CANCER GENOMICS & GENETICS RESEARCH PROGRAM

The Cancer Genomics and Genetics program (CGG) seeks to use sophisticated high throughput genomic technologies to improve our understanding of the biology of cancer and to progress the clinical management of cancer patients through the development of individualized approaches to treatment. Research in the program focuses primarily on breast, upper gastrointestinal and ovarian cancers and sarcoma, and involves some of the largest familial and population-based cancer cohorts in the world. These studies address questions of general importance to solid cancers, including inherited susceptibility to cancer and genome-wide changes in gene expression, as well as more specific questions such as prediction of response to therapy and the use of gene expression profiling for accurate cancer diagnosis.

CANCER GENETICS & GENOMICS
INVOLVEMENT OF SIAH PROTEINS IN THE TUMOUR/STROMA MICROENVIRONMENT Supervisors: Dr. Andreas Moeller & Prof David Bowtell Tumours interact with their surrounding stroma in several ways to enable the tumours to grow and eventually metastasise. Understanding the interaction of tumour cells with surrounding stroma in the tumour microenvironment is crucial to therapeutically target tumour progression. One of the key hurdles of tumour growth is to initiate the outgrowth of new blood vessels (neo-angiogenesis). The new tumour-associated blood vessels supply the tumour with oxygen and nutrients as well as enable its spread to distant organs via the blood stream. A key signal for the induction of neoangiogenesis is low oxygen levels (hypoxia) in the tumour mass. On a cellular level, hypoxia causes the production and secretion of factors like VEGF, which in turn induce neo-angiogenesis. Our group has established several breast cancer models and is investigating signal transduction in the tumour microenvironment. We have identified key enzymes that regulate the hypoxic response and we are examining their impact on neo-angiogenesis. We are currently developing drug-like molecules to some of these enzymes and are studying their efficacy in vitro and in vivo. We are now looking for motivated students (both Honours and PhD students) to strengthen our group. The projects will investigate the communication between tumour cells and their surrounding tissue. Especially the projects will focus on neo-angiogenic signals as well as immune modulator signalling. The projects are utilizing unique knockout and transgenic tumour systems and are supported by excellent mouse breeding and molecular/biochemical facilities and will be performed as part of an internationally competitive multidisciplinary team, incorporating a number of modern techniques such as microarray, quantitative real-time PCR, protein degradation assays and siRNA approaches. For more information about this project contact: Dr. Andreas Moeller, Tel: +61 3 9656 1287, E-mail: andreas.moeller@petermac.org ANALYSIS OF CANDIDATE GENES IN ACQUISITION OF CHEMORESISTANCE IN OVARIAN CANCER Supervisors: Prof David Bowtell & Dr Prue Cowin Ovarian cancer is the 5-6 most common cause of cancer death in women in Western countries, with ~800 deaths per year in Australia, with high-grade serous ovarian cancers accounting for the majority of deaths (>60%). Standard first-line chemotherapy involves a platinum agent (typically carboplatin) and a taxane.
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Whilst most women respond well to first line treatment, the disease typically recurs and, following treatment with multiple agents, acquired resistance develops followed by progression and death, usually within 5 years of initial diagnosis. We are part of the Australian Ovarian Cancer Study (AOCS), one of the largest ovarian cancer cohort studies in the world. We are also one of the two Australian projects funded through a $27 million NHMRC grant to participate in the International Cancer Genomics Consortium (ICGC). The proposed studies involves the comparison of ovarian tumour tissue collected at the time of primary surgery with samples from the same women following disease recurrence to investigate genes changes linked to acquired drug resistance and recurrent disease. Studying disease recurrence using a pairedsample approach lends considerable power to analysis. A number of genes have been linked with acquired chemoresistance in ovarian cancer, including GSTπ (Ikeda et al 2003; van der Zee et al 1995), ATP7a and ATP7B Nakayama et al 2002, 2004). Data for the copper transporter ATP7a is amongst the most compelling, where expression in primary cancer samples is associated with poor outcome. This project aims to test literature-derived candidate genes in paired samples to determine if they are associated with acquired platinum resistance. Expression levels of candidate genes will be measured by Q-PCR and IHC on TMA, and findings related to clinical outcome. There is also increasing evidence that hypermethylation of the promoter of the mismatch repair gene hMLH1 is responsible for increased resistance observed in cancer cells. To validate the methylation status of hMLH1 in primary and relapse samples, methylation specific Q-PCR will be performed. The student will learn key molecular biological techniques and will be exposed to large-scale human genetic studies that are making use of the emerging technologies, including next generation sequencing. The Bowtell lab has a very strong reputation in cancer genetics and genomics, and in fundamental studies in cancer cell biology. He/she will have the opportunity to contribute insights into one of the most clinically significant questions in ovarian cancer – the emergence of drug resistance. For more information about this project contact: Professor David Bowtell, Tel: +61 3 9656 1356, E-mail: david.bowtell@petermac.org Dr. Prue Cowin, Tel: +61 3 9656 1287, E-mail: prue.cowin@petermac.org

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SURGICAL ONCOLOGY
The Surgical Oncology Laboratory is focussed on using molecular and cellular approaches to understand the development, progression and treatment of gastrointestinal cancers. Projects are offered in the two areas of research described. UNDERSTANDING BARRETT’S OESOPHAGUS. Supervisors: Dr Nick Clemons, Assoc Prof Wayne Phillips Over the past thirty years there has been a dramatic increase in the incidence and prevalence of oesophageal adenocarcinoma in Western populations. The reason underlying the increase in this cancer is not clear but is thought to reflect an increase in the occurrence of its recognised precursor lesion, Barrett’s oesophagus. Barrett’s oesophagus is a metaplastic abnormality in which the normal stratified squamous epithelium of the oesophagus is replaced by a metaplastic intestinal-type columnar epithelium. The risk of adenocarcinoma in patients with Barrett’s oesophagus appears to be approximately 30-125-fold greater than that in the general population with an estimated incidence of 1 in 200 patient years. The origin of Barrett’s oesophagus is a matter of conjecture. There is compelling etiological evidence that acid reflux is a major contributing factor but the actual molecular and cellular mechanism underlying the phenotypic change is not clear. The group has developed novel cell culture models that allow the 3-dimensional, layered structure of the normal oesophageal lining to be reproduced in the laboratory. The student will use these models to investigate the molecular and cellular mechanisms underlying the development of Barrett’s oesophagus and/or the progression of Barrett’s oesophagus to adenocarcinoma. Understanding the biology underlying this condition will ultimately help us to design effective strategies for the management and treatment of Barrett’s oesophagus and to predict, and/or prevent, the progression of Barrett’s Oesophagus to oesophageal adenocarcinoma. For more information about this project contact: Dr Nick Clemons, Tel: 9656 1849; Email: nicholas.clemons@petermac.org Assoc. Prof. Wayne Phillips, Tel: 9656 1842; Email: wayne.phillips@petermac.org THE ROLE OF PIK3CA MUTATION IN CANCER. Supervisor: Assoc. Prof. Wayne Phillips The laboratory has a long-standing interest in the role of the phosphoinositide 3-kinase (PI3kinase) signalling pathway in the development and progression of human tumours. PI3kinase is a ubiquitous family of lipid kinases that catalyse the phosphorylation of phosphoinositides PI, PI(4)P and PI(4,5)P2 forming PI(3)P, PI(3,4)P2 and PI(3,4,5)P3 respectively. These lipid products are then able to activate a variety of downstream targets that regulate a wide range of important cellular processes including cell proliferation, migration and survival, oncogenic transformation, and intracellular trafficking of proteins. In collaboration with the Cancer Genetics laboratory, the group has demonstrated a high frequency of somatic mutations in the PI3kinase gene PIK3CA in breast, ovarian and colon cancers. Early functional studies in transfected cell lines have indicated that the tumour-associated PIK3CA mutations are activating,

resulting in enhanced lipid kinase activity and upregulation of downstream signalling events such as phosphorylation of Akt and ribosomal protein S6. However, definitive evidence demonstrating that PIK3CA mutations directly cause cancer is still lacking. Recently, the group has generated a unique mouse model with an inducible knock-in of the PIK3CAH1047R mutation. The student will use this model and induce expression of the mutation in specific tissues using transgenic and adenoviral approaches. The effect of expression of the mutation (alone and in the presence of other oncogenes) on tumour development, tissue pathology and epithelial cell function will be examined. This project will involve both in vivo studies in the whole mouse and/or in vitro studies in isolated cells. For more information about this project contact: Assoc. Prof. Wayne Phillips, Tel: 9656 1842; E-mail: wayne.phillips@petermac.org

SARCOMA GENOMICS & GENETICS
ROLE OF IMMUNOMODULATORS IN THE DEVELOPMENT & PROGRESSION OF OSTEOSARCOMA IN VIVO. Supervisors: Dr. David.Thomas, Dr. Maya Kansara The Sarcoma Genetics and Genomics laboratory studies tumours of soft tissue and bone. Osteosarcoma is the most common cancer of bone. These tumours are highly metastatic and often metastasise to lung via the hematogenous route. Treatment involves aggressive surgery with intensive adjuvant chemotherapy. Although these measures have improved prognosis, a third of those diagnosed will die from this disease. Understanding how osteosarcoma arises and persists will enable the development of targeted therapies. The skeleton and the immune system share a number of cytokines and transcription factors and therefore may mutually influence each other; the study of these cells and their interactions has been termed osteoimmunology. In this project we will investigate the interaction between the immune system and bone cancer in an in vivo mouse model of osteosarcoma. The project will use broad range techniques including mouse models of cancer, histology, cell culture, flow cytometry, and molecular profiling. For more information about this project contact: Dr. David Thomas. Tel (03) 9656 1111 david.thomas@petermac.org Dr. Maya Kansara Tel (03) maya.kansara@petermac.org 9656 1618 Email Email

REGULATION OF CELL FATE IN OSTEOSARCOMA. Supervisors: Dr. David.Thomas, Dr. Maya Kansara Osteosarcoma is the most common cancer of bone. Treatment involves aggressive surgery with intensive adjuvant chemotherapy. Although these measures have improved prognosis, a third of those diagnosed will die from this disease. Particular genes of interest in our group include RB1, Runx2, and WIF1. This project will utilise osteosarcoma cell lines, normal human bone cells, transformed normal human cells as well as primary human osteosarcoma samples to elucidate the role of the

