Ageing microenvironment mediates lymphocyte carcinogenesis and lymphoma drug resistance: From mechanisms to clinical therapy (Review).

Cellular senescence has a complex role in lymphocyte carcinogenesis and drug resistance of lymphomas. Senescent lymphoma cells combine with immunocytes to create an ageing environment that can be reprogrammed with a senescence­associated secretory phenotype, which gradually promotes therapeutic resistance. Certain signalling pathways, such as the NF­κB, Wnt and PI3K/AKT/mTOR pathways, regulate the tumour ageing microenvironment and induce the proliferation and progression of lymphoma cells. Therefore, targeting senescence­related enzymes or their signal transduction pathways may overcome radiotherapy or chemotherapy resistance and enhance the efficacy of relapsed/refractory lymphoma treatments. Mechanisms underlying drug resistance in lymphomas are complex. The ageing microenvironment is a novel factor that contributes to drug resistance in lymphomas. In terms of clinical translation, some senolytics have been used in clinical trials on patients with relapsed or refractory lymphoma. Combining immunotherapy with epigenetic drugs may achieve better therapeutic effects; however, senescent cells exhibit considerable heterogeneity and lymphoma has several subtypes. Extensive research is necessary to achieve the practical application of senolytics in relapsed or refractory lymphomas. This review summarises the mechanisms of senescence­associated drug resistance in lymphoma, as well as emerging strategies using senolytics, to overcome therapeutic resistance in lymphoma.

A pipeline for senolytics.

There is intense interest in identifying compounds that selectively kill senescent cells, termed senolytics, for ameliorating age-related comorbidities. However, screening for senolytic compounds currently relies on primary cells or cell lines where senescence is induced in vitro. Given the complexity of senescent cells across tissues and diseases, this approach may not target the senescent cells that develop under specific conditions in vivo. In this issue of the JCI, Lee et al. describe a pipeline for high-throughput drug screening of senolytic compounds where senescence was induced in vivo and identify the HSP90 inhibitor XL888 as a candidate senolytic to treat idiopathic pulmonary fibrosis.

Chemical Strategies for the Detection and Elimination of Senescent Cells.

ConspectusCellular senescence can be defined as an irreversible stopping of cell proliferation that arises in response to various stress signals. Cellular senescence is involved in diverse physiological and pathological processes in different tissues, exerting effects on processes as differentiated as embryogenesis, tissue repair and remodeling, cancer, aging, and tissue fibrosis. In addition, the development of some pathologies, aging, cancer, and other age-related diseases has been related to senescent cell accumulation. Due to the complexity of the senescence phenotype, targeting senescent cells is not trivial, is challenging, and is especially relevant for in vivo detection in age-related diseases and tissue samples. Despite the elimination of senescent cells (senolysis) using specific drugs (senolytics) that have been shown to be effective in numerous preclinical disease models, the clinical translation is still limited due to the off-target effects of current senolytics and associated toxicities. Therefore, the development of new chemical strategies aimed at detecting and eliminating senescent cells for the prevention and selective treatment of senescence-associated diseases is of great interest. Such strategies not only will contribute to a deeper understanding of this rapidly evolving field but also will delineate and inspire new possibilities for future research.In this Account, we report our recent research in the development of new chemical approaches for the detection and elimination of senescent cells based on new probes, nanoparticles, and prodrugs. The designed systems take advantage of the over-representation in senescent cells of certain biomarkers such as ß-galactosidase and lipofuscin. One- and two-photon probes, for higher tissue penetration, have been developed. Moreover, we also present a renal clearable fluorogenic probe for the in vivo detection of the ß-galactosidase activity, allowing for correlation with the senescent burden in living animals. Moreover, as an alternative to molecular-based probes, we also developed nanoparticles for senescence detection. Besides, we describe advances in new therapeutic agents to selectively eradicate senescent cells using ß-galactosidase activity-sensitive gated nanoparticles loaded with cytotoxic or senolytic agents or new prodrugs aiming to increase the selectivity and reduction of off-target toxicities of current drugs. Moreover, new advances therapies have been applied in vitro and in vivo. Studies with the probes, nanoparticles, and prodrugs have been applied in several in vitro and in vivo models of cancer, fibrosis, aging, and drug-induced cardiotoxicity in which senescence plays an important role. We discuss the benefits of these chemical strategies toward the development of more specific and sophisticated probes, nanoparticles, and prodrugs targeting senescent cells.

