Pharmacological CDK4/6 inhibition reveals a p53‐dependent senescent state with restricted toxicity
2022; Springer Nature; Volume: 41; Issue: 6 Linguagem: Inglês
10.15252/embj.2021108946
ISSN1460-2075
AutoresBoshi Wang, Marta Varela-Eirín, Simone Brandenburg, Alejandra Hernandez‐Segura, Thijmen van Vliet, Elisabeth M. Jongbloed, Saskia M. Wilting, Naoko Ohtani, Agnes Jager, Marco Demaria,
Tópico(s)Cancer-related cognitive impairment studies
ResumoArticle5 January 2022Open Access Transparent process Pharmacological CDK4/6 inhibition reveals a p53-dependent senescent state with restricted toxicity Boshi Wang Boshi Wang orcid.org/0000-0002-9257-6714 European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen (UMCG), Groningen, The Netherlands Search for more papers by this author Marta Varela-Eirin Marta Varela-Eirin orcid.org/0000-0002-0281-0678 European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen (UMCG), Groningen, The Netherlands Search for more papers by this author Simone M Brandenburg Simone M Brandenburg orcid.org/0000-0001-6144-6194 European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen (UMCG), Groningen, The Netherlands Search for more papers by this author Alejandra Hernandez-Segura Alejandra Hernandez-Segura European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen (UMCG), Groningen, The Netherlands Search for more papers by this author Thijmen van Vliet Thijmen van Vliet European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen (UMCG), Groningen, The Netherlands Search for more papers by this author Elisabeth M Jongbloed Elisabeth M Jongbloed Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Saskia M Wilting Saskia M Wilting Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Naoko Ohtani Naoko Ohtani orcid.org/0000-0001-8934-0797 Graduate School of Medicine, Osaka City University, Osaka, Japan Search for more papers by this author Agnes Jager Agnes Jager Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Marco Demaria Corresponding Author Marco Demaria m.demaria@umcg.nl orcid.org/0000-0002-8429-4813 European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen (UMCG), Groningen, The Netherlands Search for more papers by this author Boshi Wang Boshi Wang orcid.org/0000-0002-9257-6714 European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen (UMCG), Groningen, The Netherlands Search for more papers by this author Marta Varela-Eirin Marta Varela-Eirin orcid.org/0000-0002-0281-0678 European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen (UMCG), Groningen, The Netherlands Search for more papers by this author Simone M Brandenburg Simone M Brandenburg orcid.org/0000-0001-6144-6194 European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen (UMCG), Groningen, The Netherlands Search for more papers by this author Alejandra Hernandez-Segura Alejandra Hernandez-Segura European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen (UMCG), Groningen, The Netherlands Search for more papers by this author Thijmen van Vliet Thijmen van Vliet European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen (UMCG), Groningen, The Netherlands Search for more papers by this author Elisabeth M Jongbloed Elisabeth M Jongbloed Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Saskia M Wilting Saskia M Wilting Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Naoko Ohtani Naoko Ohtani orcid.org/0000-0001-8934-0797 Graduate School of Medicine, Osaka City University, Osaka, Japan Search for more papers by this author Agnes Jager Agnes Jager Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Marco Demaria Corresponding Author Marco Demaria m.demaria@umcg.nl orcid.org/0000-0002-8429-4813 European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen (UMCG), Groningen, The Netherlands Search for more papers by this author Author Information Boshi Wang1, Marta Varela-Eirin1, Simone M Brandenburg1, Alejandra Hernandez-Segura1, Thijmen van Vliet1, Elisabeth M Jongbloed2, Saskia M Wilting2, Naoko Ohtani3, Agnes Jager2 and Marco Demaria *,1 1European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen (UMCG), Groningen, The Netherlands 2Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands 3Graduate School of Medicine, Osaka City University, Osaka, Japan *Corresponding author. Tel: +31 06 52724857; E-mail: m.demaria@umcg.nl The EMBO Journal (2022)41:e108946https://doi.org/10.15252/embj.2021108946 See also: AR Barr & SE McClelland (March 2022) AbstractSynopsis Introduction Discussion Materials and Methods Data availability Acknowledgments Author contributions Disclosure statement and competing interestsSupporting InformationReferencesPDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. Metrics1MetricsTotal downloads6,734Last 6 Months1,504Total citations1Last 6 Months0View all metrics ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cellular senescence is a state of stable growth arrest and a desired outcome of tumor suppressive interventions. Treatment with many anti-cancer drugs can cause premature senescence of non-malignant cells. These therapy-induced senescent cells can have pro-tumorigenic and pro-disease functions via activation of an inflammatory secretory phenotype (SASP). Inhibitors of cyclin-dependent kinases 4/6 (CDK4/6i) have recently proven to restrain tumor growth by activating a senescence-like program in cancer cells. However, the physiological consequence of exposing the whole organism to pharmacological CDK4/6i remains poorly characterized. Here, we show that exposure to CDK4/6i induces non-malignant cells to enter a premature state of senescence dependent on p53. We observe in mice and breast cancer patients that the CDK4/6i-induced senescent program activates only a partial SASP enriched in p53 targets but lacking pro-inflammatory and NF-κB-driven components. We find that CDK4/6i-induced senescent cells do not acquire pro-tumorigenic and detrimental properties but retain the ability to promote paracrine senescence and undergo clearance. Our results demonstrate that SASP composition is exquisitely stress-dependent and a predictor for the biological functions of different senescence subsets. Synopsis Pharmacological CDK4/6 inhibitors lead to premature senescence in culture and in vivo. CDK4/6 inhibitors-induced senescence is p53-dependent and not associated with the detrimental pro-inflammatory secretory phenotype, SASP. Exposure to pharmacological CDK4/6 inhibitors (CDK4/6i) causes premature senescence. CDK4/6i-induced senescence is dependent on p53. CDK4/6i-induced senescence is well tolerated in vivo. CDK4/6i-induced senescent cells exhibit a p53-associated secretory phenotype (PASP) but lack the NF-kB-associated secretory phenotype (NASP). Introduction Unrestrained proliferation is one of the common hallmarks of cancer cells and a major target for anti-cancer interventions (Hanahan & Weinberg, 2011). Genotoxic therapies, such as chemo- and radiotherapy, cause high level of DNA damage in quickly proliferating cells and often achieve tumor suppression by leading cells into senescence—a stable form of growth arrest dependent on the upregulation of cyclin-dependent kinase (CDK) inhibitors p16 and p21 (Rodier & Campisi, 2011). A major advantage of genotoxic interventions is that they are not biased to specific molecular marks or cell types, and are effective against a broad range of tumor specimens. However, this lack of specificity and the use of systemic administration are also the basis of several short- and long-term adverse reactions, which arise from inflicting unrepairable DNA damage to non-malignant cells. In recent years, several studies have suggested that a major mechanism leading to chemotoxicity is the premature and excessive induction of non-malignant cells into senescence (Sun et al, 2012; Baar et al, 2017; Demaria et al, 2017; Murali et al, 2018; Yao et al, 2020). Genetic and pharmacological removal of non-malignant therapy-induced senescent (TIS) cells is sufficient to alleviate various therapy-associated adverse reactions including fatigue, myelosuppression, cardiomyopathy, bone loss, frailty, cancer progression, and relapse (Sun et al, 2012; Baar et al, 2017; Demaria et al, 2017; Murali