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    paterson institute for cancer research scientific report 2008

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    cover images: Main image supplied by Karim Labib and Alberto sanchez-Diaz (cell cycle Group). Budding yeast cells lacking the inn1 protein are unable to complete cytokinesis. these cells express a fusion of a green fluorescent protein to a marker of the plasma membrane, and have red fluorescent proteins attached to components of the spindle poles and actomyosin ring (sanchez-Diaz et al., nature cell Biology 2008; 10: 395). Additional images: front cover image supplied by Helen rushton, simon Woodcock and Angeliki Malliri (cell signalling Group). the image is of a mitotic spindle in fixed MDcK (Madin-Darby canine kidney) epithelial cells, which have been stained with an anti-beta tubulin antibody (green), DApi (blue) and an anti-centromere antibody (crest, red) which recognises the kinetochores of the chromosomes. the image was taken on the spinning disk confocal microscope using a 150 x lens. rear cover image supplied by Andrei ivanov and tim illidge (targeted therapy Group). Visualisation of tubulin (green) and quadripolar mitosis (DnA stained with DApi), Burkitt’s lymphoma namalwa cell after 10 Gy irradiation. issn 1740-4525 copyright 2008 © cancer research UK

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    Paterson Institute for Cancer Research Scientific Report 2008

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    Contents 4 Director’s Introduction Researchers’ pages – Paterson Institute for Cancer Research 8 Crispin Miller Applied Computational Biology and Bioinformatics 10 Geoff Margison Carcinogenesis 12 Karim Labib Cell Cycle 14 Iain Hagan Cell Division 16 Nic Jones Cell Regulation 18 Angeliki Malliri Cell Signalling 20 Caroline Dive & Malcolm Ranson Clinical and Experimental Pharmacology 22 Peter Stern Immunology 24 Nullin Divecha Inositide Laboratory 26 Tim Somervaille Leukaemia Biology 28 Georges Lacaud Stem Cell Biology 30 Valerie Kouskoff Stem Cell and Haematopoiesis 32 Akira Orimo Stromal-Tumour Interaction Researchers’ pages – The University of Manchester School of Cancer and Imaging Sciences 34 Catharine West Academic Radiation Oncology: Translational Radiobiology Group 36 Robert Hawkins & Peter Stern Biological, Immune and Gene Therapy 38 Vaskar Saha Children’s Cancer Group 40 Tim Illidge Targeted Therapy 42 Robert Hawkins Medical Oncology: Cell Therapy 44 John Gallagher Medical Oncology: Glyco-Oncology 46 Gordon Jayson Medical Oncology: Translational Angiogenesis 2 Paterson Institute for Cancer Research Scientific Report 2008

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    48 Research Services 48 Steve Bagley Advanced Imaging Facility 49 Biological Resources Unit 51 Stuart Pepper Cancer Research UK GeneChip Microarray Service 51 Morgan Blaylock Flow Cytometry Facility 52 Garry Ashton Histology 53 Steve Glover Kostoris Library 54 Mark Craven Laboratory Services 54 Maurice Cowell Logistics 54 Stuart Pepper Molecular Biology Core Facility 54 Duncan Smith MBCF Biological Mass Spectometry Facility 56 Publications 66 Seminars 68 Postgraduate Education 70 Operations Services 73 Local Engagement and Development 74 Acknowledgement of Funding 75 Career opportunities 76 How to find us Paterson Institute for Cancer Research Scientific Report 2008 3

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    Director’s Introduction Welcome to the clinical trials. He will join the Institute on the 1st 2008 Paterson February 2009 and will begin the challenging task of building a drug discovery team with major medicinal Institute annual chemistry expertise and identifying promising targets Scientific Report. As always that will be the initial focus of the centre. It is an it has been a busy year scientifically important development for us since it provides a scientific bridge between our activities and strengths in with further recruitment and consolidation of basic cancer biology and our strengths in translational the partnership represented by the research associated with early clinical trials and Manchester Cancer Research Centre. development of new biomarkers. Recruitment of new scientific leaders is essential for In contrast to these exciting new comings, sadly we saw maintaining research excellence and building up areas the going of John Gallagher, head of the Glyco- of research strength. It is particularly important for a Oncology group, who is retiring after an association research Institute such as the Paterson Institute to with the Institute of over 30 years. John is a world recruit young group leaders and to provide the expert in glyco-biology particularly with respect to support and research environment that will allow their heparan sulphates found on the surface of all cells research development to flourish. Building on a very busy recruitment schedule last year, we have recruited another Junior Group Leader, Ivan Ahel, who comes to us on the back of a very successful and productive postdoctoral period in Steve West’s laboratory at our sister Institute, the London Research Institute. Ivan’s research interests have focused on DNA repair mechanisms and in particular on novel repair proteins that contain a poly (ADP-ribose)-binding zinc finger motif (PBZ). He will continue this work when he joins us on 1st January 2009, with an emphasis on how the interaction of PBZ-containing proteins with other proteins that are modified by poly (ADP-ribosyl)-ation orchestrates the generation of complexes that function in ensuring an efficient cellular response to DNA damage. Another recruit to the Institute is Donald Ogilvie who will head the new and exciting development of a Drug Discovery Centre. This new initiative is a key component of Cancer Research UK’s strategic plan to increase its capability and activity in the development of small molecule drugs. Donald comes to us after a very distinguished career with the pharmaceutical giant AstraZeneca where he co- ordinated the development of a number of new drugs some of which are currently being tested in phase III 4 Paterson Institute for Cancer Research Scientific Report 2008

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    where they function as important co-receptors for a unit (The Derek Crowther Unit, DCU) and has as its number of growth factors including the fibroblast mission the discovery and validation of biomarkers that growth factors and hepatocyte growth factor. John’s provide indications on the efficacy and toxicity of new work has highlighted the complexity of different molecules being tested in clinical trials. This type of sulphation patterns seen with the heparan sulphates research is crucially important for modern drug and how these patterns affect such key biological development and lies at the heart of the need to processes such as stem cell differentiation and identify early in the development programme, drugs malignant transformation. John shone through with his that are likely to be effective and patients are likely to enthusiasm, dedication and above all his modesty. He respond to – in other words, the implementation of will be sorely missed. personalised medicine. If the results of such research are used to inform clinical decision making, then it has During this last year, the Clinical and Experimental to be conducted under very strict regulatory Pharmacology (CEP) group in the Institute was site- guidelines. The CEP has established itself as one of the visited by an international panel of experts. These site leading centres internationally with this capability. CEP visits provide a stringent assessment of the is lead by Caroline Dive working closely with the head international standing of the research programme in of the DCU, Malcolm Ranson. They were both very question by examining the progress and impact of the deservedly congratulated by the visiting party for research over the last five years and a view on the developing such an exciting and important research potential and importance of the proposed research programme which exemplified the power of a strong programme for the next five years. This was a and effective clinical-laboratory interface. Over the particularly important review for the Institute since next few years further development and investment in CEP is a relatively new group which is at the heart of this research area will take place, the most significant the Institute’s translational research efforts and which development being the expansion of the clinical trials has grown rapidly over the last few years. It was facility as part of the £35M development by The therefore very pleasing and reassuring that the group Christie NHS Foundation Trust. This will result in a 3- received a very positive review and whose research fold increase in the capacity of the DCU, creating one was judged to be at the international forefront. CEP of the largest early clinical trials units in the world. works very closely with the early phase clinical trials Director’s Introduction 5

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    There were a number of research highlights during the malignancies. This work will be published early in the year. The Stem Cell Biology group led by Georges New Year in the journal Molecular Cell. The Cell Cycle Lacaud characterised in detail the developmental steps laboratory headed by Karim Labib has been using yeast leading to the generation of haematopoietic cells, which as a model system to investigate the regulation of despite receiving a lot of attention from many groups DNA replication and cell division. During the last year over a number of years, remained controversial with they reported in Nature Cell Biology the discovery of a two conflicting theories predominating. One theory novel factor called Inn1 which is essential right at the hypothesised that haematopoietic cells arise from a end of the cell cycle for cytokinesis, the process mesodermal progenitor called the haemangioblast whereby the eukaryotic cell divides into two. This has whilst the other suggested that such cells arise from a to be regulated in time to ensure that it does not specialised endothelial cell that has haematopoietic occur before nuclear division, and in space to ensure potential, the haemogenic endothelium. Studies from that the division plane lies between the two nuclei Georges’s laboratory have beautifully reconciled these formed during mitosis. The characterisation of Inn1 has opposing views showing that both theories are merged provided new insight into a relatively poorly into a single developmental process where the understood yet vital process. Powerful technologies haemangioblast gives rise to a haemogenic endothelium are now available to quantitate the cellular levels of intermediate which then further differentiates to RNA transcripts through microarray analysis and generate haematopoietic cells. Furthermore, they proteins through quantitative mass spectrometry identified specific transcription factors that regulate proteomics. The Applied Computational Biology and different stages of this differentiation pathway. This Bioinformatics laboratory led by Crispin Miller has been important work will be published early in 2009 in the developing new tools to integrate microarray and journal Nature. The Cell Signalling Group led by proteomics data. A new and powerful approach was Angeliki Malliri has been studying a regulator (Tiam 1) recently refined and published in BMC Bioinformatics. of the Rac GTPase which, amongst many of its roles, These are just a few selected examples of the research functions to control cell-cell adhesion. Previous work progress that has been made over the last year. from the group had shown that deletions of Tiam1 confer resistance to the formation of both skin and The Manchester Cancer Research Centre (MCRC) intestinal tumours in appropriate mouse models. They which is now in its 3rd year continues to make good have now discovered that Tiam1 is phosphorylated and progress in integrating cancer research efforts across regulated by the oncoprotein Src, a tyrosine-kinase Manchester and realising the benefits and opportunities implicated in malignant transformation. This that the partnership between The University of phosphorylation occurs preferentially at cell-cell Manchester, The Christie NHS Foundation Trust and contacts resulting in Tiam1 degradation and disruption Cancer Research UK can provide. It is galvanising of cell adhesion. This regulation of Tiam1 is likely to be researchers to work together to develop areas of important for Src-mediated cancer cell invasion and research strength, to develop research infrastructure, to metastasis. Interestingly, in a range of different cancers, co-ordinate research training and to ensure close they found a correlation between Tiam1 alignment of basic and clinical research. Significant phosphorylation and Src-activity consistent with this advances have been made in a number of areas: early regulatory mechanism operating in tumour phase clinical trials and biomarker research as 6 Paterson Institute for Cancer Research Scientific Report 2008

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    described above; centralised tumour biobanking As always the coming year will provide many new involving five NHS Trusts across the Manchester challenges and opportunities for the Institute. The 5- conurbation; development of radiation-related research; year Institute Review will take place in June 2009. development of molecular pathology through the These reviews are obviously important for Cancer recruitment of a new Chair (Goran Landberg); Research UK since they judge the overall success and consolidation and expansion of the formal alliance with strategy of the Institute. They can also be valuable to AstraZeneca which supports research and training the Institute and it was discussions at the last review across a number of research areas. The MCRC is very that precipitated talks that lead to the development of much in line with the major initiative of Cancer the MCRC. The development of the Drug Discovery Research UK to develop a number of centres across Centre will also be high on the agenda. The big the Country where research activities are closely linked challenge will be to continue to develop the Institute with patient care and public engagement. The MCRC and support all our current research activities in a already fulfils much of what is envisaged in such a particularly difficult economic climate. centre. In December the results of the national Research Nic Jones Assessment Exercise (RAE) were announced. This Director exercise provides a comprehensive assessment of the quality of research in UK universities. It was therefore tremendous news that cancer research in Manchester was officially ranked as the best in the UK. This was a great endorsement of the research taking place in the Paterson, The University of Manchester and The Christie NHS Foundation Trust and a major boost to the MCRC partnership. Director’s Introduction 7

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    Paterson Institute for Cancer Research Applied Computational Biology & Bioinformatics Group http://www.paterson.man.ac.uk/bioinformatics Group Leader Crispin Miller Postdoctoral Fellows Research Applications Graduate Students Carla Möller-Levet (joint Programmer Manager Danny Bitton Tim Yates with Translational Andrzej Rutkowski (joint Radiobiology) with Immunology) John Hall (joint with Systems Administrator Translational Radiobiology) Zhi Cheng Wang James Bradford Bioinformatics and Computational Biology interpret these regions within the genome, in order to better exploit the data generated by new technologies such as next exist to help us make sense of the generation sequencing, SNP, exon and tiling arrays, and complex datasets generated by research in quantitative proteomics. the biosciences. In the Applied Affymetrix exon arrays DNA microarrays consist of an inert substrate, such as a glass Computational Biology and Bioinformatics slide, onto which groups of single stranded DNA molecules Group, we are interested in the are fixed at known locations. Each group, or ‘spot’, contains millions of identical molecules, which are designed so that development and application of software their sequence is complementary to that of a particular tools and analytical strategies for the transcript or region of interest. When labelled, fragmented, DNA or RNA is applied to the surface of the array it will analysis of cancer related datasets. We hybridize when it encounters a spot with the appropriate collaborate closely with other groups complementary sequence, bringing its (typically fluorescent) label with it. The hybridized array can then be imaged and working on both the clinical and the analysed using a computer to measure the relative amount of molecular biology aspects of cancer binding at each spot, thus providing an estimate of the amount of material in the original sample. Microarrays make research. it possible to generate global measurements of gene Genome annotation expression for many thousands of transcripts in parallel, or to Until recently, the genome was viewed as primarily involved look for Single Nucleotide Polymorphisms (SNPs) or changes in defining proteins, via a set of protein coding genes in DNA copy number. Unlike most microarrays, for which separated by large tracts of ‘junk’ DNA. Over the last few there is generally a one-one mapping between probe(set) years, this perspective has changed markedly. We now know and gene, Affymetrix exon arrays aim to include a separate that as much as 90% of the human genome can, in some probeset for every known and predicted exon in the entire circumstances, be transcribed, and that much of this genome. This allows differential expression to be considered transcription leads to RNA that, although never translated at the exon level, important given the prevalence of into protein, is functional in its own right. In addition, the alternative splicing. We are collaborating with a number of process of alternative splicing, by which a cell can fundamental and clinical research groups to use these systematically include or exclude parts of a gene’s sequence software tools [exonmap (Okoniewski et al. 2008); X:Map in order to yield a set of distinct gene products from a single (Yates et al. 2008)] to help explore alternative splicing in locus, has also been shown to be prevalent. Both non-coding cancer related datasets (see figure). We also make these RNA and alternative splicing have been implicated in many tools available to other researchers, and they are used cellular processes and pathways, and both are known to be internationally. Current research is focused on developing involved in a variety of human diseases, including cancer. We novel approaches for interpreting exon array data, and in are interested in developing software tools and databases particular, on generating the meaningful summaries that are (e.g. X:Map; xmap.picr.man.ac.uk) to help describe and required in order to reduce the data from the arrays’ 1.4 8 Paterson Institute for Cancer Research Scientific Report 2008

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    Visualisation of genomic data in an AJAX driven Genome Browser million probesets to a more manageable size suitable for feasibility of the approach and the quality of the data human interpretation. generated by both platforms. Most recently, we have started to combine these data with copy number information Quantitative Proteomics generated using Affymetrix SNP arrays. Tandem mass spectrometry (MS/MS) is a technique that allows proteins to be identified by fragmenting them into Analysis of archival material peptides that are then fragmented further and analysed using An issue with using microarrays for expression profiling arises a mass spectrometer. This results in a set of characteristic ion because of the need for high quality RNA samples for masses that can then be used to identify each peptide (and analysis. Many archival samples have been stored as thus their originating protein) via a database search. A Formalin-Fixed Paraffin-Embedded Tissue (FFPET), in which number of techniques such as iTRAQ allow protein the RNA has been chemically modified and/or degraded, identification to be coupled with quantitation in order to making them challenging to analyse by microarray. A measure the relative abundance of multiple proteins between collaboration with the Department of Medical Oncology at samples. Typically, this is done by averaging the signal from a the Christie Hospital and the Cancer Research UK Affymetrix protein’s individual peptides, to generate a protein level service, explored the possibility of using this material on consensus value. The path from DNA to RNA to protein is a Affymetrix GeneChip Arrays. We found that with complex one, involving many stages of regulation and control. appropriate attention to Quality Control, it was possible to We are interested in integrating quantitative protein and generate clinically relevant reliable data from these samples. mRNA level data in order to help explore the space (Linton et al. 2008). between transcription and translation. In Bitton et al. (2008), we showed for the first time that it was feasible to integrate Publications listed on page 56 transcription data generated using Affymetrix exon arrays with quantitative proteomics data from iTRAQ. The increased resolution of exon arrays makes it possible to do this at the level of individual peptides, which are then mapped back to their originating exons via the genome. When this was done, we found very high correlation between the protein and transcript data in a steady state cell line system, chosen to reduce biological variability in order to provide a suitable evaluation dataset. This demonstrates both the Research Reports 9

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    Paterson Institute for Cancer Research Carcinogenesis Group http://www.paterson.man.ac.uk/carcinogenesis Group Leader Geoff Margison Postdoctoral Fellows Scientific Officers Undergraduate Students Amna Butt Gail McGown Catia Caetano (until September) Mary Thorncroft Nick Peel Vitaly Latypov Mandy Watson (from October) Volunteer Workers Barbara Verbeek Graduate Student Jonathan Doyle (until April) Andrew Marriott Rita Matos The treatment of cancer often involves Background Alkylating agents are a family of chemicals that are used in the the use of drugs that kill cells by damaging treatment of several types of cancer including brain tumours their DNA. That some tumours do not (glioma) and skin tumours (melanoma) but there is considerable room for improvement in their effectiveness. respond to such treatments can be These agents generate a dozen different types of damage in DNA and there is increased understanding of the attributed to the presence of repair mechanisms by which some of these result in cell killing. Thus processes which can remove potentially agents such as Dacarbazine and Temozolomide generate O6- methylguanine in DNA and this kills cells via the action of the lethal damage from DNA. Understanding post replication mismatch repair system. Cell killing by this how DNA damage leads to cell death, and pathway can be prevented by the prior action of the damage reversal protein, O6-alkylguanine-DNA alkyltransferase how these repair systems process the (MGMT). This protein most probably evolved to protect damage, may provide opportunities to organisms against the toxic effects of endogenously or environmentally generated lesions in DNA. However, it can improve the effectiveness of existing also reduce the effectiveness of these chemotherapeutics and cancer therapies, and develop new agents. hence there is increasing interest in ablating the activity of MGMT in tumours in order to improve clinical outcome. Our main focus is on DNA damage and One of the strategies we have pursued has involved the repair following exposure to certain types development and use of Lomeguatrib, a drug that has now completed phase II clinical trials in combination with of alkylating agents, for example the CR- Temozolomide in malignant melanoma. UK drug, Temozolomide. Clinical trials of a MGMT removes alkyl groups from the O6-position of guanine drug that was designed to ablate one of by stoichiometric transfer to a cysteine residue in its active site, a process that results in its irreversible inactivation. We the repair processes that is critical in recently discovered, using computer based amino acid Temozolomide resistance are now sequence analysis, several proteins that have extensive homology to MGMT, but without this critical cysteine residue. essentially completed. In fission yeast, we These proteins are present in a number of organisms, found a different mechanism for dealing including E. coli and the fission yeast, S. pombe. We have named this family the alkyltransferase-like (ATL) proteins and with toxic DNA damage, and our efforts we are currently intensively investigating their modus operandi. are now directed towards the Clinical trials of Lomeguatrib characterisation of this novel repair Lomeguatrib (O6-[4-bromothenyl]guanine, previously called pathway. PaTrin-2) is one of the products of a very fruitful collaboration with Prof Brian McMurry and the late Dr Stanley McElhinney (and their group at the Chemistry 10 Paterson Institute for Cancer Research Scientific Report 2008

