INTS3 depletion phenocopied SRSF2/IDH2 promise as a drug candidate. malignancies characterised by an accumulation stress-responsive enhancer and led to dose- mutations in promoting hallmarks of of poorly differentiated myeloid blasts in bone dependent induction of ABCB1. Dynamic leukaemogenesis in myeloid differentiation cell Smithen DA, Leung LMH, Challinor M, marrow and blood. The histone demethylase induction of ABCB1 by diverse stressors, model systems. Overall, this study identified a Lawrence R, Tang H, Niculescu-Duvaz D, lysine-specific demethylase 1 (LSD1 or KDM1A) including chemotherapy, facilitated escape of pathogenic crosstalk between altered Pearce SP, Mcleary R, Lopes F, Aljarah M, has emerged as a candidate therapeutic target leukaemia cells from targeted third-generation epigenetic state and aberrant splicing in a Brown M, Johnson L, Thomson G, Marais R, in AML and a first-in-man phase 1 trial of the ABCB1 inhibition, providing an explanation for distinct subset of AML. This may have Springer C. LSD1 inhibitor ORY1001 (from Oryzon the failure of ABCB1 inhibitors in clinical trials. translational importance, given that targeted 2-Aminomethylene-5-sulfonylthiazole Genomics) recently demonstrated that LSD1 Stress-induced up regulation of ABCB1 was therapies inhibiting both mutations are in inhibitors of lysyl oxidase (LOX) and LOXL2 inhibitors promote blast cell differentiation in mitigated by combined use of pharmacologic clinical trials. show significant efficacy in delaying tumor patients, in particular those with AML associated inhibitors U0126 and ISRIB, which inhibit stress growth. with translocations targeting the Mixed Lineage signalling and have potential for use as adjuvants Journal of Medicinal Chemistry [Epub 4 Leukaemia gene. However, as for all agents in to enhance the activity of ABCB1 inhibitors. September 2019] AML, it is unlikely that LSD1 inhibitors will be highly effective as single agents. Through a Yoshimi A, Lin KT, Wiseman DH, Rahman MA, The lysyl oxidase (LOX) family of extracellular genome-wide CRISPR-Cas9 dropout screen, Pastore A, Wang B, Lee SC, Micol JB, Zhang XJ, proteins play a vital role in catalysing the the Leukaemia Biology group identified genes de Botton S, Penard-Lacronique V, Stein EM, formation of crosslinks in fibrillar elastin and and cellular pathways that collaborate with LSD1 Cho H, Miles RE, Inoue D, Albrecht TR, collagens leading to extracellular matrix (ECM) to maintain the leukaemic phenotype, and Somervaille TCP, Batta K, Amaral F, Simeoni F, stabilisation. These are known to which could be targeted by combination Wilks DP, Cargo C, Intlekofer AM, Levine RL, promote tumour progression and metastatic therapies. They identified multiple components Dvinge H, Bradley RK, Wagner EJ, Krainer AR, disease, and have thus become an attractive of the amino acid sensing arm of mTORC1 Abdel-Wahab O. therapeutic target for many types of invasive signalling as cellular sensitisers to LSD1 Coordinated alterations in RNA splicing and cancers. Following on from the Drug Discovery inhibition. Knockdown of mTORC1 components epigenetic regulation drive leukaemogenesis. Unit’s recently published work on the discovery or mTORC1 pharmacologic inhibition in Nature 2019; 574(7777):273-277. of aminomethylenethiophenes (AMTs) as combination with LSD1 inhibition synergistically potent, orally bioavailable LOX/LOXL2 induced the expression of a set of transcription Dan Wiseman, formerly of the Leukaemia inhibitors, this report details the development factor genes associated with terminal Biology group, and colleagues had previously of aminomethylenethiazoles (AMTz) as potent monocytic lineage differentiation, resulting in identified in their Christie NHS Foundation Trust inhibitors of three LOX isoforms, as well as a enhanced differentiation in both cell line and patient cohort an unappreciated frequent subseries of LOXL2-selective inhibitors. Major primary cell settings in vitro and in vivo. association between gain-of-function findings include the discovery that a thiazole Altogether, the data suggested that dual

10 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH HIGHLIGHTS 11 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH GROUPS

Organotypic brain slice (grey scale) invasion by patient-derived melanoma cells tagged with GFP (green). Illustrating melanoma cell capacity to invade into the brain tissue ex vivo.

Image supplied by Denys Holovanchuk (Molecular Oncology)

12 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE 13 www.cruk.manchester.ac.uk/Our-Research/Cancer-Biomarker-Centre Hannah Frost1 Andrew Hughes Laura Hutchinson2 Donal Landers Cancer Research UK Leanne Ogden Paul O’Regan Jenny Royle Manchester Institute Cancer Laura Stephenson Julie Stevenson Siobhan Southam2 Biomarker Centre Jason Swift Jenny Ward

Clinical Fellows Alicia Conway Victoria Foy 2019 was a busy year for the team. In February our new Good Clinical Melissa Frizziero1 Practice (GCP) facilities at Alderley Park were inspected by the MHRA. Matthew Howell

In April, we had a successful CRUK quinquenial review that resulted in Graduate Students a new name, the CRUK Manchester Institute Cancer Biomarker Alessia Catozzi Jakub Chudziak Centre (CBC). We played a significant role in the renewal and Sam Humphrey Figure 1. SCLC subtype based on NE TFs: A) PCA plot of CDX model RNAseq data. B) Multiplex immunofluorescence Alice Lallo showing co-expression of ASCL1 and NEUROD1 in a CDX model. expansion of the CRUK Lung Cancer Centre of Excellence and our Julia Ogden1 Group Leader digital Experimental Cancer Medicines Team obtained a CRUK Sarah Pearsall Caroline Dive Accelerator Award for Digital Clinical Trials. Our overall goals remain Max Schenk CDX models generated from the same patient vessel genesis. The PP team previously reported before treatment and at disease progression are VM in SCLC biopsies associated with poor Scientific Officers a) to discover, develop, validate and implement biomarkers that facilitating discovery of acquired drug prognosis. VM is present in most CDX models Deputy Group Leader Eleanor Anderson2 Ged Brady inform personalised cancer medicine; and b) to characterise and Sophie Atkinson1 chemoresistance mechanisms, including a and in two SCLC genetically engineered mouse Mahmood Ayub2 nitric-oxide activated pathway and acquired models (GEMMs) (Figure 3). Staff Scientists exploit our panel of circulating tumour cell (CTC) derived models of Samantha Barlow2 resistance by serial chemotherapy treatment of Alastair Kerr1 small cell lung cancer (SCLC) to discover new targets and test new Dominic Birch mice bearing CDX augments our SCLC ex vivo cultures often contain Elaine Kilgour Kieran Bradbury chemoresistance mechanism(s) search morphologically and phenotypically distinct cell Dominic Rothwell therapies. We developed our immune-oncology biomarker activities Nicholas Brittain2 (Figure 2). populations; neuroendocrine (NE) and non- Jonathan Tugwood Laura Booth2 and recruited Dr Alastair Kerr to lead the new Bioinformatics and neuroendocrine (NNE) cells grow in suspension Stewart Brown Associate Scientists Biostatistics (BBS) team. CBC’s highlighted study published in Nature Debbie Burt Vasculogenic mimicry in SCLC and as adherent monolayers respectively. Only Kris Frese Holly Butterworth Angiogenesis is a well characterised mechanism the minority NNE cells form characteristic VM Kathryn Simpson Medicine reported utility of ctDNA to inform selection of a patient’s Rebecca Carroll1 exploited by solid tumours to overcome limited networks when grown on matrigel (Figure 4 Phase I clinical trial and non-small cell lung cancer (NSCLC) relapse Mathew Carter blood supply and enable tumour growth. shown overleaf). In both GEMM and CDX Research Associate Alex Clipson Vasculogenic mimicry (VM) is an alternative, models, NNE cells remodel collagen in the Carlos Lopez-Garcia1 risk prediction by pulmonary vein CTC number at surgery. CBC James Copley1 tumour autonomous mechanism of de novo matrigel to form VM structures, indicating a role QA Manager contributed to Manchester’s success with the International Alliance Tony Price for Cancer Early Detection (ACED) and is supporting Cancer

Postdoctoral Fellows Research UK RadNet Manchester with biomarker sciences. Katarzyna Bloch CDX RPP Francesca Chemi 23 GEMM Adam Fletcher2 The biomarker portfolio Understanding SCLC biology and the search for NE NNE Human Mitochondria/ Ellyn Hughes1 CBC supported 19 clinical trials in 2019. The new therapy targets Sumitra Mohan Suspension Adherent DAPI pioneering CACTUS melanoma trial led by Despite multiple clinical trials, SCLC standard of Maria Peiris-Pages Ruth Stoney2 Professors Paul Lorigan and Richard Marais care remained unchanged for >3 decades until Stuart Williamson2 employs a CBC validated ctDNA assay primary the recent introduction of immunotherapy, CDX1 endpoint, reporting in real-time to instruct which benefits a minority of patients. Although 7 Bioinformaticians treatment switch from targeted- to phenotypic heterogeneity was reported 30 years Tine Descamps immunotherapy. CBC biomarker development ago, the World Health Organisation recognises a CDX RPM CD31/P RES SY NCA AS T P M Saba Ferdous also supports upcoming trials examining proton single morphological classification of SCLC. 25 GEMM Cheryl Jones CDX2 Bedirhan Kilerci1 beam therapy in the newly established Proton Molecular classification based on Katarzyna Murat1 Beam Therapy Centre at The Christie NHS neuroendocrine transcription factors (NE TFs, 1 Bethanie Neale2 Foundation Trust (CHFT). A CBC ‘first’ this year developed in 2019) may support future precision Simon Pearce involved transfer of our GCP ELISA for medicine. The Preclinical Pharmacology (PP) Matthew Roberts plasmaTie2 (pTIE2) to The CHFT Diagnostic team applied RNAseq to our 47 patient CTC William Rowe2 Biochemistry Laboratory for further validation derived explant models (CDX) and hierarchically Cong Zhou towards routine NHS use. pTIE2 dynamics inform clustered the data; principal component analysis Clinical Informaticians management of anti-angiogenic therapies, a revealed at least 4 subgroups, including those Jenny Bradford2 hypothesis examined within the CRUK funded expressing previously identified transcriptional Fouziah Butt VALTIVE1 trial in ovarian cancer; a culmination of drivers ASCL1, NEUROD1, and POU2F3 and a Figure 2. Comparison of acquired resistance developed in the patient and in the Figure 3. VM vessels in CDX and GEMMs. PAS+/CD31- VM Katherine Dempsey2 >10 years biomarker research with Professor new subgroup expressing ATOH1 (Figure 1). lab will reveal molecular mechanisms of drug resistance. vessels (pink/black arrows), CD31+ endothelial vessels (brown/ Figure 4 Paul Fitzpatrick Gordon Jayson with biomarker data modelling Roles of ATOH1 in tumour maintenance and/or red Figurearrows). RPP 3and RPM GEMMs are Rb/p53/p130 triple KO and Rb/p53 double KO with overexpression of c-Myc by the BBS team. progression and druggable downstream targets respectively. Black scale bar, 50 µm. are under investigation.

14 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE CANCER BIOMARKER CENTRE 15 CANCER BIOMARKER CENTRE (CONTINUED) funded trial of stage IIB/C melanoma patients CDX RPP that will involve ddPCR ctDNA analysis to detect early relapse/micro-metastatic disease and select 23 GEMM patients for targeted therapy. Joanne Felce NE NNE Human Mitochondria/ Alice Fickling2 ctDNA for the early detection of cancers Suspension Adherent DAPI Adam Freestone1 The NAB team continued to develop ctDNA Lynsey Franklin methylation assays and molecular barcoding Melanie Galvin Laura Glass1 approaches to increase assay sensitivity. Assay Molly Glenister-Doyle1 CDX1 evaluation is ongoing using our biobank of Weronika Golinska2 plasma samples from the Manchester Eleanor Gregory 7 Community Lung Health Study pilot with Dr Hannah Gregson2 Philip Crosbie (Manchester University NHS CDX RPM Grace Hampson CD31/P RES SY NCA Foundation Trust, MFT) where results of low dose Thomas Helps2 25 GEMM Rebekah Higgins2 AS T P M CT scans and cancer diagnoses are known. We Sarah Hilton2 are also developing nanoparticle-based Michael Hoffs CDX2 ctDNA and immune biomarkers in biopsies to approaches to enrich for ctDNA in collaboration Elizabeth Hunter1 1 Figure 5: assist selection to immunotherapy trials with Professor Kostas Kostarelos (The National Aileen Jardine Schematic of TARGET study. (Figure 5). Graphene Centre). Hana Jelassi Noel Kelso ctDNA informing treatment switching Minimal residual disease (MRD) monitoring in Luke MacGregor1 The CACTUS Trial (CirculAting Tumour DNA Simrah Mohammad NSCLC Figure 4. NNE cell undergo VM. Top row: VM networks in matrigel. Bottom row: IHC showing VM rich regions (PAS+/ Derrick Morgan gUided therapy Switch) for advanced cutaneous In collaboration with Dr Phil Crosbie and his CD31-, pink) co-localise with the NNE marker REST but not NE markers SYP and NCAM. Black scale bars, top row (bright Karen Morris melanoma phase II patients opened in May 2019. clinical team at MFT, set up of the COMPASS field), 500 µm, black scale bars, (IHC) and white scale bar (IF, 100 µm). Kamrun Nessa2 With PIs Professors Richard Marais and Paul study was completed and will be open to Anthony Oojageer Lorigan and Dr Rebecca Lee, the NAB team recruitment in January 2020. After surgery, Nicola Overton developed a primary endpoint, GCP validated, patients with community screen-detected Safwan Patel in sensing and adapting to the tumour dissemination and disease relapse. We droplet digital PCR (ddPCR) ctDNA assay NSCLC give monthly blood samples in their Rosalind Peason1 microenvironment. A deeper interrogation of enumerated PV-CTCs from 100 patients. Jackie Pierce measuring BRAF and NRAS mutations (variant community Lloyds Pharmacy for collection and cell intrinsic and environmental regulators of VM PV-CTC number was associated with lung Sabah Rashid1 allele frequencies) to instruct treatment switch transport to CBC laboratories in vans with the is underway. Figure 4 cancer specific relapse and remained an Figure 3 Alan Redfern from targeted- to immunotherapy. The expertise CRUK logo (Figure 6) to support development of Mitchell Revill independent predictor of relapse in multivariate with real-time reporting, developed during a liquid biopsy that detects relapsing disease Caroline Roberts Liquid biopsies for SCLC analysis adjusted for tumour stage. We CACTUS, is being applied to DETECTION earlier. Resected tumour tissue and nasal Jordan Roebuck The Nucleic Acids Biomarkers (NAB) team performed molecular profiling of PV-CTCs to (Circulating tumour DNA guidEd Therapy for epithelium (as a surrogate tissue) are collected at Karishma Satia developed a molecular profiling and disease query genetic links between PV-CTCs, the Figure 6: stage IIB/C BRAF or NRAS mutant- positive Daniel Slane-Tan surgery to seek early prediction of relapse. Nasal monitoring liquid biopsy workflow to support resected tumour and subsequent metastasis. In The COMPASS MRD study. Karen Smith1 mElanoma after surgiCal resecTION), a CRUK samples will be analysed in collaboration with targeted treatment of SCLC. We examined a case study, there was a higher mutation Nigel Smith Professor Avrum Spira, Boston University Medical George Thompson2 genome-wide copy number aberrations (CNA) overlap between PV-CTCs and metastasis Centre by our CRUK Lung Cancer Centre of Isabelle Thompson1 and targeted mutation analysis of 110 SCLC detected 10 months later (91%) than between Diagnosed with NSCLC Excellence (LCCE)-funded travelling postdoc, Dr Simon Topham associated genes, including those within DNA metastasis and primary tumour (79%), Kate Bloch in the Spira laboratory. Tumour and Erich van Hechnova2 damage repair pathways. We applied this suggesting that early disseminating PV-CTCs Listed for lung cancer surgery blood sample analysis will be performed in Amelie Viratham2 workflow to pre-treatment cfDNA from patients were responsible for disease relapse (Chemi et Hannah Walsh collaboration with Professor Charlie Swanton’s with extensive-stage (ES) or limited-stage (LS) al, Nat Med, 2019). We also examined TP1 Recruited to study Daniel White team at University College London within the SCLC to establish sensitivity. Tumour-related genome-wide CNA from 70 single PV-CTCs LCCE. With Dr Santiago Zelenay we will assess the changes (CNAs and/or somatic mutations) were compared to matched tumours. Two distinct Hospital Arm Community Arm Junior Translational Scientist Screen (n=100) Screen (n=50) prognostic significance of his COX2 Non-screen (n=50) Simon Hood1 detected in >93% patients with potentially populations of PV-CTCs were identified; ‘Type A’ Adjuvant inflammatory signatures in early stage tumours. chemo targetable mutations in >50% cases (Mohan et al, showed clear CNA changes in common with the TP2 Lung cancer Lung cancer TP4 to 25 surgery surgery Laboratory Manager JTO, 2019). In multivariate analysis, CNA analysis tumour, ‘Type B’ did not match the tumour. We Community blood monitoring programme Compare Blood sample every 4-6 weeks Biomarkers to inform immunotherapy trials Matthew Lancashire and mutation number significantly associated now need to elucidate the origin of ‘Type B’ cells clinical and TP3 Post surgery Post surgery Year 1 Year 2 blood based The Cells and Proteins (CAP) team expanded their clinic clinic detection of with disease stage. We explored utility of cfDNA and ask whether and how they participate in SoC post-surgical surveillance lung cancer Laboratory Support Technician 3 monthly hospital or telephone clinic ± imaging core immune biomarker ‘toolkit’. New assays analysis for disease monitoring; CNA profiles early metastatic dissemination. recurrence Andrew Stevens SoC post-surgical QoL questionnaire (6 + 12 months) include T-cell receptor sequencing (TCRseq) with showed dynamic changes through therapy to COMPASSsurveillance MRD study BRC Project Manager disease relapse. We are currently extending ctDNA to assist selection of Phase I clinical trials Jonathan Wake longitudinal analysis to 130 patients to establish if TARGET Trial (Tumour chARacterisation to Guide our approach can become a routine patient- Experimental Targeted therapy) recruitment with Programme Manager monitoring tool in the clinic. monthly Molecular Tumour Board meetings is Ekram Aidaros-Talbot ongoing with integration of clinical data, tumour Administrative Coordinator Non-small cell lung cancer (NSCLC) CTCs at mutation profiling and contemporaneous Figure 6 Suzanne Bickley surgery to predict disease relapse and explore ctDNA data utilising our visualisation tool, tumour evolution eTARGET. 1 Joined in 2019 Within the TRACERx consortium trial (TRAcking 2 Left in 2019 non-small cell lung Cancer Evolution through Feasibility stage A (100 patients) was published therapy [Rx]), we addressed whether CTCs in the (Rothwell et al, Nat Med, 2019). Stage B is pulmonary vein (PV-CTCs) of early stage NSCLC ongoing with >420 patients’ ctDNA analysed. patients are responsible for metastatic We are adding tumour mutation burden in Lloyd Pharmacy locations

16 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE CANCER BIOMARKER CENTRE 17

Figure 6 CANCER BIOMARKER CENTRE (CONTINUED)

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‘Technology’ clinical trials the NAB team, multiplex IF assays to landscape portfolio of chromogenic assays (>20 The dECMT also established several ‘technology’ Figure 7: Figure 7 the tumour microenvironment with the Tissue antibodies) enabling more detailed interrogation Plasma cytokine dynamics levels Biomarker team, and assays for PDL1 in CTCs. In of SCLC subtypes based on NE TFs, MYC family from a patient after T-cell infusion partnership with ThermoFisher, we installed the proteins (Figure 8), VM and acquired therapy. IonTorrent platform for immune biomarker chemoresistance in CDX models. The TB team is assays including TCRseq for analysis of clonality, developing tissue biomarkers applicable to Figure 8: diversity and convergence. With Professor Fiona patient biopsies within clinical trials of MYC family proteins in a SCLC CDX. Blackhall, we are assessing CTCs, ctDNA and immunotherapies linking with the CAP team. MYC (green), MYCL (red) and DAPI TCRseq in the first SCLC patients receiving The TB team will merge with the CHFT (blue), white scale bars, 100 µm. immunotherapy at the CHFT. Tissue and Academic Pathology Unit in 2020, circulating immune assays are being bringing a wealth of pathology expertise to assist implemented in melanoma, renal cancer, cancer with CBC goals. of unknown primary and extra pulmonary-neuro endocrine tumour patient studies. Bioinformatics and biostatistics The Bioinformatics and Biostatistics (BBS) team is Ex vivo co-cultures of matched peripheral blood involved in multiple collaborations and runs a lymphocytes and CTCs from SCLC patients are weekly statistics clinic to assist medical students under development as a potential platform to and clinicians (15 projects in 2019). In an ongoing predict immunotherapy responses and explore collaboration, the angiogenesis biomarker resistance mechanisms. Within iMATCH plasma Tie2 was examined in three clinical trials (Innovate Manchester Advanced Therapies culminating this year in CRUK funding to Centre Hub), we validated a multiplex ELISA translate it from GCP to routine NHS use. BBS assay to monitor cytokine storms in patients supports experimental design, for example: the receiving advanced T-cell therapies with NOTION study with digital ECMT to assess Professor Fiona Thistlethwaite (CHFT, Figure 7), feasibility of at home blood sampling to monitor and with Dr Phil Monaghan (CHFT Diagnostic patients treated with immunotherapy; TRACERx Biochemistry Laboratory) we will transfer and NEO within the LCCE to continue exploration of further develop this assay for routine clinical use. NSCLC evolution; and the EPIC study with Phase I Trialists at CHFT to access impact of patients’ With the digital ECMT, we are evaluating physiological status on clinical outcome. The feasibility of home blood sampling kits to assess BBS team implemented best practice pipelines cytokines as an early warning of adverse events including quality control metrics and is working and cytokine release syndrome in patients with CRUK MI Scientific Computing to enable receiving immunotherapy and advanced T-cell use across the Institute. The bespoke pipeline for therapies. the TARGET trial was upgraded to include the new MuTec2 mutation caller and new pipelines Tissue biomarkers for preclinical research and are in development, e.g. for the NAB team’s new clinical trials T7 workflow helping shape experimental The Tissue Biomarker (TB) team expanded their decisions and codifying robust analysis.