et al, 2018; Yao et al, 2020). The detrimental effects associated with TIS cells are mainly mediated by the secretion of various pro-inflammatory cytokines and chemokine part of the complex senescence-associated secretory phenotype (SASP; Hernandez-Segura et al, 2018b). In accordance, pro-inflammatory SASP factors are elevated in cancer patients suffering from a variety of adverse reactions to cancer therapies (Pierce et al, 2009; Ferrucci & Fabbri, 2018). Major regulator of pro-inflammatory SASP genes is NF-κB, a transcription factor mediating the response to persistent DNA damage (Rodier et al, 2009; Chien et al, 2011). However, the SASP is highly heterogeneous and regulated by additional signaling pathways, including cEBPs, NOTCH, mTOR, and SIRT1, suggesting the existence of various modules that are activated in a context-dependent fashion (Hernandez-Segura et al, 2017, 2018b). For example, cells overexpressing p16 or p53 enter a state of senescence without NF-κB signaling and production/secretion of pro-inflammatory and NF-κB-dependent SASP factors (Efeyan et al, 2007; Coppé et al, 2011; Wiley et al, 2018). Another strategy to reduce tumor growth is the use of inhibitors targeting specific mechanisms that are aberrantly used by cancer cells to fuel their unrestrained proliferation. During cell cycle progression, D-type cyclins bind to the CDK4/6, phosphorylate and inhibit the retinoblastoma (RB) tumor suppressor proteins, and derepress the activity of E2F transcription factors to allow G1-S transition. p16, encoded by the CDKN2A gene, binds to CDK4/6 and inhibits cyclin–CDK complexes. As virtually all human tumor cells carry aberrations throughout the cyclin–CDK4/6–RB–p16 axis to support their hyperproliferative state, selective CDK4/6 inhibitors (CDK4/6i) have been developed in recent years (Klein et al, 2018). The anti-cancer effects of CDK4/6i are mainly driven by the induction of cancer cells into senescence (Yoshida et al, 2016; Goel et al, 2017). CDK4/6i-treated cancer cells not only stop dividing but also activate a potent tumor immunosurveillance (Goel et al, 2017). Palbociclib (PD033291), abemaciclib (LY2835219), and ribociclib (LEE011) are approved for the treatment of hormone-sensitive and HER2-negative advanced breast cancer patients, where they have been shown to improve progression-free survival in largely metastasized breast cancer patient cohorts (Finn et al, 2016; Goetz et al, 2017). Moreover, CDK4/6i are currently investigated for treatment of different liquid and solid tumors, including as first-line treatment for lung cancer patients (Patnaik et al, 2016). Systemic administration of CDK4/6i in humans has been reported to be better tolerated than genotoxic interventions (Klein et al, 2018). In particular, asthenia (severe fatigue) represents an important obstacle for the continuous treatment with genotoxic chemotherapy but is a rare event in patients treated with CDK4/6i (Ingham & Schwartz, 2017). Recent evidences have suggested that, similar to what observed for cancer cells, prolonged treatment with CDK4/6i can promote the premature senescence of non-malignant cells (Guan et al, 2017; Hari et al, 2019). However, the phenotype of non-malignant CDK4/6i-induced senescent cells, a validation of their induction in vivo, and their contribution to potential therapy-induced adverse reactions remain largely unknown. Here, using human and mouse cells, mice, and human biopsies, we aim at defining CDK4/6i-induced senescence and its functional role. CDK4/6i treatment induces a state of cellular senescence dependent on p53 activity In accordance with previous studies (Yang et al, 2017), prolonged treatment of human primary fibroblasts (BJ) with the CDK4/6i abemaciclib was associated with a progressive loss of RB phosphorylation (Fig EV1A), downregulation of E2F2 (Fig EV1B), and increased p16 gene expression (Fig EV1C). Reduced population doubling (Fig 1A) and EdU incorporation (Fig 1B) at the end of treatment demonstrated the efficacy of abemaciclib to induce cell cycle arrest. A similar proliferative arrest was observed in human