Since time immemorial, humans have been searching for the mythical fountain of youth, a spring whose healing waters can cure all of the maladies of old age.
No one has found a cure for aging yet, but a new class of drugs called senolytics is a promising start for promoting health in old age. Senolytics are drugs designed to treat one of the root causes of aging and thus ameliorate a wide variety of age-related diseases. These drugs destroy senescent cells, damaged cells that have stopped dividing but refuse to die, instead spitting out a wide variety of potentially harmful or proinflammatory molecules that affect the tissues around them.
Studies in mice have been remarkable: senolytics seem to improve outcomes in models of several age-related diseases, including Alzheimer’s disease, bone loss, and lung disease (1–3). In at least one study, these drugs even extended lifespan in mice (4).
Clinical trials are currently in progress to determine how well these drugs will translate to humans. At the same time, many researchers caution that there are still unanswered questions about aging and senescence. There appears to be substantial variety within senescent cells, but what the subtypes are, and the exact roles each plays in health and disease, has not yet been determined.
What is senescence?
Senescent cells were first identified in 1961. Leonard Hayflick and Paul Moorhead of the Wistar Institute of Anatomy and Biology reported that normal human cells could only divide a certain number of times (5). Once that limit was reached, the cells didn’t die, but instead entered a new, nondividing state known as senescence. Hayflick later proposed that this might be a form of aging at the cellular level, and thus be relevant for whole organism aging (6).
Hayflick’s findings were largely rejected by the scientific community for more than a decade as researchers stubbornly held onto the belief that it was a lack of proper culturing conditions that caused cells to stop growing, rather than any characteristic of the cells themselves (7).
Eventually, aging and cancer researchers became interested in this phenomenon and began to study it in more detail. Researchers discovered that many different stressors cause cells to become senescent, including telomere shortening, DNA damage (including from chemotherapy or radiation treatments), oncogene activation, and oxidative stress (8). By preventing damaged or malfunctioning cells from replicating, senescence helps prevent the development of tumors, at least initially (9).
The problem is that once cells become senescent, they secrete a potent cocktail of molecules including proinflammatory factors, proteases, and growth factors. This is known as the SASP, or senescent-associated secretory phenotype. In some cases, these molecules stimulate the immune system to remove the senescent cells, but if this process fails, the SASP can induce other cells to become senescent as well and may drive cancer development and the progress of a litany of other age-related diseases, including cardiovascular disease and frailty (10–13).
Snuffing out senescence
James Kirkland, a geriatrician and endocrinologist at the Mayo Clinic, has been interested in aging ever since he was a child. He became interested in senolytics in 2004. He was inspired by a paper showing that interventions such as caloric restriction that increased health span in mice also reduced the accumulation of senescent cells that normally happens with age (14).
“That led Tamar Tchkonia and I — we were in Boston working together at the time — to debate whether this was just an association or whether there could be a causal link,” said Kirkland. Their early attempts to create a drug to selectively kill senescent cells were not successful.
A few years later at the Mayo Clinic, Kirkland and Tchkonia collaborated with fellow aging researcher Jan van Deursen to study senescence in rapidly aging (progeroid) mice. These mice not only aged prematurely, they also accumulated senescent cells in their tissues much faster than other mice (15,16).
In 2011, the team succeeded in creating a genetically modified version of these progeroid mice that allowed researchers to eliminate cells with specific senescence markers using a specific trigger drug (17). Crucially, removing these senescent cells slowed age-related disease progression in the mice, indicating a causal role for senescent cells in at least some disorders.
That same year, van Deursen, along with biotech entrepreneur Nathaniel David and Judy Campisi, biochemist at the Buck Institute for Research on Aging, founded Unity Biotechnology with the aim of discovering and commercializing senolytic agents.
Meanwhile, Kirkland and others at the Mayo Clinic continued to search for drugs to destroy senescent cells. Since senescent cells somehow manage to persist while producing a slew of chemicals that damage cells around them, Kirkland and the rest of the team hypothesized that senescent cells might be using anti-apoptotic pathways to protect themselves from internal cell death programs.