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    Department, Trinity College, Dublin). It is a very potent protein seems likely to be a specific DNA damage sensing inactivator of MGMT and it effectively sensitises human cells protein that signals to other DNA repair networks. We have and tumour xenografts to the killing effect of Temozolomide now extended these studies using native Atl1 protein in and other agents of that type. With the support of CR-UK enzyme-linked immunosorbent assays and surface plasmon Drug Development and Formulation Units and also Cancer resonance methods to quantify these molecular interactions. Research Technology, Phase I clinical trials of this drug started here at Christie Hospital in 2000 in the hands of Drs To establish if the Atl1 protein has any role in the sensitivity Malcolm Ranson and Mark Middleton (now at the Churchill of S. pombe to alkylating agents, the gene was inactivated by Hospital, Oxford). These trials established a dose gene disruption. The resulting deletants had similar growth combination of Lomeguatrib and Temozolomide for use in characteristics to the wild type strain, but had substantially Phase II trials, which have been carried out under the increased sensitivity to the growth inhibitory effects of N- auspices of KuDOS pharmaceuticals, to whom Lomeguatrib methyl-N-nitro-N-nitrosoguanidine, a methylating agent that was licensed. The clinical trials required us to develop and generates the same lesions in DNA as Temozolomide. Atl1 validate to Good Clinical Laboratory Practice standards, also protects against a number of related alkylating agents, but quantitative assays for both functionally active and total there was no detectable effect on sensitivity to the toxic MGMT protein. Malignant melanoma patients were recruited effects of other classes of DNA damaging agents. Atl1 is from several centres in the UK and Australia. They were therefore an important protein in the protection of S. pombe treated with either Temozolomide alone or a Lomeguatrib- against the toxic effects of these alkylating agents. Exactly Temozolomide combination. In addition, patients displaying how it achieves such protection has yet to be established, but, disease progression on Temozolomide (alone) therapy were crossing the atl1 deletant with deletants in established DNA treated with the Lomeguatrib-Temozolomide combination to repair pathways (epistasis analysis) has suggested that Atl1 is assess if this would reverse Temozolomide resistance. The part of, or signals to, the nucleotide excision repair pathway outcome of the trial was disappointing: neither the response and not the base excision or recombination repair pathways rates nor survival were improved in the Lomeguatrib- (see Figure). Further studies of this type along with an Temozolomide combination treated patients in comparison analysis of the proteins that interact with Atl1 to achieve with Temozolomide alone, including the group progressing on repair of the damage may confirm this. the latter. However, laboratory analyses of tumour biopsy samples collected during these studies showed that MGMT Publications listed on page 56 activity was recovering in tumour tissue very soon after treatment. Therefore a period of treatment with Lomeguatrib alone was included at the end of the combination treatment, and subsequently extended, in attempts to maintain suppression of MGMT activity. This also produced no clinical benefit. Neither did the Lomeguatrib- Temozolomide combination provide any clinical benefit in colorectal cancer patients. The reasons for this outcome have yet to be established and are the subject of much speculation. Alkyltransferase-like (ATL) proteins We previously reported the isolation of the ATL-encoding genes from E. coli and S. pombe and we named the latter, Atl1. We have shown that, in vitro, the Atl1 protein binds to methylated DNA and by doing so, can inhibit the action of MGMT. In collaboration with David Williams (University of Sheffield) we initially showed using gel shift assays that a purified fusion protein of maltose binding protein and the ATL from E. coli binds to short single- or double-stranded oligonucleotides containing a number of O6-alkyl-substituted guanines including methyl, benzyl, hydroxyethyl and 4- bromothenyl (i.e Lomeguatrib embedded into an Effects of the methylating agent N-methyl-N’-nitro-N-nitroso-guanidine oligonucleotide, which we have shown to be the most potent (MNNG) on growth in liquid culture of WT, Atl1, Rad13 and Atl1/rad13 MGMT inactivating agent so far described). However, whilst double deletant S.pombe strains. Rad13 is part of the nucleotide excision binding to these potentially toxic lesions can be repair system and the data indicate that Atl1 and Rad13 are both on demonstrated, there is no evidence of an MGMT-like alkyl this pathway. transfer, nor of glycosylase or endonuclease activity, so the Research Reports 11

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    Cell Cycle Group http://www.paterson.man.ac.uk/cellcycle Group Leader Karim Labib Postdoctoral Fellows Scientific Officer Visiting Student Giacomo de Piccoli Frederick van Deursen Bianca Targosz Luis Garcia-Rodriguez Alberto Sanchez-Diaz Graduate Students Sugopa Sengupta Asli Devrekanli Tim Maculins Hiroko Morohashi Magdalena Foltman Our group studies the mechanisms that locus of each of the essential budding yeast genes so that the encoded protein was fused to the heat-inducible degron, drive the eukaryotic cell cycle. During allowing us to analyse the effects of rapid depletion 2008 we described a novel factor called (Kanemaki et al., Nature 2003; 423: 720). In this way we found that inactivation of the budding yeast protein Ynl152w Inn1 that is required for cytokinesis in blocked cytokinesis, and we named the protein Inn1 because it is required for Ingression of the plasma membrane budding yeast. The Inn1 protein is (Sanchez-Diaz et al., 2008). associated with the contractile actomyosin Using a strain in which Inn 1 was fused to Green Fluorescent ring that defines the site at which cell Protein (GFP) we found that Inn1 is recruited at the end of division will occur at the end of mitosis. In mitosis to the site of the contractile actomyosin ring (Figure 1). Recruitment of Inn1 was dependent upon essential the absence of Inn1, the actomyosin ring components of the actomyosin ring, and we found that Inn1 still forms and contracts on schedule but co-purified from cell extracts with other previously described components of the actomysin ring such as the SH3-domain ingression of the plasma membrane does protein Hof1 and the IQGAP-domain protein Iqg1 (Sanchez- not occur, resulting in failure of cell Diaz et al., 2008). division. We found that the amino In animal cells and in yeasts, the actomyosin ring defines the terminus of the Inn1 protein forms a ‘C2- site at which cell division will occur. At the end of mitosis the ring becomes activated and then contracts into the domain’ that is essential for membrane cytoplasm, and this is coupled by an unknown mechanism to ingression, whereas the carboxy terminus ingression of the plasma membrane. We found that the actomyosin ring can still form in the absence of Inn1, and of Inn1 is required to recruit the Inn1 contraction is still initiated in all cells at the end of mitosis. In contrast, however, ingression of the plasma membrane does protein to the cleavage site. Our data not occur in the absence of Inn1 (Figure 2). The Inn1 protein indicate that the key to the function of is thus required in some way for contraction of the actomyosin ring to be coupled to ingression of the plasma Inn1 is recruitment of the C2 domain to membrane at the end of mitosis. the cleavage site, where it plays an The first 134 amino acids of the Inn1 protein are predicted to essential role by allowing ingression of the form a C2 domain, which is a class of membrane targeting plasma membrane to be coupled to domain that was first described in Protein Kinase C. The C2 domain comprises eight β-strands that form a sandwich of contraction of the actomyosin ring. two β-sheets. From one side of the sandwich, two or three In a systematic screen for new cell cycle proteins we loops protrude and these usually interact with the targets of identified a previously uncharacterised protein that is the C2 domain, which can be the head groups of membrane required for cell division. We modified the chromosomal lipids, or other proteins. We found that mutation of 12 Paterson Institute for Cancer Research Scientific Report 2008

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    conserved positively charged residues in Loop1 of the C2 domain of Inn1 blocks membrane ingression and cytokinesis, but does not prevent recruitment of the Inn1 protein to the cleavage site. This indicates that the C2 domain is essential for ingression of the plasma membrane during cytokinesis. Removal of the C2-domain blocks cytokinesis but does not prevent the regulated targeting of the rest of the protein to the cleavage site. In contrast, the remainder of Inn1 after the C2-domain is required for localisation at the cleavage site, and contains multiple ‘PXXP’ motifs that are likely to be binding sites for SH3 proteins. These findings suggested a model for the action of the Inn1 protein, whereby the majority of the protein serves to target Inn1 to the cleavage site so that the C2-domain can then fufill some essential role during cytokinesis. To test this model, we generated a diploid strain lacking one copy of the INN1 gene and in which we had fused artificially the C2-domain to the Hof1 component of the actomyosin ring. Upon sporulation of this diploid we found that haploid cells that lacked the INN1 gene and that expressed wild type HOF1 were inviable and formed chains of undivided cells, indicating a failure of cytokinesis. In contrast, however, expression of the C2-Hof1 fusion protein was able to suppress the defects normally associated with absence of Inn1, and restore ingression of the plasma membrane during cytokinesis, so that inn1∆ C2-HOF1 cells grew as well as wild type cells (Sanchez-Diaz et al., 2008). These findings indicate that the recruitment of the C2- domain of Inn1 to the cleavage site is a key requirement for ingression of the plasma membrane during cytokinesis in budding yeast. We are currently trying to understand how Inn1 is recruited to the cleavage site, how recruitment is regulated during the cell cycle, and how the C2-domain promotes ingression of the plasma membrane. Figure 2. Inn1 is required for ingression of the plasma membrane during cytokinesis in budding yeast. The picture shows timelapse images of cells Publications listed on page 57 that have just completed mitosis in the presence (a) or absence (b) of 0’ 2’ 4’ 6’ 8’ Inn1. The cells express GFP fused to the Ras2 protein that is associated with the plasma membrane, and the cells also express Myo1-Tomato. The cell in panel (b) has the heat inducible degron fused to Inn1 (inn1-td) and Myo1 -Tomato also expresses the red fluorescent protein eqFP fused to the spindle pole body component Spc42. Inn1 -GFP Me rge Figure 1. Inn1 is recruited to the actomyosin ring at the end of mitosis. The figure shows timelapse analysis of the contracting actomyosin ring in a cell that expresses Inn1-GFP and that also has the red fluorescent protein Tomato fused to the budding yeast type II myosin, Myo1. Research Reports 13

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    Paterson Institute for Cancer Research Cell Division Group http://www.paterson.man.ac.uk/celldivision Group Leader Iain Hagan Postdoctoral Fellows Scientific Officer Rotation Student Marisa Alonso-Nuñez Deepti Wilks Julian Blaser Agnes Grallert Nimesh Joseph Graduate Students Visiting Scientist Tamara Krsmanovic Elvan Boke Victor Alvarez-Tallada Marisa Madrid Dorota Feret Najma Rachidi Avinash Patel Like us cells live in an ever-changing world. MAP kinase kinase (MAPKK) Environmental stimuli to activate the MAPK (figure For cells this involves fluctuation in 1). In all organisms these exposure to nutrients, oxygen and a range cascades are activated by a MAPKKK variety of stimuli to promote of physical and chemical insults. These the phosphorylation of a environmental changes are sensed by number of targets to modify MAPKK cell metabolism and signal transduction cascades called “stress physiology in order to cope with the environmental MAPK response pathways” that alter metabolism changes. As major changes and physiology to adapt to the changing in cell physiology are needed other other targets targets other other targets targets transcription transcription to adapt to the new factors factors environment. Because the growth and environment, transcription Figure 1. A cartoon depicting the division of cancer cells deviates from factors are prime targets. organisation of a MAPK kinase The activation of these cascade normal tissues, transformed cells invariably transcription factors in turn experience heightened levels of stress. promotes the transcription of a cohort of genes to adapt to the new environment. For example, cells produce a number Understanding how stress responses work of reducing agents in response to heightened levels of and how they are triggered by oxidative stress. environmental insults can therefore Stress responses, cell division and growth control identify novel approaches to specifically In addition to changing cell constitution to tolerate stress, stress response pathways can arrest growth and division until target the stressed, cancer, cells while the adaptation is complete. Presumably this ensures fidelity leaving the unstressed cells of normal of division and minimises the long-term impact of stress, should either process be attempted whilst any damage tissue untouched. Because these stress remains un-repaired. Once the adaptive response has dealt with the novel environment, specific signalling events are response pathways are highly conserved, required to re-initiate growth and division. These recovery studying them in the relatively simple and pathways often rely upon the same stress response pathways that instigated the arrest in the first place. Quite how they highly malleable yeast can greatly do this is largely unclear at present. A broad goal of our accelerate the analysis of the more research effort is therefore to understand how stress responses exploit cell cycle regulators and growth controls to complex networks of man. regulate the timing and execution of cell division and growth following stress. We employ fission yeast as a model system MAP kinase cascades for these studies as its cell cycle controls and stress response MAP kinase cascades lie at the heart of most stress response pathways mirror those of humans. pathways. They comprise three kinases that are activated in sequence: a MAP kinase kinase kinase (MAPKKK) stimulates a 14 Paterson Institute for Cancer Research Scientific Report 2008

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    Stress responses in fission yeast The recovered tips of cells lacking the SRP MAPK Sty1 failed Two MAP kinase cascades maintain homeostasis in fission to re-establish polarised growth, as they ballooned out in a yeast. The so called “stress response pathway” (SRP) random fashion rather than pursuing the polarised growth incorporates the MAPK Sty1/Spc1. Like its mammalian that produces the linear extension of cells with an intact SRP JNK/p38 counterparts the SRP responds to a broad range of (figure 2). This indicated that SRP signalling is required to stresses, including oxidative, osmotic, heavy metal ion and both re-initiate growth and re-establish polarity. SRP signalling heat stresses. The “cell integrity pathway” (CIP) utilises the may also regulate a further aspect of recovery as cells lacking MAP kinase, Pmk1, and is required to maintain cell integrity in the SRP associated microtubule polarity factor Wsh3 were response to osmotic shock and hydrostatic pressure as well able to re-initiate growth on cue, however they could not as regulating cytokinesis. There is interplay between these select the right site at which to do so and became bent and two pathways as SRP signalling both promotes and attenuates branched following recovery (figure 2). Having identified CIP signalling depending upon the circumstances. these SRP signalling functions in growth control we can study the molecular basis of these controls to identify how stress Specific stress recovery pathways responses modulate cell structure. Phosphorylation of the conserved cell cycle regulator polo kinase on serine 402 restores both growth and division of Lessons from yeast cells after they have been arrested in a mild heat shock The ability to manipulate genes at will in a simple organism induced stress response (Petersen and Hagan, Nature 2005; whose primary purpose is simply to grow and divide is 435: 507). Curiously, serine 402 phosphorylation is not enabling us to explore the finer points of the pathways that stimulated by other stresses such as osmotic stress, even co-ordinate growth with spatial and environmental cues. This though these stresses also arrest cell growth. We therefore information informs studies in higher systems that, in turn, studied the impact of osmotic stress upon the cell growth raise models that can be most readily tested in yeast. This re- machinery over the last year. iterative cycle of comparative studies ensures that great strides are being made in understanding the molecular basis Growth in fission yeast of cell division and growth. Cell growth in fission yeast mirrors the interplay between the actin and microtubule cytoskeletons that underlies growth, Publications listed on page 57 migration and signalling in human cells. Cell extension is promoted as a consequence of polarisation of the actin sty1.Δ Wild type wsh3.Δ after recovery from stress cytoskeleton by the same GTPase cascades that promote the no stress after recovery from stress polarisation of the actin cytoskeleton to drive migration of human cells. In yeast the polarisation of the actin cytoskeleton directs the secretion of cell wall material to generate the robust polysaccharide wall that acts as an “exoskeleton” to define the rod shape of wild type fission yeast cells (figure 2). This cell wall contains a range of polysaccharides including 1−3β D-glucan that is stained by the fluorescent probe calcofluor. The selection of the site at Figure 2. Calcofluor staining of unstressed wild type cells and two mutant strains one which the actin becomes polarised to generate growth of the hour after the imposition of osmotic stress when growth patterns clearly deviate from cell tip is mediated by microtubules. Therefore, perturbation the linear extension seen following recovery of cells in which the wild type sty1+ and of the microtubule cytoskeleton, or the microtubule wsh3+ genes are present (figure 3). associated factors that communicate the inputs from microtubules to the actin cytoskeleton, cause cells to branch. Figure 3. Calcofluor staining of a Stress induced calcofluor staining mixture of cdc10.v50 cells, 30 foci SRP signalling regulates growth recovery at multiple levels (cells marked 1), 60 (cell marked We found that osmotic shock induced the deposition of 2) and 90 minutes (cells marked novel calcofluor staining structures at cell tips that persisted 3), after osmotic stress at 36°C. after cells resumed growth (arrows in figure 3). We used Bright foci of calcofluor staining 1 appear at the tip 30 minutes these landmarks to monitor the timing with which tip growth 3 after imposition of stress (arrows). resumed after the stress as foci were displaced from the tip The dots persist as new growth 2 by tip growth following recovery (figure 3 stage1>2>3). This initiates driving these landmarks established that the resumption of growth was independent progressively back towards the cell of CIP signalling as it was promoted by SRP stimulated equator (cells 1>2>3). The cell foci 1 transcription. The transcriptional nature of this recovery cycle mutation is simply used to block division for the duration of 3 pathway was underscored by its insensitivity to the mutation the experiment and so highlight of serine 402 of polo kinase to block the post-translational the apparent “movement” of the SRP response that is so critical in recovery from heat shock. foci. Research Reports 15

