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

RNA interference and drug screening approaches led to the identification of the anti‐apoptotic BCL‐2 protein family as potential therapeutic targets in senescent cells, and Navitoclax (ABT‐263) as a drug with a potent senolytic activity (Chang et al., 2016; Zhu et al., 2016). Here, we have engineered a novel prodrug, namely Nav‐Gal, by modifying the molecular structure of Navitoclax following the synthetic procedure shown in Figure S1A. To do so, Navitoclax was reacted with 2,3,4,6‐tetra‐O‐acetyl‐α‐D‐galactopyranosyl bromide (Gal) through bimolecular nucleophilic substitution (S_N2) in the presence of potassium carbonate yielding the Nav‐Gal prodrug (Figure 1b), which was comprehensively characterized by several nuclear magnetic resonance techniques (NMR), including ¹H‐NMR, ¹³C‐NMR and correlated spectroscopy (COSY) NMR, as well as by attenuated total reflectance (ATR) and high‐resolution mass spectrometry (HRMS) (Figures S1B–F). The ¹H NMR signal centred at 5.6 ppm (Figure S1B) corresponds to the anomeric proton of the attached galactose and indicates the successful covalent linkage of the monosaccharide to Navitoclax. In addition, the appearance of a signal centred at 1,749 cm⁻¹ in the ATR spectra (Figure S1E) indicates that galactose remains acetylated after purification. Correct functionalization to the nitrogen atom of bis(sulfonyl)aniline is further corroborated through the fragmentation peaks observed in the mass spectrum (Figure S1F). After extensive structural characterization, we examined the ability of Nav‐Gal to be enzymatically hydrolyzed to Navitoclax. GLB1 is the human lysosomal β‐galactosidase responsible for the SA‐β‐gal activity (Dimri et al., 1995). We used high performance liquid chromatography (HPLC) to examine the time‐dependent hydrolysis reaction of PBS (pH 7)‐DMSO (0.01%) solutions containing Nav‐Gal in the presence of recombinant GLB1 and compared the peaks with those of Navitoclax (Figure 1c). The obtained chromatograms showed the Nav‐Gal peak with the subsequent appearance of free Navitoclax signal in the presence of the β‐galactosidase activity of the lysosomal enzyme and confirmed that Nav‐Gal was completely hydrolyzed. No significant spontaneous Nav‐Gal hydrolysis was observed (data not shown).

Since senescent cells are commonly characterized by high lysosomal SA‐β‐gal activity, we hypothesized that galacto‐conjugated Navitoclax would be preferentially processed and activated in senescent cells and hence could function as a prodrug with more selective senolytic activity. To investigate this, we performed cell viability assays using a model of therapy‐induced senescence. The lung carcinoma cell line A549 was treated with cisplatin (CDDP) for 10 days, resulting in elevated SA‐β‐gal activity and expression of markers of senescence (Figure S2A–C). Cisplatin‐treated A549 cells were then subjected to increasing doses of either Navitoclax or Nav‐Gal for 72 hr. As shown in Figure 2a, the concentration of Navitoclax required to induce death in 50% of the cells, termed half maximal inhibitory concentration (IC50), after 72 hr of treatment was 1.926 µM for nonsenescent and 0.122 µM for cisplatin‐induced senescent A549 cells, while the IC50 for Nav‐Gal was 9.758 µM for nonsenescent and 0.275 µM for senescent A549 cells (Figure 2b). A similar approach was performed in a model of palbociclib‐induced senescence with a human melanoma cell line (SK‐Mel‐103) (Figure 2c,d and Figure S2A‐C). These results show (in two different models of chemotherapy‐induced senescence) that Nav‐Gal has an improved senolytic index over Navitoclax and indicates that this effect is mainly mediated by a higher degree of protection of nonsenescent cells from cytotoxic activity. In an attempt to determine whether senolytic activity and nonsenescent cell protection by Nav‐Gal were more widely observed, we then performed cell viability assays using a variety of cell lines and diverse triggers of cellular senescence. Figure 2e‐i show the effects on cell viability of both Navitoclax and Nav‐Gal at different concentrations in cisplatin‐induced senescent mouse lung adenocarcinoma (Kras^G12D/+;p53^−/− (KP) L1475(luc) cells) (Turrell et al., 2017), palbociclib‐induced senescent mouse breast cancer 4T1 cells, doxorubicin‐induced senescent human colorectal carcinoma HCT116 cells, irradiation‐induced senescent mouse lung fibroblasts (MLg) and oncogene‐induced senescent human lung fibroblasts (IMR90), versus their nonsenescent counterparts. Efficient implementation of cellular senescence using these triggers was confirmed by SA‐β‐gal staining and Western blotting for senescence markers (Figure S2A‐C). In all the human/murine cell types tested, and independently of the senescence‐inducing trigger used, Nav‐Gal showed effective senolytic activity and a significantly higher degree of protection of nonsenescent cells when compared to Navitoclax.

