Targeting senescent cells in translational medicine

Abstract

Organismal ageing is a complex process driving progressive impairment of functionality and regenerative potential of tissues. Cellular senescence is a state of stable cell cycle arrest occurring in response to damage and stress and is considered a hallmark of ageing. Senescent cells accumulate in multiple organs during ageing, contribute to tissue dysfunction and give rise to pathological manifestations. Senescence is therefore a defining feature of a variety of human age-related disorders, including cancer, and targeted elimination of these cells has recently emerged as a promising therapeutic approach to ameliorate tissue damage and promote repair and regeneration. In addition, in vivo identification of senescent cells has significant potential for early diagnosis of multiple pathologies. Here, we review existing senolytics, small molecules and drug delivery tools used in preclinical therapeutic strategies involving cellular senescence, as well as probes to trace senescent cells. We also review the clinical research landscape in senescence and discuss how identifying and targeting cellular senescence might positively affect pathological and ageing processes.

Keywords: SASP; age-related disorders; cellular senescence; senolytic drugs; senoprobes.

Figure 1. Therapeutic and diagnostic opportunities in senescence‐related disorders and during ageing

Cellular senescence is associated with multiple human disorders, offering potential interventions for targeted therapeutic and diagnostic approaches. The development of galactose‐conjugated and fluorescent probes to detect and highlight senescent cells offers an important opportunity for longitudinal monitoring of senescence in clinical trials. Pharmacologically active small compounds known as senolytics inhibit pro‐survival pathways in senescent cells leading to apoptosis, a therapeutic strategy that may additionally be enhanced by the use of immune modulators promoting natural clearance of senescent cells. Also, a variety of drugs can manipulate the SASP and its detrimental effects, thus suggesting a potential clinical use. Interventions to bypass senescence represent an interesting alternative, although this approach should be taken with caution due to the risk of uncontrolled proliferation and cancer initiation. Finally, nanoparticles encapsulating cytotoxic drugs, tracers and/or small molecules can be used as theranostic tools, both for therapeutic and diagnostic purposes. Of note, the benefits of therapeutic approaches for the prevention or elimination of senescent cells in vivo have been validated in an increasing number of conditions. Genetic manipulation to inactivate the senescence pathway or to ablate senescent cells in murine models produced (mostly) a beneficial impact irrespective of the disorder or condition investigated, including adipose atrophy, cataracts, IPF, sarcopenia, kidney dysfunction, atherosclerosis, premature ageing of the haematopoietic system, osteoarthritis, cardiomyocyte hypertrophy, loss of bone mass, type 2 diabetes, tumorigenesis, neurological disorders and natural ageing. Furthermore, clearance of senescent cells by treatment with senolytic drugs, a more clinically relevant approach, showed in vivo benefits in, among other disorders, atherosclerosis, premature ageing of the haematopoietic system, myocardial infarction, IPF, osteoarthritis, osteoporosis, type 1 diabetes, obesity‐induced metabolic syndrome and neuropsychiatric disorders, tau‐dependent pathologies, cancer and natural ageing. IPF, idiopathic pulmonary fibrosis; HSC, hematopoietic stem cells; MuSC, muscle stem cells.

Figure 2. Regulation of the cell cycle arrest and inflammatory SASP in the induction of cellular senescence and its interconnection with apoptosis