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    Paterson Institute for Cancer Research Cell Regulation Group http://www.paterson.man.ac.uk/cellregulation Group Leader Nic Jones Associate Scientists Graduate Students Scientific Officers Wolfgang Breitwieser Malgorzata Gozdecka Keren Dawson Caroline Wilkinson Orestis Mavroudis- Steve Lyons Chocholis Postdoctoral Fellows Jacek Walczynski Yujun Di Lu Zhang Clare Lawrence The MAP (mitogen activated protein) increase in tumour growth upon grafting into appropriate recipient mice. In both the embryonic phenotype and the kinase signalling pathways are central to cell growth phenotype the role of the ATF factors in the ability of the cell to respond to regulating MAPK signalling is critical. A number of dual specificity phosphatases, including MKP1, constitute major various stress conditions and in so doing downstream targets and in the mutant background their transcription is decreased resulting in loss of essential protect the cell from potential damage negative feedback regulation of MAP kinase pathways. that could arise. The highly conserved The scope for in vivo analysis using mouse knockouts is pathways are essential for a wide variety limited by the early lethality of the mutations. To circumvent of biological activities which in mammalian this difficulty we have developed a number of tissue-specific ATF2 knockouts. These models have revealed roles for ATF2 cells range from cell proliferation and in specific tissues and disease conditions: a brain specific differentiation to regulation of apoptosis; deletion of ATF2 leads to defects in hindbrain development and cerebellum functions resulting in death soon after birth their deregulation has been associated due to a respiratory defect that resembles meconium with numerous disease conditions such as aspiration syndrome; deletion of ATF2 specifically in endothelial cells leads to defects in the microvasculature of inflammation and cancer. the gut upon postnatal intestinal growth. These results emphasise the importance of ATF2 in the development and Previous efforts in our laboratory have focused on two homeostasis of mammalian tissue. mammalian MAP kinase targets, the transcription factors ATF2 and ATF7, which are both regulated by Conditional knockout models have also provided additional phosphorylation mediated by the stress activated MAPKs insights into a potential role for these factors in p38 and JNK. ATF2 and ATF7 are both members of the AP- tumourigenesis. A tumour suppressor role has been 1 family which controls the transcription of an extensive revealed in a skin tumourigenesis model using a mutant repertoire of genes including cell cycle and apoptotic mouse where ATF2 is specifically deleted in keratinocytes. regulators, numerous cytokines and growth factors. We Upon tumour initiation and promotion, the mutant animals have analysed the biological role of ATF2 and ATF7 using the demonstrate a significantly earlier onset of papillomas as genetically amenable mouse model system. Germ line well as greater numbers. The potential role of ATF2 in the mutations uncovered new insights into their role in progression of papillomas to malignant carcinomas is development: simultaneous deletion of both genes leads to currently being assessed. Likewise, irradiation of mice with embryonic lethality as a result of massive apoptosis in the ATF2 specifically deleted in T-cells results in earlier onset of embryonic liver involving both developing hepatocytes and T-cell lymphomas further supporting a tumour suppressor haematopoietic cells. Characterisation of cultured cells role. derived from mutant embryos revealed a role in limiting cell proliferation resulting in increased cell growth at high In contrast, in other tumour contexts there is indirect density. Interestingly, upon oncogenic transformation with K- evidence for ATF2 being pro-tumourigenic. In order to Ras, cells lacking ATF2 and ATF7 showed a profound assess this possibility directly, appropriate tumour models 16 Paterson Institute for Cancer Research Scientific Report 2008

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    are being developed and characterised. For example, we Phosphorylation of Atf1 by Sty1 regulates its stability; are characterising a B-cell specific ATF2 knockout model and phosphorylation results in an increase in the half-life of Atf1 analysing the effect of ATF2 deletion on lymphoma initiation and its accumulation to significantly higher levels in the cell and development following B-cell specific expression of the following stress. This increase in levels of Atf1 is critical for a c-myc oncogene. Similarly, the role of ATF2 in robust transcriptional response upon exposure to H2O2 and hepatocellular carcinoma (HCC) is being examined using for the ability of cells to mount an adaptive response two different approaches: one involves the chemical providing resistance to acute levels of osmotic stress. The induction of HCC in control and liver specific ATF2 key regulatory step in Atf1 degradation is the interaction of knockout mice which will determine whether the absence the transcription factor with the E3 ligase, SCFFbh1. This of functional ATF2 (and ATF7) leads to significant changes in interaction results in the ubiquitination of Atf1 and its onset, frequency and burden of HCC; the second utilises destruction via the proteasome. The substrate specificity of the recently developed orthotopic transplantation of the SCFFbh1 complex is determined by the F-box protein, hepatocyte precursors (hepatoblasts) to produce chimaeric Fbh1. Crucially, the interaction between Atf1 and Fbh1 is models of liver tumours. sensitive to Atf1 hyper-phosphorylation; accordingly the interaction is lost upon stress and Atf1 is stabilised. Homologues of the AP-1 family are found in all eukaryotic Consistent with these findings, disruption of the interaction organisms and their involvement in stress response is highly between Fbh1 and the rest of the SCF E3 ligase complex, conserved. Model organisms could provide useful models results in an increase in Atf1-11M protein and a rescue of for understanding the role and regulation of AP-1. Fission the phenotypes displayed by the atf1-11M mutant. yeast is a particularly pertinent model system – stress Interestingly, Fbh1 levels appear to be regulated by Atf1 responses are coordinated through the Sty1 signalling suggesting that a complex interplay exists between the F- pathway which is analogous to the mammalian p38 pathway. box protein and its target which serve to finely tune the Furthermore, many of the changes in the transcriptional abundance of the Atf1 transcription factor upon stress. profile of cells following stress is orchestrated through the Atf1 and Pap1 transcription factors which are related to Publications listed on page 57 mammalian ATF2 and cJun respectively. Stress The transcriptional changes following stress are extensive Oncogenic signals Cytokines signals and complex: a common set of genes are activated in Growth Infections response to all stresses accompanied by the activation of factors genes that are specific to the particular stress being imposed. Therefore each stress has its own transcriptional pattern resulting in the up-regulation of the appropriate defence and repair mechanisms. The complexity of the AP-1 response is best illustrated by characterising the events following exposure to oxidative stress: different patterns of transcriptional activation are seen dependent upon the exact nature of the oxidant as well as the dose Differentiation Proliferation Inflammation Apoptosis Invasion AP-1 regulates multiple functions in mammalian cells in response to a diverse Given the central role that Atf1 plays in the stress response, array of signals. we have characterised in detail its regulation and its interaction with the Sty1 kinase. Using ChIP assays we demonstrated that Atf1 is essential to target and tether the Sty1 kinase to stress-response gene promoters. Both the targeting and transcriptional activation is dependent upon Sty1 kinase activity. Surprisingly however, the key kinase target is not Atf1 itself since Sty1 is found at promoters in the presence of Atf1 protein that can no longer be phosphorylated (Atf1-11M mutant). We hypothesise that Sty1 phosphorylates and regulates another factor(s) found at the promoter, possibly a component of chromatin remodelling complexes or the polymerase complex itself. A number of approaches are being taken to identify Sty1 targets. Research Reports 17

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    Paterson Institute for Cancer Research Cell Signalling Group http://www.paterson.man.ac.uk/cellsignalling Group Leader Angeliki Malliri Postdoctoral Fellows Scientific Officer Graduate Students Sonia Castillo-Lluva Gavin White Lucy Dalton (funded by an EMBO Long- Natalie Reeves Term fellowship) Claire Rooney Helen Rushton Chong Tan (since April 2008) (since September 2008) Simon Woodcock Tumour initiation and progression result intrinsic rate of hydrolysis of bound GTP for GDP, leading to inactivation. Tiam1 (for T-lymphoma invasion and metastasis from inappropriate activation of protein) belongs to the GEF family of proteins and selectively intracellular signalling cascades. Rho-like activates Rac in response to growth factors and cell-substrate interactions. Precisely how these upstream events engage the GTPases are molecular switches in Tiam1-Rac signalling module is unclear. One possible mechanism is suggested by the observed association of Tiam1 signalling pathways that regulate with the second messenger Ras through a Ras-binding cytoskeletal and junctional organisation, as domain (RBD). Activated Ras and Tiam1 then cooperate to activate Rac (Lambert et al., Nature Cell Biol 2002; 4: 621). well as gene transcription. In this way, Rho Significantly, Tiam1-deficient cells are resistant to Ras-induced proteins influence cell morphology, cellular transformation (Malliri et al., Nature 2002; 417: 867), implying that this interaction is important for tumourigenesis. adhesion, motility, as well as cell cycle progression and cell survival. Rho proteins Tiam1/Rac signalling and tumourigenesis in vivo Mice deficient for Tiam1 are resistant to the formation of skin are transforming in vitro and are essential tumours induced by topical application of chemical for Ras-mediated in vitro transformation. carcinogens and consequent oncogenic activation of the c- Ha-Ras gene (Malliri et al., Nature 2002; 417: 867). Moreover, data has emerged to directly Tiam1-deficient tumours were not only fewer but also implicate Rho proteins in tumour initiation smaller than wild-type tumours and this correlated with increased apoptosis and reduced proliferation in carcinogen- and progression in vivo. Our group exposed Tiam1-deficient mice. Tiam1 is also a potent investigates how the activities of certain modifier of intestinal tumourigenesis (Malliri et al., J Biol Chem 2006; 281: 543). The majority of intestinal tumours are regulators of the Rho protein Rac are caused by mutations in the canonical Wnt signaling pathway, leading to aberrant expression of Wnt-responsive genes. controlled. We are also identifying Tiam1 is a Wnt-responsive gene, and is expressed in crypts of signalling events downstream of Rac that the adult mammalian intestine as well as in adenomas from patients and Min (multiple intestinal neoplasia) mice. In each modulate tumour susceptibility and of these settings, the Wnt pathway is activated. Further, by disease progression. comparing tumour development in Min mice expressing or lacking Tiam1, it was found that Tiam1 deficiency significantly Rho proteins, such as Rac1, RhoA and Cdc42, are guanine reduces the formation as well as growth of polyps in vivo nucleotide binding proteins that cycle between an inactive (Malliri et al., J Biol Chem 2006; 281: 543). GDP-bound state and an active GTP-bound state. The activity of Rho proteins is controlled by guanine nucleotide These two studies on tumourigenesis in vivo demonstrated exchange factors (GEFs) and GTPase-activating proteins that two independent oncogenic signalling pathways of major (GAPs). GEFs activate small GTPases by promoting the clinical significance (Ras and Wnt) recruit the Tiam1-Rac exchange of GDP for GTP, whereas GAPs enhance the signalling pathway by specific, albeit distinct mechanisms. In 18 Paterson Institute for Cancer Research Scientific Report 2008

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    the context of oncogenesis, activation of this signalling Tiam1 were inversely correlated with Src activity, consistent module promotes tumour initiation and growth. Moreover, with the above-mentioned post-translational regulatory this role is specific to Tiam1 since its loss cannot be mechanism operating in malignancies. Abrogating Tiam1 compensated for by other Rho GEFs. phosphorylation and degradation suppressesed Src-induced AJ disassembly. As a consequence, cells expressing a non- Tiam1/Rac signalling and the regulation of cell-cell adhesion phosphorylatable Tiam1 showed a marked decrease in The skin carcinogenesis model revealed an additional role for wound closure in response to Src. These data establish a Tiam1 in tumourigenesis. The few skin tumours arising in new paradigm for regulating local concentrations of Rho- Tiam1-deficient mice progressed more frequently to GEFs, as well as linking Tiam1-Rac signalling with a further malignancy than those in wild-type mice, suggesting that oncoprotein. Tiam1 deficiency promotes malignant conversion (Malliri et al., Nature 2002; 417: 867). Analysis of Tiam1 expression in It is increasingly apparent that Rho GEFs do more than simply skin tumours of wild-type mice revealed that benign activate Rho molecules; several studies now point to their papillomas maintained high levels of Tiam1 expression, role in influencing the choice of biological response elicited by whereas expression was reduced in squamous cell a given Rho protein. GEFs have been shown to bind to carcinomas and was completely lost in highly invasive spindle effectors directly or to scaffold proteins that complex with cell carcinomas. The increased Ras signalling associated with components of effector pathways. Thus Tiam1 interacts with advanced skin malignancies (resulting from amplification of IB2/JIP2, a scaffold that promotes Rac activation of p38 kinase the mutated Ras allele) seems to be responsible for the cascade over JNK MAP kinase cascade (Buchsbaum et al., Mol reduction or loss of Tiam1 expression in the later stages of Cell Biol 2002; 22: 4073), and also with spinophilin, a scaffold tumour progression, as demonstrated in vitro for Ras- that promotes Rac activation of p70 S6K over Pak1, a transformed MDCK cells (Zondag et al., J Cell Biol 2000; 149: different Rac effector (Buchsbaum et al., J Biol Chem 2003; 775). Thus, while Tiam1/Rac co-operate with Ras in 278: 18833). In our lab, we are using biochemical approaches establishing tumours, they antagonize Ras during tumour to identify Rac and Rac GEF interacting proteins involved in invasion. different aspects of transformation including malignant progression (acquisition of invasiveness). One probable mechanism by which Tiam1 and Rac suppress malignant progression is through their ability to stimulate Publications listed on page 57 cell–cell adhesion. In vitro studies have shown that over- expression of activated Rac or Tiam1 can promote the Sos formation of adherens junctions (AJs) and the accompanying Grb2 Src Sos induction of an epithelial-like phenotype in a number of Grb2 mesenchymal cell lines (Malliri & Collard, Curr Opin Cell Biol P MEK 2003; 15: 583). Moreover, using both RNA interference and Y Y Tiam1 Tiam1 cells derived from Tiam1-deficient mice, it has been shown ERK ERK that endogenous Tiam1 is required for both the formation as P Calpain well as the maintenance of cadherin-based adhesions (Malliri Calpain et al., J Biol Chem 2004; 279: 30092). The oncoprotein Src, a non-receptor tyrosine kinase implicated in malignant progression, potently induces epithelial–mesenchymal Tiam1 Degradation transition (EMT) by targeting AJs for dissassembly. We recently showed that direct phosphorylation of Tiam1 by Src Cell-cell adhesion is required for Src-induced EMT. Moreover, we identified a dissasembly novel post-translational mechanism of regulating Tiam1 levels. We showed that Src phosphorylates Tiam1 on tyrosine 384 Model for the regulation of Tiam1 by Src activation. Src directly phosphorylates Tiam1, (Y384). This occurs predominantly at AJs during the initial preferentially at sites of cell-cell adhesions. Phosphorylated Tiam1 recruits the Grb2-Sos stages of Src-induced EMT and creates a docking site on complex which, via MEK, increases activation of the ERK associated with Tiam1, and hence the local activation of calpain proteases at cell–cell adhesions. Calpain mediated Tiam1 for Grb2. We found that Tiam1 is constitutively proteolysis of Tiam1 results in its inactivation, reducing the activity of Rac that is necessary associated with extracellular signal-regulated kinase (ERK). to maintain cadherin adhesions. The weakening of cell–cell adhesions in this way by Src Following recruitment of the Grb2-Sos1 complex, ERK would allow increased migration of cells. becomes activated and triggers the localised degradation of Tiam1 at AJs through, in turn, activating calpain proteases. Significantly, we demonstrated that in human lung, colon, and head and neck cancers phosphorylation of Y384 of Tiam1 positively correlated with Src activity, while total levels of Research Reports 19

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    Paterson Institute for Cancer Research Clinical and Experimental Pharmacology Group http://www.paterson.man.ac.uk/cep Group Leaders Caroline Dive and Malcolm Ranson Disease Focus Team Dominic James (AZ Scientific Officers Graduate Students Leaders secondment) Karen Brookes Jane Barraclough Fiona Blackhall Tetanya Klymenko Fouziah Butt Martin Brandenburg Guy Makin Flavia Moreira Leite Martin Dawson Cristina Martin-Fernandez Andrew Renehan Lee Lancashire Olive Denenny Dimitra Micha David Moore Martin Greaves Elizabeth Sweeney Staff Scientists Christopher Morrow Grace Hampson Shaun Villa Jeff Cummings Darren Roberts Cassandra Hodgkinson Tim Ward Kathryn Simpson Karen Morris Undergraduate Student Lyndsey Priest Laura Glass Postdoctoral Fellows Clinical Fellows Robert Sloane Luke Harrison Ruth Board Nigel Smith Laboratory Aide Sarah Holt (AZ second- Emma Dean Brian Trueman Matthew Lancashire ment) Alastair Greystoke Zaira Yunis Jian Mei Hou Sarah Hughes Matthew Krebs Gireesh Kumaran The development of molecularly targeted Disease orientated translational research anticancer drugs mandates a parallel focus on lung, colorectal and paediatric development of biomarkers in order to cancers and pre-clinical studies includes give the right patient the right dose and drug target validation, drug combination schedule of treatment. CEP develops and optimisation and the impact of hypoxia on validates pharmacokinetic (PK), drug efficacy. pharmacodynamic (PD) and predictive Clinical Trials at the Christie Hospital’s DCU biomarker assays and implements Research in CEP is predicated on those novel agents entering clinical trial at the DCU. DCU typically supports c100-120 biomarker qualification within clinical trials trials with c6400 patient visits p.a. In order to meet research at the Christie Hospital’s Derek Crowther and service requirements, proposals for a new £35M Cancer Treatment Centre were developed in 2008. The new Cancer Unit (DCU) focusing on novel agents Treatment Centre will provide comprehensive facilities for targeted to apoptosis and angiogenesis. In clinical trials, experimental treatment, and service chemotherapy and is due to open in 2010. The expansion of 2008, biomarker research included the DCU within the treatment centre will make it one of the development of a robust plasma largest early clinical trials centres worldwide and integral to this development are enhanced laboratory facilities for proteomics workflow for biomarker translational research to strengthen further the CEP-DCU axis. One example of the CEP DCU axis in action is the discovery, enumeration and biomarker enhanced CR-UK Phase I trial of AEG35156 characterisation of circulating tumour cells, (Aegera Therapeutics, antisense XIAP). We reported this first in class, first in man clinical trial in the Journal of Clinical detection of oncogene mutations in Oncology (Dean et al., 2008). CEP has also developed a circulating free DNA and multiplexing suite of biomarkers to accompany ongoing early clinical trials of the BH-3 mimetic class of apoptosis promoting drugs that circulating and tissue biomarker assays. target interactions between Bcl-2 family proteins. 20 Paterson Institute for Cancer Research Scientific Report 2008

