Compounds with specific cytotoxic activity in senescent cells, or senolytics, support the causal involvement of senescence in aging and offer therapeutic interventions. Here we report the identification of Cardiac Glycosides (CGs) as a family of compounds with senolytic activity. CGs, by targeting the Na+/K+ATPase pump, cause a disbalanced electrochemical gradient within the cell causing depolarization and acidification. Senescent cells present a slightly depolarized plasma membrane and higher concentrations of H+, making them more susceptible to the action of CGs. These vulnerabilities can be exploited for therapeutic purposes as evidenced by the in vivo eradication of tumors xenografted in mice after treatment with the combination of a senogenic and a senolytic drug. The senolytic effect of CGs is also effective in the elimination of senescence-induced lung fibrosis. This experimental approach allows the identification of compounds with senolytic activity that could potentially be used to develop effective treatments against age-related diseases.

Anti-cancer therapies often result in a subset of surviving cancer cells that undergo therapy-induced senescence (TIS). Senescent cancer cells strongly modify the intratumoural microenvironment favoring immunosuppression and, thereby, tumour growth. An emerging strategy to optimise current therapies is to combine them with treatments that eliminate senescent cells. To this end, we undertook an unbiased proteomics approach to identify surface markers contributing to senescent cells immune evasion. Through this approach, we discovered that the immune checkpoint inhibitor PD-L2, but not PD-L1, is upregulated across multiple senescent human and murine cells. Importantly, blockade of PD-L2 strongly synergises with genotoxic chemotherapy, causing remission of solid tumours in mice. We show that PD-L2 inhibition prevents the persistence of chemotherapy-induced senescent cells, which exert cell-extrinsic immunomodulatory actions. In particular, upon chemotherapy, tumours deficient in PD-L2 fail to produce cytokines of the CXCL family, do not recruit myeloid-derived suppressor cells (MDSCs) and are eliminated in a CD8 T cell-dependent manner. We conclude that blockade of PD-L2 improves chemotherapy efficacy by reducing the intratumoural burden of senescent cells and their associated recruitment of immunosuppressive cells. These findings provide a novel strategy to exploit vulnerabilities arising in tumour cells as a result of therapy-induced damage and cellular senescence.

Antibody-drug conjugates (ADC) are anti-neoplastic agents recently introduced into the anti-tumor arsenal. T-DM1, a trastuzumab-based ADC that relies on lysosomal processing to release the payload, is approved for HER2-positive breast cancer. Next-generation ADCs targeting HER2, such as [vic-]trastuzumab duocarmazine (SYD985), bear linkers cleavable by lysosomal proteases and membrane-permeable drugs, mediating a bystander effect by which neighboring antigen-negative cells are eliminated. Many anti-tumor therapies, like DNA damaging agents or CDK4/6 inhibitors, can induce senescence, a cellular state characterized by stable cell cycle arrest. Another hallmark of cellular senescence is the enlargement of the lysosomal compartment. Given the relevance of the lysosome to the mechanism of action of ADCs, we hypothesized that therapies that induce senescence would potentiate the efficacy of HER2-targeting ADCs. Treatment with the DNA-damaging agent doxorubicin and CDK4/6 inhibitor induced lysosomal enlargement and senescence in several breast cancer cell lines. While senescence-inducing drugs did not increase the cytotoxic effect of ADCs on target cells, the bystander effect was enhanced when HER2-negative cells were co-cultured with HER2-low cells. Knockdown experiments demonstrated the importance of cathepsin B in the enhanced bystander effect, suggesting that cathepsin B mediates linker cleavage. In breast cancer patient-derived xenografts, a combination treatment of CDK4/6 inhibitor and SYD985 showed improved anti-tumor effects over either treatment alone. These data support the strategy of combining next-generation ADCs targeting HER2 with senescence-inducing therapies for tumors with heterogenous and low HER2 expression.