18 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE CANCER BIOMARKER CENTRE 19 www.cruk.manchester.ac.uk/Our-Research/Cancer-Inflammation-and-Immunity Stromal region triple-negative breast cancer. Profiting from a TILs TILs Response High pump2 priming grant from the Manchester B7−H3 FOXP3 Low PDL1 VEGFA 1 Response FAS PD1 BiomedicalpCR Research Centre, we have subjected CD74 0 HLA−DQA1 pMRD HLA−DRB CD11c −1 pNR CD68 tissue microarray to a pioneering methodology CCND1 CD11b −2 CMKLR1 STAT3 AKT1 CANCER INFLAMMATION HIF1A to obtain both quantitative protein and gene PTEN STAT2 Tim3 BCL2 CD4 expression levels and spatial context on a single CD86 CSF1R IFNGR1 TBX21 CD40 tissue section (Figure 1). The data has provided CXCL10 AND IMMUNITY PECAM1 4−1BB GZMB NKG7 CCL5 new insights into the inflammatory and immune IDO1 TIGIT CD3E CD8A ICAM1 tissue biology of the tumour microenvironment CD44 IFNAR1 ITGB2 CD27 CD47 PSMB10 in a cancer type of dismal prognosis in which an CXCL9 STAT1 B2M HLA−E CD45 effective treatment remains an urgent unmet VISTA Tumour bed medical need. Basic research in immunology has led to the development of TILs TILs Response High 2 B7−H3 FOXP3 Low PDL1 VEGFA 1 Response FAS practice-changing treatments such as those based on the use of PD1 pCR CD74 In0 parallel, we continue our efforts to identify HLA−DQA1 pMRD HLA−DRB CD11c −1 pNR CD68 CCND1 therapeutical targets to use in combination with CD11b −2 immune checkpoint inhibitor blocking antibodies. These therapies CMKLR1 STAT3 AKT1 HIF1A immunotherapies based on immune checkpoint PTEN STAT2 Tim3 have led to unprecedented responses in patients with late-stage BCL2 CD4 CD86 inhibitors. Aligned with our postulate that certain CSF1R IFNGR1 TBX21 CD40 CXCL10 inflammatory responses prevent effective cancers. While they continue to be tested across multiple PECAM1 4−1BB GZMB NKG7 CCL5 immunity at the tumour bed, we combined IDO1 TIGIT malignancies and in different clinical settings, it has become evident CD3E CD8A ICAM1 CD44 immune-based therapy and standard cancer IFNAR1 ITGB2 CD27 Group Leader CD47 that for nearly all cancer types only a very reduced fraction of PSMB10 treatments with specific anti-inflammatory drugs. CXCL9 STAT1 B2M HLA−E CD45 Santiago Zelenay patients experience profound and long-lasting responses. Much VISTA Using mouse models and various types of drugs more often than not patients derived transient or no benefit at all. administered in a clinically relevant manner, we Postdoctoral Fellows we have identified a limited set of inflammatory have made significant progress in teasing apart Eduardo Bonavita Figure 1 factors that can be used to define type of the underlying mechanistic basis for one of our Agrin Moeini Classifying tumour and stromal inflammatory landscape. A group of these most promising combinations. Victoria Pelly The Cancer Inflammation and Immunity group players involved in anti-cancer immune studies the principles that determine outcome responses, we continue our search for regions in the triple-negative factors, routinely co-expressed across all cancer breast cancer microenvironment. Scientific Officer types, promote immune evasion, therapy This fundamental analysis in mouse models in from immunotherapy with a focus on the instructive pathways that influence the Nuclear (blue), pan-cytokeratin Shih-Chieh Chiang resistance and progressive growth. Others by conjunction with the mining of large publicly underlying mechanisms that favour immune inflammatory profile and immunity at the (green), CD45 (red) and CD3 escape and treatment failure. Combining basic tumour site. (yellow) staining of a tissue contrast, instigate a cancer-restraining response available datasets of cancer patients has provided Graduate Students and translational research, our work has microarray containing cores from and mediate spontaneous and therapy-induced the basis for the design of two clinical trials. Both Charlotte Bell pre-treatment, needle biopsies immune-dependent control of tumours. Of led by Dr Anne Armstrong, Consultant Medical Christian Bromley uncovered broadly conserved pathways In this context, a qualitative step forward during from patients with triple-negative special interest, translating our results to human Oncologist from The Christie NHS Foundation Eimear Flanagan associated with pro-tumourigenic inflammation last year was the successful incorporation into breast cancer. (A), (C) and (E) and immune evasion. Monitoring the activity of our set of research tools of cutting-edge cancer we have found evidence that the Trust specialising in breast cancer, the trials will show the merged multiplex inflammatory status of baseline samples test the combination of non-steroidal anti- these inflammatory pathways in pre-treatment methodologies that allow a comprehensive immunofluorescence image of inflammatory drugs with immune-checkpoint patient biopsies shows promise as an indicator profiling of the tumour immune three patient cores, and (B), (D) monitored by transcriptional profiling of a of treatment outcome. Moreover, their microenvironment at the single cell level or and (F) highlight algorithmically- selected number of mediators has profound inhibitors. The first one, which will imminently therapeutical manipulation represents a enable multiplexing with spatial resolution. defined regions of tumour bed prognostic utility. Likewise, these inflammatory start recruiting patients with newly diagnosed promising approach to augment the efficacy of Collectively, the use of these technologies has and stroma. Using the Nanostring signatures predict outcome from triple-negative breast cancer, will test in a immunotherapy and other cancer treatments provided further support to our general working GeoMx platform we obtained immunotherapy in many tumour types and neoadjuvant setting the combination of transcriptomic profiles from these that rely on the anti-cancer function of the paradigm that the inflammatory response at the compare favourably when benchmarked against high-dose aspirin, a widely-used anti- different tumour current established biomarkers of response to inflammatory drug, with a checkpoint inhibitor immune system. tumour site is a vital determinant of tumour fate microenvironment avelumab. The primary objective of this trial is to and response to immunotherapy. Our data compartments. Bioinformatic immune checkpoint inhibitors. One of these Already recognised as pillars of oncology suggests that certain immune cells, even when methods were then used to gene signatures and the scoring method to determine whether co-administering aspirin with treatment, immune checkpoint drugs have classically present in very low frequency within integrate data from digital establish it from transcriptional data have been immune checkpoint inhibitors can switch the transformed the landscape of cancer tumours, can have a major effect in the overall pathology analysis, and from patented by Cancer Research UK Technologies inflammatory profile of tumours towards one that management. In cutaneous melanoma, the activation and polarisation status of multiple spatially-distinct transcriptomic Ltd. A remarkable feature is that its composition in is more favourable to T cell-mediated tumour profiling. deadliest form of skin cancer, these treatment populations, including those that are much terms of the individual gene elements was control. In the second one, for which we have modalities have become the standard of care for more prevalent intratumourally. Likewise, we defined through the characterisation of our recently secured funding from the J P Moulton patients with advanced disease, and specific have often encountered situations in our mouse genetically-engineered transplantable mouse Charitable Foundation, we will investigate combinations of checkpoint inhibitor drugs models in which, at particular points in time, the models. Yet, the gene signature predicts whether addition of anti-inflammatory drugs to result in a five-year survival rate for more than cellular immune composition of tumours can be responses in human cancers highlighting the standard of care immune-checkpoint blockade half of patients. However, the fraction of remarkably similar and yet the molecular conserved features and parallelisms of mouse enhances the efficacy of this treatment. This will complete and durable responses is much lower landscape is radically different. Of note, the latter and humans. be an exciting basket trial that will focus on three for the overwhelming majority of tumour types. more robustly anticipate the fate of those tumour types, triple-negative breast cancer, In this context, the greatest challenges in the tumours exposing the limitation of approaches Encouraged by these exciting findings, we are non-small cell lung cancer and clear cell renal immunotherapy field are the lack of effective that merely aim at monitoring the relative refining our inflammatory signature using cancer. Crucially, these three tumour types are, alternative treatments for non-responders and abundance of immune cells without establishing machine learning approaches. This bioinformatic according to our bioinformatic analysis, of suitable biomarkers to predict who those their activation status through molecular analysis has, in turn uncovered that the approach particularly promising for manipulation of the patients might be. This is particularly relevant profiling. is particularly prognostic for certain tumour types inflammatory response. This trial also constitutes given immunotherapy is very costly and has side such as lung, breast or kidney cancer. Therefore, an ideal setting to test prospectively the utility of effects, often leading to the development of These analyses combined with that of patient we are now analysing in further detail these three our inflammatory signature and to start serious toxicities even in patients with tumours datasets provided further evidence to our theory tumour types expanding our research portfolio developing it as a biomarker with diagnostic that do not respond. Under the conviction that that there is no such thing as ‘inflamed’ or considerably. Notably, we have very recently built utility. solutions to these problems require further basic ‘non-inflamed’ tumours but rather different and analysed a tissue microarray of baseline understanding of the cellular and molecular types of inflammatory ‘flavours’. In this regard, biopsies of patients newly diagnosed with

20 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE CANCER INFLAMMATION AND IMMUNITY 21 www.cruk.manchester.ac.uk/Our-Research/Cell-Division Figure 2 activity was inappropriately elevated by the abolition of PP1 recruitment to Cut12. Enhanced Polo activity probably overcomes the need for Cdc25 because it boosts Polo’s ability to inhibit CELL DIVISION Wee1 to such a degree that it completely silences Wee1. Then, without the kinase putting the phosphate onto Cdk1, there is no need for the phosphatase that normally reverses the missing phosphorylation.

Cdk1-Cyclin B activation promotes the activities The inappropriate proliferation of cancer cells can arise from of an array of mitotic protein kinases. The ensuing wave of mitotic phosphorylation drives unchecked cell division, a failure to engage cell death pathways, or chromosome condensation and the formation simultaneous changes in both. Understanding how the diverse cues and function of the division apparatus. Once “ induced replicative stress (OIRS)” in each phase of mitosis is complete, most are integrated to co-ordinate cell division and death is therefore key Figure 1: which parts of the genome are under-replicated phosphorylation events must be reversed to to understanding the biology of cancer. DNA damaging and anti- Feedback control by Polo kinase while others are amplified. OIRS increases support transition to the next phase of division, mitotic therapies owe much of their success to the checkpoint in Cdk1-Cyclin B activation at the reliance on the DNA integrity checkpoint otherwise cells remain trapped in mitosis. As a G2/M transition controls. The application of further damage, protracted mitotic arrest can trigger death, there Cdk1-Cyclin B activity is held in Group Leader pathways that ensure transition through the cell division cycle only through local radiation or systemic application of check in interphase as a is great interest in targeting the mitotic exit Iain Hagan occurs when genome integrity is guaranteed. consequence of phosphorylation DNA damaging chemotherapy, can therefore phosphatases to invoke such a mitotic arrest. The of Cdk1 by Wee1. Cdc25 removes overwhelm these checkpoint controls in tumour OIRS induced chromosome aberrations of Associate Scientist the inhibitory phosphate to cells to induce a catastrophic division while tumours perturb chromosome segregation in Agnes Grallert trigger mitosis. This trigger level We study the targets of two of these Passage through these key transitions is driven neighbouring, normal cells simply delay their mitosis to delay mitotic progression. This delay of Cdk1-Cyclin B then activates division. DNA damaging chemotherapeutic makes tumours more vulnerable to further delays Postdoctoral Fellows therapeutically important checkpoint pathways: by the activation of distinct CDK-Cyclin protein polo kinase to further boost agents and irradiation are therefore among the arising from mitotic phosphatase inhibition than Daniela Eckert the commitment to and the exit from mitosis, kinase complexes. The G2/M transition is a Cdc25 activity and inhibit Wee1 to Zoe Edwards the physical process of genome segregation. critical safeguard of genome integrity; make this transition a bi-stable most successful therapies in the clinic. This normal tissue. Lenka Halova Because the regulatory networks that control incomplete DNA replication or DNA damage switch between two distinct success has prompted intense interest in the Cynthia Li1 cell division are highly conserved, we triggers checkpoint pathways that block the states. Our work shows that prospect of compromising checkpoint controls PP1 and the protein phosphatase 2A isoforms Wei Ma complement our studies of cell division in G2/M transition to ensure that chromosomes are signalling events on the fission in tumour types that are currently radio and PP2A-B55 and PP2A-B56 play key roles in driving Pawan Singh yeast equivalent of the chemo resistant. A lead agent in this quest is the mitotic exit. PP1 acts as a monomer, whereas Neslihan Zohrap1 human cell lines with manipulation of the not segregated when incomplete or damaged. centrosome, the spindle pole Wee1 inhibitor AZD1775. PP2A enzymes are hetero-trimers comprising simple, unicellular, fission yeast in order to body, determine when this switch single scaffolding and catalytic subunits, Scientific Officers identify the key questions to ask of the The G2/M transition is driven by activation of the is flipped to trigger division. Keren Dawson1 analogous controls in the complex context of Cdk1-Cyclin B protein kinase. Wee1 related While AZD1775 is rapidly progressed through to alongside one of four different types of regulatory Eleanor Wendy Trotter human cell division cycle control. kinases inhibit Cdk1-Cyclin B during interphase Figure 2: clinical trials, we still know remarkably little about subunit. Multiple, alternatively spliced genes give by phosphorylating the catalytic Cdk1 subunit. The mitotic PP1-PP2A the basic biology of the three Wee1 family kinases the potential for hundreds of variants of Graduate Students phosphatase relay In a typical cell division cycle the G1 gap phase Removal of this phosphate by Cdc25 in the cancer cell cycle. A full understanding of PP2A-B55, PP2A-B56 in humans, whereas fission Zoe Lee PP1 and PP2A activities are all the biology of Wee1 family kinases will be key to yeast can live on one of each, or in the case of Millie Jones precedes DNA replication in S phase, before a phosphatases then promotes mitotic entry. A repressed upon entry into mitosis. identifying the optimal context for Wee1 PP2A-B55, none. second gap phase, G2, separates S from trigger level of Cdk1-Cyclin B activation The mode of PP2A repression is 1 inhibition in the clinic. We are therefore Joined in 2019 genome segregation in Mitosis (M phase) (Figure promotes a positive feedback loop that boosts unclear, however it is well 1). Growth, developmental and environmental Cdc25 and inhibits Wee1 activities to ensure that established that Cdk1-Cyclin B addressing when, where and how, these kinases After confirming that PP1, PP2A-B55 and cues determine the timing of progression mitotic commitment is a rapid and irreversible phosphorylation represses PP1 are used in human cancer cells. PP2A-B56 activities decline upon mitotic through both the point of commitment to the switch from one state (interphase) into another activity. Cyclin B destruction then commitment in fission yeast, we found that direct allows PP1 itself to auto- cell cycle in G1 phase, known as the “Restriction (division) (Figure 1). Mitotic commitment is The initial appearance of active Cdk1-Cyclin recruitment of PP1 to PP2A-B55 and PP2A-B56 catalytically remove this B on human centrosomes, before propagation re-activates these PP2A phosphatases to support Figure 1 Point”, and the transition from G2 into M. blocked when DNA replication is incomplete, or inhibitory phosphate from itself. throughout the cell, suggests that the appropriate mitotic progression/exit. Mitotic DNA has been damaged by checkpoint As PP1 is bound to the B55 pathways that boost Wee1 activity and inhibit regulatory subunit of PP2A-B55 at centrosome provides a specific inhibition of PP1 arises from direct Cdc25. this time, PP1 reactivation microenvironment to trigger the G2/M transition. phosphorylation by Cdk1-Cyclin B. The immediately restores PP2A-B55 Our studies of the fission yeast centrosome destruction of Cyclin B subsequently allows PP1 We study three core aspects of cell cycle control: activity. In contrast, PP2A-B56 is equivalent, the spindle pole body (SPB), provide to auto-dephosphorylate and restore its own unable to recruit PP1 because the biology of the Wee1 family of kinases that molecular insight into how and why this switch phosphatase activity. Reactivated PP1 then Polo kinase phosphorylates a may operate. We have shown that release of reactivates PP2A-B55. Polo phosphorylation of restrain entrance into division in response to residue within the PP1 docking Cdk1-Cyclin B or Polo kinase activity at the SPB the PP1 docking site of PP2A-B56 initially blocks stress; the role played by the centrosome in site on the regulatory B56 determining when cells commit to genome subunit. Once Polo activity will drive cells into division. In contrast, release of PP1 binding to PP2A-B56. When Polo activity segregation; and the regulation of the mitotic declines at the end of mitosis, either kinase activity at any other location around declines in mitotic exit, PP2A-B55 exit protein phosphatases whose activities PP2A-B55 can overcome Polo the cell has no impact upon division timing. Our dephosphorylates the Polo phosphorylation site activity towards this site and counteract those of the pro-mitotic kinases in attempts to define the molecular basis for such a on B56 to allow PP1 to reactivate PP2A-B56 remove the inhibitory phosphate order to drive cells out of division. striking impact have been guided by lessons from (Figure 2). We are now assessing the impact of from the PP1 docking site of B56. the SPB scaffold Cut12. Simply blocking the phosphorylation on PP2A-B55 function and Consequently, PP1 can be Tumour cells accumulate mutations to avoid the recruited to PP2A-B56 and this recruitment of protein phosphatase 1 (PP1) to seeking the phosphorylation events on PP2A + normal restraints upon passage through the second PP2A activity is Cut12 enabled us to delete the cdc25 gene complexes that are reversed by PP1 recruitment restriction point. This adaptation often generates reactivated at the end of mitosis. without compromising viability. This bypass of to restore PP2A-B55 and PP2A-B56 activities. abnormally high Cdk-Cyclin activities that Reprinted by permission from the requirement for an otherwise essential Macmillan Publishers Ltd: Nature perturb the control of DNA replication. mitotic inducer arose from the impact of the Publications listed on page 65 517:94-98, copyright 2015. Consequently, tumour cells invariably display Cut12/PP1 axis on Polo kinase activity. Polo

22 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE CELL DIVISION 23 www.cruk.manchester.ac.uk/Our-Research/Cell-Plasticity-and-Epigenetics CELL PLASTICITY & EPIGENETICS

During neoplastic development, cancer cells are constantly exposed to changing conditions within their niche of origin and further within the ecosystems encountered whilst invading novel tissues. This fast-paced environment drives a micro-evolutionary process that gives rise to distinct sub-clones within a tumour, ultimately leading to increased heterogeneity along the course of the disease. Institute Fellow Interestingly, this variability can either arise as a result of somatic Maximiliano Portal mutations acquired during tumour development or may be

Postdoctoral Fellow established due to the ability of a single genotype to produce many Yelizaveta Shlyakhtina discrete, sometimes dramatically different, phenotypes. In this

Graduate Student context, and in contrast to genetic heterogeneity, non-genetic remarkably predict which cells in the clonal clone/cluster dependent indicating that the Katherine L Moran variability may provide a dynamic source of diversity supporting the Figure 1: population will grow steadily in 3D cultures and information obtained from our single cell assays Scheme illustrating the which will fail to do so. Importantly, we have can be used as proxy to predict phenotypic rapid adaptation of cancer cells during numerous environmental phenotypic diversity found in shown that cells in a clonal population have the variations. clonal cell populations. perturbations. In our lab, we study the generation and inheritance of We have shown that populations capability to “migrate” from different phenotypic non-genetically encoded molecular traits with the aim to unravel generated from the clonal states, in some cases following constrained Ultimately, our work will develop our amplification of a single cell paths, suggesting that phenotypic diversification understanding of cellular heterogeneity and their role in the cellular response to diverse cues, such as oncogene exhibit significant phenotypic might be under strict molecular control. Notably, plasticity, and expand on the concept of transformation and the challenge with therapeutic agents. diversity, despite all cells being though we have shown the existence of non-genetic information. Importantly, it has the genetically identical. Multimodal predetermined phenotypic paths, we observed potential to reveal the molecular mechanisms analysis of these cells reveals that that cells are constantly switching from underlying these phenomena which remain there are multiple phenotypic phenotypic states thus giving the population as a largely undiscovered. A greater understanding of It is becoming increasingly apparent that variability at various levels, including the ability to metastable states present in whole an enormous evolutionary potential. cellular heterogeneity and plasticity could also individual cells within a clonal population show grow as 3D spheres and their capacity to these populations and that cells can move between states over Indeed, further analysis of individual HA1ER revolutionise how we think about evolution and significant heterogeneity, particularly in their develop resistance to cytotoxic agents. time, thus highlighting the clones by mass spectrometry show striking cancer evolution in particular, allowing the ideas response to stimuli. Strikingly, this heterogeneity Interestingly, further sub-cloning of the original dynamic nature of this system. differences between clones at the protein level of Lamarck and Darwin to be finally brought is present despite there being no genetic HA1ER cell line gives rise to populations of cells thus supporting our early observations and together into a single unified theory. variability in the population, leading us to the that only partially recapitulate the phenotypic suggesting that the observed differences at the hypothesis that the observed phenotypic heterogeneity of the parental one. In this light, transcriptome level are actually translated into differences instead rely on non-genetic the observed phenomenon may (i) either discernible phenotypes. These striking results information. Interestingly, the idea of account for temporal dynamic fluctuations in suggest that, in clonal populations, phenotypic non-genetic information and its transfer is not gene expression patterns that may cause lineage diversity is encoded at different levels other than new. Inheritance of acquired characteristics has switching – the ability of a cell to change its the “genetic” one and highlights the relevance of been a theory put forward by many scientists, molecular properties and convert to a distinct non-genetic mechanisms of adaptation to including Darwin and Lamarck, long before the phenotypic state; or (ii) represent the environmental cues. field of epigenetics was established. However, establishment of stable epigenetic states that the identity of non-genetic information carriers can be propagated over time. To explore which Following our observations, the main goal of our and potential mechanisms of information of the above possibilities could explain the lab in the upcoming years is to unravel the key transfer remain unclear. Under these premises, observed phenomenon, we developed an molecular players and mechanisms that recent technical and conceptual developments experimental/bioinformatics pipeline that orchestrate the establishment, maintenance and from our lab lead us to postulate that enables the reconstruction of cellular lineage temporal propagation of stable phenotypic states non-genetic information plays a key role in trajectories whilst concomitantly providing the within isogenic populations and investigate their maintaining phenotypic diversity. Indeed, we individual transcriptome of each cell in a single potential role in the emergence of resistance to have recently shown that individual cells step. By taking advantage of our technical therapeutic agents. In that regard, we have obtained from a clonal population (genetically development we were able to show that already gathered preliminary data that suggest, in identical cells) of RAS(G12V)-transformed cells RAS-transformed cells harbour up to 10 a fractional killing setting induced by the (HA1ER-cells) inherently display phenotypic alternative phenotypic metastable states that cytotoxic agent TRAIL, the apoptotic response is

24 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE CELL PLASTICITY & EPIGENETICS 25 www.cruk.manchester.ac.uk/Our-Research/Cell-Signalling CELL SIGNALLING

The small GTPase RAC regulates many normal cellular processes, but it can also enhance the formation and progression of cancers via mutation, overexpression or altered regulation. The aim of the Cell Signalling laboratory is therefore to find RAC-dependent Our recent data also demonstrated a role for of specific GEFs, which occurs commonly in targets, which are specific to tumour cells, and hence provide Figure 1. perinuclear RAC in the regulation of a subset of many cancers, can therefore drive different new therapeutic avenues without disrupting the necessary Model describing the role of the actin cytoskeleton known as the perinuclear oncogenic signalling pathways. CASK in spindle orientation. actin cap – thick actin bundles that run over the Group Leader physiological roles of RAC in healthy cells. CASK localises to the cell cortex, nucleus, constraining its height, guiding nuclear Role of spindle orientation in normal and where it recruits Dlg1, which in Angeliki Malliri orientation and facilitating cell migration. The abnormal epithelial cell divisions turn localises the core spindle RAC GEF STEF/TIAM2 localises to the outer In epithelial cells, the angle of cell division is Postdoctoral Fellows RAC is a cellular switch. When bound to GTP, nucleus, and at the nuclear membrane – that orientation components of LGN nuclear membrane and is required for controlled so that newly generated daughter Pauline Jeannot2 RAC binds target proteins, stimulating a range of stimulate radically different processes. and NuMA to the lateral cell membrane. There they bind maintenance of the actin cap by activating RAC. cells sit side-by-side in the epithelium and in 3D William McDaid1 cellular processes including adhesion, migration, Understanding which are pro- and anti- astral microtubules, correctly Depletion of STEF led to reduction in myosin- produce a sheet of cells surrounding a single Matteo Menotti2 division and transcription. Guanine Nucleotide tumourigenic will help determine how Aishwarya Payapilly2 orienting cell division. Disruption generated tension at the nuclear envelope, hollow centre. The angle of cell division Exchange Factors (GEFs) facilitate the switch to a aggressive or responsive to treatment particular of the interaction between CASK decreased nuclear stiffness, and ultimately depends on the orientation of the mitotic GTP-bound form from an inactive GDP-bound tumours are. and Dlg1 causes mislocalisation Scientific Officers reduced TAZ-regulated genes, due to changes in spindle. In turn, this is controlled by membrane- form. Mice lacking the RAC GEF TIAM1 were of this complex, leading to Gavin White mechanotransduction. Targeting an activated localised pools of NuMA and LGN, which bind Andrew Porter highly resistant to skin and intestinal tumour TIAM1 localises to centrosomes during mitosis, misorientation of the mitotic mutant of RAC specifically to the perinuclear astral microtubules emanating from the poles of formation induced by activation of RAS, a helping to build the mitotic spindle required for spindle. Reproduced with permission from Porter et al, region restored the actin cap in STEF-deleted the mitotic spindle. We recently established that Graduate Students common oncogenic driver. However, the few chromosome segregation. TIAM1 balances the Journal Cell Science, 2019. cells. CASK, a novel TIAM1 interactor, is a regulator of Ryan Guilbert tumours that did form were more aggressive. outward force of the motor protein Eg5, whose Joe Maltas mitotic spindle orientation (Porter et al. J Cell Sci This highlights two distinct roles for RAC effect is to increase centrosome separation. For Hannah Reed We are continuing to study the role of both 2019). We found that CASK directly interacts with signalling: stimulating tumour formation, and this function, TIAM1 needs to be phosphorylated Joshua Searle1 nuclear and perinuclear RAC signalling in and localises Dlg1, a tumour suppressor, which suppressing malignant progression. Our work by CDK1 leading to activation of RAC/PAK migration of non-small cell lung cancer (NSCLC) in turn recruits LGN and NuMA. Expressing an 1 Joined in 2019 has demonstrated how the balance between signalling. Eg5 is a potential therapeutic target as 2 cells. Lung cancer is the third most common interfering peptide and blocking the Left in 2019 these two roles is determined by the action of its inhibition leads to monopolar spindles and cancer in the UK and with over 47,000 new cases endogenous interaction between CASK and different GEFs acting at different cellular mitotic arrest; our findings suggest that loss of per year is the most common cause of cancer Dlg1, mislocalised NuMA and led to misoriented locations. TIAM1 could cause resistance to Eg5 antagonists. death. Finding new treatments for late stage cell divisions. Moreover, depletion of Dlg1 led to Our recent unpublished work has uncovered a cancers (where the 5 year survival rate is just 3%), misoriented cell division, and could only be The outcome of RAC activation depends on new role for TIAM1, regulating the duplication of and preventing early stage cancers from rescued by Dlg1 which was capable of binding sub-cellular localisation centrioles, structures at the core of each spreading, are therefore high priorities. CASK. Further, depletion of CASK in 3D culture Much early work on TIAM1 focused on its role in centrosome. Cells normally duplicate centrioles Moreover, TIAM1 and STEF/TIAM2 both contain led to the formation of multilumen structures, a strengthening cell-cell junctions, associated only once per cell cycle. Centriole a RAS-binding domain and are considered hallmark of misoriented cell divisions, which with anti-migratory effects. TIAM1 present at overduplication is common in many cancers, effectors of RAS. KRAS, one of the RAS family were reminiscent of early stages of epithelial cell-cell junctions is degraded following however, promoting aneuploidy. We observe proteins, is frequently mutated in NSCLC. We are cancers, such as ductal carcinoma in situ (DCIS), phosphorylation by the oncoprotein SRC or centriole overduplication, lagging currently looking at the role of TIAM1 and STEF/ an early form of breast cancer. This suggests that treatment with hepatocyte growth factor, chromosomes and aneuploidy in normally TIAM2 in KRAS-induced lung tumourigenesis. mutation or deletion of CASK may promote facilitating dispersal of epithelial cells. chromosomally stable cells following TIAM1 neoplastic transformation. Accordingly reduced depletion, which may explain why loss of TIAM1 Different RAC GEFs can activate RAC with CASK expression correlates with worse The precise localisation of TIAM1 at cell expression promotes malignant progression. different consequences prognosis in a number of cancer types, including junctions is important. We showed that a TIAM1 RAC GEFs are often multi-domain proteins with breast cancer. interactor, 2-Syntrophin, recruits TIAM1 to We are also continuing to study the role of β many binding partners. We recently showed adherens junctions, establishing a gradient of nuclear TIAM1, which we first described in that GEFs themselves can determine the Publications listed on page 65 RAC activity which diminishes apically. This colorectal cancer cells. Nuclear TIAM1 inhibits outcome of RAC activation. TIAM1 and P-REX1 gradient is important for tight junction migration of colorectal cancer cells by have diametrically opposite effects on cell formation. Artificially activating RAC at tight preventing the transcriptional co-activator TAZ migration through RAC: TIAM1 promotes junctions led to weaker cell-cell adhesions and from binding with its partner cell-cell adhesions to oppose cell migration disruption of normal epithelial architecture. TEAD. Interestingly, patients with high nuclear while P-REX1 actively promotes migration. TIAM1 showed significantly better survival than P-REX1 binds to Flightless-1, which is involved in Away from cell adhesions, we have uncovered those with low nuclear TIAM1 and TIAM1 levels remodelling the actin cytoskeleton to promote further locations for TIAM1 and other GEFs were significantly decreased in more advanced migration downstream of RAC. Over-expression driving RAC activity – at the centrosome, in the tumours.

