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

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.

Keywords: Navitoclax (ABT-263); cellular senescence; chemotherapy-induced senescence; lung cancer; prodrug; senolytics; thrombocytopenia.

FIGURE 1

Galacto‐conjugation of the senolytic Navitoclax into a new generation senolytic prodrug, namely Nav‐Gal, as an efficient strategy for selective senolysis. (a) Schematic representation of the mechanism of action of Nav‐Gal prodrug. Nav‐Gal is passively taken up by both nonsenescent and senescent cells. In nonsenescent cells, its conjugation with a cleavable galactose renders it inactive and unable to inhibit anti‐apoptotic proteins, such as BCL‐2, preventing the induction of apoptosis. In senescent cells, the increased lysosomal and galactosidase activity, a hallmark of cellular senescence, allows the hydrolysis of the cleavable galactose, resulting in the release of active Navitoclax into the cytoplasm of senescent cells. Free Navitoclax will inhibit anti‐apoptotic BCL‐2 proteins, which are overexpressed in senescent cells, driving specific apoptosis of these cells. (b) Chemical structures of Nav‐Gal prodrug and Navitoclax. The presence of galactopyranoside, covalently linked to the N of bis(sulfonyl)aniline as synthesized in this prodrug, hinders two key interactions: (i) π‐π interaction between the phenylthioether moiety and the bis(sulfonyl)aniline ring; and (ii) the hydrogen bond between morpholine with Tyr‐199 (Liu, Zhang, Huang, Tan, & Zhang, 2018), thereby preventing the inhibitory effect of the molecule. This moiety, the galactopyranoside, can be hydrolyzed in the presence of β‐galactosidase (cleavable galactose). (c) Chromatograms depicting hydrolysis reaction of Nav‐Gal aqueous solutions in the presence of human β‐galactosidase followed by HPLC‐UV as described in the text

FIGURE 2

The prodrug Nav‐Gal shows efficient, broad range, senolytic activity and an increased senolytic index, conferring a protective effect on nonsenescent cells. (a, b) Quantification of viability and half maximal inhibitory concentration (IC50) of (a) Navitoclax and (b) Nav‐Gal on control and cisplatin‐induced senescent A549 cells. Senolytic indices for each drug are shown in the tables below. Viability assay on A549 cells was performed as n = 5, and graphs depict one representative repeat. (c, d) Quantification of viability and maximal inhibitory concentration (IC50) of (c) Navitoclax and (d) Nav‐Gal on control and palbociclib‐induced senescent SK‐Mel‐103 cells. Senolytic indices of each drug are shown in the tables below. Viability assay on SK‐Mel‐103 cells was performed as n = 3, and graphs depict one representative repeat. (e–i) Quantification of cell viability upon Navitoclax and Nav‐Gal treatment of control and (e) cisplatin‐induced senescent KRas ^G12D/WT;p53 ^−/− lung adenocarcinoma tumour cells L1475(luc) (n = 4), (f) palbociclib‐induced senescent 4T1 cells (n = 3), (g) doxorubicin‐induced senescent HCTT116 cells (n = 3), (h) irradiation‐induced senescent MLg cells (n = 3) and (i) oncogene‐induced senescent IMR90 cells (previously treated for 72 hr with 200 nM 4‐hydroxytamoxifen (4‐OHT)) (n=3). Data in (e‐i) represent mean ± SD of replicates, and statistical significance was calculated using two‐way ANOVA ; *p < .05, **p < .01, ***p < .001; #p < .05, ##p < .01, ###p < .001

FIGURE 3

GLB1 transient downregulation prevents the senolytic activity of Nav‐Gal. (a) Representative images of SA‐β‐gal staining of control and cisplatin‐induced senescent A549 cells 48 hr after transfection with scrambled siRNA, siRNA 1 and siRNA 2. Scale bar = 200 μm. (b) Percentage of SA‐β‐gal‐positive cells in conditions presented in (a). Bars represent mean ± SD (n = 3). (c) GLB1 fold change gene expression in control and senescent A549 cells 48 hr post‐transfection with different siRNAs. (d) Representative images of SA‐β‐gal staining of control and palbociclib‐induced senescent SK‐Mel‐103 cells 48 hr after transfection with scrambled siRNA, siRNA 1 and siRNA 2. Scale bar = 200 μm. (e) Percentage of SA‐β‐gal‐positive cells in conditions presented in (d). Bars represent mean ± SD (n = 3). (f) GLB1 fold change gene expression in control and senescent SK‐Mel‐103 cells 48 hr post‐transfection with different siRNAs. (g) Quantification of cell viability upon 48 hr Navitoclax treatment of control and cisplatin‐induced senescent A549 cells (10 μM Navitoclax) (left) and control and palbociclib‐induced senescent SK‐Mel103 cells (7.5 μM Navitoclax) (right) previously transfected with different experimental siRNAs against GLB1 transcript. (h) Quantification of cell viability upon 48 hr Nav‐Gal treatment of control and cisplatin‐induced senescent A549 cells (10 μM Nav‐Gal) (left) and control and palbociclib‐induced senescent SK‐Mel103 cells (7.5 μM Nav‐Gal) (right) previously transfected with different experimental siRNAs against GLB1 transcript. Note that siRNA 1 was not functional in all the experiments and hence used as an internal negative control. All bars represent mean ± SEM (n = 3). One‐way ANOVA followed by Tukey's post‐tests were performed to calculate the significance of the results; *p < .05, **p < .01, ***p < .001

