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Galactose‐modified duocarmycin prodrugs as senolytics

duocarmycin prodrug senescence senescence‐associated β‐galactosidase senolytics

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#1 Engadin

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Posted 22 March 2020 - 11:47 PM


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S O U R C E :    Aging Cell

 

 

 

 

 

 

Abstract
 
Senescence is a stable growth arrest that impairs the replication of damaged, old or preneoplastic cells, therefore contributing to tissue homeostasis. Senescent cells accumulate during ageing and are associated with cancer, fibrosis and many age‐related pathologies. Recent evidence suggests that the selective elimination of senescent cells can be effective on the treatment of many of these senescence‐associated diseases. A universal characteristic of senescent cells is that they display elevated activity of the lysosomal β‐galactosidase, and this has been exploited as a marker for senescence (senescence‐associated β‐galactosidase activity). Consequently, we hypothesized that galactose‐modified cytotoxic prodrugs will be preferentially processed by senescent cells, resulting in their selective killing. Here, we show that different galactose‐modified duocarmycin (GMD) derivatives preferentially kill senescent cells. GMD prodrugs induce selective apoptosis of senescent cells in a lysosomal β‐galactosidase (GLB1)‐dependent manner. GMD prodrugs can eliminate a broad range of senescent cells in culture, and treatment with a GMD prodrug enhances the elimination of bystander senescent cells that accumulate upon whole‐body irradiation treatment of mice. Moreover, taking advantage of a mouse model of adamantinomatous craniopharyngioma (ACP), we show that treatment with a GMD prodrug selectively reduced the number of β‐catenin‐positive preneoplastic senescent cells. In summary, the above results make a case for testing the potential of galactose‐modified duocarmycin prodrugs to treat senescence‐related pathologies.
 
 
1 INTRODUCTION
 
Cellular senescence is a stress response that prevents the replication of old, damaged or transformed cells (Herranz & Gil, 2018). Senescence can be induced by replicative exhaustion and also by a range of insults that includes oncogenic activation, genotoxic stress or irradiation. The defining feature of senescence is a stable cell cycle arrest, but senescent cells also undergo multiple phenotypic changes including alterations in their morphology, metabolic state or chromatin arrangement (Salama, Sadaie, Hoare, & Narita, 2014). In particular, senescent cells secrete a combination of extracellular factors, the so‐called senescence‐associated secretory phenotype or SASP, which is a prominent mediator of the patho‐physiological effects of senescence (Acosta et al., 2008, 2013; Coppe, Desprez, Krtolica, & Campisi, 2010; Kuilman & Peeper, 2009).
 
Despite that the acute induction of senescence limits fibrosis and protects against cancer progression, the abnormal accumulation of senescent cells with age or in diseased tissues is detrimental (Munoz‐Espin & Serrano, 2014). Interestingly, evidence drawn from genetic models has shown that eliminating senescent cells increases lifespan, improves healthspan and benefits the outcomes of a wide range of diseases (Baker et al., 2011, 2016; Childs et al., 2016, 2017). These studies have led to a collective effort to identify “senolytics,” drugs that selectively kill senescent cells. Several senolytics have been identified including dasatinib and quercetin (Zhu et al., 2015), piperlongumine (Wang et al., 2016), FOXO4‐interfering peptides (Baar et al., 2017), HSP90 inhibitors (Fuhrmann‐Stroissnigg et al., 2017), cardiac glycosides (Guerrero et al., 2019; Triana‐Martinez et al., 2019) or the Bcl2 family inhibitors ABT‐263 (navitoclax) and ABT‐737 (Chen et al., 2015; Yosef et al., 2016; Zhu et al., 2016). Currently, Bcl2 family inhibitors have become the gold standard on senolysis. Bcl2 family inhibitors eliminate a range of senescent cells in vivo and reproduce the effects observed in transgenic mice modelling senescence ablation (Ovadya & Krizhanovsky, 2018). However, ABT‐263 causes severe thrombocytopenia and neutropenia, what might complicate its use on the clinic. Moreover, it is becoming evident that specific senolytics might be necessary to eliminate different types of senescent cells. Therefore, there is a need to identify additional drugs with senolytic properties.
 