26 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE CELL SIGNALLING 27 www.cruk.manchester.ac.uk/Our-Research/Drug-Discovery DRUG DISCOVERY

Biological studies to improve disease linkage are Across all our projects, we work to ensure that Informatics & Modelling advancing at the DDU and through a our DDU projects are integrated with Professor Matthew Roberts1 The last year has been a time of consolidation for a new Drug collaboration with Professor Stephen Taylor at Dive’s biomarker discovery programme, with a Graduate Students The University of Manchester. number of joint appointments, so that all Discovery Unit. Many new academic collaborations have been Elizabeth Hogg3 nominated targets have selection and predictive fostered with our colleagues in the CRUK Manchester Institute and James Stratford In the longer term, our portfolio is being biomarkers. We also delighted to work closely Mihaela Ficu1 repopulated from the impressive number of with the excellent committed clinicians in the The University of Manchester, resulting in validated new cancer drug 1 Megan Mylrea early targets which have been sourced from our Christie NHS Foundation Trust. target projects. Senior Group Leader collaborators, such as Undergraduate Students Hannah Fortune2 Professors Richard Marais, Caroline Dive, Iain Publications listed on page 65 Alex Anderton1 Hagan, Dr Claus Jorgensen and beyond, with Director Clinically, our pan-RAF programme completed progression, resistance to treatment and leveraged additional expertise. We are Caroline Springer its Phase I clinical trial at The Christie and Royal recurrence in many cancers. Current therapies EA to Caroline Springer particularly excited by the opportunity for Marsden NHS Foundation Trusts. This orally target the bulk of tumour cells, but CSCs escape Jayne Fowler discovery of inhibitors for lung cancer, given the Head of Medicinal Chemistry bioavailable, well-tolerated panRAF/SRC treatment resulting in tumour regrowth and academic and clinical expertise present in the Dan Niculescu-Duvaz 1Joined in 2019 inhibitor was developed in collaboration with 2 Manchester Institute, the Christie NHS Allan Jordan2 treatment failure. Thus, there is an urgent need Left in 2019 Professor Richard Marais and is designed to treat for new discoveries to target the CSCs within 3Joint with Systems Oncology Foundation Trust and the CRUK Lung Cancer Head of Biochemistry mutant BRAF and mutant RAS melanoma. This tumours, for use in combination with the Centre of Excellence. Graeme Thomson project has been licenced to Basilea standard of care drugs. We are pursuing a lead Pharmaceutica who are leading on future trials. optimisation drug discovery programme to Head of Cellular Pharmacology Our RET pre-clinical candidate development target CSCs. We have discovered potent, Graeme Walker programme has also been licenced and we selective inhibitors of our CSC target, which have Head of Informatics & Modelling anticipate that our RET inhibitor will enter promising pharmacokinetic profiles (Figure 1). Rae Lawrence first-in-human trials next year. Our medicinal chemistry has been greatly supported by internal computational chemistry Figure 1. Chemists The main cause of death in cancer is due to and by crystallography in collaboration with A potent and selective CRUK MI Mohammed Aljarah metastasis and we are assessing our lysyl oxidase Leicester University (funded by a CRUK designed ligand bound to a target Michael Brown (LOX) inhibitors in cancer models of metastases. Accelerator Award). We are also collaborating involved in CSC growth. Vikki Clayton2 LOX is an enzyme that regulates cross-linking of with Professor Marais and The University of Mark Dodsworth2 Efthymios Gavriil1 structural proteins in the extracellular matrix and Manchester based breast cancer expert Dr Paula Jackson1 plays a critical role in metastasis as well as in the Robert Clarke, in elucidating the biology of our Laura Johnson2 tumour growth in many cancers. LOX is a target. Chris Kershaw validated therapeutic target, and our aim, in Leo Leung collaboration with Professor Marais, is to Our gene-directed enzyme prodrug therapy Rebecca Newton discover first-in-class, orally bioavailable small (GDEPT) programme uses novel newly Ali Raoof Kate Smith2 molecule LOX inhibitors. We have discovered engineered vaccinia viral vectors, in Deborah Smithen LOX inhibitors with good pharmacokinetic collaboration with Professor Richard Marais, to properties and have progressed our LOX drug target tumours selectively and produce a unique Bioscientists discovery programme to late lead optimisation. bacterial enzyme locally, which is able to convert Alex Baker2 Our inhibitors show therapeutic activity in many subsequently administered prodrugs to 2 Habiba Begum different primary tumour models as well as cytotoxic drugs thus killing the tumour cells. We Elizabeth Blaikley anti-metastatic efficacy in preclinical models. have now shown highly selective tumour Aimee Blair2 Mairi Challinor Professor Marais’ group has discovered new targeting with our viral vectors from a single Andrew Clifton biology concerning LOX interaction with systemic administration, which accompanies Emily Crowley signalling pathways, which provides good long-term tumour xenograft growth reductions Zoi Diamantopoulou2 biological rationale for combining targeted in preclinical models. Joanna Grabarek agents with our LOX inhibitors. We are currently 2 Louise Griffiths selecting the best drug candidates to progress to Our partnership with IDEAYA Bioscience on our Nicola Hamilton toxicology studies before moving into early Poly(ADP-ribose) glycohydrolase (PARG) Ben Hodgson Paul Kelly clinical trials in patients, as monotherapy and in program continues to progress. In this Filipa Lopes combinations. collaborative effort IDEAYA have taken on Gareth Prosser1 responsibility for the further chemical Rebecca Holland1 Cancer stem cells (CSCs) are a subset of tumour optimisation and development of these series, Robert McLeary cells with the ability to perpetuate cancer growth looking to improve DMPK properties in order to Alice Pilborough indefinitely. CSCs are involved in tumour facilitate demonstration of in vivo activity. Alex Stowell

28 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE DRUG DISCOVERY 29 www.cruk.manchester.ac.uk/Our-Research/Head-and-Neck-Cancer-Biology HEAD AND NECK CANCER BIOLOGY

Lentiviral infection: Cas13d-EGFP

LDV PCR FACS sort: ARIA3 Genetic alterations in the genomic profiles of head and neck tumour Screen samples can be targeted for drug therapies. The focus of the Head and Neck Cancer Biology group is on salivary gland cancers as there is a significant unmet need in this patient group. Our primary aim is No Virus Negative Control Cas13d-EGFP (n=2) GFP expression GFP expression GFP expression to develop a new biological understanding of how changes at the All Single Cells All Single Cells GFP+ population All Single Cells All Single Cells GFP+ population All Single Cells All Single Cells GFP+ population

gene and protein level result in tumour growth and metastasis. Novel X5M1 X5M1 X5M1 Institute Fellow insights provided by this approach will allow drugs to be screened Robert Metcalf using cell lines and mouse models. Those drugs which are found to be effective in laboratory studies will be taken forwards to clinical X6 X6 X6 Count Count Count fluorescence fluorescence trials in patients with the goal of improving patient survival. - - Auto Auto X11 X11 X11 Initial research focuses on adenoid cystic identifying mechanisms through which MYB carcinoma (ACC), in collaboration with Professor drives tumour growth and metastasis, and to Caroline Dive and the Cancer Biomaker Centre. develop predictive and prognostic biomarkers to ACC is a salivary gland cancer that metastasises optimise patient treatment. GFP GFP GFP GFP GFP GFP in > 50% of patients leading to a median survival of 3 years with no effective drug therapies. Due Chromatin immunoprecipitation sequencing to chromosomal rearrangements, 90% of ACC has identified MYB downstream targets that are GFP+ population sorted GFPhi population sorted and tumours are driven by overexpression of enriched for genes controlling cell cycle and implanted n=1/model implanted n=1/model myeloblastosis (MYB) transcription factor gene regulation. As the cell cycle is dysregulated family members: a-MYB and c-MYB. The overall through multiple mechanisms in most human goal is to develop new therapies for ACC by tumours and offers avenues for therapeutic targeting, our initial focus is to determine whether Findings can be rapidly translated from this Figure 2. and how MYB overexpression impacts on cell research to develop biomarker led clinical trials Flow cytometry and FACS sorting cycle control in ACC. for this patient group. As there is a high risk of Lentiviral infection: Cas13d-EGFP of GFP positive ACC PDX cells metastasis and no standard drug therapies, this Figure 1. following short term ex vivo Proof of principle lentiviral Image: Low Light, Microscope, x10, at day 7 culture and lentiviral infection For functional studies to identify critical effector research could have immediate global impact in infection of ACC patient derived with Cas13-EGFP expressing genes downstream of MYB, we are developing the ACC patient population. The aim is to develop xenograft models (X5M1, X6 and vector enables re-implantation of isogenic MYB knock-down/knock-out ACC a platform to broaden research focus across X11) with Cas13-EGFP expressing Cas13 modified cells in vivo. models using shRNA and CRISPR/Cas9 or other rare sub-types of head and neck cancers. In vector following short term ex No Virus Negative Control Cas13d-EGFP vivo culture confirmed by GFP dCas9-KRAB followed by lentiviral mediated MYB addition, understanding of MYB biology in ACC expression on fluorescent Phase GFP Phase GFP rescue. To validate candidates identified through provided by this study may provide insight into microscopy images. this approach as therapeutic targets, in vitro and other solid tumours where MYB is frequently in vivo drug screening will be performed in overexpressed including sub-groups of patients models of ACC. As metastasis is a defining feature with breast and colorectal cancers. X5M1 of ACC, in vivo and ex vivo fluorescence/ luminescence imaging studies in isogenic Publications listed on page 66 models will be used to identify functional predictors of metastasis. Fundamental X6 discoveries in ACC biology will be translated to patients using tumour/liquid biopsies to identify and validate prognostic and predictive biomarkers.

X11

BF/GFP images at 7 days post lentiviral infection (n=2)

30 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE HEAD AND NECK CANCER BIOLOGY 31 www.cruk.manchester.ac.uk/Our-Research/Leukaemia-Biology

Figure 1: ABCB1 enhancer landscape ChIPseq tracks for H3K27Ac LEUKAEMIA BIOLOGY surrounding ABCB1 (chr7:87,495,508-87,626,404; hg38) in human K562 AML cells. Putative enhancers (E1-4) are highlighted in blue. P indicates the promoter. To generate resistant K562 AML cells, drug sensitive lines 1-3 were exposed to progressively escalating The goal of the team is to identify new disease mechanisms in acute concentrations of daunorubicin myeloid leukaemia (AML) and candidate therapeutic targets for over a three-month period. Deepti Wilks over the last decade. Remarkably, Identification of genes and cellular pathways development through to the clinic, aiming for patient benefit. This we also found that in primary human AML, whose loss of function collaborates or synergises year we published two studies along these lines. In the first, we exposure of fresh blast cells to daunorubicin with pharmacologic inhibition of LSD1 to reported our discovery and functional evaluation of the enhancer activates the stress-responsive enhancer leading promote differentiation represents an attractive to dose-dependent induction of ABCB1. strategy for uncovering novel drug combinations landscape controlling expression of ABCB1, the most important Furthermore, this dynamic induction of ABCB1 for testing in early phase trials. This research Group Leader chemotherapy resistance gene in AML. In the second, we reported was observed in response to diverse stressors, question led us to make use of CRISPR Tim Somervaille including chemotherapy. Such chemotherapy- technology. Using a genome wide loss-of- our discovery by CRISPR screening that dual treatment of AML cells induced up regulation of ABCB1 facilitates escape function CRISPR-Cas9 screening approach we Figure 2: Postdoctoral Fellows with inhibitors of the histone demethylase LSD1 and inhibitors of of leukaemia cells from targeted third-generation identified multiple components of the amino Combined pharmacologic Alba Maiques-Diaz2 ABCB1 inhibition, providing a novel explanation acid sensing arm of mTORC1 signalling - RRAGA, mTORC1 leads to synergistic induction of a terminal differentiation inhibition of LSD1 and mTORC1 Isabel Romero-Camarero in AML cells for the failure of ABCB1 inhibitors in clinical trials: MLST8, WDR24 and LAMTOR2 - as cellular Bettina Wingelhofer programme, and loss of leukaemia cell proliferative potential. Indicative cytospin images show chemotherapy drugs acutely induce their own sensitisers to LSD1 inhibition. This was confirmed Hannah Seberg1 THP1 AML cell morphology cellular resistance mechanism. by knockdown of certain mTORC1 components following 120hrs of treatment as well as mTORC1 pharmacologic inhibition. In Clinical Research Fellows Resistance of leukaemia cells, including ABCB1 inhibitors: previous trials have assumed respectively with DMSO vehicle, Mark Williams In experiments aiming to identify a particular, we found that dual treatment of AML leukaemia stem cells (LSC) with disease constant expression. Advances in enhancer LSD1 inhibitor OG-86, mTORC1 John Chadwick pharmacologic method of inhibiting this process, cells with an LSD1 inhibitor and the mTORC1 inhibitor RAD001 or the reconstituting activity, to the chemotherapy biology have established that these distal we discovered that stress-induced up-regulation inhibitor rapamycin potently enhanced combination. Bioinformatician drugs used in standard induction and regulatory elements govern cell-type specific of ABCB1 could be mitigated by combined use of differentiation in both cell line and primary AML Fabio Amaral consolidation regimens is the most common gene expression and frequently respond to pharmacologic inhibitors U0126 and ISRIB. cell settings, in vitro and in vivo, and substantially cause of treatment failure in acute myeloid environmental conditions and homeostatic Combinatorial use of these drugs, or their reduced the frequency of clonogenic primary Senior Scientific Officer leukaemia (AML). While a number of resistance perturbations. Critically, the enhancer landscape analogues, hold promise for testing in early phase human AML cells in a modelled minimal residual Gary Spencer mechanisms have been proposed over the of ABCB1 has yet to be defined. clinical trials as adjuvants to enhance the activity disease setting. We observed synergistic up- Scientific Officer years, the most significant and experimentally of ABCB1 inhibitors. regulation of a set of transcription factor genes Priyanka Bhattacharya2 well established is high expression of the ABCB1 In work led by Mark Williams, a clinical research associated with terminal monocytic lineage drug efflux pump (also known as MDR1 or fellow in the group funded jointly by a Kay In a second study led jointly by Bettina differentiation. Taken together our data indicate Graduate Students P-glycoprotein). This cell membrane pump Kendall Junior Research Fellowship and Cancer Wingelhofer and Gauri Deb, respectively current that dual mTORC1 and LSD1 inhibition is an Fabrizio Simeoni actively exports anthracyclines and other Research UK, we have recently identified and and former postdocs in the group, we extended attractive candidate combination approach for Francesco Camera chemotherapy drugs from the interior of cells functionally validated the network of enhancers our long-standing interest in the histone enhanced differentiation in MLL-translocated Bradley Revell1 and its level of expression predicts for treatment that controls expression of ABCB1. We show that Naseer Basma1 demethylase Lysine Specific Demethylase 1 AML for evaluation in early phase clinical trials. failure in AML. More generally, ABCB1 is also both brief and prolonged exposure of myeloid (LSD1) as a candidate therapeutic target in AML. 1 Joined in 2019 highly expressed in many poor risk malignancies leukaemia cells to daunorubicin activates an Pre-clinical studies have demonstrated that LSD1 Publications listed on page 66 2 Left in 2019 (e.g. ovarian cancer) as well as in normal gut, integrated stress response (ISR)-like contributes to the differentiation block that is the liver, kidney and the blood-brain barrier. transcriptional program to induce ABCB1. cardinal feature of AML. We have previously Inhibitors of ABCB1 have been tested in clinical Using approaches such as chromatin reported that LSD1 knockdown or LSD1 trials in AML but with limited success. In view of immunoprecipitation with next generation pharmacologic inhibition promotes its significant role in the disease, the rationale for sequencing, 4C-sequencing and CRISPR differentiation of, in particular, AML cells with targeting ABCB1 remains a strong one. inhibition we were able to demonstrate that the chromosomal translocations targeting MLL. Furthermore, given the abundance of preclinical mechanism of ABCB1 up-regulation involved Development of more potent and specific evidence supporting a role for ABCB1 in drug remodelling and activation of an ATF4-bound, tranylcypromine-derivative inhibitors such as resistance, the failure in clinical trials of inhibitors stress-responsive enhancer in intron 4 of ABCB1. iadademstat (ORY-1001, from Oryzon Genomics) of ABCB1 has not been adequately explained. Protracted cellular stress primed this stress has facilitated early phase clinical trials, which are responsive enhancer for rapid increases in ongoing. In patients with AML iadademstat is well A prerequisite for the design of new therapeutic activity following re-exposure of cells to tolerated and induces molecular and strategies is a greater understanding of the daunorubicin, providing an epigenetic memory morphological differentiation of blast cells in cancer-specific regulation of ABCB1 and its role of prior drug treatment. leukaemias driven by MLL gene rearrangements. in drug resistance. Specifically, until now it has been unclear how ABCB1 expression is We extended our studies from cell lines into While these early clinical trial results are established and maintained in human AML. primary patient AML cells, making use of our encouraging, all effective treatments in AML are Whether expression is constitutive or dynamic is excellent Haematological Malignancy Biobank, currently delivered in combination regimens. of critical relevance to the clinical application of which has been generated and curated by

32 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE LEUKAEMIA BIOLOGY 33 www.cruk.manchester.ac.uk/Our-Research/Molecular-Oncology

Figure 1: Example of phylogenetic tree of the T cell receptor. MOLECULAR ONCOLOGY Phylogenetic tree of the T cell receptor in-frame rearrangements obtained from ctDNA of one patient treated with immunotherapy for metastatic melanoma. Each leaf represents a distinct sequence, and colours indicate the different V, D, and J gene from individual As members of the CRUK MI community, our aim is to expand our rearrangements (the number knowledge of the basic biology underlying tumorigenesis in next to each leaf refers to the sequence count in the pool). The melanoma, breast and prostate cancer and translate this to patient distance scale is proportional to benefit. Working in a multidisciplinary team composed of cell and the number of different nucleotides between the molecular biologists as well as clinicians ensures that our discoveries sequences. are exploited in the fields of early detection of disease, personalised Group Leader medicine and the development of novel therapeutic approaches. Richard Marais

Associate Scientists Immunotherapy has changed the way that could anticipate which patients would set of 10 genes associated with stroma driven by quite different genetic events, and we Nathalie Dhomen melanoma is treated and in particular, immune respond to treatment. Moreover, expansion of a remodelling that were consistently upregulated recapitulate these in our mouse models. Valeria Pavet checkpoint inhibitors (ICI) have made a major specific subset of immune-effector peripheral T in tumours responding to therapy. Additionally, a Oncogene specificity is also observed in contribution to increased survival rate for cells occurred only in the patients that Senior Clinical Scientist 7-gene proliferation signature was different types of skin cancer, as different patients with advanced stage disease. However, responded to therapy, suggesting that these Martin Cook Figure 2: downregulated in responding tumours. We promote the transformation of many metastatic melanoma patients still face a cells could be responsible for mediating the Stroma remodelling and reduced validated that our gene signatures could identify melanocytes into melanoma and keratinocytes Senior Research Scientist significant mortality risk, in part due to our anti-tumour effects. Since these events are cell division define durable which patients would respond within 3-4 weeks into squamous and basal cell carcinomas. Adele Green limited insight into who will respond to therapy prognostic for response, occur within 3 weeks of response to therapy with of starting treatment with ICI in two independent Indeed, oncogene specificity is observed in and how to unleash the potential of ICI in starting immunotherapy and can be measured anti-PD-1 in melanoma. (Top) patient cohorts (Figure 2). This study shows that many tissues, and together with teams from the Postdoctoral Fellows combination with other treatments for the via minimally invasive liquid biopsies, they may Stroma score defining the level of Franziska Baenke2 our preclinical model can reveal insights into US and The Netherlands, we hypothesised that benefit of all patients. Designing more effective be able to provide an early response assessment expression of the 10-gene Candelaria Bracalente signature identified in our mouse human disease that are difficult to identify in the understanding the biological circuits that 1 clinical trials begins with increased for melanoma patients, and therefore have the Daphné Brisard model (left) and validated in two human population itself. promote or prevent malignant transformation of understanding of the biological impact of potential to allow personalised delivery of Alessio Cannistraci independent human cohorts particular cell types by different oncogene Elena Galvani therapies on both the cancer cells and the immunotherapy care. (middle, right). (Bottom) In the past year, we have expanded the scope of drivers will increase our knowledge of the Pauline Hascoët tumour microenvironment (TME). To that end, Proliferation score defining the our studies by generating a new mouse model malignant process and support the Sjors Kas we examined how ICI treatment affected T cell Patient-derived materials have helped us to expression of the 7-gene panel 1 for uveal melanoma, a rare melanoma subtype development of new therapeutic approaches. Patricia Pacios-Centeno behaviour in responding and non-responding understand better the dynamic interactions identified in our mouse model HaoRan Tang2 that arises in the melanocytes of the eye. Despite We are delighted that this ambitious hypothesis . between tumours and the immune system. (left) and validated in two Lucas Trucco independent human cohorts developing from the same cell of origin and research plan was selected to be one 2 However, our ability to detect subtle changes Joshua Tweedy (middle, right). (melanocytes), uveal and skin melanoma are of the CRUK Grand Challenges in 2019 T cells are components of the immune system that could impact therapy response is hampered (www.specificancer.org). Bioinformaticians that protect us from pathogens. ICIs re- by the diversity and plasticity of the immune Piyushkumar Mundra programme T cells, boosting their ability to kill system in the human population, and its shaping Where possible, we translate our findings into cancer cells. T cells recognise cancer cells by diverse variables including genetic Clinical Fellows patient benefit, and so we are excited to have through specific proteins on their surface called background, lifestyle and prior lines of treatment. Pablo Garcia Martinez secured funding for DETECTION, a clinical trial T cell receptors (TCR), which are generated by Conversely, tractable and easy to manipulate Zena Salih to assess if longitudinal monitoring of tumour somatic recombination of the TCR locus to models, such as transplantable mouse tumours, Sara Valpione DNA in the blood (ctDNA) can reveal minimal create the antigen recognition repertoire xenografts and organoids, lack normal tumour residual disease after curative intent surgery in Scientific Officers variability needed for effective immune function. heterogeneity, a co-evolved inflammatory stage IIB/C melanoma patients (CRUK Christopher Chester Since each TCR is specific to an individual T cell environment and the TME. We previously Experimental Medicine Award programme). Darryl Coles clone, we studied how immunotherapy affected reported that our BRAFV600E/UVR mouse Megan Grant DETECTION will also determine if ctDNA- the evolution of the TCR repertoire by melanoma model recapitulates the cardinal Clare McManus2 guided earlier treatment improves patient 1 sequencing TCRs in the T cells in the blood of genomic features of human melanoma, Jessica Walker outcomes. Alongside CAcTUS (CirculAting patients receiving ICI. In parallel, we analysed the including a UVR-induced mutational signature, Tumour DNA gUided therapy Switch), our Translational Research TCR repertoire in the DNA released into the high C-to-T load and recurrent mutations in the ongoing trial (opened in April 2019) to investigate Project Manager blood by T cells that have died in the course of same top 10 genes as occur in human if ctDNA can guide the switch between targeted Jackie Mitchell fighting against cancer cells (Figure 1). Our data cutaneous melanoma. Our mice have a fully and immune therapy in advanced cutaneous suggest that there is a dynamic awakening of the native immune system and the tumours develop Graduate Students melanoma patients, DETECTION builds on our immune system under the selective pressure of a normal TME, but can be controlled for genetic Luke Chisholm discoveries that ctDNA can be used to monitor immunotherapy, which was revealed by clonal and environmental variables that cannot be Denys Holovanchuk melanoma burden and progression in patients. Matthew Wilson expansion and contraction of specific T cells controlled when working with human-derived within three weeks of the first cycle of ICI samples. We used our model to evaluate the Publications listed on page 67 1 Joined in 2019 treatment. Critically, we identified a signature biological features of durable response to ICI 2 Left in 2019 within these early peripheral T cell responses targeting the PD-1 checkpoint and identified a

34 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE MOLECULAR ONCOLOGY 35 www.cruk.manchester.ac.uk/Our-Research/Prostate-Oncobiology PROSTATE ONCOBIOLOGY

Prostate cancer is a heterogeneous disease, both clinically and biologically. New therapies have produced some clinical successes but a substantial subset of patients progress to incurable castration- resistant PCa (CRPC). Importantly, it is yet not possible to predict which patient will develop aggressive tumours versus more indolent cases. Therefore, the work of our group aims at understanding the Group Leader early stages of PCa development and to identify and characterise Esther Baena cells of PCa origin to facilitate the development of better therapies.

