Galacto-conjugation of Navitoclax as an efficient strategy to increase senolytic specificity and reduce platelet toxicity.

Miguel Rovira,Araceli Lérida-Viso ,Beatriz Lozano‐Torres,Juan F Blandez,Carla P Martins,Estela González-Gualda ,David Macías ,Gary J Doherty,Hui-Ling Ou ,Sofía Mirón-Barroso ,Andrea Bernardos,Daniel Muñoz‐Espín,Manuel Serrano,Félix Sancenón,Marta Páez-Ribes ,Ramón Martínez‐Máñez,Ljiljana Fruk,Cristina González-López ,Joanne S Wilson ,Zhenguang Zhang

doi:10.1111/acel.13142

Abstract

Pharmacologically active compounds with preferential cytotoxic activity for senescent cells, known as senolytics, can ameliorate or even revert pathological manifestations of senescence in numerous preclinical mouse disease models, including cancer models. However, translation of senolytic therapies to human disease is hampered by their suboptimal specificity for senescent cells and important toxicities that narrow their therapeutic windows. We have previously shown that the high levels of senescence‐associated lysosomal β‐galactosidase (SA‐β‐gal) found within senescent cells can be exploited to specifically release tracers and cytotoxic cargoes from galactose‐encapsulated nanoparticles within these cells. Here, we show that galacto‐conjugation of the BCL‐2 family inhibitor Navitoclax results in a potent senolytic prodrug (Nav‐Gal), that can be preferentially activated by SA‐β‐gal activity in a wide range of cell types. Nav‐Gal selectively induces senescent cell apoptosis and has a higher senolytic index than Navitoclax (through reduced activation in nonsenescent cells). Nav‐Gal enhances the cytotoxicity of standard senescence‐inducing chemotherapy (cisplatin) in human A549 lung cancer cells. Concomitant treatment with cisplatin and Nav‐Gal in vivo results in the eradication of senescent lung cancer cells and significantly reduces tumour growth. Importantly, galacto‐conjugation reduces Navitoclax‐induced platelet apoptosis in human and murine blood samples treated ex vivo, and thrombocytopenia at therapeutically effective concentrations in murine lung cancer models. Taken together, we provide a potentially versatile strategy for generating effective senolytic prodrugs with reduced toxicities.

Highlights

Senescence is an evolutionarily conserved cellular response to severe stress and damage characterized by stable cell cycle arrest, upregulation of pro-survival signalling pathways and the induction of a complex secretory phenotype, termed the senescence-associated secretory phenotype (SASP) (Gorgoulis et al, 2019)
Using a variety of model systems, we show that galacto-conjugation of Navitoclax, which we name Nav-galactopyranosyl bromide (Gal), results in a prodrug with selective, pro-apoptotic senolytic activity released in senescent cells that is dependent on GLB1 activity
The use of novel genetically engineered mouse models has demonstrated that the selective elimination of senescent cells attenuates a number of age-related pathologies and promotes the healthspan and lifespan of mice (Baker et al, 2016)

Summary

| INTRODUCTION

Senescence is an evolutionarily conserved cellular response to severe stress and damage characterized by stable cell cycle arrest, upregulation of pro-survival signalling pathways and the induction of a complex secretory phenotype, termed the senescence-associated secretory phenotype (SASP) (Gorgoulis et al, 2019). Taken together, these results demonstrate that the combination of senescence-inducing therapy with senotherapy is highly effective in inhibiting tumour growth in vivo, providing preclinical proof-of-principle of the therapeutic benefits of using Nav-Gal as a potent prodrug with senolytic activity. We found that Nav-Gal reduced platelet toxicity, either in combination with cisplatin or as monotherapy, when administered in a sequential manner (Figure S4A–C), providing further evidence of platelet protection in vivo These results confirm that the galacto-conjugation of Navitoclax to produce the Nav-Gal prodrug serves as an effective strategy to decrease Navitoclax-driven thrombocytopenia at physiologically relevant concentrations capable of halting cancer growth.

| DISCUSSION

Findings

| EXPERIMENTAL PROCEDURES