An alternative strategy for targeting senescence is to exploit properties that differentiate senescent from normal cells. In this regard, the senescence‐associated β‐galactosidase activity (SA‐β‐gal) is one of the more conserved and defining characteristics of senescent cells. Senescent cells present an increased lysosomal mass (Kurz, Decary, Hong, & Erusalimsky, 2000). As a result, senescent cells display elevated levels of lysosomal enzymes such as β‐galactosidase (encoded by GLB1 (Dimri et al., 1995)) or α‐fucosidases (Hildebrand et al., 2013). Indeed, it has been shown that galacto‐oligosaccharide encapsulated nanoparticles (GalNP) preferentially release their content on senescent cells (Agostini et al., 2012). Consequently, this GalNP can be used in combination with different cargos to either image or kill senescent cells (Munoz‐Espin et al., 2018).
 
Galactose modification has been frequently used to improve the pharmacokinetic properties or the delivery of existing drugs. In addition, galactose modification can be used to generate prodrugs that rely on E. coli β‐galactosidase for controlled activation (Melisi, Curcio, Luongo, Morelli, & Rimoli, 2011). When combined with antibody‐linked β‐galactosidase, this approach is known as antibody‐directed enzyme prodrug therapy (ADEPT) (Bagshawe, 2006; Tietze & Schmuck, 2011). In ADEPT, a conjugate of a tumour‐specific antibody and an enzyme, such as β‐galactosidase, is combined with the application of a hardly cytotoxic prodrug. By means of the enzyme in the conjugate, the prodrug is selectively cleaved in cancer cells leading to the formation of a highly cytotoxic compound. Several of these galactose‐modified cytotoxic prodrugs have been described (Leenders et al., 1999). A class of such prodrugs are galactose‐modified duocarmycin (GMD) derivatives (Tietze, Major, & Schuberth, 2006). Duocarmycins are a group of antineoplastic agents with low picomolar potency. They are thought to act by binding and alkylating double‐stranded DNA in AT‐rich regions of the minor groove (Boger, Johnson, & Yun, 1994; Tietze et al., 2006; Tietze, Schuster, Krewer, & Schuberth, 2009), but alternative mechanisms of action have been proposed to account for the cytotoxic effects of duocarmycin dimers (Wirth, Schmuck, Tietze, & Sieber, 2012).
 
Here, we investigated whether galactose‐modified prodrugs can preferentially kill senescent cells. We have assessed several GMD derivatives and confirmed their senolytic potential in cell culture, ex vivo and in vivo. Given the increasing list of senescence‐associated diseases and the benefits of senolytic treatment, we propose that GMD derivatives and, more generally, galactose‐modified prodrugs are a new class of senolytic compounds and they should be tested to assess their therapeutic potential.
 