To determine whether the senolytic effect of the prodrug Nav‐Gal depends on the increased lysosomal β‐galactosidase activity of senescent cells, siRNAs were used to knock‐down the expression of GLB1 in A549 and SK‐Mel‐103 cells. As shown in Figure 3a‐f, siRNA2 efficiently downregulated the transcription of GLB1 at 48 hr post‐transfection and resulted in a significantly decreased number of SA‐β‐galactosidase‐positive cells in both cell lines, when compared to scrambled siRNA and siRNA 1. While the killing efficiency of Navitoclax was maintained in the senescent cells in both cell lines (Figure 3g), transient downregulation of GLB1 significantly prevented the senolytic activity of Nav‐Gal (Figure 3h) in A549 and SK‐Mel‐103 cells, indicating that the selective senolysis of the prodrug is (at least) partially driven by the increased GLB1 expression of senescent cells.

The use of Navitoclax has been described to induce apoptosis preferentially in senescent cells through inhibition of the BCL‐2‐regulated pathway (Zhu et al., 2016). To assess whether Nav‐Gal and Navitoclax killed senescent cells by the same end mechanism, cisplatin‐induced senescent lung cancer A549 cells and palbociclib‐induced senescent melanoma SK‐Mel‐103 cells were treated with Navitoclax or Nav‐Gal (or vehicle) in parallel, with automated real‐time measurement of apoptosis study using a live‐cell analysis system. Figure 4a,b and Figure S3A–D show that treatment with either Navitoclax or Nav‐Gal induced apoptosis preferentially in chemotherapy‐induced senescent cells (as inferred from an increased annexin V signal). Importantly, and consistent with the results above (Figure 2a,b), the induction of apoptosis was lower in control nonsenescent cells treated with Nav‐Gal compared with Navitoclax (both at a high dose; 10 μM), particularly at later time points. While over 70% of nonsenescent A549 cells presented a strong signal for annexin V after 36 hr of treatment with Navitoclax, this was true for only ~30% of the cells after treatment with the same dose of Nav‐Gal (Figure 4a,b and Figure S3A). Consistent effects were observed with the melanoma cell line SK‐Mel‐103 (Figure S3B–D). These results indicate that Nav‐Gal kills senescent cells by inducing apoptosis and that it protects against nonsenescent cell death to a higher extent than Navitoclax.

