Pharmacological Inhibition of Centrosome Clustering by ... · Centrosome ampliﬁcation is a hallmark of virtually all types of cancers, including solid tumors and hematologic malignan-cies

Therapeutics, Targets, and Chemical Biology

Pharmacological Inhibition of CentrosomeClustering by Slingshot-Mediated CofilinActivation and Actin Cortex DestabilizationGleb Konotop1, Elena Bausch1, Tomoaki Nagai2, Andrey Turchinovich3, Natalia Becker4,Axel Benner4, Michael Boutros5, KensakuMizuno2, Alwin Kr€amer6, andMarc Steffen Raab1

Abstract

Centrosome amplification is a hallmark of virtually all typesof cancers, including solid tumors and hematologic malignan-cies. Cancer cells with extra centrosomes use centrosome clus-tering (CC) to allow for successful division. Because normalcells do not rely on this mechanism, CC is regarded as apromising target to selectively eradicate cells harboring super-numerary centrosomes. To identify novel inhibitors of CC, wedeveloped a cell-based high-throughput screen that reportsdifferential drug cytotoxicity for isogenic cell populations withdifferent centrosome contents. We identified CP-673451 andcrenolanib, two chemically related compounds originallydeveloped for the inhibition of platelet-derived growth factorreceptor b (PDGFR-b), as robust inhibitors of CC with selectivecytotoxicity for cells with extra centrosomes. We demonstrate

that these compounds induce mitotic spindle multipolarity byactivation of the actin-severing protein cofilin, leading todestabilization of the cortical actin network, and provide evi-dence that this activation is dependent on slingshot phospha-tases 1 and 2 but unrelated to PDGFR-b inhibition. Morespecifically, we found that although both compounds attenu-ated PDGF-BB–induced signaling, they significantly enhancedthe phosphorylation of PDGFR-b downstream effectors, Aktand MEK, in almost all tested cancer cell lines under physio-logic conditions. In summary, our data reveal a novel mech-anism of CC inhibition depending on cofilin-mediated corti-cal actin destabilization and identify two clinically relevantcompounds interfering with this tumor cell–specific target.Cancer Res; 76(22); 6690–700. �2016 AACR.

IntroductionCentrosomes are cytoplasmic organelles composed of a pair

of centrioles, which nucleate and anchor microtubules. Cen-trosomes act as microtubule-organizing centers in animal cellsand play a key role in mitotic fidelity by securing bipolarmitotic spindle formation and equal chromosome segregation(1, 2). The number of centrosomes is tightly regulated byensuring that centrosomes are duplicated exactly once per cellcycle (3).

Centrosome amplification (CA) is found in most types ofcancers. Although it is still not clear whether CA is a cause or aconsequence of tumor initiation and progression, extra centro-somes strongly correlate with chromosomal instability, clinicalaggressiveness, and adverse clinical outcome in several tumortypes (4–10). Cancer cells carrying supernumerary centrosomesescape detrimental multipolar divisions by coalescing multiplecentrosomes into two functional spindle poles, a processknown as centrosome clustering (CC; ref. 11). CC contributesto chromosome segregation errors by generating merotelicmicrotubule–kinetochore attachment errors, leading to tolera-ble levels of genomic instability (12). Because most healthytissues have normal centrosome content, they do not rely onCC for successful division, which makes this mechanism apromising therapeutic target.

In addition to microtubule motor proteins, including dynein,Ncd/HSET, and Eg5 (11, 13–15), a role for cortical actin inCCwasinitially suggested by a genome-wide RNAi screen inDrosophila S2cells, where depletion of several components of the actin cyto-skeleton led to CC inhibition (13). Also, depletion of the actin-associated protein MISP destabilized attachments between astralmicrotubules and the actin cortex, led to defects in spindleorientation, and increased the incidence of multipolar spindlesin cells with CA (16). Finally, CC requires a functional spindleassembly checkpoint (SAC) to provide the necessary time foreffective centrosome coalescence (13, 14, 17).