 
2 RESULTS
 
2.1 A galactose‐modified duocarmycin prodrug with senolytic properties
 
The natural antibiotic duocarmycin is a highly cytostatic compound (Boger & Johnson, 1995). A series of glycosidic derivatives of duocarmycin have been previously developed to be used as prodrugs in the context of antibody‐directed enzyme prodrug therapy (ADEPT) (Tietze, Hof, Muller, Krewer, & Schuberth, 2010; Tietze et al., 2009). Given that senescent cells display elevated levels of SA‐β‐galactosidase activity, we hypothesized that galactose‐modified cytotoxic prodrugs will be preferentially processed by senescent cells, resulting in their selective killing. To test this hypothesis, we took advantage of a galactose‐modified duocarmycin (GMD) prodrug (referred as prodrug A, JHB75B) previously described (Tietze et al., 2009). We analysed the effects that a seco‐duocarmycin analogue dimer (duocarmycin SA) and its galactose derivative (prodrug A) had on the survival of IMR90 ER:RAS cells, a model of oncogene‐induced senescence (OIS). Activation of the ER:RAS fusion with 4‐hydroxy‐tamoxifen (4OHT) induces senescence in IMR90 ER:RAS cells (Georgilis et al., 2018). Treatment with duocarmycin SA was equally effective in killing normal and senescent cells, with the exception of a small selectivity towards senescent cells at the lower concentrations (Figure 1a). In contrast, when we treated IMR90 ER:RAS cells with prodrug A (differing only in the addition of two galactose groups that inactivate it), we observed the preferential elimination of senescent cells (Figure 1b and Figure S1a). Duocarmycins are known to bind and alkylate DNA in AT‐rich regions of the minor groove and induce cell death in a way dependent of DNA replication (Boger et al., 1994; Tietze et al., 2006, 2009) We checked that senescent cells were growth arrested at the time of the drug treatment (Figure S1b). This shows that the effect observed is not due to hyperreplication of cells during early stages of OIS and suggests that the prodrug might act by some of the alternative cytotoxic mechanisms described for duocarmycin dimers (Wirth et al., 2012). Treatment with prodrug A induced caspase 3/7 activity on senescent cells (Figure 1c), and the selective death of these cells was prevented with a pan‐caspase inhibitor (Figure 1d). The above results suggest that GMD prodrugs can behave as senolytics by selectively inducing apoptosis on senescent cells.
 
 
acel13133-fig-0001-m.jpg
 
 
 
Figure 1.  A galactose‐modified duocarmycin prodrug with senolytic properties. (a) Molecular structure of seco‐duocarmycin analog dimer (JHB71A, left). IMR90 ER:RAS cells were treated with DMSO or with 4OHT (4‐hydroxy‐tamoxifen) for 6 days to induce OIS. Cells were treated with the indicated concentrations of seco‐duocarmycin analog dimer for 72 hr. Cell numbers were quantified using DAPI staining, and percentage of survival cells are plotted (right) (n = 4). (b) Molecular structure of a galactose‐modified prodrug derivative of seco‐duocarmycin analog dimer (JHB75B, referred as prodrug A, left). Cells were treated with prodrug A for 72 hr as described before (n = 4). © Treatment of senescent cells with a GMD prodrug triggers caspase‐3/7 activity. IMR90 ER:RAS were treated with 4OHT or vehicle (DMSO) for 6 days to induce senescence. 2.5 μM prodrug was then added together with NucLight Rapid Red Reagent for cell labelling and Caspase‐3/7 reagent for apoptosis (IncuCyte). Caspase 3/7 activity was measured at 4‐hr intervals. (d) After 6‐day treatment with 4OHT or vehicle (DMSO), IMR90 ER:RAS were treated with 1 μM ABT‐263 or 2.5 μM prodrug A for 72 hr in the presence or absence of the pan‐caspase inhibitor Q‐VD‐OPh (n = 4). All statistical significances were calculated using unpaired Student's t tests. All error bars represent mean ± s.d; n represents independent experiments.; ns, not significant; *p < .05; **p < .01; ***p < .001, ****p < .0001
 
 
 
2.2 Senolytic properties of prodrug A depend on the lysosomal β‐galactosidase
 
We had hypothesized that GMD prodrugs could behave as senolytics due to the higher SA‐β‐galactosidase activity of senescent cells. To investigate whether the levels of β‐galactosidase activity correlate with the sensitivity of senescent cells to GMD prodrugs, we induced senescence in IMR90 ER:RAS cells. Afterwards, we treated control or senescent cells with 2.5 µM prodrug A and used a fluorescent substrate (DDAO) to quantify SA‐β‐galactosidase activity at single‐cell resolution (Figure 2a). IMR90 ER:RAS cells undergoing OIS had more cells with higher SA‐β‐galactosidase levels, as it was evident when analysing cell intensities (Figure 2b), or when using threshold system to quantify the percentage of SA‐β‐galactosidase‐positive cells (Figure 2c) or divide the cells in negative, high and highest for SA‐β‐galactosidase (Figure S2). While treatment with 2.5 µM prodrug A selective killed the majority of the senescent cells (Figure S1a), it was interesting to observe that the senescent cells that survived display lower levels of SA‐β‐galactosidase activity (Figure 2a–c and Figure S2), linking the senolytic selectivity of prodrug A with the SA‐β‐galactosidase activity.
 