Effective anti‐cancer treatments have enhanced clinically beneficial effects when given in combination, including in NSCLC. We therefore next sought to ascertain the efficacy of concurrent cisplatin and Nav‐Gal treatment in vitro. We first used clonogenic assays to determine the efficacy of the combination of senescence‐inducing cisplatin and Navitoclax, Nav‐Gal or vehicle, administered concomitantly to A549 cells. Compared with monotherapies, the combination of cisplatin and a senolytic drug was substantially more effective at inhibiting cell proliferation (Figure 4c‐f). From 0 to 2 µM cisplatin, as the concentration of both Navitoclax and Nav‐Gal increases, additional impairment of cell growth was observed. There was also a significant reduction in the concentration of Nav‐Gal required to produce growth inhibition in 1 µM cisplatin versus 0 µM cisplatin (Figure S3G). Similar to the IC50 experiments (Figure 2a,b), a higher dose of Nav‐Gal than Navitoclax was required to achieve the same effect, likely due to the requirement for Nav‐Gal processing and activation by lysosomal β‐galactosidase activity. While both senolytic drugs showed an additive effect in combination with cisplatin, increasing doses of Nav‐Gal exhibited coefficients of drug interactions (CDIs) <1 and closer to 0 in combination with 1 μM cisplatin (Figure 4g), which suggests that the combination of drugs may be synergistic (Zhao et al., 2014). Of note, these observations were also validated in the context of a sequential treatment, where colonies were first exposed to increasing concentrations of cisplatin for 7 days, followed by 7 days of senotherapy (Figure S3E,F).

In order to validate the efficiency of the prodrug Nav‐Gal in combination with senescence‐inducing chemotherapy in vivo, we used a model whereby A549 cells were transplanted subcutaneously into the flanks of severe combined immunodeficient (SCID) mice. In a first approach, we aimed to assess the induction of senescence in tumour‐bearing mice treated with cisplatin. Histological analyses of the tumours collected after treatment showed evidence of senescence induction upon cisplatin administration (as inferred from SA‐β‐gal positivity) (Figure 5a). The effects of concomitant treatment with cisplatin and each senolytic were then investigated. Once tumours reached an average volume of 100 mm³, mice were treated with cisplatin and daily doses of Navitoclax and Nav‐Gal, alone or in combination, for 17 days (as shown in Figure 5b). Importantly, both drugs significantly improved the tumour growth inhibition of cisplatin (Figure 5c). The combination of cisplatin with either Navitoclax or Nav‐Gal had comparable effects on tumour growth inhibition, but both showed statistically significant differences with control and monotherapy groups. In contrast, Navitoclax and Nav‐Gal had no appreciable effect on tumour growth when administered in the absence of cisplatin, indicating that their therapeutic activities require concomitant induction of senescence by cisplatin in this model system. Consistently, histological analyses of the tumours revealed that cisplatin treatment results in increased p21 positivity (correlating with Western blot analyses of cisplatin‐treated cells in vitro, Figure S2C) and decreased Ki67 positivity, which together constitute a hallmark of senescent cells (Figure 5d,e). Treatment with cisplatin and either Navitoclax or Nav‐Gal exhibited reduced levels of p21 and Ki67 positivity, alongside a strong TUNEL signal, strongly suggesting that apoptosis of senescent cells facilitates the anti‐tumour effect. Additionally, a therapeutic effect of both Navitoclax and Nav‐Gal in tumour growth was observed in a sequential treatment after one week of cisplatin administration, suggesting a potential application as adjuvant therapy for clearing senescent cells (Figure S4A,B).

To obtain further evidence of the therapeutic potential of Nav‐Gal in combination with senescence‐inducing chemotherapy in a more physiological context, we used an orthotopic model of NSCLC. Here, wild‐type C57BL/6J mice were transplanted (via tail vein injection) with a syngeneic luciferase‐expressing KP lung adenocarcinoma cell line (L1475luc) (Turrell et al.,). Once tumours were established in the lungs, baseline luciferase signals were obtained and mice were then treated for 10 days with cisplatin alone, cisplatin and Nav‐Gal in combination, or their vehicles in combination, using the experimental scheme shown in FigureA. Representative images of the luciferase signal at initial‐ and end‐points are shown in FigureB. Luciferase signal monitoring demonstrated that concomitant treatment of the mice with cisplatin and Nav‐Gal significantly decreased tumour burden (FigureC) compared with cisplatin monotherapy. Histological analysis of the lungs showed high burden of senescent cells only in cisplatin‐treated mice (as evidenced by increased SA‐β‐gal and p21 levels), and decreased SA‐β‐gal and p21 levels and higher TUNEL staining in mice concomitantly treated with cisplatin and Nav‐Gal, indicating enhanced apoptosis of senescent cells (representative areas are shown in FigureD). 2017 S5 S5 S5 S5

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.