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HIF-b is constitutively expressed and is involved in several transcriptional systems whereas the two HIF-a subunits are specific to the hypoxic pathway. • The true frequency of multifocal / multicentric breast cancers is unknown. The technique for screening is the novel methodology of high resolution melting analysis which has been recently developed in this Laboratory and allows rapid and sensitive 'in-tube" typing within a few hours. Peter Mac Page 3 of 21 May 2010 .org Email Email DISTINGUISHING RECURRENT BREAST TUMOURS FROM DE NOVO PRIMARY BREAST CARCINOMAS Supervisors: Prof Stephen Fox & A/Prof Alexander Dobrovic Profiling tumours for a series of cancer specific changes will allow the ready determination of the relationships between primary and recurrent/new primary breast cancers. (MacManus MP et al. Tel: +61 3 9656 1515 stephen. Currently we do not have the ability to accurately identify whether a new breast tumour arising in the breast is a new primary breast cancer or a recurrence of a previous tumour. molecular biology and biochemical studies. allowing rapid targeting and degradation by the proteasome pathway via the von Hippel-Lindau (VHL) protein and the ubiquitin E3 ligase complex.Thomas@Petermac. For more information about this project contact: Professor Stephen Fox.org Dr. Maya Kansara Tel (03) 9656 1618 Maya.Thomas. Tel (03) 9656 1111 David. Hypothesis. in hypoxia. Morphology alone is not adequate in distinguishing between truly synchronous tumours and multiple deposits of the same tumour. Email: alexander. translocation to the nucleus where it is able to bind HIF-b. This is essential since their treatments are very different. In some patients complete tumor regression is achieved allowing sphincter preservation. These markers have been selected because the readout is only minimally affected by the presence of contaminating normal tissue.fox@petermac.org Email: MOLECULAR PATHOLOGY IDENTIFYING THE PATIENTS LIKELY TO RESPOND TO NEOADJUVANT RADIOTHERAPY OF THE RECTUM Supervisor: Prof Stephen Fox Hypothesis: Expression of hypoxia and downstream signalling pathways will identify patients with rectal carcinoma likely to respond to primary chemo-radiation. Clinical issues: • Patients with breast cancer are at increased risk of a second breast cancer and also from local relapses in the breast. Michael McManus We seek to understand why about 1% of patients with apparently-incurable non-small cell lung cancer have experienced long-term survival and even cure after low dose palliative radiotherapy at Peter Mac. The ability to predict tumor response from such treatments would therefore improve the selection of patients for such treatments. The project will use broad range techniques including cell culture. Cancer. It is treated by radiotherapy before resection to try and shrink the tumour to improve survival and reduce local recurrence. Clinical issues: Rectal carcinoma is a significant cause of mortality and morbidity in Australia. This results in one oxygen incorporated into the prolyl residue of HIF-a. • Recurrent breast cancer is considered a metastasis but not all recurrences have similar biological behaviour and therefore a "recurrence" breast tumour classification system on which to base treatment decisions is needed. The complex then recruits co-activators that bind specific DNA hypoxia response elements (HREs) resulting in increased mRNA transcription. For more information about this project contact: Prof Stephen Fox. David.org UNDERSTANDING THE MOLECULAR MECHANISM OF LONG TERM SURVIVAL AFTER RADIOTHERAPY FOR LUNG CANCER Supervisors: A/Prof Alexander Dobrovic & Dr. This issue is of importance since staging and treatment decisions are based on these observations. This initiative builds on a highly successful research project. However. That study attained widespread publicity in the medical and lay press and media. The HIF complex is composed of a heterodimer of HIF-1a (or HIF-2a) and HIF-b (also known as aryl-hydrocarbon nuclear translocator). as frequently occurs within tumours.dobrovic@petermac. cell biology. both internationally and in Australia.org Email: Assoc. and was discussed in articles in the Lancet and in Nature Clinical Reviews. Research Division. Tel: +61 3 9656 1807. In normoxic conditions the HIF-a units are unstable since two prolyl residues within the oxygen-dependent degradation domains of HIF-a subunits are hydroxylated by prolyl hydroxylases and di-oxygen as a co-substrate.fox@petermac. For more information about this project contact: Dr. Tumour specific markers based on mutation and methylation will be used to compare primary and ipsilateral second presentation.above genes and others in the development of osteosarcoma. including the Health Report with Norman Swann. 2006 106:1110-6). The transcriptional complex hypoxia inducible factor (HIF) has emerged as a key regulator mediating many cellular responses necessary to adapt to changes in oxygen tension. The efficacy of radiotherapy is determined by the degree of tissue hypoxia (oxygen tension) and hypoxia is associated with an aggressive tumour phenotype. This is likely to become more important with the further molecular characterisation of breast cancer for targeted treatment. The tumours in these patients are likely to have a defect with in DNA repair or in DNA damage 2011 Project Summaries. there is insufficient oxygen to allow this process resulting in HIF-a stabilisation.. A further level of control is achieved through hydroxylation of an asparagine residue by factor inhibitor of HIF (FIH) at the C terminus of HIF-1a that interferes with CBP binding and thus transcription. Professor Alexander Dobrovic. Tel: +61 3 9656 1515 stephen.Kansara@Petermac.

Email: alexander.org 2011 Project Summaries.sensing pathways which underlies their extraordinary radiosensitivity. particularly lung adenocarcinoma.Methylation profiling for methylated DNA repair genes: As the amount of DNA from the tumours will be limited and the DNA undergoes further degradation during bisulphite modification. selected anti. CHK2.and pro-apoptotic genes. Research Division. and FANCF. HIF1 alpha to assess hypoxia and genes that Ramaswamy reported as being associated with metastasis in adenocarcinoma. This defect/these defects will also occur in some patients treated with conventional radiotherapy. Peter Mac Page 4 of 21 May 2010 . Experimental plan . Inactivating mutations at any of these genes is likely to lead to radioresistance and would be more likely to be found in the control population. PTEN (exons 5-8). Tel: +61 3 9656 1807. Mutation status of genes determining sensitivity to radiation: DNA will be screened for p53 (exons 5-8). and PIK3Ca (exons 9. BRCA1. Methylation of genes involved in the detection and repair of damage caused by ionising radiation will be assessed. a whole genome amplification method specific for bisulphite modified DNA will be used.dobrovic@petermac. Profiling expression: We will use a panel of 190 genes including all known DNA repair genes. These include ATM. 20) mutations using high resolution melting. For more information about this project contact: A/Prof Alexander Dobrovic.

despite a considerable wealth of information on its key role in immune homoeostasis and immune surveillance. Inappropriate regulation of cell death contributes to the pathology of various diseases including cancer. primary cell isolation. intact wild type CL and CL from granzyme B deficient mice to determine whether removing key regulators of specific apoptosis pathways can alter the death from apoptosis to alternative death mechanisms. Interestingly. Purified perforin and granzyme B have been shown to induce mitochondrial damage. Charles Robin Perforin is a cytolytic protein. The student will investigate and compare the morphological and biochemical features of death of these lines induced by purified granzyme B. so that rational choices can be made on the most appropriate (that is. Tel: +61 3 9656 1326. It is envisaged that the student will identify the key proteins involved in CL-mediated apoptosis and potentially identify novel pathways by which tumour cells can be killed. perforin. cells that express specific viral proteins or mutated proteins that are integral to apoptosis are still killed by CL. we are defining the molecular means by which new classes of anti-cancer drugs kill cancer cells. Perforin is a mysterious protein. which regulates cytotoxicity of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Our lab has developed a bank of primary lymphomas arising from various gene deficient animals. FHL is characterised by severe enlargement of the lymphoid organs.voskoboinik@petermac. The University of Melbourne. the evolution of putative functional domains of perforin will be investigated.trapani@petermac. This project will investigate how CL kill target cells in which key proteins involved in the mitochondrial pathway to apoptosis have been deleted. biochemistry and immunology techniques. CL mediated killing. or the inability of an immune cell to process perforin leads to a fatal disease in neonates. Little is known about how intact CL kill their target cell. apaf. Email: joe. but only recently experimental methodologies have been developed to investigate its properties. For more information about this project contact: Prof Joe Trapani. Ilia Voskoboinik. several perforin homologue genes are being identified within a single genome of various types of fish and frogs. Tel: +61 3 9656 1657. CANCER CELL DEATH LABORATORY Cytotoxic lymphocytes (CL) are specific cells of the immune system that seek out and kill dangerous cells. most potent and least toxic) cancer chemotherapy for a patient.CANCER IMMUNOLOGY RESEARCH PROGRAM The Cancer Immunology Program is identifying ways in which the immune system can be harnessed to prevent and control cancer. while single copies of the gene exist in higher organisms. apoptosis assays. We are interested in the very early stages of how immune cells can pick up and respond to the presence of cancer cells. For more information about this project contact: Prof Joe Trapani. Further. Email: joe. including Bid. and time-lapse microscopy. resulting in a specific form of cell death known as apoptosis. These hypotheses will be tested experimentally by expressing perforin homologs in immune cells and testing their ability to kill target cells. Email: ilia. The lack of perforin expression or the loss of function due to detrimental mutations. We have demonstrated that specific toxins made by “killer T cells” can prevent the onset of certain cancers (immune surveillance).org Dr. Applicants are expected to complete major in genetics and biochemistry. Ilia Voskoboinik. These cells destroy virus infected or transformed cell targets. We investigate the mechanisms by which cytotoxic lymphocytes kill their targets. It was discovered more than 20 years ago. Prof Joe Trapani & Dr. We have recently established experimental models and developed specific techniques to investigate the exact nature of how intact CL kill their target cells. familial haemophagocytic lymphohistiocytosis (FHL). Understanding the structure and properties of perforin will help to elucidate the mechanism of cytotoxicity of CTL and NK cell.trapani@petermac. Research Division. cell biology. Tel: +61 3 9656 1326. These studies are expected to further elucidate the evolution of the immune system and will provide key information on the mechanism and structure of a key player. We have found that some CL that lack granzyme B can still kill their targets but cannot induce apoptosis. A KEY REGULATOR OF IMMUNE HOMOEOSTASIS.org 2011 Project Summaries. Very little is known about the mechanism and structure of perforin. The starting point of the project will be a comparative genomic analysis of perforin homologues from evolutionarily distant species. Supervisors: Dr. This poses the question as to how CL specifically induce apoptosis and how CL can kill cells in which apoptosis is blocked. ROLE OF MITOCHONDRIAL PATHWAY TO APOPTOSIS IN CYTOTOXIC LYMPHOCYTE MEDIATED KILLING Supervisors: Prof Joe Trapani One mechanism by which cytotoxic lymphocytes (CL) kill their target cells is via granule exocytosis. and are developing genetic technologies to modify and expand the activity of these cells to treat established malignancies. During this project the student will develop skills in tissue culture. The project involves bioinformatics and a wide range of genetic. which are infiltrated by activated macrophages and T cells.org BRIDGING THE EVOLUTION AND FUNCTION OF PERFORIN. During this process CL deliver perforin and granzyme B to the target cell. and caspase-9. On the basis of these analyses. Current advances in genome projects have made available the DNA sequences from many evolutionarily distant species for comparative analysis. Peter Mac Page 5 of 21 May 2010 . The project is offered jointly by Cancer Immunology Program at Peter MacCallum Cancer Centre and Department of Genetics. In addition.

