Mitochondrial ubiquitin dynamics and apoptotic cell death.

The ability of cells to undergo suicide or apoptosis plays a very important role in the development and maintenance of a healthy body as well as in disease. This ensures that infected or dysfunctional cells are eliminated before causing too much havoc. The decision over life and death is happening at the mitochondria. These are the power stations of the cell, one major source of the energy currency, ATP, that is needed for life. Just like our nuclear power stations, this activity comes with a certain risk and damaged mitochondria leak toxic particles called reactive oxygen species (ROS) that are harmful to the cell. Therefore a fail-safe mechanism exists that safely engulfs such damaged mitochondria in a double-membrane and moves them to the waste-disposal factory of the cell, the lysosome, where they can be dismantled. This process is called Mitophagy, derived from the greek "phagos", meaning eating. Failure to undergo mitophagy is believed to be one of the causes of neuronal cell death seen in patients suffering from Parkinson's disease (PD). Two proteins critical to this process, which are lost or mutated in some forms of PD are Parkin and PINK1. Together they decorate the outer surface of damaged mitochondria with a small tag called ubiquitin, that marks them out for destruction. We are interested in another protein that is constantly wedged into the surface of mitochondria and has the ability to remove ubiquitin. We call such proteins deubiquitylases or DUBs, and this particular one is called USP30. Depleting USP30 from cells that suffer from a loss of Parkin or PINK1 restores their ability to clear away damaged mitochondria. This means that inhibitors against USP30 may have value in the treatment of Parkinson's disease. Interestingly, we have found that removing USP30 also sensitises cells to undergo faster suicide, which we can trigger with a class of death-inducing drugs called BH3-mimetics. These drugs have already been used in the treatment of cancer to promote cell death of rogue tumour cells. However, many cancer cells are insensitive or resistant to these drugs. We believe that targeting USP30 function may provide a means to improve the efficacy of these drugs. In this programme of work we will study the way USP30 controls cell death and revisit its role in mitophagy, to pave the way for the development of drugs that can exploit this dual aspect of USP30 function in cancer and Parkinson's disease. In addition, we will also study two further DUBs, called USP9X and USP24, which have also been suggested to promote cell death and may therefore provide alternative targets for combinatorial therapies with other death inducing drugs, like the BH3-mimetics.

Mitochondria play an important role in the orchestration of apoptotic cell death. Synthetic BH3-mimetics promote apoptosis and have shown some success in the clinic. However, additional approaches are required to improve their efficacy and overcome resistance. Mitochondrial dysfunction has been linked to neurodegeneration, in particular to Parkinson's disease (PD), which is characterised by the loss of dopaminergic neurons from the substantia nigra. Two PD-associated genes, the ubiquitin E3-ligase Parkin and PINK1 are involved in the safe disposal of damaged mitochondria by mitophagy. Depletion of the unique mitochondrial deubiquitylase (DUB), USP30, has been shown to promote mitophagy in cells with defective Parkin or PINK1 and rescue motor function and dopamine levels in a corresponding fly model. We have recently shown that USP30 depletion also sensitises cancer cells to BH3-mimetics, expanding the clinical potential of a future USP30 inhibitor.

We will generate CRISPR/CAS9 dependent knock-out and/or catalytically inactive knock-in cell lines and use a range of proteomic and cell biological approaches to a) identify new substrates of USP30 and explore its role in Parkin-independent mitophagy, b) explore the synergism between USP30-silencing and a toolkit of BH3-mimetics within a panel of cell lines and c) elucidate the mechanism of action of USP30 in modulating apoptotic cell death. Whilst this proposal is focused on USP30 as the preeminent mitochondrial DUB, we will in parallel evaluate USP9X and USP24, two DUBs which have been proposed to stabilise MCL1, thus contributing to a major mechanism of resistance to BH3-mimetics.

This is primarily a basic research project addressing fundamental questions of the role of reversible ubiquitylation in apoptotic cell death and mitophagy. However, both of these processes play important roles in neurodegenerative disease and cancer, i.e. disease settings in dire need of new treatment regimes. Our research will thus be of interest to both the academic and industrial sectors.

Industrial impact:
DUBs, which are at the center of our proposal, are now recognized as potential new drug targets in a range of disease settings. Proof of principle for selective inhibitors has now been established, and a number of Pharma and Biotech companies have active drug discovery programmes focusing on this class of enzymes (Genentech, Millenium, Mission Therapeutics). USP30 has been proposed as a potential drug target for the treatment of Parkinson's disease. Our recent results suggest an additional role for future USP30 inhibitors in combination therapies with BH3-mimetic compounds, that have already been used in the clinic. Such combinatorial approaches may provide a means to extend the efficacy of extensively trialed inhibitors navitoclax or venetoclax beyond the chronic lymphocyte leukemia (CLL) setting, to other, e.g. solid, tumours, which so far have shown resistance towards these inhibitors. Our work will have an impact on drug discovery programmes by providing a rationale for USP30 (and potentially USP9X and USP24) as drug targets in cancer therapies. Furthermore our proteomics component of the work, in particular the search for USP30 substrates, will provide potential biomarkers and pharmacodynamic markers that are essential for progressing prospective inhibitors into clinical trials, whether they are targeted towards neurological disease or towards cancer.

Note that we already have existing links with industry, which have a strong interest in developing inhibitors against DUBs, in particular with Forma Therapeutics, a major US Biotech company and with Mission Therapeutics, a UK-based Biotech focusing on DUBs.

Economic & Social Impact:
Our work will contribute to the development, testing and production of drugs, which generates business and job opportunities within the UK. Neurodegenerative disease and Cancer constitute two of the major health issues impacting on the quality of life of people in this country, and impose a major burden of care on the NHS in an aging society. In the long-term, our work on USP30 may influence treatment strategies for Parkinson's disease and Cancer patients. Such developments will benefit industry, but also the NHS and ultimately patients (i.e. the public).
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Public/Education/Training:
We will communicate our findings to the public via the University web-site and press releases when appropriate. Our laboratory hosts tours for visitors (both adults and local schoolchildren) interested in the work of the Liverpool Cancer Centre. An immediate impact of the research lies in specialised skills training of research staff associated with the project, who will gain further experience in genetic engineering: the PDRA will participate in the first CRISPR Workshop at the Innovative Genomics Initiative at Berkley, USA, and benefit from state of the art technological advice from our collaboration with Jacob Corn, scientific lead of this new Institute. The programme of work will also further hone the following key skills of the PDRA as well as the technician: a) handling of large datasets, b) time management, c) strategic decision making.

The University of Liverpool HR department runs a host of personal development courses and online resources grouped under a professional development toolkit. The Universities of Liverpool and Manchester are piloting a cross-institutional mentor network in support of the Athena SWAN Charter. Mentors are required to attend a training session and mentees are able to self-select a mentor based on their individual development goals.