Harnessing E2F1 Regulation for Prosenescence Therapy in P53-Defective Cancer Cells

Published OnlineFirst May 1, 2014; DOI: 10.1158/1078-0432.CCR-13-1942

Clinical Cancer Molecular Pathways Research

Molecular Pathways: Harnessing E2F1 Regulation for Prosenescence Therapy in p53-Defective Cancer Cells

Anni Laine1 and Jukka Westermarck1,2

Abstract Induction of terminal proliferation arrest, senescence, is important for in vivo tumor-suppressive function of p53. Moreover, p53-mutant cells are highly resistant to senescence induction by either oncogenic signaling during cellular transformation or in response to different therapies. Senescence resistance in p53-mutant cells has been attributed mostly to inhibition of the checkpoint function of p53 in response to senescence-inducing stress signals. Here, we review very recent evidence that offers an alternative explanation for senescence resistance in p53-defective cancer cells: p21-mediated E2F1 expression. We discuss the potential relevance of these findings for senescence-inducing therapies and highlight cyclin-dependent kinases (CDK) and mechanisms downstream of retinoblastoma protein (RB) as prospective prosenescence therapeutic targets. In particular, we discuss recent findings indicating an important role for the E2F1–CIP2A feedback loop in causing senescence resistance in p53-compromised cancer cells. We further propose that targeting of the E2F1–CIP2A feedback loop could provide a prosenescence therapeutic approach that is effective in both p53-deficient and RB-deficient cancer cells, which together constitute the great majority of all cancer cells. Diagnostic evaluation of the described senescence resistance mechanisms in human tumors might also be informative for patient stratification for already existing therapies. Clin Cancer Res; 20(14); 3644–50. 2014 AACR.

Background expression of oncogenes or inactivation of tumor suppres- Replicative senescence is a cellular phenomenon by sors (1). Originally, oncogene-induced senescence (OIS) which normal cells are induced to terminal growth arrest was identified in mutant oncogenic HRAS–transfected after multiple replications in vitro (1). Recently, two re- human and mouse fibroblasts (4). Later, OIS and tumor search groups showed that replicative senescence is not suppressor inactivation–induced senescence were reported only associated with cellular aging but also contributes to to prevent transformation in the context of several different tissue remodeling during embryonic mammalian devel- oncogenes and tumor suppressors in many tissue types both in vitro in vivo opment (2, 3). In cell culture conditions, senescent cells and (5). For example, in a mutant oncogenic KRAS often show clear morphologic changes characterized by -driven mouse model, multiple senescent markers are cell flattening, enlargement, and multinucleated appear- expressed in premalignant lung and pancreas tumors, ance (1). In addition to morphologic changes, several whereas malignant lung and pancreas adenocarcinomas biomarkerscanbeusedtodetectsenescentcellsin vitro show robust proliferation (6). These studies clearly indicate and in vivo. As comprehensively reviewed by Kuilman and that for malignant transformation, cells must become resis- colleagues (1), many of these markers are cell specific and tant to both apoptotic cell death and senescence induction. BRAF tissue specific, and it has been a challenge to identify In humans, oncogenic mutation in induces senes- markers that would reliably indicate for senescence cence in premalignant nevi and arrests malignant melano- induction in in vivo tissues. ma progression (7). Also, senescence has been detected in In addition to replicative senescence, another type of early-stage human prostate cancer lesions, further indicat- senescence can occur in response to different types of stress ing that senescence is a restrictive phenomenon during stimuli, such as DNA damage, oxidative stress, and acute human carcinogenesis (8). p53 and its downstream effector pathway in Authors' Affiliations: 1Turku Centre for Biotechnology, University of Turku senescence regulation and Åbo Akademi University; and 2Department of Pathology, University of Turku, Turku, Finland The tumor suppressor p53 is involved in senescence induction by various senescence-inducing stimuli (9). Con- Corresponding Author: Jukka Westermarck, Turku Centre for Biotech- nology, University of Turku and Åbo Akademi University, Tykistokatu€ 6, sistent with this, ectopic activation of p53 has been shown Turku 20520, Finland. Phone: 358-23338621; Fax: 358-23338000; E-mail: to inhibit tumor initiation and tumor maintenance by jukka.westermarck@utu.fi inducing senescence in vivo (10, 11). In fact, most recent doi: 10.1158/1078-0432.CCR-13-1942 studies indicate that even though apoptosis induction is 2014 American Association for Cancer Research. the classical response to acute p53 induction, it may be