Cell-permeable small molecules that exclusively eradicate cellswith extra centrosomes might be promising tools for targetedcancer therapy. CC can be inhibited by molecules that interferewith MT dynamics, such as taxanes, Vinca alkaloids, or the

1Max-Eder Research Group "Experimental Therapies for HematologicMalignancies", German Cancer Research Center (DKFZ) and Depart-ment of Internal Medicine V, University of Heidelberg, Heidelberg,Germany. 2Department of Biomolecular Sciences, Graduate Schoolof Life Sciences, Tohoku University, Sendai, Miyagi, Japan. 3MolecularEpidemiology Group, German Cancer Research Center, Heidelberg,Germany. 4Division of Biostatistics, German Cancer Research Center,Heidelberg,Germany. 5DivisionofSignalingandFunctionalGenomics,Medical Faculty Mannheim, German Cancer Research Center andUniversity of Heidelberg, Heidelberg, Germany. 6Clinical CooperationUnitMolecular Hematology/Oncology,GermanCancerResearchCen-ter and Department of Internal Medicine V, University of Heidelberg,Heidelberg, Germany.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Alwin Kr€amer, German Cancer Research Center, ImNeuenheimer Feld 280, Heidelberg 69120, Germany. Phone: 4962-2142-1440;Fax: 4962-2142-1444; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-1144

�2016 American Association for Cancer Research.

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noscapinoid EM011 (18–20). However, these drugs are notselective for cells with supernumerary centrosomes. Moleculeswith increased selectivity include griseofulvin and its derivativesand HSET inhibitors, which effectively decluster multiple centro-somes, but lead at higher concentrations to the formation ofmultipolar spindles with acentriolar poles (13, 21–24).

Experimentally, cells with extra centrosomes can be obtainedby increasing the expression levels of key components of thecentriole replicationmachinery, such as Polo-like kinase 4 (PLK4)or the scaffolding proteins HsSAS-6 and STIL (25–30).

In this study, we employed a novel small-molecule screeningstrategy based on a differential viability readout between twoisogenic cell populations with different centrosome content toidentify CP-673451 and crenolanib, two class III receptor tyrosinekinase (RTK) inhibitors, as CC inhibitors. We demonstrate thatthe inhibition of CC was attributed to activation of the actin-severing protein, cofilin, which constitutes a novel mechanism ofcortical actin-mediated CC inhibition. Furthermore, our worksheds light on the mechanisms of CP-673451 and crenolanib-induced cofilin activation mediated by the slingshot phospha-tases (SSH) SSH1 and SSH2.

Materials and MethodsDetailed experimental procedures are included in the Supple-

mentary Data.

Cells and reagentsTo generate EGFP-PLK4-U2OS, human osteosarcoma cells

carrying the regulatory plasmid pcDNA6/TR were transfectedwith ToPuro-EGFP-PLK4. Plasmid generation is described inSupplementary Data. EGFP-PLK4-U2OS and H2B-mCherry-a-tubulin-EGFP-HeLa (31) cells were cultivated in DMEM þGlutaMAX (Life Technologies) supplemented with 10% FCS(Biochrom). All unmodified cancer cell lines were obtained fromATCC and authenticated by MCA (2014). For PDGF-BB stimu-lation, starved cells (0% FCS, 24 hours) were pretreated with drugor vehicle for 3 hours and stimulated with 500 mg/mL PDGF-BB(Biotrend) for 15 minutes. Inhibitors included LIMKi3 (Merck),damnacanthal (Enzo), griseofulvin (Sigma), BYL719, CP-673451, and crenolanib (Selleckchem). Cells were synchronizedwith 100 ng/mL nocodazole (24 hours) or 2 mmol/L thymidine(16–18 hours; Sigma).