primary fibroblasts WI38 treated with abemaciclib or another CDK4/6i, palbociclib (Fig EV1D). Importantly, drug withdrawal was not sufficient to restore proliferation of CDK4/6i-treated cells (Figs 1B and EV1E and F), particularly after an 8-day treatment at 1 μM (Fig EV1G–I). CDK4/6i-treated cells assumed a flattened and enlarged morphology (Fig EV1J), activated the senescence-associated-β-galactosidase (SA-β-gal) (Fig EV1K), and maintained high level of the endogenous CDK4/6i p16 (Fig EV1L), all features that can also be observed in cells treated with other anti-cancer and senescence-inducing agents such as the genotoxic drug doxorubicin (Demaria et al, 2017). RNA-sequencing analysis of cells 8 days post-treatment revealed that downregulation of cell cycle genes (Fig EV1M), upregulation of lysosomal-associated pathways (Table EV1), and activation of a "core" transcriptional signature of senescence previously identified in our laboratory (Hernandez-Segura et al, 2017) (Fig EV1N) were observed in both abemaciclib- and doxorubicin-treated cells. Interestingly, RNA-sequencing data suggested that abemaciclib-treated BJ cells differentially expressed several p53 transcriptional targets related to cell cycle (Spurgers et al, 2006; Fischer, 2017; Fig EV1O). In accordance, we observed an increased nuclear localization of p53 in abemaciclib-treated BJ cells (Fig EV1P), while ChIP analyses revealed that abemaciclib enhanced p53 binding to the promoter regions of its target genes CDKN1A (p21), MDM2, and GADD45A, which was similar to what observed in cells treated with doxorubicin or nutlin-3a (Fig 1C). In addition, BJ cells with stable expression of a luciferase reporter construct (p53LUC) confirmed gradual activation of p53 transcriptional activity upon exposure to abemaciclib, similar to what was observed in cells treated with the Mdm2 inhibitor nutlin-3a (Fig 1D). To evaluate whether p53 was essential to orchestrate the irreversible cell cycle arrest in response to CDK4/6i, we analyzed various models with impaired p53 expression or activity. BJ cells transduced with a genetic suppressor element (GSE) against p53 (Mittelman & Gudkov, 1999) or with shRNA lentiviral particles (Fig EV2A) assumed enlarged morphology (Fig EV2B) and activated a temporary arrest (Fig EV2C) during abemaciclib or palbociclib treatment, comparable to what observed in wild-type cells. However, in the absence of p53, cells were able to restore proliferation after drug removal, as measured by colony formation assays (Figs 1E and EV2D) and EdU incorporation (Fig 1F). Similarly, mouse dermal fibroblasts (MDFs) derived from p53 knockout mice (Fig 1 G and H) and wild-type mouse embryonic fibroblasts (MEFs) with inactive p53 (Fig EV2E) also failed to enter irreversible growth arrest upon exposure to abemaciclib. The bypass of irreversible growth arrest in p53-deficient BJ cells was associated with a failure in modulating the expression of several p53 transcriptional targets related to cell cycle upon treatment with abemaciclib (Fig 1I). Finally, to further evaluate the dependence of CDK4/6-induced irreversible growth arrest on p53 transcriptional activity, we exposed BJ cells to pifithrin-α (PFT-α), a reversible inhibitor of p53-dependent transcription (Sohn et al, 2009). The concomitant treatment abemaciclib/PFT-α allowed a substantial number of BJ cells to bypass the irreversible growth arrest (Fig EV2F). Taken together, these data suggest that CDK4/6i induce non-malignant cells to a state of a senescence-associated permanent growth arrest dependent on p53 transcriptional activity. Click here to expand this figure. Figure EV1. Prolonged CDK4/6i treatment induces p53-dependent cellular senescence A–C. Human fibroblasts (BJ) were treated with vehicle (water for 8 times in 24 h) or abemaciclib (1 μM for 1 or 8 times in 24 h). Protein was isolated from vehicle or abemaciclib-treated cells and immunoblotted for p-Rb, Rb, and actin (A). RNA was isolated from treated cells, and mRNA levels of E2F2 gene (B) and p16 gene (C) were quantified by qPCR relative to tubulin (internal control). n = 3 independent experiments. D–F. 8-day population doubling of WI38 cells treated with vehicle, palbociclib, or abemaciclib (both 1 μM for 8 times in 24 h; n = 3 independent experiments) (D). At 8 dpt, treated WI38 cells were incubated with EdU for 10 h and stained (scale bar, 150 μm; n = 6 samples from 3 independent experiments) (E). 