MINI REVIEW

published: 22 January 2020

doi: 10.3389/fcell.2019.00367

Senolytics: A Translational Bridge

Between Cellular Senescence and
Organismal Aging
Harikrishnan Thoppil 1,2 and Karl Riabowol 1,2*
1
Arnie Charbonneau Cancer Institute, Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB,
Canada, 2 Arnie Charbonneau Cancer Institute, Department of Oncology, University of Calgary, Calgary, AB, Canada

Aging is defined as a progressive decrease in physiological function accompanied

by a steady increase in mortality. The antagonistic pleiotropy theory proposes that
aging is largely due to the natural selection of genes and pathways that increase
fitness and decrease mortality early in life but contribute to deleterious effects and
pathologies later in life. Cellular senescence is one such mechanism, which results in
a permanent cell cycle arrest that has been described as a mechanism to limit cancer
cell growth. However, recent studies have also suggested a dark side of senescence
in which a build-up of senescent cells with age leads to increased inflammation due
Edited by: to a senescence-associated secretory phenotype (SASP). This phenotype that includes
Vassilis G. Gorgoulis,
many cytokines promotes tumorigenesis and can exhaust the pool of immune cells in
National and Kapodistrian University
of Athens, Greece the body. Studies clearing senescent cells from mice using the p16-based transgene
Reviewed by: INK-ATTAC have shown that senescent cells can impact both organismal aging and
Chiaki Takahashi, lifespan. Here we discuss these advances that have resulted in the development of
Kanazawa University, Japan
Brian Gabrielli,
a whole new class of compounds known as senolytics, some of which are currently
The University of Queensland, undergoing clinical trials in humans for treating a variety of age-related pathologies such
Australia
as osteoarthritis.
*Correspondence:
Karl Riabowol Keywords: aging, senescence, lifespan, senolytics, senomorphics
karl@ucalgary.ca

Specialty section: AGING

This article was submitted to
Cell Growth and Division, Limited Replicative lifespan was first described by Hayflick and Moorhead (1961), who showed that
a section of the journal human diploid fibroblasts have a finite capacity for replication after which they enter a metabolically
Frontiers in Cell and Developmental active but irreversibly arrested proliferative state. Aging has been extensively studied in model
Biology
organisms such as Caenorhabditis elegans and is affected by a variety of factors such as stress,
Received: 28 October 2019 nutrient intake, sex, and gene expression (Moskalev et al., 2015). Several genes have been discovered
Accepted: 16 December 2019
in these relatively simple model organisms such as DAF-2 (insulin/insulin-like growth factor-1
Published: 22 January 2020
receptor homolog) which, when mutated results in almost doubling the lifespan of C. elegans
Citation: (Kenyon, 2011). Success in manipulating lifespan in these model organisms ushered in a massive
Thoppil H and Riabowol K (2020)
hunt to discover more aging-related genes, mechanisms and therapeutic compounds. At present,
Senolytics: A Translational Bridge
Between Cellular Senescence
The DrugAge database of aging-related drugs lists around 567 distinct chemical perturbagens that
and Organismal Aging. can significantly increase lifespan in a subset of non-disease models spanning over 30 species
Front. Cell Dev. Biol. 7:367. (Barardo et al., 2017). Clinical trials such as The Metformin in Longevity Study (MILES) have been
doi: 10.3389/fcell.2019.00367 launched recently to assess the anti-aging potential of metformin in delaying age-related ailments

Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 January 2020 | Volume 7 | Article 367
Thoppil and Riabowol Senolytics in Cellular and Organismal Aging