Postdoctoral Fellows Valentina Ubertini2 Maria Roel Sánchez The initiation of aggressive tumours and CRPC LY6D is a gene with yet no established role in than ultrasound. Importantly, our study identified recently published work mpMRI non-visible involves the existence of so-called cancer- prostate development or cancer. It is a member Figure 1: that even with this superior technique >10% tumours could be regarded as genomically Scientific Officer initiating cells, with the ability to self-renew, to of the Ly6/uPAR family, characterised by their LY6D is expressed from early potentially significant PCas are missed by this aggressive and could potentially give rise to Pengbo Wang survive anti-tumoural treatments and to interact roles in cell proliferation, cell-cell interaction, stages of human prostate cancer. approach because they are not detected by lethal clones. Thus, our study further emphasises with niche-cells. These properties are required immune cell maturation, and cytokine Magenta (LY6D), pan-cytokeratin Graduate Students mpMRI. We hypothesised that the genomic the complexities of diagnosis in clinically for asymmetric cell divisions, ultimately production, which are all essential components (green), nuclear (Dapi). Ivana Steiner makeup of these ‘invisible’ lesions could provide localised PCa. Importantly, it highlights the Alexander Du Feu contributing to the heterogeneity of PCa. of tumour initiation and progression. We are important insights into their metastatic capacity shortcomings of this new diagnostic method, Alexandra Hendry1 Irrespective of recurrent mutations found in PCa currently defining the functional role of LY6D for and help to assess their potential lethality. To using mpMRI on its own as a triage test, and the cells, recent evidence suggests a key role of tumorigenesis and tumour maintenance, which address this question, we have recently need of additional biomarkers to inform patient Clinical Fellow cell-of-origin and their reprogrammed niche for so far remains unknown. Our in vitro and in vivo completed a study correlating genomics and diagnosis and prediction of treatment response. Amin Ali1 + disease progression. data showed that LY6D cells in the luminal mpMRI in men undergoing radical Thus, our study has the potential of being lineage represent luminal progenitors inherently 1 Joined in 2019 prostatectomy in order to elucidate the genomic practice changing by refining diagnostic testing 2 Left in 2019 We have identified inherently castration-resistant resistant to androgen deprivation and enriched characteristics of mpMRI visible and non-visible in PCa. cellular subpopulations in the prostate, defined organoid-forming multipotent luminal tumours and to assess the inter-relationship. We by their unique cell-surface markers, using single progenitors. Taken together, these findings found that the intra-tumour heterogeneity Our studies thereby advance patient cell profiling and functional characterisation by suggest that LY6D expression correlates with within visible mpMRI lesion bears the risk of stratification and set a pipeline to develop novel organoid-culture and in situ lineage tracing PCa initiation and progression to castration- misclassifying patients when using genomic therapeutics. Further studies are warranted to analysis in mouse models. In particular, our resistant growth from the luminal lineage. biomarkers from a single biopsy. For instance, determine the cellular composition of tumours studies define LY6D as a marker for prostate Importantly, in support of this hypothesis, considering a previously validated threshold for during progression and their association with progenitors and castration-resistant luminal analysis of human PCa cohorts revealed that the percentage of genomic aberrations mpMRI visibility, as well as the precise role of cells, which may serve as a prognostic marker for higher LY6D expression levels is associated with (PGA≥7.49%) of two cores obtained from the LY6D in prostate epithelial heterogeneity, PCa aggressive prostate cancer (Figure 1). For this, we more aggressive disease and worse outcomes, same visible lesion, one of the cores can classify initiation and progression to adenocarcinoma, compared the mouse prostate of non-treated suggesting that LY6D may serve as a prognostic the patient as low risk, while the other core can to validate its utility as a novel prognostic marker mice (hormone-naïve) with those that biomarker for advanced PCa. classify the patient as high risk. Similarly, intra- for patient stratification, and to tailor specific underwent androgen-deprivation upon surgical tumour transcriptomic heterogeneity within therapies targeting CR LY6D+ cells as novel castration. Our single cell expression analysis In a complimentary study combining genomic visible mpMRI lesions can also lead to therapeutic target for patients with aggressive revealed an unexpected molecular and multiparametric imaging analysis of misclassification. As a result, single mpMRI disease. heterogeneity in the prostate luminal lineage high-risk prostate cancer patients, we have targeted biopsy poorly reflects the genomic and also to a lesser degree in the basal lineage. characterised the radiogenomic landscape of heterogeneity of PCas and may therefore be We found that a subset of prostate cells in the multifocal prostate cancer. The diagnosis of unsuitable to assess the patients risk based on luminal lineage co-express multiple basal and prostate cancer is based on imaging studies, genomic analyses. Contrarily, limiting biopsies luminal markers including androgen-driven followed by ultrasound-guided biopsies of to mpMRI visible-only lesions may genes together with prostate stem/progenitor suspicious lesions, which remain the gold- underestimate the patients individual risk for marker genes. An important observation from standard therapeutic procedure. However, more disease progression and metastasis. Our study our study is that many castration-resistant (CR) recently, there is a trend towards the use of showed that PCa tumours undetected by prostate cells in the luminal lineage exhibit a multiparametric (mp)MRI to trigger biopsies in mpMRI can harbour genomic alterations, which bi-lineage expression signature similar to that of PCa patients, combined with genetic testing to are commonly seen in metastatic castration intermediate cells, raising a possibility that such refine risk stratification. mpMRI is supposed to resistant prostate cancer (mCRPC), including RB1 intermediate cells may be intrinsically CR. provide superior images of higher resolution and TP53 loss, and MYC amplification. In our

36 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE PROSTATE ONCOBIOLOGY 37 www.cruk.manchester.ac.uk/Our-Research/Skin-Cancer-and-Ageing SKIN CANCER AND AGEING

The primary focus of our studies is melanoma and squamous cell carcinoma (SCC), two forms of skin cancer that predominate in the elderly population and usually arise over skin that has endured sun exposure and/or ageing. Melanoma affects over 12,000 people and causes over 2,000 premature deaths each year in the UK, and 85% of deaths occur in patients older than 60. Age of the melanoma patient Institute Fellow at diagnosis is the most powerful predictor of outcome together Amaya Virós with primary tumour thickness.

Postdoctoral Fellow Tim Budden One factor underpinning the increased melanomas that arise predominantly from Clinical Fellow incidence of melanoma in the elderly is that as intra-epidermal melanocytes breach the Sarah Craig we age we acquire more ultraviolet (UV)-driven dermo-epidermal basal membrane to invade damage. However, although more melanomas the deeper cutaneous tissue and prepare for Graduate Student appear in the elderly population and more lymphatic, local and neurovascular spread from and molecular subtypes, and we have found sex bias in the metastatic incidence and course of Shilpa Gurung frequently occur at anatomic sites that have the deeper cutaneous structures. One intriguing Figure 1: each subtype presents unique features of ageing. disease, and suspect an even stronger bias accumulated sun damage over the years, they aspect of melanoma progression in the elderly is Left panel: Non-exposed skin Additionally, we have now described the towards more aggressive male disease during the Pre-Doctoral Clinical from the arm of a 20 year old. The molecular aspects that define melanoma in the latter stages of progression. We are therefore Academic Fellow can also arise at rarely exposed sites, with a less the higher likelihood of spreading via the dermis shows abundant, dense elderly patient, a work that is under revision. For performing comprehensive analyses in mouse Charles Earnshaw clear-cut relationship to chronic UV exposure. vascular system, and one possibility could be the collagen (light pink bundles). The this major aim of our lab, we have looked at the and human cuSCC to understand the clinical The aged skin anatomy and function varies structural and functional changes in the aged epidermis projects multiple greatly depending on the amount of sun cutaneous microenvironment are responsible epithelial extensions into the mutation rates, mutation types, pattern of DNA differences we observe. damage that has accumulated over the years, for this phenomenon. We are investigating how underlying connective tissue of damage accumulated in melanoma cells during with chronically damaged sites presenting aged keratinocytes, fibroblasts and adipocytes the dermis. Right panel: ageing, the sex of patients and the relationship to Publications listed on page 67 greater tissue decay, more wrinkles and more modify early cutaneous melanoma proliferation, Chronically sun damaged skin of survival. We have identified and patented the the lower limb of a 70 year-old UV-driven mutations in the aged cells. invasion, migration and metastasis. Skin from unique DNA features that reliably identify old female. The epidermis is patients at high risk of melanoma-specific death. sun-protected sites becomes paucicellular flattened, the amount of collagen Our lab aims to understand what drives skin across the 3 layers, and there is less connective is greatly reduced in the dermis, We are working to validate this in a larger human ageing (intrinsic, due to chronological age, and tissue in the dermis, leading to thinning across all and instead of the architecturally cohort, using our markers as a predictor tool in extrinsic due to UV) and how these differences the layers of the skin. In contrast, skin from preserved collagen bundles we clinical practice, to stratify patients at high risk of affect skin cancer onset, skin cancer progression sun-exposed sites present a predominant observe degraded connective death. Moreover, we are investigating if these tissue (solar elastosis, star). and outcome. We are exploring how these pattern of cell loss and dermal connective tissue markers can identify elderly patients who will specific changes in the architecture and degradation, where collagen fibre networks better respond to checkpoint inhibitor treatment. cellularity of aged skin can be exploited disappear to form globules of degraded collagen therapeutically. We have new evidence that the and other fibres that form the connective We have established a new line of research earliest stages of disease, and the tumour extracellular matrix. investigating the sex bias in the molecular microenvironment, are critical to plan patient landscape of cutaneous squamous cell surveillance and care, and we are hoping to Further to our on-going work on how aged skin carcinoma (cuSCC). Approximately 50,000 new contribute new strategies. Previous work has cells in the microenvironment contribute to cases of cuSCC are diagnosed yearly in the UK, shown that multiple cells composing the melanoma, we have completed a study representing a significant cost to healthcare. melanoma tumour environment, co-opted from dissecting how the physical degradation of the Patients present cuSCC at anatomic sites that the healthy aged skin, can contribute to disease extracellular matrix that occurs during ageing have accumulated greater level of UV damage. severity. Fibroblasts, innate immune cells, affects early tumour cell movement and skin The capacity of primary cuSCC to metastasise is adaptive immune cells, keratinocytes and homeostasis. We have now established low, but primary SCC incidence is so high in our adipocytes are all known to crosstalk with multinational clinical collaborations that will population that the overall number of metastatic melanocytes and/or melanoma cells. Our lab is allow us to explore the aged microenvironment SCC cases is considerable. The incidence of SCC addressing the role of healthy aged cutaneous in human tissue. is 2-fold greater in men than women, and this bias cells in promoting or inhibiting melanoma is assumed to arise from greater male exposure to progression at the earliest stages of disease, and In addition to the aged skin microenvironment, UV, from increased surface skin area of the ears investigating how their contribution varies we focus on how aged melanoma cells differ in and scalp of men compared to women, and due according to anatomic site. We are particularly their behaviour from young melanoma cells. to a delay in aged men seeking clinical care. We interested in the primary tumour stage, when Melanoma comprises multiple epidemiological have performed a large human audit investigating

38 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE SKIN CANCER AND AGEING 39 www.cruk.manchester.ac.uk/Our-Research/Stem-Cell-Biology FigureFigure 1 1 FigureFigure 2 2

FigureK5+ 1 K8+ K5+ K8+ AFigure 2 STEM CELL BIOLOGY UGS isolation Explant culture K8 K5+ K8+ K5+ K8+ A K5 UGS isolation Explant culture (7 days) Mature explant E15.5 K8 DAPI K5 B (7 days) Mature explant

CDH1 E15.5

The genes encoding for core binding factors AML1/RUNX1 and DAPI CBF are frequently rearranged or mutated in human leukaemias B NKX3.1 β CDH1 such as acute myeloid leukaemia (AML) and acute lymphoblastic RUNX1 RUNX1 NKX3.1 leukaemia (ALL). Consistent with its implication in leukaemia, the DAPI NKX3.1

transcription factor RUNX1 has also been shown to be critical for RUNX1 RUNX1

haematopoietic development. Beyond its major regulatory role in DAPI challenged prostate epithelial plasticity by strong Runx1 expression in the stratifiedNKX3.1 Group Leader haematopoiesis, RUNX1 has been more recently implicated in Figure 1: performing surgical castration experiments. urogenital epithelium, and lower levels in a Georges Lacaud various non-haematopoietic cancers. In this context, we investigated Whole-mount Strikingly, we found Runx1 expression in the specified subset of prostatic epithelial cells immunofluorescent staining majority of castration resistant cells. In addition, characterised by high levels of Nkx3.1 and p63. the expression and potential role of RUNX1 in normal prostate and of mouse prostate organoids Postdoctoral Fellows Runx1 was expressed, both in intact and Throughout the development of the prostate, derived from Runx1 expressing Neo Wen Hao prostate cancer in collaboration with the groups of Dr Esther Baena regressed prostate, and in a subset of cells which RUNX1+ cells were mainly localised in the Divya Malik cells. does not express Nkx3.1, a widely studied proximal region of the ducts, which later Muhammad Maqbool at the CRUK Manchester Institute and Prof Karen Blyth at the CRUK Beatson Institute. Figure 2. regulator of prostate development also known to corresponds to the peri-urethral region. Using a Scientific Officers (A) Schematic of the UGS explant mark cells with both castration-resistant and urogenital sinus explant culture model to study Michael Lie-a-Ling culture model to study early regenerative properties. Molecular profiling of embryonic prostate development ex vivo, we Rahima Patel1 events of prostate organogenesis the different compartment of the prostate by were able to show that the majority of epithelial Prostate Cancer able to survive the process. This approach has (B) Co-immunofluorescent + Prostate Cancer (PCa) is the most commonly allowed the identification of progenitor staining for RUNX1/NKX3.1/CDH1 single cell RNA sequencing before and after expansion takes place in the NKX3.1 Graduate Students surgical castration revealed that high Runx1 compartment, found in the distal part of the Renaud Mevel diagnosed cancer in men, and while its ‘castration-resistant’ cell populations within the after 7 days in UGS explant + Ewan Selkirk incidence continues to rise, it is currently adult murine prostate, characterised by their culture. RUNX1 and NKX3.1 mark expression marks an independent luminal ducts, while RUNX1 cells remain largely Muhammad Fadlullah Wilmot impossible to discriminate with accuracy the ability to survive in the absence of androgens distinct cellular subsets. lineage in intact prostates, which has a close quiescent at the centre/proximal region of the difference between indolent and more and to fully regenerate an intact adult prostate transcriptomic profile with castration resistant explant (Figure 2). These results show that during 1 Joined in 2019 aggressive forms of PCa, such as the after re-administration of testosterone. Thus, luminal cells. We then hypothesised that the prostate development, Runx1 marks a population + + development of metastatic castration-resistant there is currently a need to better characterise increased proportion of RUNX1 cells following of cells already distinct from the NKX3.1 lineage, PCa. This lack of prognostic biomarkers the extensive heterogeneity within the epithelial castration could be a consequence of either an enriched in the peri-urethral area, which is likely represents a central clinical challenge to avoid a compartment of the prostate, in order to induction of Runx1 expression or the existence of to correspond to the mature peri-urethral + + worsening problem of over-diagnosis and improve our understanding of the processes intrinsically castration resistant RUNX1 cells RUNX1 luminal cells found in the adult prostate. -treatment. In PCa patients, androgen associated with prostate tumorigenesis. present in the epithelium of intact mice. Using an deprivation therapy (ADT) is frequently used as in vivo lineage-marking approach, we were able Overall, the study of Runx1 expression in the + an adjunct to radiation therapy by targeting RUNX1 in normal prostate to demonstrate that RUNX1 luminal cells are mouse prostate has allowed us to characterise tumour cells that rely on androgens for their Using fluorescent reporter mouse models, we intrinsically castration resistant and maintain their specific lineages that reside in the prostate growth. However, despite the therapeutic found that Runx1 was expressed in a large identity after androgen deprivation. However, it epithelium. In particular, we found that Runx1 benefits of targeting androgen receptor proportion of basal cells, but also strongly was also evident that the regressed prostate is marks a population of adult luminal cells with signalling, cancer cells are able to adapt and expressed in a subset of luminal cells. In situ composed of cells which were not initially intrinsic castration resistant potential. Further survive, resulting in disease recurrence after a analysis revealed that Runx1+ luminal cells are expressing Runx1 in the epithelium of intact mice, studies are now warranted to assess whether few months or years. Understanding the origin particularly abundant in the peri-urethral region meaning that androgen deprivation also leads to oncogenic events occurring within this cellular of these resistant cells and the mechanisms of the mouse prostate, which is thought to be upregulation of Runx1 expression in these other subset would lead to aggressive castration underlying the acquisition of castration- enriched in stem and progenitor cells. To further castration resistant populations. We then resistant tumours. Also, the potential functional resistance represent therefore major goals to evaluate the biological potential of Runx1 administered ectopic testosterone to the mice role played by Runx1 in prostate epithelial cells improve PCa treatment. expressing cells we used an ex vivo prostate back to physiological levels to stimulate epithelial would need to be studied further, notably to organoid culture assay. We found that RUNX1+ regeneration and evaluated the consequences evaluate to which extent it participates in prostate + The epithelium of the prostate is composed of luminal cells were consistently forming on lineage marked RUNX1 cells. In this setup, we tumorigenesis. Finally, it would be important to three epithelial cell types: luminal, basal and rare organoids more efficiently than the remainder were unable to identify clonal expansion in the determine whether RUNX1 could be used as a neuroendocrine cells. Luminal cells form a layer fractions. Also, RUNX1+ luminal cells presented a fully regenerated prostate, suggesting that clinical marker of PCa in patient samples. of polarised tall columnar cells producing strong potential at forming organoids with a intrinsically castration-resistant Runx1 expressing prostatic secretions and are largely dependent hollow structure, rich in Keratin 8 expression cells do not proliferate in response to androgen Publications listed on page 67 on androgen receptor signalling, unlike the (Figure 1), suggesting that Runx1 is marking a signalling. supportive basal cell layer which is located specific subpopulation of luminal cells with between the luminal cells and the surrounding biased differentiation potential. Our results led us to interrogate the pattern of stroma. Surprisingly, while surgical castration of Runx1 expression during the establishment of the wild-type mice results in considerable cell death In order to further characterise the biological prostate epithelial lineages. During in vivo within the luminal layer, a small cellular subset is potential of Runx1 expressing cells, we embryonic prostate development, we found

40 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE STEM CELL BIOLOGY 41 www.cruk.manchester.ac.uk/Our-Research/Systems-Oncology are identifying targetable pathways in the tumour Reciprocal Signal Flux stroma and optimising their use for combination therapy. ECM SYSTEMS ONCOLOGY Delivering personalised medicine in PDA Personalised therapy, the subscription of a Erk/Akt therapy that is matched to specific characteristics of individual tumours, has benefitted cancer patients enormously, but is still not available to patients with PDA. In an effort to improve ECM treatment options and patient selection in PDA, Tumours are complex ecosystems where cancer cells are Immune regulation we are involved in establishing a national infrastructure, PRECISION-Panc, where individual embedded within a complex stromal reaction comprising multiple Erk/Akt tumours are subjected to molecular profiling infiltrating cell types and pathological changes to the extracellular such that patients can be matched with selected matrix. The aim of the Systems Oncology laboratory is to determine treatments. These clinical trials are underpinned by the development of biomarkers and pre- and define the rule-set by which tumour cells conscribe host cells to Immune regulation clinical research to further refine treatment support tumour growth and resistance to therapies. Understanding strategies targeting specific dependencies. Together with collaborators at The Christie NHS Group Leader these rules will enable development of synergistic combination Akt Foundation Trust and Central Manchester NHS Claus Jørgensen therapies targeting both tumour cell intrinsic dependencies as well Basal Signal Flux Foundation Trust we have implemented methodologies for isolation and expansion of Postdoctoral Fellows as their extrinsic dependencies on stromal reciprocal signals. catalogue, isolate and characterise individual primary tumour cells in 3-dimensional cultures Celia Cintas1 (also known as organoids). Matching the tumour Adrian Gomez1 Figure 1: stromal elements (including both cellular and microenvironment to mimic the cellular and Brian Lee Pancreatic Ductal Adenocarcinoma fibroblasts (Figure 1). We observed that individual Model of heterotypic interactions extracellular components). The critical aim of Nasir Haider1 biophysical constraints of the patient tumour, Pancreatic Ductal Adenocarcinoma (PDA) is a tumour cell clones instigate diverse stromal across heterogeneous tumour these analyses is to determine whether individual Joanna Kelly1 cells and stromal fibroblasts. these models may be used to define optimal dismal disease with an average five-year survival behaviour, where some tumour cell clones stromal cell populations (or extracellular matrix Stephen Kershaw3 Clonal tumour cell populations components) differentially alter the tumour cell combination of therapies and biomarkers to Alexander Kononov1 rate of 9%. This is due to a combination of late induce fibroblasts to regulate the extracellular drive diverse phenotypic phenotype and whether this results in a further develop clinical trials. Giulia Veluscek2 diagnosis, the aggressive nature of the cancer matrix and others induce fibroblasts to be more responses in stromal fibroblasts, differential sensitivity to therapy. Using a and a lack of effective therapy. Consequently, immune regulatory. Interestingly, while stromal which in turn differentially Scientific Officer while PDA only is the 11th most common cells appeared more diverse, the ensuing engage tumour cell signalling to combination of proteomics and transcriptomics Xiaohong Zhang occurring cancer in the UK, it is currently the 4th reciprocal effect on individual tumour cells lead normalise output. analyses we are defining the key pathways largest contributor to cancer related deaths. The to normalisation (phenotypic convergence). For regulating tumour cell resistance. In parallel we Graduate Students Christopher Below4 most frequent occurring genetic mutations have example, the transcriptional programmes Figure 2: Catherine Felton1 been identified with activating mutations in the regulated in the tumour cells were less diverse Pancreatic cancer organoids and Felix Heider1 oncogene KRAS and inactivation of the tumour (more similar) in the presence of stromal stromal cells. Elizabeth Hogg3 suppressors CDKN2A, SMAD4 and TP53. fibroblasts. Moreover the activity of the Colin Hutton regulatory MAPK signalling pathway was Amy McCarthy2 PDA is characterised by an extensive normalised between tumour cell clones, when GFP+ Fibroblast iRFP+ Cancer Organoid Clinical Fellow desmoplastic reaction, which makes up 80% of they were allowed to interact with stromal Konstatinos Georgiadis5 the tumour volume on average. Here, an fibroblasts. Consequently stromal interactions abundant and pathological remodelled had diverse impact on the effect of specific 1Joined in 2019 extracellular matrix increases the tissue stiffness therapeutic inhibitors, such as MEK inhibitors. 2Left in 2019 and restricts perfusion to limit therapeutic Together these data suggest that interactions 3Joint with Caroline Springer, delivery. In addition, there is an abundant between tumour and stromal cells need to be DDU infiltrate of fibroblasts and myeloid cells, which carefully considered when devising therapeutic 4Joint with Martin Humphries, University of Manchester together create an immune-privileged strategies and that stromal targeting may have an 5Joint with Caroline Dive, CBC environment and confer resistance to therapy. heterogeneous effect in a complex tumour and Juan Valle, Christie NHS Genetic heterogeneity of tumours is associated ecosystem. Foundation Trust with aggressive behaviour and rapid development of therapeutic resistance. Defining and targeting the tumour However, it is less clear whether heterogeneous microenvironment in PDA populations of tumour cells also establish Understanding the role of the microenvironment distinct reciprocal interactions with stromal cells. in shaping the therapeutic response across This is an important question as heterogeneous selected patient populations is critical to define interactions across tumour and stromal cell whether approaches targeting the tumour populations increase functional plasticity and stroma should be delivered in a personalised may therefore impact development and manner, or whether a broader non-selective implementation strategies for stromal-targeting approach can be taken. In order to define therapies. In order to interrogate such interdependencies between tumour and interactions we recently studied a set of single stromal cells it is critical to map the cellular and cell derived populations of pancreatic cancer extracellular components of the cells and their interdependencies with stromal microenvironment. We have therefore started to

42 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE SYSTEMS ONCOLOGY 43 www.cruk.manchester.ac.uk/Our-Research/Transcriptional-Networks-in-Lung-Cancer TRANSCRIPTIONAL NETWORKS IN LUNG CANCER

Lung cancer is the primary cause of cancer-related mortality worldwide. The major focus of 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 and cell biology. MicroRNAs (miRNAs) are single stranded RNAs of Group Leader 19-25nt in length that negatively regulate gene expression by Michela Garofalo translational inhibition or degradation of the mRNA targets. MiRNAs

Postdoctoral Fellows are differentially expressed in almost all types of human cancers Lei Shi versus the normal tissue counterpart and are key players in cancer Tiziana Monteverde onset and progression. Scientific Officer Peter Magee

Graduate Students To investigate whether KRAS, one of the most harbouring K-Ras mutations, although so far, no Lucy Ginn1 mutated oncogenes in lung adenocarcinoma, specific drug demonstrated efficacy. One of our Manuela La Montagna was able to modulate miRNAs expression, we goals was to identify K-RAS-modulated miRNAs Athanasios Paliouras2 overexpressed wild-type and mutant KRAS that by targeting molecules involved in the RAS (KRASG12D) in non-small cell lung cancer (NSCLC) pathway can be employed as therapeutic tools 1 Joined in 2019 cells. We identified two miRNAs, miR-30c and in lung cancer. By overexpressing wild-type or 2Left in 2019 miR-21, significantly upregulated in wild-type mutant KRAS (KRASG12D) and using inducible successful examples is Echinoderm Microtubule who developed resistance in the clinic and and mutant forms and showed that miR-30c and human and mouse cell lines, we analysed Figure 1. Working Model. Like-4-Anaplastic Lymphoma Kinase (EML4- therefore they could be potential biomarkers of miR-21 induce cell proliferation and enhance KRAS-regulated miRNAs in NSCLC. We showed KRAS activates miR-30c and ALK)-mutant NSCLC, which affects 4-5% of lung resistance to ALK inhibitors. In summary, our miR-21 through the transcription migration/invasion by inhibiting crucial tumour that miR-30c and miR-21 are significantly cancer patients. Several EML4-ALK inhibitors study takes advantage of the transcriptional factor ELK1, which in turn by have already been approved by the FDA, namely addiction hypothesis to propose a new suppressor genes. Systemic delivery of upregulated by both KRAS isoforms and induce downregulating NF1, RASA1, anti-miR-21 in combination with cisplatin in vivo drug resistance and enhance cell migration/ RASSF8 and BID, regulates KRAS, crizotinib, ceritinib, alectinib, brigatinib and treatment strategy for a subset of patients with suppressed the initiation of lung tumours in a invasion through the inhibition of important NF-kB and the intrinsic apoptotic lorlatinib. Even though the objective response acquired resistance to first, second and third- mouse model of lung cancer. A subset of lung tumour suppressor genes, such as RASA1 and pathway, inducing lung rate for the ALK inhibitors crizotinib and alectinib generation ALK inhibitors. adenocarcinomas is driven by the EML4-ALK RASSF8. MiR-30c and miR-21 levels were tumorigenesis and inhibiting in the clinic surpasses 60%, patients typically translocation. While ALK inhibitors in the clinic elevated in tumours from patients that apoptosis in NSCLC. MiR-30c and develop resistance to these inhibitors and miR-21 are released into the relapse soon thereafter. lead to excellent initial responses, acquired underwent surgical resection of early stage bloodstream and could be resistance to these inhibitors due to on-target NSCLC compared to normal lung and in plasma potential biomarkers for early mutations, or parallel pathway alterations, from the same patients. Systemic delivery of NSCLC detection. In order to mimic the context of acquired represents a major clinical challenge. We LNA-anti-miR-21 in combination with cisplatin in resistance to ALK inhibitors in vitro, we utilised discovered that EML4-ALK cells with acquired vivo suppressed the development of lung cell lines with acquired resistance to crizotinib resistance to crizotinib, ceritinib or alectinib tumours in a KRASG12D-driven genetic mouse (CrizR), ceritinib (CeritR) and alectinib (AlecR) by overexpress specific cyclin dependent kinases model of lung cancer. Mechanistically, we long-term exposure to these drugs. RNA-seq (CDKs) and CDK inhibitors halt tumour growth demonstrated that ELK1 is responsible for identified a cell cycle dysregulation in crizotinib- and robustly induce apoptosis in this setting. miR-30c and miR-21 transcriptional activation by resistant cells, evidenced by an upregulation of direct binding to the miRNA proximal promoter CDKs and their partner Cyclins. Following this KRAS and non-coding RNAs regions (Figure 1). In summary, our study defines observation, we treated EML4-ALK drug- The proto-oncongene RAS encodes three that miR-30c and miR-21 may be valid resistant cells with different CDK inhibitors. different RAS proteins: HRAS, NRAS and KRAS, biomarkers for early NSCLC detection and their These compounds robustly induce apoptosis regulated by guanine nucleotide exchange silencing could be beneficial for therapeutic through downregulation of anti-apoptotic factors (GEFs), which stimulate RAS activation applications. genes. Importantly, alvocidib reduced tumour through GDP for GTP exchange, and by GTPase- progression in vivo in xenograft mouse models. activating proteins (GAPs), which catalyse the ALK-EML4 lung tumours Furthermore, we found that two microRNAs, hydrolysis of GTP to GDP to switch off the KRAS In NSCLC small molecule inhibitors for mutant miR-25 and miR-30c, are upregulated in signalling. Mutations in KRAS are very frequent in kinases have offered unprecedented success in crizotinib-resistant cells and in plasma of patients NSCLC (~30%) and in lung adenocarcinoma the management of disease. One of the most

44 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE TRANSCRIPTIONAL NETWORKS IN LUNG CANCER 45 http://www.cruk.manchester.ac.uk/Our-Research/Translational-Oncogenomics Figure 1. Figure 2. TRANSLATIONAL ONCOGENOMICS

With an estimated 1.3 million new cases identified worldwide in 2018, prostate cancer is the most frequently diagnosed male cancer in the Western world, and in the UK, there are approximately 11,700 prostate cancer deaths per year.