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    Biomarker Research in CEP in 2008 is exemplified by the of the tumour suppressor PTEN or activating mutations in following highlights i) application of cytokeratin 18 (CK18) the PIK3CA gene that encodes the catalytic p110α subunit of based circulating biomarkers of cell death as PI-3K. The PI-3K signalling pathway is considered to be a pharmacodynamic biomarkers; ii) demonstration of the tractable drug target in cancer treatment, however, the potential of circulating tumour cells (CTCs) as precise impact of PIK3CA mutations on drug responsiveness pharmacodynamic and predictive biomarkers; iii) the potential remains incompletely defined. In order to begin to address predictive utility of oncogene mutation detection in circulating the clinical utility of PIK3CA mutation detection as a free DNA and iv) the development of multiplexed circulating circulating biomarker and within our CR-UK/AstraZeneca biomarkers of angiogenesis. Clinical Pharmacology Fellowship Scheme and with experts at DxS diagnostics, we participated in the generation of a (i) CK18 based circulating biomarkers of cell death novel assay based on Amplification Refractory Mutation In collaboration with Abbott Laboratories, we demonstrated System (ARMS) allele-specific PCR and Scorpion primers using a small cell lung cancer (SCLC) xenograft model that (DxS Diagnostics) to detect the 4 most common mutations the BH-3 mimetic ABT 737 provoked tumour cell apoptosis in the PIK3CA gene. The resultant high throughput, that was reported by the appearance of full length and multiplexed ARMS assays of these PIKC3A mutations are caspase cleaved CK18 in the blood stream as assessed by more sensitive than sequencing (Board et al., 2008a). Our M65 and M30 ELISAs respectively (Micha et al., 2008). These first and ongoing clinical study in metastatic breast cancer circulating biomarkers were elevated rapidly after drug suggests that there is a high concordance rate between administration before detectable tumour shrinkage and plasma and tumour PIK3CA incidence. The assay will be informed on appropriate blood sampling times for the applied in 2009 to trials of PI-3K and mTOR inhibitors to ongoing Phase I clinical trial where biomarker analysis is assess its potential to predict drug responses. underway. (iv) Multiplexing circulating biomarkers of angiogenesis (ii) Circulating Tumour Cells Molecular therapies targeting tumour vasculature have We evaluated CTCs as a useful biomarker for trials of BH-3 recently achieved clinical recognition through randomised mimetics in SCLC. In preparation for upcoming trials, CTC trials of conventional therapy with or without Vascular number was evaluated in patients on standard chemotherapy Endothelial Growth Factor (VEGF). However, prospective using the Veridex CellSearch system; CTC numbers identification of patients who will benefit from VEGF decreased post drug treatment in accordance with patient inhibitors has not yet been achieved and remains a significant response in cycle 1 of therapy. The levels of drug targets in obstacle to the approval of this type of drug on the NHS. CTCs may serve to predict which patients will respond to There are plentiful potential circulating pharmacodynamic BH-3 mimetics thus assays were developed to identify and/or predictive biomarkers of angiogenesis but it is not yet amplification of bcl-2 (a potential marker of drug sensitivity, clear how to select an optimal biomarker panel for trial measured using FISH, see figure) and upregulation of Mcl-1 implementation. CEP working with Gordon Jayson’s (a potential biomarker of drug resistance for the BH-3 Translational Angiogenesis team has used the Endogen mimetic ABT 263, by immunohistochemistry). Efforts are Searchlight platform to develop and validate multiplexed underway using the Metagenex ISET system that separates ELISAs that can now be used in upcoming trials of anti- CTCs from white blood cells on the basis to size exclusion to angiogenic therapies using minimal blood volumes. Of critical isolate RNA from purified CTCs of sufficient quantity and importance, and ongoing is the integration of these circulating quality to undertake transcript profiling with the goal to biomarkers with imaging biomarkers to obtain a discover signatures of drug resistance and sensitivity and comprehensive picture of patient drug responses where identify novel drug targets. RECIST criteria may not be sufficiently informative. (iii) Oncogene Mutations in Circulating Free (cf) DNA Publications listed on page 57 A number of tumour specific mutations e.g. within EGFR, B- RAF and K-RAS, can predict response to certain novel therapeutics and progression free survival. Mutation detection is traditionally performed on archival tissue samples which may no longer reflect current tumour biology, and can be difficult to obtain, especially within the context of clinical trials. Reliable detection of tumour specific mutations within cfDNA provides an alternative that is minimally invasive, can be performed on serial samples and potentially, provides a ‘real time’ assessment of the tumour mutation status to guide clinical decision making. We have optimised methods for extracting circulating DNA and Standard Operating Procedures were produced for blood processing and for DNA extraction (Board et al., 2008b). Deregulation of the PI-3K pathway in cancer is prevalent most notably with loss Bcl-2 amplification in SCLC CTCs Research Reports 21

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    Paterson Institute for Cancer Research Immunology Group http://www.paterson.man.ac.uk/immunology Group Leader Peter L. Stern Postdoctoral Fellows Scientific Officers Graduate Students Fernanda Castro Kate Mulryan Mariam Al-Muftah Owen McGinn Debbie Burt (with David Gilham) Tom Southgate (seconded to BIGT) Georgi Marinov Andrzej Rutkowski Clinical Fellows (with Crispin Miller) Emma Crosbie (Walport Fellow) Sai Daayana (with Henry Kitchener) Christy Ralph (with Robert Hawkins) mononuclear cells (PBMC) samples from baseline until after The Immunology Group has a major goal the fourth vaccination at 14 weeks using a proliferation assay of understanding and exploiting the with 5T4-Fc fusion protein, overlapping 32mer 5T4 peptides, function and/or expression of 5T4 MVA-LacZ and MVA-5T4 infected autologous monocytes. Significant responses to 32mer peptide pools were seen in 8 oncofoetal molecules in the context of of 19 CRC patients by time of surgery and 13 of 19 who cancer associated changes in motility, and received 2 more vaccinations by week 14. We also assessed the levels of systemic T regulatory cells, plasma cytokine adhesion contributing to metastasis. levels, phenotype of tumour infiltrating lymphocytes including Successful translational studies have led to T regulatory cells and tumour MHC class I loss of expression. ongoing Phase 3 clinical trials of 5T4 Approximately half of the patients showed phenotypes consistent with relative immune suppression and/or escape directed immunotherapies. This report highlighting the complexity of positive and negative factors details studies of both animal model and challenging any simple correlation with clinical outcome. human immune responses to 5T4 antigen Mouse models of 5T4 immunotherapy aimed at improving both vaccine and We have shown that transient depletion of T regulatory cells antibody delivered therapies for cancer. In (Tregs) can enhance 5T4 immunity in renal cell carcinoma patients (Thistlethwaite et al., 2008), suggesting that Treg addition progress on the development of modulation might improve the efficacy of the 5T4 vaccine immunotherapies for HPV associated (TroVax). To investigate the role of Tregs in 5T4 immunity we cancers are reported. utilised 5T4 knockout (KO) C57BL6 mice to map the 5T4 T cell repertoire. By immunising wild type (WT) and 5T4 KO Immune regulation and escape in TroVax vaccinated patients mice with a single dose of a replication defective adenovirus We have recently reported the results of a phase II trial in vaccine encoding murine 5T4 (Ad-m5T4) and re-stimulating which two TroVax (MVA-5T4) vaccinations were given to these primed cells in vitro with overlapping 32-mers, we patients both pre- and post-surgical resection of liver identified m5T4 derived epitopes recognized by CD4 and metastases secondary to colorectal cancer (CRC). 5T4- CD8 T cells using interferon gamma (IFNγ) ELISPOT. 5T4 specific cellular responses were assessed at entry and two KO mice generated strong and high avidity 5T4-specific CD4 weeks after each vaccination by proliferation of fresh and CD8 T cell responses. In WT mice, 5T4-specific CD4 T lymphocytes and ELISA for antibody responses; 18 of 19 cells were either absent or anergized and the CD8 T cell CRC patients mounted a 5T4-specific cellular and/or humoral response weaker and lower avidity. The latter might result response (see also Biological, Immune and Gene Therapy from a lack of help and/or active suppression by Tregs. To Report). We have now completed a comparison of individual look for evidence of a natural 5T4 specific Treg cell and between patient responses over the course of the population, CD4+CD25+ T cells were isolated from naïve treatments using cryopreserved peripheral blood 22 Paterson Institute for Cancer Research Scientific Report 2008

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    WT and 5T4 KO animals and their suppressive activity on infiltration were more likely to respond clinically following 5T4 primed KO effector cells assessed by IFNγ ELISPOT. HPV vaccination. This investigation has used Our data demonstrate that WT CD4+CD25+ T cells have a immunohistochemistry to investigate the levels and types of stronger specific suppressive activity than 5T4 KO VIN infiltrating immune cells including Tregs, as the latter have CD4+CD25+ T cells (p < 0.01). Ongoing studies are been shown to be associated with poor clinical responses in evaluating methods to selectively modulate 5T4 specific Tregs various cancers. The imiquimod treatment is characterised by as a means to improve efficacy of 5T4 vaccines in the clinic. increased local infiltration of CD8 and CD4 T cells but in non-responders (failure to clear VIN on vulvoscopy or Immunotherapy of HPV associated VIN histology) this is accompanied by increased Tregs whereas in The introduction, in autumn 2008, of UK wide human the responders (clearance of VIN on vulvoscopy and papillomavirus (HPV) vaccination programmes for prevention histology) these are at significantly lower density. In the of cervical cancer represents a magnificent dividend for the imiquimod/PDT trial, responders had significantly increased national and international HPV research community. UK pre-existing lympho-proliferative responses to the HPV 16 based scientists and clinicians are continuing to make compared to non-responders but there was no stimulation of important contributions to the challenges of implementation HPV immunity with treatment. Following vaccination in the and education (Garland et al., 2008). In addition, we have imiquimod/vaccine trial, patients showing clinical responses continued to develop therapeutic approaches for treatment had significantly increased lympho-proliferation to the vaccine of HPV associated neoplasia including high grade vulval compared to the non-responders. Imiquimod followed by intraepithelial neoplasia (VIN). The chronic nature of this PDT or TA-CIN both show promise as non surgical therapies condition might be explained by a balance between the for VIN. As a correlation between pre-existing HPV response, consequences of the virus infection and immune control (see response to vaccination and clinical response was noted, any figure). To improve the management of VIN, we undertook 2 therapeutic anti-HPV treatment could be very valuable. phase II studies using a topical immunomodulator cream – Non-responders showed higher level of T-regulatory cells in imiquimod followed by either photodynamic therapy (PDT) situ consistent with their negative prognostic value in some or therapeutic HPV vaccination with TA-CIN, a fusion protein cancers. The therapeutic impact of treatment may depend of HPV16L2E6E7. The initial use of imiquimod in this study on the differential immune response of responders and non- was designed to create a more favorable local environment responders. characterized by increased T-cell infiltration for the subsequent PDT especially, because the response to the Publications listed on page 58 latter has been associated with increased CD8 T-cell infiltration. In addition, other previous work had shown that patients with lesions containing a higher density of T cell Research Reports 23

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    Paterson Institute for Cancer Research Inositide Laboratory http://www.paterson.man.ac.uk/ Group Leader Nullin Divecha Associate Scientist Postdoctoral Fellows Scientific Officer David Jones Iman van den Bout Yvette Bultsma Laura Norton Graduate Students Willem-Jan Keune Lilly Sommer Phosphoinositides are a family of lipid metastasis suppressive mechanism. It is second messengers interlinked by the likely that tumour cells must by-pass this activities of an extensive and highly process to become metastatic. regulated network of kinases and Interestingly a number of oncogenes that phosphatases that modulate suppress cell death in response to loss of phosphoinositide levels in response to extracellular matrix signalling also suppress environmental changes. Alterations in the decrease in PtdIns(4,5)P2 and are phosphoinositide levels can regulate many consequently able to maintain growth different cancer-relevant cellular pathways factor signalling. including survival, proliferation, migration, Understanding how PtdIns(4,5)P2 levels are regulated and cell substratum interactions and how changes in PtdIns(4,5)P2 are transduced in to regulating transcription. PtdIns(4,5)P2 is at the heart cellular function. PtdIns(4,5)P2 is present in the plasma membrane where its of phosphoinositide signalling, being the levels can be regulated in response to receptor activation. substrate for both phosphatidylinositol-3- PtdIns(4,5)P2 is also synthesised within the nucleus where its kinase (PI3K) and phospholipase C (PIC). levels can be regulated distinctly from the plasma membrane pool. Understanding how these two pools of lipids are The PI3K /PTEN pathway is deregulated in controlled and which cellular pathways they regulate tumours promoting cell survival and represents a major goal of the laboratory. PtdIns(4,5)P2 can proliferation through the activation of PKB be synthesised by the action of two distinct but related kinases (collectively termed PIP kinases). PIP5Ks and flux through the PIC pathway is phosphorylate PtdIns4P on the 5’ position, while PIP4Ks upregulated in human tumours. phosphorylate PtdIns5P on the 4’ position. It is likely that PIP5Ks are the major producers of PtdIns(4,5)P2 while the Furthermore PtdIns(4,5)P2 is itself a role of PIP4K may be to regulate cellular PtdIns5P levels regulator of cytoskeletal dynamics, cell and/or a distinct minor pool of PtdIns(4,5)P2. PIP5Ks are survival pathways and cell polarity (see upregulated in nearly all cancer cell lines tested and overexpression of PIP5K can induce dramatic changes in cell Panbianco et al., 2008). Cells monitor morphology, increase migratory capacity and attenuate PtdIns(4,5)P2 levels and a decrease in their apoptosis in response to cellular stressors. PIP4Ks on the other hand appear to regulate the levels of cellular PtdIns5P levels can lead to the induction of which can impinge on the activity of the tumour suppressor apoptosis. As PtdIns(4,5)P2 levels are protein p53 and a number of other transcriptional regulators sensitive to extracellular matrix signalling, that are implicated in the development of cancer and on PKB activation. the induction of apoptosis in response to decreases in PtdIns(4,5)P2 may constitute a 24 Paterson Institute for Cancer Research Scientific Report 2008

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    One approach to understanding how PIP kinases are proteins as these likely represent downstream lipid regulated regulated is to elucidate how they may be post-translationally proteins. In collaboration with Clive D. Santos (PROBE modified and what proteins they interact with. Post proteomic platform, Dept of Biomedicine University of translational modifications such as phosphorylation, Bergen) isolated nuclei have been treated with reagents that acetylation, methylation or ubiquitination can induce changes tightly interact with phosphoinositides and therefore are able in enzymatic activity, localisation, interaction partners and to dissociate endogenous proteins that are bound to nuclear stability. Using mass spectrometry we have identified 12 phosphoinositides. The released proteins have been analysed different sites of phosphorylation on PIP5K and 3 sites of by mass spectrometry and include transcriptional regulators phosphorylation and 8 sites of acetylation on PIP4Kβ. We and chromatin remodelling complexes. Candidate proteins have generated modification-specific antibodies and are using are being tested for their phosphoinositide binding these to elucidate how and when the sites of modification characteristics. are regulated and what the impact of modification is on the subcellular localisation and activity of the enzymes. Using PIP5K as a target for drug development. methods such as mass spectrometry and yeast two hybrid we In collaboration with CRT (Cancer Research Technology) we are investigating what proteins interact with PIP kinases. For have screened and identified small molecular weight example the small molecular weight G protein Rac interacts compounds that are able to inhibit PIP5K activity. Chemical with and regulates the localisation of PIP5K. We have modification and structure-function studies have identified identified an allele of PIP5Kα that has attenuated ability to important chemical moieties on these small molecules which interact with Rac and demonstrated that this mutant protein have led to the development of more potent PIP5K no longer localises to the membrane. In vivo PIP5K activity inhibitors. The rationale behind PIP5K as a cancer relevant modulates focal adhesion stability required during neuronal target is shown in the figure. PtdIns(4,5)P2 is the substrate for retraction, an important process in guiding neurones to their PI-3-kinase which regulates the oncogenic activity of PKB. We targets. This role of PIP5K is blocked if its interaction with hypothesised that depletion of PtdIns(4,5)P2, using an inhibitor Rac is attenuated. We have constructed a targeting vector to to PIP5K, may compromise the receptor mediated synthesis knock in this mutation to study the role of the interaction of PtdIns(3,4,5)P3 and may therefore attenuate the activation between PIP5K and Rac in vivo. We have also identified a of PKB. The inset shows that treatment with one of our number of proteins that interact with PIP4Kβ. We are PIP5K inhibitors attenuates both insulin (ins) and H2O2 investigating whether these interacting proteins are regulated induced PKB activation. Future experiments will concentrate by or regulate the activity or the localisation of PIP4Kβ on driving this concept forward to define which types of cancer cells may be sensitive to inhibition of PIP5K. To define the array of pathways that are regulated by PtdIns(4,5)P2 and PtdIns5P we are identifying lipid interacting Publications listed on page 59 PIP5K Ras CRT-PIP5K inhibitors PI3K PtdIns(3,4,5)P3 PtdIns(4,5)P2 PTEN P-473PKB P-308PKB PKB c Ins H2O2 c Ins H2O2 + migration proliferation survival Inhibitors of PIP5K activity suppress growth factor and oxidative stress induced PKB activation. PIP5K mediates the synthesis of PtdIns(4,5)P2 which can be used by the PI-3-kinase (PI3K) for the synthesis of PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 regulates the activation of the oncogene PKB. Activation of PKB attenuates apoptosis pathways and increases proliferative pathways. Pretreatment of cells with CRT inhibitors attenuate PIP5K activity in vivo and block the activation of PKB as assessed using antibodies against specific phosphorylated residues that are indicative of the upregulation of PKB activity (473 and 308). Research Reports 25

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    Paterson Institute for Cancer Research Leukaemia Biology Group http://www.paterson.man.ac.uk/leukaemia Group Leader Tim Somervaille Postdoctoral Fellow Scientific Officer Graduate Student Xu Huang Gary Spencer William Harris The cancer stem cell (CSC) model posits a quarter of cells within the leukaemia clone and exhibit mature myeloid immunophenotypes (Somervaille and Cleary, that many human malignancies consist of Cancer Cell 2006; 10: 257). Secondly, protocols that enhance two functionally distinct cell types: (i) engraftment of human leukaemia cells in xenogeneic transplant assays demonstrate the presence of LSCs in CSCs, which are self-renewing cells with leukaemia cell sub-populations previously considered to be devoid of them. the capacity to initiate, sustain and expand the disease, and (ii) non-self-renewing Since LSCs may be more numerous and mature than originally proposed, the nature and generality of the progeny cells, derived from CSCs through hierarchical organization of malignancies has recently been differentiation, which may make up the questioned. However, consistent with the CSC model, only a subset of AML cells have clonogenic potential in in vitro bulk of the tumour and account for assays, and human AML blast cells undergo differentiation in disease symptomatology. In order for vivo to mature granulocytes, as may murine LSCs initiated by MLL-AF9 (Somervaille and Cleary, Cancer Cell 2006; 10: 257). malignancies to be cured, it may be necessary and sufficient to exclusively To further elucidate the hierarchical disposition of AML, a major goal is to identify transcriptional programs, genes and eliminate CSCs. Consequently, there is pathways that specifically correlate with and promote the considerable interest in further retention of LSCs within the self-renewing compartment of leukaemias. It is not known whether such LSC maintenance understanding the biologic and molecular programs are synonymous with programs responsible for properties of these cells, by comparison leukaemia initiation, for example Hoxa/Meis in MLL leukaemogenesis. It is also not clear whether they share with both their non-self-renewing features with transcriptional programs expressed in adult or embryonic stem cells (ESCs), or whether there is a downstream progeny and their normal relationship with genes and pathways implicated in the adult stem cell counterparts. function of AML stem cells such as NFκB, phosphatidylinositide-3-kinase, CTNNB1, Bmi1, Pten and Junb. CSCs (also called tumour-propagating or tumour-initiating cells) were first formally described in human acute myeloid In work performed at the Paterson Institute in the past year leukaemia (AML) as rare cells that share an by members of the Leukaemia Biology group, and also immunophenotype with normal hematopoietic stem cells previously by Tim Somervaille at Stanford University in the (HSCs). This paradigm has recently been substantially revised United States, we investigated the genetic determinants that based on two significant observations. First, in murine models maintain LSC frequencies and leukaemia cell hierarchies using of human leukaemia induced by the MLL-AF9 oncogene, self- a mouse model that faithfully recapitulates many of the renewing leukaemia stem cells (LSCs) may account for up to pathologic features of AML induced by chromosomal 26 Paterson Institute for Cancer Research Scientific Report 2008

