Early detection test for women with the BRCA1 and BRCA2 gene

Women with hereditary mutations in the BRCA1 or BRCA2 genes have an elevated risk of developing ovarian cancers. Surgery to remove the ovaries and fallopian tubes (salpingo-oophorectomy) can reduce the risk of developing ovarian cancer by up to 80 per cent. As a result, many women elect to have surgery as a risk-reducing strategy. However, most women who have surgery have no evidence of cancer and surgically-induced menopause increases their risk of other diseases, including osteoporosis and cardiovascular disease. A method to determine the need for surgery in this group of ‘at risk’ women is required.

The team is trialling a new diagnostic test for the detection of early-stage ovarian cancers in women carrying hereditary mutations in the BRCA1 or BRCA2 genes. Unlike genetic testing, which indicates only a predisposition to develop cancer, this new test identifies biological changes associated with the early stages of cancer formation. By using this test to detect early-stage cancers, most women could be spared the risks of surgery and its associated side-effects.

The goal is to establish this test in a regular screening program. Successful implementation will greatly reduce the number of women undergoing surgical intervention, allowing the majority to lead full, healthy lives.

Collaborators | Monash Medical Centre, Melbourne; Royal Adelaide Hospital, Adelaide

Supporters | The Ovarian Cancer Research Foundation

Role of Cell Death and pore forming proteins in Type 2 Diabetes

Dietary danger molecules, such as the saturated fatty acid palmitate, trigger the activation of the pro-inflammatory cytokine, interleukin-1β (IL-1β), via NLRP3 inflammasome activation. Based on our past work (Lawlor KE et al. Nature Commun 2015 6:282) we are examining the contribution of extrinsic apoptotic caspase-8 and necroptotic MLKL activity to pathogenic NLRP3 inflammasome activation and Type 2 Diabetes development. Gasdermins (GSDMs) are a family of membrane pore-forming proteins that have recently been defined as proteolytic caspase substrates that induce an inflammatory form of lytic cell death called pyroptosis (Kayagaki N et al. Nature 2015 526:666; Orning P et al. Science 2018 362:1064, Wang Y et al. Nature 2017 547:99). The aim of this project is to define how pyroptotic cell death contributes to NLRP3 inflammasome activation and/or DAMP release to worsen obesity-induced Type 2 Diabetes This project offers the opportunity to be trained in a variety of techniques, including cell culture, Western blotting, inflammasome/cell death assays, ELISA, qPCR, tissue analysis – histology, flow cytometry, serum/liver/adipose metabolic assays, Type 2 diabetes preclinical models.

Collaborators: James Murphy, Seth Masters, James Vince (WEHI), Andrew Murphy (Baker Heart and Diabetes Institute)

Identifying mitochondrial factors that activate inflammatory signalling

Mitochondrial (“intrinsic” BCL-2 family regulated) apoptosis has long been thought to be immunologically silent. However, using small molecule inhibitors of pro-survival BCL-2 family members, we have recently discovered that mitochondrial apoptosis can induce a cascade of events that culminate in activation of the NOD-like receptor protein 3 (NLRP3) inflammasome and pro-inflammatory cytokine, Interleukin-1beta (Vince JE Cell Reports 2018). In this project we will further characterise this pathway and test whether its activation alters cancer progression in vivo. This project will use our novel gene knockout macrophages and specific targeted drugs, as well as a range of cell biology and biochemical/molecular approaches (e.g. inflammasome/cell death assays, ELISA, Western blotting, CRISPR Cas9 gene editing screens, proteomics).

Collaborators: Gabriela Brumatti, James Vince (WEHI)

Mitochondrial apoptosis and inflammasome activation

Macrophages are innate immune cells that detect environmental, pathogen or host cellular danger molecules, and initiate appropriate immune responses. We have recently discovered that targeting pro-survival proteins BCL-XL and MCL-1 in macrophages induces apoptosis to clear microbial infection (Speir M et al. Nature Microbiology 2016) and also triggers inflammation via activation of the NOD-like receptor 3 (NLRP3) inflammasome and Interleukin-1beta (Vince JE et al. Cell Reports 2018). This project aims to define novel regulators of this pathway and investigate how these proteins alter pathogen clearance (Deo P et al. Nature Microbiology 2020). This project will use our novel gene knockout macrophages and specific targeted drugs, as well as a range of cell biology and biochemical/molecular approaches (e.g. inflammasome/cell death assays, ELISA, Western blotting, Q-PCR, over-expression systems, CRISPR Cas9 gene editing, infectious preclinical models).

Collaborators: Richard Ferrero (Hudson Institute), James Vince, Marco Herold, David Huang (WEHI), Thomas Naderer, Benjamin Kile (Monash University), Kate Stacey (UQ)

Defining regulators of cell death and inflammasome activation

Pattern recognition receptors, including Toll-like receptors (TLRs) and NOD-like receptors (NLRs) are key components of the innate immune response. They sense microbial, host-derived and environmental danger molecules, and induce inflammatory signalling responses, via inflammasomes and other molecular complexes. We recently defined how deficiency in the cell death inhibitory protein XIAP sensitises innate immune cells to TLR-induced NLRP3 inflammasome activation (Lawlor KE et al. Nature Comms 2015, Lawlor KE* et al. Cell Reports 2017). Moreover, mutation of other cell death regulatory machinery drives hyperinflammation (Lalaoui N et al. 2020 Nature, Rickard JA et al. Elife 2014). The aim of this project is to further define molecules, like XIAP, that regulate this alternative inflammasome pathway. This project offers the opportunity to be trained in a variety of techniques, including cell culture, Western blotting/immunoprecipitation, proteomics, overexpression/CRISPR Cas9 gene editing, flow cytometry, ELISA and qPCR.