The key challenge in management of this disease with a poor prognosis in prostate cancer, and this Group Leader is to accurately assess the risk of progression to could be due to acquisition of a more metastatic metastasis in each patient. Low risk, indolent phenotype and/or resistance to radiotherapy. Robert Bristow tumours with favourable pathology may never Our work has shown that hypoxia is frequently tumours, and raises the intriguing possibility that offered treatments specifically targeting the progress, and in such cases active surveillance is associated with a range of unfavourable Scientific Officers Figure 1. hypoxia may be a feature of the tumour primary tumour. Since the disease has spread, a treatment option. Intermediate risk cancers that pathophysiological features. These include the Steve Lyons Next generation sequencing microenvironment that impacts tumour systemic treatments are required to control the remain localised to the prostate are potentially presence of an adverse sub-pathology, IDC-CA Alicja Partridge1 studies have identified the evolution, and perhaps selects for specific disseminated malignancies. However, recent curable with either surgery or radiotherapy (intraductal carcinoma with cribriform genomic alterations correlated alterations. This process may be facilitated by findings from the STAMPEDE trial demonstrate a Postdoctoral Fellows approaches. However, high risk cancers have an architecture), a propensity to metastasise, and with poor outcomes in prostate inefficient DNA repair since hypoxia is known to significant improvement in disease-free survival Richard Rebello increased probability of producing incurable high rates of copy-number aberration. Indeed we cancer. By working with primary Christoph Oing2 lead to downregulation of several repair proteins in patients with oligo-metastatic disease (≤4 metastases which may be undetectable at the have shown previously that combined indices of cell cultures isolated from patients, we aim to characterise including MLH1, MSH2, RAD51 and BRCA2. detectable metastases), who receive radiotherapy point of diagnosis. Here, localised therapy genomic instability and hypoxia can improve Graduate Students the roles of specific gene Further work involving experimental studies is to the prostate. This striking finding suggests that (radiotherapy/surgery) is combined with systemic accuracy of prognosis in following either radical Alexandru Suvac alterations in driving phenotypes now required to shed light onto the molecular the primary tumour continues to contribute to Ronnie Rodrigues treatments to target secondary disease. Although prostatectomy or radiotherapy. Therefore, the relevant to the disease. mechanisms at play in hypoxic tumours. metastatic spread over a long period – perhaps Jack Ashton initially effective, androgen deprivation therapy is relationship of hypoxia to genome stability is a Immortalised primary cells will be by releasing further tumour cells into the often evaded, and an incurable metastatic major theme in our work. engineered to recapitulate the Our laboratory studies aim to address the circulation, or by expressing soluble factors that 1Joined in 2019 castrate-resistant prostate cancer emerges. prostate cancer genome using a 2Left in 2019 range of molecular techniques. interplay between hypoxia and the function of prime pre-metastatic niches. Our lab previously In our latest bioinformatic analysis (Bhandari et al. The engineered cells can then be potentially oncogenic genomic aberrations. reported that the co-presence of tumour hypoxia The current method of quantifying risk in prostate Nature , 2019), we have taken advantage used to provide insights into the Primary prostate epithelial cells form the basis of (based on mRNA signatures or needle electrode cancer involves measurement of PSA (Prostate of well characterised gene expression scoring responses to hypoxia, genome our isogenic cell culture models. Once measurements) and genomic instability Specific Antigen) levels in serum, pathological systems to estimate levels of hypoxia in a cohort stability and DNA repair, immortalised by expression of the telomerase synergistically portend rapid relapse after primary assessment of biopsy material to produce a of 1188 tumours from 27 cancer types together tumorigenesis and drug gene, these cells provide the ideal basis for treatment for prostate cancer, supporting the Gleason Score, and staging of the disease using with matched normal tissue controls. This resistance. further genetic engineering since they have an concept that a hostile tumour microenvironment the internationally recognised TNM (Tumour, analysis detected subsets of patients with Figure 2. unaltered genome that remains stable during may select for or drive adaptation of a distinctive Node, Metastasis) staging system. Yet despite the elevated hypoxia in 23 of the 27 cancer types, and The HYPROGEN study will culture. We have used the latest genome genomic profile and rapid failure due to occult use of stringent clinical criteria to place patients tumours consistently had higher hypoxia scores provide unique insight into engineering techniques to exogenously express, metastases. This raises the possibility that hypoxic into prognostic groups, 30-50% of men can still than normal tissue. Notably, we found that the prostate cancer metastasis. By or mutate those genes we identified as being regions present in primary tumours play an fail precision radiotherapy or surgery due to local extent of hypoxia varies substantially across taking biopsies from patients with more frequently altered in hypoxic cells, important role in the development of metastatic resistance and/or systemic spread. Clearly, there patients within cancer types. This striking oligo-metastatic disease prior to prioritising PTEN and MYC. In addition, we are disease. The HYPROGEN trial aims to characterise is a need to develop new biomarkers that give an variability suggests assessment of hypoxia could treatment, we aim to also investigating the role of BRCA2, a DNA repair the role of hypoxia in driving the early metastatic insight into heterogeneity of outcomes in provide valuable information for use in comprehensively characterise the processes involved in the gene that when mutated in carrier families, spread of tumours. Patients with oligo-metastatic prostate cancer patients. In addition, new personalised medicine strategies. Correlating transition from localised to significantly increases the risk of developing disease will be recruited to the study prior to biomarkers that predict patient responses to the hypoxia scores with genomic features, we found systemic cancer. The tracer aggressive prostate cancer. We are currently receiving treatment. Hypoxia will be assessed by various treatment options will also be required. hypoxia associated with increased genome molecule Pimonidazole will be analysing the phenotypes of these cells with means of the tracer molecule, pimonidazole, To this end, recent advances have shown that instability as measured by the percent genome used to assess the impact of respect to genome stability, DNA repair, which will be administered before biopsies are detailed understanding of the genomic/ alteration criterion. Alterations such as deletions hypoxia in driving extra-prostatic transformation and the response to hypoxia. taken from both the primary tumour and selected transcriptomic landscape of prostate cancer and duplications were significantly higher in spread and genomic instability. We will compare the molecular Given the clear correlation between hypoxia and bone metastases. Genomics and transcriptomics together with knowledge of the degree of hypoxic tumours, as was the number of single profiles of matched primary and genome instability observed in patient biopsy will then be applied to provide insights into the hypoxia within tumours can provide important nucleotide variants/Mb. At the individual gene metastatic tumours, to identify material, it will be important to test whether relationship between hypoxia, genomic instability insights into the aggressiveness and likely level, hypoxia was associated with significantly novel features and correlate these exposure to chronic or cycling hypoxia is and metastatic spread. We will test whether early prognosis of localised prostate cancer. more frequent alteration of several oncogenes with hypoxia levels. The sufficient to drive mutational processes. metastases, circulating tumour cells and hypoxic and tumour suppressors including TP53, MYC prevalence of circulating tumour regions of the primary tumour share distinct Hypoxia and genome instability – signs of and PTEN. Furthermore, the mutation profile of cells and their contribution to Illuminating the genomic landscape of hypoxia- genomic and/or gene expression profiles. aggression in prostate cancer hypoxic tumours is consistent with inefficient secondary tumour formation will also be assessed. driven early metastatic prostate cancer – In many solid tumours, regions of acute or mismatch repair and homologous HYPROGEN (HYpoxia-driven PROstate cancer Publications listed on page 68 chronic hypoxia develop as proliferating tumour recombination repair of DNA. This hypoxia- GENomics) cells outstrip the ability of a poorly organised associated genome instability is in agreement Patients unlucky enough to have metastatic vasculature to supply oxygen. Hypoxia correlates with our previous analysis of a separate cohort of prostate cancer at the point of diagnosis are rarely

46 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE TRANSLATIONAL ONCOGENOMICS 47 www.cruk.manchester.ac.uk/Our-Research/Tumour-Suppressors TUMOUR SUPPRESSORS

Cell proliferation The most frequently mutated gene in all cancers is the tumour Aneuploidy/

suppressor p53 (TP53). In our lab, we are interested in the different genomic instability Cancer mutations in TP53 and the functional consequences of these mutant p53 cell mutations in lung cancers.

P53 is a tumour suppressor that plays a pivotal indicated that although the mutant p53 proteins Institute Fellow role in detecting a variety of stresses and helps often lose wildtype function, many are more with deciding how cells are to react to cellular harmful than a simple loss of p53 function/ Patricia Muller Cancer stress. If the stress is minimal, p53 will help in activity. Mutant p53 proteins gain the ability to Cancer Cancer allowing for cell survival and repair of stress- inhibit the p53 family members p63 and p73 (see cell cell Postdoctoral Fellow Cancer cell Yannick von Grabowiecki related damage. Alternatively, if the stress is too review Hall & Muller, Int J Mol Sci 2019) and the cell Cancer much, p53 will initiate an apoptotic program to microRNA machine protein Dicer to promote Drug Graduate Student destruct the damaged cell. In order to invasion and metastasis. This inhibition leads to Callum Hall orchestrate this, p53 acts as a transcription factor an enhanced recycling of integrins and growth that can regulate hundreds, if not thousands of factor receptors to the cell membrane, mediated Cell engulfment genes dependent on its levels of expression, by RCP (Rab Coupling Protein). In a screen to epigenetic modifications and a variety of other detect mutant p53 dependent RCP-interacting Cell Motility factors. Ultimately, these p53 functions prevent proteins, we identified drug transporters that the formation of DNA mutations and therefore mediate the efflux of chemotoxic drugs out of prevent tumour formation. However, TP53 itself cancer cells. We are following up on these data Drug Resistance is the most frequently mutated gene in human and are investigating a role for these proteins and binding domain of p53 are more prone to mutate function (Figure 1). Pilot data in our lab cancers, illustrating its importance as a tumour RCP in mediating mutant p53-dependent Figure 2. in SCLC compared to all cancers. These data furthermore suggest that these conformational suppressor. Remarkably, almost any amino acid chemoresistance. Mutations in p53 can result in the suggest selective advantages for these mutations changes are associated with a phenotype that we in the p53 protein can be found mutated in expression of mutant p53 to occur in SCLC. Although many of the hotspot usually see in mutant p53 expressing cells (Figure proteins. Many of these proteins human cancers, but mutations are more The mutation frequency for p53 in all cancers is mutations have been thoroughly researched for 2). Certain mutants have been characterised as Figure 2 Gain-of-function ofhave lostmutant some or all of theirp53 WT common in the DNA binding region of the about 60%, but can be variable in individual functions. In addition, many gain their role in invasion, chemoresistance and ‘unfolded’, whereas others appear normally molecule. In addition, there are a few hotspots cancers. In non-smallMutations cell lung cancer (NSCLC) in p53 can resultnovel in functions the involved expression in metastasis, of mutant not much is known p53 about proteins. these Manyfolded. Most of likely these this differentiation proteins is too black have lost some or all of that have been more thoroughly studied, the frequency is about 30%, but in small cell lung various aspects of mutations specifically. In collaboration with CBC, and white and gradations of unfolding exist. This Figure 1. although these comprise only 10-25% of all the cancer (SCLC) p53 istheir mutated inWT nearly functions. all cases In addition,tumourigenesis. many Depicted gainare novelwe have created functions a library of these involved selected in variouscould mean that aspects certain p53 mutant of tumourigenesis proteins . Depicted are The folding states of p53 p53 missense mutations that are found in (90% or more). When looking at the mutation those functions that we are mutations and we are investigating if the might be more vulnerable to metal-mediated a) Under normal conditions, p53 cancers. frequency in SCLC comparedthose to functions all cancers, that we arestudying studying in the lab. in themutations lab. that occur in SCLC are beneficial to the unfolding than others, which will have functional is folded to interact with the DNA. Upon exposure to certain metals, there are clear differences that cannot be solely growth, metastasis and survival of these tumour implications in cancers in which abnormal metal p53 is partially unfolded, which Mutations in p53 can result in loss of p53 attributed to the ‘smokers’ mutation signature. In cells. levels are present. We are currently characterising causes it to lose DNA binding expression, or expression of mutant p53 particular, mutations in the N-terminus, the which metals affect p53, which mutants are most capacity and makes it inactive for proteins. Previous data from our lab has C-terminus and at certain regions in the DNA Finally, we identified that specific metals can vulnerable for this and what are the functional normal WT function. The folding change the function of wildtype p53 and make it consequences of metal exposure on tumours in states can be detected by two behave like a mutant p53 by promoting invasion vivo. antibodies PAb1620 and PAb240. and metastasis or binding to mutant-specific PAb1620 recognises multiple a b interacting proteins. Smokers’ lungs have been Publications listed on page 68 epitopes that are in close “wild type” (WT) conformation ‘Unfolded’ conformation metal proximity when folded, but too exposed to a variety of metals and in many far apart to be recognised by this IP: cancers increased metal levels can be detected. antibody upon unfolding. PAb240 PAb 1620 Certain metals can cause a conformational recognises a cryptic epitope that change in p53 that renders it impaired in normal is only accessible when the p53 Metal PAb 240 molecule is unfolded. Picture exposure adapted from Wang PL et al All p53 (Oncogene, 2001). b) Western blots of immunoprecipitations with these antibodies under native conditions, allowing to detect folded and unfolded p53 upon metal exposure.

48 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE TUMOUR SUPPRESSORS 49 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES

50 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE 51 www.cruk.manchester.ac.uk/Research bringing the facility right up to date with the Biological Resources Unit latest technology available. Q1 of 2019 saw the Transgenic Breeding extensive optimisation of key proteomic Jennifer Hughes, Daniel Bennett, Tim Bloor, Carl application workflows on the new systems. Conway1, Ali Jammoul1, James McDonald, Wesley 1 1 RESEARCH SERVICES Excitingly, the expected performance Moore , Kerry O'Shea, Rose Storey, Natalie Varley , Martin Vincent, Mark Willington1 ,2 enhancements these systems offer over the previous generation of instruments they 1Joined in 2019 2Left in 2019 replaced were exceeded in practice. For example, our absolute sensitivity of protein The BRU Transgenic Breeding Team breeds detection was improved by more than a mice to meet the requirements of CRUK MI magnitude using the Orbitrap Lumos, allowing researchers. Services offered in the last year As the following reports show, 2019 has been a very productive year us to characterise proteins whose abundance have included rederivations using fresh and for the scientific core facilities. Achievements have been in a variety was too low to detect at all previously. We also frozen sperm and embryos, pairing mice for exploited a new type of liquid chromatography breeding, monitoring timed matings, recording of different areas, which demonstrates the breadth of activity in the chemistry, micro Pillar Array, in collaboration and weaning litters, ear snipping for core facilities. with PharmaFluidics. The use of these highly identification and genotyping, managing the ordered laser etched micro pillars containing an genotyping service (using an external service outer porous shell grafted with C18 groups provider), translating and transferring genotyping resulted in a significant increase in our peak Chief Laboratory Officer new mouse models, but has also contributed results, monitoring tumour prone lines for onset capacity (the number of analytes we can fully Stuart Pepper to the broader scientific community by co- of symptoms and cryopreservation of lines that Chief Laboratory Officer resolve in a given time) and boosted sensitivity by organising a national meeting on CRISPR can be archived. In accordance with Home approximately three-fold. The characteristics of Stuart Pepper In both Biological Mass Spectrometry and technologies. Office requirements, the mice are closely this new chemistry allow us to more Scientific Computing there has been a focus on monitored in order to ensure high welfare comprehensively profile complex and low developing workflows and services on new Next generation sequencing has been a core standards. abundance proteomes. The hardware and hardware, leading to some exciting new service for some time, but there is continued chemistry enhancements were carefully capabilities, including an enhanced virtualisation development on specific applications. Over the The breeding facility is housed in a clean unit exploited with a bespoke set of method infrastructure. These two teams have also last year there has been a significant expansion with a high health status and is kept free from development projects applied to our suite of worked closely with the newly expanded in single cell sequencing projects at the Institute, common mouse pathogens, which means that applications for qualitative and quantitative Computational Biology team to develop end to which the Molecular Biology Core Facility have new transgenic mouse lines from external analyses of proteins and their post-translational end solutions for mass spec data generation, supported. Towards the end of the year the sources cannot be brought in directly as live modifications. This has resulted in additional storage and analysis. As technologies have MBCF took delivery of a NovaSeq instrument, mice. New lines coming from external sources applications being offered to our users, taking evolved this close interaction between the which will be used to further develop instead have to be transferred in as either advantage of the full abilities of the new traditional core facility areas has become vital sequencing services in 2020. embryos or sperm and thoroughly health hardware. and it has been great to see over the last year screened in order to ensure that the resulting how closely the teams in IT, Sci Com, Mass Spec A key consideration when developing services is offspring are pathogen free. When live mice are This year saw the uptake of label-free, TMT and and the Molecular Biology Core Facility have balancing the provision of full services, where received they are housed in separate quarantine SILAC based quantitative applications in both worked together to provide seamless end to end the facility do all sample processing and training facilities during the rederivation process. protein profiling and phospho-protein profiling support for research projects. for users to gain expertise in particular domains to great affect. 2019 also saw a step techniques. The Histology facility blends these Twelve staff members currently provide change in the way proteomic datasets are The collaborative approach is also well two approaches particularly well; by combining day-to-day care for 141 different transgenic computationally processed. The Scientific developed within the Visualisation, Imaging and provision of services with extensive training, our mouse lines spread across approximately 2,300 Computing team have performed a wonderful Irradiation team who work closely with both early career scientists gain valuable expertise, cages in a facility located within The University of job of migrating our data processing pipelines Histology and the BRU Experimental Facility to and we are able to achieve sample throughput Manchester main campus. The last year has onto a new virtual machine environment they support research activity. A new high throughput on expensive equipment that could not be been busy with 52 new breeding lines being built and manage on our behalf. Their hard work scanner has come online this year, and the work achieved with a small Histology team. started and 44 lines being closed. Thirteen of and application has resulted in our ability to has begun with Histology to further expand our the 52 new lines have been rederived, using share high performance computing resources capability for multiplex staining during 2020. Overall the core facilities have been involved in a embryos and live mice sent from the USA and across multiple workflows without the need to Support for multiplex analysis of single cells has diverse range of activities supporting research UK. Another four of the new lines have been replicate this resource for every software also developed in the FACS service as the Helios projects across the Institute. With new transferred from TPF, three have been rederived pipeline we use. We can therefore run our data platform has been used as the basis for a robust equipment and newly appointed staff the next from in-house frozen stocks and the remainder analysis applications at exceptionally high immuno-profiling platform. year should also prove to be highly successful. have been generated by crossing mice from performance levels and trial new software existing lines. Mice are transferred on request in applications without costly outlay for new The Experimental Facility have also carried out twice weekly shipments to the BRU hardware. Timely to this job’s completion, was some excellent work developing ultrasound Biological Mass Spectrometry Experimental Team at Alderley Park. After the appointment of a new Bioinformatician to guided injection of tumour cells, a technique Duncan Smith, Yvonne Connolly transfer a minimum of one week acclimatisation the Computational Biology Support team in late that delivers 3Rs benefits and scientific benefits is required before mice can be enrolled in 2019. The new role is shared between MBCF and in equal measure. The BRU Transgenic Breeding 2019 saw the re-launch our in-house MS experiments. As well as shipping to Alderley MS and the role will provide professional team have had an overhaul of operations with services on the Alderley Park site. The facility Park, this year we have also shipped live mice to bioinformatics support to the MS facility users the introduction of Tick@LAb, software that benefits from access to three cutting-edge mass Switzerland, USA and North Korea, and have sent proteomic data analysis needs. improves the efficiency of operation of the entire spectrometers (Orbitrap Lumos, Q-Exactive HFX frozen embryos to USA and Netherlands.

facility. Our Transgenic Production Facility has and a 6600 Q-ToF). Both Orbitrap based systems continued to be highly successful at delivering were new acquisitions at the end of 2018,

52 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES 53 RESEARCH SERVICES (CONTINUED) mice in plastic tunnels causes them less stress mycoplasma and mouse hepatitis virus (MHV). than the traditional method of picking them up Detection is carried out using highly sensitive by the tail so this is something we want to and specific reverse transcription coupled strongly consider as a welfare improvement. QPCR. Apart from the obvious reason of wanting Another initiative is refining our injectable to be certain which cell line one is working with 2019 saw the introduction of the specialist Experimental Services anaesthetic doses for our tumour-targeted journals and funding bodies frequently ask for mouse management system, going live in Team Leader: Lisa Doar irradiation (X-ray) work and also developing the evidence that cell lines used in research have February 2020. Records for all live mice in equipment we use to restrain the mice for the been authenticated. The most common established transgenic colonies were transferred 2019 has been a busy year for the BRU procedure so that we can use inhalation approach to authenticating cell lines is with a in the initial upload, with remainder of records Experimental Facility, with the workload anaesthesia instead of injectable anaesthesia, multiplex PCR assay, which detects highly being added soon after. The system is now continuing to increase, which is a great outcome which has a much faster recovery time and is variable short tandem repeat (STR) regions in the being used for keeping detailed animal stock following the disruption of the past two years better tolerated by the mice. genome. The Core’s Cell Line Authentication records, including breeding details and since the fire. Service currently uses a commercial kit that genotype results, tracking health problems, and In terms of technical development, we have also examines a total of 21 loci across the genome. In is also used for sending and reporting Outside of the day-to-day workload, the team been working on utilising our ultrasound system addition, we have recently explored the completion of tasks. We are moving over to has been involved in giving talks, attending to enable us to carry out image guided injection application of Next Generation Sequencing using the system for semi-automated transfer of training and symposiums, and developing 3Rs of tumour cells into different organs as an (NGS) in the analysis of STRs as well as single genotyping results and other functionalities may initiatives (reduction, refinement and alternative to relying on surgery. We are yet to nucleotide polymorphisms (SNPs) for cell be explored. One major advantage of moving replacement). We have given talks on some of trial this in a live study but the developmental authentication. over to the new system will be that mouse our interesting models and model development work is going well and we are looking at carrying details should be much more easily searchable work, which is an important part of our work out injections into the pancreas and also into the NGS related method development and across all lines, allowing easy identification of because the development of a good refinement bladder wall via this method. The process is validation remain a major focus for the Core’s subsets within the population. Similarly tasks should be shared more widely with other considerably quicker to carry out than surgery as services. Over the last year especially, we can also be tracked or searched, allowing both institutes. The team have also taken part in some well as being much less invasive for the mice, experienced a rapid uptake of single cell RNA researchers and technicians to track progress public engagement events and have been however it does require great technical skill so sequencing projects. The service currently offers more easily. Although edits can be made to the involved in training organised by Understanding training will need to be undertaken. two high throughput SC-RNA sequencing data on the system, changes will be tracked and Animal Research (UAR) in how to talk to school methods, SMART-Seq2 as well as 10x Genomics viewable within the history functionality, leading children about animal research. Alongside our normal day job, the team have Technology. The latter, based on single cell to both greater transparency and greater been putting time and effort into the plans for compartmentalisation using nano-liter sized gel security. In terms of 3Rs initiatives, we are currently our new BRU facility which will be built as part of beads in emulsion (GEM) has proved very working on rolling out tunnel handling of mice the Paterson Redevelopment Project. We have successful as it enables the simultaneous across the facility. It is thought that handling the had support from some companies experienced analysis of thousands of single cells in every in designing and building several other animal experiment. Through comparative validation facilities. Following much discussion, we now tests we showed that the GEM based single cell A CDX derived from a patient have the facility design detailing exactly where partition results in highly reproducible gene with small cell lung cancer was screened for the most commonly every piece of equipment will be located in each expression profiles. To date the service has expressed transcription factors. room. The opportunity to design our animal processed a wide variety of input samples, It is shown to mostly express facility and the planning process has been a ranging from cultured cell lines to primary tissue ASCL1 (in green), but also a few rewarding experience. including blood, bone marrow and tumour dispersed single cells expressing biopsies. In further developments in SC-RNA NEUROD1 (in red), that do not sequencing the service also incorporated express ASCL1. Molecular Biology Core and Computational methods for sample multiplexing (cell hashing) Image supplied by Alessia Biology Support Facility as well as feature barcoding (CITE-Seq) that Catozzi (Cancer Biomarker Wolfgang Breitwieser, Andzhela Abu Rashed, enables the simultaneous analysis of the Centre) Chris Clark, Bonnie Evans, Rachel Horner, Dave transcriptome and panels of protein markers in Lee1, Amy Priestman, Sudhakar Sahoo, Robert single cell populations. The annotation of the Sellers, and John Weightman heterogeneous cell types from primary tissue poses interesting new challenges. To this end, a 1Joined in 2019 range of novel bioinformatics analysis tools has been assessed and acquired by Computational Cell culture is a fundamental tool of many Biology Support. molecular biology research activities. It is therefore crucial that cultured cells are The Computational Biology Support team is unmistakably identified and are free from responsible for genomics and transcriptomics common pathogens such as mycoplasma. All analysis for projects undertaken by groups at the scientists at the MI are strongly encouraged to MI. Crucially, the team is also tasked with the test their cells upon receipt of new lines and establishment and benchmarking of regularly during culturing. The Molecular bioinformatics workflows for analysis pipelines, Biology Core has been offering a mycoplasma e.g. for bulk and single cell RNA, ChIP, whole screening service for a number of years using a genome, and targeted sequencing. Starting at PCR method and has, in the last year, switched the end of 2018, the CBS has undergone to a highly reliable QPCR detection method. In substantial changes in the team structure that addition, prior to injection into mice, all cell line has resulted in the merger with the Molecular samples are routinely screened for both Biology Core. Apart from the continued support