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    translocations of the MLL gene, which occur in about 5-10% conclusion that CSCs may be aberrantly self-renewing of human AMLs. Confirming recent speculation that CSC downstream progenitor cells whose frequency in human frequency may differ between distinct tumour types, LSC malignant disease correlates with and dictates prognosis. frequency in AML was found to vary substantially according to the initiating MLL oncogene. This feature, and the The transcription/chromatin regulatory factors Myb, Hmgb3 observation that LSC frequency varies within the leukaemia and Cbx5 are critical components of the LSC hierarchical cell hierarchy, was used to derive a transcriptional program maintenance program and suffice for Hoxa/Meis-independent for LSC hierarchical maintenance. The program indicates that immortalization of myeloid progenitors when co-expressed, MLL LSCs are maintained in a self-renewing state by co- establishing the cooperative and essential role of an ESC-like option of a transcriptional program that shares features with LSC maintenance program ancillary to the leukaemia initiating ESCs and is transiently expressed in normal myeloid MLL/Hox/Meis program. This work is currently in press in Cell precursors rather than HSCs or mature neutrophils. Stem Cell and will be published early in 2009. Furthermore, the shared transcriptional features of LSCs, ESCs, normal mid-myeloid lineage cells, and a diverse set of Publications listed on page 60 poor prognosis human malignancies supports the broader Figure 1. Murine acute myeloid leukaemia initiated by MLL-AF1p. Figure 2. In vitro semi-solid culture of murine acute myeloid leukaemia cells generates colonies of cells. The single cells that initiate these colonies have leukaemic stem cell potential. Research Reports 27

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    Paterson Institute for Cancer Research Stem Cell Biology Group http://www.paterson.man.ac.uk/stemcellbiology/ Group Leader Georges Lacaud Postdoctoral Fellows Scientific Officers Graduate Students Cristina Ferreras Catherine Gavin Monika Antkiewicz Christophe Lancrin Ting Zheng Patrycja Sroczynska Michael Lie-A-Ling Olga Tsoulaki Flor Perez-Campo The transcription factor AML1/RUNX1 is alone leukemia in mice. However an alternatively spliced form of AML1-ETO has recently been shown to cause a frequent target of gene rearrangements following retroviral transfer a rapid development of leukemia and mutations in human acute in mice. Based on this new finding, we are developing an animal model in which the expression of this form of AML1- myelogenous leukemia (AML) and acute ETO is inducible. This new tool will allow us to study the lymphoblastic leukemia (ALL). Consistent molecular events leading upon expression of AML-ETO to the development of leukemia. with its implication in leukemias, RUNX1 has also been shown to be critical for Early haematopoietic development The earliest site of blood cells development in the mouse haematopoietic development. The MOZ embryo is the yolk sac where blood islands, consisting of haematopoietic cells surrounded by a layer of angioblasts, gene is involved in three independent develop at approximately day 7.5 of gestation. The parallel myeloid chromosomal translocations development of these two lineages in close association provided the basis for the hypothesis that they arise from a fusing MOZ to the partner genes CBP, common precursor, a cell called the haemangioblast. A P300 or TIF2. Our group studies the conflicting theory instead associates the first haematopoietic cells to a phenotypically differentiated endothelial cell with function of MOZ and RUNXI in haematopoietic potential, i.e. a haemogenic endothelium. haematopoietic development and Support for the haemangioblast concept was initially provided by the identification during embryonic stem (ES) maintenance with the aim to better cells differentiation of a clonal precursor, the blast colony- understand how alterations of these forming cell (BL-CFC), which gives rise after 4 days to blast colonies with both endothelial and haematopoietic potential. functions lead to leukemogenesis. We use Although recent studies have now provided evidence for the complementary approaches such as in vitro presence of this bipotential precursor in vivo, the precise mechanism of generation of haematopoietic cells from the differentiation of mouse embryonic stem haemangioblast still remains completely unknown. (ES) cells and in vivo mouse models. A new model of haematopoietic development We performed a series of studies to determine the cellular RUNx1 and leukemia. and molecular events leading to the generation of blast Human acute leukemias are characterized by the presence of colony from BL-CFC. Our data demonstrate that the recurrent chromosomal abnormalities, which frequently result haemangioblast generates haematopoietic cells through the in the formation of chimeric transcription factors. The core formation of a haemogenic endothelium intermediate, binding factors AML1/RUNX1 and CBFβ are the most providing the first direct link between these two precursor frequent targets of these genetic alterations. The t(8;21) populations. This haemogenic endothelial cell population is translocation resulting in AML1-ETO fusion and the inv(16) transiently generated during blast development and is also generating the SHMMC-CBFβ fusion accounts together for detected in gastrulating embryos. At the molecular level, we more than 20% of all the AML cases. Animal models have have demonstrated that the transcription factor SCL/TAL1 is indicated that the full length AML-ETO, expressed either indispensable for the establishment of this haemogenic upon viral transfer or as a transgene, is not able to induce endothelium cell population from the haemangioblast 28 Paterson Institute for Cancer Research Scientific Report 2008

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    whereas RUNX1/AML1 is critical for generation of stage than expected. Subsequent CSFR-1 expression haematopoietic cells from this haemogenic endothelium. requires prior PU.1 expression and RUNX1 binding. Once a These results indicate that the two a priori conflicting theories stable transcriptional haematopoietic circuit has been on the origin of haematopoietic development, established, RUNX1 is in contrast dispensable. These results haemangioblast and haemogenic endothelium, can be merged indicate stage specific functions and requirements for RUNX1 into a single linear developmental process leading to the in the development and maintenance of the haematopoietic formation of the first committed haematopoietic precursors. program. Transcriptional targets of RUNx1/AML1 Function of the HAT activity of MOZ Our initial studies revealed a profound defect in the potential The MOZ gene is involved in leukemia in three independent of the RUNX1-/- ES cells to generate blast colonies. More myeloid chromosomal translocations fusing MOZ to the recently we demonstrated that RUNX1 is critical for the partner genes CBP, P300 or TIF2. All these genes encode generation of definitive haematopoietic from haemogenic enzymes containing a histone acetyl transferase domain endothelium during the formation of blast colonies. RUNX1 (HAT) suggesting that aberrant modification of histones or is likely to regulate the expression of an important set of other factors could provide the first step in the route to genes at this stage of development. To identify these genes, oncogenicity. We specifically addressed the role of the HAT we compared the patterns of gene expression of activity of MOZ during haematopoiesis by generating a haemangioblast-enriched-cell-populations or haemangioblast- mouse strain that carries a single amino acid change in the derived-cell-populations from either RUNX1 deficient or HAT domain of MOZ. Analysis of these mice has revealed a RUNX1 competent ES cells. We further validated the profound defect in haematopoiesis. The numbers of differential expression of candidates on samples generated haematopoietic stem cells and their potential is dramatically from the ES/EB system and further documented the affected in homozygous mice. These in vivo results were regulation by RUNX1 of the transcription of several of these confirmed with ES cells mutated for the HAT activity of MOZ genes by promoter assays or chromatin immunoprecipitation. as again less haematopoietic precursors are generated with We are currently evaluating the specific function of some of the mutated ES cells. Altogether these results demonstrate these genes at the onset of haematopoietic development and the critical role of MOZ driven acetylation in the balance testing their potential to rescue haematopoietic development between proliferation and differentiation during in absence of RUNX1. We have selected previously haematopoiesis. We are currently investigating the precise uncharacterized transcriptional target genes of RUNX1 and molecular and cellular mechanisms affected in absence of the have initiated a series of experiments, such as conditional HAT activity of MOZ. knock-out and knock-in, to determine the pattern of Publications listed on page 60 expression and function of these new genes. Expression and Function of RUNx1/AML1 isoforms RUNX1/AML1 is expressed as multiple naturally occurring spliced isoforms that generate proteins with distinct activities on target promoters. We have generated ES cells containing a reporter gene knock-in in the different isoforms and produced knock-outs altering the specific expression of these isoforms. We have demonstrated that the expression of these isoforms is differentially regulated during early haematopoietic development both in vitro and in vivo and that their expression defines specific stages of haematopoietic development. We are investigating the biological potential of cells expressing the respective isoforms and the function of each isoform. Figure 1. May Grumwald/Giemsa staining of haematopoietic precursors generated upon in vitro differentiation of mouse ES cells. RUNx1 and chromatin remodelling We have examined in collaboration with the group of Constanze Bonifer (Institute for Molecular Medicine, Scl / Tal1 Runx1 / AML1 University of Leeds) the molecular mechanisms leading to the expression of the transcription factor PU.1 and its target Colony-Stimulating-Factor 1 Receptor gene (CSF1R) at the Haemangioblast Haemogenic Blast Colony onset of haematopoietic development. Our results indicate (BL-CFC) Endothelium Haematopoietic precursors that chromatin remodeling at the Pu.1 locus is initiated by Figure 2. Model of haematopoietic development. transient binding of RUNX1 at an earlier developmental Research Reports 29

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    Paterson Institute for Cancer Research Stem Cell and Haematopoiesis Group http://www.paterson.man.ac.uk/sch Group Leader Valerie Kouskoff Postdoctoral Fellows Scientific Officer Graduate Students Arnaud Gandillet Stella Pearson Katalin Boros Alicia Gonzalez-Serrano Guilherme Costa Sarah Lewis The emergence of haematopoietic cells expression by multi-colour flow-cytometry and the quantitative analysis of biological potential using clonogenic during embryonic life occurs soon after replating assays. The first known progenitor specified to the gastrulation. A tight coordination of blood program is the haemangioblast. This precursor is characterized by its expression of Flk1, the VEGF (vascular proliferation, differentiation and migration endothelial growth factor) receptor 2, and its ability to during the generation of these first blood generate both primitive and definitive haematopoietic cells ensures the proper survival and precursors as well as endothelium and smooth muscle lineages. The hemangioblast, in presence of VEGF, give rise to growth of the developing embryo. fully committed blood precursors, characterized by their Understanding the molecular mechanisms expression of CD41, the alpha2b integrin chain. Using that control the formation of these blood microarray expression profiling of subpopulations at various stages of differentiation, we have identified novel cell surface precursors from the mesodermal germ markers and transcription factors implicated in the layer is the major focus of our laboratory. specification of blood precursors from the mesodermal germ layer. Several lines of evidence suggest that during adult life leukemogenesis can result Multi-parameter analysis of blood specification. from the re-initiation of an embryonic In our effort to define novel cell surface markers expressed during the specification of the haematopoietic lineages, we program or from the inappropriate identified CD40 and Icam2 as two molecules substantially up- expression of genes controlling critical regulated upon haemangioblast commitment. Both genes code for proteins expressed on the cell surface of adult steps of this embryonic program. A clear leukocytes and are actively implicated in immunological understanding of the molecular responses. To define a possible function for these two mechanisms orchestrating the onset of molecules at the onset of blood formation, we first investigated their pattern of expression during the haematopoietic specification should help differentiation of ES cells to blood progenitors. Interestingly, us to better define the basis of de- we observed a progressive and sequential up-regulation of regulated proliferation and differentiation these two cell surface molecules during the maturation of mesodermal precursors to fully committed blood cells. observed in haematological malignancies. Figure 1 illustrates the dramatic shift in CD40 and Icam2 A model system to study haematopoietic specification. profile observed between day 3 and 4 of ES cell The in vitro differentiation of Embryonic Stem (ES) cells offers differentiation. Detailed studies allowed us to establish that a powerful approach to advance our understanding of many low levels of CD40 expression specifically define the Flk1+ developmental processes. Murine ES cells can be induced to haemangioblast subpopulation. As these precursors differentiate and generate primitive and definitive differentiated to generate blood-restricted precursors Icam2 haematopoietic precursors, an in vitro process that accurately expression is switched on while CD40 expression becomes recapitulates the in vivo development of yolk sac significantly higher. In multi-parameter analysis, the integration haematopoiesis. This progressive differentiation, leading to of CD40 and Icam2 expression pattern into previously the formation of fully mature blood cells, can be monitored defined pathway of blood differentiation allowed us to by the measurement of gene expression via real-time further refine and identify discrete steps during the polymerase chain reaction (PCR), the analysis of cell surface specification of mesoderm into fully restricted blood cells. 30 Paterson Institute for Cancer Research Scientific Report 2008

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    Control of blood precursor differentiation by Sox genes: colonies blast-like in appearance while the number of mature The microarray profiling analyses also led us to investigate the myeloid or erythroid colonies was strongly decreased. possible function of Sox transcription factors in the overall Further analysis revealed that enforced expression of either orchestration of blood cell development. Sox genes belong gene promoted a dramatic increase in cell proliferation to the HMG (High Mobility Group) superfamily; they are coupled with an arrest in differentiation toward mature blood highly conserved throughout evolution and implicated in the cells. Doxycycline removal led to the down-regulation of regulation of many developmental processes. Sox genes are Sox7/18 expression, resulting in a progressive reduction of subdivided into 7 groups according to their respective degree cell proliferation and promoting the maturation toward all of homology in both their HMG boxes and trans-activation myeloid and erythroid lineages. Insight into the molecular domains. Sox7, 17 and 18 are all members of the F group program sustained or initiated by Sox7 and Sox18 revealed and became the focus of our investigation. Our expression the activation of the canonical Wnt pathway (figure2). We analysis revealed the sharp up-regulation of Sox7 and Sox18 are now further dissecting this molecular program promoting expression at the onset of haematopoietic development. the self-renewal of early haematopoietic precursors. However, upon further differentiation to generate fully Interestingly, the outcome of the mis-regulation of these two committed blood cells and endothelium, the expression of Sox genes is highly reminiscent of the leukemogenesis both Sox7 and Sox18 was down-regulated. To address the process in which immature blood progenitors loose their significance of this transient expression, we assessed the effect differentiation potential while acquiring an uncontrolled of sustained Sox7 or Sox18 expression during capacity to proliferate. haematopoietic differentiation using a doxycycline inducible ES cell system. Enforced expression of either gene using a Publications listed on page 60 progenitor replating assay resulted in the generation of Figure 1. Profiles depicting CD40 and Icam2 expression in differentiated Embryonic Stem cells. Embryonic stem cells were induced to differentiate and analyzed at day 3, 3.5 and 4 of the differentiation time course for their relative expression of CD40 and Icam2 by flow- cytometry. Figure 2. Immunofluorescence detection for intra- cellular β-catenin in haematopoietic precursors expressing Sox7. Purified haematopoietic precursors were cultured in the presence of doxycycline to induce Sox7 expression and analyzed by immuno- fluorescence for β-catenin (in green) and α-tubulin (in red). DAPI (in blue) marks the nucleus (Magnification 100x). Research Reports 31

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    Paterson Institute for Cancer Research Stromal-Tumour Interaction Group http://www.paterson.man.ac.uk/stromal Group Leader Akira Orimo Postdoctoral Fellow Scientific Officer Graduate Student Yasushi Kojima Kieran Mellody Ahmet Acar Human tumours are highly complex microenvironment. The significant contribution of stroma to the development of a wide variety of tumours has been tissues and the non-neoplastic cell supported by extensive clinical evidence; this contribution is compartment of tumours, which is often highlighted by the higher incidence of tumour formation in tissues exhibiting a chronically inflamed stroma as well as termed the “stroma”, is itself quite those undergoing various types of wound healing, in which the stroma plays a central role. Use of mouse models of complex histologically. Carcinoma cells tumorigenesis also reveals that stromal cells, notably initially recruit and/or activate these inflammatory cells, vascular cells, and fibroblasts, actively support tumour growth. various stromal non-neoplastic cells, including fibroblasts, myofibroblasts, Large numbers of myofibroblasts, which are characterized by their production of α-smooth muscle actin (α-SMA), have immune cells, endothelial cells, bone been observed repeatedly in the stroma of the majority of marrow-derived cells, etc. The resulting invasive human breast cancers. However, the specific contributions of these cells to tumour progression are poorly stromal cells reciprocate by fostering defined. Myofibroblasts also exist in areas of wound healing carcinoma cell growth and survival during and chronic inflammation and are often portrayed as “activated fibroblasts” that play crucial roles in wound repair; the course of tumour progression. myofibroblasts possess greatly increased contractile ability, Studying the heterotypic interactions promote angiogenesis, and stimulate epithelial cell growth through the production of ECM and the secretion of growth between the neoplastic cells and the factor and cytokines. The striking histological resemblance of supporting stroma is believed to be tumour stroma and the stroma present in sites of wound healing, both containing large numbers of myofibroblasts, essential for understanding nature of a raises the following questions: are myofibroblasts present in tumour biologically equivalent to those observed in wound bulk of carcinoma mass. During 2008, we healing or, alternatively, do tumour-associated myofibroblasts studied 1) how tumour-associated stroma acquire “cancer-specific alterations” that distinguish them from those present in wounds? Such questions still remained to be becomes altered and co-evolves with investigated. tumour cells during the course of tumour Stromal fibroblasts, termed carcinoma-associated fibroblasts progression 2) how the stroma facilitates (CAFs), were extracted from human carcinomas and these progression of tumour and 3) what cells include collectively both fibroblastic and myofibroblastic cell populations. CAFs are known to substantially promote specific stroma-derived signal is crucial in growth of nearby carcinoma cells co-injected into promoting tumour invasion and immunodeficient mice. This striking tumour-promoting property was indeed observed in CAFs extracted from metastasis. various different types of human carcinomas. Independent of those, we demonstrated that fibroblasts present in the Tumour-promoting roles of carcinoma-associated invasive human mammary carcinoma mass are biologically fibroblasts (CAFs) very different from their counterparts located outside Neoplastic epithelial cells coexist in carcinomas with a tumour masses and from mammary stromal fibroblasts biologically complex stroma composed of various types of prepared from reduction mammoplasties in several mesenchymal cells as well as extracellular matrix (ECM), both important functional respects; of which create the complexity of the tumour (i) CAFs extracted from invasive human breast carcinomas 32 Paterson Institute for Cancer Research Scientific Report 2008