apoptosis and phagocytosis assays. ELISA. Students will have access to excellent infrastructure and will learn skills in mouse experimentation. Supervisors: Dr. Supervisors: Dr. multi-color flow cytometry. These experiments will provide an insight into the metastatic progression of breast cancer and will be the first of their kind. We are offering PhD studies to now systemically determine an immune signature of dying tumour cells and the role of various myeloid cell populations in mediating the anti-tumour activity of conventional and novel cancer therapies. Significantly. the frequency of metastasis will be assessed in experiments where the immune cell lineages which populate the metastatic site have been ablated. We are seeking an Honours student to pursue a project focused on these cellular interactions and their relevance to inflammation.smyth@petermac. the immune cell lineages will be investigated in great detail. and other basic cellular immunology techniques. NKT cells can produce significant quantities of cytokines very rapidly. We have generated a unique breast cancer model. Ultimately the goal is to understand the identity. For more information about this project contact: Dr Daniel Andrews.CELLULAR IMMUNITY LABORATORY INNATE CYTOKINE NETWORKS AND INFLAMMATION. For more information about this project contact: Dr. leukocyte isolation and transfer. Reference relevant to this project: Lyakh. and basic molecular techniques. Research Division. Mark Smyth. E-mail: mark. utilizing fluorescently marked immune and tumours cells. Andreas Moeller & Prof. Subsets of NKT cells exist. For more information about this project contact: Prof. (2008) Immunol Rev. commonly called metastasis. Students will have access to excellent infrastructure and will acquire skills in mouse tumour model experimentation. bone marrow chimeras and orthotopic breast cancer models will be used to determine the composition of the immune cell infiltrate into the metastatic sites as well as their functions. These subsets produce different cytokines upon stimulation. Dan Andrews and Prof. septic shock is diagnosed in approximately 750. tumour cells are interacting with the surrounding environment. Other studies of ours illustrate the optimal therapeutic efficacy of agonistic antibodies to the TRAIL death receptor requires host CD11c leukocyte function. gamma delta T (gdT) cells are known to play a significant role in innate immunity. Mark Smyth The main cause of morbidity in breast cancer patients is caused by the spread of the diseases to distant sites. Interest is focused upon IL-1 family members and IL-17. Mark Smyth Innate immune cells include NKT and gamma delta T cells. We found that immune cell infiltration into metastatic sites is directed and orchestrated by primary tumours. to assess the immune cell infiltrate at metastatic sites in breast cancer. In this project. Tel: +61 3 9656 3728. E-mail: mark. We are utilizing several models of inflammation and sepsis in mice to determine the key cytokine networks regulated by these unique innate lymphocyte subsets and their relationship to NK cells. which is thought to be how NKT cells mediate diverse functions. These immune cells generate a microenvironment aiding further metastatic tumour progression. which have been ascribed different properties. Using FACS and Immuno-histology. which are induced by tumours to populate the metastatic site. will be assessed for their cytokine production. The cytokine producing capabilities of gdT cells is now becoming more widely recognized as is the role these can play in shaping immunity. and other basic cellular immunology techniques. IHC.org CANCER IMMUNOTHERAPY RESEARCH LABORATORY 2011 Project Summaries. Mark Smyth The therapeutic efficacy of anticancer chemotherapies may depend on the capacity of dendritic cells (DC) to present antigens from dying cancer cells and to prime tumor-specific interferon-g-producing T lymphocytes. ROLE AND FUNCTION OF IMMUNE CELLS AT SITES OF METASTASIS IN BREAST CANCER. leukocyte isolation. Recently we have shown that some anticancer chemotherapies that are successful in immunocompetent mice are inefficient against tumors established in mice defective in various components of the NLRP3dependent caspase-1 activation complex ("inflammasome"). Tel: +61 3 9656 1752. E-mail: Daniel. a pro-inflammatory cytokine whose production was originally ascribed to a lineage of helper T cells termed Th-17.org THE ROLE OF DC IN TUMOUR THERAPIES. role and function of immune cells at the metastatic site and explore the potential to use this information to reduce metastatic tumour burden in breast cancer patients. L et al. While the role of monocyte derived cells at the primary site of breast cancer is slowly being understood there is very little known about the role and composition of immune cells at metastatic sites. Immune cells are key mediators of anti-tumour and protumour functions at both the primary and metastatic site. Similarly to NKT cells.org THE IDENTITY. cytometric bead array. Mark Smyth. PhD students will have access to unique reagents/mice and excellent infrastructure and will acquire skills in mouse tumour model experimentation. Various genetargeted mice specifically deficient in various subsets of DC or molecules of the inflammasome will be available. immune cell isolation. multicolor flow cytometry. Nicole Haynes & Prof. making them potent regulators of immune responses during infection and cancer. Supervisors: Dr.moeller@petermac.Tel: +61 3 9656 3728. At the original site of the primary tumour as well as at metastatic sites. Andreas Moeller.org Prof. 226:112.andrews@petermac. E-mail: andreas. Isolated immune cells.000 patients/year in North America and Europe and around 31% of these patients die making understanding of the molecular mechanisms operating during inflammation of significant importance. multi-colour flow cytometry. Peter Mac Page 6 of 21 May 2010 . Furthermore.smyth@petermac. Tel: +61 3 9656 1287.

However. Indeed. Trapani JA. Several gene formats have been compared by laboratories around the world for their ability to trigger T cells to engage and destroy tumour cells. Supernatural T cells: genetic modification of T cells for cancer therapy. Research Division. Such methods are laborious and time consuming.Sc. dendritic cells and several types of T cells. which results in slow progress towards the generation of optimal tumourspecific T cells for the adoptive immunotherapy of cancer. The project will involve: (1) The use of molecular biology to generate a DNA library. Particular cancers being addressed in this section are kidney cancer. flow cytometry.314:126-9. Dudley ME. but the immune system is often tolerant of tumours. chemotherapy and radiotherapy. In this regard. breast cancer and multiple myeloma. We seek to turn this disease-fighting capacity against cancer cells by using anti-cancer genes to endow immune cells with the ability to recognize and destroy tumour cells. Nevertheless. Smyth MJ. Wunderlich JR. Blood 2002. References: 1. Morgan RA. This process is termed genetic redirection. immunotherapy offers great promise as a treatment for cancer. (Hons) Supervisor: Dr. This library approach should enable the rapid testing of many hundreds of different receptor formats to select the best for cancer immunotherapy. effective lymphocytes specific for tumour antigens are often not present within the immune repertoire of patients.The goal of our laboratory is to develop effective immunotherapies for cancer. investigators are continuing to try different signalling domains in the cytoplasmic region of chimeric genes. Science 2006. PhD or B.2. Hence. We are seeking students for B. CD27 and 41BB.100: 3155-63. Phil Darcy Adoptive immunotherapy involving genetic modification of T cells with TCRab genes or single-chain (scFv) chimeric receptors is emerging as a promising approach to specifically direct their activity toward tumour cells1. cytokine and proliferation assays. in which final preparations of genes and cells are made ready for use in clinical trials. in which different immune system cells are used including natural killer cells. the development of other treatment options is a high priority.5:928-40. that have been successfully used to redirect T cell function against tumour cells. 2 Kershaw MH. and despite advances in the major treatment options such as surgery.(Hons) and/or PhD studies into the following projects: GENERATING SUPER CANCER-KILLING LYMPHOCYTES USING A RECOMBINANT COMBINATORIAL LIBRARY. Peter Mac Page 7 of 21 May 2010 . Phillip Darcy Approximately 1 in 3 Australians develop cancer at some stage in their lives. Nat Rev Immunol 2005. In order to enhance the activity of T cells against tumour cells. Darcy PK. The student will also become competent in tissue culture (retroviral transduction of T cells and tumour cells) and handling of mice. Gene constructs. et al. Nevertheless. Indeed several studies in patients have shown a good correlation between persistence of adoptively transferred T cells and their anti-tumour effects. (2) The genetic modification of human T cells using this library and retroviral vector technology. immune system cells protect us from a formidable onslaught of infectious disease throughout our lives. where molecular biology techniques are employed to create the most potent cancer killing molecules. (3) Comparing the functional responses of genetically modified T cell clones against tumour cells using cytotoxicity and cytokine secretion assays. (4) Clinical translation. 3 Haynes NM. A potential way to improve this approach is to increase their survival and persistence. the aim of the current research project is to construct a recombinant library of T cell signalling molecules and use it to genetically modify human T cells to endow them with enhanced anti-cancer function. we propose to test whether incorporation of these various signaling molecules from the TNF receptor family into our existing chimeric receptor design (tripartite receptor) can enhance survival and anti-tumour activity of gene-modified T cells. Reference of interest: MH Kershaw et al.Sc. Signaling molecules from the TNF receptor family that include OX-40. (3) Combination therapies. genetic modification of T cells can be used to generate lymphocytes with the ability to recognize and respond against tumour cells. the magnitude of the T cell response has been suboptimal and does not approach that possible against non-self antigens such as viral proteins. Recently we have shown that chimeric receptors comprising the co-stimulatory CD28 signaling domain linked in tandem with the CD3-z domain (scFv-CD28-z) could optimally trigger T cell function in vitro and in vivo3. despite some success of genetically redirected T cells against tumour cells. Professor Michael Kershaw and Dr. these gene-engineered T cells were less effective against established subcutaneous disease. The latest chimeric formats link scFv through hinge and transmembrane regions to several signalling domains including the zeta chain of the CD3 complex and the costimulatory domain from CD28. Supervisors: Assoc. et al. are often chimeric and encode extracellular domains consisting of single-chain antibody (scFv) specific for tumour antigen linked to intracellular domains able to trigger T cell activation. To overcome the limitations of this methodology. Nature Reviews Immunology 2005 Dec. sequencing. play an important role in both survival of T cells following activation and generation of an effective memory response.4. Teng MW. ELISA. (2) Cell selection. Clearly. Teng MW. There is considerable power in the many billions of circulating blood cells that comprise the immune system.5(12):928-40 GENERATION OF A TRIPARTITE CHIMERIC SINGLE CHAIN RECEPTOR FOR ENHANCING CANCER THERAPY. Studies in the lab can be divided into four main areas: (1) Anti-cancer gene design. almost 50% of cancer patients die of their disease. 2011 Project Summaries. previous methods involve selection of candidate molecules followed by genetic cloning and functional characterisation in T cells. However. We are looking for a highly motivated student who is interested in developing effective treatments for cancer. Indeed. where gene-modified immune cells are combined with drugs or vaccines to produce the optimal anti-tumour treatment. The project will involve a number of molecular and biochemical methods including DNA cloning. in this project.

For more information about this project contact: 2011 Project Summaries. As single agents. Second. et al. cell culture. In addition. The techniques used will include working with mouse models. these are either currently in use for the treatment of MM or are in clinical development. Ricky Johnstone. These studies will assess the importance of genetic information in guiding the treatment of human AML and determine if genetically engineered mouse models of human cancer can accurately predict therapy response in patients. For more information about this project contact: Prof. isolated and functionally characterized.org DEVELOPMENT OF GENETICALLY ENGINEERED MOUSE MODELS OF AML TO STUDY TUMORIGENESIS AND RESPONSE TO NOVEL THERAPEUTICS. particularly among 20-40 year-olds where it is the most common cause of cancer death. Tel: 9656 3727. ricky. or bortezomib and MD5-1 can cure mice bearing cancers that are resistant to either agent used as a monotherapy. As a result. and strategies explored to prevent oncogenesis in different contexts. Tel: 9656 3727. Trapani JA. a combination of bortezomib and HDACi has been shown to synergistically kill a variety of tumor cell lines. the incidence of melanoma is increasing in Australia and deaths attributable to the disease are projected to increase accordingly. Using sophisticated mouse models. microarray-based gene expression studies.org Email: MELANOMA RESEARCH LABORATORY Melanoma is a common cancer and a source of significant morbidity and mortality in our community. Supervisor: Prof Ricky Johnstone The genetic heterogeneity of cancer influences the trajectory of tumor progression and may underlie clinical variation in therapy response. For more information about these projects contact: Prof. Compounding the increasing disease and economic burden imposed by melanoma in our community is the lack of effective therapies for patients with advanced disease. Peter Mac Page 8 of 21 May 2010 . melanoma causes the second/third most years of lost productive life of all cancers in males/females. Ricky Johnstone. through use of a highly efficient model of human melanoma progression that replicates closely the biology of this disease in patients. Disturbingly. melanocytes at different stages of development will be conditionally tagged. often through the aberrant recruitment of epigenetic modifying enzymes such as histone deacetylases and methyltransferases. We and others have recently demonstrated that a combination of vorinostat and the agonistic antiTRAIL receptor monoclonal antibody (mAb) MD5-1. Complementary studies of human melanocyte development will also be performed. The primary aims of this project are to utilize a novel mouse model of MM to assess the therapeutic effects of combining novel tumor cell-selective apoptosisinducing small molecules and biological agents. through improving understanding of normal melanocyte development. we aim to identify mechanisms of melanomagenesis and thereby develop strategies for improved disease prevention. Supervisor: Prof Ricky Johnstone Multiple myeloma (MM) is an incurable disease and there is clearly an unmet medical need for new therapeutic options. MLL is fused with one of over 60 distinct partner genes through chromosomal translocations in various human acute leukemias. CHARACTERIZATION OF NORMAL MELANOCYTE DEVELOPMENT Supervisors: Dr. Moreover. Moreover. resulting in the formation of multiple MLL fusion proteins (MLL-FPs). The agents under investigation are the histone deacetylase inhibitor (HDACi) vorinostat. The close links between our research program and the clinical research activities of the Peter Mac Melanoma Unit enable rapid clinical translation of our lab discoveries in order to help patients. The oncogenic effects of various genetic and environmental stimuli on melanocyte lineage subpopulations will then be studied. we aim to identify mechanisms of melanoma propagation and metastasis.johnstone@petermac. these mouse models will be used to test the efficacy of epigenetic modifying agents such as histone dacetylase inhibitors and histone methyltransferase inhibitors as well as conventional chemotherapeutic drugs. To model such heterogeneity. and quantitative PCR. bortezomib and activators of the TRAIL pathway used alone or more likely in combination will provide therapeutic benefit for patients with MM. Using retoviral gene transduction of hemopoietic stem cells to express diverse MLL-FPs we will produce mice that develop AML driven by different ongogenic fusion proteins. GENE REGULATION LABORATORY DEVELOPING NEW THERAPEUTIC STRATEGIES TO TREAT MULTIPLE MYELOMA. We will additionally dissect the molecular and biological events that underpin the single agent and combination therapy effects. the proteosome inhibitor bortezomib and activators of the TRAIL death receptor pathway. we aim to produce genetically and pathologically accurate mouse models of common forms of human acute myeloid leukemia (AML) expressing oncogenic fusion proteins involving the mixed lineage leukemia gene (MLL). Mark Shackleton and A/Prof Grant McArthur This project will adapt classical stem cell biology techniques developed in other solid organ systems (Nature 439:84) to the study of normal melanocyte development.4 Haynes NM. Teng MW. We hypothesise that novel anti-cancer agents such as vorinostat. First. These mice will be utilized to study disease onset and progression and determine the oncogenic potential of different MLL-FPs.johnstone@petermac. J Immunol 2002. MLL-FPs are capable of leukemic transformation and dysregulation of multiple genes.169:5780. Email: ricky. Research Division. Our research program seeks to address the problem of melanoma using two approaches. flow cytometry and cell sorting. we propose that a detailed understanding of the molecular mechanisms of action of novel anti-cancer agents will lead to the development of rational combination strategies that will provide significantly greater therapeutic benefit than single agent therapy.