Thrombocytopenia is a major dose‐limiting, and clinically important, toxicity that is produced by Navitoclax in human patients (Cang et al., 2015; Wilson et al., 2010). A potential benefit for galacto‐conjugation of senolytic drugs is the reduction of their associated toxicities, and the potential widening of their therapeutic windows. To determine whether galacto‐conjugation affected platelet toxicity, we performed ex vivo experiments with both human and murine blood samples where we exposed the whole blood to increasing concentrations of either Navitoclax or Nav‐Gal, using fluorescein‐labelled annexin V to identify apoptotic platelets by flow cytometry ((Vogler et al., 2011); Figure S6A,B). As observed in Figure 6a,b for human blood and in Figure 6c,d for mouse blood, when samples were exposed to the same concentrations of Navitoclax and Nav‐Gal, the proportion of platelets with annexin V signal was significantly higher after exposure to Navitoclax than with Nav‐Gal. This effect persists in both models, despite a significant difference in platelet sensitivity to BCL‐2 family inhibition between each model.

To determine whether Nav‐Gal also protected platelet levels at therapeutic doses in vivo, we first examined the platelet count of wild‐type C57BL/6J treated daily with senotherapy for a total of 10 days (Figure 6e). As shown in Figure 6f, daily administration of Navitoclax resulted in severe thrombocytopenia after only 5 days of treatment, independently of the route of administration. Remarkably, Nav‐Gal treatment did not cause thrombocytopenia in the mice and led to platelet count levels comparable to those of vehicle‐treated individuals, showing reduced platelet toxicity compared to Navitoclax. In order to validate our findings in the context of an in vivo cancer model, the platelet count was also assessed at end‐points of the concomitant treatment of xenograft‐bearing mice described in Figure 5b‐d. Notably, as shown in Figure 6g,h, no significant additional thrombocytopenia was observed in the group treated with cisplatin and Nav‐Gal (cf. Navitoclax). We also 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. Altogether, we conclude that galacto‐conjugated Navitoclax is effective in clearing senescent cells in vivo and can reduce its associated toxicities.

For the synthesis of Nav‐Gal prodrug, 40 mg (0.04 mmol) of Navitoclax (Eurodiagnostico), 25 mg (0.06 mmol) of 2,3,4,6‐tetra‐O‐acetyl‐α‐D‐galactopyranosyl‐bromide (Sigma) and 10.5 mg (0.07 mmol) of K₂CO₃ (Sigma) were mixed. Anhydrous acetonitrile (Sigma) was added, and the mixture was stirred at 70°C for 3 hr under argon‐enriched atmosphere. The solvent was eliminated under vacuum. The product was purified by flash chromatography on silica gel (Sigma), from hexane‐ethylacetate (3:7 v/v; Scharlab) to hexane‐ethylacetate (7:3 v/v) used as eluent. Purified Nav‐Gal was obtained as a yellow powder in 35% yield. For large stocks used in mouse experiments, the reactions were performed in different batches. Once the desired amount was obtained, all batches were pooled and purified on a single flash chromatography column as described above.

Nav‐Gal was characterized by conventional techniques. For samples preparation, pure Nav‐Gal was dissolved in deuterochloroform (CDCl₃; Sigma) and transferred to a Wilma NMR tube (Sigma). Samples were immediately analyzed by proton nuclear magnetic resonance (¹H NMR), Carbon‐13 nuclear magnetic resonance (¹³C NMR) and homonuclear bidimensional correlated spectroscopy (COSY NMR). NMR spectra were acquired with a Bruker FT‐NMR Avance 400 (Ettlingen, Germany) at 300 K and analyzed with MestReNova 6.0 software. Chemical shifts are expressed as δH (ppm) relative tetramethylsilane (TMS). Attenuated total reflectance (ATR) was performed in a Bruker Tensor 27 FT‐IR (Ettlingen, Germany) at room temperature and analyzed with OPUS Data Collection Program (V 1.1). Finally, high resolution mass spectrometry (HRMS) was recorded with an ABSciex TRIPLETOF T5600.