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    report also suggests that stromal fibroblasts that have undergone p53 loss are clonally selected during tumour progression, yielding a highly proliferative stroma. However, another report indicates that myofibroblasts isolated from human mammary breast carcinomas exhibit no detectable genetic alterations, as gauged by array CGH and SNP array analyses; this suggests that any stably maintained phenotype may depend on epigenetic modifications of the genome, such as DNA methylation. Alternatively, the stabilization of their phenotype may depend on some type of positive-feedback signalling of the sort CAFs stimulate tumour angiogenesis created by autocrine signalling loops. Sections from MCF-7-ras human breast tumours containing various fibroblasts were stained by anti-CD31 antibody (d, e, and f) or by Masson’s trichrome (a, b, and c). We note that our CAFs show no detectable aneuploidy as Scale bar, 100 μm. (Orimo et al., Cell 2005; 121:335). determined by karyotype analysis, no anchorage-independent growth in culture, and no tumorigenicity in vivo. Moreover, are more competent than normal fibroblasts in enhancing some of the CAFs begin to senesce after 15 PDs in culture, tumour growth by comingled breast cancer cells. similar to the behaviour of normal human stromal fibroblasts. (ii) CAFs include larger populations of myofibroblasts, which exhibit high levels of α-SMA expression and increased We speculate roles of the epi/genetic alteration(s) in collagen contractility. regulating behaviour of CAFs as the following: (iii) When comingled with a line of human breast cancer cells, 1) the alterations acquired during the course of tumour CAFs give rise to highly vascularized tumours in contrast to progression must be a rare event that provokes stable the poorly vascularized tumours generated by admixed reprogramming in the resulting fibroblasts in tumours. Once normal stromal fibroblasts (see figure). established, they help initiate and maintain CAFs phenotypes (iv) CAFs release increased levels of SDF-1 (stromal cell- in a stable fashion. derived factor-1) which is responsible for recruiting 2) they can be, however, not eligible to transform these endothelial progenitor cells (EPCs) into a tumour mass, fibroblasts into tumourigenic cells. thereby boosting tumour angiogenesis. 3) only particular fractions of CAFs are present as bona fide In addition, the SDF-1 secreted from CAFs enhances tumour cells that acquired the stable alterations in tumours, since growth by direct paracrine stimulation via the CXCR4 CAFs are basically composed of heterogenous cell receptor displayed by human breast carcinoma cells, thereby populations by constantly recruiting into tumour-associated revealing a second role for stromal SDF-1 in promoting stroma locally preexisting mesenchymal cells and/or bone tumour progression in vivo. marrow-derived hematopoietic cells. Just after the (v) Both the tumour-enhancing and myofibroblastic recruitment, the latter would be converted into tumour- properties of CAFs are stably retained by these cells in the supporting CAFs in the absence of acquisition of the stable absence of ongoing contact with breast carcinoma cells. alterations. 4) these CAFs thus depend largely on ongoing signaling from Evolution of tumour stromal fibroblasts in tumours nearby carcinoma cells in supporting the carcinoma growth, CAFs retain their myofibroblastic properties and tumour- while stably altered CAFs depend on the alterations that promoting phenotypes, even after they have been passaged regulate them in a cell-autonomous fashion. This thought for ten population doublings (PDs) in vitro without ongoing together with the description above reflects the notion that contact with carcinoma cells. Accordingly, even though the the stable alterations may not encourage the resulting CAFs appear to have initially acquired a myofibroblastic fibroblasts to clonally expand. phenotype under the influence of carcinoma cells, once it is 5) out-growth of the CAFs would be also under the control acquired, they display this trait in the absence of further by carcinoma cells; this schema enables the latter to signalling from the carcinoma cells. Unanswered by these orchestrate virtually all mesenchymal cells in tumours, observations are (i) how do CAFs acquire and maintain their facilitating to develop eventually full-blown tumour. activated, tumour-enhancing phenotypes? (ii) might CAFs harbour genetic and/or epigenetic alterations that act to Studying cross-talk between tumour cells and mesenchymal confer their unique phenotypes? cells during tumour progression could help understand nature of biology of human carcinomas and facilitate to Some reports indicate that stromal regions microdissected develop novel stroma-targeted therapeutic approaches. from human breast cancers exhibit a high frequency of genetic alterations, such as chromosomal regions of loss of heterozygosity (LOH) and somatic mutations. A recent Research Reports 33

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    Paterson Institute for Cancer Research Academic Radiation Oncology: Translational Radiobiology Group Group Leader Catharine M.L. West Postdoctoral Fellows Clinical Fellows Graduate Student John Hall Guy Betts Stephanie Donaldson (from September 2008 Catriona Douglas (joint with Imaging Science) joint with Applied Karim Sillah Computational Biology & (until October 2008) Undergraduate Students Bioinformatics) Lisa Joseph (to August 2008) Carla Möller-Levet Scientific Officers (joint with Applied Joely Irlam-Jones Scientific Administrator Computational Biology & Helen Valentine Rebecca Elliott Bioinformatics) Research Nurse Kathryn Fellows The Translational Radiobiology Group aims tumour oxygenation, one of which involves measuring the level of tumour expression of hypoxia-related proteins. to investigate approaches for predicting Carbonic anhydrase 9 (Ca9) is a transmembrane glycoprotein how patients respond to radiation and which is expressed in some types of normal tissue, such as exploit developments in high throughput duodenal, jejunal, hepatic and pancreatic tissue. The protein is widely expressed in some tumours including head and neck technologies to carry out translational squamous cell carcinoma. It is a hypoxia-inducible protein research in clinical trials. The radiobiologist and tumour expression has been linked with oxygenation status. We were the first group to associate tumour Hal Gray highlighted the importance of expression with a poor prognosis following radiotherapy in tumour hypoxia as a potential factor cervix tumours. Subsequent work by the group has also limiting the success of radiotherapy in the shown a relationship between high expression in a homogeneous group of oropharyngeal cancers and poor 1950s. His observations led to hypoxia outcome following radiotherapy. dominating radiotherapy-related research Work over the past year investigated expression of Ca9 in a for many years. Therefore, hypoxia – now large series of larynx cancers (Catriona Douglas & Helen widely recognised as a key factor driving Valentine in collaboration with Mr Jarrod Homer and Dr Nick cancer development, progression and Slevin). From a database of 423 patients who underwent potentially curative radiotherapy, scores for tumour CA9 treatment resistance - is a large focus for expression were available for 310, a considerably larger research by the group. number of patients than any series reported in the literature. High (≥10%; n=109) vs low (<10%; n=201) expression was associated with a worse locoregional recurrence-free (P= Hypoxia in head and neck cancer 0.032; figure 1) and cancer specific (P=0.040) survival on Each year about 650,000 people are diagnosed with cancer univariate analysis. Associations of high expression with a of the head and neck worldwide and 350,000 people will die poor prognosis were retained on multivariate analysis for from the disease. Over the last 20 years, the overall 5-year both local control (HR=2.16, 95% CI=1.07−4.34; p=0.031) survival rate has remained at ~50% despite significant and cancer specific survival (HR=2.53; 95% CI=1.01-6.34; advances in surgery and oncology practice. There is a need p=0.048). This is an important finding clinically because to increase understanding of biological factors associated with surgery is an option for treating the disease and is associated a poor prognosis and to improve the individualisation of with a similar outcome as radiotherapy. Radiotherapy is treatment in order to increase survival. There is considerable generally preferred because it results in better preservation evidence that high levels of tumour hypoxia are associated of organ function, i.e. voice, but if local recurrence occurs with a poor prognosis in patients with head and neck cancer. following treatment a total laryngectomy is usually carried Various approaches are being studied to assess the level of out. 34 Paterson Institute for Cancer Research Scientific Report 2008

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    Development of a hypoxia-associated gene signature in head and neck cancer With our collaborators in Oxford (Prof Adrian Harris, Dr Francesca Buffa) and Applied Computational Biology & Bioinformatics (Dr Crispin Miller) we have continued work on our hypoxia-associated gene expression signature (Guy Betts & Carla Möller-Levet). Over the past year the signature has been streamlined and reduced from 99 to 26 genes, and a comparison made of Exon and Affymetrix U133plus2 GeneChip data. With the recent award of an MRC Biomarker grant, the group is looking forward to validating and qualifying the signature using a PCR approach in multiple clinical datasets. RAPPER and VORTEx-BIOBANK The Translational Radiobiology group co-ordinates the biobanking associated with several national radiotherapy trials. RAPPER (Radiogenomics: Assessment of Polymorphisms for Predicting the Effects of Radiotherapy) is collecting samples from a number of national trials. The CR- UK funded project aims to explore associations between genetic variation expressed as single nucleotide polymorphisms and radiation toxicity. This year we exceeded our planned recruitment of 2,200 patients with breast, prostate, gynaecological or rectal cancer (figure 2). Day-to- Figure 1. Kaplan-Meier survival curve showing the locoregional recurrence-free survival of day administration is carried out by Rebecca Elliott and 310 patients with squamous cell carcinoma of the larynx who underwent radiotherapy. Patients are stratified according to high (≥10% positive tumour cells) vs low tumour Ca9 Kathryn Fellows recruited patients at the Christie Hospital. expression. Examples of tumours with low and high expression of Ca9 are also shown. Collaborators include Drs Neil Burnet & Alison Dunning (Cambridge), Prof Søren Bentzen (Wisconsin) and numerous clinical oncologists locally and nationally. Sample collection will continue from national radiotherapy trials for a second validation phase of genotyping. The first sample for VORTEX- BIOBANK (Joely Irlam-Jones) was received in 2007 and we have now banked 36 matched fresh tumour and normal tissue samples and 32 blood samples. Publications listed on page 60 Figure 2. RAPPER study target and cumulative accrual. Research Reports 35

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    Paterson Institute for Cancer Research Biological, Immune and Gene Therapy Group Group Leaders Robert E. Hawkins, Peter L. Stern Senior Clinical Research Clinical Research Fellows Undergraduate Student Fellow Christy Ralph Sameena Khan Fiona Thistlethwaite Sai Daayana (with HC Kitchener) Research Nurse Senior Fellow Alaaeldin Shablak Andrea Byatt David Gilham Saladin Sawan (with Catherine Holland) Administrators Postdoctoral Fellows Nicola Hudson Eyad Elkord Scientific Officers Shira Baram (Clinical Immunotherapy) Debbie Burt (seconded Dominic Rothwell from Immunology) (Molecular Monitoring) Hayley Batha Ryan Guest (at NBS) both trials patients receive conditioning chemotherapy prior In September, the Cancer Immunotherapy to an autologous infusion of CIR T cells and the patients Laboratory in TRF2 opened with its design subsequently receive intravenous IL2 to support the in vivo survival and expansion of the CIR T-cells. The first trial targets including the capacity for GCLP evaluation. CEA in patients with solid tumours that have been shown to This demands the establishment of a express CEA. Recruitment to the first cohort of patients in this trial is now complete and well underway for the second range of validated immune and molecular cohort. More recently a second trial targeting CD19 in NHL based assays for monitoring clinical trials has opened and a number of patients have been recruited to the first cohort. In both trials, prior to use for treatment, the and is also linked to the development of a autologous expanded CIR T cells are extensively phenotyped GMP cell processing facility. Dr Eyad by flow cytometry to allow for correlation with any subsequent engraftment. A validated assay based on Elkord is leading on the cellular quantitative PCR allows the detection of the level of transduced cells in each patient. The first results indicate that immunology component and Dr Dominic following transfusion of the expanded CIR T cells, their levels Rothwell on molecular aspects. The first initially drop but a transient return can been seen following the end of IL-2 treatment; one of three patients showed UK trials of gene modified cell therapy persistence of transduced T cells beyond 6-weeks post have now opened at The Christie, infusion. Ongoing secondary and scientific assays include molecular monitoring of clonal populations with T cell targeting carcinoembryonic antigen (CEA) spectratyping and the assessment of retroviral integration via in CEA-expressing solid tumours and LAM-PCR. CD19 in Non-Hodgkin lymphoma (NHL) An MVA-based vaccine targeting the oncofoetal antigen patients. Studies are continuing on human 5T4 in patients undergoing surgical resection of colorectal cancer liver metastases immune responses to 5T4 antigen We investigated the use of a therapeutic vaccine, TroVax in providing the platform for further patients undergoing surgical resection of colorectal cancer liver metastases. Systemic immunity generated by vaccination improvements in vaccine and antibody before and after resection of metastases was measured in delivered therapies for cancer. addition to assessing safety and analyzing the function and phenotype of tumour-associated lymphocytes. Twenty patients were scheduled to receive two TroVax vaccinations Chimeric immune receptor (CIR) T-cell trials at 2-week intervals pre-operatively and two post-operatively; Tumour antigen specific CIRs enable T cells to target and if immune responses were detected two further vaccinations specifically destroy tumour cells in a MHC-independent were offered. Blood was taken at trial entry and two weeks manner. Two first-in-man phase I clinical trials of CIR T-cell after each vaccination; tumour biopsies were collected at trials are now open and recruiting patients at The Christie. In 36 Paterson Institute for Cancer Research Scientific Report 2008

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    surgery. 5T4-specific cellular responses were assessed by after five cycles. Four patients had stable disease on CT scan; lymphocyte proliferation and ELISPOT, with antibody two had clinically beneficial tumour shrinkage; one has an responses by ELISA. Immunohistochemistry characterized ongoing response with marked reduction of tumour markers. the phenotype of tumour infiltrating lymphocytes. Seventeen Changes to lymphocyte phenotype (measured by flow of 19 colorectal cancer patients showed 5T4 expression in cytometry) and lymphocyte proliferative responses to 5T4 the liver metastases or surrounding stroma and 18 mounted tumour antigen with CTLA4 blockade were assessed. By day a 5T4-specific cellular and/or humoral response. In patients 15, parallel rises in expression of FoxP3 (Treg marker), who received at least four vaccinations and potentially CTLA4 and another immunomodulatory target, PD1, were curative surgery (n=15), those with above median 5T4- observed. By day 60 most of these rises have returned to specific proliferative responses or T cell infiltration into the near baseline levels, but increased CTLA4 expression in resected tumour showed significantly longer survival CD4+CD25low cells was sustained. These complex patterns compared to those with below median responses (figure). of expression are being further explored functionally. Seven of 8 patients that had pre-existing proliferative Importantly, enhanced proliferative T cell responses to specific responses to 5T4 were longer term survivors; these patients 5T4 peptide pools were seen in 7 of 13 patients. This is showed significantly higher proliferative responses following consistent with anti-CTLA4 releasing potentially useful anti- vaccination than those who subsequently died. These data tumour immunity. Responses to other tumour antigens such suggest that the magnitude of 5T4 proliferative responses and as CEA are in progress. Analysis of the cellular mechanisms the density of CD3 cells in colorectal cancer liver metastases of anti-tumour effect of anti-CTLA-4 monoclonal antibodies are associated with longer survival. These observations has provided conflicting results in cancer patients. We are warrant more studies to identify the precise underlying currently investigating whether the antitumor effect of CTLA- mechanisms. 4 blockade is due to increased T cell activation or inhibition of Treg activity. Immune regulation and cancer therapy The immune system can recognise cancer, but immune driven 5T4 antibody targeted superantigen therapy cure is rare. Cytotoxic T lymphocyte-associated antigen 4 We are leading a Phase III trial of targeted superantigen (CTLA4), a crucial inhibitor of T cell activation, has a role in therapy based on our previous clinical/translational studies. regulating host immune responses against cancer. It is Extended immune analyses are addressing several questions. expressed by natural regulatory T cells (Treg) and by other T What is the level and activity of superantigen specific T cell in cells on activation. CTLA4 blockade can induce immune the treated patients? This involves direct detection of the driven cure in some animal models, and has shown promising patients’ drug reactive T cells by flow cytometry and results in phase I and II clinical trials in melanoma patients. superantigen antibody dependent cell-mediated cytotoxicity Second-line chemotherapy in advanced oesophageal and of tumour cells. Is there any bystander effect on 5T4 specific gastric cancers has poor response rates which are rarely immunity? Such 5T4 specific immunity is monitored using sustained and high levels of toxicity; new treatment strategies 5T4 peptide lymphocyte proliferation or by specific ELISA for are needed. Thus, 18 adult patients with metastatic antibodies. Ultimately the goal is to discover whether the oesophageal or gastric adenocarcinomas received CP- active immunity correlates with clinical outcome? 675,206 (Pfizer), a human monoclonal antibody against CTLA4 in a Phase II clinical trial. Twelve received a single Publications listed on page 61 cycle, five two cycles, and one patient remains on treatment Kaplan-Meier overall survival curves for patients stratified according to below or above median anti-5T4 immune responses and CD3+ T cell infiltration into the tumor. The curves show 5T4 antibody responses (A), 5T4 proliferative responses (B), com- bined 5T4 proliferative and antibody responses (C), CD3+ T cell infiltration into the tumor (D), and combined 5T4 proliferative responses, 5T4 anti- body responses and CD3+ T cell infil- tration (E) (Elkord et al., 2008b). Research Reports 37

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    Paterson Institute for Cancer Research Children’s Cancer Group Group Leader Vaskar Saha Postdoctoral Fellows Clinical Trials Manager Administrators Clare Dempsey Catriona Parker Neelofer Charfare Mark Holland Christine Connor Jizhong Liu Scientific Officers Seema Alexander Clinical Research Fellow Naina Patel Shekhar Krishnan Ashish Masurekar The CR-UK Children’s Cancer Group show the same minimal residual disease patterns. Moreover, the efficacy of Mitoxantrone appears to be related to a low moved to the Paterson at the end of 2006 relapse rate post transplant. This suggests that Mitoxantrone from Queen Mary University London. may be more effective in eradicating cellular niches where ALL cells may escape therapeutic annihilation (see below). Initially located within the Kay Kendall This is the first ever trial in relapsed childhood ALL to pro- vide an answer to a randomised question. Overall the trial laboratory in the Institute, we have now has produced some of the best results ever achieved in this relocated to dedicated space on the 2nd cohort of children. Last year we also completed the first Pan-European phase II trial in childhood ALL with the drug floor. Our research focuses on Clofarabine. As the drug showed a beneficial effect, the high understanding the biological basis for the risk arm of ALLR3 is now being amended to incorporate Clofarabine. This will open in early 2009 and form the basis differences in the therapeutic response in of a Pan-European trial EuReALL in 2011 at which time we children with Acute Lymphoblastic will close ALLR3. We also coordinate in the UK, on behalf of a European interstudy group, a clinical trial of Imatinib in chil- Leukaemia (ALL). The group conducts dren with Philadelphia positive ALL in the UK. This trial is also international clinical trials, and obtains expected to run till 2011. clinical material nationwide from children with ALL undergoing therapy. Clinical material is processed and stored at the Centre for Integrated Genomic Research at the University of Manchester. This material, linked to clinical data, provides the basis for our laboratory research. Clinical Trials In 2008, Carly Leighton left and Catriona Parker joined the team as Clinical Trials Manager. Our flagship clinical trial, ALLR3, is for children with relapsed ALL. In that, we had asked a randomised question of the efficacy of Mitoxantrone (test drug) over Idarubicin. The randomisation was stopped in January of this year, earlier than planned, as the test drug Figure 1. Overall survival of children, treated on the ALL R3 protocol for relapsed Mitoxantrone proved to be superior to the standard drug ALL by randomised drug. (figure 1). Curiously, the differences in outcome do not cor- relate to the kinetics of disease clearance, as both drugs 38 Paterson Institute for Cancer Research Scientific Report 2008