Differential viability readoutEGFP-PLK4-U2OS cells were split into two populations and

incubated with 2 mg/mL tetracycline (Sigma) or vehicle. After 2days, induced and noninduced cells were seeded in 384-well or96-well plates and rested (24 hours) prior to small-moleculeaddition. After 5-day exposure, cell viabilities were determinedwith CellTiter-Glo (Promega).

Statistical analysisResults are given as mean percentages� SD. Significances were

calculated by two-tailed t test or two-way ANOVA methods.

ImmunoblottingCell lysis and immunoblotting was performed according to

standard protocols. Antibodies used were as follows: cofilin, phos-pho-cofilin, phospho-Akt, phospho-MEK1/2, phospho-LIMK1/LIMK2, LIMK1, LIMK2, and SSH1 (CST); GFP, MCM7, PDGFR-b;

HRP-conjugated secondary antibodies (Santa Cruz Biotechnology);a-tubulin (Sigma); Eg5 (BD), and phospho-Eg5 (Novus).

Time-lapse microscopy and image acquisitionTime-lapse microscopy was performed on a Zeiss Cell Observer.

Z1 under controlled environmental conditions. The numbers oftotalmitotic cells counted are indicated over each bar. Fluorescencemicroscopy was performed as described previously (16) using aZeiss Axiovert 200M. Antibodies used were as follows: Eg5 (BD);CP110 (Acris); g-tubulin (Exbio); pericentrin (Abcam); AlexaFluor488 or 568-conjugated secondary antibodies (Molecular Probes).

In vitro kinase assayKinase assay was performed as described previously (32).

ResultsEstablishment of a cell-based high-throughput screening assayfor the identification of small-molecule inhibitors of CC

To identify novel inhibitors of CC, we developed a cell-basedscreening assay that reports on the differential effects of smallmolecules on the viability of two isogenic cell populationswith different centrosome content. Specifically, we engineereda human osteosarcoma cell line (U2OS) to conditionally over-express EGFP-tagged PLK4 (EGFP-PLK4) from a tetracycline-inducible promoter. Under noninduced conditions, only 2%to 3% of EGFP-PLK4-U2OS cells harbored aberrant centrosomenumbers (i.e., >2 g-tubulin signals), whereas 48 hours afterinduction, over 80% of cells exhibited CA, which remained stablefor several days despite tetracycline withdrawal (Fig. 1A–C).Induced EGFP-PLK4-U2OS cells were CC proficient, as 98.8 �0.7% of cells underwent bipolar cell division (n¼ 1,783). To testthe suitability of EGFP-PLK4-U2OS cells for viability-based high-throughput screening, we treated control and induced cells withincreasing concentrations of griseofulvin, an inhibitor of CC (21),for 5 days and subsequently measured the viabilities of both cellpopulations using a luminescence reporter assay based on quan-tification of ATP. As expected, griseofulvin induced more cytotox-icity inEGFP-PLK4-U2OScellswithCAas comparedwith cellswithnormal centrosome content (Fig. 1D). Furthermore, live cell imag-ing demonstrated that treatment of induced EGFP-PLK4-U2OScells with 4 mmol/L griseofulvin (i.e., the concentration with thelargest viability difference between control and induced cells)increased the rate of multipolar divisions by more than 5-fold incomparison with DMSO (Fig. 1E). More than 80% of the progenyof multipolar divisions underwent cell death, in comparison withonly 17% of the progeny of bipolar divisions (Fig. 1F).