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Senescence Resistance Driven by Inactivated p53

senescence, rather than apoptosis induction, that mediates they all feed to E2F1 regulation. We further discuss the tumor suppressor function of p53 in vivo (12, 13). In early potential of these mechanisms as prosenescence therapy research, both p53 and its downstream RB activity were targets. identified as limiting factors in human cellular transformation by forcing cells into cellular senescence (14). This could p21 mediates p53-induced senescence be explained by findings that upon oncogene-induced Because p53 has a well-established role in senescence signaling, p53 is stabilized, leading to activation of the induction, it is not a surprise that the downstream targets p53 downstream pathway, p53/p21/CDK2/RB/E2F1 (Fig. of p53 are important senescence regulators (9). Activation 1; ref. 9). In fact, it has even been demonstrated that p53 of p53 results in induction of direct p53 transcriptional activation by a conditional allele in mouse tumors is not target p21, which further inhibits cyclin-dependent sufficient to induce tumor regression in the absence of Ras- kinase 2 (CDK2)/cyclin E complex, leading to RB-medi- driven oncogenic stress (15). ated E2F1 inhibition (Fig. 1; refs. 17, 18). In turn, and Whereas activation of wild-type p53 results in senescence relevant for the model proposed in this review, p53 induction, genetic p53 inhibition can overcome oncogenic inhibition in cancer cells leads to increased p21 expres- mutant BRAF-induced senescence in mouse lung leading to sion and consequent activation of E2F1. We do realize tumor progression (16). In addition, p53 inactivation has that other downstream effectors of p53 than p21 may also been shown to overcome Pten inactivation–induced senes- contribute to senescence regulation, but for the clarity of cence in mouse prostate leading to cancer progression (8). presentation, those mechanisms are not discussed here. Thus far, the senescence resistance of p53-defective cancer The critical role for p21 in senescence regulation, even cells has been attributed to inhibition of p53 checkpoint independently of p53, was supported by two recent function in response to OIS. However, an alternative expla- articles describing p21-dependent programmed senes- nation for senescence resistance of p53-mutant cancer cells cence during mouse development (2, 3). As a further could be that p53 inactivation actively drives mechanisms support to the central role for p21 in senescence, recent of senescence resistance. In this review, we discuss the latter studies have demonstrated p21-induced senescence in explanation and uncover that common between these p53 p53-defective cancer cell lines (17, 19–21) and tumors inhibition–driven senescence resistance mechanisms is that in vivo (22, 23).

Mechanisms of regulation Prosenescence in cancer therapy regimen

p53 inhibition: MDM2 activity, p53 Mutant p53 mutations reactivators SCF/Skp2 inhibitors p21 inhibition: p53 inhibition p21 Guanylate cyclase p16 inhibition: genetic deletion p16 inhibitors CDK activation: p16 and CDK4/6 CDK2 CDK inhibitors p21 inactivation

RB inhibition: CDK activity, RB mutations, promoter methylation Senescence Proliferation E2F1 activation: RB E2F1 Vinca alkaloids, phosphorylation, transcription, RNAi therapy CIP2A expression P PP2A inhibition: PP2A inhibitor PP2A PP2A reactivators proteins, mutations

Signaling inhibitors, CIP2A activation: transcription CIP2A RNAi therapy

Figure 1. Regulation and therapeutic targeting of E2F1-driven cancer cell senescence resistance. Activity and expression of transcription factor E2F1 is regulated by upstream tumor suppressors p53, p16, and RB and by positive feedback loop between E2F1 and its target gene CIP2A. CIP2A inhibits activity of tumor suppressor PP2A resulting in increased E2F1 expression and senescence resistance. Blue color denotes proteins and regulatory connections that in normal cells promote senescence induction (blue enlarged multinucleated cell), whereas regulatory mechanisms driving senescence resistance and increased proliferation in cancer cells are denoted by red color. Left (red), mechanisms by which the depicted proteins are deregulated in cancer cells and thus promote senescence resistance. Right (blue), potential pharmacologic approaches to target the depicted proteins for prosenescence cancer therapies.

