Mechanisms of cellular senescence and the use of repurposed drugs as senotherapeutic compounds

Abstract

The mechanisms of cellular ageing underpin the pathologies of many age-related diseases, but are not yet fully understood. Cellular senescence and mRNA splicing factor dysregulation are particularly interesting as recent evidence suggests they may be druggable targets. Compounds that are already prescribed for other diseases may be “senotherapeutic” with the ability to affect cellular senescence. Methods for improving existing models and techniques for assessing senescence were developed, including the improvement of human in vitro tissue culture models, techniques for medium-throughput senescence screening and a tool for analysis of fluorescence imaging of nuclear staining (FINS). Diseases of premature ageing, progeroid syndromes, represent an important avenue of research for treating disease and for basic ageing science. mRNA splicing factor dysregulation was found to occur in progeroid cells, providing more evidence supporting it as a hallmark of cellular ageing. Progeroid cells were affected by trametinib treatment, a drug known to reverse some aspects of senescence in normal circumstances, but the more severe phenotypes were not rescued. This suggested that senomorphic compounds can only rescue less senescent cell cultures. As many compounds have never been investigated for senotherapeutic ability, a medium-throughput screen studied 240 drugs for altered gene expression of the senescence marker, CDKN2A. 32 compounds were screened further for senescence-associated beta galactosidase (SAB) activity (the “gold standard” measure of senescence). Compounds that either decreased or induced senescence were identified for further validation. Bioinformatic association of structure and function identified a 3 potential common molecular substructure within compounds that decreased CDKN2A gene expression. Some synthetic female hormones affected senescence in the screen, so were used in further experiments to measure senescence and aspects of the senescence phenotype in male and female treated cells. Such treatment affected senescence in male cells and some senescence characteristics in both sexes, suggesting a sex-specific senomorphic effect and that sex differences in the context of ageing and cellular senescence may be important. This thesis contributes several new methods for studying human-relevant models of cellular ageing, evidence of the basic mechanisms of cellular ageing, potential candidates for the treatment of age-related disease, and identifies sex as being an important factor in the response of cells to senotherapeutic treatment. Senotherapeutic compounds that can target the underlying mechanisms of cellular ageing could treat many different diseases.