Identification of CP-673451 and crenolanib as inhibitors of CCTo identify new cell-permeable molecules that target CC, we

screened two small-molecule libraries consisting of 843 FDA-approved compounds and 273 kinase inhibitors (Fig. 2A).The FDA-approved library was screened at a concentration of 10mmol/L, whereas the kinase inhibitor library was screened at threedifferent concentrations (100 nmol/L, 1 mmol/L, and 10 mmol/L)because of the concentration-dependent target specificity ofmanykinase inhibitors. Hits were ordered according to their CC inhi-bition (CCI) index, calculated as ratio of viabilities betweencontrol and induced cells, and normalized to the viability ratioof vehicle-treated populations. Thus, a positive CCI index indi-cated that a smallmolecule compromised the viability of inducedcells with CA over that of noninduced controls. For further

Cofilin Activation Inhibits Centrosome Clustering

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evaluation, we chose the kinase inhibitors, CP-673451 and CP-868596(crenolanib),because theyexhibited thehighestCCI indexvalues and due to their structural homology (Fig. 2B and C; Sup-plementary Table S1). Both compounds are composed of amino-piperidine-, quinoline- and benzimidazole-ring systems andtermed quinolinobenzimidazoles. Detailed dose–response viabil-ity analyses revealed that the presence of supernumerary centro-somes reduced IC50 values of CP-673451 and crenolanib from1.6to 0.6 mmol/L and 1.2 to 0.6 mmol/L, respectively (Fig. 2D).

CP-673451 and crenolanib inhibit CCConsistent with the increased cytotoxicity seen in cells with

CA, live cell imaging demonstrated that CP-673451 and cre-

nolanib increased the percentage of multipolar divisions ofinduced EGFP-PLK4-U2OS cells by approximately 3-fold at 1mmol/L and 5-fold at 2 mmol/L (Fig. 3A). To test whethermultipolar divisions were caused by centrosome declustering,we treated control and induced EGFP-PLK4-U2OS cells withincreasing concentrations of both compounds and quantifiedthe percentage of multipolar telophases, resulting in morethan two daughter cells. As expected, both drugs increasedthe rate of multipolar telophases in a dose-dependent manner,reaching maxima of about 20% at 2 mmol/L. The percentageof multipolar telophases in control cells remained less than 2%,indicating that only cells carrying supernumerary centrosomeswere prone to multipolar cell division (Fig. 3B and C).

Figure 1.

Performance assessment of EGFP-PLK4-U2OS cells for high-throughput small-molecule screening.A, schematic overview of the screening concept. Induced (þTet)and noninduced (�Tet) EGFP-PLK4-U2OS cells are exposed to small molecules. Induction of spindle multipolarity by CC inhibitors (CCI) will selectively impairsurvival of cells with CA. B, mean percentages � SD of EGFP-PLK4-U2OS cells with more than two g-tubulin signals. Tetracycline (Tet) was removed 48 hoursafter induction. C, noninduced cells (counts/sample� 500, averaged from two independent experiments).C, representative images of noninduced (left) and induced(right) EGFP-PLK4-U2OS cells. Cells were treated with vehicle (�Tet) or tetracycline (þTet) for 48 hours and stained for g-tubulin (red), CP110 (green),and DNA (blue) 96 hours postinduction. Scale bar, 10 mm. D, dose–response curves � SD comparing relative viabilities of induced (þTet) and noninduced (�Tet)EGFP-PLK4-U2OS cells after 5 days of exposure to griseofulvin (1–7 mmol/L; � , P < 0.02; �� , P < 0.01; n ¼ 3). E, time-lapse imaging over 48 hours showing averagepercentages of multipolar divisions in induced EGFP-PLK4-U2OS cells after exposure to 4 mmol/L griseofuilvin (GF) from two independent experiments. F, fate ofprogeny resulting from bipolar and multipolar divisions of induced EGFP-PLK4-U2OS cells after exposure to 4 mmol/L griseofulvin. Daughter cells weretracked by time-lapse microscopy for up to 48 hours.

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Importantly, virtually all multipolar telophases exhibited cen-trioles at each pole (100/101 for 1 mmol/L CP-673451, 91/91for 1 mmol/L crenolanib), emphasizing the inhibition of CC byboth compounds (Fig. 3D). Neither CP-673451 nor crenolanibcaused centrosome amplification (Supplementary Fig. S1).

