Ashley Saathoff

Aging is a primary cause of chronic disease and multimorbidity. The foremost contributing factor to this process is DNA damage from either environmental or internal factors, manifesting as genomic instability, epigenetic remodeling, and cellular senescence. Persistent activation of DNA damage repair pathways alters the epigenetic landscape and chromosomal structure, leading to dysregulated gene expression and cellular ex-differentiation into a metabolically active but growth arrested state known as cellular senescence. Senescent cells (SnCs) accumulate in many tissues in an age dependent manner and have been found to be important drivers of chronic disease primarily due to their pathologic, proinflammatory secretory phenotype. Biologics called senotherapeutics, which selectively clear SnCs have shown tremendous efficacy in slowing the development of or attenuating chronic disease in many pre-clinical models by improving many age-associated phenotypes. However, targeting SnCs doesn’t target the genomic and epigenomic alterations that drive deleterious aging hallmarks. Partial reprogramming, through ectopic expression of the Yamanaka factors, has been shown to alleviate many aging phenotypes through epigenomic and transcriptional rejuvenation to a younger state, highlighting that targeting the epigenome is a promising strategy for healthy aging. We hypothesize that targeting key enzymes involved in modulating the aged and senescent epigenome, as well as those involved in partial reprogramming, will be efficacious in alleviating many aging phenotypes simultaneously. We reasoned that because genotoxic stress is a robust phenotype of aging and a key driver in the development of senescence and the important role that histone deacetylases (HDAC) 1 & 2 play in chromatin remodeling during DNA damage repair, that targeting HDACs may be able to both selectively clear SnCs and promote cellular rejuvenation. We screened a library of HDAC modulating small molecules for their ability to either selectively target SnCs for apoptosis or suppress their pathological pro-inflammatory phenotype. To do this we conducted a phenotypic drug screening assay that takes advantage of senescence-associated β-galactosidase activity, one of the most robust markers of cellular senescence, that allows fluorescent detection of SnCs. Through our drug screening efforts, we identified a host of HDAC inhibitors and confirmed our results through gene expression analysis using quantitative polymerase chain reactions measuring the relative expression of key senescence markers. Additionally, single-cell RNA-seq data suggests that our identified senotherapeutics potentially target specific subsets of SnCs. Further research using primary cells from young and old humans and mice will be required to examine their potential in rejuvenating age cells prior to in vivo testing.