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    Laboratory with Peter Stern’s group to develop an animal model to vali- Naina Patel with the help of Shekhar Krishnan has identified date this observation. Seema Alexander has developed a novel mechanism of resistance to the key anti-leukaemic lentiviral strategies to investigate the relationship of lysosomal drug Asparaginase. The lysosomal cysteine proteases CTSB proteases, such as AEP, in the invasion process. Her initial and AEP both cleave and inactivate the drug. Naina has experiments suggest that though AEP expressing cell lines identified the cleavage sequence and shown that mutation of are invasive, AEP per se is not responsible for this phenome- the first cleavage site is sufficient to protect the drug. non. Clare Dempsey is working with a leukaemia-associated Shekhar has shown that active AEP appears to be localised in fusion transcript known to be associated with endosomal a distinct lysosomal compartment found at the periphery of trafficking. She has transduced cell lines and haematopoietic the cell (figure 2). Working with Paul Bates’ group at the stem cells with a lentivirus expressing CALM-AFIO and will London Research Institute, Naina is continuing to modify now examine its leukaemogenic potential and its effect on Asparaginase to make it less degradable, less toxic and more cargo trafficking. As endocytosis and signalling are dependent effective. This work has led to a successful grant application on the microenvironment, Jizhong Liu is investigating the from the Leukaemia Research Fund to correlate the expres- interactions between normal and malignant haematopoietic sion of these proteases with clinical response to therapy. cells and mesenchymal tissue. He has established a 3D bone This is the first biomarker study to be carried out in children marrow culture system to study these interactions. His with ALL in the UK and will be one of the largest of its kind investigations suggest that mesenchymal cells protect ever carried out. This work is being carried out by Ashish haematopoietic stem cells (HSCs) from toxic stimuli. It is Masurekar and Jizhong Liu and is co-ordinated by Catriona likely that a subpopulation of ALL cells is able to mimic HSCs Parker. and find refuge in mesenchymal niches. As discussed earlier, this may be the explanation of the differential effect of Our work has uncovered the presence of large lysosomes in Mitoxantrone in ALLR3. We hope that the 3D model that ALL cells. There is evidence from other cancers that lysoso- Jizhong has developed will help us answer this question. mal proteases participate in mechanisms that promote cell survival. These include processes such as cell migration inva- Overall, as a translational research group, we continue to sion and autophagy. Mark Holland has identified ALL cell conduct hypothesis-driven laboratory research, based on lines that invade across matrigel and endothelial barriers. He observations made on patients undergoing clinical trials. has taken a global proteomic approach to investigate the Finally, we are pleased to report that both Frederik van Delft changes in the plasma membrane proteome of the invading and Shai Senderovich successfully defended their doctoral cells. With the help of Professor Anthony Whetton and dissertations in 2008. Duncan Smith, using SILAC and iTRAQ, Mark has identified an actin-related signature of invasion. He is now working Publications listed on page 62 Figure 2. Mature active AEP in SD1 cells accumulates towards the cell periphery in discrete acidic LAMP1-positive compartments. A) Immunostaining localises AEP in peripheral acidic compartments labelled by an acidotropic lysosomal probe. AEP immunostaining in formalin-fixed cytospins of SD1 cells pre-incubated with the cell-permeable LysoTracker Red DND- 99 lysosomal probe (LyTr). The large yellow punctate fluorescence in the images represents overlay of green (AEP) and red (lysosomal probe) fluorescence (Magnification ×100, Optivar 1.6). B) Live cell labelling using a fluorescent cell- permeable activity-based probe (AEP-ABP) localises active AEP in peripheral LAMP1- positive vesicles. LAMP-1 immunostaining in formalin-fixed cytospins of SD1 cells pre- incubated with a fluorescent cell-permeable AEP activity-based probe (AEP-ABP). Images indicate active AEP (red) at the cell periphery, localised in what appear to be large globular vesicles ringed by LAMP-1 (green) (Magnification ×100, Optivar 1.6). DAPI (blue) stains the nucleus, Trans refers to brightfield images. Research Reports 39

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    Paterson Institute for Cancer Research Targeted Therapy Group Group Leader Tim Illidge Preclinical Group Graduate Students Research nurses Postdoctoral Fellows Waleed Alduaij Caroline Hamer James Hainsworth Monique Melis Susan Neeson Jamie Honeychurch Andrei Ivanov Clinical Radioimmunotherapy Ruth Swann Group Senior Clinical Scientist Maureen Zivanovic Clinical Fellow Clinical scientist Nick Brown (dosimetry) Jill Tipping The goal of the Targeted Therapy Group is In contrast type II mAb such as tositumomab (B1) are generally potent at inducing cell death in target cells. We to define the optimal way to combine observed increased tumour cell death with tositumomab and radiotherapy (RT) with immunotherapy in RT, which was not seen with rituximab and RT. This increased tumour cytotoxicity was reversed with the MEK inhibitors the treatment of cancer by enhancing our (U0126, PD98059) as well as siRNA targeting MEK1 or understanding of the underlying MEK2. Furthermore the addition of U0126 reversed the loss of clonogenic survival triggered by combining tositumomab mechanisms of action. This will be with RT. Phosphorylated ERK1/2 (pERK) was found to achieved by the specific objectives which accumulate in the nucleus following tositumomab and the nuclear accumulation of pERK was greatly enhanced in are i) to investigate how the recognition of combination with RT. In contrast rituximab caused early ERK phosphorylation but this was not sustained and remained radiotherapy (RT) induced tumour cell within the cytoplasm suggesting that this might underlie the death by different antigen presenting cells lack of additive tumour cell death. In summary our data indicate that activation and nuclear accumulation of pERK in the tumour microenvironment can appear to be required to produce the synergistic effect impact on the ensuing immune response; produced by combining tositumomab and RT (Ivanov et al., 2008). ii) to investigate the role of myeloid derived suppressor cells (MDSC) in Recently, in collaboration with Dr Mark Cragg’s group in Southampton we have investigated a new form of mAb tumour regrowth after RT and to develop induced cell death in B-cell lymphomas. Using both strategies to enhance RT tumour control lymphoma cell lines and primary chronic lymphocytic leukemia (CLL) cells, we have demonstrated for the first time by modifying host immune response after the importance of lysosome-mediated cell death for antibody RT and iii) to translate our experimental therapy elicited by clinically relevant mAb directed against two different target antigens namely CD20 and HLA DR research findings into developing early (figure). By virtue of a detailed and kinetic approach we have phase clinical trials determined that death is preceded by homotypic adhesion with both adhesion and death being dependent upon actin redistribution. Malignant B cells, undergoing homotypic Mechanisms of action of radioimmunotherapy (RIT) adhesion, actively communicate via ~ 5 nm wide temporary Our recent work has focused on the molecular mechanisms inter-cytoplasmic bridges. of action of RIT induced tumour cell death in vitro. We have investigated the downstream signalling events in a variety of The formation of these channels is accompanied by the human B cell lymphomas after treatment with RT and anti- mutual exchange of plasma membrane components and CD20 mAb using a number of clinically relevant anti-CD20 importantly the extent of plasma membrane swapping mAb. Anti-CD20 mAb can be broadly sub-divided into correlates with the extent of cell death induced by both anti- either type I (eg. rituximab) or type II (eg. tositumomab). CD20 and anti-HLA DR mAb. To our knowledge there are Rituximab and other Type I anti-CD20 mAb engage no known precedents for these phenomena recorded for complement effectively and cause target cell lysis these cell types. The fact that similar findings have been 40 Paterson Institute for Cancer Research Scientific Report 2008

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    recorded after treatment with functionally different mAb accurately determine serum rituximab concentrations has direct to CD20 and HLA DR antigens suggests that the been established as a national reference laboratory resource. phenomena observed may be the general mechanism of The first study to use the validated serum rituximab assay is lymphoma cell killing by activating antibodies. The latter is the NCRI Phase III study which compares the policy of potentially of great interest as it provides a potential means “Watch and Wait” versus rituximab (Grace Hampson is to bypass the often dysregulated apoptotic death pathways of funded by a 2008 CR-UK TRICC grant awarded to tumour cells allowing for effective tumour cell killing in the Illidge/Dive). We plan to apply this assay to more national presence of apoptotic inhibition. Interestingly both of the clinical studies in the forthcoming year. mAbs studied evoke lysosomal non-apoptotic cell death pathway and this is likely to go some way to explain their Early Phase Clinical Trials of Radioimmunotherapy efficacy in vivo. This experimental work has been selected for The clinical RIT group has made considerable progress over an oral presentation at ASH 2008 and has been submitted to the last few years in leading early phase clinical trial design the Journal of Clinical Investigation after successful pre- both nationally and internationally, with a substantial portfolio submission enquiry. of early phase clinical trials. The highlight of the year was the publication in Blood of the Phase I/II dose escalation RIT Immune response to RT induced dying tumour cells study. This study was the first of its kind to investigate the The work in this research area has focused on enhancing the effect that induction therapy (4 weekly infusions of 375 therapeutic potential of RT by investigating combining RT mg/m2 rituximab) has on the subsequent efficacy and toxicity with immunotherapeutic approaches. Our recent work has of anti-CD20 RIT in relapsed indolent B cell Lymphoma focused on understanding the nature of the host immune (Illidge et al., 2008). Induction therapy with rituximab was response to RT induced tumour cell death found to significantly increase the effective half-life of 131I- rituximab and higher serum levels of rituximab at week 6, Over the last few years we have successfully developed a after rituximab induction therapy, correlated with an number of powerful tools that will in the future programme increased effective half-life of the radioimmunoconjugates facilitate the exploration of the possible roles of subsets of professional antigen presenting cells (APC) in the immune An important observation we made from this study was that response to tumour cell death induced by RT. We have induction therapy with multiple doses of rituximab did not focused our attentions on two types of APC, namely appear to compromise the clinical efficacy or increase toxicity macrophages (MΦ) and Dendritic cells (DC). Recently we of subsequent 131I-rituximab RIT. The overall response rate have demonstrated that by manipulating MΦ within the (ORR) was 94%, with complete response (CR) rate 50%. The tumour microenvironment we can induce protective anti- median time to progression was 20 months, significantly tumour CD8 T-cell responses with anti-CD40 against longer than for the last qualifying chemotherapy with ongoing irradiated lymphoma cells that are in themselves poorly durable remission of more than 60 months. Fractionation of immunogenic. In these studies we have shown the potential 131 I-rituximab allowed cumulative whole body doses of over importance of MΦ in cellular vaccination and have 120 cGy, around 60% greater than those previously achieved demonstrated that depletion of MΦ using clodronate- with a single administration of a murine encapsulated liposomes considerably enhances primary radioimmunconjugate, to be delivered without significant vaccination efficacy in the presence of adjuvant anti-CD40 hematological toxicity. mAb (Honeychurch et al., submitted). Publications listed on page 62 Our results demonstrate that in order to induce a protective Involvement of immune response, additional host immune stimulation is lysosomes in required and that depletion of MΦ populations can improve antibody-induced cell tumour cellular vaccination strategies (Honeychurch et al., death. 1 hour after addition of mAb - 2008). Lysotracker red added. Blue – DAPI Clinical Translational applications of the laboratory research nucleus; Green – programme GFP labeled Actin; There have been a number of major successes in translational Red – Lysotracker. research that have resulted directly from, or are closely related to this CR-UK laboratory programme of work. Anti-idiotype against Rituximab (serum ritxumab assay) The serum rituximab ELISA assay that we previously developed has now been validated to GCLP in collaboration with the Clinical and Experimental Pharmacology Group (CEP). A robust, reliable and reproducible ELISA which can Research Reports 41

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    Paterson Institute for Cancer Research Medical Oncology: Cell Therapy Group Group Leader Robert Hawkins Senior Fellow Jennifer Loconto Graduate Students David Gilham Vivien Watson Grazyna Lipowska-Bhalla Erik Alcantar Orozco Postdoctoral Fellows Scientific Officers Mariam Al-Muftah (with John Bridgeman (from July) Allison Robinson Immunology) Eleanor Cheadle Vicky Sheard Simon Dovedi Hayley Batha (until February) (until October) The Cell Therapy Group has focused upon Mutations in the charged amino-acids reduce the ability of the chimeric receptor to interact with the TCR; consequently, manipulating the immune system of the this significantly reduces the sensitivity of the chimeric patient in order to drive the eradication of receptor function suggesting that the formation of the TCR- chimeric receptor complex is critical for optimal anti-tumour tumours. This manipulation involves the activity of the T cell. However, important questions arise from this observation. Does the chimeric receptor interfere insertion of a gene encoding a tumour with the activity of the normal T cell? Could there be safety targeting receptor into patient’s T-cells issues associated with the long-term formation of the TCR/chimeric receptor complex? To explore these which then can kill tumour cells. This questions, we have developed two model systems (based approach is now in clinical trial in upon our clinical trials) to investigate the potency of chimeric receptors and to assess the long-term safety of gene- Manchester (targeting gastro-intestinal modified T cell therapy. cancer and B-cell lymphoma). Our Genetic Modification of Mouse T-cells current work is based upon using model In order to test chimeric receptor technology in suitable systems to try and understand the key tumour models, we have had to develop and improve protocols to allow high levels of gene transfer into primary factors that may influence this approach in mouse T cells. For reasons which are not entirely the patient with the aim of improving the understood, mouse T cells tend to be more refractory to the viral gene transfer systems which work efficiently in human T treatment. cells. We have optimised retroviral gene transfer into primary mouse T cells and have established culture conditions which The Chimeric Immune Receptor: Molecular Interactions permit the expansion of these T cells to high numbers ex vivo with the T cell Receptor for use in adoptive transfer experiments. We have passed on The gene that is introduced into the patient’s T cells encodes our methods to collaborators who have confirmed the for a protein called a Chimeric Immune Receptor. This robustness of the protocol (Prof. Zelig Eshhar, Weizmann receptor consists of an extracellular domain that can bind to Institute, Israel). Using these improved protocols, we are target proteins present on the cell surface of the target cell currently examining how the culture condition and duration and is fused to the transmembrane and cytoplasmic domain of culture impacts upon T cell persistence and anti-tumour of the CD3ζ receptor; which is one of the components of activity in vivo. the T cell receptor (TCR) complex. Natural CD3ζ proteins bind to the TCR through electrostatic interactions between Models of gastrointestinal cancer and B-cell lymphoma. charged amino-acids present within their transmembrane Our first trial (sponsored by Cancer Research UK) involves domains. We have shown that our chimeric receptor is also generating T cells with specificity for CEA through the incorporated within the TCR complex present on the surface expression of a chimeric immune receptor called MFEz. The of the gene-modified T cell by biochemical and by second (sponsored by the Kay Kendall Leukaemia Fund) Fluorescence Resonance Energy Transfer (FRET, Figure 1). involves targeting B cell lymphoma using T cells armed with a chimeric receptor specific for the CD19 protein (CD19z). In 42 Paterson Institute for Cancer Research Scientific Report 2008

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    order to obtain regulatory approval for these trials, MFE23.CD3z we have generated model systems for both CEA and CD19 which have demonstrated the efficacy of human T cells targeting the relevant tumour types in immuno-compromised mouse models. Alexa 555 (CIR) Alexa 647 (TCR) Raw FRET Merge In order to further understand and to improve the approach, we are using mouse T cells armed with MFE23.htm.CD3z chimeric receptors to target mouse tumours expressing the relevant protein target. In an initial set of experiments, we showed that T cells injected close to the site of tumour proved more much effective at challenging the growth of CEA+ tumours Alexa 555 (CIR) Alexa 647 (TCR) Raw FRET Merge than T cells given systemically. This suggests that Figure. 1 FRET Analysis of TCR-CIR Interactions – Jurkat T cells expressing “TCR-interacting” getting the T cells to the site of the tumour is critical MFE23.CD3ζ and “TCR non-interacting” MFE23.htm.CD3ζ were stained with Alexa-647 conjugated- anti-TCR antibodies and Alexa-555 conjugated CEA protein. Cells were imaged using an Axiovert-Time for the therapy to succeed. However, immuno- lapse microscope incorporating Metamorph software. FRET signal is observed in cells expressing the compromised mice (i.e. mice lacking a functional MFE23.CD3ζ CIR but not the MFE23.htm.CD3ζ CIR. immune system) bearing established CEA+ tumours Tumour Infiltrating lymphocytes can be effectively treated using gene-modified T cells. These Aside from the genetic modification approach, we are actively observations suggest that the presence of competing immune investigating the isolation of antigen specific T cells from the cells may inhibit the functionality of the gene-modified T cells. tumours of patients. This approach has been pioneered by Consequently, conditioning of the patient (i.e. transiently Dr Steven Rosenberg (NCI, Washington DC) in malignant depleting the immune system) prior to T cell infusion may be melanoma with some spectacular clinical responses. In important in driving an effective anti-tumour response. collaboration with Mr Gary Ross (Dept. of Surgery) and Dr Paul Lorigan (Medical Oncology), we have tested biopsy Indeed, in our CEA model system, mice receiving either samples from ten melanoma patients with approximately 120 chemotherapy or radiotherapy prior to T cell infusion TIL cultures being initiated. Of these, approximately 50% showed significantly reduced levels of tumour growth generated sufficient T cells for testing with only one patient of compared to animals treated with T cells alone (Figure 2). In the ten failing to generate any TIL cultures. Importantly, of the the B cell lymphoma model, the combination of human first three patients tested, two have generated TILs that are CD19z T cells with chemotherapy effectively treated the Raji able to respond against autologous tumour cells. We are B cell lymphoma in vivo (Cheadle et al., 2008) while the further developing this protocol in order to move towards combination of chemotherapy and mouse CD19z T cells was clinical testing of melanoma TILs in Manchester within the optimal in eradicating a long term (13 day) established mouse next two years. B-cell lymphoma in immune competent mice (Cheadle et al., in press). Importantly, these studies demonstrate that Summary achieving high levels of circulating gene-modified T cells is an The focus of the Cell Therapy group over the last twelve important factor to predict for anti-tumour response. These months has been upon the development of model systems studies support the central aim of both the CEA and CD19 to test adoptive T cell therapies. These models are now clinical trials which is to determine which therapeutic advancing our knowledge of gene modified T cell biology conditions can achieve high levels of circulating gene-modified which will translate into improved future clinical protocols. T cells in the patient. Publications listed on page 63 Figure 2. Improved tumour free survival of mice involves pre-conditioning of the mice prior to infusion of tumour specific engineered T cells. Research Reports 43