3 × 103 treated WI38 cells were replated in 6-well dish, cultured for 8 days, and stained with 0.2% crystal violet (n = 3 independent experiments) (F). G, H. Cells were treated with vehicle or abemaciclib (1 μM for 1 or 4 or 8 times in 24 h), after drug withdraw, either replated for colony formation assay (n = 3 independent experiments) (G) or incubated with EdU for 10 h, and stained at 8 dpt (n = 9 samples from 3 independent experiments) (H). I. Cells were treated with vehicle or abemaciclib (250 nM or 500 nM or 1 μM for 8 times in 24 h); after drug withdraw, the cells were replated for colony formation assay (n = 3 independent experiments). J. Representative phase-contrast images of BJ or WI38 cells at the end of each drug treatment (scale bar, 1 mm; n = 3 independent experiments). K. At 8 dpt, treated BJ cells were fixed and stained for SA-β-gal and quantified (scale bar, 1 mm; n = 3 independent experiments). L. Whole-cell lysate of treated BJ cells was used to immunoblot for p16 (n = 3 independent experiments). M–O. RNA-sequencing was performed with human fibroblasts (BJ) treated with vehicle (water for 8 times in 24 h) or abemaciclib (1 μM for 8 times in 24 h) (n = 3 independent samples, sequenced together). Heatmap of cell cycle genes (M), senescence signature (N), and p53-repressed (red) and p53-activated (blue) cell cycle-related genes (O) calculated from RNA-seq datasets of cells 8 dpt relative to the vehicle-treated group. P. Cells were treated with vehicle or abemaciclib (1 μM for 8 times in 24 h); after drug withdraw, the nuclear fraction was isolated and protein extracted for Western blotting. Lamin A/C was used as the marker of nucleus and loading control (n = 3 independent experiments). Data information: Data are means ± SD. Two-way ANOVA (B, C, and H). One-way ANOVA (D, E, and K). ***P < 0.001. Download figure Download PowerPoint Figure 1. CDK4/6i treatment induces a state of cellular senescence dependent on p53 activity A, B. Human fibroblasts (BJ) were treated with vehicle (water for 8 times in 24 h) or abemaciclib (1 μM for 8 times in 24 h). The population doubling over the 8-day treatment is plotted (A). 1 or 8 dpt, cells were incubated with EdU for 20 h and stained and quantified (n = 9 samples from 3 independent experiments; scale bar, 150 μm) (B). C. Chromatin was extracted from BJ cells treated with vehicle or abemaciclib (1 μM for 8 times in 24 h) or nutlin-3a (10 μM for 8 times in 24 h) or doxorubicin (250 nM for 24 h), and ChIP assays using an antibody against p53 were performed. qRT–PCR was performed using primers amplifying promoter regions of CDKN1A, GADD45A, and MDM2 genes containing p53 binding sites. Values indicate fold enrichment relative to the vehicle group (n = 3 independent experiments). D. BJ cells transduced with a p53 reporter were treated with nutlin-3a (10 μM, positive control) or abemaciclib (1 μM for 1 or 4 or 8 times in 24 h), and luciferase activity was measured after treatments at the indicated time points (n = 3 independent experiments). E. 3 × 103 BJ cells of the indicated genotypes were replated after vehicle or abemaciclib treatment (1 μM) and stained with 0.2% crystal violet 8 dpt (n = 3 independent experiments). F. 8 dpt, treated BJ cells were incubated with EdU for 20 h and stained (n = 9 samples from 3 independent experiments). G. 3 × 103 mouse dermal fibroblasts (MDFs) of the indicated genotypes were replated after vehicle or abemaciclib treatment (4 μM for 8 times in 24 h) and stained with 0.2% crystal violet 8 dpt (n = 3 independent experiments). H. 8 dpt, treated MDFs were incubated with EdU for 20 h and stained (n = 9 samples from 3 independent experiments). I. RNA was isolated from vehicle- or abemaciclib (1 μM)-treated scramble/shp53 BJ cells, and mRNA for the indicated genes was quantified by qRT–PCR relative to tubulin (n = 3 independent experiments). Data information: Data are means ± SD. Unpaired Student's t-test (A and H). One-way ANOVA (B, D, and F). Two-way ANOVA (C and I). *P < 0.05, **P < 0.01, and ***P < 0.001. dpt, days post-treatment. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. CDK4/6i induces cellular senescence dependent on p53 Immunoblot of p53 on cells of indicated genotypes and treatments. Representative images of p53GSE BJ cells at the end of the 8-day indicated treatments (scale bar, 1 mm; n = 3 independent experiments). 8-day population doubling of treated p53GSE BJ cells (n = 3 independent experiments). 3 × 103 vehicle or palbociclib-treated (1 μM for 8 times in 24 h) scramble/shp53/ p53GSE BJ cells were replated in 6-well dishes, cultured for 8 days, and stained with 0.2% crystal violet (n = 3 independent experiments). 3 × 103 vehicle- or abemaciclib-treated (4 μM for 8 times in 24 h) WT/p53GSE MEFs were replated in 6-well dishes, cultured for 8 days and stained with 0.2% crystal violet (n = 3 independent experiments). BJ cells were treated with abemaciclib ± pifithrin-α and EdU staining performed at 8 dpt (n = 9 samples from 3 independent experiments). Data information: Data are means ± SD. One-way ANOVA (C). Unpaired two-tailed t-test (F). ***P < 0.001. Download figure Download PowerPoint CDK4/6i promotes premature senescent cells in vivo without toxicity To determine whether induction of normal cells into senescence upon treatment with CDK4/6i happens in vivo, we exposed mice to a clinically relevant and tumor-suppressive dose of abemaciclib. At first, the anti-neoplastic effects of this dose (50 mg/kg/day of abemaciclib for 7 consecutive days) were validated in our laboratory using a model of breast cancer (Fig EV3A–C). Then, we treated cancer-free p16-3MR mice, which harbor a Renilla luciferase (RL) reporter gene driven by the p16 promoter (Demaria et al, 2014), with abemaciclib or doxorubicin. Abemaciclib- and doxorubicin-treated mice showed a similar enhanced bioluminescent signal (Fig 2A and B), and comparable induction of p16 (Fig 2C) and SA-β-gal in kidneys (Fig 2D and E). To evaluate whether induction to senescence by CDK4/6i treatment depends on p53 also in vivo, we exposed p16-3MR mice to a co-treatment abemaciclib/PFT-α. The presence of the p53 transcriptional inhibitor PFT-α prevented abemaciclib-mediated induction of whole-body luminescence (Fig 2F) and of p16 and p21 expression in kidney (Fig 2G), suggesting senescence bypass. Therapy-induced senescence is an important promoter of several adverse reactions to treatment (Wang et al, 2020) independent from tumorigenesis. Among those, evidences from preclinical and clinical contexts have highlighted the correlation between levels of senescence and severe fatigue (Wang et al, 2020). To evaluate whether such detrimental effect is also exerted by CDK4/6i-induced senescent cells, we compared the physical activity of cancer-free mice exposed to abemaciclib or doxorubicin (Fig 2A–E). As previously shown (Demaria et al, 2017), treatment with doxorubicin severely affected strength and endurance, as measured by rotarod assay (Fig 2H), grip strength (Fig 2I), and hanging time (Fig 2J) at both 7 and 14 days after treatment. In contrast, mice treated with abemaciclib did not show any apparent reduction of physical performance in comparison with vehicle-treated cohorts (Fig 2 H–J). Weight and blood counts of abemaciclib-treated mice remained similar to control mice, while doxorubicin-treated animals showed substantial weight loss (Fig 2K), reduced number of red blood cells (Fig 2L), a slight decrease in the total number of leukocytes (Fig 2M), and impaired proportion of B cells, granulocytes, and macrophages (Fig 2N). Together, these data suggest that treatment with CDK4/6i induces a well-tolerated state of senescence in vivo. Click here to expand this figure. Figure EV3. Senescence-inducing dose of abemaciclib inhibits tumor growth Scheme of abemaciclib treatments for MMTV-PyMT-firefly breast cancer