For hydrolysis reaction studies, aqueous solutions of Navitoclax and Nav‐Gal at a concentration of 10^–5 M (pH 7) in 0.01% DMSO were prepared. Human β‐galactosidase (Biotechne, R&D Systems) was then added to Nav‐Gal solutions to a final concentration of 8 ng/µl, and chromatograms were acquired after complete reaction (λ_exc = 365 nm) with a Waters 1525 binary HPLC pump equipped with a Waters 2990 diode array detector. Chromatograms were obtained using Empower 3 software. Conditions: KromasilC18 column, 0.9 ml/min, (H₂O (0.1% acetic acid)): MeOH gradient elution: 50:50 3 min, 40:60 7 min, 30:70 10 min, 20:80 5 min, 10:90 3 min, 0:100 3 min. Data analysis was performed using OriginPro8 software.

The human lung carcinoma cell line A549 was obtained from the European Collection of Authenticated Cell Cultures (ECACC). SK‐MEL‐103 (human melanoma), 4T1 (breast cancer), HCT116 (human colorectal carcinoma) and MLg (mouse lung fibroblastic) cell lines were obtained from the American Type Culture Collection (ATCC). The murine KP lung adenocarcinoma cell line L1475 was derived from KrasLSL‐G12D/+;p53Fx/Fx mice (Jackson et al., 2005) and transduced with MSCV‐luciferase‐hygromycin retrovirus, as previously described (Turrell et al., 2017). A549, SK‐MEL‐103, 4T1 and L1475(luc) cell lines were maintained in DMEM, and supplemented with 10% FBS (Sigma). HCT116 cells were grown in McCoy's 5A medium (Thermo Fisher Scientific) supplemented with 10% FBS. MLg cells were cultured in DMEM‐F‐12 culture media (Sigma) supplemented with 2 mM l‐Glutamine and 10% FBS. ER:Mek IMR90 cells were cultured in phenol red‐free DMEM (Sigma) supplemented with 10% FBS, 2 mM l‐Glutamine and 1 mM sodium pyruvate (Sigma). All cells were incubated in 20% O₂ and 5% CO₂ at 37°C. Cells were routinely tested for mycoplasma using the universal Mycoplasma Detection Kit (ATCC) or by RNA‐capture ELISA.

For senescence induction, A549 cells were treated with 15 µM of cisplatin (Stratech) for 10 days. SK‐MEL‐103 and 4T1 cells were treated with 5 µM palbociclib (Eurodiagnostico) for 7 days. HCT116 cells were treated with 100 nM doxorubicin (Carbosynth) for 72 hr. MLg cells were irradiated with 10 Gy and used for viability assays 10 days later. ER:Mek IMR90 cells were treated with 200 nM 4‐hydroxytamoxifen for 72 hr and used for viability assays 2 days later.

For experiments with cells, cisplatin (Stratech) was reconstituted in sterile PBS; palbociclib (Eurodiagnostico), Navitoclax (Stratech) and Nav‐Gal were reconstituted in DMSO. For in vivo experiments, cisplatin was reconstituted in saline; Navitoclax was formulated in 10% ethanol, 30% polyethylene glycol 400 and 60% Phosal PG, and Nav‐Gal was reconstituted in DMSO and further diluted in saline.

For transient downregulation of GLB1, a total of 30,000 control or 50,000 senescent A549 and SK‐MEL‐103 cells were plated per well in a 24‐well plate. The next day, cells were transfected with TriFECTa^® Kit DsiRNA Duplex siRNAs (Integrated DNA Technologies) hs.Ri.GLB1.13.1 (siRNA1), hs.Ri.GLB1.13.3 (siRNA2) or scrambled siRNA, using Lipotectamine RNAiMAX Reagent (Thermo Fisher Scientific) as per manufacturer's instructions. After 48 hr, RNA was extracted using the RNeasy Mini Kit (Qiagen) and cDNA was synthesized with the High‐Capacity RNA‐to‐cDNA™ Kit (Thermo Fisher Scientific).