This hypothesis proved true. By analyzing gene transcription, the researchers showed that senescent cells turned on several pathways that associated with apoptosis resistance. They tested dozens of approved drugs and natural products known to inhibit these pathways and found a combination of two agents (dasatinib and quercetin, now known as D+Q) that seemed to selectively target and eliminate senescent cells (18).
In mice, this combination reduced markers of senescent cells, as well as ameliorated impairments in models of age-related physical dysfunction and fibrotic pulmonary disease (3,4,19). Importantly, the mice also tolerated the drug combination, at least in the short term. Since senescent cells take time to accumulate, senolytics can be given intermittently, which likely reduces the risk of side effects, said Kirkland. Once the drugs proved relatively safe and effective in mice, it was time to test them in humans.
Into the clinic
Like so many people, Miranda Orr, a neuroscientist at Wake Forest University School of Medicine, has personal experience with Alzheimer’s disease. She has devoted her work to figuring out how the disease progresses and how doctors might treat it.
Initially, she had focused on tau proteins, which accumulate and interfere with neuronal function in Alzheimer’s disease. But during her time as a postdoctoral researcher, she attended a lecture on senescence. “That was really where the lightbulb moment occurred,” she said. “When cells become senescent — these are stressed cells that don't actually die — they are essentially in the right place at the right time for pathology and dysfunction of tissues, which is very similar to what happens in Alzheimer's disease.” Cells that have aggregated tau don’t often die, but they closely correlate with disease severity. Her team is exploring senescence as a potential explanation for if and how these cells might worsen the neurodegeneration around them.
Her suspicion of a relationship between senescence and tau pathology turned out to be right; in postmortem brain tissue samples from Alzheimer’s patients, neurons with tau protein tangles expressed genes associated with senescent cells. In mouse models of Alzheimer’s disease, treatment with the senolytic D+Q combo reduced tangle density as well as lessened the loss of neurons that occurs as the disease progresses over time (20).
Now, Orr is taking this experimental therapy into clinical trials. In an open-label pilot study, five patients with early-stage Alzheimer’s disease received D+Q treatment. The main goal of the pilot was to determine if these drugs penetrated into the central nervous system, which they did. The drugs showed up in patients’ cerebrospinal fluid. While this is an important first step, larger trials will be needed to determine if this combination effectively slows the progress of this devastating disease. Researchers are currently recruiting participants for a placebo-controlled phase II trial (21).
More than one way to kill a cell
While the D+Q combination is one of the best-studied senolytic therapies, it’s far from the only strategy that researchers use to try to eliminate senescent cells. Around the same time that Kirkland and his colleagues discovered D+Q, Daohong Zhou, an aging and cancer researcher now at the University of Texas Health Science Center, was working on another method for killing senescent cells using an experimental cancer drug called navitoclax (22).
Like D+Q, navitoclax targets anti-apoptotic pathways that senescent cells use to keep themselves alive. Specifically, it inhibits the anti-apoptotic proteins Bcl-2 and Bcl-xL. Zhou discovered that in mice, navitoclax reduced senescent cells in both muscle and bone marrow and prevented the premature aging often seen after total body irradiation. In humans, this type of irradiation can be used to treat lymphoma and leukemia.
Zhou joined Unity Biotechnology as a scientific cofounder in 2014. The company had been developing a senolytic agent for knee osteoarthritis that worked by inducing apoptosis in senescent cells. That drug failed in phase II clinical trials. They then turned to another senolytic agent targeting Bcl-xL (23).
They’ve had encouraging results. Unity’s Bcl-xL inhibitor, UBX1325, succeeded at improving vision in a phase II trial for diabetic macular edema and is also being tested for age-related macular degeneration (24).
Bcl-xL inhibitors have a fatal flaw that prevents them from being used for many other age-related diseases: they kill platelets. This means that they’re too toxic for systemic administration. Unity circumvents this problem by using local administration: they inject UBX1325 directly into the eye.
Zhou didn’t want to give up on Bcl-xL inhibitors for other diseases for which local administration isn’t possible though. He determined to figure out how to keep these drugs from killing platelets. He came across the work of biochemists Craig Crews at Yale University and Jay Bradner at Novartis on PROTACs (proteolysis-targeting chimeras) and decided to see if this technology could solve his problem.