FIGURE 4

The galacto‐conjugated prodrug Nav‐Gal shows an enhanced effect when combined to senescence‐inducing cisplatin treatment and a lower induction of apoptosis of nonsenescent cells. (a) Representative images of cell viability showing staining for annexin V (red) of control or cisplatin‐induced senescent A549 cells, exposed to vehicle, Navitoclax (10 μM) or Nav‐Gal (10 μM) treatment over time. Red scale bar = 300 μm; black scale bar = 100 μm. (b) Average percentage of annexin V‐positive cells in control and cisplatin‐induced senescent A549 cells exposed to vehicle (top), Navitoclax (10 μM; middle) or Nav‐Gal (10 μM; bottom) treatment over time. Data represent mean ± SD (n = 3), where for each biological repeat the percentage of annexin V‐positive cells was calculated in 3 independent technical repeats per experimental condition. Statistical significance was calculated using two‐tailed Student's t tests; *p < .05, **p < .001. (c, d) Representative images of clonogenic survival of A549 cells exposed to increasing concomitant concentrations of cisplatin (CDDP) and (c) Navitoclax or (d) Nav‐Gal as specified in axis. (e, f) Numerical heat map representation of normalized mean clonogenic potential after 10 days of increasing concomitant treatment with CDDP and (e) Navitoclax or (f) Nav‐Gal, where 1 = maximum clonogenic potential corresponding to untreated condition (n = 3). (g) Co‐coefficient of drug interaction (CDI) value trend of Navitoclax and Nav‐Gal across 0.5 μM cisplatin treatment (left) and 1 μM cisplatin treatment (right). CDI < 1 (yellow area) indicates a synergistic effect, where CDI values closer to 0 correlate to higher synergy between the drugs concomitantly used. Data represent mean ± SD (n = 3)

FIGURE 5

Concomitant treatment with the prodrug Nav‐Gal and cisplatin significantly inhibits tumour growth in a human lung cancer xenograft mouse model. (a) Representative images of A549 xenografts stained for SA‐β‐Gal activity (in blue) after treatment with cisplatin or vehicle. (b) Schematic representation of concomitant treatment on A549 xenograft‐bearing mice. (c) Tumour volume of A549 xenografts in mice concomitantly treated with cisplatin and Navitoclax or Nav‐Gal (as described in (b); n ≥ 10 tumours per group. Data represent mean ± SEM. (d) Representative histological images of tumours at the end of concomitant treatment, stained for Ki67 and p21 expression, and labelled using TUNEL staining. Scale bar = 100 μm. (e) Percentage of Ki67‐ (top), p21‐ (middle) and TUNEL‐positive (bottom) cells in tumours from animals treated with vehicle, cisplatin, or cisplatin and Navitoclax or Nav‐Gal concomitantly (n ≥ 5 tumours per group). For quantification, a total of 4 fields per tumour was analyzed, covering most of the total tumour area. Two‐way ANOVA followed by Bonferroni post‐tests was performed to calculate the significance of the results; *p < .05; **p < .01; ***p < .001

FIGURE 6

Nav‐Gal reduces platelet apoptosis in human and mouse blood ex vivo, and prevents thrombocytopenia in mice treated concomitantly with chemotherapy, compared to Navitoclax. (a) Blood from healthy human volunteers was collected and treated ex vivo with 60, 20 and 6.66 μM Navitoclax or Nav‐Gal as described. Apoptosis was analyzed after annexin V‐FITC antibody incubation by flow cytometry. Graphs show proportion of apoptotic platelets upon each treatment based on scatter signals and annexin V expression signal after gating for CD41‐positive cells. (b) Percentage of annexin V‐positive platelets in human blood after treatment with 60, 20 and 6.66 μM Navitoclax or Nav‐Gal. Bars represent mean ± SD (n = 3). (c) Blood from wild‐type C57BL/6J mice was collected and treated ex vivo with 0.72, 0.24 and 0.08 μM Navitoclax or Nav‐Gal as described. Apoptosis was analyzed after annexin V‐FITC antibody incubation by flow cytometry. Graphs show proportion of apoptotic platelets upon each treatment based on scatter signals and annexin V expression signal after gating for CD41‐positive cells. (d) Percentage of annexin V‐positive platelets in mouse blood after treatment with 0.72, 0.24 and 0.08 μM Navitoclax or Nav‐Gal. Bars represent mean ± SD (n = 5). (e) Wild‐type C57BL/6J mice were treated daily with Navitoclax by oral gavage (o.g.) (100 mg/kg body weight), with Navitoclax administered by i.p. injection (85 mg/kg body weight) or with Nav‐Gal by o.g administration (85 mg/kg body weight) for 10 consecutive days. Blood was collected on day 0, 5 and 10 by superficial vessel puncture and platelet count was analyzed. (f) Platelet count on day 0, 5 and 10 during the treatment of wild‐type C57BL/6J mice in each experimental condition as described in (e) (vehicle, n = 4; Navitoclax o.g., n = 9; Navitoclax i.p., n = 8; Nav‐Gal o.g., n = 8). Bars represent mean ± SEM. (g) SCID mice bearing A549‐derived xenografts were treated with cisplatin (CDDP, 1.5 mg/kg body weight three times a week) and concomitant daily senotherapy (Navitoclax [100 mg/kg body weight] or Nav‐Gal [85 mg/kg body weight]) as shown in this schematic representation. Blood was collected after treatment by cardiac puncture and, platelet count was analyzed. (h) Platelet count in each experimental condition described in (g) upon end of treatment in vivo (vehicle and CDDP, n = 5; CDDP + Navitoclax, n = 4; CDDP + Nav‐Gal, n = 6). Data represent mean ± SEM. Two‐way ANOVA followed by Bonferroni post‐tests or one‐tailed t tests was performed to calculate the significance of the results; * p < .05; ***p < .001; ****p < .0001