Characterization of human senescent cell biomarkers for clinical trials

Abstract

There is an increasing need for blood-based biomarkers of senescent cell burden to facilitate selection of participants for clinical trials. Potential candidates include p16Ink4a expression in peripheral blood T-cells and circulating protein concentrations of the senescence-associated secretory phenotype (SASP). p16Ink4a is encoded by the CDKN2A locus, which produces six variant transcripts in humans, two of which encode homologous p16 proteins: p16Inka4a, encoded by p16_variant 1, and p16{gamma}, encoded by p16_variant 5. While distinct quantitative polymerase chain reaction primers can be designed for p16_variant 5, primers for p16_variant 1 also measure p16_variant 5 (p16_variant 1+5). In a recent clinical trial evaluating effects of the senolytic combination, dasatinib + quercetin (D+Q), on bone metabolism in postmenopausal women, we found that women in the highest tertile for T-cell expression of p16_variant 5 had the most robust skeletal responses to D+Q. Importantly, assessment of p16_variant 5 was more predictive of these responses than p16_variant 1+5. Here, we provide a comprehensive in vitro and in vivo characterization of p16_variant 5 expression. In vitro, p16_variant 1 increased rapidly (week 1) following the induction of DNA damage, whereas p16_variant 5 increased later (week 4), consistent with the latter being more specific for an established senescent state. Further analysis of our clinical trial data identified a SASP panel in plasma that correlated with p16_variant 5 expression in T-cells and performed as well in identifying postmenopausal women with a positive skeletal response to D+Q. Collectively, our findings support that the assessment of T-cell p16_variant 5 expression may be more specific for senescence and provide further support for this assay as a biomarker for selecting participants in clinical trials of senolytic interventions. In addition, our data indicate that correlated plasma SASP markers could be used in lieu of the more technically challenging T-cell p16 assay. Finally, the ability to identify individuals with a beneficial skeletal response to D+Q using two different measures of senescent cell burden (i.e., the T-cell p16 assay and the SASP score) provides further support for the hypothesis that the underlying senescent cell burden dictates the clinical response to a senolytic intervention.