Nature 456:593. By comparing the malignant and molecular properties of sister clonal tumors. Cell 138:822. Email: mark. et al. PNAS. Efficient tumor formation by single human melanoma cells. More recently. References relevant to these projects: Quintana E. Email: mark. and have generated exciting preliminary data to indicate the involvement of polarity in these processes. Mark Shackleton. and molecular studies such as SNP genotyping. Vaillant F. NextGen sequencing and functional genomics. and is controlled by a group of proteins including the Scribble and Par3 complex. The project involves a wide range of techniques. The project will involve some animal experimentation. This assay offers a unique opportunity to study human cancer biology.org of lymphocytes. By orchestrating cell fates such as self renewal potential.oliaro@petermac. mouse handing and surgery. 2006). CD46 has previously been shown to interact with members of the polarity network. Tel: +61 3 9656 1657. Science. 2007) . to determine whether the polarity proteins influence the development or progression of leukemia. and to establish how phenotypes in these mice relate to lymphocyte polarity. DNA methylation analysis.Dr. function and fate in human T and NK cells (Oliaro et al. in the regulation of asymmetric cell division of T lymphocytes. et al. Shackleton M. including measles virus.org THE ROLE OF SIGNALING AND POLARITY PROTEINS IN ASYMMETRIC CELL DIVISION OF T LYMPHOCYTES Supervisors: Dr Jane Oliaro and Dr Sarah Russell Our laboratory studies the role of signaling and polarity proteins in T lymphocyte biology. Shackleton M. Generation of a functional mammary gland from a single stem cell. Peter Mac Page 9 of 21 May 2010 . For more information about this project contact: Dr. Jane Oliaro. whether signaling through CD46 affects this process. Sarah Russell. a process termed asymmetric cell division (ACD). PhD projects are available to use these mouse models to investigate the effects of deletion of individual polarity proteins on the development of hematopoietic cells. This observation provides a potential mechanism for generating the diversity of T lymphocytes required for an effective immune response. CD46. Email: sarah. Polarity . we demonstrated that asymmetric cell division of T lymphocytes in response to antigen presentation may be used to generate effector and memory T lymphocytes (Chang et al. including animal experimentation. and suggests that a conserved mechanism based on asymmetric cell division also exists in immune cells. The project will focus on how polarity proteins control asymmetric cell division. control of ACD can play important roles in the initiation and prognosis of epithelial cancers. this approach enables direct correlation of tumor phenotype and genotype/epigenotype in a way that is likely to reveal those molecular aberrations that are functionally relevant to malignant progression and potentially target-able by modern therapeutic approaches. Mark Shackleton. immunological synapse formation and cytotoxic activity during an immune response. For more information about this project contact: Dr. Cancer cell heterogeneity: clonal evolution and cancer stem cells. ACD is classically considered to occur in the cells of solid tissues. Multiple projects within this framework are planned. Tel: 9656 5235. multi-parameter flow cytometry and state-of-the-art microscopy. We have now developed a number of mouse models with which to test the role of polarity and asymmetric cell division in hematopoiesis. Mark Shackleton and A/Prof Grant McArthur This project will use a novel human melanoma tumorigenesis assay (Nature 456:593) to study how melanomas progress once they have formed. and signaling through CD46 can affect T lymphocyte polarity. and both fixed and live imaging of cells using our state of the art microscopy facilities. Quintana E. immunity and leukemogenesis. Tel: 9656 5235. Morrison SJ. and what the consequences are for T lymphocyte fate and function.shackleton@petermac. Nature 439:84. Recent studies from a number of groups have led to a working model by which these genes exert tumour suppressive effects by coordinating ACD of epithelial cells. involving a wide range of techniques: working with fresh human tumor specimens and analyzing clinical data. immunostaining and flow cytometry.a process regulated by polarity proteins in other cell types. Our laboratory has recently obtained exciting new data to indicate that a similar process controls the fate decisions 2011 Project Summaries. genetics and epigenetics at the clonal level.is critical for T lymphocyte functions such as migration.shackleton@petermac.russell@petermac.org IDENTIFICATION OF DETERMINANTS OF MELANOMA PROGRESSION Supervisors: Dr. For more information about this project contact: Dr. An Honours project is available to investigate the role of polarity proteins and the cell surface receptor. many of which have been identified as tumour suppressor genes in Drosophila. Shackleton M.org IMMUNE SIGNALLING LABORATORY THE ROLE OF POLARITY PROTEINS AND ASYMMETRIC CELL DIVISION IN LYMPHOCYTE FATE DECISIONS AND CANCER Supervisor: Dr Sarah Russell Cellular diversity during development is often generated through the segregation of cell fate determinants into one daughter cell upon cell division. immunological techniques such as tissue culture and flow cytometry. Tel: +61 3 9656 3727. Research Division.or the compartmentalisation of proteins within a cell . Email: jane. and pathogens that bind to CD46 may utilise this to alter T lymphocyte responses. CD46 is a receptor for a number of pathogens.

this project focuses on understanding the role of regulatory T and Th-17 cells in myeloma patients prior and during therapy. Uniquely. and even evade chemotherapy. and mouse bone marrow transplantation. The research seeks to answer questions in humans regarding the pathways to malignant cell death and the successful induction of an anti-tumor immune response for on-going disease control. We also demonstrated a significant increase in regulatory T cells during therapy. the HITRL lab collaborates with CIR program groups to translate basic therapy concepts in the mouse. The HITRL lab performs research on patients receiving novel immunotherapeutics for a range of hematological malignancies. but combination immunotherapy is required to maintain long-term immune control over residual disease. the HSC has an intrinsic cell orientation (or polarity). Email: stephen. fabricate and integrate the microstructures and valves. Tel: +61 3 9656 3576. Our project aims to understand the molecular pathways that govern HSC self-renewal with the longer-term goal of applying this knowledge to the clinical settings of ex-vivo HSC expansion for bone marrow transplant and impairing cancer cell growth. A range of microfabrication techniques will be used to design.org DEVELOPMENT OF A MICROFLUIDIC BIOREACTOR USING NOVEL DYNAMIC VALVING FOR CELL MANIPULATION Supervisor: Dr Sarah Russell The elucidation of biological signalling pathways for diverse processes such as cancer and immunity has recently been revolutionized by access to highthroughput functional genomic screens. CD4 T CELL MYELOMA POLARIZATION IN MULTIPLE Supervisors: Dr Paul Neeson & Assoc Prof David Ritchie Our recent studies of multiple myeloma patients during lenalidomide and dexamethasone therapy have highlighted the importance of the CD4 T cell compartment in mediating cellular driven anti-myeloma responses. In addition. The student will primarily work at the Centre for MicroPhotonics. lenalidomide is a potent inhibitor of IL-6 production in the myeloma bone marrow microenvironment.russell@petermac. while another group has reported an increase in another subset of regulatory cells (called Th-17 cells) in myeloma patients. CO2 laser cutting. The novel immunotherapeutics are derived from either the CIR program or from biotech/pharmaceutical companies. Such screens have created a currently unmet need for microfluidics devices that will enable very high throughput (parallelism) and fast analysis time. regulatory T cells engage in a reciprocally regulated relationship primarily guided by the presence or absence of IL-6.” Specific Aims:1. The HITRL lab has direct access to patient samples collected during the course of trials with immunotherapeutics and uses these samples to answer questions regarding disease and immune system response. Tel: +61 3 9656 3727. COMBINATION IMMUNOTHERAPY IN LONG-TERM IMMUNE CONTROL Supervisors: Dr Paul Neeson & Assoc Prof David Ritchie “That anti-CD20 plus CHOP chemotherapy are required to regress spontaneous DLBCL tumours in transgenic mice. we have identified two genes. This project will investigate the molecular details by which these two genes achieve this change in both mouse and human HSC fate using cell culture. Sarah Russell. through pre-clinical models and into human clinical trials.POLARITY AND ASYMMETRIC CELL DIVISION IN HAEMATOPOIETIC STEM CELL SELF-RENEWAL Supervisors: Dr Stephen Ting and Dr Sarah Russell The daily and lifelong regeneration of the blood system is dependent on haematopoietic stem cells (HSCs). The aim of this project is to fabricate microfluidic devices with which to control and monitor the response of cells to genomic and pharmacological intervention. 2. 2011 Project Summaries. immunotherapeutics have been trialed in preclinical models before translation into the clinic. For more information about this project contact: Dr. and their effect on the patient’s immunological response to myeloma and hence patient outcome post therapy. live cell microscopy. Email: sarah. To demonstrate that a Bcl-6 protein vaccine plus agonistic CD40 antibody delays development of lymphoproliferative disease and subsequent DLBCL in the IuHABCL6 mouse. Therefore. however their precise mode of action in humans remains unclear. which have the unique ability to proliferate and differentiate into all blood cellular elements (multipotency) whilst maintaining their HSC identity (self-renewal). Novel mechanisms for trapping and releasing cells based on dynamic valving using pneumatically operated valves will be developed. Peter Mac Page 10 of 21 May 2010 . We shall also initiate studies as to whether these two genes can alter the clinical outcome of various mouse models of blood cancers such as leukemia and lymphoma. hot embossing and soft lithography. Stephen Ting. Via a candidate gene approach focusing on genes that are functional in cell polarity and a novel in vitro to in vivo HSC expansion assay. Research Division. Frequently. isolation of HSCs.org HAEMATOLOGY IMMUNOLOGY TRANSLATIONAL RESEARCH LABORATORY The Haematology Immunology Translational Research Lab (HITRL) is a collaboration between the Cancer Immunology Research program and the clinical Hematology Dept at Peter Mac. Ap2a2 and Gpsm2 that are able to alter HSC fate. To demonstrate that an immune response to Bcl-6 can be raised in the BALB/c mouse and that a Bcl-6 protein vaccine plus anti-CD40 antibody combination therapy can prevent and regress the growth of a Bcl-6 expressing A20lymphoma. During this process. These unique stem cell properties of multipotency and selfrenewal may be a mechanism by which cancer cells are maintained or expand. but will also interact with biologists at the Peter MacCallum Cancer Centre to assess the applicability of the microfabricated devices. A mechanism by which self-renewal can be achieved is via the segregation of cell fate determinants at the time of HSC division. For more information about this project contact: Dr.ting@petermac. including femtosecond pulse laser etching. Interestingly.