Gene expression of GLB1 and ACTB genes was measured by quantitative real‐time PCR performed on a QuantStudio thermocycler (Applied Biosystems) following Luna^® Universal qPCR Master Mix (New England Biolabs) protocol and amplification parameters, and using predesigned KiCqStart^® SYBR^® Green Primers H_GLB1_1 and H_ACTB_1 pairs (Sigma‐Aldrich). Relative quantification was carried out using 2−ΔΔCt methodology.

For A549, SK‐MEL‐103, 4T1, HCT116 and ER:Mek IMR90 cell lines, control and senescent cells were seeded in flat‐bottom‐clear 96‐well plates at a density of 6,000–8,000 and 4,000–6,000 cells per well, respectively. The following day cells were treated with serial dilutions of Nav‐Gal or Navitoclax in 0.2% FBS‐containing media. Viability was assessed 48 or 72 hr later after two hours of incubation at 37°C with CellTiter‐Glo^® Luminescent Cell Viability Assay (Promega) or CellTiter‐Blue^® Cell Viability Reagent (Promega). Raw data were obtained by measuring luminescence in a VICTOR Multilabel Plate Reader (Pelkin Elmer) or fluorescence at an excitation/emission wavelength of 560 nm/590 nm in an Infinite 200 PRO Multimode Spectrophotometer (TECAN).

For MLg cells, control and senescent cells were seeded in a 12‐well plate at a density of 80,000 and 60,000 cells per well respectively, and cells were treated with three to five different increasing concentrations of Nav‐Gal or Navitoclax on the following day. After 72 hr, viability was assessed with CellTiter‐Blue^® Cell Viability Reagent (Promega) as described above.

To determine the induction of apoptosis after the treatment with Navitoclax and Nav‐Gal, 4,000 senescent and 6,000 nonsenescent A549 or SK‐Mel‐103 cells were seeded in 96‐well plates. 24 hr later, annexin V Fluorescent Reagent (Essen Bioscience) was added to the media, and after a first scan, the cells were treated with vehicle (DMSO), 11 µM Navitoclax or 11 µM Nav‐Gal for 48 hr. Images were collected every 2 hr with an Incucyte^® S3 Live‐Cell Analysis System microscope (Essen Bioscience) over time. A total of 2 pictures per well were analyzed using the IncuCyte ZOOM™ software analyser, and total and annexin V‐positive cells were counted using ImageJ software.

For clonogenic potential analysis, 10,000 A549 cells were seeded per well in a 6‐well plate and allowed to attach overnight. In a first approach (concomitant treatment), cells were then treated with increasing and concomitant concentrations of cisplatin and Navitoclax or Nav‐Gal. In a second approach (sequential treatment), cells were first treated with increasing concentrations of cisplatin for 7 days, and then Navitoclax or Nav‐Gal were added for 7 additional days. Fresh drug was added every 2–3 days, and 10 days later, colonies were washed with PBS and fixed with 4% PFA for 10 min. They were then permeabilized using ice‐cold methanol for 20 min, and left to dry. Colonies were then stained with 0.5% crystal violet for 30 min, and excess was removed by thoroughly washing three times with deionised water. Colonies were imaged using a BioRad GelDoc XR+ machine (BioRad Technologies). To calculate mean clonogenic potential, staining of colonies was subsequently diluted in 10% acetic acid and optical density (OD) was measured at 595 nm using an Infinite 200 PRO Multimode Spectrophotometer (PECAN) microplate reader. The blank (10% acetic acid) was subtracted from each OD measurement, and the average value for each experimental condition was calculated and normalized against clonogenic potential value obtained in no‐treatment condition.