Collaborators: Paul Hertzog (Hudson Institute), James Vince, John Silke, Najoua Lalaoui (WEHI), Monica Yabal (Munich)

SRY and male bias in Parkinson’s disease

A drug to stop brain cell death in Parkinson’s disease.

Parkinson’s disease is the second most common neurodegenerative disease with 32 Australians newly diagnosed every day. So there is a critical need to stop the disease from progressing. Current therapies only alleviate symptoms. In several experimental models of PD (human, mouse and rat neuronal cell lines, and toxin-induced rat models) we showed that the SRY is ectopically expressed at abnormally high levels1.  Our so-called ‘anti-sense SRY’ gene therapy specifically targeted SRY and reduced levels of protein to near-physiological; in so doing, the drug slowed cell death and consequent movement deficits in rat models2. The SRY gene sequence differs considerably between animal species. The current drug formulation targets the sequence of the rat version of the SRY gene but only 40% of the gene sequence is similar between humans and rats. The project involves the design and optimisation in vitro of a humanised version of our DNA-based drug.  Nextgen variant antisense SRY DNA oligos will be synthesised and tested in human neuronal cell lines and SRY levels and transcriptional activity analysed.

Role of cancer stem cells in tumour progression

Cancer “stem cells” are hypothesised to control multiple aspects of tumour behaviour, including progression, recurrence and clinical response to therapy. Therapies designed to eliminate the CSC subset are likely to be key in achieving effective, sustained remission.

We have identified a key population of stem-like cells in ovarian cancer that control the invasive behaviour of ovarian cancers in vitro and in vivo. This project is focused on understanding the molecular nature of these cells, and the pre-clinical development of a novel monoclonal antibody therapy for directed tumour regression.

Therapeutic targeting of the colon cancer epigenome

Project Description: Therapeutic targeting of the colon cancer epigenome

Colorectal cancer (CRC) is the second most common malignancy in Australia with over 17,000 cases diagnosed a year. Wnt/b-catenin signaling pathway plays a pivotal role in the development of CRC. 90% of CRC contains genetic mutations that lead to the hyperactivation of Wnt/b-catenin pathway, while abrogating β-catenin function results in the loss of tumorigenic potential of Wnt-active CRC cells. However, the transduction of Wnt/b-catenin pathway in CRC, especially at the transcriptional and epigenetic levels, remains incompletely understood. This project uses genome editing technology to systematically investigate the regulators of Wnt/b-catenin signaling in CRC cells and evaluate their merits in CRC therapy.

Defining the role of Mediator kinase in cancer

Targeting Mediator kinase CDK8 and CDK19 in normal and cancer states

CDK8 and its paralog CDK19 (collectively known as Mediator kinase) transduce b-catenin and other transcriptional programmes as part of a multi-meric transcriptional complex called Mediator

Targeting Mediator kinase will provide therapeutic benefit for colon cancer and other Wnt driven malignancies. Specific aims of this project will use sophisticated mouse models and genetically engineered cell lines to:

1) To characterise the function of Mediator kinase components Cdk8 and Cdk19 in normal tissue homeostasis and intestinal tumourigenesis in vivo. Using inducible and conditional Mediator kinase genetically engineered knockout mice, we will characterise the contribution of Mediator kinase function in normal intestinal homeostasis and malignancy.

2) To investigate Mediator kinase inhibition in clinically relevant colon cancer models. We will utilise both genetic and pharmacological CDK8/19 inhibitors to examine the consequences of Mediator kinase loss in clinically meaningful cancer models (cell lines, organoids and patient-derived xenografts).

3) To identify and characterise CDK8/19 kinase substrates that mediate its oncogenic activity.  Using innovative proteomic approaches coupled with powerful genetic and chemical reagents, we will identify and characterise CDK8/19 kinase targets in b-catenin driven malignancy.

Overcoming chemoresistance in colon cancer

Functional Genomic Screens to Identify Drivers of Chemoresistance

Patients with late-stage colorectal cancer (CRC), especially metastatic CRC, have poor survival rates compared with their early-stage counterparts. This is partly due to the development of resistance to chemotherapy agents – which are used as standard treatment for late-stage CRC. Occurrence of chemoresistance, reduces treatment options and increases the risk of recurrence.

Understanding the molecular mechanisms involved in chemoresistance will enable clinicians and researchers to design strategies to reduce or prevent chemoresistance, reverse chemoresistance after it has occurred, predict chemoresistance potential and finally to design better chemotherapy agents. All these benefits will ultimately increase survival rates.

This project aims to create a comprehensive catalog of genes that are involved in conferring chemoresistance in CRC which will enable us to describe the biological interactions and pathways that enable tumor cells to resist chemotherapy.

As a pilot project we have carried out a whole genome CRISPR negative selection screen on RKO cells, a CRC cell line which are resistant to Oxaliplatin – a component of both FOLFOX and CapeOx,  the two most common chemo treatment regimens. The CRISPR screen was conducted in such a way that it would disrupt all the genes in the genome, one gene per clone. If this disrupted gene is essential for chemoresistance, the clone would have been removed from the population. By looking at the genes which have thus been “selected out”, we hope to identify potential genes that are critical mediators chemoresistance.