Paul Neeson.org 2011 Project Summaries. graft versus tumour (GVT) effect and graft versus host disease (GVHD). To closely dissect the generation.mouse model (Rag-Hu) to explore the above aims. Research Division. These studies will use the recently developed human lymphopoiesis reconstituted Rag2-/-γc-/. Peter Mac Page 11 of 21 May 2010 .neeson@petermac. Professor David Ritchie. Email: david.3. We will examine the generation of central memory T cells derived in the setting of either autologous antigenspecific T cell immunotherapy or allogeneic T cell engraftment. Tel: +61 3 9656 3657. 2. persistence and functional capacity of T cell immune responses.org Assoc. To demonstrate that conventional therapy using a debulking regimen plus combination immunotherapy are required to regress established DLBCL and maintain long-term remission. Email: paul.ritchie@petermac. THE ROLE OF TUMOUR OR ALLOGENIC ANTIGEN IN TUMOR CONTROL Supervisors: Dr Paul Neeson & Assoc Prof David Ritchie “That in vivo re-exposure to tumour or allogeneic antigen is required for development of effector memory T cells (TEM) and central memory T cells (TCM) from adoptively transferred (AT) autologous or allogeneic T cells and is essential for ongoing tumor control. To examine the impact of the co-stimulatory therapies IL-21 and anti-CD137 on the enhancement of antitumor effects of adoptive T cell therapy or on the development of GVHD or both. For more information about these project contact: Dr.” Specific Aims:1.

Loss of tissue architecture and proliferation control are hallmarks of cancer. Two distinct major lines of investigations are currently being pursued in the laboratory. Research Division. Richardson H.org Our laboratory has developed a collaborative research program with Drs Helena Richardson’s and Sarah 2011 Project Summaries. This project aims to translate the information obtained in Drosophila tumour screens to mammalian systems and the study of human cancer. et al. Nature Reviews Cancer. proliferation and migration in 2D and 3D cultures using non-transformed epithelial cell line models.Genetic screens conducted in Helena Richardson’s laboratory have identified a number of genes that can cooperate with scribble mutants or the Ras oncogene to produce tumours in Drosophila.org CELL CYCLE & CANCER GENETICS The focus of the Cell Cycle and Cancer Genetics laboratory has been to elucidate the molecular mechanisms that regulate tissue homeostasis in vivo.org Dr Tony Brumby. Cooperation between loss of cell polarity and oncogenes in epithelial tumour development (see details below). we are investigating how radiation kills cells and ways to improve the application of this therapy to patients. 2003. Second. The project will examine biological activity of mammalian homologues of newly identified Drosophila oncogenes and tumour suppressors in the regulation of cell polarity. Brumby AM. to address fundamental cancer questions: 1. How do cell growth regulators function in tumour progression Our lab is a dynamic lab.richardson@petermac. Bioessays. Second. uses sophisticated genetic and cell biological analysis of the animal model system. the ability of these tumour cells to metastasise through multiple tissue layers. these genes appear to normally coordinate the establishment and maintenance of polarity. Peter Mac Page 12 of 21 May 2010 . discs-large (dlg) and lethal giant larvae (lgl). and b.. The cell cycle and development lab. we are developing a program of research addressing how maintenance of the architecture and polarity of cells within a tissue can regulate cell number and behaviour in vivo. 2003. This will be followed by in vivo studies using sensitised mouse cancer models. In Drosophila. Dow LE. 22:5769-79. Humbert P. 5:626-639. EMBO J. and a molecular understanding of how these processes are disrupted in cancer should lead to the development of novel therapeutic strategies. These studies will provide new insights into the development of mammalian cancer References: 1. Brumby AM. Oncogene. This project will suit those who enjoy working on model systems that can be readily manipulated. 4. For more information about this project contact: Dr. Email: helena. Since radiotherapy is a major treatment modality for cancer. the property of a cell to organise itself into a spatially asymmetric structure necessary for its function.. Email: tony. 25:542-53. the vinegar fly Drosophila. Russell S. Richardson HE.org Email: CELL CYCLE & DEVELOPMENT ANALYSIS OF COOPERATING ONCOGENESIS a. cell proliferation and tumour progression 3. a central issue in normal development and the etiology of cancer. mammalian homologues will be tested for cooperativity with Scribble knockdown or expression of oncogenic Ras in transformation and tumourigenicity in vitro. Tel: +61 3 9656 1466. as a model for human leukemia. 2003 22:9225-30.CELL BIOLOGY RESEARCH PROGRAM Diverse areas of cellular and molecular biology important in cancer are being explored within this program. Brumby AM. CHARACTERIZATION OF SCRIBBLE ASSOCIATED GENES IN CANCER Supervisors: Richardson Dr. 2. Tel: +61 3 9656 3526. 2005. patrick. loss of scrib. How does is cell proliferation regulated in a whole animal 2. dlg or lgl function in epithelial cells gives rise to tumorous overgrowth characterised by loss of polarity. impaired cell cycle exit. the signalling pathways within tumour cells and host cells adjacent to tumours that drive tumour growth and metastasis and the importance of the aberrant regulation of apoptosis in tumour cells.humbert@petermac. Patrick Humbert. Initial studies carried out in our laboratory provide proof of principle that we can exploit Drosophila findings to good effect in mammalian epithelial systems. We are looking at regulation of the cell cycle. these tumours have many of all the hallmarks of human cancer. Cooperation between oncogenes or tumour suppressors in blood cell tumour formation.brumby@petermac. For more information about this project contact: Dr Helena Richardson. Tel: +61 3 9656 1466. Russell’s laboratories to elucidate the role of a novel class of tumour suppressor genes first identified in Drosophila that include scribble (scrib). invasion into adjacent tissue and when transplanted into a novel host Drosophila. Email: helena. AND Helena Dr Helena Richardson. How does the shape of cell affect cell growth.richardson@petermac. Patrick Humbert & Dr. First. Richardson HE. 3. multilayering. Together. with researchers at all stages of their career. In particular. Tel: +61 3 9656 1466. we are examining how proliferative control within a differentiating tissue contributes to tissue homeostasis. Senior postdoc Tony Brumby has extensive experience in working on Drosophila and in supervising students.

One newly identified signaling pathway that our laboratory studies is the Salvador-Warts-Hippo pathway. below). This unique metastasis model represents a significant advance in the field. Research in our laboratory over the past 2 years has led to the development of new tumour lines that aggressively metastasise to brain from the mammary gland. 2004. Fly. Patrick Humbert.org METASTASIS RESEARCH LABORATORY BREAST CANCER METASTASIS TO BRAIN. Clark AJ. This pathway controls organ size by restricting cells from growing and dividing excessively. Zhang et al. For more information about this project contact: Dr. Cell 114. Mills. By studying various aspects of this pathway we aim to understand how organ size is correctly specified during development. Tapon (2007). Harvey et al. Humbert P. However. Clark AJ. References from our laboratory relevant to the project: 1. 137. 172. Current therapies for breast cancer metastasis remain ineffective against brain metastasis. N. References: 1. J Cell Biol. Cell 110.org • • To understand the mechanisms underlying the genesis of human cancers. 457-467. migration and invasion assays). Cancer Res. 8. 467-478. Claire Milton.milton@petermac. In the majority of cases. 95-105. The project will make use of a variety of techniques ranging from basic cell culture and protein chemistry to in vitro functional assays and in vivo animal studies.org Dr. Tel: +61 3 9656 1291. K. 25:542-53. This erythroid culture system now allows for the first time the systematic genetic analysis of the asymmetric division associated with terminal differentiation of erythrocytes. Research Division. (2009). Harvey et al. 7. (2008). Kieran Harvey. Pfleger et al. We have set up a powerful in vitro erythroid differentiation system and used it to characterize the defective asymmetric division (“enucleation”) of Rb-deficient erythroid cells. Blood 104:1324-1326 2. J Cell Biol. the incidence of brain metastasis in advanced breast cancer patients is rapidly increasing due to better disease management and longer survival in affected patients. 6033-6041. lung and liver. For more information about this project contact: Dr. integrin and other receptors expression) and functionally (in vitro adhesion. Bennett and K. Russell S. 691-696. (2010). We also use the fly to study the mechanism by which these genes function. et al. 7. which controls organ size during development (reviewed in reference 5. The full pattern of metastasis in vivo will also be evaluated using PCR and fluorescence-based assays. CHARACTERISATION OF A NEW PRE-CLINICAL MOUSE MODEL Supervisor: Dr. Moreover. Tel: +61 3 9656 3526.F. In addition to bone. 5. Milton et al. K. Harvey (2006). to discover and investigate genes involved in cancer. 2003. 4.humbert@petermac. K.C. Development. will give rise to the functional erythrocyte population required to provide oxygen to the organism. Doyle KM. 6.F. The aim will be to characterise the model phenotypically (cell morphology. Bioessays. 25. K. Tel: +61 3 9656 3621. F. For more information about this project contact: CELL GROWTH & PROLIFERATION THE HIPPO SIGNALLING PATHWAY IN ORGAN SIZE CONTROL AND HUMAN CANCER Supervisor: Dr. X. properties central to the formation of cancer. Peter Mac Page 13 of 21 May 2010 . 809-815. Kieran Harvey In the Cell Growth and Proliferation laboratory we use the model organism. (2007). (2003). The Salvador-Warts-Hippo pathway is conserved in humans and several studies from our laboratory and others have implicated this pathway in the genesis of human cancer.RESEARCH AIMS: HOW DO RED BLOOD CELLS ENUCLEATE? Supervisor: Dr. the molecular pathways regulating metastasis to brain are largely unknown. These studies will provide fundamental insights into the regulation of erythroid differentiation. 1. Email: kieran. Drosophila melanogaster (vinegar fly). To understand the mechanism by which different signaling pathways specify the size of developing organs.F. 3. the “enucleated” daughter cell. due in part to the lack of clinically relevant model in which to study disease progression. 69. Doyle KM. Richardson H.harvey@petermac. Nat Rev Cancer. Patrick Humbert Project description .The terminal differentiation of erythroid cells provides an extreme example of asymmetric division where the final division gives rise to one daughter cell containing all of the genetic material whilst the other daughter cell. (2006). C. Email: patrick. C. Harvey and N.M. proliferation. 2101-2110. Normand Pouliot Breast cancer affects 1 in 10 women in Australia and is the leading cause of cancer related death. death is due to the development of secondary tumours compromising the function of distant organs. For the first time we have the opportunity to identify genes specifically associated with metastasis to brain and a clinically relevant model in which to test the function of selected metastasis genes and/or novel therapies. Approximately 70% of human disease genes are conserved in flies. Email: claire. 16. Humbert PO. 2011 Project Summaries. brain is a common site of metastasis in advanced breast cancer patients. Our approach is to identify genes involved in cancerouslike growth in flies and then use human and mouse models to determine whether the human counterparts of these fly cancer genes have a role in human cancer. This project aims to characterize erythroid “enucleation” and to use pharmacological and RNA interference approach to identify the molecular pathways involved in this terminal asymmetric division. and how deregulation of this pathway contributes to human cancer. Curr Biol. 735-43. 9.F. 182-191. making it an excellent model for these studies. Iazzolino RM and Humbert PO (2006) Blood 108:886-895 3. 2. (2002). Tapon et al.

