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Cellular senescence is a primary aging process and tumor suppressive mechanism characterized by irreversible growth arrest, apoptosis resistance, production of a senescence-associated secretory phenotype (SASP), mitochondrial dysfunction, and alterations in DNA and chromatin. In preclinical aging models, accumulation of senescent cells is associated with multiple chronic diseases and disorders, geriatric syndromes, multimorbidity, and accelerated aging phenotypes. In animals, genetic and pharmacologic reduction of senescent cell burden results in the prevention, delay, and/or alleviation of a variety of aging-related diseases and sequelae. Early clinical trials have thus far focused on safety and target engagement of senolytic agents that clear senescent cells. We hypothesize that these pharmacologic interventions may have transformative effects on geriatric medicine.
Targeting Cellular Senescence as an Intervention in Primary Aging
Cellular and molecular processes that account for primary aging include chronic, low-grade, non-microbial inflammation, cellular senescence (see Glossary), accumulation of damaged macromolecules (DNA, proteins, carbohydrates, lipids), as well as stem and progenitor cell dysfunction [1]. These changes lead to pathophysiological manifestations of tissue atrophy and loss, denervation, and hypertrophy; decreases in perfusion, responsiveness to external signals (i.e., stimuli and stressors), and regenerative responses to injury; fatty transformation, and fibrosis; and changes in homeostatic set points. At the level of the organism, clinical findings present as functional decline, decreased resilience, increased vulnerability, reduced survival, and multimorbidity.
Substantial progress has been made towards targeting cellular senescence as a fundamental aging process with encouraging results that support the geroscience hypothesis [2]. In preclinical models, clearance of senescent cells results in improvement in age-related phenotypes, including geriatric syndromes, chronic diseases, and poor resilience [3., 4., 5., 6., 7., 8., 9., 10., 11., 12., 13., 14., 15.]. Interventions that genetically or pharmacologically remove senescent cells in animal models of aging offer proof-of-concept that they can be translated for human conditions associated with aging, especially those with heavy senescent cell burden (see Clinician’s Corner). There are many potential interventions proposed to intervene in primary aging processes that may be distinct from cellular senescence; however, these processes are not necessarily mutually exclusive and, in fact, may be interdependent (Figure 1). This suggests that any intervention that targets a single fundamental aging process could affect multiple processes that impact aging.
Figure 1. Interdependency of Fundamental Aging Processes.
Cellular senescence can occur in many cell types, including stem and progenitor cells, causing impairments in differentiated function and tissue regeneration. Through production of the senescence-associated secretory phenotype (SASP), chronic inflammation can mediate deteriorative changes and result in the accumulation of damaged macromolecules. The SASP can also promote paracrine and systemic bystander effects (Image 1) that cause stem and progenitor cell dysfunction. This schema is a framework that can be tested in a hypothesis-driven fashion.
Cellular Senescence
Cellular senescence has been described as a major mechanism for aging at the cellular level, although likely not the only mechanism. There are multiple inducers of cellular senescence, including DNA damage, reactive metabolites, inflammation, oncogenes, mitogens, proteotoxic stress, and damage-associated molecular patterns [16., 17., 18.]. A DNA damage response (DDR) is central for the induction of cellular senescence [19]. The DDR can be caused by telomere shortening or dysfunction, mutations, radiation, and alkylating agents. Reactive metabolites include reactive oxygen species, fatty acids, elevated glucose, and ceramides. Protein aggregation, unfolded protein responses, and mTOR activation are associated with proteotoxic stress and loss of proteostasis. These inducers ultimately mediate the cellular senescence phenotype through mechanisms that converge on pathways that activate p16INK4a/Rb and/or p53/p21, depending on the inducer and cell type [20]. Hallmarks of the cellular senescence phenotype include irreversible growth arrest, a SASP (Box 1), apoptosis resistance, mitochondrial dysfunction [21], persistent DNA damage foci [22], and epigenetic remodeling, which includes formation of senescence-associated heterochromatin foci [23]. Transposons (‘jumping genes’) can be produced within senescent cells that re-insert into senescent cell DNA, contributing to the senescent cell phenotype and exacerbating their SASP [24,25].