 
acel13133-fig-0002-m.jpg
 
 
 
Figure 2.  Galactose‐modified duocarmycin prodrugs preferentially target senescent cells with high SA‐β‐galactosidase activity. (a) Representative pictures of fluorescent SA‐β‐galactosidase staining in IMR90 ER:RAS cells treated with prodrug A or vehicle. Scale bar, 100 µm. (b) Single‐cell intensities value for DDAO galactoside in a representative well of a 96‐well plate seeded with IMR90 ER:RAS cells treated with DMSO or 4OHT. Grey dotted line indicates quantification cut‐off. Cells were considered positives for SA‐β‐gal when their cell intensity was > 165. © Quantification of SA‐β‐galactosidase activity in IMR90 ER:RAS cells treated with prodrug A or vehicle. Statistical significance was calculated using two‐way ANOVA. (d‐g) The senolytic properties of prodrug A depend on the lysosomal β‐galactosidase (GLB1). (d) Experimental set‐up. (e) Representative pictures of cytochemical SA‐β‐Gal staining in IMR90 ER:RAS infected with different shRNAs against GLB1 or an empty vector and (f) quantification (n = 3). Statistical significance was calculated using one‐way ANOVA. (g) Quantification of cell survival of senescent and control IMR90 ER:RAS infected with different shRNAs targeting GLB1 or an empty vector and treated with ABT‐263 or prodrug A for 3 days (n = 3). Statistical significance was calculated using two‐tailed, Student's t test. All error bars represent mean ± SD; n represents independent experiments; ns, not significant; *p < .05; **p < .01; ***p < .001, ****p < .0001
 
 
 
The increase in β‐galactosidase observed on senescent cells is due to an increase in lysosomal mass (Kurz et al., 2000) resulting in higher activity of the lysosomal β‐galactosidase (encoded by GLB1) (Lee et al., 2006). To further prove that the senolytic activity of GMD prodrugs is dependent on SA‐β‐galactosidase, we took advantage of three independent shRNAs to knock down GLB1 (Figure 2d). Knock‐down of GLB1 in IMR90 ER:RAS cells resulted in decreased SA‐β‐galactosidase activity, but it did not impact the growth arrest or the induction of p16INK4a observed during OIS (Figure 2e,f and Figure S3a,b). Taking advantage of these cells, we observed that GLB1 knock‐down did not affect the senolytic potential of ABT‐263 but ablated the ability of prodrug A to selectively kill senescent cells (Figure 2g and Figure S3c). In summary, our data suggest that GMD prodrugs trigger apoptosis of senescent cells in a GLB1‐dependent manner.
 
 
2.3 Galactose‐modified duocarmycin prodrugs are broad‐spectrum senolytics
 
To understand the extent to which GMD prodrugs behave as senolytics, we assessed the effect that prodrug A has on several types of senescent cells. To this end, we took advantage of IMR90 cells and induced senescence by etoposide or doxorubicin treatment, irradiation, or serial passage (Figure S4a–d). In all those instances, treatment with prodrug A resulted in the selective elimination of senescent cells (Figure 3a–d). Moreover, to evaluate whether the senolytic effects of prodrug A were restricted to IMR90 cells or also observed in other cell types, we took advantage of human mammary epithelial cells able to undergo OIS upon Ras activation (HMEC ER:RAS, Figure S4e). Prodrug A was also able to selectively kill HMEC senescent cells (Figure 3e), suggesting that its senolytic effects were not cell type restricted. Finally, we wanted to examine whether the senolytic properties were specific of prodrug A, or the general concept (conversion of other cytotoxic drugs in galactose‐modified prodrugs) was wider. To this end, we took advantage of two previously described GMD prodrugs, JHB76B and JHB35B (Tietze et al., 2009, 2010). Both drugs were also effective in selectively eliminating senescent cells (Figure 3f and Figure S5), suggesting that the generation of galactose‐modified prodrugs might be a general route to design senolytic compounds.
 