Senolysis of gemcitabine-induced senescent human pancreatic cancer cells.

INTRODUCTION: Gemcitabine (GEM) is often used to treat pancreatic cancer. Many anti-cancer drugs induce cancer cell death, but some cells survive after cell cycle arrest. Such a response to DNA damage is termed cellular senescence. Certain drugs, including the Bcl-2-family inhibitor ABT-263, kill senescent cells; this is termed senolysis. In this study, we examined the therapeutic benefits of ABT-263 in GEM-induced senescence of human pancreatic cancer cells. METHODS AND RESULTS: Of four pancreatic cancer cell lines (PANC-1, AsPC-1, CFPAC-1, and PANC10.05), GEM induced senescent features in PANC-1 and AsPC-1 cells, including increases in the cell sizes and expression levels of mRNAs encoding interleukin (IL)-6/IL-8 and induction of ß-galactosidase. Successive treatment with GEM and ABT-263 triggered apoptosis in PANC-1 and AsPC-1 cells and suppressed colony formation significantly. Senolysis of GEM-induced senescent pancreatic cancer cells by ABT-263 was triggered by a Bcl-xL inhibitor, but not by a Bcl-2 inhibitor, suggesting a central role for Bcl-xL in senolysis. In a xenograft mouse model, combined treatment with GEM and ABT-737 (an ABT-263 analog exhibiting the same specificity) suppressed in vivo growth of AsPC-1 significantly. CONCLUSION: Together, our results indicate that sequential treatment with GEM and senolytic drugs effectively kill human pancreatic cancer cells.

Senolytic and senomorphic agent procyanidin C1 alleviates structural and functional decline in the aged retina.

Increased cellular senescence burden contributes in part to age-related organ dysfunction and pathologies. In our study, using mouse models of natural aging, we observed structural and functional decline in the aged retina, which was accompanied by the accumulation of senescent cells and senescence-associated secretory phenotype factors. We further validated the senolytic and senomorphic properties of procyanidin C1 (PCC1) both in vitro and in vivo, the long-term treatment of which ameliorated age-related retinal impairment. Through high-throughput single-cell RNA sequencing (scRNA-seq), we comprehensively characterized the retinal landscape after PCC1 administration and deciphered the molecular basis underlying the senescence burden increment and elimination. By exploring the scRNA-seq database of age-related retinal disorders, we revealed the role of cellular senescence and the therapeutic potential of PCC1 in these pathologies. Overall, these results indicate the therapeutic effects of PCC1 on the aged retina and its potential use for treating age-related retinal disorders.

Selective Elimination of Senescent Cancer Cells by Galacto-Modified PROTACs.

Although the selective and effective clearance of senescent cancer cells can improve cancer treatment, their development is confronted by many challenges. As part of efforts designed to overcome these problems, prodrugs, whose design is based on senescence-associated ß-galactosidase (SA-ß-gal), have been developed to selectively eliminate senescent cells. However, chemotherapies relying on targeted molecular inhibitors as senolytic drugs can induce drug resistance. In the current investigation, we devised a new strategy for selective degradation of target proteins in senescent cancer cells that utilizes a prodrug composed of the SA-ß-gal substrate galactose (galacto) and the proteolysis-targeting chimeras (PROTACs) as senolytic agents. Prodrugs Gal-ARV-771 and Gal-MS99 were found to display senolytic indexes higher than those of ARV-771 and MS99. Significantly, results of in vivo studies utilizing a human lung A549 xenograft mouse model demonstrated that concomitant treatment with etoposide and Gal-ARV-771 leads to a significant inhibition of tumor growth without eliciting significant toxicity.