pericyte involvement can predict cancer progression and disease-free survival in patients despite treatment with current cancer therapies. it is also clear that stem cell properties such as selfrenewal and tissue replacement can be enhanced by cellular and molecular regulators found in the microenvironment. Tel: +61 3 9656 1285. Tel: +61 3 96561285. Our recent studies using a unique model of breast cancer have revealed that cancer cells growing in bone suppress an immune defence pathway called the Type I interferon pathway. Pritinder Kaur. invasion and proliferation).anderson@petermac.org CHARACTERISING THE FUNCTION OF LAMININ-511 RECEPTORS IN BREAST CANCER METASTASIS Supervisors: Dr. Normand Pouliot and Dr Robin Anderson Our laboratory focuses on the identification and characterisation of genes involved in the spread of breast tumours to distant organs (metastasis). Investigate the role of specific candidate molecules synthesised and secreted by pericytes identified in our laboratory. pericytes. Our studies provide key evidence that breast cancer cells have the ability to modulate the immune response to avoid being recognised and eliminated. Email: normand. We are looking to extend these studies. The mechanisms of breast cancer spread to bone are largely unknown. Human skin epidermis is a constantly renewing tissue. although this remains poorly characterized. that appears to play an important role in this process (1). These activities are dependent on the interaction of tumour cells with laminin511 via cell surface receptors called integrins. For more information about this project contact: Dr Belinda Parker. 2005 2. References: 1.. confocal and light microscopy. molecular skills (RNA inhibition. Tel: +61 3 96561284. flow cytometry. In epithelial cancers.Dr. laminin-511 promotes cell adhesion. some basic protein chemistry (SDS-PAGE. ovarian and breast cancer. isolated using specific antibodies and flow cytometry can enhance the proliferative capacity of human keratinocyte stem cells and their committed progeny independent of angiogenesis. and Dr. real time PCR.. real time qPCR). A dermal cell type i. Normand Pouliot. including histological analysis. This allows breast tumour cells to survive in bone and grow into lethal tumours that are currently untreatable. Peter Mac Page 14 of 21 May 2010 . Tel: +61 3 9656 1285. Email: robin. Technically. Research Division. The second part of the project will seek to characterise/identify the novel α3bx integrin dimer using standard protein chemistry approaches. Email: belinda. Holger Schlüter. that will improve the growth of skin cells. cell culture. migration and protease-dependent invasion thereby facilitating homing of tumour cells to bone and other organs in vivo (2). However. Our lab recently demonstrated in long-term tissue reconstitution assays that the greatest capability of tissue reconstitution resides within the candidate keratinocyte stem cells. Two projects are available for PhD students with the following aims: 1. and that restoration of this pathway blocks cancer spread. immunoblots) and animal work.org MECHANISMS OF IMMUNE ESCAPE BY BREAST CANCER CELLS TO ALLOW SURVIVAL AND SPREAD TO BONE Supervisors: Dr Belinda Parker and Dr Robin Anderson Over 80% of patients who die from their breast cancer succumb due to the development of metastatic disease. cell 2011 Project Summaries. 2. More recent unpublished work implicates the α6β4 integrin dimer and a potentially novel integrin composed of the α3 subunit and an unknown β partner (α3βx). In the course of our studies we have identified a basement membrane protein. WOUND HEALING AND CANCER IN HUMAN SKIN Supervisors: Dr. In culture. The primary aim of this project will be to down-regulate the expression of α6β4 integrin receptor in highly metastatic cells by RNA inhibition and to characterise the functional consequence of receptor down-regulation in in vitro assays and on metastasis in vivo. A broad range of techniques will be utilised for this PhD project. Recent work from our laboratory identified several integrins mediating breast tumour cell adhesion but the critical integrin mediating metastasis to bone remains unclear (2).org EPITHELIAL STEM CELL BIOLOGY EPIDERMAL STEM CELLS AND THEIR MICROENVIRONMENT: INVESTIGATION OF THE MOLECULAR REGULATORS OF NORMAL SKIN REPLACEMENT. in vitro and in vivo metastasis assays.pouliot@petermac. Through both national and international collaborations. Chia et al. 2007 For more information about this project contact: Dr. immunohistochemistry and fluorescence imaging. Both projects use techniques routinely employed in our laboratory such as tissue culture of primary cells. This project aims to identify the immune responses that are activated in response to this pathway and if restoration of such responses is critical in blocking the spread of breast cancer to bone. Investigate the role of the same molecules secreted by pericytes that promote tumour growth in models of skin. laminin-511. where the combined action of relatively quiescent keratinocyte stem cells and their committed progeny undergo tightly regulated proliferation to replace normal skin tissue.pouliot@petermac. the project will make use of a variety of laboratory skills including standard cell culture and in vitro functional assays (adhesion. Normand Pouliot.e. A hallmark of stem cells is the capacity to regenerate the tissue of origin for a prolonged period of time and to constantly self-renew.org Dr Robin Anderson. animal models of breast cancer. migration. Eckhardt et al. Email: normand. we have access to rare models of breast cancer and the tools required to study the role of Type I IFNs at an internationally competitive level.parker@petermac. immunoprecipitation.

Tel: 9656 3713. Published studies demonstrated a pronounced effect of methylproamine analogue Hoechst 33342 on the DNA synthesis and cell cycle progression. standard molecular and cell biological methods like cloning or SDS-PAGE.g. and subsequent irradiation in different phases of cell cycle (G0/G1.org Dr. and in this context. has identified new. Dr Alesia Ivashkevich & Professor Roger Martin The normal tissue damage associated with radiotherapy has motivated the development at Peter Mac of a new class of DNA-binding radioprotecting drugs that could be applied topically to normal tissues at risk. Tel: roger. For example the bacterial enzyme formamidopyrimidine DNA-glycosylase (FPG) recognises oxidised base lesions such as 8-oxo-7. Many of these questions concern the mechanism of radioprotection.martin@petermac.org Email: Email: MOLECULAR RADIATION BIOLOGY PROTECTION DAMAGE BY ANALOGUES OF RADIATION-INDUCED METHYLPROAMINE AND • using the plasmid model and DNA in cultured cells. what are the cellular subpopulations that are responsible for radiation-induced toxicity. Therefore the efficiency of radioprotection of cells irradiated in different phases of cell cycle may provide information on mechanisms of radioprotection. Understand the role of stem cells and their environment in promoting wound healing 4. Endonuclease III (ENDOIII) recognises oxidised pyrimidines. as well as microscopy (light and electron) and mouse models of tissue regeneration. improved analogues. In the single cell comet assay. While protecting cells from lethal consequences of irradiation. Cytotoxicity and radioprotection by methylproamine and analogues will be evaluated by assay of clonogenic survival. apparently by reducing radiation-induced DNA damage. The results of these studies will have the following impact on human skin biology: 1. S. Research Division. For studies of DNA breaks in cultured cells. Pritinder Kaur. In the context of radioprotection of normal tissues such as gastrointestinal mucosa and skin. holger. In the proposed project. and proof-of-principle of topical radioprotection has been established in mouse oral mucosa. Radiobiology of radioprotection. Our research program has raised a number of more fundamental questions each of which represent potential post-graduate research projects.kaur@petermac. Understand how stem cells escape from regulatory mechanisms that prevent cancer development 3.org 9656 1290. 8dihydro guanine (8-oxoGua) and inserts a DNA single-stranded break at the site of damage. whereas FPG recognises a variety of different base lesions. This project would involve studies of DNA damage and protection in both purified DNA • For more information about this project contact: Professor Roger Martin. ovarian & breast For more information about this project contact: Dr.g. These changes may result in toxicity or affect the repair of radiation induced DNA damage. Methylproamine. A continuing program of lead optimization. Email: 2011 Project Summaries. Two-dimension alternating electric field provides separation of large DNA molecules that is not achievable in conventional gel electrophoresis. G2/M). in collaboration with Associate Professor Jonathan White at the University of Melbourne School of Chemistry. In parallel with measurement of radiation induced DNA strand breaks. In pulsed field gel electrophoresis experiments. Tel: 9656 3714. DNA-binding radioprotectors may cause toxicity and other cellular effects at higher concentrations. The conventional agarose gel electrophoresis will be used to assay DNA breaks in plasmid model. the pulsed field gel electrophoresis and the single cell gel electrophoresis (comet assay) will be used. Increase our understanding of how stem cells and their environment manage to routinely replace skin cells 2. It is also well established that cells in different phases of cell cycle exhibit various sensitivity to ionising radiation. the lead compound. burns patients. The mechanistic questions/issues include: • Protection of radiation-induced DNA damage by methylproamine. Subsiduary questions concern the spectrum of chemical lesions involved ( eg strand breaks and various types of base damage). The project will involve synchronisation of cells by contact inhibition or nutritional starvation and further release into fresh medium. Extension of the evidence in support of the hypothesis that radioprotection is due to repair of initial radiation-induced DNA damage. pritinder. and which of these are susceptible to radioprotection? Cell cycle dependent radioprotection and effect of methylproamine on cell cycle progression. various types of DNA base damage will be assayed by subjecting irradiated DNA with various endonucleases that convert base lesions to breaks. Improve current methods for expanding skin cells for transplantion onto patients with large skin deficits e. human 8-hydroxyguanine DNAglycosylase 1 (hOGG1) is specific for 8-oxoGua. the effect of methylproamine and other analogues on the cell cycle progression of non-irradiated cells will be investigated using FACS (fluorescence activated cell sorting) technique. including thymine glycol and uracil glycol. Pavel Lobachevsky. 5. Peter Mac Page 15 of 21 May 2010 .sorting using a fluorescence-activated cell sorter. Finally. our collection of methylproamine analogues with a range of radioprotective efficacies are an important resource.schlueter@petermac. Improve diagnosis of patients with aggressive epithelial cancers e. the DNA electrophoresis profiles are obtained for individual cells embedded in the gel using fluorescence microscopy. Holger Schlüter. Supervisors: Dr. cells are embedded in agarose plugs before irradiation and DNA extraction to prevent DNA damage associated with DNA handling in solution. and the extent to which each of these is “repaired’ or suppressed by DNA-bound radioprotector. cancer and wound healing. reduces radiation induced cell kill at low concentrations.

Tel: 9656 pavel. the conjugate-receptor complex is degraded. biodistribution in an animal model. namely phosphorylation of histone H2AX to form γ-H2AX. These ligands have the iodine atom positioned at varying distances from the axis of the DNA helix.martin@petermac.org Exploration of various new targeting protein/receptor systems for tumour imaging/therapy. Dr Alesia Ivashkevitch & Professor Roger Martin OF In the context of the radioprotector program described above.org 1357. The experimental work will involve radio. intracellular distribution. Tel: 9656 pavel. and this will be evaluated as the basis for an assay of radiosensitivity of radiotherapy patients. The assay is facilitated by software developed by Dr Lobachevsky for automatic counting of γ-H2AX foci. Our ultimate ambition is to apply and translate the knowledge we are 2011 Project Summaries. Email: Email: Dr. The focus of this strategy is a conjugate of the radioactive DNA ligand linked to a tumour targeting protein specific for an appropriate cell surface receptor. it has obvious application for design of individualised radiotherapy. 1357. Adoption of the general strategy for imaging-only applications requires re-design of the DNA ligands so that the iodine atom is positioned away from the DNA helix so as to minimise DNA damage (as distinct from maximising DNA damage for combined therapy/PET imaging objective). Email: THE POTENTIAL OF DNA LIGANDS LABELLED WITH AUGER-EMITTERS FOR ENDORADIOTHERAPY AND IMAGING OF TUMOURS. Pavel Lobachevsky. If the assay is validated. and quantitation of the relative amounts of intact. The experimental work will involve: • preparation of 125I-labelled DNA ligands. Email: Email: • • • • Dr. Peter Mac Page 16 of 21 May 2010 . with release of the labelled ligand and its translocation and binding to nuclear DNA. This project involves collaborators at Peter Mac (Professor Stephen Fox and Associate Professor Trevor Leung) and NIH in USA (Drs William Bonner and Olga Sedelnikova). but a more recent development is the integration of this requirement into a general tumour targeting strategy.lobachevsky@petermac.and conjugation chemistry techniques.org EVALUATION OF RADIOSENSITIVITY RADIOTHERAPY PATIENTS. radiotoxicity. Pavel Lobachevsky.martin@petermac. purification of the radioactive ligands by preparative HPLC incubation of labelled ligands with plasmid DNA agarose gel electrophoresis of the plasmid DNA samples. Fluorescent foci are detectable in cells by immunofluorescence using labelled γ-H2AX-antibodies. 1357. In collaboration with the CRC for Bioimaging development. Research Division. Pavel Lobachevsky. Professor Roger Martin Auger-emitters are a special class of radioisotopes characterized by highly focused radiochemical damage. Somatostatin receptors are overexpressed in a range of tumours. Supervisors: Dr. Preliminary studies have established a radiation doseresponse for isolated human lymphocytes irradiated ex vivo. particularly DNA double-strand breaks (DNAdsbs). For more information about this project contact: Professor Roger Martin. Radiolabelled octreotide has been used for imaging somatostatin receptor-positive tumours. For more information about this project contact: Professor Roger Martin. by iododestannylation of the trialkyl-tin precursors synthesised by collaborators in Associate-Professor Jonathan White’s lab at Bio21. as well as in conjunction with Auger therapy. linear and nicked plasmid species.lobachevsky@petermac. headed by Professor Rod Hicks. Tel: 9656 pavel. Pavel Lobachevsky. As well as targeting the radioisotope to DNA for maximal cytotoxic damage. and computation of the DNA breakage efficiency (DNSdsbs per decay) for each labelled ligand. Pavel Lobachevsky. Potential opportunities for post-graduate reseach projects are in two distinct arenas: Design and evaluation of new labelled DNA ligands. retains binding affinity to somatostatin receptors and is much more resistant to degradation than somatostatin. The combination of both positron and Auger electron emission in the decay scheme of 124I presents very special opportunities. It is suggested to use iodine-125 as Auger emitting isotope due to its large half-life time (60. the DNA acts as a “sink” to accumulate labelled ligand during multiple cycles of receptor-mediated endocytosis. This project will focus on a new ligand designed to exhibit minimal DNA damage. The overall aim of the program is to establish the relationship between distance of the iodine atom from DNA and the extent of DNA breakage.4 days) and convenience for research purposes. a synthetic analogue of somatostatin. as well as cell culture based receptor binding and clonogenic assays. After internalisation. The assay is based on a very early event in the response of cells to radiation-induced DNA damage. The general requirement to position an Auger-emitter very close to DNA to fully exploit the cytotoxic potential of Auger emission is well-established. Supervisors: Dr. In collaboration with Dr Jonathan White at the University of Melbourne’s School of Chemistry. somatostatin receptor system and octreotide derivatives as targeting peptides. The aim of this project is to conjugate the DNA ligand labelled with an Auger electron emitting isotope to octreotide and investigate biological properties of the conjugate such as affinity to somastotatin receptors. within minutes after irradiation. Tel: roger. new iodinated DNA ligands are being designed and synthesised.org 9656 1357.lobachevsky@petermac. a relatively new assay of radiation-induced DNA damage has been established in the lab. The positron-emission feature of 124I enables this “sink” effect to be exploited in the context of PET imaging generally. purification of labelled ligands and conjugates by HPLC. Tel: roger. Octreotide. the current emphasis is on the TUMOUR SUPPRESSION LABORATORY The major goal of our research is to identify the key regulatory nodes in tumour suppression.org 9656 1357.Dr.