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    Paterson Institute for Cancer Research Medical Oncology: Glyco-Oncology Group Group Leader John Gallagher Senior Fellow Scientific Officers Graduate Student Malcolm Lyon Jon Deakin Annie Wat Nijole Gasiunas Postdoctoral Fellows Graham Rushton Rebecca Baldwin Claire Johnson Chris Robinson Cancer cells are characterised by metastasis and poor disease prognosis. There is a strong case for HGF/SF-MET as a target in anti-cancer therapy. Our aim progressive growth and the capacity to has been to elucidate how GAGs are recognised by HGF/SF, invade and colonise distant sites. Heparan and how they act as co-receptors, to exploit this as a potential route to the development of inhibitors of co- sulphate (HS) is a cell surface co-receptor receptor function and thus HGF/SF action. This is for many of the growth and migration complicated by the unusually high affinity of HGF/SF for both heparan sulphate (HS)/heparin and dermatan sulphate (DS), factors involved in the dissemination and two GAG types which differ significantly in structure. Recently we have completed a two-pronged experimental vascularisation of human tumours. Our approach, whereby we have tested the binding and activating research has shown that patterns of properties of two different groups of molecules: (i) a wide array of natural and modified GAGs/sulfated polysaccharides sulphation along the HS chain act as (Catlow et al., 2008), and (ii) a panel of minimal-binding binding sites for growth factors and enable tetrasaccharide sequences of variable but well-defined sulfation patterns. their efficient engagement with tyrosine kinase receptors. Using novel analytical These studies revealed that binding required a minimum of two sulphate groups within a tetrasaccharide, in combination methods we have investigated the with the presence of iduronate resides, but surprisingly is molecular design and function of HS in independent of sulphate positioning. Indeed, using a newly- developed technique, combining our previous gel mobility different cell types, including embryonic shift assay (GMSA) (Lyon et al., J Biol Chem 2004; 279: stem (ES) cells. Our recent findings 43560) with reverse-phase-HPLC, we have now been able to show conclusively that all heparin tetrasaccharide isomers, indicate that the unique domain structure that contain two sulphates at either N-, 2-O- or 6-O- and conformational flexibility of HS drives positions (see figure) bind equally well, and identically to a DS tetrasaccharide that is 4-O-sulphated (Deakin et al., in press). the assembly of ligand-receptor signalling Thus the ability to bind either HS/heparin or DS appears to arise from a non-specific ability to accommodate a variety of complexes on the plasma membrane sulphate configurations in iduronate-containing GAGs. Absolute affinities correlate with the overall level of Hepatocyte Growth Factor/Scatter Factor (HGF/SF): a sulphation, suggesting a major role for electrostatic single, primary co-receptor binding site with dual interactions in complex formation. glycosaminoglycan specificities HGF/SF is a potent mitogen and migration factor for In collaboration with Dr Dusan Uhrín (University of epithelial and endothelial cells. It acts via a dual receptor Edinburgh), we were able to show that the NMR chemical system of the MET tyrosine kinase receptor and the shifts induced in the truncated NK1 variant of HGF/SF by glycosaminoglycan (GAG) chains of proteoglycan co- titration with either heparin or DS oligosaccharides were receptors. Excessive HGF/SF-MET signalling occurs in many very similar, strongly indicating that both these GAGs bind to carcinomas, sarcomas and also multiple myeloma, where the a single site of relatively low specificity (Deakin et al.). These degree of elevation correlates with tumour invasiveness, 44 Paterson Institute for Cancer Research Scientific Report 2008

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    results will hopefully impact upon the future development of these saccharides elicit a potent FGF2 mitogenic response HGF/SF inhibitors based upon mimics of GAG co-receptors which correlates with their ability to dimerise FGF2 in a co- which we described in our 2007 Report (Raiber et al., Bioorg operative manner. These results, together with parallel studies Med Chem Lett 2007; 17: 6321). on acidic FGF (FGF1), have revealed that co-operative ligand dimerisation by heparin and HS is likely to be the main Heparan sulphate in the regulation of embryonic stem cell driving force in assembly of FGF signalling complexes on the (ES) cell differentiation cell surface. The predicted stoichiometry of these complexes ES cells are a valuable model to study the role of HS during from size exclusion chromatography (SEC) is 2:2:1 FGF: development. ES cell differentiation is accompanied by FGFR: HS where FGFR is the FGF-receptor dramatic alterations in HS sulphation that enable each cell/organ to respond characteristically to extrinsic signals in Investigations of these complexes by multi-angle light the form of peptide growth factors and morphogens. In scattering (MALLS) available at the Biomolecular Analysis previous reports we described the alterations in HS that Facility (University of Manchester) has yielded data that occurred during neural differentiation. To highlight the strongly support the stoichiometries predicted from SEC. A essential requirement for HS for progression along the neural particularly interesting observation was that saccharide lineage we have characterised the differentiation potential of concentration had no effect on the stoichiometry of FGF- HS-deficient Ext1-/- ES cells. These cells were found to be heparin complexes. This is what we would expect from our defective in both neural and mesodermal/haematopoietic published model of co-operative ligand dimerisation. differentiation (see below), mimicking the block in (Robinson et al., J Biol Chem 2005; 280: 42274). Further development seen in HS-deficient mouse embryos. studies by our collaborators Professor Tom Blundell and Alan However, remarkably differentiation could be restored by the Brown in Cambridge on the thermodynamic properties of addition of soluble HS or heparin oligosaccharides to the monomeric and dimeric FGF-heparin complexes using growth media. These important findings open the way for isothermal titration calorimetry (ITC) have largely confirmed investigating whether different HS species can dictate cell fate the co-operative binding model. Overall these studies decisions in ES cells. highlight an unexpected structural plasticity of HS and heparin that enables a monomeric ligand like FGF to induce a We have also characterised the dynamic modifications in HS conformational change that creates a highly favourable sulphation motifs during mesoderm specification and blood proximal site for a second FGF. This HS-bound dimeric FGF formation (Baldwin et al., 2008). This paper highlighted the efficiently recruits two receptors that then deliver signals expression of a specific HS epitope (detected by antibody across the cell membrane. HS4C3) that is selectively and transiently expressed during formation of the haemangioblast, a cell expressing Flk1 (VEGF Key collaborators Disaccharide Units in Heparan Sulphate receptor) which is capable of forming both blood and Valerie Kouskoff and Georges endothelial progeny. Functional assays revealed that cells Lacaud (Stem Cell Biology and positive for HS4C3 and Fk1 had a dramatically increased Haematopoiesis Groups), CH O [ H A) 2 ability to form blood cells compared to those expressing Flk1 Gordon Jayson (Translational SO 3 - O O alone. The development of these cells is dependant upon the Angiogenesis Group), Catherine COO -O OH O OH HS-binding growth factor VEGF, suggesting this HS epitope Merry and Brian Bigger H NHSO 3 - O[ may be important for correct signaling of VEGF. In vivo (University of Manchester), Tom SO 3 - studies showed remarkable correlation with in vitro findings, Blundell and Ermanno Gherardi GlcNSO (+/- 6-OSO ) 1 – 4 IdoA, 2-OSO 3 3 3 with expression of the HS4C3 epitope restricted to newly (University of Cambridge), formed mesodermal tissues during gastrulation. We believe Dušan Uhrín (University of this is the first time a defined HS epitope has been implicated Edinburgh), Anne Dell and B) CH OH 2 COO - in a specific developmental pathway and that this additionally Berangere Tissot (Imperial O O O provides a novel enrichment technique for the isolation of College, London), Jim Wilkinson OH O OH haemangioblasts from mixed differentiated ES cell cultures. (University of Salford), Gerdy OH NHAc This work was done in collaboration with Valerie Kouskoff, ten Dam (University of Georges Lacaud and Catherine Merry. Nijmegen, Netherlands) and GlcNH.Ac 1 – 4 GlcA Marios Stavridis (University of Co-operative dimerisation of Fibroblast Growth Factor Dundee). The heparan sulphate (HS) chain is made up of sulphated (A) and non-sulphated (B) disaccharide (FGF) on Heparan Sulphate drives the assembly of units; these units occur in a series of clusters or FGF/FGF-Receptor signalling complexes Publications listed on page 63 domains rather than being randomly distributed. Heparan sulphate is a mandatory co-receptor for the Growth factors such as HGF/SF and the FGFs bind to the sulphated domains but recognise different fibroblast growth factors (FGFs). Heparin saccharides of sulphation patterns. Pattern recognition by growth defined length (8-to-10 sugars) and sulphated at the N-, 2- factors is essential for the co-receptor function of and 6- positions (see figure) were used as chemical analogues HS. of the sulphated regions of HS. At very low concentrations, Research Reports 45

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    Paterson Institute for Cancer Research Medical Oncology: Translational Angiogenesis Group Group Leader Gordon Jayson Senior Fellows Clinical Fellows Postgraduate Student Egle Avizienyte Gireesh Kumaran Karl Broburg Claire Mitchell Postdoctoral Fellows Nishanth Murukesh Rotation Student Marek Barath Kelvin Wilkinson Claire Cole Scientific Officers Steen Hansen Alison Backen Karen Brookes Graham Rushton Over the last year the group has patients at each dose level we were able to increase the statistical power of the study and, through measurements of progressed in two directions; the GCLP tumour volumetrics, were able to detect a dose-level vs validation and implementation of imaging volumetric response. This is important as the traditional method of reporting radiological evaluation of tumour and blood borne biomarkers for anti- response, RECIST, failed to detect this relationship. Thus our findings highlight tumour volumetric measurements as a angiogenic agents and the second potential novel biomarker for mechanism based therapeutics. approach has been to develop research In a second programme we have developed and validated to platforms that will assess the contribution the standards of GCLP multiplex and singleplex assays of of heparan sulfate proteoglycans in human sixteen angiogenic proteins. Traditional assays are based on singleplex ELISAs that are costly and consume significant epithelial ovarian cancer. The translational amounts of plasma. By multiplexing nine proteins, for the first proteoglycans programme has emerged time, we have impacted significantly on the cost, time and amount of plasma needed for these assays. The protocols from the basic science studies that our have been submitted for publication and will be used in group has performed over the last few forthcoming evaluations of plasma samples in ICON7, a randomised trial of carboplatin and paclitaxel with and years within Professor John Gallagher’s without bevacizumab in the first line treatment of ovarian laboratory. cancer. Additional samples that will be evaluated will be obtained from randomised trials of anti-angiogenic agents in pancreatic and colorectal cancer. Finally, within the next Biomarkers for Anti-Angiogenic therapy (Collaborators: twelve months we will apply the technology within an in- Dive, Jackson, Parker) house study that unites the single and multiplex studies Over the last few years the group has identified and developed in the last year, with our longstanding imaging evaluated a series of imaging based biomarkers for anti- programme. angiogenic therapy. Within the last year we have reported a phase I trial of a novel anti-VEGFR2 di-Fab compound (Ton et Recognising that the data obtained from imaging would be al., 2007 Clin Cancer Res; 13: 7113), which differed from significantly augmented through the inclusion of other broader spectrum VEGF inhibitors in that it did not measurements of circulating anti-angiogenic biomarkers we impact on Ktrans, the endothelial permeability-surface area have nearly completed a study where we have compared product. On the other hand, biopsy of hemangiomata that dynamic MR and dynamic CT measurements of patients were induced by the compound confirmed that the undergoing treatment for ovarian cancer. This trial will be compound was present in areas of non-phosphorylated finished within the next few months and the data are receptor, that is, that the biopsy data were compatible with revealing novel insights into the relationship between imaging the proposed mechanism of action. By changing the and circulating biomarkers. In a second combination traditional phase I drug design to expand the number of 46 Paterson Institute for Cancer Research Scientific Report 2008

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    biomarker study, we function as the Chief Investigator site (in Conditioned medium generated by defined ovarian cancer conjunction with Oxford) for the first-into-man study of cell lines showed significant differences in their ability to GSAO, a tri-valent arsenical anti-angiogenic agent, conducted induce endothelial tubule formation in three dimensional under the auspices of the Cancer Research UK New Agents setting. We are in the process of evaluating HS composition Committee. in HUVECs cultured in conditioned medium from different ovarian cancer cell lines to determine whether angiogenic Contribution of Heparan Sulfate Proteoglycans to Epithelial factors generated by cancer cells affect HS composition on Ovarian Cancer Angiogenesis (Collaborators: Gallagher, endothelial cells. Gardiner) The heparan sulfate (HS) proteoglycans are responsible for Activity of heparin, HS and heparin oligosaccharides the regulation of biological activity of several angiogenic and Over the last few years we have developed the organic anti-angiogenic cytokines. In previous studies we chemistry to make heparan sulfate oligosaccharides of demonstrated that FGF2 was dependent on HS for its defined structure. These are being evaluated in assays of biological activity; that the cytokine induces mitosis in vitro and HUVEC proliferation and tubule formation. Initial studies are angiogenesis in vivo and that both phenotypes can be aimed at identifying the optimum length and overall sulfation inhibited by soluble heparin octasaccharides. Using a novel pattern that are most effective in these assays. The chemistry molecular probe we demonstrated that ovarian cancer has the potential to generate species with defined sulfation endothelium has the capacity to activate FGF2 and in the pattern enabling us to refine the structures that mediate current studies our aim was to understand this process in growth factor activation and thereby to further our greater detail. understanding of growth factor activation and signaling. We are currently analysing the distribution of sulfated HS in We are translating our basic science HS programme towards ovarian serous cancers and normal ovarian tissues. Our the clinic in two ways: it is clear that certain moieties in the preliminary data show that N-sulfated HS is localized HS chain are responsible for growth factor activation and specifically to the ovarian tumour vascular network, but not using retroviral expression of RNAi for particular HS to tumour tissue, while sulfated HS staining in normal ovaries synthetic enzymes our aim is to identify critical enzymes that is predominantly detected in the perivascular and stromal generate the moieties essential for growth factor activation. areas (see figure). We have a large panel of characterised Lastly, we have developed a technique that analyses the antibodies that specifically recognise sulfation (collaboration composition of HS in very small amounts of biological with Dr. G ten Dam, Nijmegen Center for Molecular Life samples. This technique will be used to start to analyse Sciences). Using these antibodies we were able to show that samples provided both from the surgical and non-surgical HS sulfated at different positions of hexosamine is mostly oncological environments. confined to the vascular endothelial cells in ovarian tumours. In addition, the antibody recognising 6-O sulfated HS species Publications listed on page 64 highlighted perivascular region of tumour vasculature. HS sulfation specific antibodies stained normal ovarian tissue predominantly at stroma and basal side of the vessels. Our programme has hitherto focused on FGF2 and we aimed to define the cytokines involved in ovarian cancer angiogenesis in greater detail. We are currently screening a panel of ovarian cancer cell lines for the expression of angiogenic factors using angiogenesis antibody arrays and multiplex ELISAs. We have already discovered high levels of FGF2 and IL-8 in a subset of ovarian cancer cell lines. Using human umbilical vein endothelial cells (HUVECs) we have established in vitro assays to evaluate endothelial cell ability to form tubules either on Matrigel or in fibrin or collagen gels. Relationship between sulfated HS and endothelium. Normal ovarian tis- sue was fluorescently stained with antibodies that recognise the endothe- lium (von Willebrand factor, vW) and sulfated heparan sulfate (H). The data show that the perivascular layer contains sulfated heparan sulfate. Research Reports 47

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    Research Services Head of Research Services Jenny Varley The Paterson Institute prides itself on the quality of its Research Services which support all research activities based within the Institute. 2008 has seen some significant changes notably the closure of the Transmission Electron Microscopy (TEM) Service and the transfer of the CR-UK Microarray Service from Research Services to Institute management. Advanced Imaging Facility Head: Steve Bagley Over the year we have considerably extended the imaging capabilities of the Institute with the installation and Early in 2008 Steve Murray left the Institute and this development of new equipment. The requirement for precipitated a review of the requirements for a TEM service. complex imaging techniques has increased considerably to Given the costs of maintaining, housing and staffing the facility embrace a range of applications. Extensive examination of together with very limited and intermittent requirements for cellular morphology, quantification of protein levels, TEM, the decision was made to transfer the equipment to quantification of turnover rates and interactions between the University, with access available to Institute Groups as multiple low light signals are now all covered in the portfolio required. of supported technologies. Some much-needed refurbishments were completed at the A Nipkow spinning disk confocal microscope was introduced beginning of the year, giving better facilities for the Molecular early in the year. This technology is particularly good for Biology Core Facility, the Flow Cytometry Facility and for the imaging multiple fluorescent proteins in a 3-D volume. The Mass Spectrometers. We also increased the space for the scanning technology it employs enables the user to record Histology Facility because of both an increased workload and absolute localization in multiple channels in 3-D and so the inclusion of the MCRC Biobank within their remit. generate a detailed map of spatial interactions. The extended sensitivity of this system and high-end optics permit the To maintain cutting edge services we need to constantly visualization of very faint signals that would be below the upgrade or replace equipment, and this year has been no threshold of a conventional wide-field microscope. This exception. Details of each new development are given in the enhanced sensitivity also reduces the levels of illumination appropriate section below, but installation of a new Nipkow required for imaging, which in turn reduces photo-bleaching spinning disk confocal microscope, a Zeiss Mirax whole slide and thus greatly extends the periods over which a sample can scanning system, a Nano Acquity UPLC system and LTQ- be imaged. The system also supports the visualization of Orbitrap XL, an XY clone laser, an LSRII and a BD InFlux multiple XY positions so that thousands of cells can be cytometer, and imminently a new laser capture imaged during a single investigation, considerably reducing the microdissection system have all taken place in the last twelve amount of microscope time required to gain statistical valid months. datasets. The system is currently being employed for studies ranging from yeast cell division to the dynamics of the cytoskeleton during the migration and adhesion of mammalian cells. A whole slide imaging system has transformed histological imaging within the Institute. Over 8.5 TB of data have been generated by nine research groups over the 10 months up to October 2008. The system scans both tissue sections and 48 Paterson Institute for Cancer Research Scientific Report 2008

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