The Differential Effect of Senolytics on SASP Cytokine Secretion and Regulation of EMT by CAFs.

The tumor microenvironment (TME) plays an essential role in tumor progression and in modulating tumor response to anticancer therapy. Cellular senescence leads to a switch in the cell secretome, characterized by the senescence-associated secretory phenotype (SASP), which may regulate tumorigenesis. Senolytic therapy is considered a novel anticancer strategy that eliminates the deleterious effects of senescent cells in the TME. Here, we show that two different types of senolytic drugs, despite efficiently depleting senescent cells, have opposite effects on cancer-associated fibroblasts (CAFs) and their ability to regulate epithelial-mesenchymal transition (EMT). We found that senolytic drugs, navitoclax and the combination of dasatinib/quercetin, reduced the number of spontaneously senescent and TNF-induced senescent CAFs. Despite the depletion of senescent cells, the combination of dasatinib/quercetin versus navitoclax increased the secretion of the SASP pro-inflammatory cytokine IL-6. This differential effect correlated with the promotion of enhanced migration and EMT in MC38 colorectal cancer cells. Our results demonstrate that some senolytics may have side effects unrelated to their senolytic activity and may promote tumorigenesis. We argue for more careful and extensive studies of the effects of senolytics on various aspects of tumor progression and tumor resistance to therapy before the senolytic strategy is implemented in the clinic.

Cellular senescence of granulosa cells in the pathogenesis of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of reproductive age, but its pathology has not been fully characterized and the optimal treatment strategy remains unclear. Cellular senescence is a permanent state of cell-cycle arrest that can be induced by multiple stresses. Senescent cells contribute to the pathogenesis of various diseases, owing to an alteration in secretory profile, termed 'senescence-associated secretory phenotype' (SASP), including with respect to pro-inflammatory cytokines. Senolytics, a class of drugs that selectively eliminate senescent cells, are now being used clinically, and a combination of dasatinib and quercetin (DQ) has been extensively used as a senolytic. We aimed to investigate whether cellular senescence is involved in the pathology of PCOS and whether DQ treatment has beneficial effects in patients with PCOS. We obtained ovaries from patients with or without PCOS, and established a mouse model of PCOS by injecting dehydroepiandrosterone. The expression of the senescence markers p16INK4a, p21, p53, γH2AX, and senescence-associated ß-galactosidase and the SASP-related factor interleukin-6 was significantly higher in the ovaries of patients with PCOS and PCOS mice than in controls. To evaluate the effects of hyperandrogenism and DQ on cellular senescence in vitro, we stimulated cultured human granulosa cells (GCs) with testosterone and treated them with DQ. The expression of markers of senescence and a SASP-related factor was increased by testosterone, and DQ reduced this increase. DQ reduced the expression of markers of senescence and a SASP-related factor in the ovaries of PCOS mice and improved their morphology. These results indicate that cellular senescence occurs in PCOS. Hyperandrogenism causes cellular senescence in GCs in PCOS, and senolytic treatment reduces the accumulation of senescent GCs and improves ovarian morphology under hyperandrogenism. Thus, DQ might represent a novel therapy for PCOS.

Senolytic therapeutics: An emerging treatment modality for osteoarthritis.

Osteoarthritis (OA), a chronic joint disease affecting millions of people aged over 65 years, is the main musculoskeletal cause of diminished joint mobility in the elderly. It is characterized by lingering pain and increasing deterioration of articular cartilage. Aging and accumulation of senescent cells (SCs) in the joints are frequently associated with OA. Apoptosis resistance; irreversible cell cycle arrest; increased p16INK4a expression, secretion of senescence-associated secretory phenotype factors, senescence-associated ß-galactosidase levels, secretion of extracellular vesicles, and levels of reactive oxygen and reactive nitrogen species; and mitochondrial dysregulation are some common changes in cellular senescence in joint tissues. Development of OA correlates with an increase in the density of SCs in joint tissues. Senescence-associated secretory phenotype has been linked to OA and cartilage breakdown. Senolytics and therapeutic pharmaceuticals are being focused upon for OA management. SCs can be selectively eliminated or killed by senolytics to halt the pathogenesis and progression of OA. Comprehensive understanding of how aging affects joint dysfunction will benefit OA patients. Here, we discuss age-related mechanisms associated with OA pathogenesis and senolytics as an emerging modality in the management of age-related SCs and pathogenesis of OA in preclinical and clinical studies.