in humans (Crandall, 2015; Piskovatska et al., 2019). One of hallmarks of the senescence phenotype, although no markers
the essential components for studying aging and age-related appear to be universal for all types of senescent cells. Therefore,
diseases in humans are biomarkers that are indicative of to ensure the accurate identification of senescent cells it has been
chronological age (Calimport et al., 2019). Recently it has been recommended by the International Cell Senescence Association
shown that DNA methylation patterns show a strong correlation that multiple markers be used in a three-step senescence
with chronological age and this “epigenetic clock” is effective identification protocol (Gorgoulis et al., 2019).
in predicting all-cause mortality with age (Horvath, 2013).
Analyzing cancer tissues with this epigenetic clock, composed of
methylation levels from 353 CpGs, indicated that tissues from MECHANISMS AND STIMULI OF
cancer patients treated with various therapies appeared to be an SENESCENCE
average of 36 years older compared to the actual chronological
age of the patients, while induced pluripotent stem cells (iPSCs) A wide variety of stimuli affecting multiple molecular pathways
from the same individuals showed resetting of the clock to are involved in the induction of a senescence based irreversible
an epigenetic age of zero. However, the biological mechanisms arrest in a state resembling G0 of the cell cycle in mammalian
behind this epigenetic biomarker remain unknown, especially, cells (Kuilman et al., 2010; Muñoz-Espín and Serrano, 2014).
due to a lack of correlation with gene expression data. These pathways are broadly classified into activating either
p16 (Alcorta et al., 1996) or p21 (Brown et al., 1997) via
the p53 tumor suppresser (Atadja et al., 1995; Vaziri et al.,
SENESCENCE 1997), and these signals converge to produce high levels
of active hypophosphorylated Rb (Retinoblastoma) Tumor
Senescence refers to a state of permanent proliferative Suppressor (Chicas et al., 2010; Rajarajacholan et al., 2013).
arrest characterized by insensitivity to growth factors and The tumor suppressive nature of cellular senescence is quite
mitogens (Kuilman et al., 2010). One of the mechanisms evident from the fact that tampering with levels of several
that regulate this insensitivity is dysregulation of normal of these oncogenes/tumor suppressors can lead to an escape
endocytosis (Wheaton et al., 2001; Rajarajacholan et al., from senescence (Campisi, 1997). Broadly speaking, senescence
2013). Senescent cells, were shown to overexpress caveolins, a can be triggered by numerous stresses including DNA-damage,
major component of endocytosis apparatus which prevented oxidative stress, and oncogene induced stress. DNA-damage
their ability to phosphorylate Erk-1/2 phosphorylation post induced senescent cells are primarily observed in cancer patients
EGF stimulation which was recovered by downregulation via post administration of radiotherapy and chemotherapy (Robles
antisense-oligonucleotides. Similar suppression of Erk-1/2 et al., 1999; Gewirtz et al., 2008) and treatment results in
activation was also observed in non-senescent cells post caveolin accelerated telomere loss (Unryn et al., 2006). Radiotherapy and
overexpression (Park, 2002; Park et al., 2002). In cell culture, Chemotherapy not only affect cancer tissue but also induce
as observed by Hayflick and Moorhead (1961), a senescence senescence in surrounding healthy tissue. Several observations
state is achieved upon repeated passaging, and as shown later, have confirmed that exposure to topoisomerase inhibitors such
it is mainly due to shortening of telomeric DNA found at the as doxorubicin and etoposide induces senescence in primary
end of chromosomes (Harley et al., 1990) that activates an diploid human fibroblasts in vitro (Robles et al., 1999). Studies
ataxia-telangiectasia mutated (ATM) (Vaziri et al., 1997) and have also shown that proteins of the SASP that are released from
p53-mediated (Atadja et al., 1995) DNA damage response. these surrounding senescent cells can induce an epithelial to
This was hypothesized first as the end replication problem; mesenchymal transition (EMT) and hence promote malignancy
as a consequence of semi-conservative DNA replication and in normally non-aggressive human breast cancer cell lines
later confirmed by Blackburn, Greider, and Szostak (Lundblad (Coppé et al., 2008). Another important player in inducing
and Szostak, 1989; Blackburn, 1991). Senescent cells typically senescence is ING1a from the INhibitor of Growth family of
have an enlarged morphology and are most widely detected epigenetic regulators. Originally identified in 1996 by subtractive
histochemically by an increased β-galactosidase activity known hybridization between cDNAs from normal mammary epithelial
as senescence-associated β-galactosidase (SA-βGAL) (Itahana cells and transformed breast cancer cells, the ING family of
et al., 2007), which is correlated to increased autophagy (Young genes are evolutionary conserved and ING proteins primarily
et al., 2009). Other biomarkers of senescence include increased localize to the nucleus (Garkavtsev et al., 1996). Our lab has
expression of common senescence mediators such as p16, shown that the ING1a isoform that is expressed in aging,
p21, p53, and p47ING1a (Kuilman et al., 2010; Rajarajacholan but not low passage fibroblasts induces senescence when
et al., 2013). However, not all types of senescence result ectopically expressed several-fold faster (24–36 h) compared
from telomere depletion. For example, up to 50% of mouse to other senescence inducing agents such as t-BHP (t-butyl
embryonic fibroblasts (MEF’s) exhibit a senescence phenotype hydroperoxide) and doxorubicin (Rajarajacholan and Riabowol,
after a mere five passages resulting from oxygen sensitivity due 2015). We have shown that inhibition of endocytosis is one
to ROS-induced DNA damage (Parrinello et al., 2003). These of the major mechanisms by which ING1a induces senescence
interdependent features of cell cycle withdrawal, macromolecular (Rajarajacholan et al., 2013; Rajarajacholan and Riabowol,
damage, dysregulated metabolism and an altered senescence- 2015). This occurs via affecting at least three independent
associated secretory phenotype (SASP) have been described as members of the Rb pathway including Rb, p16INK4a , and