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RB-E2F1 as a nodal point of senescence sensitivity mouse model (17). Rescue experiments demonstrated that regulation in p53-defective cancer cells senescence induction either by p53 activation, or by direct As mentioned above, CDK2 inhibition mediates senes- adenoviral overexpression of p21, was fully dependent on cence signaling from p21 to RB. In support of an important the capacity of these stimuli to downregulate CIP2A expres- role for CDK2 activity in mediating senescence resistance, sion (17). Importantly, CIP2A depletion also induced inhibition of CDK2 was shown to induce senescence in p53- senescence in breast cancer cells with impaired p53 or RB deficient human leukemic tumor cells and in MYC-driven in function (17). Moreover, CIP2A was identified as a novel vitro and in vivo transformation models (24, 25). Hydbring direct target gene for E2F1, explaining how high E2F1 and colleagues further presented evidence that direct MYC expression in p53-mutant cancer cells drives senescence phosphorylation by CDK2 was important for senescence resistance (17). In line with these results, also ectopic evasion (25). CDK2 phosphorylates tumor suppressor ret- expression of E2F1 rescues the senescence phenotype in inoblastoma protein (RB), resulting in release of transcrip- cancer cells (38, 39, 41) and similarly to CIP2A, E2F1 tion factor E2F1 from RB/E2F1 complex (Fig. 1; ref. 26). RB knockout mouse shows reduced tumorigenesis in MMTV- protein family proteins, RB (p105), p107, and p130, have neu model (17, 35). In addition to breast cancer, loss of overlapping functions in cell-cycle control (26) and have CIP2A has been reported to induce the senescent pheno- compensatory and complementary roles in senescence reg- type in gastric cancer and in esophageal squamous cell ulation (27, 28). For example, in the absence of functional carcinoma cell lines (45, 46). Importantly, high CIP2A RB, p107 was shown to primarily mediate irradiation- expression predicts for poor patient survival in more than induced senescence in prostate cancer cells (29). On the a dozen different human cancer types (44). Together other hand, the most recent data with human fibroblasts these results indicate that a senescence-inhibiting role show that RB, but not p107 or p130, has an essential role in for CIP2A may be generalized to the majority of human oncogene-induced senescence in human cells (30) impli- cancer types. cating for a cell type specificity for requirement of different The molecular function of CIP2A is to inhibit phospha- RB family members in senescence regulation in experimen- tase activity of a tumor suppressor phosphatase complex tal conditions. Importantly, unlike RB, mutations in p107 PP2A (44, 47). Different PP2A complexes regulate serine/ or in p130 are rare in human cancer, strengthening the threonine phosphorylation of numerous cellular targets important role for RB in tumor suppression among mem- implicated in a variety of physiologic and cellular processes bers of the RB protein family (26). Relevant for the model (48). Although the role of PP2A is not thus far widely proposed in this review, a tumor suppressor VHL induced studied in the context of senescence, the emerging evidence RB-dependent senescence in vivo even in the absence of does indicate that certain PP2A complexes do promote functional p53 (31). In addition, in human cancer cells, senescence (49). Related to the role of PP2A in CIP2A- including those that are p53 deficient, reactivation of RB mediated senescence resistance, inhibition of the B55a induces the senescence phenotype (32). subunit of PP2A was identified to promote stability of The target of RB, E2F1, is a transcription factor whose E2F1 phosphorylated on serine 364 and to prevent p53- expression is increased in many human cancers (33). The induced downregulation of E2F1 (17). Together these E2F protein family consists of 8 transcription factors (E2F1– results indicate that E2F1–CIP2A interplay involving PP2A 8) that are important regulators of the cell cycle and cell (Fig. 1) is an important novel mechanism regulating senes- fate (33). Even though E2F proteins have overlapping cel- cence sensitivity of human cancer cells. Notably, these lular functions, they differ in their biologic roles, for results do not exclude the possibility of a role for other instance, in mammary development and in mammary PP2A and CIP2A targets than E2F1 in the regulation of carcinogenesis (34, 35). Excellent recent reviews underline senescence resistance in cancer cells. specific functions of E2F family members (36, 37), but – hereafter, we only concentrate in discussing the role of Clinical Translational Advances E2F1 in regulating senescence induction in cancer cells. Notably, although traditionally considered as apoptosis- Related to the role of E2F1 in regulating OIS, several pub- inducing agents, many of the currently used chemothera- lications have reported downregulation of E2F1 in senes- pies most probably exert their therapeutic effect, at least cent cells (38, 39). More importantly, inhibition of E2F1 partly, by senescence induction (50). For example, signif- induces the senescence phenotype in cancer and in immor- icant induction of several senescence-associated gene talized cells (17, 33, 40, 41). Recent studies have charac- expression was detected in prostate tumors after mitoxan- terized E2F1 target genes relevant for senescence regulation trone chemotherapy, and senescence-associated b-galacto- (30, 42). However, despite RB proteins, the mechanisms by side positivity was reported in 41% of breast tumors after which E2F1 expression and activity are regulated during neoadjuvant chemotherapy, including cyclophosphamide, senescence regulation have been elusive. Recently, we dem- doxorubicin, and 5-fluorouracil (50–52). On the basis of onstrated a previously unidentified positive feedback loop these and other studies suggesting an important role for between E2F1 and another human oncoprotein CIP2A (43, senescence induction in cancer therapy responses, an 44) and demonstrated that this feedback mechanism pro- increasing interest has been expressed in development of motes tumorigenesis by suppressing senescence induction prosenescence cancer therapeutic strategies (9, 50). The in breast cancer cell lines and in a HER2-driven breast cancer rationale of driving cancer cells to senescence, rather than