A PROTAC is a small molecule that has two binding sites; at one end, a binding site latches onto the target protein, and the other binding site attaches to an E3 ubiquitin ligase. Once both are attached, the ligase marks the target protein for destruction by the cell’s housekeeping system.
Conveniently for scientists, different types of cells have different types of E3 ubiquitin ligases. By designing a PROTAC that targeted Bcl-xL and a ligase that was rare in platelets but common in other cell types, Zhou and his collaborator Guangrong Zheng, a medicinal chemist at the University of Florida, created a drug that caused the degradation of Bcl-xL in senescent cells, which killed them, while leaving platelets relatively unharmed (25). Zhou and Zheng founded Dialectic Therapeutics in order to continue developing these drugs, and one of these PROTACs, known as DT2216, has entered a phase I clinical trial for patients with relapsed or refractory cancers.
There are multiple ways that this drug might help treat cancer. Cancer cells, like senescent cells, may also upregulate anti-apoptotic pathways to help them resist death caused by chemotherapeutics. Therefore, degrading Bcl-xL may increase their vulnerability to chemotherapy. At least in animal models, this seems to hold true; DT2216 increased tumor sensitivity to traditional chemotherapies (26,27).
Chemotherapy can cause cancer cells to become senescent. In the short term, this means that the cell no longer divides, but in the longer term, the chemicals secreted by senescent cells can promote tumor recurrence (28). Killing these senescent cells with senolytics may help avoid this fate.
Meanwhile, dozens of other biotech start ups are testing other approaches to senolytic therapy. Numeric Biotech’s leading drug candidate disrupts the binding of two proteins called FOXO4 and p53, releasing p53 to induce apoptosis in senescent cells. Scientists at Deciduous Therapeutics develop drugs that stimulate the immune system to kill senescent cells. Oisín Biotechnologies is developing a gene therapy to induce apoptosis in cells that express the senescence marker p16.
Others are trying to mitigate the harmful effects of senescent cells without outright killing them. Researchers at Geras Bio are pursuing SASP inhibitors, drugs intended to block the harmful proinflammatory chemicals produced by senescent cells. Atropos Therapeutics scientists are developing therapies that will prevent cells from becoming senescent in the first place.
A more complex picture of senescence
While many researchers focus on eliminating senescent cells, others focus on understanding their fundamental biology. Increasingly, researchers appreciate the substantial heterogeneity of these cells, but the factors that contribute to their differences and their roles in disease are not yet fully understood.
The heterogeneity of senescent cells has huge clinical significance. We have to understand exactly which cells are targeted by these different transgenic models and different senolytics.
- Ming Xu, University of Connecticut
Kirkland admits that senescent cells can’t necessarily be lumped together. “The exact nature of senescence depends on the cell type that became senescent, how long it was senescent, what induced senescence, and the microenvironment. So, senescence is a cell fate like differentiation or replication, so it's very hard to define necessarily what a senescent cell is the same way it’s hard to define what a differentiated cell is. A differentiated neuron is different from a differentiated fat cell.”
While it’s relatively straightforward to define a senescent cell in a dish, markers of senescence in the cells of living beings are much harder to pin down. For example, a high level of p16 expression is often used as a marker of senescence, but not all senescent cells express p16, and some cells with strong p16 expression don’t actually seem to be senescent (29). Therefore, a transgenic mouse model that allows scientists to eliminate p16 expressing cells leaves an unknown number of senescent cells behind.
“The heterogeneity of senescent cells has huge clinical significance,” said Ming Xu, an aging researcher at the University of Connecticut. “We have to understand exactly which cells are targeted by these different transgenic models and different senolytics.” Improving the understanding of different populations could help scientists understand their roles in various diseases and develop drugs to target the appropriate populations.
The paradox of senescence
Scientists have largely examined the harmful effects of senescent cells but for all the bad press they get, senescent cells — or at least cells that have markers traditionally associated with senescence — can sometimes be beneficial.
Cells with markers of senescence seem to be important during embryonic development, but can also promote healing and regeneration in the skin, heart, and lungs (30–33). Paradoxically, other studies indicated that senescent cells may actually damage those very same tissues and that removing them is beneficial (34). How can scientists reconcile these seemingly opposite effects?