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Thoppil and Riabowol Senolytics in Cellular and Organismal Aging

FIGURE 1 | Schematic representation of the link between cellular senescence and aging with examples of SASP Factors, Senolytics and Senomorphics and their
targets of action.

p57Kip2 . ING1a induces expression of Intersectin 2, subsequently delayed onset of sarcopenia, cataracts, significantly better exercise
altering the stoichiometry of the endocytosis apparatus and test scores and reduction in other age-related pathologies. Later
blocking signaling by mitogens (Rajarajacholan et al., 2013). This studies confirmed the beneficial effects of senescent cell removal
represents a novel mechanism of senescence induction that is in wild type mice that showed increased median lifespan,
independent of telomere attrition and DNA damage signaling. delayed tumorigenesis and attenuated age-related multi-organ
deterioration (Baker et al., 2016). Removal of senescent cells in
mice has also been shown to attenuate markers of age-associated
CELLULAR SENESCENCE AND neurodegenerative diseases such as tau hyperphosphorylation
ORGANISMAL AGING and neurofibrillary tangle deposition (Bussian et al., 2018). Since
it is likely that even a small percentage of senescent cells can
One of the hallmarks of mammalian aging is the accumulation of promote substantial deleterious effects, there may be natural
irreversibly arrested senescent cells in various tissues (Figure 1). mechanisms that clear these senescent cells in vivo to maintain
In a 2006 primate study, it was observed that senescent cells, overall fitness. One primary response to p53 activation in murine
as estimated by ATM activation do accumulate and can reach liver carcinoma was found to be the accumulation of senescent
over 15% of the total cell population in aged individuals (Herbig cells, which were subsequently shown to be cleared by the innate
et al., 2006). In contrast to the vast majority of in vitro studies, immune response (Xue et al., 2011). NK cells were also found
this was one of the first studies showing a clear association to eliminate senescent cells in vivo by perforin (Prf) mediated
between aging and the accumulation of senescent cells in vivo. granule exocytosis (Sagiv et al., 2013) and Prf1−/− mice showed
Although this established a strong correlation, efforts were a 4X accumulation of senescent cells and a 21% decrease in
underway to establish causation between the accumulation of median lifespan, which was alleviated by senescent cell clearance
senescent cells and aging in vivo. In 2011, the Kirkland lab showed (Ovadya et al., 2018).
that removing p16Ink4a positive senescent cells delayed age- Further evidence regarding the connection between cellular
related disorders and increased healthspan in a BubR1 progeroid senescence and organismal aging comes from accelerated
accelerated model of aging mice (Baker et al., 2011). This was aging syndromes such as HGPS (Hutchinson-Gilford progeria
accomplished using a novel transgene composed of a p16Ink4a syndrome). A higher proportion of HGPS fibroblasts are SA-
promoter driving a FKBP–Caspase-8 fusion protein, which is β-Gal positive compared to fibroblasts from normal, age-matched
selectively expressed in senescent cells and can be activated by controls, and they can be returned to a normal replicative state
the synthetic drug, AP20187. These transgenic mice showed by exogenous expression of the TERT subunit of telomerase

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Thoppil and Riabowol Senolytics in Cellular and Organismal Aging

