DNA Methylation Signatures of Cellular Senescence Are Not Reversed by Senolytic Treatment.

Senescence, age, and mortality risk intersect at a small subset of the DNA methylome.

• Only 9,363 CpGs out of 396,333 analyzed (2.4%) were identified at the intersection of senescence, age, and mortality risk.
• This overlap represents a small proportion of age-related CpGs, which the authors hypothesize explains why existing epigenetic clocks show inconsistent associations with cellular senescence.
• The analysis was designed to identify a core senescence signal conserved across different cell types and different senescence inducers.

Three epigenetic clocks were generated using the senescence-related CpG subset, trained to predict in vitro senescence, age, and mortality, respectively.

• The clocks were trained on the 9,363 CpGs at the intersection of senescence, age, and mortality signals.
• The three predictors were designed to predict: (1) in vitro senescence, (2) chronological age, and (3) mortality risk.
• These clocks were constructed specifically to better monitor senescence and senolytic treatment compared to traditional epigenetic clocks.

All three senescence-focused epigenetic clocks stayed the same or accelerated after senolytic treatment in both in vivo and in vitro data.

• None of the three clocks showed reductions following senolytic treatment.
• This pattern was observed in both in vivo and in vitro experimental settings.
• The authors describe this result as 'surprising,' as the clocks were designed to focus on senescence-relevant CpGs and were expected to be more sensitive to senolytic interventions than traditional clocks.
• Prior studies had already found that existing epigenetic clocks show 'no reductions after senolytic treatment,' and this study extended that finding to senescence-specific clocks.

Existing epigenetic clocks show inconsistent associations with cellular senescence.

• Prior studies found that existing epigenetic clocks show inconsistent associations with cellular senescence and no reductions after senolytic treatment.
• The authors hypothesize this is because senescence-related CpGs constitute a small proportion of the age-related CpGs used in standard clocks.
• This inconsistency motivated the development of senescence-focused clocks in the current study.

The findings challenge the assumption that aging biomarkers decrease after geroscience interventions.

• The authors state the results 'call into question whether cellular senescence can be captured by DNA methylation.'
• The findings also 'challenge the assumption that aging biomarkers decrease after geroscience interventions.'
• Epigenetic clocks are described as 'commonly used aging biomarkers based on DNA methylation that predict long-term morbidity and mortality risk,' making this challenge broadly relevant to the field.
• Increased cellular senescence with age is posited to contribute to age-related disease and mortality, making the inability to capture it with DNA methylation a significant finding.