Identifying factors that restore DNA repair in ageing cells through WRN reactivation

Active: 2020.03.01 - 2021.09.30
UK SPINE Scientific Liaison: Monica Spisar

Premature ageing is seen in a number of human heritable syndromes that result from mutation in key DNA repair factors, strongly suggesting that failure to repair the genome is causative in driving ageing and age related disease. Evidence supporting this hypothesis comes from studies of normal ageing, where senescent cells are found to accumulate with age, and cause at least some of the diseases of ageing. Senescent cells accumulate DNA damage foci which persist over time, suggesting failure either to initiate or complete DNA repair. However, the molecular basis underlying why senescent cells fail to repair DNA damage is not known.

Human premature ageing Werner syndrome (WS) recapitulates many features of normal human ageing across multiple tissues. WRN, the gene mutated in WS, is one of a very few genes with single nucleotide polymorphisms that consistently link with longevity in genome wide association studies, suggesting that its importance is not limited to the rare recessive Werner syndrome but is widely applicable across ageing human populations.

Despite its well known roles in ageing and DNA metabolism, remarkably little is known about WRN as a DNA-protective factor in normal ageing and, in particular, in cell senescence. This work will identify novel means to promote DNA repair in cells by restoring ‘young’ levels of WRN function. Two complementary approaches will be deployed: reactivation of gene expression and post-translational stabilisation of the protein. The findings from this project will support development of anti-ageing drugs acting via restoration of DNA repair in ageing cells.

A sensitive assay to detect methylation changes in the WRN promoter will be developed to support establishing baseline assessment of WRN promoter methylation, mRNA and protein levels across the cell life course. From human patient samples, new information on WRN status in ageing and inflammation will be generated. A small molecule epigenetic chemical probe library (and, where relevant, other drug libraries) will be screened for restoration of WRN expression in cells expressing low levels of WRN. Hits achieving elevated WRN mRNA will be verified to confirm the impact of these drug-like molecules on WRN expression in cultured cells and will be tested in proliferating primary human skin fibroblasts exposed to DNA damaging agents. Drugs that lead to improved viability will be tested for geroprotective effects.

Separately, phage display will be used to identify peptide motifs that bind to WRN and MDM2. The sequence of phage peptides (those similar to the MDM2 protein sequence, or based on the strongest consensus sequences) showing activity in a peptide-based competitive ELISA assay will be used as the basis for synthesis of cell-penetrating peptides (CPPs) capable of preventing/disrupting WRN-MDM2 interactions in vitro in subsequent assays.

The CPPs designed to disrupt WRN-MDM2 interactions will be incubated with cells; levels of WRN protein and WRN localisation, as well as any cytotoxicity, will be assessed. This will be repeated following exposure to DNA damaging agents, with analysis of viability and WRN protein. Recovery of cell proliferation and DNA damage foci will be assessed. Increased WRN levels in treated versus control cells will indicate that the peptides stabilise functional WRN and prevent breakdown following DNA damage responses.

Active drugs or peptides identified will be tested for ability to prevent onset of cell senescence following DNA damage, thereby identifying early stage leads for novel anti-ageing drug discovery based upon restoration of DNA repair capacity.