(Benson et al., 2010). Similarly, expressing progerin, the mutant which attenuate the expression of cytokines such as IL-6
lamin-A protein known to cause HGPS, in primary cells obtained (Interleukin-6) that make up the SASP. UBX0101, a compound
from healthy individuals result in expression of senescent from the Unity Biotechnology pipeline is another promising
markers (Cao et al., 2011). These results suggest that progerin- senolytic that inhibits the MDM2/p53 interaction and is currently
induced telomere dysfunction may result in premature cellular undergoing Phase 1 clinical trial against osteoarthritis. UBX0101
senescence leading to the accelerated aging symptoms observed administration was reported to decrease classical osteoarthritis-
in HGPS patients. related phenotypes such as cartilage erosion and joint pain in
mice by p53-mediated clearing of senescent cells. This study
suggested that senescent cells might promote osteoarthritis (Jeon
SENOLYTICS AND SENOMORPHICS et al., 2017), another medically relevant age-related pathology.
Heat Shock Protein 90 (HSP90) might represent another
Substantial evidence in the last decade connecting senescent cell target for senolysis. This protein is one member of a large
accumulation, age-related ailments, and roles in lifespan and family of chaperones that help other proteins to fold and refold
healthspan fueled the search for therapeutic compounds that after cell stress and is indirectly involved in a wide variety
could selectively target senescent cells. A transcriptomic analysis of cellular processes such as DNA repair, heat stress response
between senescent cells and proliferating cells showed increased and neurodegenerative pathologies (Schopf et al., 2017). While
expression of pro-survival/anti-apoptotic genes such as Bcl-xL HSP90 levels remain relatively similar in senescent and non-
(B-cell lymphoma-extra-large) a member of the Bcl-2 family senescent cells, AKT (Protein Kinase B), which is a downstream
of proteins that regulates programmed cell death by blocking effector of HSP90 is highly expressed in senescent cells. Inhibiting
caspase activation (Zhu et al., 2016). This provided evidence to HSP90 destabilizes AKT and results in increased apoptosis
support the observation that senescent cells accumulate with age (Karkoulis et al., 2013). HSP90 inhibitors such as 17-DMAG
by being resistant to a variety of stresses that would normally were discovered through a screen for senolytic compounds
induce apoptosis (Wang, 1995). Consistent with this idea, siRNAs based on SA-βGAL positive cell count as an endpoint. 17-
to reduce Bcl-xL expression selectively reduced survival and DMAG was further shown to be senolytic in vivo by decreasing
viability in senescent cells while not affecting proliferating cells p16 positive cells and extending healthspan in Ercc1−/1 mice
(Zhu et al., 2015). Quercetin and Dasatinib were obtained as (Fuhrmann-Stroissnigg et al., 2017).
hits from a drug screen based on these observations (Hickson
et al., 2019). Quercetin: a flavonoid and Dasatinib: an anticancer
agent, are known inhibitors of a variety of tyrosine kinases THE NEXT GENERATION OF
(Huang et al., 1999; Montero et al., 2011). These compounds SENOLYTICS
form one of the first discovered members of the senolytic class
of drugs that selectively induce apoptosis in senescent cells. Four Since senescence is a complex phenotype involving multiple
years after their initial identification as candidate senolytics, a pathways and proteins, it is unlikely that a single senolytic
Dasatinib + Quercetin combination was reported to decrease the compound targeting a single protein will be able to eliminate all
senescent cell burden in humans as part of a Phase-1 clinical types of senescent cells. For example, Quercetin is more selective
trial in diabetic kidney disease patients (Hickson et al., 2019). against senescent human endothelial cells, while Dasatinib is far
This 2019 study was the first peer-reviewed study to demonstrate more effective against senescent human primary preadipocytes
the efficacy of senolytics to decrease senescent cell burden in (Hickson et al., 2019). Similarly, while Navitoclax, a senolytic
humans. This was carried out after an initial pilot study in agent targeting the Bcl-2 family of proteins is selective against
early 2019 in 14 idiopathic pulmonary fibrosis (IPF) patients senescent human umbilical vein epithelial cells (HUVECs) and
was completed to evaluate the feasibility of implementing a IMR90 human lung fibroblasts, it is not particularly effective
senolytic treatment (Justice et al., 2019). What now remains to be against primary human preadipocytes (Zhu et al., 2016). Senolytic
determined is whether future clinical trials will demonstrate any compounds that target senescent cells more broadly while
positive medical outcomes resulting from decreased senescent maintaining low cytotoxicity by increased selectivity solely for
cell burden in diabetes and other age-associated ailments. cells that are truly senescent are therefore of great interest!
Another senolytic strategy is the inhibition of the interaction Elevated activity of the lysosomal β-galactosidase, which is a
between Mouse Double Minute 2 (MDM2) and p53 that usually nearly universal characteristic of senescent cells (Lee et al., 2006)
results in the ubiquitination and proteasomal degradation of and has been exploited as a senescence biomarker can also be
p53 (Nag et al., 2013; Wade et al., 2013). The MDM2 protein utilized for drug selectivity against a wide range of senescent cells.
acts as an E3 ubiquitin ligase and facilitates p53 degradation. One example is a drug delivery system utilizing encapsulation
MDM2 contains two promoters: P1 and P2. P1 is constitutively of cytotoxic drugs with galacto-oligosaccharides (Muñoz-Espín
active at low levels while the P2 promoter has p53 binding et al., 2018). These capsules that can be loaded with a variety
sites and acts as a negative regulator of p53 (Hollerer et al., of cytotoxic compounds, take advantage of the high senescent
2019). The MDM2 antagonists Nutlin-3a and MI-63 have cell lysosomal β-galactosidase activity to preferentially release
been shown to increase p53 levels and attenuate the secretory their cargo within senescent cells. A very recent study also
phenotype of senescent cells (SASP) (Wiley et al., 2018). These used a similar strategy involving galactose-modified prodrug.
represent a new class of compounds known as “Senomorphics,” Here, preferential processing of galactose-modified duocarmycin