Senolytics prevent age-associated changes in female mice brain.

PURPOSE: Considering that the combination of dasatinib and quercetin (D + Q) demonstrated a neuroprotective action, as well as that females experience a decline in hormonal levels during aging and this is linked to increased susceptibility to Alzheimer's disease, in this study we evaluated the effect of D + Q on inflammatory and oxidative stress markers and on acetylcholinesterase and Na+, K+-ATPase activities in brain of female mice. METHODS: Female C57BL/6 mice were divided in Control and D (5 mg/kg) + Q (50 mg/kg) treated. Treatment was administered via gavage for three consecutive days every two weeks starting at 30 days of age. The animals were euthanized at 6 months of age and at 14 months of age. RESULTS: Results indicate an increase in reactive species (RS), thiol content and lipid peroxidation followed by a reduction in nitrite levels and superoxide dismutase, catalase and glutathione S-transferase activity in the brain of control animals with age. D+Q protected against age-associated increase in RS and catalase activity reduction. Acetylcholinesterase activity was increased, while Na+, K+-ATPase activity was reduced at 14 months of age and D+Q prevented this reduction. CONCLUSION: These data demonstrate that D+Q can protect against age-associated neurochemical alterations in the female brain.

An in vivo screening platform identifies senolytic compounds that target p16INK4a+ fibroblasts in lung fibrosis.

The appearance of senescent cells in age-related diseases has spurred the search for compounds that can target senescent cells in tissues, termed senolytics. However, a major caveat with current senolytic screens is the use of cell lines as targets where senescence is induced in vitro, which does not necessarily reflect the identity and function of pathogenic senescent cells in vivo. Here, we developed a new pipeline leveraging a fluorescent murine reporter that allows for isolation and quantification of p16Ink4a+ cells in diseased tissues. By high-throughput screening in vitro, precision-cut lung slice (PCLS) screening ex vivo, and phenotypic screening in vivo, we identified a HSP90 inhibitor, XL888, as a potent senolytic in tissue fibrosis. XL888 treatment eliminated pathogenic p16Ink4a+ fibroblasts in a murine model of lung fibrosis and reduced fibrotic burden. Finally, XL888 preferentially targeted p16INK4a-hi human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF), and reduced p16INK4a+ fibroblasts from IPF PCLS ex vivo. This study provides proof of concept for a platform where p16INK4a+ cells are directly isolated from diseased tissues to identify compounds with in vivo and ex vivo efficacy in mice and humans, respectively, and provides a senolytic screening platform for other age-related diseases.

Senolytic drugs dasatinib and quercetin combined with Carboplatin or Olaparib reduced the peritoneal and adipose tissue metastasis of ovarian cancer.

Chemotherapy and targeted drugs-induced senescent ovarian cancer cells that accumulate in peritoneal adipose tissue contribute significantly to chronic inflammation, disrupt homeostasis, and may fuel various aspects of cancer progression. However, the pro-senescence effects of chemotherapy and targeted drugs on adipose derived stem cells (ADSCs) within peritoneal adipose tissue remain poorly understood. In this study, we show that the first-line chemotherapy and targeted drugs can induce the cellular senescence of ADSCs in vitro and increase the aging of peritoneal adipose tissue in vivo. These treatments significantly promoted the dysregulation of glucose and lipid metabolism, including insulin resistance and liver lipid accumulation. Our study shows that dasatinib and quercetin, as senolytics, effectively restore glucose homeostasis in mice with ovarian cancer and significantly reduce adipose tissue aging. Importantly, combining these drugs with Carboplatin or Olaparib results in a marked decrease in both peritoneal and adipose tissue metastasis of ovarian cancer cells. Mechanistically, we revealed that there is crosstalk between ovarian cancer cells and senescent ADSCs. The crosstalk increases inflammatory cytokines and chemokines production in ADSCs and notably upregulates chemokine receptors on cancer cells. Collectively, these data indicate that senescent ADSCs induced by chemotherapy and targeted therapy drugs impair adipose tissue function. However, the senolytic drugs dasatinib and quercetin, can significantly ameliorate organ aging and damage induced by these treatments. Notably, dasatinib and quercetin combined with Carboplatin or Olaparib reduced the peritoneal and adipose tissue metastasis of ovarian cancer, ultimately benefiting the mice undergoing chemotherapy and targeted therapy.