To analyze the synergistic effect of the concomitant treatment of cisplatin and the senolytics Navitoclax and Nav‐Gal, we calculated the co‐coefficient of drug interaction (CDI) using the normalized clonogenic potential values of the individual and concomitant treatments at different concentrations. CDIs were calculated as ratios following the formula CDI = (AB)/(AxB), where AB is the clonogenic potential value of the concomitant treatment with the two drugs combined at a specific concentration, and A and B are the clonogenic potential values of the individual treatments of each drug at such concentration. Following this formula, a CDI > 1 indicates that the drugs are antagonistic, a CDI = 1 indicates that the drugs are additive and a CDI < 1 indicates that the drugs are synergistic, with values closer to 0 indicating higher synergy between the drugs. This effect can depend on the ratio and molarity of drugs used, and for this reason, CDIs were calculated and plotted at different molar ratios of cisplatin and Navitoclax or Nav‐Gal.

Cell lysis was performed using RIPA buffer (Sigma) supplemented with phosphatase inhibitors (PhosSTOP™ EASYpak Phosphatase Inhibitors Cocktail, Roche) and protease inhibitors (cOmplete™ Protease Inhibitor Cocktail, Roche). Proteins were quantified and separated by SDS‐PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) according to standard protocols. Membranes were immunoblotted with antibodies against p21 and p53 from Santa Cruz Biotechnology, and phospho‐Rb (pRBS780) from Cell Signaling. After incubation with the primary antibody overnight, membranes were washed and incubated with secondary HRP‐conjugated AffiniPure antibodies (Jackson ImmunoResearch) for 1 hr at room temperature and subsequently incubated with Enhanced Chemiluminescence Detection solution (Amersham). Membranes were placed on X‐ray films and processed using a Xograph Compact X4 automatic processor.

All mice were treated in strict accordance with the local ethical committee (University of Cambridge Licence Review Committee) and the UK Home Office guidelines. For the orthotopic model experiment, 5–6 weeks old C57BL/6J mice were transplanted in the lungs with 3 × 10⁵ syngeneic luciferase‐expressing KP (L1475(luc)) lung tumour cells via tail‐vein injection. All mice were maintained in ventilated cages within a specific pathogen free animal facility. Luminescence values were recorded 5 days later after i.p. injection with D‐luciferin (150 mg/kg body weight, PerkinElmer) using an IVIS Spectrum Zenogen machine (Caliper Life Sciences) and analyzed with Living Image software, and then randomized into the different experimental groups. Mice were treated with either vehicle, 1 mg/kg CDDP (via i.p. injection) every 3 days and 85 mg/kg Nav‐Gal (via i.p. injection) for two consecutive days after each CDDP administration. Luminescence values were recorded again as described above on day 5 and 15, when treatment was finished, all mice were culled by anaesthetic overdose, and lungs were collected for subsequent histological analyses.

To establish subcutaneous tumour xenografts, 5–7 weeks old SCID (CB17‐Prkdc^scid/J) females were injected subcutaneously with 4 × 10⁶ A549 cells in each flank. Tumours were measured with callipers every 2–3 days, and the tumour volume was calculated with the formula length × width²/2. When tumour volume reached an average of 100 mm³, mice were randomized and assigned to one of the control or therapy groups. Therapy was initiated with either vehicle, 1.5 mg/kg cisplatin (CDDP, via intraperitoneal (i.p.) injection) three times per week, 100 mg/kg Navitoclax (via oral gavage) 5 days ON/2 days OFF, 85 mg/kg Nav‐Gal (via i.p. injection) 5 days ON/2 days OFF, or a combination of the mentioned drugs. Mice were culled by cervical dislocation after 3 weeks of treatment.

For platelet toxicity analysis, wild‐type C57BL/6J mice were treated daily with 100 mg/kg Navitoclax (via oral gavage), 85 mg/kg Navitoclax (via i.p. injection) or 85 mg/kg Nav‐Gal (via i.p. injection) for 10 consecutive days.

