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Senolytics, small molecules targeting cellular senescence, have emerged as potential therapeutics to enhance health span. However, their impact on epigenetic age remains unstudied. This study aimed to assess the effects of Dasatinib and Quercetin (DQ) senolytic treatment on DNA methylation (DNAm), epigenetic age, and immune cell subsets. In a Phase I pilot study, 19 participants received DQ for 6 months, with DNAm measured at baseline, 3 months, and 6 months. Significant increases in epigenetic age acceleration were observed in first-generation epigenetic clocks and mitotic clocks at 3 and 6 months, along with a notable decrease in telomere length. However, no significant differences were observed in second and third-generation clocks. Building upon these findings, a subsequent investigation evaluated the combination of DQ with Fisetin (DQF), a well-known antioxidant and antiaging senolytic molecule. After one year, 19 participants (including 10 from the initial study) received DQF for 6 months, with DNAm assessed at baseline and 6 months. Remarkably, the addition of Fisetin to the treatment resulted in non-significant increases in epigenetic age acceleration, suggesting a potential mitigating effect of Fisetin on the impact of DQ on epigenetic aging. Furthermore, our analyses unveiled notable differences in immune cell proportions between the DQ and DQF treatment groups, providing a biological basis for the divergent patterns observed in the evolution of epigenetic clocks. These findings warrant further research to validate and comprehensively understand the implications of these combined interventions.

In addition to reducing fracture risk, zoledronic acid has been found in some studies to decrease mortality in humans and extend lifespan and healthspan in animals. Because senescent cells accumulate with aging and contribute to multiple co-morbidities, the non-skeletal actions of zoledronic acid could be due to senolytic (killing of senescent cells) or senomorphic (inhibition of the secretion of the senescence-associated secretory phenotype [SASP]) actions. To test this, we first performed in vitro senescence assays using human lung fibroblasts and DNA repair-deficient mouse embryonic fibroblasts, which demonstrated that zoledronic acid killed senescent cells with minimal effects on non-senescent cells. Next, in aged mice treated with zoledronic acid or vehicle for 8 weeks, zoledronic acid significantly reduced circulating SASP factors, including CCL7, IL-1β, TNFRSF1A, and TGFβ1 and improved grip strength. Analysis of publicly available RNAseq data from CD115+ (CSF1R/c-fms+) pre-osteoclastic cells isolated from mice treated with zoledronic acid demonstrated a significant downregulation of senescence/SASP genes (SenMayo). To establish that these cells are potential senolytic/senomorphic targets of zoledronic acid, we used single cell proteomic analysis (cytometry by time of flight [CyTOF]) and demonstrated that zoledronic acid significantly reduced the number of pre-osteoclastic (CD115+/CD3e-/Ly6G-/CD45R-) cells and decreased protein levels of p16, p21, and SASP markers in these cells without affecting other immune cell populations. Collectively, our findings demonstrate that zoledronic acid has senolytic effects in vitro and modulates senescence/SASP biomarkers in vivo . These data point to the need for additional studies testing zoledronic acid and/or other bisphosphonate derivatives for senotherapeutic efficacy.

The accumulation of senescent cells has an important role in the phenotypical changes observed in ageing and in many age-related pathologies. Thus, the strategies designed to prevent these effects, collectively known as senotherapies, have a strong clinical potential. Senolytics are a type of senotherapy aimed at specifically eliminating senescent cells from tissues. Several small molecule compounds with senolytic properties have already been identified, but their specificity and range of action are variable. Because of this, potential novel senolytics are being actively investigated. Given the involvement of HDACs and the PI3K pathway in senescence, we hypothesized that the dual inhibitor CUDC-907, a drug already in clinical trials for its antineoplastic effects, could have senolytic effects. Here, we show that CUDC-907 was indeed able to selectively induce apoptosis in cells driven to senesce by p53 expression, but not when senescence happened in the absence of p53. Consistent with this, CUDC-907 showed senolytic properties in different models of stress-induced senescence. Our results also indicate that the senolytic functions of CUDC-907 depend on the inhibitory effects of both HDACs and PI3K, which leads to an increase in p53 and a reduction in BH3 pro-survival proteins. Taken together, our results show that CUDC-907 has the potential to be a clinically relevant senolytic in pathological conditions in which stress-induced senescence is involved.

The aging of the immune system, or immunosenescence, was recently verified to have a causal role in driving the aging of solid organs, while the senolytic elimination of senescent immune cells was found to effectively delay systemic aging. Our recent study also showed that immune cells in severely dystrophic muscles develop senescence-like phenotypes, including the increased expression of senescence-associated secretory phenotype (SASP) factors and senescence markers. Here we further investigated whether the specific clearance of senescent immune cells in dystrophic muscle may effectively improve the function of muscle stem cells and the phenotypes of dystrophic muscle. We observed increased percentage of senescent cells in macrophages from mdx /utro(-/-) mice (a murine model for muscular dystrophy disease, dystrophin-/-; utrophin-/-), while the treatment of mdx /utro(-/-) macrophages with senolytic drug fisetin resulted in reduced number of senescent cells. We administrated fisetin to mdx /utro(-/-) mice for 4 weeks, and observed obviously reduced number of senescent immune cells, restored number of muscle cells, and improve muscle phenotypes. In conclusion, our results reveal that senescent immune cells, such as macrophages, are greatly involved in the development of muscle dystrophy by impacting the function of muscle stem cells, and the senolytic ablation of these senescent cells with fisetin can be an effective therapeutic strategy for improving function of muscle stem cells and phenotypes of dystrophic muscles.