mouse model in vivo. Female p16-3MR mice bearing MMTV-PyMT-firefly tumors in mammary fat pad were treated with vehicle (PBS, 7 consecutive days) or abemaciclib (50 mg/kg in PBS, 7 consecutive days). The mice were injected with D-Luciferin, and bioluminescence was visualized/quantified by the IVIS spectrum in vivo imaging system before and after abemaciclib treatments, as shown by representative images and quantification (n = 5 mice/group). Excised tumors and quantification of tumor weights (n = 5 mice/group). Data information: Data are means ± SD. Unpaired two-tailed t-test (B and C). *P < 0.05 and **P < 0.01. Download figure Download PowerPoint Figure 2. CDK4/6i promotes premature senescent cells in vivo without toxicity A–E. p16-3MR mice were treated with vehicle (PBS, 7 consecutive days), doxorubicin (5 mg/kg, 3 consecutive days), or abemaciclib (50 mg/kg, 7 consecutive days). n = 6 mice/group. 14 dpt, bioluminescence was visualized and quantified by the IVIS spectrum in vivo imaging system, as shown by representative bioluminescence images (A) and quantification (B). RNA was isolated from kidneys, and mRNA encoding p16 was quantified by qRT–PCR (C). Representative images (D) to visualize SA-β-gal activities in vehicle-, doxorubicin-, or abemaciclib-treated mouse kidney sections at 15 dpt (arrows indicated positive area; scale bar, 1 mm; n = 3 mice/group) and quantified (E). F. p16-3MR mice were treated with abemaciclib (50 mg/kg, 7 consecutive days) ± pifithrin-α (2 mg/kg, 7 consecutive days). Values indicate the ratio of post-treatment bioluminescence to pretreatment (n = 5 mice/group). G. RNA was isolated from abemaciclib ± pifithrin-α treated kidneys, and mRNA encoding p16 and p21 genes was quantified by qRT–PCR and normalized on tubulin (n = 5 mice/group). H–N. For the doxorubicin- or abemaciclib-treated mice (n = 6 mice/group), physical performance was measured by rotarod assay at 15 dpt (H), and grip strength meter at 7 dpt and 14 dpt (I), and hanging tests were performed at 7 dpt and 14 dpt and normalized to weights (J). (K) Relative weight changes were calculated at 7 dpt and 14 dpt. Red blood cells (L) and white blood cells (M) were counted at 15 dpt. Percentage of T cells, B cells, granulocytes, and macrophages were determined by flow cytometry analysis (N). Data information: Data are means ± SD. One-way ANOVA (B, C, E, H, L, and M). Unpaired Student's t-test. (F). Two-way ANOVA (G, I, J, K, and N). *P < 0.05, **P < 0.01, and ***P < 0.001, N.S. = not significant. dpt, days post-treatment. Download figure Download PowerPoint CDK4/6i-induced senescence lacks NF-κB activity and NF-κB-driven SASP components Abemaciclib-treated cells showed increased levels of mitochondrial ROS, which could explain higher p53 activity (Fig EV4A). However, the level of DNA damage response (DDR) signaling was absent from CDK4/6i-induced senescence (Fig 3A). In senescent cells, DDR signaling is a major activator of NF-κB, which in normal cells is dispensable for the cell cycle arrest but acts as a strong inducer of pro-inflammatory SASP factors (Coppé et al, 2008; Chien et al, 2011). Interestingly, cells induced to senescence by palbociclib or abemaciclib did not enhance NF-κB activity, as quantified via a luminescent reporter system, whereas a strong upregulation was observed in cells exposed to either doxorubicin or paclitaxel (Fig 3B). This was in accordance with RNA-sequencing data, which indicated that a vast majority of NF-κB-associated SASP genes were up-regulated in genotoxic stress-induced senescent cells (doxorubicin), while these genes were not differentially regulated or even downregulated during CDK4/6i-induced senescence (Fig 3C and D and Table EV2). qPCR (Fig 3E), ELISAs (Fig 3F) and cytokine array (Fig 3G) validated and confirmed that in primary human fibroblasts, only genotoxic drugs, but not CDK4/6i, were able to promote the production and secretion of factor part of the NF-κB-associated secretory phenotype (from now called NASP) such as IL-6, CXCL1, CCL5, and MMP1. This difference was also observed in cell types other than
Referência(s)