54 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES 55 RESEARCH SERVICES (CONTINUED) Sophisticated labelling techniques including the A new mouse model has been established and development of multiplex panels of antibodies, histologically characterised. In addition, the mRNA in situ hybridisation and the use of both environment of the eye and the presence of mRNA in situ hybridisation and protein tumour infiltration is being studied requiring immunohistochemistry on single tissue sections whole skull processing in addition to cranial- for the Institute’s research groups, the merger generated reporter mouse models that enable are routinely available and automated. The use caudal spatial information from the spinal cord. has produced a marked improvement in speed the studies of live cell imaging, cell lineage of DNA barcoded antibodies and their Specialised tissue processing and multiplex of quality control and validation of sequence run tracing, etc. simultaneous amplification prior to imaging is immunohistochemistry and the development of data coming off the instruments. This has proved another labelling technology the unit is currently panels of antibodies have been instrumental in of enormous benefit to maintain quality in Together with other transgenic facilities within evaluating. This allows for spatial, quantitative this study. standard services and in the validation of new the UK, we started the LASA Animal Science and tumor immune-profiling studies. methods. In addition to providing a helpful Transgenic Section. During 2019, we organised The Stem Cell Biology group have continued to bridge between the wet lab and the service two technical workshops about surgical The high throughput routine apply multiplex immunohistochemistry to a users, the integration of the Computational innovations and CRISPR technology in the immunohistochemistry service, troubleshooting variety of tissue samples, including mouse Biology Support has also helped in the generation of transgenic mouse models. These and antibody validation services once again prostate organoids and urogenital systems, and interactions with other services, e.g. Scientific two technical meetings, and the others to come continue to see high demand, together with an human patient tissue microarrays in order to Computing. Pleasingly, the team has, over the next year, provide the right environment to build increase in the numbers of researchers further characterise the expression of RUNX1 in course of the last year, grown in number and has an active community of transgenic accessing the automated platforms individually. the context of other markers. completed a multitude of analysis projects and technologists in the UK. The unit continues to be used routinely for contributed to numerous publications. Crucially, phenotyping of CDX models on our automated Another project looking at the assessment of the service has recruited a Senior Computational platforms ensuring consistency, reproducibility metastatic incidence and molecular traits of Biologist to fill a gap in support for tandem mass Histology and standardisation. brain metastases from melanoma in xenograft spectrometry data generated by the Institute’s Garry Ashton, Caron Abbey, Marta Madureira da models of cutaneous melanoma has used the Biological Mass Spectrometry Facility. This post Graca, Usman Mahmood, Emma Watson, Research projects involving the use of biobank immunohistochemistry service. Working in the assesses and maintains existing analysis Katherine Lally, Deepti Wilks (Haematological material processed through the facility core facility the PhD student was able to identify workflows, e.g. Mascot, with a mission to Malignancy Biobank), David Millard1, Keren continues to increase. A dedicated scientific a unique melanoma cell population with develop new pipelines and guide users in the Dawson2 officer is responsible for ensuring the unit is relatively high ability to colonise the brain analysis of all proteomics data. compliant with current human tissue legislation. (compared to its isogenic counterpart). 1Joined in 2019 2Left in 2019 In addition to FFPE and frozen tissue samples, Additionally, laser capture microdissection the number of blood, bone marrow and plasma allowed the extraction of genetic material Transgenic Production Facility By offering a full range of both routine and samples from haematological malignancy specifically from metastatic tissue regions Natalia Moncaut, Athina Papaemmanouil, Satish advanced histological services, the Histology patients continues to increase. allowing gene expression analysis. Arcot-Jayaram1, Mark Willington2 core facility underpins oncology research across the CRUK MI. In addition to the Alderley Park One interest of the Molecular Oncology group is More generally the Histology core facility has 1Joined in 2019 2Left in 2019 facility there is also a small facility housed within uveal melanoma, cancer of the eye arising from been involved in determining the invasion of the OCRB. Together both of these facilities allow the melanocytes of the uveal tract inside the eye. cells in organotypic assays in addition to The Transgenic Production Facility (TPF) is a core for the adoption of tissue-based experimental facility that offers a comprehensive service in the approaches across a large number of both basic generation of genetically engineered mouse and translational research groups. Hematoxylin and eosin (H&E) lines. TPF mission is to assist the researcher not staining of mouse lung tissue only by providing cutting-edge transgenic In 2019 recruitment continued with a new showing ‘heart’ shape formed services but to also advise them and if desired scientific officer appointed, continuing to by lung epithelial cells. carry out all steps of transgenic model design expand and develop the laser capture Image supplied by Charlotte Bell and generation on collaborative basis. microdissection service with downstream (Cancer Inflammation and extraction of both RNA and DNA. Both archival Immunity) The way we understand, classify and treat FFPE and fresh frozen samples are routinely cancer has been transformed by next generation evaluated. Like previous years, focus remains on sequencing of cancer patient genomes. In this the training and continued professional context, the discovery and development of development of staff ensuring the unit continues CRISPR-Cas-based genome editing has to be at the forefront with technological provided a powerful means to test the functional developments whilst also offering a relevance of human cancer-associated comprehensive and flexible service relevant alterations. In this context, we use this versatile across all research themes. technology to achieve the targeted introduction of patient-specific mutations in genetically In routine practice, both human and mouse engineered cancer mouse models in a cost- tissues, in addition to organotypic assays, effective and straightforward manner. This year, spheroids, agar plugs and cell pellets, continue we have collaborated with a number of research to be evaluated together with fresh vibratome groups using this approach and generated tissue sections (50–250μm) for ex vivo cultures several mouse models with constitutive and of tumours to evaluate and develop three conditional point mutations that will allow the dimensional studies. Requests for special stains researchers to evaluate the impact of such have seen an increase with Masson Trichrome, mutations in cancer development, progression PAS and reticulin stains still commonly and response to treatment. Also, using random requested. integration and targeted transgenesis we

56 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES 57 RESEARCH SERVICES (CONTINUED) analysis of sequencing data has been mass cytometry (Helios/CyTOF) within the accelerated by the integration of a new FPGA facility became the primary role of Antonia (Field-Programmable Gate Array) into Phoenix. Banyard. The Illumina DRAGEN (Dynamic Read Analysis for GENomics) Bio-IT Platform was purchased in Education and staff training became a strong verifying cellular findings in mouse models and Architect and provides programming as well as cooperation with the Molecular Biology core theme, with staff training a high priority. In ex vivo models for several groups. HPC support and assists scientists in facility and provides accurate, ultra-rapid addition to our usual training commitments, the implementing bioinformatic analysis pipelines. secondary analysis of sequencing data, making it facility took a strategic decision to give Finally, the Prostate Oncobiology group are In April, Rishi Ramgolam left the Institute after he a perfect addition to the existing platform. researchers greater access to the facility’s cell interested in characterising a subpopulation of successfully implemented the analysis pipelines sorters. Through an in-house “super user” luminal prostate progenitors and their role in for the clinical TARGET trial and migrated them In recent years, it has emerged that the steadily training scheme, users were trained to operate prostate tumourigenesis and treatment from the former Troodon HPC system to growing demand for virtual servers for compute, the facility’s cell sorters allowing the extension of resistance. Multiplexing has again been used to Phoenix. data and memory intensive workloads the hours of operation. immunophenotype and study cellular spatial necessitated the acquisition of a more powerful distribution. In addition, in situ hybridisation This year, SciCom focused on improving the and scalable virtualisation solution. Thus, During the past year 146 researchers and 32 allowing for gene expression analysis or the infrastructure and initiating new projects to build SciCom designed and purchased a virtualisation groups have used the facility’s resources. In study of translocation events in the murine the CRUK MI scientific computing platform for platform using high performance computing addition to supporting researchers, the facility prostate tissue has also been employed. the future. At the beginning of the year we hardware and Open Virtualisation (oVirt) VM has invested significant development time in expanded the storage capacity of the central management software. After the successful improving sample preparation and workflows, research storage by 1PB and its backup capacity installation, a project was initiated for migrating particularly those used in the newly established Scientific Computing by 1.5PB, increasing its total capacity to 2PB and all VMs to the new platform, including the virtual mass cytometry service. Marek Dynowski, Kevin Doyle, Nadeem Baig1, 3PB respectively. Afterwards, we took crucial servers for the HD-SCA project between the The sorting service spent a significant amount of Stephen Kitcatt1, Rishi Ramgolam2, ZhiCheng steps to develop Phoenix, CRUK MI’s High Cancer Biomarker Centre and Peter Kuhn’s lab at time establishing optimal sorting conditions for Wang Performance Computing Cluster, into a the University of Southern California. The problematic tissues such as prostate tissue. 1Joined in 2019 2Left in 2019 comprehensive data analysis platform. It now opportunity was also used for consolidating the These sample preparations contain an provides convenient tools for every aspect of the virtual machines with high availability but low assortment of cells encompassing huge ranges The high staff turnover, currently very common data analysis life cycle and gives users the option performance requirements, by transferring them of cell sizes that are difficult to resolve from in IT, continues to be a major challenge for the to subsequently analyse their data, which was to the Core IT infrastructure. The new cellular debris and cell aggregates, making Scientific Computing core facility (SciCom). processed and stored on Phoenix using an technology enables SciCom to replace some obtaining pure populations challenging. We Therefore, we were pleased to hire two new R-Web GUI (http://rstudio.scicom.cruk. costly Windows workstations with powerful VMs have also worked closely with the Molecular software architects in 2019. In January, Nadeem manchester.ac.uk). This reduces risks and speeds that users access through remote desktop Biology Core Facility to optimise single cell Baig joined the facility to work on the Octopus up the analysis process, since data no longer has connection. Several of these Windows VMs were sorting procedures, dramatically reducing assay Pre-processing framework, and Stephen Kitcatt to be transferred between local computers and set up for the Biological Mass Spectrometry and costs. joined in October. Stephen is a Senior Software Phoenix. The pre-processing and downstream the Visualisation, Irradiation & Analysis core facilities in close cooperation with the Core IT Flow Cytometry is considered the gold standard team. For example, the server component of the for evaluation of marker expression in Organotypic brain slice invasion HALO image analysis platform, which is immunological samples and recently the by patient-derived melanoma cells tagged. Red: melanoma administered by the IT team on SciCom IT addition of the Helios has considerably cells, green: brain-resident infrastructure. The cooperation between the strengthened our ability to probe deeply into astrocytes, blue: cell nuclei teams was further intensified by the publication which immune cells respond in the presence of (DAPI). Illustrating melanoma- of a common service catalogue for CRUK MI. cancerous cells. This technology allows brain interactions during brain researchers to investigate how these immune invasion by melanoma cells. cells are recruited and how they interact with Flow Cytometry each other and with cancer cells. We have Image supplied by Denys Holovanchuk (Molecular Jeff Barry, Antonia Banyard, Helen Carlin1, Jack developed phenotyping antibody panels for Oncology) Eastham¹, Michele Fresneda Alarcon¹, Yorsa murine and human cells. This powerful Elagili2 technique uses over 40 markers, producing information rich datasets. We are working 1Left in 2019 2Joined in 2019 closely with bioinformaticians to create powerful workflows to phenotype blood cells and tissue The Flow Cytometry facility’s remit is to provide cells for clinical trials and mouse models. This state-of-the-art instrumentation for both basic technique will enable our scientists to potentially science and translational cancer studies. The determine patient stratification for clinical facility also has a wider remit to work alongside treatments and elucidate pathways in mouse researchers to develop new approaches and to models to better understand progression of provide expertise and training. many different cancer types.

This year the facility has undergone a significant In the forthcoming year, the facility’s aims are to re-organisation. From April 2019 the Flow build a strong, responsive flow team and to Cytometry facility separated from Advanced continue to collaborate with researchers to Imaging (now Visualisation, Irradiation & develop and apply high content flow and mass Analysis), with Jeff Barry becoming the service cytometry applications. To further these manager. The operation and development of objectives, the facility is looking to renew some

58 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES 59 RESEARCH SERVICES (CONTINUED)

Live image of melanoma cells on the decline and so equipment that has rapidly become the convention for a wide range invading into collagen become surplus has been reconditioned and, in of research questions. The Facility team have some cases repurposed. The ability of the Facility had a busy year, as demonstrated by the extent Image supplied by Andrew to accommodate new equipment modalities to which instruments have been utilised: over Porter (Cell Signalling) and respond to research requirements is 2,000 hours for high-end microscopy; 34,000 essential as it endeavours to continually adapt to hours for incubator-based microscopy; 1,800 new demands in automation and analysis. hours for high content screening; over 24,000 slides processed for whole-slide histology In vivo and in vitro irradiation was enhanced in imaging; and over 80 training sessions were 2017 with the purchase of cabinet irradiators that held. permit customisation of dose and allow use of filtering to attenuate the beam. In collaboration Design of the new laboratories within the plans with BRU and Targeted Therapy (within Division for the redevelopment of the Paterson site has of Cancer Sciences), a recent development has taken a considerable amount of effort this year, been the introduction of aluminium filters to however we are coming to the end of the permit modification of the X-ray beam profile, planning phase and look forward to the start of reducing the penetrating ability of the beam and building work next year. hence a measurable reduction in dose with depth. The application of beam filtering refines For the coming year, plans are in place to research efforts and improves welfare of in vivo develop technology to increase the levels of model systems held under an X-ray. Over the multiplexing tissues from the routine five to coming year in collaboration with BRU new seven labels, through a collaboration between methods of location-based dosimetry will be the research groups and the Histology facility. explored, techniques for comparing irradiation This technology will enable complex pathways systems across research sites and completion of and interdependences to be mathematically a novel shielding system. modelled across a range of tissue and cancer types. In addition, there are also new avenues of The Facility operates under the philosophy of investigation into corelative technologies, such training and enabling researchers to be able to as imaging histology and combining mass operate the equipment and to assist in cytometry data, molecular biology outputs and performing data processing and analysis. The histology mapping, developments and extent of training this year has increased in refinements in irradiation technologies and response to demand, for levels of training from working with Scientific Computing for the basic through to advanced. Super-resolution implementation of more machine learning imaging is becoming a commonplace enabling algorithms. of its older systems to benefit from new and permitted new forms of imaging tissue, such as technology and 3D high content screening has novel instrumentation, ensuring we remain at extended focus and polarisation. There are plans the fore front of technological advancements. to introduce another system to automate mega-slide imaging, a technology that enables Populations of chronically Visualisation, Irradiation & Analysis larger tissue sections to be accessed and UV-damaged dermal fibroblasts Steve Bagley, Alex Baker, Isabel Peset Martin1, evaluated that will permit a greater are plated on FITC-labelled Heather Woodhouse1, Kang Zeng understanding of phenotype and heterogeneity. collagen (green) coated wells and With the assistance of Scientific Computing and stained with Hoechst (Nuclei - 1Left in 2019 core IT, additional licenses of histological image blue) and Phalloidin (Actin – red) to assess their collagen analysis software have been introduced as the degradation activity. 20x The facility has undergone expansion and majority of histology that is digitised is processed repositioning in response to both fundamental using tissue-based and single cell phenotypic Image supplied by Charles and translational research requirements, being analysis. Earnshaw (Skin Cancer and concerned not just with photon imaging but also Ageing) a range of other modalities. A large proportion of As automated imaging and screening becomes the facility’s efforts have always occupied the vital to research output, an additional incubator- space between the Institute facilities, such as based microscope was purchased. Within the with Histology with whole slide imaging and Facility there has been a migration from routine using high content screening to validate flow microscopy where a researcher is in front of a cytometry sorting. However, in vivo support for microscope to capture a few fields of view, to imaging and irradiation has occupied a large systems that permit the generation of large 2D proportion of this year. and 3D data sets, statistical relevant analysis and in a format that is collaborative. As a The purchase of an additional histology whole consequence, the use of widefield slide imaging system has aided throughput and environmental chamber-based microscopy is

60 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH SERVICES 61 CANCER RESEARCH UK MANCHESTER INSTITUTE PUBLICATIONS AND ADMINISTRATION

Tissue microarray cores from triple-negative breast cancer needle biopsies were subjected to multiplex immunofluorescence. Nuclei are labelled in blue; cancer cells labelled in green with a pan-cytokeratin antibody. CD45, expressed by all immune cells, is in red, and CD3, expressed only by T cells, is in yellow.

Image supplied by Christian Bromley (Cancer Inflammation and Immunity)

62 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE 63 Zhou C, Taylor S, Tugwood J, Simpson K, Rudin CM, Poirier JT, Byers LA, Dive C, Jayson GC, Symonds P, Paul J, Davidson S, Dowlati A, George J, Heymach JV, Johnson Carty K, McCartney E, Rai D, Dive C, West C. JE, Lehman JM, MacPherson D, Massion PP, RESEARCH (2019) Minna JD, Oliver TG, Quaranta V, Sage J, Dynamics of circulating vascular endothelial Thomas RK, Vakoc CR, Gazdar AF. (2019) PUBLICATIONS growth factor-A predict benefit from Molecular subtypes of small cell lung cancer: a antiangiogenic cediranib in metastatic or synthesis of human and mouse model data. recurrent cervical cancer patients. Nature Review Cancer, 19(5):289-297. British Journal of Clinical Pharmacology, 85(8):1781-1789. Cell Division (page 22) Vassileva V, Stribbling SM, Barnes C, Carroll L, Iain Hagan Braga M, Abrahams J, Heinzmann K, Haegeman C, MacFarlane M, Simpson KL, Refereed research publications Cancer Biomarker Centre Corcoran RB, Siu LL, Bardelli A. (2019) Dive C, Honeychurch J, Illidge TM, Aboagye Forte GM, Davie E, Lie S, Franz-Wachtel M, (page 14) How liquid biopsies can change clinical practice in oncology. EO. (2019) Ovens AJ, Wang T, Oakhill JS, Maček B, Hagan Caroline Dive Evaluation of apoptosis imaging biomarkers in Annals of Oncology, 30(10):1580-1590. IM, Petersen J. (2019) a genetic model of cell death. Import of extracellular ATP in yeast and man Refereed research publications EJNMMI Research, 9(1):18. modulates AMPK and TORC1 signalling. Chemi F, Rothwell DG, McGranahan N, Gulati Tinsley N, Zhou C, Tan G, Rack S, Lorigan P, Blackhall F, Krebs M, Carter L, Thistlethwaite F, Journal of Cell Science, 132(7). pii: jcs223925. S, Abbosh C, Pearce SP, Zhou C, Wilson GA, Harle ASM, Blackhall FH, Molassiotis A, Yorke Jamal-Hanjani M, Birkbak N, Pierce J, Kim CS, Graham D, Cook N. Cumulative antibiotic use significantly J, Dockry R, Holt KJ, Yuill D, Baker K, Smith JA. Ferdous S, Burt DJ, Slane-Tan D, Gomes F, (2019) Moore D, Shah R, Al Bakir M, Hiley C, Veeriah decreases efficacy of checkpoint inhibitors in Cell Signalling (page 26) patients with advanced cancer. Cough in patients with lung cancer: a S, Summers Y, Crosbie P, Ward S, Mesquita B, longitudinal observational study of Angeliki Malliri Oncologist [Epub 10 July 2019] Dynowski M, Biswas D, Tugwood J, Blackhall characterization and clinical associations. F, Miller C, Hackshaw A, Brady G, Swanton C, Chest, 155(1):103-113. Refereed research publications Dive C; TRACERx Consortium. (2019) Lindsay CR, Blackhall FH, Carmel A, Porter AP, White GRM, Mack NA, Malliri A. Fernandez-Gutierrez F, Gazzaniga P, Groen Pulmonary venous circulating tumor cell Mohan S, Foy V, Ayub M, Leong HS, Schofield (2019) dissemination before tumor resection and HJM, Hiltermann TJN, Krebs MG, Loges S, The interaction between CASK and the López-López R, Muinelo-Romay L, Pantel K, P, Sahoo S, Descamps T, Kilerci B, Smith NK, disease relapse. Carter M, Priest L, Zhou C, Carr TH, Miller C, tumour suppressor Dlg1 regulates mitotic Nature Medicine, 25(10):1534-1539. Priest L, Riethdorf S, Rossi E, Terstappen L, spindle orientation in mammalian epithelia. Wikman H, Soria JC, Farace F, Renehan A, Dive Faivre-Finn C, Blackhall F, Rothwell DG, Dive C, Brady G. Journal of Cell Science, 132(14). pii: jcs230086. Kirwan CC, Descamps T, Castle J. C, Besse B, Michiels S. (2019) EPAC-lung: pooled analysis of circulating Profiling of circulating free DNA using targeted Circulating tumour cells and and genome wide sequencing in patients with hypercoagulability: a lethal relationship in tumour cells in advanced non-small cell lung cancer. Small Cell Lung Cancer. Drug Discovery (page 28) metastatic breast cancer. [Epub 16 October Caroline Springer European Journal of Cancer, 117:60-68. Journal of Thoracic Oncology Clinical and Translational Oncology [Epub 31 2019] August 2019] Refereed research publications Marti FEM, Jayson GC, Manoharan P, Cornwell MJ, Thomson GJ, Coates J, O'Connor J, Renehan AG, Backen AC, Mistry Rothwell DG, Ayub M, Cook N, Thistlethwaite Geary B, Walker MJ, Snow JT, Lee DCH, Belotserkovskaya R, Waddell ID, Jackson SP, H, Ortega F, Li K, Simpson KL, Allen J, Connell F, Carter L, Dean E, Smith N, Villa S, Dransfield Pernemalm M, Maleki-Dizaji S, Azadbakht N, Galanty Y. (2019) J, Underhill S, Misra V, Williams KJ, Stratford I, J, Clipson A, White D, Nessa K, Ferdous S, Apostolidou S, Barnes J, Krysiak P, Shah R, Small-molecule inhibition of UBE2T/FANCL- Jackson A, Dive C, Saunders MP. (2019) Howell M, Gupta A, Kilerci B, Mohan S, Frese K, Booton R, Dive C, Crosbie PA, Whetton AD. mediated ubiquitylation in the fanconi anemia Novel phase I trial design to evaluate the Gulati S, Miller C, Jordan A, Eaton H, Hickson (2019) N, O'Brien C, Graham D, Kelly C, Aruketty S, pathway. Identification of a biomarker panel for early addition of cediranib or selumetinib to preoperative chemoradiotherapy for locally Metcalf R, Chiramel J, Tinsley N, Vickers AJ, ACS Chemical Biology, 14(10):2148-2154. detection of lung cancer patients. Kurup R, Frost H, Stevenson J, Southam S, Journal of Proteome Research, 18(9):3369- advanced rectal cancer: the DREAMtherapy trial. Landers D, Wallace A, Marais R, Hughes AM, Smithen DA, Leung LMH, Challinor M, 3382. Brady G, Dive C, Krebs MG. (2019) Lawrence R, Tang H, Niculescu-Duvaz D, European Journal of Cancer, 117:48-59. Utility of ctDNA to support patient selection for Pearce SP, Mcleary R, Lopes F, Aljarah M, Mohan S, Ayub M, Rothwell DG, Gulati S, early phase clinical trials: the TARGET study. Brown M, Johnson L, Thomson G, Marais R, Tay RY, Fernández-Gutiérrez F, Foy V, Burns K, Kilerci B, Hollebecque A, Sun Leong H, Smith , 25(5):738-743. Springer C. Pierce J, Morris K, Priest L, Tugwood J, Nature Medicine NK, Sahoo S, Descamps T, Zhou C, Hubner 2-Aminomethylene-5-sulfonylthiazole Ashcroft L, Lindsay CR, Faivre-Finn C, Dive C, RA, McNamara MG, Lamarca A, Valle JW, Dive Inhibitors of Lysyl Oxidase (LOX) and LOXL2 Blackhall F. (2019) Other publications C, Brady G. (2019) Show Significant Efficacy in Delaying Tumor Prognostic value of circulating tumour cells in Conway AM, Mitchell C, Cook N. (2019) Analysis of circulating cell-free DNA identifies Challenge of the unknown: how can we Growth. KRAS copy number gain and mutation as a limited-stage small-cell lung cancer: analysis of the concurrent once-daily versus twice- improve clinical outcomes in cancer of Journal of Medicinal Chemistry, [Epub 4 novel prognostic marker in Pancreatic cancer. unknown primary? September 2019] Scientific Reports, 9(1):11610. daily radiotherapy (CONVERT) randomised controlled trial. Journal of Clinical Oncology, 37(23):2089- 2090. Leung L, Niculescu-Duvaz D, Smithen D, Annals of Oncology, 30(7):1114-1120. Siravegna G, Mussolin B, Venesio T, Marsoni S, Lopes F, Callens C, McLeary R, Saturno G, Seoane J, Dive C, Papadopoulos N, Kopetz S,