Aging is a risk factor for the development of osteoarthritis (OA), a progressive joint disease leading to cartilage damage, pain, and loss of function. In a mouse model of OA, senolytic drugs to selectively clear senescent cells (SnCs) that accumulate with injury or aging yielded a chondroprotective effect; however, this therapeutic benefit was limited in aged mice. Due to inconsistency between cartilage destruction and pain-associated symptoms, we studied the therapeutic effect of senolytics on joint pain in spontaneous OA. We orally treated 21- and 22-month old mice with an ABT263 and Dasatinib and Quercetin (D+Q) drug combination. Selective elimination of the SnCs that accumulated in the articular cartilage and synovium by these two drugs did not alter cartilage degeneration and abnormal bone changes during spontaneous OA progression. Treatment reduced thermal and mechanical hyperalgesia associated with OA and peripheral sensitization through decreased expression of axon guidance proteins (nerve growth factor NGF/TrkA) and nociceptive neuron (calcitonin gene-related peptide, CGRP) projection to the synovium, subchondral bone marrow, and dorsal root ganglion, and knee joint angiogenesis. Selective removal of the SnCs from in vitro cultures of synovial cells from human OA patients also decreased expression of senescent markers, axonal growth-promoting factors, such as NGF, and angiogenesis markers. We suggest that systemic administration of ABT263 and D+Q is an exciting therapeutic approach to age-related OA pain.

Accumulation of senescent cells in tissues with advancing age participates in the pathogenesis of several human age-associated diseases. Specific senescent secretome, the resistance of senescent cells to apoptotic stimuli, and lack of immune system response contribute to the accumulation of senescent cells and their adverse effects in tissues. Inhibition of antiapoptotic machinery, augmented in senescent cells, by BCL-2 protein family inhibitors represents a promising approach to eliminate senescent cells from tissues. This study aimed to explore synergistic and selective senolytic effects of anti-apoptotic BCL-2 family targeting compounds, particularly BH3 mimetics. Using human non-transformed cells RPE-1, BJ, and MRC-5 brought to ionizing radiation-, oncogene-, drug-induced and replicative senescence, we found synergy in combining MCL-1 selective inhibitors with other BH3 mimetics. In an attempt to uncover the mechanism of such synergy, we revealed that the surviving subpopulation of cells resistant to individually applied ABT-737/ABT-263, MIK665, ABT-199, and S63845 BCL-2 family inhibitors showed elevated MCL-1 compared to untreated control cells indicating the presence of a subset of cells expressing high MCL-1 levels and, therefore, resistant to BCL-2 inhibitors within the original population of senescent cells. Overall, we found that combining BCL-2 inhibitors can be beneficial for eliminating senescent cells, thereby enabling use of lower, potentially less toxic, doses of drugs compared to monotherapy, thereby overcoming the resistance of the subpopulation of senescent cells to monotherapy.

Cell senescence is a process that causes growth arrest and the release of a senescence associated secretory phenotype (SASP), characterized by secretion of chemokines, cytokines, cell growth factors and metalloproteases, leading to a tissue condition that may precipitate cancers and neurodegenerative processes. With the recent pandemic of coronavirus, senolytic drugs are being considered as possible therapeutic tools to reduce the virulence of SARS-CoV-2. In the last few years, our research group showed that lithium carbonate at microdose levels was able to stabilize memory and change neuropathological characteristics of Alzheimer's disease (AD). In the present work, we present evidence that low-dose lithium can reduce the SASP of human iPSCs-derived astrocytes following acute treatment, suggesting that microdose lithium could protect cells from senescence and development of aging-related conditions. With the present findings, a perspective of the potential use of low-dose lithium in old patients from the "high risk group" for COVID-19 (with hypertension, diabetes and chronic obstructive pulmonary disease) is presented.

COVID-19 is not deadly early in life, but mortality increases exponentially with age, which is the strongest predictor of mortality. Mortality is higher in men than in women, because men age faster, and it is especially high in patients with age-related diseases, such as diabetes and hypertension, because these diseases are manifestations of aging and a measure of biological age. At its deepest level, aging (a program-like continuation of developmental growth) is driven by inappropriately high cellular functioning. The hyperfunction theory of quasi-programmed aging explains why COVID-19 vulnerability (lethality) is an age-dependent syndrome, linking it to other age-related diseases. It also explains inflammaging and immunosenescence, hyperinflammation, hyperthrombosis, and cytokine storms, all of which are associated with COVID-19 vulnerability. Anti-aging interventions, such as rapamycin, may slow aging and age-related diseases, potentially decreasing COVID-19 vulnerability.