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apoptosis, is that there is increasing evidence that apoptosis- types, such as leukemia, melanoma, and breast cancer (55). inducing regimens increase the risk for secondary malig- Dinaciclib does induce apoptosis in tumor cell lines, and nancies and mutational evolution of those cancer cells that this could be explained by its activity toward multiple CDKs are spared from apoptosis. However, it should be noted (56). However, dinaciclib also reduces phosphorylation of that a recent mouse study indicated that p53-driven RB in vitro in osteosarcoma cells and in vivo in ovarian cancer senescence induction in tumors might actually protect cell xenografts (56, 57), and therefore it would be important tumor cells from chemotherapy-induced apoptosis (53). to validate whether it has senescence-inducing activity in This can be explained by the fact that senescent cells human tumors. persist in tumors but are not sensitive to DNA-damaging Whereas CDK2 mediates the effects of p21 in senes- therapies due to cell-cycle arrest (9, 50). On the other cence regulation, CDK4 and CDK6 are important med- hand, clearance of senescent cells from tumors by innate iators of senescence resistance in cancer cells with inacti- immune cells has been shown repeatedly (9, 10). On the vated tumor suppressor p16 (Fig. 1; ref. 58). A common basis of these findings, it is evident that further studies are featureofalltheseCDKsisthattheyregulateRBandthus needed to clarify the therapeutic potential of prosenes- they have a common target relevant for E2F1 and senescence strategies separately in each cancer therapy setting. cence regulation. In a mouse model, inhibition of CDK4, but not CDK6 or CDK2, induces senescence in advanced Harnessing p53 downstream effectors for KRAS-driven non–small cell lung carcinoma (NSCLC; prosenescence therapy ref. 59). Another CDK inhibitor in clinical trials, palbo- Several mouse models have indicated that p53 reactiva- ciclib (PD-0332991), inhibits CDK4 and CDK6 down- tion would be an efficient prosenescence therapeutic strat- stream of p16 and induces senescence in melanoma and egy (10–13). However, p53 is mutationally inactivated in a glioblastoma cells in vitro (60, 61) and very efficiently very large fraction of human cancers, and therefore thera- suppressed growth of preformed intracranial glioblasto- peutic strategies to activate senescence via wild-type p53 are ma xenografts (61). The importance of p16-mediated unlikely to be efficient in most human tumors. To overcome senescence regulation in tumorigenesis is supported by this serious shortcoming, several strategies to reactivate a fact that p16-encoding gene INK4A is frequently mutat- mutated p53 have been developed, and these strategies ed in human cancer (1). have been reviewed recently (54). Importantly, an alterna- Importantly, in glioblastoma cells, depletion of RB con- tive therapeutic strategy to induce senescence in p53-com- ferred senescence resistance to palbociclib, further demon- promised tumor cells would be to target the mechanisms strating