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.

Targeting NIK in haematological malignancies

Project Outline:

Targeting NIK in haematological malignancies

The NF-kB pathway plays a crucial role in B cell homeostasis and function.  Transcriptional activity of NF-kB is mediated by a number of homo and hetero-dimeric complexes containing p50 (NF-kB1) or p52 (NF-kB2). The rate limiting step in the canonical pathway is the degradation of inhibitor of kappa-B alpha (IkBa) which under steady state condition holds p50 containing complexes in the cytoplasm.  In contrast, the non-canonical NF-kB pathway is driven by NIK (Map3k14) dependent processing of p100 to p52 resulting in translocation of dimer complexes containing p52 to enter the nucleus and drive transcription. Despite a number of high profile publications describing a non-redundant role for NF-kB in a number of B cell malignancies, efforts so far in targeting the canonical pathway have failed possibly because of an ongoing requirement of this pathway in a number of organs. Studies done so far on the non-canonical pathway indicate that this pathway is required for the development of stroma in primary and secondary lymphoid organs but less important for the homeostasis of mature immune cells. The non-canonical pathway is aberrantly activated in a number of haematological malignancies as described below.

Multiple Myeloma (MM):

Multiple myeloma (MM) is a chronic and incurable plasma cell malignancy.  Despite immense heterogeneity in clinical presentation and genetic events that determine outcome, a common feature of all MM is aberrant activation of NF-kB. Most myeloma patient samples and cell lines have activation of the canonical and/or the alternate pathway. Mutations in upstream regulators of the non-canonical pathway like TRAF3, TRAF2, cIAP1/2 (BIRC2/3) resulting in constitutive activation of NIK are common in MM.

Diffuse Large B Cell Lymphoma (DLBCL):

DLBCL, one of the most common types of B-cell lymphoma with two main subtypes based on cell of origin classification: germinal centre B-Cell (GCB)-like DLBCL and activated B-Cell (ABC)-like DLBCL. ABC-like DLBCLs are characterised by constitutive NF-kB signalling. Some recent work has highlighted the importance of non-canonical NF-kB pathway is GCB-DLBCL that overexpress BCl6 consistent with their cell of origin. 

Chronic Lymphocytic Leukaemia (CLL):

CLL, which is characterised by clonal expansion of B cells is the most common form of leukaemia in adults. The B cell receptor plays a critical role in driving proliferation of CLL cells. Most CLLs have active Akt/mTOR, ERK and NF-kB signalling pathways as a consequence of the B cell receptor (BCR) being constitutively ON in both an antigen dependent and independent manner. BIRC3 (cIAP2) mutations are often linked with un-favourable prognosis independent of other genetic lesions. Birc3 is an E3 ubiquitin ligase which suppresses non-canonical NF-kB activation. CLLs harbouring BIRC3 mutations have increased NIK levels resulting in constitutively activated non-canonical NF-kB underscored by constitutive p100 processing to p52, resulting in increased expression of the pro-survival molecule Bcl-XL (an NF-kB dependent gene) rendering them less sensitive to conventional chemotherapy.

Mantle Cell lymphoma (MCL):

MCL is a rare B-cell lymphoma that can be aggressive or indolent and often have constitutive activation of BCR signalling. A recent study has reported that MCLs with constitutive canonical NF-κB signalling and low p52 levels (non-canonical) are more sensitive to ibrutinib (BTK inhibitor) treatment than MCLs with genetic abnormalities that result in the activation of the non-canonical NF-κB pathway.

These findings strongly support the hypothesis that the non-canonical NF-κB pathway is associated with lymphomagenesis and resistance to therapy. We therefore postulate that inhibition of the alternative NF-κB pathway may offer a novel targeted therapeutic strategy for diverse B-Cell malignancies and could improve the response to other therapeutic approaches.

 

Collaborators:

Dr Pasquale Fedele
Dr Michael Low
Dr Raffi Gugasyan