To analyze platelet count in mice, blood was collected by cardiac puncture during isoflurane anaesthesia in xenograft experimental mice, and by superficial vessel puncture in wild‐type C57BL/6J experimental mice. Blood was collected into anticoagulating Microvette^® tubes (Sarstedt), and platelet levels were immediately measured using a Scil Vet abc Plus hematology analyzer (Horiba).

SA‐β‐Gal staining was performed in frozen tissue sections using the Senescence β‐Galactosidase Staining kit (Cell Signaling), following the manufacturer instructions. Briefly, whole tissue was fixed at RT for 15 min with a 2% formaldehyde and 0.2% glutaraldehyde, washed and incubated for 6 hr at 37°C with the staining solution containing X‐gal in N‐N‐dimethylformamide (pH 6.0). Tissues were subsequently counterstained with nuclear fast red, dehydrated and mounted. For immunohistochemistry, 5 mm paraffin sections were deparaffinized and re‐hydrated, and slides were incubated with anti‐p21 (Abcam) and Ki67 (Abcam) antibodies at 4°C overnight, or processed for TUNEL staining using the DeadEnd™ fluorometric TUNEL System (Promega) as per manufacturer's instructions. Immunohistological reaction was of p21, and Ki67 was developed using 3,3‐diaminobenzidine tetrahydrochloride (DAB), and nuclei counterstained with haematoxylin. For immunofluorescence, frozen tissue sections were either processed for TUNEL staining using the above mentioned kit or alternatively fixed in 4% PFA for 10 min, permeabilized with 0.5% Triton™ X‐100 for 3 min and subsequently blocked with Normal Donkey Serum for 1 hr at room temperature. They were then probed with anti‐p21 (Abcam) and Ki67 (Abcam) antibodies at 4°C overnight. The following day, sections were incubated with Anti‐Rat IgG 555 and Anti‐Rabbit IgG 488 secondary antibodies (Cell Signaling Technologies), briefly incubated in DAPI‐containing PBS, and mounted using Fluoromount‐G™ Mounting Medium. Images were obtained using an AxioScan Slide Scanner (Zeiss) and processed with ZEN 2 Blue Edition software (Zeiss). Positive signal for p21, Ki67 and TUNEL was quantified with ImageJ.

For human platelets apoptosis assay, blood from three healthy volunteers was extracted according the UPV ethics procedures and processed essentially as shown in Vogler et al., 2011). For mouse platelet apoptosis assay, blood from five wild‐type C57BL/6J mice was collected by cardiac puncture in accordance with the University of Cambridge ethical committee and the UK Home Office guidelines. Samples were collected in nonvacuum citrate tubes (DH medical material), and blood in control tubes was diluted in Tyrodes Buffer. Blood in treated samples was mixed with 2X drug solution (either Navitoclax or Nav‐Gal) in RPMI (10% FBS). Control and treated specimens were incubated for 5 hr in 20% O₂ and 5% CO₂ at 37°C. Treated samples were diluted in Tyrodes Buffer, and then all specimens were mixed with Annexin Binding Buffer (Thermo Fisher Scientific) and incubated for 10 min with Annexin V‐FITC (Immunostep) and CD‐41‐PE (Immunostep) conjugated antibodies. A23187 (Sigma), a calcium ionophore, was also added in parallel control samples to induce cell death and be used as a positive control. Samples were finally diluted in Annexin Binding Buffer (Thermo Fisher Scientific), and data were acquired using an FC500 MPL Flow Cytometer (Beckman‐Coulter) for human samples and an LSR Fortessa cell analyzer running the FACSDiva software (BD Biosciences) for mouse samples. FlowJo 10.3 software was used to analyze the results.

Statistical analyses were performed as described in the figure legend for each experiment. Statistical significance was determined by one‐ and two‐way ANOVA, Student's t tests and Bonferroni post hoc tests using Prism 5 software (GraphPad) as indicated. A p‐value below .05 was considered significant and indicated with asterisk: *p < .05, **p < .01 and ***p < .005.