A New Gene Set Identifies Senescent Cells and Predicts Senescence-Associated Pathways Across Tissues

Although cellular senescence is increasingly recognized as driving multiple age-related co-morbidities through the senescence-associated secretory phenotype (SASP), in vivo senescent cell identification, particularly in bulk or single cell RNA-sequencing (scRNA-seq) data remains challenging. Here, we generated a novel gene set (SenMayo) and first validated its enrichment in bone biopsies from two aged human cohorts. SenMayo also identified senescent cells in aged murine brain tissue, demonstrating applicability across tissues and species. For direct validation, we demonstrated significant reductions in SenMayo in bone following genetic clearance of senescent cells in mice, with similar findings in adipose tissue from humans in a pilot study of pharmacological senescent cell clearance. In direct comparisons, SenMayo outperformed all six existing senescence/SASP gene sets in identifying senescent cells across tissues and in demonstrating responses to senescent cell clearance. We next used SenMayo to identify senescent hematopoietic or mesenchymal cells at the single cell level from publicly available human and murine bone marrow/bone scRNA-seq data and identified monocytic and osteolineage cells, respectively, as showing the highest levels of senescence/SASP genes. Using pseudotime and cellular communication patterns, we found senescent hematopoietic and mesenchymal cells communicated with other cells through common pathways, including the Macrophage Migration Inhibitory Factor (MIF) pathway, which has been implicated not only in inflammation but also in immune evasion, an important property of senescent cells. Thus, SenMayo identifies senescent cells across tissues and species with high fidelity. Moreover, using this senescence panel, we were able to characterize senescent cells at the single cell level and identify key intercellular signaling pathways associated with these cells, which may be particularly useful for evolving efforts to map senescent cells (e.g., SenNet). In addition, SenMayo represents a potentially clinically applicable panel for monitoring senescent cell burden with aging and other conditions as well as in studies of senolytic drugs.

A New Gene Set Identifies Senescent Cells and Predicts Senescence-Associated Pathways Across Tissues

Although cellular senescence is increasingly recognized as driving multiple age-related co-morbidities through the senescence-associated secretory phenotype (SASP), in vivo senescent cell identification, particularly in bulk or single cell RNA-sequencing (scRNA-seq) data remains challenging. Here, we generated a novel gene set (SenMayo) and first validated its enrichment in bone biopsies from two aged human cohorts. SenMayo also identified senescent cells in aged murine brain tissue, demonstrating applicability across tissues and species. For direct validation, we demonstrated significant reductions in SenMayo in bone following genetic clearance of senescent cells in mice, with similar findings in adipose tissue from humans in a pilot study of pharmacological senescent cell clearance. In direct comparisons, SenMayo outperformed all six existing senescence/SASP gene sets in identifying senescent cells across tissues and in demonstrating responses to senescent cell clearance. We next used SenMayo to identify senescent hematopoietic or mesenchymal cells at the single cell level from publicly available human and murine bone marrow/bone scRNA-seq data and identified monocytic and osteolineage cells, respectively, as showing the highest levels of senescence/SASP genes. Using pseudotime and cellular communication patterns, we found senescent hematopoietic and mesenchymal cells communicated with other cells through common pathways, including the Macrophage Migration Inhibitory Factor (MIF) pathway, which has been implicated not only in inflammation but also in immune evasion, an important property of senescent cells. Thus, SenMayo identifies senescent cells across tissues and species with high fidelity. Moreover, using this senescence panel, we were able to characterize senescent cells at the single cell level and identify key intercellular signaling pathways associated with these cells, which may be particularly useful for evolving efforts to map senescent cells (e.g., SenNet). In addition, SenMayo represents a potentially clinically applicable panel for monitoring senescent cell burden with aging and other conditions as well as in studies of senolytic drugs.