a requirement of RB for sensitivity to CDK4/6 that promote senescence resistance due to p53 inactivation inhibition (61). Instead, in melanoma cells, CDK4/6 inhi- (Fig. 1). Potential such strategies are discussed below. bition was shown to inhibit phosphorylation of FOXM1, which was shown to be partly independent of RB and was Senescence induction in cancer cells with compromised critical for CDK4/6 inhibitor–induced senescence (60). p53 function by targeting of p21 and CDKs However, a recent study demonstrated that FOXO transcrip- As described above, ectopic activation of p21 in p53- tion factors directly stimulate E2F1 expression (62), further mutant cells drives cancer cells effectively to senescence suggesting that despite differences in upstream regulation, and this is dependent on downregulation of E2F1–CIP2A E2F1 has a critical role in senescence sensitivity regulation feedback loop (Fig. 1; refs. 17, 19–21). Previously, an by CDK inhibitors. On the basis of these results, CDK inhibitor of guanylate cyclase, LY8583, was demonstrated inhibitors seem to present a promising prosenescence ther- to increase p21 expression 2-fold and to induce senescence apy option in several types of cancers with compromised in p53-compromised cancer cells in vitro (19). Similarly, p53 function. two chemical compounds inhibiting SCF-Skp2 complex, MLN4924 and SZL-P1–41, were recently identified to Senescence induction in cancer cells with compromised induce p21 expression followed by senescence in p53- p53 and RB function by targeting of E2F1–CIP2A deficient cancer cells in vitro (22, 23). MLN4924 and SZL- feedback loop P1-41 were further shown to significantly inhibit tumor Similar to p53, RB is also inactivated in a large fraction of growth of preformed human prostate and lung cancer human cancers, and on the basis of recent functional xenografts (22, 23), suggesting that Skp2 inhibitors may analysis, this leads to resistance to senescence induction represent a promising novel class of prosenescence thera- by either RAS-driven replicative stress or by inhibition of peutics in p53-defective cancer cells. CDK4/6 or VHL (30, 31, 61). Importantly, Chicas and CDK2 activity is increased in p53-mutant cancers due to colleagues recently demonstrated that senescence resist- p21 inhibition, and either genetic or pharmacologic CDK2 ance in RB / cells was associated with selective dysregula- inhibition drives senescence in various cell types, including tion of E2F1 target genes involved in senescence regulation a p53-null human cancer cell line in vitro (24, 25). Thereby, (30). Together with very recent data that ectopic activation pharmacologic inhibition of CDK2 could be an attractive of E2F1–CIP2A feedback loop can rescue senescence phe- approach for prosenescence therapy. Notably, a novel CDK notype in cancer cells (17, 38, 39, 41), these results indi- inhibitor, dinaciclib (MK-7965), most potently inhibiting cate that targeting of this feedback loop could provide a CDK2 and CDK5, is under clinical trials for several cancer prosenescence therapy approach that is effective in both