 
acel13133-fig-0003-m.jpg
 
 
 
Figure 3.  Galactose‐modified duocarmycin prodrugs are broad‐spectrum senolytics. (a–d) Quantification of cell survival after treatment with prodrug A in IMR90 undergoing different types of senescence. Senescence was induced by treatment with 50 μM etoposide (a, n = 4), 0.5 μM doxorubicin (b, n = 4) or 7.5 Gy irradiation (c, n = 3). In (d), the effect of prodrug A on replicative senescence of IMR90 cells (passage 12 versus passage 22) (n = 4) was assessed. (e) Quantification of cell survival after treatment with prodrug A in HMEC ER:RAS, human mammary epithelial cells expressing hTERT that undergo senescence upon activation of ER:RAS by 4OHT treatment (n = 3). (f) Quantification of cell survival after treatment with two other galactose‐modified duocarmycin derivatives, JHB76B and JHB35B, in the context of oncogene‐induced senescence in IMR90 ER:RAS (n = 4). An extended version of this figure including additional drug concentrations is shown in Figure S2. All statistical significances were calculated using unpaired Student's t tests. All error bars represent mean ± SD; n represents independent experiments; ns, not significant; *p < .05; **p < .01; ***p < .001
 
 
 
2.4 Prodrug A exerts a bystander effect
 
The above experiments suggest that senescent cells preferentially convert GMD prodrugs into their active duocarmycin derivatives. Since duocarmycins have strong cytostatic properties (Boger & Johnson, 1995), we wonder whether the conversion of GMD prodrugs into their active compounds could result in the bystander killing of normal cells. To assess this, we took advantage of cocultures of normal and senescent cells expressing different fluorescent proteins (Figure 4a). We then treated the cocultures with prodrug A and observed that while lower concentrations killed a subset of senescent cells without affecting to the normal cells, at higher concentrations, a subset of the normal cells also died (Figure 4b,c). Interestingly, a higher percentage of senescent cells survived in cocultures (Figure 4c) that in monocultures (Figure 1b) treated with 5 µM prodrug A, further suggesting the existence of a bystander effect.
 
 
acel13133-fig-0004-m.jpg
 
 
 
Figure 4.  Bystander effect caused by prodrug A in cocultures of normal and senescent cells. (a) Experimental set‐up. (b) Representative pictures of the co‐culture experiment of IMR90 ER:RAS (GFP positive) with wild‐type IMR90 (RFP positive) cells. © Quantification of cell survival for each population of the co‐culture experiment after treatment with different concentrations of prodrug A or vehicle (n = 3). Statistical significance was calculated using unpaired Student's t tests. All error bars represent mean ± s.d; n represents independent experiments; ns, not significant; *p < .05; **p < .01; ***p < .001
 
 
 
2.5 Prodrug A eliminates senescent cells in vivo
 
Chemotherapy and radiotherapy are amongst the most common anticancer treatments. Irradiation, chemotherapy and even some targeted anticancer drugs, all induce senescence (Schmitt et al., 2002; Wang et al., 2017). Although induction of tumour senescence explains the anticancer properties of these treatments, the generation of bystander senescent cells is responsible for their side effects (Demaria et al., 2017). To assess whether prodrug A could eliminate these senescent cells, we first irradiated mice and upon a latency period to allow for the accumulation of senescent cells, treated them with prodrug A, ABT263 or vehicle (Figure 5a). Treatment with prodrug A or ABT‐263 resulted in a reduced presence of senescent cells in lung as assessed using SA‐β‐galactosidase activity (Figure 5b,c). Furthermore, a similar trend was observed when we assessed the expression of Cdkn1a (that encodes for p21Cip1) or the SASP components Il6 and Cxcl1 (Figure 5d).
 