Box 1
The Senescence-Associated Secretory Phenotype (SASP)
The SASP consists of proinflammatory cytokines, chemokines, and extracellular matrix-degrading proteins that have deleterious paracrine and systemic effects, including inducing senescence in otherwise healthy cells as a bystander effect [11,95., 96., 97.]. Factors that are comprised in the SASP vary depending on the cell type from which senescent cells were derived, the inducer(s) of senescence, microenvironmental signals, and the approach to their suppression. The SASP can be modified or normalized using glucocorticoids, rapamycin, metformin, reverse transcriptase inhibitors (e.g., lamivudine) or JAK1/2 inhibitors [13,27,55,98., 99., 100.]. Depending on the kind of inhibition, not all components of the SASP are downregulated. Identification of cell-specific SASP factors may provide unique signatures of senescent cells that could be used as surrogate biomarkers for target engagement and clinical intervention. Unlike senolytic agents, which can be given intermittently to reduce senescent cell burden, SASP inhibitors, senomodulators, or senostatic agents that modify or suppress the SASP may need to be given on a more regular schedule, since senescent cells are not being cleared.
Although senescent cells may have beneficial effects in a transient fashion (Box 2), they accumulate chronically in virtually all tissues with aging, promoting detrimental consequences. The smallest amount of senescent cell burden required to promote local tissue dysfunction and/or have deleterious effects at distant locations is unknown. However, a relatively low abundance of senescent cells (e.g., ~10–15%) are present in the skin of old primates [26], and transplanting even a small number of senescent cells into younger mice is sufficient to cause tissue dysfunction [12]. Strategies for decreasing senescent cell burden include targeting networks of antiapoptotic regulators that confer senescent cell survival, reducing the inflammatory SASP, and genetically clearing senescent cells by activating apoptotic signals driven by p16Ink4a or p21Cip1 promoter elements in transgenic animals [3., 4., 5.,8,11., 12., 13., 14.,27., 28., 29., 30., 31., 32., 33., 34.].
Box 2
Potential Beneficial Effects of Cellular Senescence
Cellular senescence is a tumor suppressive mechanism [101], but it may have other beneficial physiological roles in wound healing and in development (i.e., embryogenesis). By contrast to senescent cells that accumulate slowly with aging, other senescent-like cells may function transiently in a remodeling role, with their removal facilitated by the immune system. Senescence may also be beneficial in limiting the fibrotic response produced secondary to liver damage [102] and in promoting stem cell function by inducing cell plasticity and facilitating tissue regeneration [103]. Activation of p16Ink4a in pancreatic beta cells promotes glucose-stimulated insulin release and, in diabetic mice, results in better glucose control [104]. The SASP may reinforce senescent cell growth arrest of precancerous cells through an autocrine feedback loop [105., 106., 107.]. It may also induce senescence of neighboring non-cancerous (but dividing) cells through a paracrine mechanism that protects against the same stressors that increase the risk of malignant transformation [95,108,109]. The effects of senescence are likely to be context-specific and pleiotropic and so intermittent clearance of senescent cells may help to avoid targeting of those that are beneficial in the short term.
Genetic Models of Senescent Cell Clearance
Elimination of even a relatively small proportion of existing senescent cells (~30% reduction) using a ‘suicide’ transgene, INK-ATTAC, that permits inducible elimination of p16Ink4a-expressing senescent cells upon administration of a drug (AP20187; Figure 2), extends health-span and prevents the development of multiple age-related morbidities in both progeroid and normal chronologically aged mice [3,8,11,35]. Using mice harboring the p16Ink4a promoter-driven ATTAC suicide transgene (Figure 2), it is possible to test the contributions of p16Ink4a- driven cellular senescence in mediating age-related phenotypes.
Figure 2. A Genetic Model for Senescent Cell Clearance.
Upper panel: schematic diagram of the INK-ATTAC construct. Lower panel: mechanism of apoptosis activation in p16Ink4a-positive senescent cells upon administration of AP20187 to INK-ATTAC mice. A fusion protein consisting of FK506-binding protein (FKBP) and caspase 8 (CASP8) is driven by a transcriptionally active element of the p16Ink4a promoter in senescent cells. A small-molecule compound (AP20187) without known off-target effects causes dimerization of FKBP-CASP8 and results in caspase-dependent apoptosis of senescent cells that are positive for p16Ink4a. Monitoring of p16Ink4a-positive senescent cells is accomplished by markers such as enhanced green fluorescent protein (EGFP). Use of internal ribosome entry sites (IRES) improves detection of auto-fluorescent reporter gene products such EGFP. Figure based on information first described by Baker et al. [3].