A Need for Refined Senescence Biomarkers and Measures of Senolytics in the Brain.

Cellular senescence contributes to Alzheimer's disease (AD) pathogenesis. Treatments that remove senescent cells, senolytics, improve brain outcomes in AD mice with amyloid-ß or tau deposition. 3xTgAD mice develop both AD neuropathologies; however, Ng et al. report low p16INK4a-associated senescence in the brain. Senolytic treatment by genetic removal; dasatinib with quercetin (D+Q), which enter the brain; and ABT-263 with limited brain penetrance all reduced AD neuropathology. Refined measures of senescence and brain exposure would help clarify the benefits of senolytics despite low p16INK4a-associated senescence and potential limited brain penetrance.

Computational identification of natural senotherapeutic compounds that mimic dasatinib based on gene expression data.

The major risk factor for chronic disease is chronological age, and age-related chronic diseases account for the majority of deaths worldwide. Targeting senescent cells that accumulate in disease-related tissues presents a strategy to reduce disease burden and to increase healthspan. The senolytic combination of the tyrosine-kinase inhibitor dasatinib and the flavonol quercetin is frequently used in clinical trials aiming to eliminate senescent cells. Here, our goal was to computationally identify natural senotherapeutic repurposing candidates that may substitute dasatinib based on their similarity in gene expression effects. The natural senolytic piperlongumine (a compound found in long pepper), and the natural senomorphics parthenolide, phloretin and curcumin (found in various edible plants) were identified as potential substitutes of dasatinib. The gene expression changes underlying the repositioning highlight apoptosis-related genes and pathways. The four compounds, and in particular the top-runner piperlongumine, may be combined with quercetin to obtain natural formulas emulating the dasatinib + quercetin formula.

Repurposing Drugs for Senotherapeutic Effect: Potential Senomorphic Effects of Female Synthetic Hormones.

Repurposing previously approved drugs may fast track the route to the clinic for potential senotherapeutics and improves the inefficiency of the clinical drug development pipeline. We performed a repurposing screen of 240 clinically approved molecules in human primary dermal fibroblasts for their effects on CDKN2A expression. Molecules demonstrating effects on CDKN2A expression underwent secondary screening for senescence-associated beta galactosidase (SAB) activity, based on effect size, direction, and/or molecule identity. Selected molecules then underwent a more detailed assessment of senescence phenotypes including proliferation, apoptosis, DNA damage, senescence-associated secretory phenotype (SASP) expression, and regulators of alternative splicing. A selection of the molecules demonstrating effects on senescence were then used in a new bioinformatic structure-function screen to identify common structural motifs. In total, 90 molecules displayed altered CDKN2A expression at one or other dose, of which 15 also displayed effects on SAB positivity in primary human dermal fibroblasts. Of these, 3 were associated with increased SAB activity, and 11 with reduced activity. The female synthetic sex hormones-diethylstilboestrol, ethynyl estradiol and levonorgestrel-were all associated with a reduction in aspects of the senescence phenotype in male cells, with no effects visible in female cells. Finally, we identified that the 30 compounds that decreased CDKN2A activity the most had a common substructure linked to this function. Our results suggest that several drugs licensed for other indications may warrant exploration as future senotherapies, but that different donors and potentially different sexes may respond differently to senotherapeutic compounds. This underlines the importance of considering donor-related characteristics when designing drug screening platforms.