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Thoppil and Riabowol Senolytics in Cellular and Organismal Aging

(GMD), a cytotoxic conjugate, was used to eliminate a wide development of transgenes and chemical perturbagens, have
variety of senescent cell types (Guerrero et al., 2019). Targeting finally given us the tools to selectively manipulate the quantity
cell surface proteins enriched in senescent cells such as DPP4 of senescent cells in vivo. Thorough characterization of their
(dipeptidyl peptidase 4) can also be used to enhance selectivity deleterious secretory phenotype, that varies in different cell
(Kim et al., 2017). Another cell surface protein, the CD9 receptor types, continues to improve our understanding of how they
has also been used to target senescent cells and their secretory contribute to the wide variety of age-related diseases observed
phenotype with anti-CD9 monoclonal antibody encapsulated, in our populations. Finally, as the first Senolytics enter clinical
lactose-wrapped nanoparticles, loaded with rapamycin as the trials, we are on the cusp of establishing a translational bridge
payload (Thapa et al., 2017). These nanoparticles have dual between cellular senescence and organismal aging (Muñoz-
selectivity toward senescent cells based on both increased Espín and Serrano, 2014; Calimport et al., 2019; Gorgoulis
CD9 receptors on the cell surface and increased lysosomal et al., 2019) that may help ameliorate the burden of many age-
β-galactosidase expression. related pathologies.

CONCLUSION AUTHOR CONTRIBUTIONS

The role of cellular senescence in organismal aging has been well All authors listed have made a substantial, direct and intellectual
established in the past two decades in a variety of organisms contribution to the work, and approved it for publication.
(Fick et al., 2012; Heidinger et al., 2012; Muñoz-Lorente et al.,
2019). From initial thoughts indicating that senescence served
primarily as an anti-cancer mechanism, we note the emerging FUNDING
view of senescence also acting as a driving force behind a
wide variety of age-related pathologies (Fernandes et al., 2016). This work was supported by a grant to KR from the Canadian
More recent studies in the last decade, which resulted in the Institutes of Health Research (MOP-133646).

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Thoppil and Riabowol Senolytics in Cellular and Organismal Aging

Young, A. R. J., Narita, M., Ferreira, M., Kirschner, K., Sadaie, M., Darot, J. F. J., Conflict of Interest: The authors declare that the research was conducted in the
et al. (2009). Autophagy mediates the mitotic senescence transition. Genes Dev. absence of any commercial or financial relationships that could be construed as a
23, 798–803. doi: 10.1101/gad.519709 potential conflict of interest.
Zhu, Y., Tchkonia, T., Fuhrmann-Stroissnigg, H., Dai, H. M., Ling, Y. Y., Stout,
M. B., et al. (2016). Identification of a novel senolytic agent, navitoclax, targeting Copyright © 2020 Thoppil and Riabowol. This is an open-access article distributed
the Bcl-2 family of anti-apoptotic factors. Aging Cell 15, 428–435. doi: 10.1111/ under the terms of the Creative Commons Attribution License (CC BY). The use,
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(2015). The Achilles’ heel of senescent cells: from transcriptome to senolytic in this journal is cited, in accordance with accepted academic practice. No use,
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