Haupt. Alkalay-snir. Haupt. Email: ygal. suggesting that tumour suppression by PML depends on the status of p53 (Haupt et al. Surprisingly. Two projects are outlined below. A.. M. For more information about these projects contact: Assoc. Y. Alsheich-Bartok.... This link suggests that deregulation of E6AP will affect tumour development by deregulating tumour suppression. Maya... we have now shown that PML also activates the function of mutant p53. THE ROLE OF THE E3-LIGASE E6AP IN CANCER DEVELOPMENT. Cancer Research 69. are controlled in normal and cancer cells. but other projects are available including those utilizing genomic and proteomic approaches. Nature 387. I. The aim of this project is to explore the regulation of PML by mutant p53. the nature of this interaction is profoundly distinct from its wild type counterpart.. M. Damalas. To better treat cancer we need to understand how tumour suppressors. 2. S.. Muller. In an insidious manner the mutant form of p53 acts to promote cancer. Alsheich-Bartok. Mizrahi. K. Y. Appella. but also confer new oncogenic properties (gain of function) that render growth advantage to the cancer cell. Blandino. R. MDM2 promotes the rapid degradation of p53. 2009). For this purpose we are developing mouse models in which E6AP expression will be either eliminated. Peter Mac Page 17 of 21 May 2010 . (in press). Y. The second part of the project will focus on exploring the mechanisms that are involved in this regulation. 3. 2009).. and Haupt.. Cell Death Diff. such as p53. such as damage to DNA. Silberman. Promyelocytic Leukemia Protein is required for the gain of function by mutant p53. We showed that PML is essential for the oncogenic activities of mutant p53. Wt p53 activation is critically dependent upon interaction with partner proteins. and to define its significance to cancer development in human cancer and in mouse models of cancer. and Oren. The project will also involve testing the role of E6AP in cancer development in animal models.. p53 and PML. We previously explored the mechanism controlling the stability of the p53 protein (Haupt et al. T. This would be achieved by measuring the status of PML and its function in cancer cells expressing or lacking mutant p53. Significantly. Louria-Hayon. PML is an activator of wild type p53 (Alsheich-Bartok et al. (1997). Alsheich-Bartok.. More recently. Scheffner. Saito.. This will include measurement of mRNA as well as protein levels and localization. our understanding of how mutant p53 imposes its oncogenic functions. and Haupt. E6AP promotes the degradation of the PML tumour suppressor. 1997). REGULATION OF THE TUMOUR SUPPRESSOR PML Supervisor: Professor Ygal Haupt A key regulator of p53 is the tumour suppressor promyelocytic leukemia protein (PML). Y. Y. (2008).. O. (2009). However. and Haupt. Supervisor: Professor Ygal Haupt Mutation in p53 is the most common known genetic event leading to cancer. 3653-3661 4.. The aim of this project is to explore the interplay between mutant p53 and these partners at the biochemical and cellular levels. Relevant Publications: 1.. cellular and biochemical techniques. S... or elevated in an inducible manner. Our recent findings demonstrate that some of these partners also regulate mutant p53. This work will parallel other project in the lab where the involvement of E6AP in human cancer is tested. In more than 50% of all human cancers.. M. Interestingly. I. Regulation of p53 by CK1 in response to DNA damage is enhanced by PML..haupt@petermac.. p53 acts to eliminate cells of cancerous potential by halting their growth. Y. Grossman. Upon activation.deriving from our basic research to the cancer clinic. S. we discovered how the stability of PML is regulated (LuoriaHayon et al. O. REGULATION OF MUTANT P53: IMPLICATION TO CANCER DEVELOPMENT.. I.. O. Matentzoglu. Levav-Cohen. S. E. Oncogene 27.org 2011 Project Summaries. (2009). G. with emphasis on cellular aging. 2008). Supervisor: Professor Ygal Haupt We have recently linked the E3 ligase E6AP to two key tumour suppressors. It plays a key role in the cellular response to stress conditions.. The project will involve a variety of molecular. Kazaz.. Professor Ygal Haupt. Tel: 9656 5871. I. and how these are regulated are largely unknown.. Research Division. it loses its anticancer properties through direct mutations. 4818-4826. -H. In addition the student will be exposed to animal models of cancer. Berger. A. Our preliminary findings suggest that mutant p53 also regulates PML itself as part of its oncogenic potential. M. We are seeking Honours and PhD students to undertake projects on this area of research. Jiang. 296-299. One approach to studying the regulation of mutant p53 is to compare it to that of wild type (wt) p53. S. Voorhoeve. Haupt. di Agostino. Mutations in p53 not only disrupt its tumour suppressive functions. The most important agent of the body for fighting cancer is the tumour suppressor p53. The aim of this project is to define the mechanisms by which E6AP affects tumour growth.

We will use a combination of the above approaches to define the specific temporal requirement for activation of Pol I transcription initiation and elongation as cells leave Go and also during the ongoing cell cycle. Another interesting related project underway is to examine the Pol I independent roles of UBF by microarray and ChIP on chip. We will examine if the loss of UBF during differentiation also leads to a decreased number of active genes and altered r-chromatin. Peter Mac Page 18 of 21 May 2010 .org 2011 Project Summaries. (i) Epigenetic Mechanism of UBF action: To define the effect of decreased UBF expression on the r-chromatin status and Pol I transcription. Rb. For more information about this project contact: Assoc. Myc). USA) will explore this question further using a version of UBF targeted via a lac repressor fusion protein to a heterochromatic. Our data suggest they are nucleosomal.org TEMPORAL ACTIVATION OF Pol I TRANSCRIPTION IN PROLIFERATING AND DIFFERENTIATED CELLS: GROWTH FACTOR KINASES VS CYCLIN DEPENDENT KINASES Supervisors: Ross Hannan and Rick Pearson The molecular mechanism(s) that activate Pol I transcription are still a matter for debate. in genetic mouse models of cancer and in embryonic stem cells (the later being of considerable interest as there is considerable anecdotal evidence that Pol I transcription is wired differently in stem cells). and MEFS null for many of the above genes. As part of this project we will create novel molecular beacons to measure by Pol I transcription in individual cells by florescence microscopy which will allow for the first time to follow rDNA transcription during the ongoing cell cycle and how it responds to inhibitors of various signaling pathways. Supervisor: Ross Hannan Collaborating with Sui Huang (Northwestern Medical School Chicago) and Lawrence (Oklahoma Medical School) The RNA Polymerase-1 specific nucleolar transcription factor UBF belongs to the sequence non-specific class of HMG1-box proteins. One interpretation of this data is that UBF binding to high affinity sites across the rDNA underpins NOR morphology. We have begun studies to examine this possibility. Ras/Raf/ERK. Specifically.. Canada) and we are making inducible transgenic RNAi approaches.hannan@petermac. c-MYC and cell cycle control. p53. UBF is thought to induce a chromatin-like structure termed the “enhancersome” in which is thought to be essential for the assembly of the preinitiation complex (PIC) at the promoters of ribosomal genes. Email: ross. Moreover. Professor Ross Hannan. Hannan and McArthur) with extensive and complementary expertise in areas ranging from proteomics and protein chemistry. or at least associated with histones. the availability of specific inhibitors for cyclin-dependent growth factor kinases.. (ii) Epigenetic regulation of Pol I transcription during differentiation and development: To determine whether changes in UBF expression are a physiologically relevant mechanism by which Pol I transcription is regulated in mammalian cells and whether the decreased expression of UBF is a prerequisite for loss of rDNA transcription during differentiation and quiescence. ERK. Experiments in collaboration with Sui Huang (Northwestern Medical School. through signal transduction and cell biology to the regulation of gene transcription and protein translation. The animals will allow us to look at the consequences of manipulating UBF levels and number of active genes during development. We will determine whether enforced expression of UBF can delay or block the down regulation of rDNA transcription and differentiation. and regulation by cell cycle kinases (CDK2/4/6) have been implicated in the process (reviewed in 6). Both direct activation of Pol I transcription by growth factor dependent kinases (eg. PI3K and JNK) interaction with oncogenes and tumor suppressors (eg. amplified chromosome region containing lac operator repeats. we have demonstrated that UBF expression drops dramatically during differentiation of skeletal muscle. Through DNA binding of its HMG boxes. We will examine whether enforced expression of UBF in differentiated cells (either cardiomycytes or differentiated granulocytes) can lead to alterations in r-chromatin and the number of transcriptionally competent genes. For more information about this project contact: Assoc. The important role ascribed to UBF in regulation of rDNA transcription is reflected by observations that key growth/proliferation regulatory pathways PI3K/mTOR/S6. Email: ross.hannan@petermac. We have developed conditional KO of UBF with our collaborator Tom Moss (Quebec. Data from yeast suggest they are not. These studies are essentially an extension of the work currently under review in JCB. PIC formation (4). We will compare this to the activation of Pol I transcription in differentiated cells. This will allow us to undertake a detailed structure function analysis of UBF to differentiate the domains in this factor required for gene activation and chromatin remodeling etc. An additional long-term experiment will be crystallize UBF1 and UBF2 on the rDNA to obtain structural data to explain their differing abilities to regulate chromatin. Professor Ross Hannan. Research Division. ie UBF modulates r-chromatin. A particularly important question to come from these studies is whether active rDNA repeats are nucleosomal or not. and more recently in granulocytes which correlates with decreased rDNA transcription. converge on this factor (1-3). cardiac muscle. Tel: +61 3 9656 1747. or other than. Tel: +61 3 1747. EPIGENETIC REGULATION OF Pol I TRANSCRIPTION BY UBF. recent studies demonstrate that UBF DNA binding in vivo is not restricted to the promoter but is also found on multiple sites across the entire transcribed rDNA repeat suggesting roles for UBF in addition to. We have also developed a UBF rescue assay whereby we can rescue UBF RNAi mediated knockdown with RNAi resistant versions of UBF. both initiation and elongation steps have been proposed to be rate limiting (7) With the advent of inducible RNAi approaches to rapidly silence the above kinases. Interestingly. perhaps in a lexisomal state. USA) and Lawrence Rothblum (Oklahoma Medical School. Mammalian studies are unclear. it is now possible to take a holistic approach to elucidate the mechanism of activation and maintenance of Pol I transcription in mammalian cells.GROWTH CONTROL AND DIFFERENTIATION PROGRAM The Growth Control and Differentiation Program consists of three groups (Pearson.