64 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE RESEARCH PUBLICATIONS 65 RESEARCH PUBLICATIONS (CONTINUED) Pearson S, Blance R, Somervaille TCP, Whetton Oudit D, Dawson MA, Hicks RJ, Lorigan P, AD, Pierce A. (2019) McArthur GA, Marais R, Wong SQ, Dawson SJ. AXL inhibition extinguishes primitive JAK2 (2019) mutated myeloproliferative neoplasm Prediction and monitoring of relapse in stage III progenitor cells. melanoma using circulating tumor DNA. Davies L, Aljarah M, Brown M, Johnson L, Utility of ctDNA to support patient selection for HemaSphere, 3(3):e233. Annals of Oncology, 30(5):804-814. Zambon A, Chambers T, Ménard D, Bayliss N, early phase clinical trials: the TARGET study. Knight R, Fish L, Lawrence R, Challinor M, Tang Nature Medicine, 25(5):738-743. Other publications Rothwell DG, Ayub M, Cook N, Thistlethwaite H, Marais R, Springer C. (2019) Wingelhofer B, Somervaille TCP. (2019) F, Carter L, Dean E, Smith N, Villa S, Dransfield Anti-metastatic inhibitors of lysyl oxidase (LOX): Emerging epigenetic therapeutic targets in J, Clipson A, White D, Nessa K, Ferdous S, design and structure-activity relationships. Leukaemia Biology (page 32) acute myeloid leukemia. Howell M, Gupta A, Kilerci B, Mohan S, Frese K, Journal of Medicinal Chemistry, 62(12):5863- Tim Somervaille Frontiers in Oncology, 9:850. Gulati S, Miller C, Jordan A, Eaton H, Hickson 5884. N, O'Brien C, Graham D, Kelly C, Aruketty S, Refereed research publications Gilding LN, Somervaille TCP. (2019) Metcalf R, Chiramel J, Tinsley N, Vickers AJ, Jain A, Agostini LC, McCarthy GA, Chand SN, Williams MS, Amaral FM, Simeoni F, The diverse consequences of FOXC1 Kurup R, Frost H, Stevenson J, Southam S, Ramirez A, Nevler A, Cozzitorto J, Schultz CW, Somervaille TC. deregulation in cancer. Landers D, Wallace A, Marais R, Hughes AM, Lowder CY, Smith KM, Waddell ID, Raitses- A stress-responsive enhancer induces dynamic Cancers (Basel), 11(2). pii: E184. Brady G, Dive C, Krebs MG. (2019) Gurevich M, Stossel C, Gorman YG, Atias D, drug resistance in acute myeloid leukemia. Utility of ctDNA to support patient selection for Yeo CJ, Winter JM, Olive KP, Golan T, Pishvaian Journal of Clinical Investigation[Epub 26 early phase clinical trials: the TARGET study. MJ, Ogilvie D, James DI, Jordan AM, Brody JR. November 2019] (page 34) Nature Medicine, 25(5):738-743. (2019) Molecular Oncology Richard Marais Poly (ADP) ribose glycohydrolase can be Deb G, Wingelhofer B, Amaral FMR, Maiques- effectively targeted in pancreatic cancer. Diaz A, Chadwick JA, Spencer GJ, Williams EL, Refereed research publications Cancer Research, 79(17):4491-4502. Skin Cancer and Ageing Leong HS, Maes T, Somervaille TCP. Smithen DA, Leung LMH, Challinor M, (page 38) Pre-clinical activity of combined LSD1 and Lawrence R, Tang H, Niculescu-Duvaz D, Amaya Virós Pillay N, Tighe A, Nelson L, Littler S, Coulson- mTORC1 inhibition in MLL-translocated acute Pearce SP, Mcleary R, Lopes F, Aljarah M, Gilmer C, Bah N, Golder A, Bakker B, Spierings myeloid leukaemia. Brown M, Johnson L, Thomson G, Marais R, Refereed research publications DCJ, James DI, Smith KM, Jordan AM, Morgan Leukemia [Epub 28 November 2019] Springer C. Angela Moreno, Esperanza Manrique-Silva, RD, Ogilvie DJ, Foijer F, Jackson DA, Taylor SS. 2-Aminomethylene-5-sulfonylthiazole Amaya Virós, Celia Requena, Onofre (2019) Yoshimi A, Lin KT, Wiseman DH, Rahman MA, Inhibitors of Lysyl Oxidase (LOX) and LOXL2 Sanmartín, Víctor Traves, Eduardo Nagore. DNA replication vulnerabilities render ovarian Pastore A, Wang B, Lee SC, Micol JB, Zhang XJ, Show Significant Efficacy in Delaying Tumor (2019) cancer cells sensitive to poly(adp-ribose) de Botton S, Penard-Lacronique V, Stein EM, Growth. Histologic features associated with an invasive glycohydrolase inhibitors. Cho H, Miles RE, Inoue D, Albrecht TR, Journal of Medicinal Chemistry [Epub 4 component in lentigo maligna lesions. Cancer Cell, 35(3):519-533. Somervaille TCP, Batta K, Amaral F, Simeoni F, September 2019] JAMA Dermatology, 155(7): 782–788. Wilks DP, Cargo C, Intlekofer AM, Levine RL, Acton B, Small HF, Smith KM, McGonagle A, Dvinge H, Bradley RK, Wagner EJ, Krainer AR, Leung L, Niculescu-Duvaz D, Smithen D, Lopes Stowell AIJ, James DI, Hamilton NM, Hamilton Abdel-Wahab O. (2019) F, Callens C, McLeary R, Saturno G, Davies L, N, Hitchin JR, Hutton CP, Waddell ID, Ogilvie Stem Cell Biology (page 40) Coordinated alterations in RNA splicing and Aljarah M, Brown M, Johnson L, Zambon A, DJ, Jordan AM. (2019) Georges Lacaud epigenetic regulation drive leukaemogenesis. Chambers T, Ménard D, Bayliss N, Knight R, Fluoromethylcyclopropylamine derivatives as Nature, 574(7777):273-277. Fish L, Lawrence R, Challinor M, Tang H, Marais potential in vivo toxicophores - A cautionary Refereed research publications R, Springer C. (2019) Miyamoto C, Kojo S, Yamashita M, Moro K, disclosure. Paris J, Morgan M, Campos J, Spencer GJ, Anti-metastatic inhibitors of lysyl oxidase (LOX): Lacaud G, Shiroguchi K, Taniuchi I, Ebihara T. Bioorganic & Medicinal Chemistry Letters, Shmakova A, Ivanova I, Mapperley C, Lawson design and structure-activity relationships. (2019) 29(4):560-562. H, Wotherspoon DA, Sepulveda C, Vukovic M, Journal of Medicinal Chemistry, 62(12):5863- Runx/Cbf complexes protect group 2 innate Allen L, Sarapuu A, Tavosanis A, Guitart AV, β 5884. lymphoid cells from exhausted-like Villacreces A, Much C, Choe J, Azar A, van de hyporesponsiveness during allergic airway Head and Neck Cancer Lagemaat LN, Vernimmen D, Nehme A, Rodrigues M, Koning L, Coupland SE, inflammation. Mazurier F, Somervaille TCP, Gregory RI, Biology (page 30) Jochemsen AG, Marais R, Stern MH, Valente A, Nature Communications, 10(1):447. Robert Metcalf O'Carroll D, Kranc KR. (2019) Barnhill R, Cassoux N, Evans A, Galloway I, Targeting the RNA m6A reader YTHDF2 Jager MJ, Kapiteijn E, Romanowska-Dixon B, Vijayabaskar MS, Goode DK, Obier N, selectively compromises cancer stem cells in Refereed research publications Ryll B, Roman-Roman S, Piperno-Neumann S; Lichtinger M, Emmett AML, Abidin FNZ, Shar N, acute myeloid leukemia. Rothwell DG, Ayub M, Cook N, Thistlethwaite UM Cure 2020 Consortium. (2019) Hannah R, Assi SA, Lie-A-Ling M, Gottgens B, Cell Stem Cell, 25(1):137-148.e6. F, Carter L, Dean E, Smith N, Villa S, Dransfield So Close, yet so Far: Discrepancies between Lacaud G, Kouskoff V, Bonifer C, Westhead DR. J, Clipson A, White D, Nessa K, Ferdous S, Uveal and Other Melanomas. A Position Paper (2019) Lin CH, Wang Z, Duque-Afonso J, Wong SH, Howell M, Gupta A, Kilerci B, Mohan S, Frese K, from UM Cure 2020. Identification of gene specific cis-regulatory Demeter J, Loktev AV, Somervaille TCP, Gulati S, Miller C, Jordan A, Eaton H, Hickson Cancers (Basel), 11(7). pii: E1032. elements during differentiation of mouse Jackson PK, Cleary ML. (2019) N, O'Brien C, Graham D, Kelly C, Aruketty S, embryonic stem cells: An integrative approach Oligomeric self-association contributes to E2A- Metcalf R, Chiramel J, Tinsley N, Vickers AJ, Tan L, Sandhu S, Lee RJ, Li J, Callahan J, Ftouni using high-throughput datasets. PBX1-mediated oncogenesis. Kurup R, Frost H, Stevenson J, Southam S, S, Dhomen N, Middlehurst P, Wallace A, PLoS Computational Biology, 15(11):e1007337. Landers D, Wallace A, Marais R, Hughes AM, Scientific Reports, 9(1):4915. Raleigh J, Hatzimihalis A, Henderson MA, Brady G, Dive C, Krebs MG. (2019) Shackleton M, Haydon A, Mar V, Gyorki DE,

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Other publications Tumour Supressors (page 48) Mevel R, Draper JE, Lie-A-Ling M, Kouskoff V, Patricia Muller Lacaud G. (2019) RUNX transcription factors: orchestrators of Other publications development. Mackay HL, Muller PAJ. (2019) There was a reduced number of PhD theses submitted in 2019 due , 146(17). pii: dev148296. Development Biological relevance of cell-in-cell in cancers. to extensions to the studies of a number students as a consequence Biochemical Society Transactions, 47(2):725- Menegatti S, de Kruijf M, Garcia-Alegria E, 732. of the Paterson Building fire in 2017. Lacaud G, Kouskoff V. Transcriptional control of blood cell Hall C, Muller PAJ. (2019) emergence. The diverse functions of mutant 53, its family FEBS Letters593(23): 3304–3315 members and isoforms in cancer. International Journal of Molecular Sciences, 20(24), 6188. Amy McCarthy Translational Oncogenomics Systems Oncology (page 46) Rob Bristow Investigating heterogeneity in tumour-stroma interactions in Select additional pancreatic ductal adenocarcinoma Refereed research publications publications Salem A, Little RA, Latif A, Featherstone AK, Babur M, Peset I, Cheung S, Watson Y, Memon D, Bi J, Miller CJ. (2019) Tessyman V, Mistry H, Ashton G, Behan C, In silico prediction of housekeeping long Matthews JC, Asselin MC, Bristow RG, Jackson intergenic non-coding RNAs reveals HKlincR1 A, Parker GJM, Faivre-Finn C, Williams KJ, as an essential player in lung cancer cell Athanasios Rafail (Sakis) Paliouras O'Connor JPB. (2019) survival. Transcriptional Networks in Lung Cancer Oxygen-enhanced MRI is feasible, repeatable, Scientific Reports, 9(1):7372. and detects radiotherapy-induced change in Investigating the mechanisms of acquired resistance to ALK hypoxia in xenograft models and in patients Hadjidemetriou M, McAdam S, Garner G, inhibitors in EML4-ALK-driven lung cancer with non-small cell lung cancer. Thackeray C, Knight D, Smith D, Al-Ahmady Z, Clinical Cancer Research, 25(13):3818-3829. Mazza M, Rogan J, Clamp A, Kostarelos K. (2019) Bhandari V, Hoey C, Liu LY, Lalonde E, Ray J, The human in vivo biomolecule corona onto Livingstone J, Lesurf R, Shiah YJ, Vujcic T, PEGylated liposomes: a proof-of-concept Huang X, Espiritu SMG, Heisler LE, Yousif F, clinical study. Huang V, Yamaguchi TN, Yao CQ, Sabelnykova Advanced Materials, 31(4):e1803335. VY, Fraser M, Chua MLK, van der Kwast T, Liu SK, Boutros PC, Bristow RG. (2019) Matsumura Y, Ito Y, Mezawa Y, Sulidan K, Daigo Molecular landmarks of tumor hypoxia across Y, Hiraga T, Mogushi K, Wali N, Suzuki H, Itoh T, cancer types. Miyagi Y, Yokose T, Shimizu S, Takano A, Terao Nature Genetics, 51(2):308-318. Y, Saeki H, Ozawa M, Abe M, Takeda S, Okumura K, Habu S, Hino O, Takeda K, Other publications Hamada M, Orimo A. (2019) Taylor RA, Fraser M, Rebello RJ, Boutros PC, Stromal fibroblasts induce metastatic tumor Murphy DG, Bristow RG, Risbridger GP. (2019) cell clusters via epithelial-mesenchymal The influence of BRCA2 mutation on localized plasticity. prostate cancer. Life Science Alliance, 2(4). pii: e201900425. Nature Reviews Urology, 16(5):281-290. von Schuckmann LA, Hughes MCB, Lee R, Rebello RJ, Oing C, Gillessen S, Bristow RG. Lorigan P, Khosrotehrani K, Smithers BM, (2019) Green AC. (2019) TP53 and Prognosis in mCRPC Survival: Biology Survival of patients with early invasive or Coincidence? melanoma down-staged under the new eighth Clinical Cancer Research, 25(6):1699-1701. edition of the American Joint Committee on Cancer staging system. Journal of the American Academy of Dermatology, 80(1):272-274.

68 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE THESES 69 EXTERNAL SEMINAR

SPEAKERS 2019 Actin fibres (stained red with the dye phalloidin) in 3D

Image supplied by Andrew Porter (Cell Signalling)

The seminar series that we run is vital for the Institute, connecting world-class researchers across the broad spectrum of cancer research. We have enjoyed another successful year for scientific 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. The postdoctoral researchers at the Institute also give weekly seminars which are very well attended and help to integrate the entire cancer research efforts of the Institute.

Massimiliano Mazzone Pamela Kreeger Lukas Dow Zuzana Koledová VIB Leuven Centre for Cancer Biology University of Wisconsin-Madison Weill Cornell Medicine Masaryk University

Kairbaan Hodivala-Dilke Andrew Fry James O’Connor Hasan Korkaya Barts Cancer Institute University of Leicester University of Manchester Augusta University

René Bernards Gerard Evan Madelon Maurice María del Mar Vivanco Netherlands Cancer Institute University of Cambridge UMC Utrecht CIC bioGUNE

Pierre Close Steve Jackson Sara Zanivan Carla van Gils GIGA Institute The Gurdon Institute Beatson Institute UMC Utrecht

Manuela Tosin Shozeb Haider Marleen Kok Karin E. de Visser University of Warwick UCL Netherlands Cancer Institute Netherlands Cancer Institute

Julian Downward Kenneth Pienta Maite Huarte Cristina Branco Francis Crick Institute Johns Hopkins University Hospital CIMA, University of Navarra Queen’s University Belfast

Miguel Ángel del Pozo Elke van Oudenhove Charlotte Coles Spanish National Centre for Cardiovascular Perlmutter Cancer Centre Breast Cancer Now Seminars Cambridge University Hospitals Research Qi Chen Therese Sørlie Richard Bayliss University of California, Riverside Oslo University Hospital University of Leeds Anton Berns Cathrin Brisken Sam Butterworth Netherlands Cancer Institute Swiss Federal Institute of Technology Lausanne University of Manchester Walid Khaled Jeff Pollard David Tuveson University of Cambridge University of Edinburgh Cold Spring Harbour Laboratory

70 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE EXTERNAL SEMINAR SPEAKERS 2019 71 OPERATIONS

sites. They support a varied programme of Over the past year, the HR Department national and international speakers. Details continued to deliver a high quality, proactive The Operations team provides a suite of support services that can be found at www.cruk.manchester.ac.uk/ service to the Institute. The department seminars. provides advice and guidance to managers facilitate the smooth running of the Institute. There are four senior and staff on all employment related matters operational managers. Caroline Wilkinson is the Chief Operating such as recruitment, onboarding, policy Officer with responsibility for scientific administration and Finance and Purchasing guidance, employment legislation and best Mike Berne, David Jenkins, Denise Owen, practice. communications. She also acts as the primary point of operational Muhammad Raja, Vikki Rosheski, Debbie contact within the Institute for both The University of Manchester Trunkfield The department’s main focus in 2019 was the development and launch of our online Chief Operating Officer and Cancer Research UK. Rachel Powell is the Chief Human With the transition to Alderley Park finally candidate management system JobMarker, Caroline Wilkinson Resources Officer and oversees a team providing general human complete, operational activity began to return which went live in June. The implementation resources support and recruitment services. Stuart Pepper is the to pre-fire levels which brought with it of this system has streamlined the recruitment Chief Laboratory Officer and has oversight of general laboratory increased financial activity. The finance team and selection processes at the Institute, began a restructure of activity and therefore enhancing the candidates’ overall management across the Institute as well as responsibility for the IT responsibilities as we look to provide a more experience. and Health and Safety teams. Mike Berne is the Chief Finance comprehensive and efficient financial service Officer who manages the work of the Institute’s finance and to the group leaders. The department has also transferred wholly over to e-personnel files to ensure that we purchasing team. The Institute continues to support the Director continue to align to GDPR requirements and and the management of the £30m budget best practice. Chief Laboratory Officer while providing costs and advice for new This year we welcomed a number of new General Administration Team Stuart Pepper research proposals and contracts for all of our The department has been visible at a number members to the team. Chris Bamber joined us Ruth Cox, Samantha Brandolani1, Maria Belen groups. We have also started to review activity of career fairs such as Nature Careers and as a Health and Safety Advisor; Samantha Conti2, Jayne Fowler, Delydd Jones within the core facilities to try and improve the recruitment events held at Alderley Park. Brandolani was recruited as maternity cover 1 Joined in 2019 financial management and systems currently During 2019, we completed 136 recruitment for Ruth Cox; Andrew Haines started as a 2 Joint with the Scientific Administration team implemented. We have been awarded 10 new rounds and successfully appointed 79 Recruitment Officer; and Wayne Howarth research grants totalling nearly £4m within the individuals to enhance the work of the joined the Logistics team. We said goodbye to This year we welcomed Samantha Brandolani year as well as a number of new commercial Institute. This was compared to 126 Hong Mach, Tom Bolton, Stephen Keane and as Executive Assistant to the Institute Director contracts and renewed funding against recruitment rounds in 2018; an increase of 7.5% Tony Dawson. We wish them well in their as she covers the maternity leave of Ruth Cox. existing grants in one year. In addition, we have coordinated future endeavours and thank them for their Samantha also manages the Institute 148 security screenings for individuals at many years of excellent service at the Institute. Administration team which comprises of Belen The exit from the European Union has had an Alderley Park and inducted 116 individuals on The Operations team has integrated well into Chief Finance Officer Conti, who undertakes the role of Executive extensive impact on the finance team this year the Alderley Park Site Induction. life at our interim home of Alderley Park whilst Assistant to the Senior Management Team, with work being carried out around avoiding Mike Berne continuing to support activity at our other sites Jayne Fowler as Executive Assistant to the disruption of service during the Brexit period. In 2019 the department facilitated the as we work on arrangements for our eventual Director of the Drug Discovery Unit, and In addition to this, we have a continuing successful promotion of 10 individuals. The return to our main base in Withington. Major Delydd Jones who is our Administration responsibility to adhere to financial regulations Institute has continued our commitment to projects across the team for the year included Services Coordinator. and procedures, including those outlined in develop our staff and ensure that Personal examining the Institute’s results from the our research grants and contracts. Given that Development Reviews (Contribution Reviews) biannual University staff survey, holding As a team they support the Director and the we collaborate with a number of European are undertaken. feedback sessions with staff and preparing an Institute Faculty in the current, temporary entities, there is a lot of work to be undertaken action plan in response. We developed and home of Alderley Park. They are responsible to assess the impact of research funding We have continued our commitment to joint deployed a new online portal to streamline our for the organisation of several annual events, changes stemming from the UK’s exit. partnership working with the union, which has recruitment process. The team continued to including the Summer Party, Institute resulted in the revision of several HR policies work on operational preparations for a Chief Human Resources Officer Christmas party, and a whole host of guest and procedures. We have also worked closely possible no-deal scenario following the exit of speakers to deliver lectures over the course of Human Resources with CRUK and The University of Manchester, Rachel Powell the UK from the EU and carried out a rolling the year along with the Institute Colloquium. Rachel Powell, Laura Bayliff, Rachel Craven, plus the Institute is also a member of a review and update of our general business Andrew Haines1, Julie Jarratt, Laura Jones, research-based Pay Club, which consists of 11 continuity plans. They ensure that staff at both Alderley Park and Emma Lloyd, David Stanier2 other research institutes. We also held a series of staff meetings along with the Senior OCRB are able to participate in the visits and ¹ Joined in 2019 view the seminars using a video-link between 2 Joint with the Scientific Administration team Management Team to discuss the results from

72 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE OPERATIONS 73 OPERATIONS (CONTINUED) in our large -80oC freezer room (which • Organising the delivery of clean general houses many valuable research samples). We and tissue culture lab coats across the AP have also reviewed the risk control measures in site relation to CRUK MI activities, including MSC testing, isoflurane monitoring in the Biological Working with the Health and Safety Manager, the staff survey; this provided an opportunity CRUK MI resilient wired and wireless network Resources Unit and airborne xylene levels in the Lab Services Head coordinates the for staff to understand the work undertaken by infrastructure across all CRUK MI research the Histology facility areas. maintenance and testing of the microbiological the Institute and enable further developments facilities at Alderley Park and the OCRB. safety cabinets and the lab water systems at AP. in employee relations at the Institute. We also have been involved in the Paterson Our email service is MS-Exchange based and is replacement building design process, In conjunction with the Chief Laboratory Officer, the Lab Services Head reports and In the meantime, the Institute’s Gender Pay currently hosted by The University of attending meetings and advising and tracks facility management concerns raised in Gap has decreased this year from 12.4 % to Manchester. This will transition to O365 during discussing our requirements with the laboratories and in shared spaces such as cold 10.5% and we will continue to monitor this the coming year as part of the University's digital designers. The range of issues discussed has rooms, freezer rooms, dark rooms and the closely. The Institute is committed to working transformation project.Supporting multi-site been large and included ventilation and towards the Athena Swan accreditation and operation and remote working is a challenge, room. Over the last year, the cooling requirements, fume cupboard Freezer Monitoring System that protects our this will be a priority over the next 12 months. however, we have deployed network specifications, drainage needs, electrical stored samples was expanded to include more We have continued to provide support our EU monitoring to rapidly identify the source of any supply requirements, space requirements for freezers and fridges. staff during the uncertain time as the UK outages. We also make greater use of services and the general layout of laboratories prepares to leave the European Union. automated deployment tools to deploy new and offices. This will continue in the In addition, the Lab Services Head continues to client computers. Also, our adoption of 'self- forthcoming months. administer the revised lab waste removal Next year, the focus will be on the Athena service' application installation now enables account at AP and manages the cleaning team Swan accreditation, review of the Personal research staff to resolve a significant number of We have also undertaken more routine health based at OCRB. Development Reviews and the recruitment of IT Service Requests themselves. and safety activities, including the new research groups in line with the Institute’s implementation of a full inspection schedule research strategy. In the past year, we also developed further of our laboratory areas, the production of Logistics collaboration with our Scientific Computing remedial action plans and their close-out. We Andrew Lloyd, Michael Alcock, Edward Fitzroy, and Visualisation, Irradiation and Analysis will further analyse these shortly to identify any Nigel Fletcher, Sedia Fofana, William Glover, Information Technology groups on various aspects of IT service Institute wide learning opportunities and Wayne Howarth1, Stephen Keane2, Jonathan Steve Royle, Matthew Young, Hong Mach1, provision with a view to adopting a more unified report them to our Health and Safety Lloyd, Robin Sherratt, Tony Woollam Brian Poole approach going forward. Committee. 1Joined in 2019 2Left in 2019 ¹ Left in 2019 Planning work is now progressing at pace to Over the past year, the Logistics teams based shape our return to our former 'home' in The CRUK Manchester Institute core IT team Laboratory Services at Oglesby Cacner Research Building and Withington based on The Christie NHS provides a wide range of IT support services to Mark Craven, Busola Atuegbe1, Corinne Hand, Alderley Park have continued to deliver Foundation Trust site, in a new custom-built over 400 research and support staff, currently Tony Dawson2, Petra Kubinova and Christine successfully a high quality and reactive service cancer research building to be shared between spread across several sites. This includes staffed Whitehurst to the Institute. The University of Manchester, including CRUK 1 2 service desks on our two main sites at Alderley Joined in 2019 Left in 2019 MI, and The Christie. Park and the Oglesby Cancer Research The service includes the receipting, checking, Building, where we provide 'drop-in' Service During 2019, Lab Services continued to support booking in and distribution of goods ordered Desks providing hardware and software support the research buildings across the site with a by staff. In the OCRB, the team facilitate the Safety and Facilities Management and advice. main base at the OCRB where the glass washers delivery of dry ice where they also monitor the Colin Gleeson and autoclaves are based. From here they gas cylinders and replace them as necessary. supply sterile glassware, plastics and bespoke We manage over 600 desktop computers, The team also monitor the liquid nitrogen Health and Safety microbiological media to scientists at the OCRB comprising a mixture of Windows PC's and levels in the cell storage tanks and will Colin Gleeson, Chris Bamber1 and the expanded sites at the Kay Kendall Laptops, Apple Macs and Mac Book's, plus a replenish these when required. 1 laboratory, WMIC Building and Incubator growing number of 'tablet' devices, mainly Joined in 2019 Building. Apple iPad's and iPhones. All centrally Researchers can order central stores stock authenticated, with access to a central file-store, We have focused on a range of issues over the items via the intranet, which can be collected past year. These have included the The team support the scientists at AP, alongside a server farm and network printing. All desktop, the on-site VWR team, with sterile plastics and or distributed by the Logistics team. Included development of a risk control assurance portable and handheld devices are built on bespoke microbiological media. in this system are the enzymes and media Windows 10, Mac-OS Mojave or OSX standard programme with particular focus on the stored in the Institute freezers at the OCRB services provided by the landlord of Alderley images to facilitate their on-going In partnership with the Logistics team, Lab (Sigma, Life tech, Promega, New England Bio Park. In so doing, we have secured the management. Services continue to deliver items from OCRB to labs, and Qiagen). New products have been provision of the landlord’s building fire risk other sites via the daily shuttle van service. They introduced throughout the year. We have Our core IT infrastructure comprises an assessments, including the testing of fire also continue to support the research groups in continued to make savings by buying in bulk enterprise-class file storage facility with a alarms, planned fire evacuations, the testing of other ways: from our suppliers. o o capacity in the region of 400Tb for our research cold room (+4 C and -20 C) alarms, liquid data. This is based on a replicated design, nitrogen room low-oxygen alarms, RCD • Maintaining and servicing two The team based at OCRB support waste hosted in two geographically separate testing, the testing of fixed LEV, the removal of photographic dark rooms at OCRB and AP removal and are implementing new recycling datacentres to provide a resilient, high waste by the landlord’s contractors, and the • Providing a drop in monthly pipette clinic initiatives. Recycling at Alderley Park continues availability, redundant, and fit for purpose, maintenance of critical equipment controlling at OCRB and AP storage facility. This is connected by a dedicated environmental parameters such as the cooling

