Susana Llanos

The COVID-19 pandemic highlighted the need for the rapid development of antiviral therapies. Viral RNA-dependent RNA polymerases (RdRp) are promising targets, and numerous virtual screenings for potential inhibitors were conducted without validation of the identified hits. Here we have tested a set of presumed RdRp inhibitors in biochemical assays based on fluorometric detection of RdRp activity or on the electrophoretic separation or RdRp products. We find that fluorometric detection of RdRp activity is unreliable as a screening method because many small compounds interfere with fluorophore binding to dsRNA, and this effect is enhanced by the Mg²⁺ metal ions used by nucleic acid polymerases. The fact that fluorimetric detection of RdRp activity leads to false-positive hits underscores the requirement for independent validation methods. We also show that suramin, one of the proposed RdRp inhibitors that could be validated biochemically, is a multi-polymerase inhibitor. While this does not hinder its potential as an antiviral agent, it cannot be considered an specific inhibitor of SARS-CoV-2 RdRp.

Chemotherapy often generates intratumoral senescent cancer cells that strongly modify the tumor microenvironment, favoring immunosuppression and tumor growth. We discovered, through an unbiased proteomics screen, that the immune checkpoint inhibitor programmed cell death 1 ligand 2 (PD-L2) is highly upregulated upon induction of senescence in different types of cancer cells. PD-L2 is not required for cells to undergo senescence, but it is critical for senescent cells to evade the immune system and persist intratumorally. Indeed, after chemotherapy, PD-L2-deficient senescent cancer cells are rapidly eliminated and tumors do not produce the senescence-associated chemokines CXCL1 and CXCL2. Accordingly, PD-L2-deficient pancreatic tumors fail to recruit myeloid-derived suppressor cells and undergo regression driven by CD8 T cells after chemotherapy. Finally, antibody-mediated blockade of PD-L2 strongly synergizes with chemotherapy causing remission of mammary tumors in mice. The combination of chemotherapy with anti-PD-L2 provides a therapeutic strategy that exploits vulnerabilities arising from therapy-induced senescence.

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.

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Interstrand crosslinks (ICLs) are DNA lesions frequently induced by chemotherapy that interfere with essential processes such as replication and transcription. ICL repair may be initiated by the convergence of two replication forks at the crosslink, which results in a termination-like DNA structure recognized and processed by the Fanconi Anemia (FA) pathway. An alternative possibility to generate a suitable substrate for ICL repair involves “ICL traverse”, a DNA damage tolerance mechanism in which a single fork arriving at the ICL can skip the lesion and restart DNA synthesis from a downstream point. This reaction requires FANCM translocase, the BLM/TOP3A/RMI1-2 (BTR) complex and other factors. Here we report that PrimPol, the second primase-polymerase identified in mammalian cells after Polα/Primase, interacts with BTR and participates in the ICL traverse reaction. A functional complementation assay reveals that the primase activity of PrimPol is required, confirming the need for re-priming events during ICL traverse. Genetic ablation of PRIMPOL strongly impaired this tolerance mechanism, making cells more dependent on fork convergence to initiate ICL repair. PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs, opening the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents.

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.

Palbociclib is a selective inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6) approved for the treatment of some cancers. The main mechanism of action of palbociclib is to induce cell cycle arrest and senescence on responsive cells. Here, we report that palbociclib concentrates in intracellular acidic vesicles, where it can be readily observed due to its intrinsic fluorescence, and it is released from these vesicles upon dilution or washing out of the extracellular medium. This reversible storage of drugs into acidic vesicles is generally known as lysosomal trapping and, based on this, we uncover novel properties of palbociclib. In particular, a short exposure of cells to palbociclib is sufficient to produce a stable cell-cycle arrest and long-term senescence. Moreover, after washing out the drug, palbociclib-treated cells release the drug to the medium and this conditioned medium is active on susceptible cells. Interestingly, cancer cells resistant to palbociclib also accumulate and release the drug producing paracrine senescence on susceptible cells. Finally, other lysosomotropic drugs, such as chloroquine, interfere with the accumulation of palbociclib into lysosomes, thereby reducing the minimal dose of palbociclib required for cell-cycle arrest and senescence. In summary, lysosomal trapping explains the prolonged temporal activity of palbociclib, the paracrine activity of exposed cells, and the cooperation with lysosomotropic drugs. These are important features that may help to improve the therapeutic dosing and efficacy of palbociclib. Finally, two other clinically approved CDK4/6 inhibitors, ribociclib and abemaciclib, present a similar behavior as palbociclib, suggesting that lysosomal trapping is a property common to all three clinically-approved CDK4/6 inhibitors.

Senescent cells accumulate in multiple aging‐associated diseases, and eliminating these cells has recently emerged as a promising therapeutic approach. Here, we take advantage of the high lysosomal β‐galactosidase activity of senescent cells to design a drug delivery system based on the encapsulation of drugs with galacto‐oligosaccharides. We show that gal‐encapsulated fluorophores are preferentially released within senescent cells in mice. In a model of chemotherapy‐induced senescence, gal‐encapsulated cytotoxic drugs target senescent tumor cells and improve tumor xenograft regression in combination with palbociclib. Moreover, in a model of pulmonary fibrosis in mice, gal‐encapsulated cytotoxics target senescent cells, reducing collagen deposition and restoring pulmonary function. Finally, gal‐encapsulation reduces the toxic side effects of the cytotoxic drugs. Drug delivery into senescent cells opens new diagnostic and therapeutic applications for senescence‐associated disorders.