Mol Cell Biol 23(23). This project aims to take advantage of a novel small molecule inhibitor of AKT to address the following objectives: (i) To establish the importance of AKT activity/signaling in c-MYC activation of ribosome biogenesis. Tel: 96563758 Email: Karen.A. et al. 332-337 (2004) 3. Dr Kathy Jastrzebski and Dr Rick Pearson The goal of the Pfizer Cancer genomics program is to generate Proteomic. Prof. We are currently profiling ovarian tumour cells and assessing the 2011 Project Summaries. References relevant to this project 1. the fascinating mechanisms that control cell size have resisted molecular genetic insight. In yeast. Drug sensitive cells.000 genes. deregulation of MYC signaling is common in human tumors. In metazoans. Email: response of these cells to a PI3Kinase/mTOR inhibitor.sheppard@petermac. fitness. Fewell GD.11:975-82. Consequently. Research Division. cell and molecular biology and the use of mouse models of cancer. Two types of screens will be employed: (i) Screens for regulators of Pol I transcription based on a 45S reporter. Furthermore.org ANALYSIS OF THE ROLE OF AKT SIGNALING IN CMYC DRIVEN TUMORIGENESIS Supervisors: Assoc.Dr Rick Pearson. Our recent studies have shown that the AKT/mTOR pathway is also essential for ribosome biogenesis (3). Tel: 9656 1247. Metabolomic and Genomic profiles of human cancer cells and use this information to predict drug effectiveness in patients. To achieve this aim. H. Tel: +61 3 9656 1247. and function. in particular in Burkitts B cell lymphoma and its specific over-expression in B cells leads to rapid lymphoma development in the Eµ -MYC mouse model (2).org SCREENS THAT CONNECT GROWTH TO PROLIFERATION AND MODULATE rDNA TRANSCRIPTION AND CELL SIZE Supervisors: Ross Hannan and Rick Pearson The problem was most succinctly put in a recent review by Mike Tyers (8) “Size is a fundamental attribute impacting cellular design. Prof. AKT promotes lymphoma development and drug resistance in this mouse model. References relevant to this project: 1. Wendel. Gene targets will then be confirmed and their ability to induce resistance will be tested in normal (immortalized) ovarian cells and in a panel of ovarian tumour cells to determine if a single hit confers resistance or if other mutations are required.hannan@petermac. 212-20. ovarian tumour cell lines will be screened for their sensitivity to the inhibitor. Tel: kate. This project will use a broad range of techniques including cell culture.M. These studies will aid in understanding pathways involved in drug resistance and potential novel targets for combinational therapy. thus making it an excellent therapeutic target for this disease.pearson@petermac. 2006 Nov. Email: rick. p424-30.pearson@petermac. for the large part the signalling pathways and A FUNCTIONAL GENOMIC SCREEN TO IDENTIFY GENES ASSOCIATED WITH DRUG RESISTANCE IN OVARIAN CANCER Supervisors: Dr Karen Sheppard. Nature 428. Int J Gynecol Cancer. M. 2. Schmitt K. This would then allow personalized patient treatment and limit the use of ineffective drugs and their potential side effects. et al. EMBO J 23 (16). Rick Pearson. 2005 15 Suppl 3: p. (iii) To provide a mechanistic rationale for using AKT inhibitor in c-MYC driven tumors. Drug Discov Today. G. molecular biology and functional genomics. Despite a long history of study. division is delayed until a critical size has been achieved.” We plan to use this library to functionally screen the entire human genome to identify genes that couple growth to proliferation and which regulate rDNA transcription and cell size. Hannan.org Dr Kate Hannan. Poortinga. For more information about this project contact: Dr Karen Sheppard. Bookman. Dr Kate Hannan Increased cell growth is a key feature of transformed cells and absolutely requires sustained increases in the synthesis of functional ribosomes. Email: rick. (ii) To establish the importance of AKT activity/signaling in c-MYC induced tumor formation. This is particularly important. This screen is based on the observation that despite a relatively good understanding of a few core components of the Pol I transcription apparatus.org 9656 1279. but in certain cell types extracellular signals may independently induce growth and division. demonstrating a functional interaction between MYC and AKT (2). as dysregulation of this pathway is one of the most common events in human cancer. State-of-the-art high through-put screening of genes responsible for conferring resistance will be performed using the OPEN Biosystems shRNAmir library which allows systematic silencing of more than 25. Peter Mac Page 19 of 21 May 2010 . transduced with the library will be treated with drug and those that proliferate (ie are now resistant) will then be selected and the gene(s) required for sensitivity identified using Next Generation sequencing. The aim of this project is to identify genes which confer resistance to PI3Kinase /mTOR inhibition in ovarian tumour cells. This project will employ an extensive suite of techniques including biochemical analysis of signaling pathways. Size homeostasis requires a doubling of cell mass with each division. K. 8862-8877 (2003) For more information about this project contact: Assoc. 3325-3335 (2004) 2. To date the only factor identified to be sufficient to modulate all steps required for ribosome biogenesis is the proto-oncogene and transcriptional regulator c-MYC (1). The PI3K/AKT/mTOR pathway is up-regulated in 30% of ovarian cancers and has been implicated as a major determinant of oncogenic transformation. 3. cell cycles can be actively coupled to growth.G et al. Shaw & Cantley Nature 2006 441. Rick Pearson.

2011 Project Summaries. Mol. To identify regulators of rDNA transcription. 5.org Assoc Prof Grant McArthur. Hannan. Dramatic responses to PLX4032 have been observed in patients with metastatic melanoma expressing V600E B-Raf. For more information about this project contact: Dr Petranel Ferrao. T. The results of this research work will elucidate the potential benefit of combination therapies in the treatment of metastatic melanoma. To address this hypothesis we will use a genome-wide screen using shRNAmirs and monitor cell size to identify genes required to recouple the AKT large size phenotype to a proliferative advantage.hannan@petermac. in an attempt to avert acquired resistance to the drug as a single agent therapy. For more information about this project contact: A/Prof Ross Hannan. some growth factors / genes are able to drive increased proliferation. currently in Phase 1 clinical trials at the Peter Mac.org 9656 1806. Stefanovsky et al. Research Division. Curr Opin Genet Dev (2004) 14. Tel: 03 petranel. Cell. Tel: +61 3 1247. Mol Cell (2001) 8:1063-73 2. Why for this later group the increased growth doesn’t lead to increased proliferation is not clear but it suggests that additional signals are needed. Another set of growth factors / genes drive increased growth which doesn’t lead to increased proliferation but rather larger cells that divide either at the same rate or a slower rate than the parental cells. Importantly. characterization of c-MYC transcriptional targets indicates that the majority of genes regulated by this factor are associated with various aspects of ribosome assembly and function (1). to address our central hypothesis we will reduce the levels of UBF by shRNA knockdown in Eµ-MYC lymphoma cells and examine the impact on the biology of the lymphomas. Thus. Email: ross. Finally we will examine the mechanism by which the manipulation of UBF expression by c-MYC regulates rDNA transcription. (2006) 21(5):629-39. In contrast. et al.hannan@petermac. We have found that overexpression of AKT increases cell growth in the immortalised human fibroblasts (BJ cells) without driving proliferation.org References 1. Moss. Email: IS INCREASED RIBOGENESIS IS SUFFICIENT BY ITSELF OR IN COOPERATION WITH OTHER ONCOGENIC MUTATIONS TO PROMOTE TUMOUR PROGRESSION IN VIVO. some patients are acquiring specific resistance to the drug.ferrao@petermac. Tyers Current Biology (2004) 14 : R1014-R1027 9.. Clontech) and Pol1shRNAi-Red knockdown construct will be selected by FACS for clones that exhibit half maximal red florescence (HEK-PolI-shRNAI-RedMed) compared to control cells expressing the cherry red vector alone. Email: grant. the transcription of the 45S rRNA gene which gives rise to the 28S. Therefore we propose that UBF may also be limiting for malignant transformation induced by c-MYC. As an extension of this focused study we will also address the crucial question of whether increased ribogenesis is sufficient by itself or in cooperation with other oncogenic mutations to promote tumour progression in vivo. Biol. Supervisors: Ross Hannan and Grant McArthur The transcriptional activator c-MYC plays a prominent role in cancer and has been implicated in the regulation of fundamental cellular events such as proliferation. EMBO J (2004) 23:3325-35 4. 7. To test this we will prepare transgenic mice expressing UBF under control of the immunoglobulin enhancer to confer constitutive high level expression in the B-cell lineage and examine the mice for evidence of malignancy. Stable cell lines co expressing the Cherry red expression vector (pmCherry1. The extensive approach would include a cDNA expression library screen. Mais et al. Interestingly. Yuan et al Mol Cell (2005) 1 :77-87 6. Email: ross. Directed analysis would include genomic and proteomic analysis of V600E B-Raf melanoma cell lines exhibiting intrinsic resistance to the drug in vitro. an increasing body of research supports a role for the deregulation of ribogenesis in the initiation of malignant transformation (2-4). Tel: +61 3 9656 1747. Taken together these data lead to the intriguing hypothesis that many of the phenotypic effects of c-MYC including its oncogenic potential are related to its ability to modulate rRNA synthesis and ribosome function.pearson@petermac. overexpression of AKT in transformed BJs leads to increased proliferation at a normal size. Stegmeier et al PNAS 1 (2005) 102:13212-13217. Stefenovsky et al Mol Cell. (ii) Screens that re-couple growth to proliferation: This screen is based on the following observations. is a specific inhibitor of the mutant V600E B-Raf protein. Peter Mac Page 20 of 21 May 2010 . There are several methodologies available in the laboratory to identify and predict mechanisms of drug resistance to PLX4032. The efficacy of selected combinational therapies with PLX4032 will be assessed using drug response assays. (2003) 23:8862-77 3. 210-7.mcarthur@petermac.org MECHANISMS OF RESISTANCE TO A B-RAF INHIBITOR DRUG Supervisors: Dr Petranel Ferrao and Assoc. 8. Moreover the abundance of this factor is limiting for the ability of c-MYC to activate rDNA transcription. additional mutations are required to convert increased PI3K/AKTdependentsignalling into a proliferative advantage. differentiation and apoptosis. Poortinga et al. Tel: +61 3 9656 1616. For more information about this project contact: Assoc. Such clones when screened with the shRNAmir library will allow for the identification of repressors (increased red fluorescence) or activators (reduced red fluorescence) of Pol I transcription. We hypothesise that in tumour cells.org Dr Rick Pearson.downstream protein complexes that regulate mammalian rDNA transcription are poorly defined. Professor Grant McArthur A Plexxikon compound. We were the first to demonstrate that c-MYC can regulate the key component and major rate-limiting step in ribosome biogenesis (ribogenesis). Tel: +61 3 9656 1747. PLX4032. Professor Ross Hannan.8S and 18S rRNAs subsequently verified by others. Email: rick. Our studies indicate that one mechanism by which c-MYC modulates 45S rRNA synthesis is through direct regulation of the Pol I transcription factor UBF. This proposal will directly test this central hypothesis using a transgenic model of B-cell lymphoma (Em-MYC) in which malignancy is dependent on over expression of c-MYC (10). resulting in a large cell phenotype. Genes and Development (2005) 19 :50-64 5. However. that is to say they drive growth which leads to cells that cycle faster at the same size as the parental cells.

Education & Communication Coordinator (Research) Peter MacCallum Cancer Centre. 11.owen@petermac. Victoria.org http://www. 25(7):1522-33 RESEARCH EDUCATION PROGRAM For more information about research opportunities in the Research Division contact: Dr Caroline Owen. Peter Mac Page 21 of 21 May 2010 .org/research 2011 Project Summaries. East Melbourne. Goodfellow et al (2006) EMBO J.petermac. 533-8. Adams et al (1985) Nature 318. Australia 3002 Tel: +61 3 9656 1930 Email: caroline. St.10. Andrew’s Place. Research Division.

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