The selective removal of senescent cells by senolytics is suggested as a potential approach to reverse aging and extend lifespan. Using high-throughput screening with replicative senescence of human diploid fibroblasts (HDFs), we identified a novel senolytic drug R406 that showed selective toxicity in senescent cells. Using flow cytometry and caspase expression analysis, we confirmed that R406 caused apoptotic cell death along with morphological changes in senescent cells. Interestingly, R406 altered the cell survival-related molecular processes including the inhibition of phosphorylation of the focal adhesion kinase (FAK) and p38 mitogen-activated protein kinase (MAPK) in senescent cells. This pattern was not observed in other known senolytic agent ABT263. Correspondingly, apoptotic cell death in senescent cells was induced by simultaneously blocking the FAK and p38 pathways. Taken together, we suggest that R406 acts as a senolytic drug by inducing apoptosis and reducing cell attachment capacity.

Cellular senescence is a hallmark of aging, whose onset is linked to a series of both cell and non-cell autonomous processes, leading to several consequences for the organism. To date, several senescence routes have been identified, which play a fundamental role in development, tumor suppression and aging, among other processes. The positive and/or negative effects of senescent cells are directly related to the time that they remain in the organism. Short-term (acute) senescent cells are associated with positive effects; once they have executed their actions, immune cells are recruited to remove them. In contrast, long-term (chronic) senescent cells are associated with disease; they secrete pro-inflammatory and pro-tumorigenic factors in a state known as senescence-associated secretory phenotype (SASP). In recent years, cellular senescence has become the center of attention for the treatment of aging-related diseases. Current therapies are focused on elimination of senescent cell functions in three main ways: i) use of senolytics; ii) inhibition of SASP; and iii) improvement of immune system functions against senescent cells (immunosurveillance). In addition, some anti-cancer therapies are based on the induction of senescence in tumor cells. However, these senescent-like cancer cells must be subsequently cleared to avoid a chronic pro-tumorigenic state. Here is a summary of different scenarios, depending on the therapy used, with a discussion of the pros and cons of each scenario.

Therapeutic strategies targeting the hallmarks of aging can be broadly grouped into four categories, namely systemic (blood) factors, metabolic manipulation (diet regimens and dietary restriction mimetics), suppression of cellular senescence (senolytics), and cellular reprogramming, which likely have common characteristics and mechanisms of action. In evaluating the potential synergism of combining such strategies, however, we should consider the possibility of constraining trade-off phenotypes such as impairment in wound healing and immune response, tissue dysfunction and tumorigenesis. Moreover, we are rapidly learning that the benefit/risk ratio of aging-targeted interventions largely depends on intra- and inter-individual variations of susceptibility to the healthspan-, resilience-, and/or lifespan-promoting effects of the interventions. Here, we exemplify how computationally-generated proxies of the efficacy of a given lifespan/healthspan-promoting approach can predict the impact of baseline epigenetic heterogeneity on the positive outcomes of ketogenic diet and mTOR inhibition as single or combined anti-aging strategies. We therefore propose that stochastic biomathematical modeling and computational simulation platforms should be developed as in silico strategies to accelerate the performance of clinical trials targeting human aging, and to provide personalized approaches and robust biomarkers of healthy aging at the individual-to-population levels.

Is aging a disease? It does not matter because aging is already treated using a combination of several clinically-available drugs, including rapamycin. Whether aging is a disease depends on arbitrary definitions of both disease and aging. For treatment purposes, aging is a deadly disease (or more generally, pre-disease), despite being a normal continuation of normal organismal growth. It must and, importantly, can be successfully treated, thereby delaying classic age-related diseases such as cancer, cardiovascular and metabolic diseases, and neurodegeneration.

Senescent cells accumulate with aging and at sites of pathology in multiple chronic diseases. Senolytics are drugs that selectively promote apoptosis of senescent cells by temporarily disabling the pro-survival pathways that enable senescent cells to resist the pro-apoptotic, pro-inflammatory factors that they themselves secrete. Reducing senescent cell burden by genetic approaches or by administering senolytics delays or alleviates multiple age- and disease-related adverse phenotypes in preclinical models. Reported senolytics include dasatinib, quercetin, navitoclax (ABT263), and piperlongumine. Here we report that fisetin, a naturally-occurring flavone with low toxicity, and A1331852 and A1155463, selective BCL-X L inhibitors that may have less hematological toxicity than the less specific BCL-2 family inhibitor navitoclax, are senolytic. Fisetin selectively induces apoptosis in senescent but not proliferating human umbilical vein endothelial cells (HUVECs). It is not senolytic in senescent IMR90 cells, a human lung fibroblast strain, or primary human preadipocytes. A1331852 and A1155463 are senolytic in HUVECs and IMR90 cells, but not preadipocytes. These agents may be better candidates for eventual translation into clinical interventions than some existing senolytics, such as navitoclax, which is associated with hematological toxicity.