Current models of senescent cell clearance have been limited to the mouse. However, the rat has tremendous potential for use in geroscience research because its aging physiology is closer to humans than mice (especially with respect to onset of certain age-related conditions). Also, its larger size permits serial sampling and imaging studies that are not feasible in the mouse [36., 37., 38., 39., 40., 41.]. The rat is particularly amendable to measurements of cognitive-behavioral, endocrine (e.g., glucose tolerance), muscle, and cardiovascular endpoints and there is extensive historical data on physiological changes with aging [42., 43., 44., 45., 46.]. With advancements in genetic modification technologies in the rat [47., 48., 49., 50., 51.], it is now possible to create rat models of senescent cell clearance. For example, it would be very useful to produce a rat model where p16Ink4a-positive cells can selectively be eliminated. Results using this rat model would more closely model the effects of cell senescence (and its abrogation) toward human physiology.
Senotherapeutic Agents to Reduce Senescent Cell Burden
While genetic models of senescent cell clearance provide proof-of-principle for the health and life-span benefits of reducing the burden of old cells and the SASP, translation into humans has relied on the identification and development of senotherapeutic drugs (although efforts to develop senolytics actually began before and independently from developing the transgenic mice). Depending on whether these agents mostly promote senescent cell death or normalization of the SASP (i.e., without killing senescent cells), they have been described as senolytics or SASP inhibitors/senomorphics/senostatics, respectively (Figure 3, Key Figure) [12., 13., 14.,27., 28., 29., 30.,32,34,52., 53., 54., 55., 56.].
Figure 3. Key Figure. Pharmacologic Reduction in Senescent Cell Burden.
Senescence-associated secretory phenotype (SASP) inhibitors (senomorphic/senostatic agents) do not kill senescent cells but suppress or normalize the SASP. Immune clearance mitigates accumulation of senescent cells but diminishes with aging. Senolytic agents kill senescent cells by targeting apoptosis resistance pathways and may also improve immune clearance by removing senescent immune cells.
Senescent cells can resist apoptotic stimuli, thus increasing the likelihood of their survival through antiapoptotic defenses (Box 3). In some respects, senescent cells are like nondividing cancer cells, including apoptosis resistance and metabolic shifts [7,11,28,57., 58., 59.]. Many first-generation senolytics are in fact repurposed cancer therapeutics. Others are bioflavonoids. Senolytics have been used to prevent, delay, or alleviate a plethora of age-onset diseases and syndromes in preclinical models. Combinations of senolytics that target more than one pathway conferring apoptosis resistance will conceptually clear larger numbers and types of senescent cell subpopulations; this appears to be the case for the combination of the first generation senolytics dasatinib and quercetin [59]. Dasatinib is an orally bioavailable synthetic small molecule-inhibitor of SRC-family protein-tyrosine kinases, used in the therapy of chronic myelogenous leukemia that is positive for the Philadelphia chromosome. Quercetin is a bioflavonoid and antioxidant found in many food sources.
Box 3
Apoptosis Resistance Networks
The networks that senescent cells employ to become apoptosis resistant include those involving ephrins/dependence receptors, PI3K/AKT, Bcl-2 (Bcl-xl, Bcl-2, Bcl-w), p53/FOXO4/p21/Serpine (PAI-1&2), HIF-1α, and HSP90 [28,32]. Senolytics target different senescent cell populations, depending on the pathways used to confer apoptosis resistance [59]. For example, dasatinib interferes with apoptosis resistance networks containing dependence receptors, Src and tyrosine kinases, and targets senescent primary preadipocytes (i.e., adipose-derived stem cells). Quercetin interferes with networks containing the Bcl-2 family, p53/p21/Serpine, and PI3K/AKT and targets senescent endothelial cells and mesenchymal stem cells. In combination, dasatinib and quercetin target the senescent cell types that they are individually effective against, but also target senescent lung and embryonic fibroblasts.