Scientists don’t yet have a definitive answer, but they have lots of hypotheses. Time is likely important, said Bill Keyes, a cell biologist at the Institute of Genetics and Molecular and Cellular Biology. In the short term, said Keyes, exposure to the SASP (the chemicals produced by senescent cells) can be beneficial: neighboring cells may become more stem cell-like, promoting repair and regeneration.
In an ideal world, the proinflammatory factors produced by the senescent cell summon cells in the immune system to remove it, ultimately removing the SASP factors as well. “Under these circumstances, senescence is a way of preventing the damaged cell from proliferating, but also signaling and orchestrating its own removal and replacement,” said Keyes.
In aging and disease, this process may break down; the senescent cells aren’t removed so they keep bombarding their neighbors with SASP chemicals, which can have negative effects.
Prolonged exposure to the SASP may turn on too many stem cell markers in surrounding cells. In some cases, researchers hypothesized that this “stemness” was interpreted by the receiving cells as being a tumor initiating signal, so the cells turned on senescence to prevent tumor formation. However, if cancer cells that cannot become senescent are exposed to the SASP for long periods, they can promote tumor growth.
At this point, Keyes said that it’s not entirely clear whether the senescent cell remains the same with time or whether it undergoes further changes that may make it more harmful.
Marco Demaria, a cellular aging scientist at the European Research Institute for the Biology of Aging, agrees that time is important. However, Demaria’s work shows that time isn’t the only important factor: cells may activate different senescence programs involving unique patterns of gene activation and chemical secretion (different SASPs) in response to various stressors.
For example, when researchers in Demaria’s lab treated cells with the cancer drug abemaciclib, cells exhibited many features of senescence but produced different chemicals than other senescent cells. Since this senescence program seemed to be controlled by the transcription factor p53, Demaria dubbed the secreted factors the p53-associated secretory phenotype (PASP). The PASP lacked many of the proinflammatory signalling molecules usually produced by senescent cells; Demaria showed that NF-κB regulated these inflammatory factors induced by other types of cancer drugs. This secretory profile was named the NASP, and appeared to be more harmful than the PASP (35).
“[We think] that the PASP covers most of the beneficial functions of senescent cells, while the NASP is mostly detrimental,” said Demaria, although he noted that further explorations are currently in progress to determine how widely applicable this is. “We are trying to validate this hypothesis in various in vivo settings.”
Too soon or just in time?
Given that there’s still so much scientists don’t know about senescence, including the precise contexts in which it may be either beneficial or harmful, is it too soon for senolytics to be tested in humans?
Overall, researchers say no: many believe that experimenters should move forward, albeit carefully.
There's beneficial senescence and detrimental senescence. And the concern is that maybe senolytics will target both. But we've never really checked senolytics in the beneficial population, and we don't know how similar the two populations of senescence are.
- Bill Keyes, Institute of Genetics and Molecular and Cellular Biology
Tien Peng, a cell biologist at the University of California, San Francisco who identified benefits of senescent cells in the lung, said he doesn’t think it’s too early to test senolytics in humans. “We should look at the potential unintended effects of eliminating these cells,” said Peng. “All drugs have unintended side effects; that doesn’t prevent us from using them. We should consider the context in which the benefits would outweigh those risks.”
Weighing risks and benefits is important, agreed Xu. He noted that many of the clinical trials currently underway involve drugs with relatively good safety profiles or are attempting to treat serious diseases that are otherwise incurable. However, he also emphasized that acting cautiously in terms of safety is crucial as rushing into clinical trials with an unsafe drug would harm not only patients, but could also set back the field as a whole.
Even though first generation senolytics are already in human trials, that doesn’t mean that researchers should stop developing our understanding of the basic biology of senescent cells. Improving understanding will be important for developing better, more targeted drugs.
“There's beneficial senescence and detrimental senescence,” said Keyes. “And the concern is that maybe senolytics will target both. But we've never really checked senolytics in the beneficial population, and we don't know how similar the two populations of senescence are. I think with better understanding of this, we should be able to design better drugs, or at least screen for ones targeting the detrimental senescence. I think it’s all coming.”
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