A Conversation with ChatGPT on Contentious Issues in Senescence and Cancer Research.

Artificial intelligence (AI) platforms, such as Generative Pretrained Transformer (ChatGPT), have achieved a high degree of popularity within the scientific community due to their utility in providing evidence-based reviews of the literature. However, the accuracy and reliability of the information output and the ability to provide critical analysis of the literature, especially with respect to highly controversial issues, has generally not been evaluated. In this work, we arranged a question/answer session with ChatGPT regarding several unresolved questions in the field of cancer research relating to therapy-induced senescence (TIS), including the topics of senescence reversibility, its connection to tumor dormancy, and the pharmacology of the newly emerging drug class of senolytics. ChatGPT generally provided responses consistent with the available literature, although occasionally overlooking essential components of the current understanding of the role of TIS in cancer biology and treatment. Although ChatGPT, and similar AI platforms, have utility in providing an accurate evidence-based review of the literature, their outputs should still be considered carefully, especially with respect to unresolved issues in tumor biology. SIGNIFICANCE STATEMENT: Artificial Intelligence platforms have provided great utility for researchers to investigate biomedical literature in a prompt manner. However, several issues arise when it comes to certain unresolved biological questions, especially in the cancer field. This work provided a discussion with ChatGPT regarding some of the yet-to-be-fully-elucidated conundrums of the role of therapy-induced senescence in cancer treatment and highlights the strengths and weaknesses in utilizing such platforms for analyzing the scientific literature on this topic.

Radiotherapy-Induced Astrocyte Senescence Promotes an Immunosuppressive Microenvironment in Glioblastoma to Facilitate Tumor Regrowth.

Accumulating evidence suggests that changes in the tumor microenvironment caused by radiotherapy are closely related to the recurrence of glioma. However, the mechanisms by which such radiation-induced changes are involved in tumor regrowth have not yet been fully investigated. In the present study, how cranial irradiation-induced senescence in non-neoplastic brain cells contributes to glioma progression is explored. It is observed that senescent brain cells facilitated tumor regrowth by enhancing the peripheral recruitment of myeloid inflammatory cells in glioblastoma. Further, it is identified that astrocytes are one of the most susceptible senescent populations and that they promoted chemokine secretion in glioma cells via the senescence-associated secretory phenotype. By using senolytic agents after radiotherapy to eliminate these senescent cells substantially prolonged survival time in preclinical models. The findings suggest the tumor-promoting role of senescent astrocytes in the irradiated glioma microenvironment and emphasize the translational relevance of senolytic agents for enhancing the efficacy of radiotherapy in gliomas.

Senolytic treatment fails to improve ovarian reserve or fertility in female mice.

Senescent cell number increases with age in different tissues, leading to greater senescent cell load, proinflammatory stress, and tissue dysfunction. In the current study, we tested the efficacy of senolytic drugs to reduce ovarian senescence and improve fertility in reproductive age female mice. In the first experiment, 1-month-old C57BL/6 female mice were treated every other week with D + Q (n = 24) or placebo (n = 24). At 3 and 6 months of age, female mice were mated with untreated males to evaluate pregnancy rate and litter size. In the second experiment, 6-month-old C57BL/6 female mice were treated monthly with D + Q (n = 30), fisetin (n = 30), or placebo (n = 30). Females were treated once a month until 11 months of age, then they were mated with untreated males for 30 days to evaluate pregnancy rate and litter size. In the first experiment, D + Q treatment did not affect pregnancy rate (P = 0.68), litter size (P = 0.58), or ovarian reserve (P > 0.05). Lipofuscin staining was lower in females treated with D + Q (P = 0.04), but expression of senescence genes in ovaries was similar. In the second experiment, D + Q or fisetin treatment also did not affect pregnancy rate (P = 0.37), litter size (P = 0.20), or ovarian reserve (P > 0.05). Lipofuscin staining (P = 0.008) and macrophage infiltration (P = 0.002) was lower in fisetin treated females. Overall, treatment with D + Q or fisetin did not affect ovarian reserve or fertility but did decrease some senescence markers in the ovary.