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p53-deficient and RB-deficient cancer cells, which together stress and to promote transformation (68). Finally, as constitute the great majority of all cancer cells. different PP2A complexes mediate senescence induction Although targeting of the E2F1–CIP2A feedback loop and regulate E2F1 (49), testing of efficacy of small-mol- appears to be an attractive approach for prosenescence ecule activators of PP2A (17, 47) as novel prosenescence therapy, tools to target this loop selectively are currently therapeutic agents might be warranted. limited. Inhibitors targeting the E2F family of proteins have been generated; however, they are not specific for certain Future Directions E2F proteins (63, 64). Interestingly, one of the traditional In sum, the literature reviewed here reveals that senes- classes of chemotherapies, vinca alkaloids, inhibits E2F1 cence resistance in p53-defective cancer cells results from at expression and effectively induces senescence in human least two different mechanisms: (i) loss of checkpoint breast cancer cells in vitro (17, 65). Although the exact function of p53 in response to senescence inducing signals mechanism by which vinca alkaloids inhibit E2F1 remains and (ii) activation of the E2F1–CIP2A feedback loop due to be determined, it occurs at the level of mRNA expression to inhibited p21 function. On the basis of these findings, and does not involve either p53 or p21 (17). Vinca alkaloid further development of small-molecule activators of p21 vinorelbine-induced senescence was preceded by downre- and inhibitors of CDKs may provide opportunities for gulation of both E2F1 and CIP2A expression, and CIP2A development of prosenescence therapies against p53- deficiency further enhanced the E2F1 inhibition by vinor- mutant tumors. Moreover, the emerging indications for the elbine (17). Furthermore, low CIP2A expression in human essential role for E2F1 and CIP2A in mediating senescence breast tumors predicted for favorable survival in patients resistance in both p53- and RB-compromised tumors treated with vinorelbine (P ¼ 0.019; ref. 17). These results should boost development of therapeutics against these identify vinca alkaloids as potential clinical inhibitors of previously undruggable proteins. This could involve mod- E2F1-mediated senescence resistance and use of CIP2A ern drug development approaches, such as allosteric pro- diagnostics for patient stratification to vinca alkaloid–based tein–interaction inhibitors, or RNAi therapy. Finally, the prosenescence therapy. presented evidence suggests that diagnostic evaluation of Notably, CIP2A is one of the most frequently over- the RB, E2F1, and CIP2A status of newly diagnosed cancers expressed oncoproteins across human cancer types, and might help stratification of patients for therapies with inhibition of CIP2A by siRNA results in significant reduc- already existing therapeutic agents with demonstrated pro- tion of xenograft growth of cervical cancer, head and neck senescence activity, such as vinca alkaloids (17, 65). squamous cell carcinoma, breast cancer, and bladder in vivo urothelial cell carcinoma cells (44).Thereby,and Disclosure of Potential Conflicts of Interest encouraged by recent success in a clinical lipid nanopar- J. Westermarck has ownership interest (including patents) in CIP2A RNAi ticle (LNP)-formulated RNAi therapy trial targeting VEGF therapy. No potential conflicts of interest were disclosed by the other author. and kinesin spindle protein (KSP) in patients with hepatic and extrahepatic tumors with Alnylam (66), RNAi ther- Authors' Contributions apy against CIP2A or E2F1 could be proposed as a future Conception and design: A. Laine, J. Westermarck Analysis and interpretation of data (e.g., statistical analysis, biosta- approach for prosenescence therapy in various cancer tistics, computational analysis): A. Laine types. Naturally, development of a small-molecule inhib- Writing, review, and or revision of the manuscript: A. Laine, itor against CIP2A or CIP2A–E2F1 interaction, if feasible, J. Westermarck Administrative, technical, or material support (i.e., reporting or orga- could provide an opportunity for prosenescence therapy. nizing data, constructing databases): A. Laine Moreover, targeting upstream regulators of CIP2A expression could be another alternative for prosenescence ther- Acknowledgments apy. Of thus far identified regulators of CIP2A expression The authors apologize to their colleagues whose work could not be cited (Chk1, ETS1, JNK2, or MYC; ref. 44), checkpoint kinase because of space limitations. Chk1 could be the most promising candidate to be targeted for prosenescence therapy, as Chk1 inhibitors are already in clinical trials (55). We recently demonstrat- Grant Support This work was supported by grants from the Academy of Finland ed that inhibition of constitutive Chk1 activity in various (8217676 and 122546; to J. Westermarck), the Sigrid Juselius Foundation cancer cell lines reduces CIP2A expression resulting in (J. Westermarck), and the Foundation for the Finnish Cancer Institute PP2A reactivation and potent inhibition of malignant cell (J. Westermarck). growth (67), whereas increased Chk1 expression was Received January 10, 2014; revised April 10, 2014; accepted April 14, 2014; shown to protect against oncogene-induced replicative published OnlineFirst May 1, 2014.

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3650 Clin Cancer Res; 20(14) July 15, 2014 Clinical Cancer Research

Molecular Pathways: Harnessing E2F1 Regulation for Prosenescence Therapy in p53-Defective Cancer Cells

Anni Laine and Jukka Westermarck

Clin Cancer Res 2014;20:3644-3650. Published OnlineFirst May 1, 2014.

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