Introduction to Senescence and Senolytic Drugs

Introduction

Oncogenic proliferative signals are coupled to a variety of growth inhibitory processes, such as the induction of apoptotic cell death and stable cell-cycle arrest, in phenomena termed cellular senescence. Both apoptosis and cellular senescence are considered to serve as important safeguards against neoplasia. However, unlike apoptotic cells, senescent cells are viable for long periods of time and thereby accumulate with age in various organs and tissues. Moreover, it has recently become apparent that senescent cells are not merely non-dividing, but eventually develop a secretory profile composed of pro-inflammatory cytokines, chemokines, and extracellular matrix-degrading proteases, a typical signature termed the senescence-associated secretory phenotype (SASP). Although SASP reportedly plays some beneficial roles, it also exhibits deleterious side effects such as chronic-inflammation and/or tumourigenesis, depending on the biological context. Thus, although cellular senescence primarily acts as a tumour suppression mechanism, the accumulation of senescent cells in aged tissues may eventually promote the age-related decline of organ function and/or associated diseases, such as cancer. Indeed, the clearance of p16 INK4a-positive senescent cells from aged transgenic mice reportedly delays the onset of various age-related dysfunctions, such as sarcopenia, cataracts, atherosclerosis, loss of adipose tissue, and tumourigenesis, thus extending the healthy lifespan. Along similar lines, the elimination of therapy-induced senescent cells reduced several side-effects of chemotherapy and even cancer recurrence in mice. Thus, it is anticipated that the removal of senescent cells could prevent the toxicity of anticancer treatments and enhance the therapeutic benefits.

Senolytic drugs, which specifically induce cell death in senescent cells, are likely to represent a new therapeutic avenue, and several candidate drugs were identified using a bioinformatics approach. However, the senolytic drugs identified to date were not discovered by a truly unbiased high-throughput screening (HTS) method, and thus appear to have limitations in clinical applications. For example, in a phase II study of ABT263 (a specific inhibitor of the anti-apoptotic proteins BCL2 and BCL-xL) for the treatment of advanced and recurrent small-cell lung carcinoma patients, transient thrombocytopenia and neutropenia were reported as side-effects. Furthermore, the combination of dasatinib and quercetin (D + Q), another previously reported senolytic drug, apparently exhibited remarkable cell-type specificity, although its mechanisms of action remain obscure. Therefore, the identification of more effective senolytic drugs and the elucidation of their mechanisms of action are required, towards a better strategy for the removal of senescent cells in vivo.

In this study, we identify bromodomain and extra-terminal domain (BET) family protein inhibitor (BETi) as a promising senolytic drug. The blockade of BRD4, a BET family protein, by chemical inhibitors or RNA interference robustly provokes senolysis. This is due, at least in part, to the combined effects of the attenuation of non-homologous end joining (NHEJ) repair and the activation of autophagic pathway in senescent cells. These results reveal the cellular vulnerability of senescent cells, and provide valuable insights into the resistance of senescent cells to death and possibilities for its control.

Results

High-throughput screening of senolytic drugs

To identify more effective senolytic drugs, we conducted an unbiased HTS of a chemical compound library consisting of around 47,000 small molecules, in an open innovation drug discovery program for academic researchers sponsored by the Takeda Pharmaceutical Company (RINGO-T project). Early passage human diploid fibroblasts (HDFs) expressing a tamoxifen-regulated form of oncogenic Ras were treated with or without 4-hydroxytamoxifen (4-OHT) for 8 days, and then incubated with each chemical compound of the library for 72 h. A cell viability analysis based on measurements of the ATP levels and Caspase3/7 activity revealed that 15 small molecules preferentially induce cell death in Ras-induced senescent cells, as compared to the control non-senescent cells. Intriguingly, although we were unable to find previously reported senolytic drugs in our HTS, four top ranking hit molecules (JQ1, I-BET151, I-BET762, and PFI-1) turned out to be inhibitors of bromodomain and extra-terminal (BET) family proteins, and are currently in advanced clinical development as a new class of therapeutics for the treatment of human cancers.

Fig. 1: HTS identified BET inhibitors as potent senolytic compounds.

a Outline of the high-throughput screening (HTS) procedure for identifying senolytic compound. IMR90-ER:Ras cells were stimulated with or without 4OHT for 7 days to induce senescence. These cells were plated on 384-well plates and incubated for another 24 hrs, followed by treatment with Takeda chemical compound library consisting of 47, 000 small molecules for 3 days. These cells were then subjected to cell viability analysis using CellTiter-Glo Luminescent Cell Viability assay system (Promega), followed by apoptosis analysis using Caspase-Glo3/7 Assay system (Promega). b–e Early passage pre-senescent (control) normal human diploid fibroblasts (TIG-3 cells) were rendered senescent by serial passage (replicative senescence), treatment with 250 ng/ml doxorubicin for 10 days (+DXR) (b) or infection with retrovirus encoding oncogenic Ras (+HRasV12) (d). These senescent cells and control pre-senescent cells (control (b) and +Vector (d)) were then incubated with increasing concentration of ARV825 indicated at right for 4 days. Relative cell number was counted throughout the experiments and representative photographs of the cells in the indicated culture conditions are shown at the bottom of the b, d. For all graphs, error bars indicate mean ± s.d. (n = 3) (b, d). Cells treated with or without 10 nM ARV825 for 4 days were subjected to western blotting using the antibodies shown on the right (c, e). β-actin was used as a loading control. The representative data from three independent experiments were shown (b–e). Source data are provided as a Source Data file.

To further confirm and extend our HTS results, the senolytic activities of JQ1 (the most frequently used BET inhibitor (BETi) in published studies), OTX015 (the most frequently used BET inhibitor (BETi) in published studies), OTX015