In terms of alleviating age-related phenotypes, minimizing the SASP using Janus kinase/signal transducer and activator of transcription (JAK/STAT) inhibition is very similar to eliminating or reducing the burden of senescent cells genetically (e.g., using INK-ATTAC mice) or pharmacologically with senolytic agents [4,11,13,60]. Single or intermittent doses of senolytics appear to attenuate at least some age- or senescence-related conditions, which may not be the case for SASP-inhibitors, since they do not clear senescent cells. This is related to the time it takes for senescent cells to reaccumulate after they are removed. Based on studies in salamander limb regeneration [61] and in bleomycin-induced senescence in young mice [62], turnover of senescent cells is estimated to be about 4 weeks. Thus, intermittent treatment with senolytics may be a reasonable approach in humans, potentially reducing side effects. Because senescent cells do not replicate, concerns over possible drug resistance seem unlikely. At younger ages, senescent cells are cleared by the host immune system [63., 64., 65., 66.]; thus, to the extent that senotherapeutic or other agents may also delay or mitigate immune senescence or adverse effects of non-immune senescent cells on immune cell function, clearance of senescent cells may be further optimized (Figure 3).
The use of senotherapeutic drugs could potentially be effective in treating a variety of conditions primarily or secondarily mediated by accumulation of senescent cells [6,33,67., 68., 69., 70., 71., 72., 73., 74., 75., 76., 77., 78., 79., 80., 81., 82., 83., 84., 85., 86., 87.]. These include multimorbidity, conditions of premature or accelerated aging (e.g., childhood cancer survivors, bone marrow transplant survivors, progeroid syndromes, diabetes due to obesity, human immunodeficiency virus-related dementia and frailty, premature ovarian failure), conditions with localized cellular senescence (e.g., osteoarthritis, glaucoma, macular degeneration, fracture nonunion, radiotherapy-induced bystander effects, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease and tobacco-related lung injury, atherosclerosis, preeclampsia, hyperoxia-induced neonatal lung injury, and airway hyper-reactivity), and the geriatric syndromes. Otherwise fatal conditions for which pathogenesis is related to high senescent cell burden are also candidates for treatment with senotherapeutics (e.g., idiopathic pulmonary fibrosis, primary sclerosing cholangitis). Senolytics and/or senomorphics could also be used prophylactically in the setting of clinical stressors that are known to generate senescent cells, such as chemotherapy, radiotherapy, elective surgery, bone marrow transplantation, rehabilitation after myocardial infarction, and hypoxia-reoxygenation, as well as other stresses associated with arteriovenous fistulae for hemodialysis.
Early results from small pilot clinical studies suggest minimal adverse effects of senotherapeutic agents and clearance of senescent cells in humans [88., 89., 90., 91.]. However, with the first generation senolytic dasatinib, contraindications exist, including QTc interval >450 ms, hepatic insufficiency, drug interactions with CYP3A4 inhibitors, CYP3A4 inducers, antacids, H2 antagonists/proton pump inhibitors, and other cautions as indicated by the full prescribing information. At doses and frequency used for anticancer effects, dasatinib can cause myelosuppression, bleeding-related events, fluid retention, cardiovascular events, pulmonary arterial hypertension, QT prolongation, severe dermatologic reactions, cell lysis syndrome, and embryo-fetal toxicity, but it is uncertain how these potential complications will relate to the intermittent and infrequent administration of dasatinib used for senescent cell clearance. Similarly, the first generation senolytic quercetin has contraindications with respect to potential drug–drug interactions (e.g., strong inhibitors of CYP3A4) and common side effects (e.g., headache), but again it is not clear how these possible adverse events will be manifested with intermittent dosing regimens.
On-going clinical trials will continue to primarily address safety and target engagement of senescent cells, with secondary endpoints that may give insight into efficacy of senolytics and senomorphics. See Resources for representative clinical trials that are assessing senotherapeutic drugsi-v. Larger clinical trials will be needed to better understand the potential for senolytics and senomorphics as therapeutic interventions and to more fully evaluate off-target effects. Limitations of current clinical trials include small recruitment sizes, the paucity of measurable, clinically relevant outcomes appropriate for older adults, as well as the need for biomarkers that follow pharmacokinetic, pharmacodynamic, and mechanistic properties of study drugs and that could serve as surrogate endpoints [92., 93., 94.].
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Edited by Engadin, 20 May 2020 - 04:22 PM.