(A) Most senescence‐inducing triggers converge in the activation of the cell cycle inhibitor pathways p53/p21 and/or p16^{INK 4a}. These result in the inhibition of cyclin‐dependent kinase 1 (CDK1), CDK2, CDK4 and CDK6, which prevents the phosphorylation of the retinoblastoma protein (RB), leading to the suppression of S‐phase genes and an ensuing stable cell cycle arrest. DNA‐damaging triggers activate the DNA damage response (DDR) pathway resulting in the activation of p53 and p21. Ageing and epigenetic derepression of the Ink4a/ARF locus also lead to the activation of cell cycle inhibitors p16 and p21. ROS lead to the activation of the MAPK signalling pathway and its downstream effector p38. The aberrant expression of oncogenes or the loss of tumour suppressors leads to p53 activation through the Ras‐Raf‐MEK‐ERK or AKT signalling pathways, and TGFβ, and important factor of the SASP, leads to p15, p21 and p27 upregulation via SMAD signalling. Other triggers such as developmental cues and polyploidy activate the AKT, SMAD and/or Ras‐Raf‐MEK‐ERK pathway for p21 upregulation, while processes such as cell fusion signal through the DDR for p53 activation. In response to damage and different types of stress high levels of p53 with specific post‐translational modifications (such as acetylated K117 and E177) target DNMT3a, a suppressor of p21 and senescence, and trigger the apoptotic programme by upregulating PUMA and NOXA, which in turn activate the caspase cascade leading to cell death. (B) SASP implementation is orchestrated by the activation of the transcription factors NF‐κB and C/EBPβ through upstream signalling pathways. DNA‐damaging agents, ROS and OIS, generally activate the expression of SASP TFs via the AKT and/or the Ras‐Raf‐MEK‐ERK axis. In addition, DNA fragments are also known to trigger the activation of the cGAS/STING signalling, resulting in the activation of the IRF3 TF and subsequent transcription of Type 1 IFN. OIS‐derived SASP is dynamic and can also be orchestrated by NOTCH signalling, a process that restrains the inflammatory secretion by inhibiting C/EBPβ at initial stages, and allows the activation of SASP‐related super enhancers through NF‐κB later on. Accumulating increased levels of TFs reinforce the senescent phenotype through autocrine and paracrine signalling. SASP‐derived inflammatory chemokines such as IL‐6 and IL‐8 promote epigenetic modifications reinforcing the cell cycle arrest through the JAK/STAT cascade, while IL‐1α stimulates the activity of NF‐κB and C/EBPβ promoting a positive feedback loop on the secretion of other cytokines. Finally, senescence promotes survival networks by the regulation anti‐apoptotic pathways. This includes PI3K‐AKT signalling, which can inhibit pro‐apoptotic BAD and FOXO1/3, and phosphorylate caspase‐9; anti‐apoptotic FOXO4, that is present in senescent cells and interacts with p53; and NF‐κB, that may also promote survival responses by transcriptional induction of anti‐apoptotic proteins of the Bcl‐2 family. ATM/ATR, ataxia‐telangiectasia mutated and Rad3‐related homologue; IFN, interferon; OIS, oncogene‐induced senescence; ROS, reactive oxygen species; SASP, senescence‐associated secretory phenotype; TFs, transcription factors; TS, tumour suppressor.

Figure 3. Therapeutic approaches targeting cellular senescence

To prevent the deleterious effects of cellular senescence, four different strategies can be potentially implemented. The inhibition of pro‐survival pathways by the use of apoptosis‐inducing drugs is a leading approach. First and second generation of inhibitors of the BCL‐2 cell death regulator family of proteins can induce selective apoptosis of senescent cells. Targeting senescence metabolism through glycolysis blockade and attenuation of ATM, HDAC, FOXO4 activities as well as the PI3K cascade have also been reported as effective approaches. A second strategy is the activation of the immune system against senescent cells to stimulate their clearance. Enhancing the cytotoxic activity of NK against senescent cells, and manipulating the humoral innate immunity with the use of antibodies against receptors, such as DPP4 and vimentin, are proposed attractive strategies. Thirdly, manipulation of the SASP without compromising the cell cycle arrest of senescent cells has also proven beneficial in particular settings. A large number of molecules can interfere with NF‐κB and C/EBPβ transcriptional activities or their upstream regulators, dampening the expression of SASP factors, such as IL‐1, IL‐6 and IL‐8, and thus reducing the senescence‐derived inflammatory milieu. Lastly, genetic and epigenetic manipulation of cells, including the induction of reprogramming, have been proposed as a means of bypassing or reverting the state of cellular senescence, although these approaches should be taken with caution given the potential risk of cancer initiation. BCL‐2, B‐cell lymphoma 2; CAR, chimeric antigen receptor; GzmB, granzyme B; HDAC, histone deacetylase; HSP90, heat shock protein 90; i4F, inducible four Yamanaka factors; LSD1, lysine‐specific histone demethylase 1A; MICA, MHC class I polypeptide‐related sequence A; NK, natural killer; TERT, telomerase reverse transcriptase.

Figure 4. Novel diagnostic and therapeutic approaches for targeting senescent cells: probes and nanoparticles

(A) Representative structural images of some of the novel tools developed for the detection and targeting of senescent cells. Diagnostic probes are either fluorescent or chromogenic and can be detected upon β‐galactosidase catalytic reaction. Nanocarriers are loaded or tagged with either fluorescent particles (such as rhodamine) or drugs/senolytics (doxorubicin, navitoclax, rapamycin) for different clinical interventions. Most of the senescence‐directed nanoparticles are coated or conjugated to galactose‐derived residues or have been designed to bind to specific receptors. (B) Tracking the β‐galactosidase activity of senescent cells is one of the commonest strategies for the development of probes and nanoparticles. The enzymatic activity cleaves galactose residues conjugated to endocytosed probes or nanoparticles and allows the release of carriers or the emission of colour/fluorescence within the lysosomal compartment. Other developed tools can bind to receptors present on the membrane to either allow the detection of senescent cells (Nano‐MIPs) or subsequently become endocytosed and processed by β‐galactosidase activity (CD9‐mAb‐coated nanoparticles). B2M, beta‐2 microglobulin; β‐gal, β‐galactosidase; GalNPs, 6‐mer galacto‐oligosaccharides‐conjugated nanoparticles; GosNPs, galacto‐oligosaccharides‐conjugated nanoparticles; MIPs, molecularly imprinted particles; NPs, nanoparticles; PEG, polyethylene glycol.