74 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE OPERATIONS 75 OPERATIONS (CONTINUED) David Stanier has continued to oversee the In 2019, our AWERB met formally on five Institute’s transport arrangements between occasions with two additional meetings Alderley Park and the Oglesby Cancer involving all licensees at the Institute. Two of Research Building. This latter site is home to these meetings were also attended by our two of our research teams who have remained Home Office Inspector. During the year, five to develop with the CRUK MI Logistics team Administration team that is overseen by close to the Christie Hospital site for applications to the Home Office for new working together with APL waste teams. Caroline Wilkinson, Chief Operating Officer for operational reasons. He also co-ordinates Project Licences and 12 applications for the Institute, who also acts as the main point of several aspects of on-boarding new starters amendments to existing Project Licences were One of the key tasks carried out by the team contact for both The University of Manchester including data protection training and health reviewed by the AWERB. Significant input is over the past year is the transport of samples and Cancer Research UK. The team have and safety induction sessions. David is the provided to the applicants by the Home Office and goods. Using the OCRB as the main base continued to work with colleagues at Alderley Institute’s Information Governance Co- Liaison Coordinator (HOLC), Named Veterinary and drop off point, the team have supported Park to ensure that we are integrated with ordinator supporting Caroline Wilkinson as the Surgeon (NVS) and Named Animal Care and the movement of samples and goods processes at our interim location. Institute’s Information Governance Guardian. Welfare Officer (NACWO) at the draft stage but between various research locations/core This year David has worked closely with review by the wider AWERB membership facility groups. The team continue to collect Gill Campbell is our Grants Adviser who colleagues at The University of Manchester’s contributes to further improvements. Licences samples from the Christie NHS Foundation provides support for the Institute’s researchers Information Governance Office to implement have incorporated a number of techniques Trust CTU on a daily basis and support the to apply for external funding to supplement a new information governance software tool new to CRUK MI this year (e.g. ultrasound collection of mice from the Incubator Building their core CRUK allocation. A total of 39 grant to track information assets. He has also guided injection into the pancreas; injection of twice weekly. The team have also worked applications were submitted in 2019, with 10 conducted several audits to ensure that the tumour cells into the brain of mice under alongside the Lab Service team in delivering being successfully funded, bringing in the Institute continues to follow best practice with anaesthesia). At our mouse breeding facility sterile media and glassware and returning additional sum of over £4m to the Institute. Of respect to protecting our data. we have introduced a new database system to empty items. The team are currently particular note is the CRUK Experimental manage the tracking of the breeding supporting staff based in the Incubator Medicine Award to Richard Marais and Christie Steve Morgan continues in his role at the programme. This will result in efficiencies in Building, WMIC building, Michael Smith NHS Foundation Trust Consultant Medical Oglesby Cancer Research Building where he our breeding programme. We have actively building, Tumour Implantation Facility and the Oncologist Paul Lorigan, which will fund their works closely with staff from the University’s participated in workshops, or given invited Christie Biobank team located in the KK DETECTION trial to use circulating tumour Faculty of Biology, Medicine and Health to presentations at national level (involving the laboratories. Next year there are plans to DNA to guide treatment for metastatic ensure that reception runs smoothly and to NC3Rs, RSPCA and LASA) on our practices and support the research lab based in the Proton melanoma. Another significant success was operate the Institute’s switchboard. improvements that we have introduced. We Beam facility. the CRUK Accelerator Award to Caroline Dive are currently trialling a tunnel handling and Andrew Hughes, funding their technique for mice, which is recommended to programme “UpSMART” that aims to digitalise Animal Welfare provide welfare advantages to the mice and Electronics experimental cancer medicine centres across Caroline Wilkinson, Establishment Licence the quality of data generated compared to Yunis Al-hassan, Tony Woollam the UK, Italy and Spain. Georges Lacaud also Holder, Simon Poucher, Regulatory Liaison traditional means of handling. The NVS received funding from the charity Bloodwise, and Training Officer, Janet Watson, Animal provides invaluable advice on all animal As part of the Institute’s electrical and fire to investigate MOZ histone acetyl transferase Welfare & Ethical Review Body (AWERB) Chair, husbandry and procedural technique matters, safety strategies, the Electronics Team have activity in leukaemia. The grant application Stuart Pepper, Deputy AWERB Chair including this year improved anaesthesia PAT tested well over a thousand pieces of process is ably supported by our Grants practice, screening of biological agents to electrical equipment across multiple sites. This Committee, chaired by Iain Hagan, who review The Institute upholds the highest standards of minimise risk of adverse reactions, after care includes PAT testing newly acquired all proposals and provide critical feedback. welfare for the laboratory mice used in our following tumour removal and dosing to the equipment. In addition, the Institute’s research. All animal research activities are lungs via the airways. A total of 30,413 mice electronics engineer has assembled many Tom Bolton is the Institute’s Web Developer. In conducted in full compliance with the Animals were used at the Institute in regulated items of new equipment and repaired and 2019, he worked closely with the Human (Scientific Procedures) Act 1986 (ASPA) and are procedures under the Act in 2019. calibrated multiple items of scientific research Resources team to produce a bespoke online scrutinised by the Institute’s Animal Welfare equipment. In a number of cases repairs are application system for staff recruitment for the and Ethical Review Body (AWERB). This Licensees are required by law to report any carried out at electronic component level. This Institute, which was successfully deployed consists of experienced animal husbandry unforeseen adverse effects on animals or repair facility provides a significant economic resulting in streamlining the Institute’s staff, a veterinary surgeon, Institute scientists, a breaches of the controls and limits in their benefit to the Institute in that unnecessary recruitment process. Tom moved on in the statistician and lay members. The AWERB licence. Thirty-four incidents were self- expenditure on replacement equipment is autumn after several successful years at the supports all staff involved with animal research, reported to the Home Office in the year. From avoided. The Institute electronics engineer Institute and we wish him all the best in the ensuring the provision of appropriate these incidents, the majority were mice found also tracks Institute equipment which is under next phase of his career. management structures and processes, staff dead overnight unexpectedly after being warranty, service contract or in-house repair. training, the facilities for the care and use of assessed as being well the previous day (thirty Again, this provides a significant economic Julie Edwards is the Postgraduate Education mice, and encouraging implementation of the in total). Eight mice failed to recover from benefit to the Institute. Manager and has continued to help our 3Rs’ principles (replacement, reduction and short term general anaesthesia. All incidents students with the transition to Alderley Park, refinement of animals). It also reviews the were satisfactorily resolved with the inspector and in particular, with arranging extensions for ethics of proposed collaborations and all grant with suitable adjustments made where Scientific Administration those whose PhDs were interrupted by the fire applications involving animal research. One of relevant. There were also incidents whereby Caroline Wilkinson, Tom Bolton1, Gillian at the Paterson Building in 2017. Julie attended our Group Leader scientists has been twenty mice on a breeding programme were Campbell, Maria Belen Conti3, Julie Edwards, a successful careers event at Alderley Park for appointed this year to the Review Panel for not culled within a specified time frame. There Steve Morgan, David Stanier2 University of Manchester students and co- grant awarding at the National Centre for the were no welfare issues for these mice 1Left in 2019 2Joint with HR 3Joint with the General ordinated successful rounds of recruitment for Replacement, Refinement & Reduction of however. The AWERB reviewed these reports Administration team our next cohort of graduate students. Animals in Research (NC3Rs). and the learning shared across licensees. The Institute has a small Scientific

76 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE OPERATIONS 77 OPERATIONS (CONTINUED)

The CRUK MI AWERB has continued to interact translation and commercialisation, as well as therapies to satisfy the unmet clinical needs of exciting licensing opportunities originating with other establishments through the North providing clearer and more streamlined cancer patients. Martyn continues to be from the Cancer Research UK Manchester West AWERB Hub, the Establishment Licence interfaces with other teams across R&I with involved with the management of Institute that continue to attract commercial Holder (ELH), and HOLC attend regular whom we collaborate to achieve our joint collaborations with Pharmaceutical partners partners. We look forward to building on our meetings with the Home Office Animals in goals of progressing CRUK science. This is such as Basilea and AZ and also the filing and successes and continuing to work closely with Science Regulation Unit (ASRU) and the ELH enabling us to build deeper and more strategic management of a number of patent the Cancer Research UK funded researchers in sits on the national ELH forum and also helps relationships with our funded centres, applications for the Drug Discovery Unit to Manchester under the new CP structure to train new Establishment Licence Holders. The Institutes and Universities, as well as improving protect novel compounds resulting from advance discoveries to beat cancer in the AWERBs of CRUK MI, AstraZeneca and Agenda internal information flow and collaboration. their research. years ahead. Life Sciences, all based on the Alderley Park site, continue to hold joint meetings to share CRUK is aware that the ability to translate new CP is also currently actively managing a broad ideas, presentations and training opportunities discoveries in to patient benefit has not portfolio of development programmes and – in October, the three organisations held a progressed at the same pace as discovery second joint 3Rs’ poster event. research. This disconnect is linked to several factors related to academic culture, Fulfilling our commitment under the entrepreneurial mindset and the skills required Lung cancer cells in 3D Concordat on Openness on Animals to move discoveries forward. The CRUK-PACE Research, CRUK MI staff have talked about our team was set up to understand how CRUK Image supplied by Andrew research with mice to the public at a Research could Promote an Academic Culture of Porter (Cell Signalling) Spotlight event, as well as Institute Open Days; Entrepreneurship within our research a workshop run by the Understanding Animal community. The team has produced an Research organisation helped our staff and entrepreneurial programme to promote an students to communicate how we work with academic culture where entrepreneurship is mice in our research, and the care with which incentivised, enabled and rewarded. As part of we do this, to the general public at these this initiative we organised a very successful events. CRUK Innovation Summit on the Alderley Park site in October 2019 to facilitate this within the CRUK Manchester Institute. To further build on Cancer Research UK Commercial this, an Innovation and Translation Officer is Partnerships being recruited to focus specifically on Martyn Bottomley building a culture of innovation and translation at the CRUK Manchester Institute. This post Cancer Research UK Commercial Partnerships will work closely with the CP Team and the (CP) Team is a specialist oncology-focused CRUK MI. development and commercialisation team, which is part of Cancer Research UK’s By arrangement with The University of Research and Innovation Directorate. The CP Manchester, CRUK owns and is responsible for Team aims to maximise patient benefit from the development and commercialisation of CRUK-funded research worldwide by intellectual property arising from CRUK funded advancing research discoveries into research at The University of Manchester. To development with pharmaceutical and effectively facilitate this, Martyn Bottomley, a biotechnology parties. We aim to bridge the CRUK CP Translation Lead is based within gap between cutting edge academic research Manchester and is currently hot desking at the and industrial development of cancer OCRB, UMIP and Alderley Park to work closely therapeutics, medical technologies and with the staff funded by CRUK at The University diagnostics. We achieve this by working of Manchester. Martyn offers access to closely with prestigious international research oncology-focused expertise in technology institutes, such as the Cancer Research UK evaluations, patent applications and Manchester Institute, and funding bodies to management, funding for development, develop, protect and commercialise commercialisation, drug discovery, market oncology-related discoveries. intelligence, and project management. He also works closely with UMIP, The University of Following on from a reorganisation in April Manchester technology transfer organisation. 2018, the CP Team continues to work in functionally distinct sub-teams in order to Martyn continues to work very closely with the provide greater strength, depth and Drug Discovery Unit (DDU) based at Alderley accountability in our core activities supporting Park to facilitate the development of drug

78 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE OPERATIONS 79 www.cruk.manchester.ac.uk/education POSTGRADUATE EDUCATION

give them a broad understanding of many students, as well as poster presentations from a aspects of cancer research and basic biology. In range of scientists across the Institute covering The Cancer Research UK Manchester Institute (CRUK MI) offers a addition, we hold a series of weekly all aspects of cancer research. Poster prizes are postgraduate degree (PhD) for students interested in a career postdoctoral research seminars and awarded, including the Lizzy Hitchman Prize for attendance from PhD students is also an the best poster presented by a PhD student or involving cancer research. The Institute considers education of both integral part of their learning. While students clinical fellow. In 2019, the prize went to PhD research and clinician scientists to be a major investment in the themselves are asked to give talks at key points student Fabrizio Simeoni from the Leukaemia future of cancer research, and has an excellent track record of during their PhD, they also have opportunities Biology group for his PhD work describing how to present their work at lab meetings and during FOXC1 blocks terminal differentiation in acute launching careers in basic, translational and clinical research. As part student forums within the Institute. myeloid leukaemia cells. Postgraduate of this commitment, we have an active postgraduate programme Education Manager that provides students and clinical research fellows of outstanding STAy (Science TakeAway) is a Committee run by There was further success for Fabrizio who Julie Edwards junior scientists and students in the Institute helped to organise the first BACR PhD cancer potential the opportunity to study for a cancer-related PhD degree. with the aim of providing a forum for discussion conference which took place in December This is achieved through a training programme that aims to improve and training related to research, 2018. He was one of six who formed the BACR effectiveness in research, provide professional and management communication of scientific engagement and PhD Student Conference Organising development of social and networking Committee compiling an exciting programme skills and enhance career development. Our PhD students have opportunities. STAy are keen to encourage of talks by leading cancer scientists, attracting exceptional employment prospects following graduation, with the networking, career progression and personal over 100 participants from across the UK. This great majority (>95%) continuing in academia, industry or healthcare, growth of early-career researchers, and with was a fantastic opportunity for Fabrizio who this in mind, in January 2019 the STAy contributed to the grant application resulting in and securing positions in destinations across the UK, Europe and Committee were given the opportunity to host a £5000 award from CRUK to help fund this event. Postgraduate Tutor the USA. a number of external seminars. External speakers were invited to not only give a Angeliki Malliri scientific talk on their research, but also a short Cancer Research UK contributes towards an In 2019, we welcomed nine graduate students develop the presentation skills which are so talk about their own career pathway and hold annual International PhD Student Cancer and one clinical research fellow to our PhD fundamental to the majority of careers in small group discussions with audience Conference (IPSCC) allowing high calibre programme, working in a variety of fields science and elsewhere. Graduate training is members. students (typically in 2nd - 4th years) from top including leukaemia, pancreatic cancer, drug monitored by the Education Committee, cancer research institutes across Europe to discovery, lung cancer, prostate cancer, signal staffed by the Institute’s group leaders and In 2019, STAy and healthcare professionals organise and present at their own scientific transdution and cancer biomarkers. It was also student representatives (see below). A main from the Christie NHS Foundation Trust jointly conference. The conference is organised by particularly gratifying to see that, over the past supervisor and a second or co-supervisor are organised a World Cancer Day Event, bringing students for students from core participating twelve months, three of our PhD students and nominated for each student, who are able to together people with a wide range of cancer institutes; London Research Institute (LRI), Clinical Research Fellows had published first provide additional advice and consultation on experience, researchers, patients and health Cambridge Institute (CI), Beatson Institute Postgraduate Director author original papers in the British Journal of both academic and non-academic matters. care teams. The evening presented a number (BICR), Netherlands Cancer Institute (NKI), and Chair of the Education Pharmacology, Scientific Reports, and The Each student is also assigned an advisor (similar of short talks, discussion activities with the European School of Molecular Medicine, Milan Committee Journal of Clinical Investigation. First author to a personal tutor on an undergraduate opportunity to share perspectives on life, (SEMM, IFOM & IFEO), and the German Cancer Tim Somervaille review articles were also published in FEBS programme) whose role is to provide impartial cancer and research. Other activities over the Research Centre (DKFZ). Letters and Development. support and advice in a pastoral capacity. last year have included pub quizzes, bowling, Further support is also available individually speed networking, science media training, a The 13th IPSCC was organised by students at The Cancer Research UK Manchester Graduate from the Director of Postgraduate Education, grant writing seminar and a mini-research the National Cancer Institute, Amsterdam from Programme Postgraduate Tutor, Postgraduate Manager, or spotlight conference. 12-14 June 2019. The CRUK MI was represented We aim for each student to receive high quality collectively as the Education Committee by 23 students, the highest number of training in scientific research through an Administration Group. The CRUK Manchester Institute Colloquium attendees from a single institute. The intellectually demanding but achievable takes place annually in September, and is an conference was attended by over 100 students research programme. Each project is peer- The CRUK MI runs an external seminar series excellent opportunity for our new intake of from the participating institutes with 16 panel reviewed in advance and monitored featuring talks from many of the leading students to meet other established PhD selected student talks and 86 posters over two throughout its course through a mixture of oral scientists in cancer research, which our students, members of the Institute, including and a half days. The forum provided a unique presentations, written reports and progress students are encouraged to attend. The group leaders, postdoctoral fellows, and opportunity for PhD students to present their meetings. These modes of assessment are speakers are internationally renowned scientific officers. This forum communicates up work via a poster or oral presentation, network designed not only to provide formal points at scientists and we consider it essential that our to date science in the form of oral presentations with some of Europe’s best cancer research which progress (of both the student and the students are exposed to outstanding research given by group leaders and second year PhD students, and engage in organised career project) can be monitored, but also to help from leaders in different disciplines, which will

80 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE POSTGRADUATE EDUCATION 81 POSTGRADUATE EDUCATION (CONTINUED)

sessions providing advice on future career leaders, the Chief Operating Officer, the options. Students enjoyed keynote lectures Postgraduate Tutor and the Postgraduate from Dr Andrea van Elsas, Aduro Biotech; Prof Education Manager from the CRUK Manchester Karin de Visser, NKI; Dr Nicholas Navin, MD Institute. Anderson Cancer Center; and Prof Ben Feringa, Rijks Universiteit Broningen. Our goal is for every student to have a project that is both achievable and intellectually We were delighted that Maximilian Schenk from stimulating and demanding. Projects and CRUK Manchester Institute Cancer Biomarker students are monitored by the Education Centre was awarded the overall prize for best Committee, which makes sure that the oral presentation showcasing his work on proposed plan of research is suitable, and that chemoresistance in small cell lung cancer. progress is made consistently throughout the Christian Bromley based in the Cancer course of the studentship. Various assessments Inflammation and Immunity group, was throughout the studentship, including regular awarded the prize for one of the top three talks, progress meetings and written reports, poster presentations “Combining cancer are vital to ensuring successful completion and promoting and cancer-inhibitory inflammation graduation for the PhD degree. Such signatures to predict cancer patient outcome”. assessments help to not only monitor progress, but also help to develop performance and The social events during the conference presentations skills. included a pub quiz, networking social event and a walking dinner with a guided tour around Education Committee Members Amsterdam, which were all well received. The event was a great success - especially for the Tim Somervaille CRUK MI Manchester Institute. Postgraduate Director and Chair Angeliki Malliri PhD studentships Postgraduate Tutor All of our CRUK core funded studentships are of Richard Marais four years’ duration, and consist of an approved Ex-Officio Member research project in one of our core funded Wolfgang Breitwieser research groups. Some students have joint Julie Edwards supervisors in different groups, fostering Postgraduate Manager important collaborations and providing exposure to different disciplines. Recruitment is Claus Jørgensen highly competitive, with 300-500 applicants Elaine Kilgour1 competing for around four-eight places each Georges Lacaud year. Interviews are typically conducted Jonathan Tugwood2 annually over a two-day period in early January. Amaya Viros1 Caroline Wilkinson Our students benefit from access to advanced state-of-the-art facilities, including advanced Student Representatives imaging, biological mass spectrometry, flow cytometry, histology and next generation Jakub Chudziak2 Callum Hall sequencing. Our research groups offer PhD Organotypic brain slice (grey scale) invasion by patient-derived melanoma cells tagged with mCherry (orange). Illustrating melanoma cell studentships and projects covering the entire Ryan Guilbert1 capacity to invade into the brain tissue ex vivo. breadth of research within the Institute 1 Image supplied by Denys Holovanchuk (Molecular Oncology) currently based over two sites at Alderley Park, Joined in 2019 2 Cheshire and the Olgesby Cancer Research Left in 2019 Building, Manchester.

Education Committee 2019 The Education Committee acts for postgraduate students and consists of group

82 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE POSTGRADUATE EDUCATION 83 Images clockwise from top left:

Staff Engagement Day: Nic Jones, Caroline Dive and Rob Bristow chat to the Re-Write Cancer fundraising team about why the new building is critical to research progress

Staff Engagement 2: Steve Bagley explains the work of the Imaging, Irradiation and Analysis team to CRUK fundraising staff

60 Year Celebration: Rob Bristow celebrates 60 years of fundraising with the Nelson & Barrowford Fundraising Committee

Research Spotlight: A public event organised in collaboration with the STAy Committee

Research Spotlight: A public event organised in collaboration with the STAy Committee

research and hosted the CRUK Engagement Charitable Fund, the Institute was delighted to Team’s Escape the Lab activity. welcome fundraising colleagues from CRUK to Alderley Park for an exclusive tour of the labs to CANCER RESEARCH UK’S Following the event, our scientists also attended see our research in action. Scientists from the CRUK’s national Relay for Life Summit, inspiring Cancer Biomarker Centre, the Drug Discovery RESEARCH ENGAGEMENT supporters from across the UK through the Unit and the Imaging, Irradiation and Analysis research taking place at the Institute and team welcomed CRUK staff in to the labs, giving thanking them for their support. a crucial insight in to the work taking place which may help to inspire prospective donors of the Cancer Researcher UK’s Research Engagement Team brings Senior Group Leader Rob Bristow celebrated campaign. CRUK-funded research to life for its supporters, the public and its with the Nelson & Barrowford Fundraising staff, working regionally with researchers to develop face-to-face Committee in May, where he thanked them for Huge thanks go to all the volunteer group 60 years of determined fundraising and on leaders, researchers, scientists and staff who engagement opportunities. The team drives local interaction with reaching a £1million milestone. Rob shared with donate their time, energy and enthusiasm to life-saving research through compelling research content. supporters and guests the impact their support our engagement activities. fundraising efforts make to patients’ lives and the latest progress happening in Manchester. Research Engagement Manager Relay for Life Stockport: Following Rob’s address, a member of the Tim Hudson Almost 2000 people interacted with the work of present their work to a lay audience of 60 Some of the scientists and the Manchester Institute during 2019, through members of the public via presentations and families who took part in the committee (who are all over 80 years old) the work of the CRUK Research Engagement posters. Scientists from across the MI, together fundraising event commented that whilst they often think of Manager based in Manchester. with other colleagues from The University of stepping down, they now felt inspired to keep on Manchester, spoke to the public about their fundraising. During nine visits to Institute labs at Alderley Park research. The evening culminated in a keynote and the Oglesby Cancer Research Building, over presentation by Institute Fellow, Patricia Muller. Senior Group Leader Iain Hagan celebrated with 160 donors, fundraisers, volunteers, corporate volunteers from across the North West at a partners and CRUK staff had the opportunity to Our scientists also took their work out to the regional Flame of Hope event, during which Iain gain a close-up view of the research their work community at 18 external events. thanked the supporters for their contributions helps to fund. whilst illustrating how their efforts are translated Relay for Life Stockport took place in the height in to critical research. A highlight came in October when, with support of the summer, featuring the team of researchers from CRUK, members of the MI STAy Committee from the Manchester Institute and University of With the launch of the Re-Write Cancer staged Research Spotlight: Manchester’s Early Manchester, captained by Steve Lyons. As well campaign to raise £20million towards the rebuild Career Researchers, a public event giving PhD as joining in the festivities on the day, MI of the Paterson Building, alongside The students and Scientific Officers a chance to scientists and staff shared stories about their University of Manchester and The Christie

84 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE CANCER RESEARCH UK'S RESEARCH ENGAGEMENT 85 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 2019 was The Cancer Research UK Manchester Institute has a strong £29.6m. The major source of this funding was awarded by Cancer programme of basic and translational research. There are close Research UK (CRUK) via a core grant of £13.6m plus additional links with clinical and translational research groups throughout strategic funding of £6.3m. This funding enables the various the Christie Hospital site. scientific groups and service units within the Institute to carry out their research. The Institute offers excellent laboratory facilities In addition to postgraduate and postdoctoral and outstanding core facilities, including opportunities, the Institute is seeking to recruit molecular biology services, next generation outstanding candidates to the positions of The infrastructure of the CRUK Manchester • European Organisation for Cancer Research sequencing, real-time PCR, mass spectrometry, Junior and Senior Group Leaders. The Institute is funded by HEFCE generated income and Treatment of Cancer flow cytometry, histology, advanced imaging, packages provided are extremely attractive at a cost of £2.1m. • European Research Council and a biological resources unit. Details of all and commensurate with the experience of • Fondation ARC pour la Recherche sur le groups and facilities are given in this report, the applicant, with significant funding for The balance of the Institute’s funding is received Cancer and can guide interested parties to the personnel, recurrent expenditure and from a number of additional sources. The • GlaxoSmithKline appropriate contacts. equipment. Junior Group Leaders are research carried out through these additional • Harry J Lloyd Charitable Trust appointed for an initial six-year period with projects enhances and supports the research • John Swallow Fellowship Opportunities exist at a number of levels in the a review between five and six years for undertaken by the core funding. • Kay Kendal Leukaemia Fund Institute. We have a well-established consideration of promotion to Senior Group • Leo Pharma Foundation programme of degrees by research which is Leader, with Senior Group Leaders appointed These sources are as follows: • Menarini Biomarkers Singapore described in the section on Postgraduate to non-time limited positions. • Merck Education. We encourage applications from • Amgen • Moulton Charitable Trust suitably qualified graduates to apply to join Specific vacancies can be found on our web • Angle Inc • National Institute of Health Research either the PhD or MD programmes. Graduates pages (http://www.cruk.manchester.ac.uk/ • Astex Pharmaceuticals • Ono Pharmaceuticals with a first or 2.1 honours degree in a biological Opportunities/Opportunities-Home) but • Astra Zeneca • Pancreatic Cancer Research Fund science can apply each year to train for a suitably qualified and enthusiastic individuals • Bioven • Pickering Leukaemia Research four-year PhD in one of our research should contact the Institute at any time to • Bloodwise • Prostate Cancer UK laboratories. The University of Manchester enquire about career possibilities. • Carrick Therapeutics • Rosetrees Trust offers a wide range of training for new and • CellCentric • Taiho Oncology Inc existing students which provides opportunities • Christie Hospital NHS Foundation Trust • The US Department of Health and Human to acquire skills that will complement the • Clearbridge Biomedicals Services research programme and help achieve personal • CRT Pioneer Fund • Wellcome Trust and career development goals. At the Institute, • David & Ruth Lewis Trust • Worldwide Cancer Research we also ensure that postgraduate students are • Euclises Pharmaceuticals Inc provided with high quality, relevant and • European Commission We are immensely grateful to all our sponsors. appropriate training alongside development opportunities. The Institute also has a well- developed process for ensuring excellent CRUK MANCHESTER INSTITUTE FUNDING 2019 pastoral care and mentoring for all students. Postdoctoral applicants of high calibre are regularly sought. Although Postdoctoral Fellows will be encouraged to apply for their 25.4% KEY own fellowships, funded positions are available for outstanding candidates. Interested applicants should contact the Group Leaders CRUK Core Grant directly, with details of their research interests and recent experience. 46% CRUK Strategic Funding

HEFCE

7.3% Other Sources

21.3%

86 SCIENTIFIC REPORT 2019 CANCER RESEARCH UK MANCHESTER INSTITUTE CAREER OPPORTUNITIES AT THE CRUK MANCHESTER INSTITUTE 87 CONTACT DETAILS

M6 to Preston, Carlisle, Scotland M62 to Leeds M6 M66 Rochdale

Bury M62 Bolton

M61 ISSN 1479-0378 Wigan Oldham Copyright © 2019 Cancer Research UK

M60 Edited by: Caroline Wilkinson Gillian Campbell M602 Manchester Ashton-Under-Lyne City Centre

M6 M67 M62 A57 to Sheeld 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: [email protected] 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/

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The University of Manchester Alderley Park SK10 4TG United Kingdom Telephone +44(0) 161 446 3156 www.cruk.manchester.ac.uk

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