 
acel13133-fig-0005-m.jpg
 
Figure 5.  Prodrug A reduces the numbers of senescent cells accumulating after whole‐body irradiation. (a) Experimental design of the whole‐body irradiation‐induced senescence experiment. Mice (n = 4/5 per group) were irradiated with 6 Gray to induce senescence. Two months later, mice were treated with vehicle, prodrug A (JHB75B) or ABT‐263 for 4 consecutive days, before being culled for analysis. (b, c) Representative pictures of lung cryosections (b) and quantification of the lung area positive for SA‐β‐Gal staining ©. (d) Expression levels of Cdkn1a, Il6 and Cxcl1 in lungs of nonirradiated mice or irradiated mice treated with prodrug A, ABT‐263 or vehicle. Statistical significance was calculated using unpaired Student's t test. Data represent mean ± SD; n represents number of mice; ns, not significant; *p < .05; **p < .01

 

 

 

2.6 Galactose‐modified prodrugs eliminate preneoplastic senescent cells

 
OIS is primarily considered as a tumour suppressive mechanism (Collado et al., 2005), but senescent cells present in the tumour microenvironment can also drive tumour progression (Gonzalez‐Meljem, Apps, Fraser, & Martinez‐Barbera, 2018). We have previously demonstrated in mouse models of adamantinomatous craniopharyngioma (ACP), a pituitary paediatric tumour, that clusters of cells that accumulate nucleo‐cytoplasmic β‐catenin are senescent and drive tumour progression in a paracrine manner (Gonzalez‐Meljem et al., 2017). Indeed, β‐catenin‐positive cells are ki67 negative, express p21Cip1 and high levels of the lysosomal β‐galactosidase GLB1 (Figure S6a) (Gonzalez‐Meljem et al., 2017). To understand if GMD prodrugs could eliminate these pro‐tumourigenic senescent clusters, we used the Hesx1Cre/+;Ctnnb1lox(ex3)/+ ACP mouse model. We have used this system before to assess the senolytic properties of cardiac glycosides (Guerrero et al., 2019). Tumoural cluster‐containing embryonic pituitaries were cultured ex vivo with vehicle or prodrug A (Figure 6a). Treatment with prodrug A preferentially eliminated the β‐catenin‐accumulating senescent cell clusters, without affecting other cell types in the pituitary such as synaptophysin + cells (Figure 6b–d). Co‐staining with an antibody recognizing cleaved caspase 3 showed that prodrug A predominantly induced apoptosis of senescent cluster cells (Figure 6e and Figure S6b). The above results suggest that GMD prodrugs could be also used to eliminate preneoplastic senescent cells.
 
 
acel13133-fig-0006-m.jpg
 
 
 
Figure 6.  Galactose‐modified duocarmycin prodrug eliminates preneoplastic senescent lesions. (a) Experimental design for the senolytic experiment in the Hesx1Cre/+;Ctnnb1lox(ex3)/+ mouse model of adamantinomatous craniopharyngioma (ACP). Tumoural pituitaries from 18.5dpc Hesx1Cre/+;Ctnnb1lox(ex3)/+ embryos were cultured in the presence of prodrug A at the indicated concentrations or vehicle (DMSO) and processed for analysis after 72 hr. (b) Immunofluorescence staining against β‐catenin (green) and synaptophysin (red) is shown. Synaptophysin is a marker of the normal hormone‐producing cells in the pituitary gland. Scale bar, 50μm. © Quantification of β‐catenin‐accumulating cells after treatment with different concentrations of prodrug A or vehicle (n = 6–12). (d) Quantification of synaptophysin‐positive cells after treatment with different concentrations of prodrug A or vehicle (n = 6–12). (e) Quantification of β‐catenin‐accumulating cells positive for cleaved caspase‐3 after treatment with different concentrations of prodrug A or vehicle (n = 6–12). All statistical significances were calculated using nonparametric ANOVA with Dunn's post hoc comparison. All error bars represent mean ± SD; n represents number of pituitaries; ns, not significant; *p < .05; **p < .01; ***p < .001
 
 
 
 
 
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Also tagged with one or more of these keywords: duocarmycin, prodrug, senescence, senescence‐associated β‐galactosidase, senolytics

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