Effects and Related Mechanisms of the Senolytic Agent ABT-263 on the Survival of Irradiated A549 and Ca9-22 Cancer Cells

Senolytic agents eliminate senescent cells and are expected to reduce senescent cell-mediated adverse effects in cancer therapy. However, the effects of senolytic agents on the survival of irradiated cancer cells remain unknown. Here, the effects of the senolytic agent ABT-263 on the survival of irradiated A549 and Ca9-22 cancer cells were investigated. ABT-263 was added to the culture medium after irradiation. SA-β-gal activity and cell size, which are hallmarks of cell senescence, were evaluated using a flow cytometer. The colony-forming assay and annexin V staining were performed to test cell survival. We first confirmed that radiation increased the proportion of cells with high SA-β-gal activity and that ABT-263 decreased it. Of note, ABT-263 decreased the survival of irradiated cancer cells and increased the proportion of radiation-induced annexin V+ cells. Furthermore, the caspase inhibitor suppressed the ABT-263-induced decrease in the survival of irradiated cells. Intriguingly, ABT-263 decreased the proportion of SA-β-gal low-activity/large cells in the irradiated A549 cells, which was recovered by the caspase inhibitor. Together, these findings suggest that populations maintaining the ability to proliferate existed among the irradiated cancer cells showing senescence-related features and that ABT-263 eliminated the population, which led to decreased survival of irradiated cancer cells.

Targeting cellular senescence with novel senotherapeutics by design to extend healthspan

Senescent cells accumulate with age in various tissues and organs, leading to the decline in tissue function and deterioration of many age-related diseases and aging. Senolytics have emerged as an effective therapeutic approach to eliminate senescent cells to improve aging phenotypes and associated co-morbidities. Despite their promising potential, only a handful of senolytics have been reported, including a natural flavonoid fisetin discovered by our group. Fisetin has been shown to reduce senescence, suppress age-related pathology, and extend healthspan in aged mice. However, its moderate potency, potential mutagenic risk and poor bioavailability have limited its further clinical applications. By leveraging drug design, medicinal chemistry and high-content imaging analysis, we have successfully optimized the senolytic activity of fisetin, leading to the identification of two improved fisetin senolytic analogs (FAs) with reduced toxicity in non-senescent cells. The improved senolytic activity of these FAs was demonstrated in murine and human senescent cell models as well as in accelerated aging and naturally aged mouse models. The analysis of the senolytic activity of the FAs as well as several other recently identified senolytics, including a senolytic lipid, will be presented.

Senolytics in a Model of Alzheimer's Disease

The therapeutic effects of senescent cell killing with senolytics in neurodegeneration mouse models poise this strategy as an intervention candidate for Alzheimer’s Disease (AD). However, it is unclear whether senolytic therapies for AD are translatable to human cells. To determine whether senolytics could be a viable therapeutic for AD, we have treated long-term mixed human neuron/astrocyte primary cultures with amyloid beta oligomers (ABO), which we have shown to induce a phenotype consistent with senescence in neurons. Fifteen days after ABO treatment, we administered Navitoclax (Nav) and the natural killer cell-line NK92, which are known to selectively kill senescent cells in the periphery. Following treatment, we assessed senescence markers in our cultures as well as senescent cell killing selectivity through cleaved Caspase 3 quantification. Our preliminary data show that Nav (8, 4, and 0.5uM) kills both control and ABO treated cells. NK92 cells (10 to 1 effector to target ratio) also kill some control cells, suggesting there is not a clear cut mechanism by which NK92 cells can distinguish senescent from non-senescent neurons or astrocytes. Although analysis of selective killing is ongoing, off-target killing indicates that we need more refined senolytic strategies to implement their safe human use.

Sexual dimorphic responses of C57BL/6 mice to Fisetin or Dasatinib and Quercetin cocktail oral treatment

Senolytic treatment in aged mice clears senescent cell burden leading to functional improvements. We hypothesized that administering senotherapeutics in young adulthood of mice would slow physiological markers of aging through mid-life. C57BL/6 mice were treated monthly with either Fisetin or a Dasatinib (D) plus Quercetin (Q) cocktail from 4-13 months of age. Fisetin treated male mice had reduced senescence-associated secretory phenotype (SASP), enhanced glucose and energy metabolism, improved cognitive performance, and increased hippocampal expression of adiponectin 1 receptor and glucose transporter 4. D+Q treated females had increased SASP expression along with accumulation of white adipose tissue, reduced energy metabolism, and cognitive performance. Senotherapeutics in young adulthood, has beneficial, negligible, or detrimental effects in mice dependent upon sex and treatment.