Because a SAC-mediated mitotic delay is required for CC(13, 17), we addressed whether CP-673451 and crenolanibaffect the timing of mitosis. Fluorescence time-lapse microsco-py of dividing HeLa cells, stably expressing H2B-mCherry anda-tubulin-EGFP, revealed that at 1 mmol/L, crenolanibincreased the duration of mitosis by about 2-fold, while CP-673451 did not delay mitosis. These effects were more prom-inent at 2 mmol/L, leading to 2- and 3-fold mitosis prolonga-tion for CP-673451 and crenolanib, respectively (Supplemen-tary Fig. S2). These data indicate that inhibition of CC was notcaused by SAC inactivation.

Finally, we tested the effect of CP-673451 and crenolanibon CC in various cancer and nontransformed cell linesthat harbor varying degrees of spontaneous CA as well as in

3Flag-STIL-HCT116, another cell line with inducible CA result-ing from conditional STIL overexpression (SupplementaryTable S2). Both compounds increased the rates of multipolartelophases by at least 2-fold in all cell lines with CA, includingnonmalignant MCF10A cells, which harbor about 10% CA. Asexpected, no significant multipolarity was observed in BJ fibro-blasts, which do not contain extra centrosomes. Taken together,these observations suggest that both compounds act as inhi-bitors of CC in all cell lines tested and thereby preferentiallyaffect cells that carry supernumerary centrosomes.

Depletion of PDGFR-b has no effect on CCCP-673451 and crenolanib are potent inhibitors of platelet-

derived growth factor receptor b (PDGFR-b; refs. 33, 34). Becauseboth molecules share PDGFR-b as their main target, we nextsought to analyze the effects of RNAi-mediated PDGFR-b deple-tion on CC. Surprisingly, downregulation of PDGFR-b did notincrease the percentage of multipolar divisions in EGFP-PLK4-U2OS cells with CA (Fig. 3E), indicating that inhibition of CC

Figure 2.

CP-673451 and crenolanib show selective lethality toward cells with CA. A, screening timeline. B, scatter plot showing the screening results of the 1 mmol/Lkinase inhibitor library screen. Positive hits with a CCI index >0.3 and adjusted P < 0.05 are highlighted in black. CP-673451 and crenolanib scored the highestvalues. C, molecular structures of CP-673451 (1-(2-(5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl)quinolin-8-yl)piperidin-4-amine) and crenolanib(1-(2-(5-((3-methyloxetan-3-yl)methoxy)-1H-benzo[d]imidazol-1-yl)quinolin-8-yl)piperidin-4-amine). D, dose–response curves � SD comparing relativeviabilities of induced (þTet) and noninduced (�Tet) EGFP-PLK4-U2OS cells after 5 days of exposure to increasing concentrations of CP-673451 and crenolanib(�� , P < 0.01; �� , P < 0.001; n ¼ 3).






caused by both quinolinobenzimidazoles was not mediated byimpaired PDGFR-b signaling.

CP-673451 and crenolanib affect the organization of the actincytoskeleton

U2OS cells treated with 1 to 4 mmol/L CP-673451 or creno-lanib showed a ruffled cell surface as a sign for alterations of thecortical actin cytoskeleton. Phalloidin-FITC staining of the actincytoskeleton revealed that both compounds markedly affectthe morphology of stress fibers and overall actin organization(Fig. 4A). Drug concentrations (1 mmol/L) led to the appear-ance of bundled actin networks instead of characteristic stressfibers. Strikingly, treatment of U2OS cells with 4 mmol/L of

both drugs led to a complete disorganization of stress fibers andthe appearance of aberrant F-actin arrangements. Similar resultswere obtained in other cell lines, including MDA-MB-231,LOVO, and HCT116 (data not shown).

CP-673451 and crenolanib activate cofilinThe observed rearrangements of the actin cytoskeleton indi-

cated that the compounds might affect the regulation of actindynamics. Rapid actin remodeling in response to extracellularstimuli is elicited by the activation of cofilin, which is regulatedby an inhibitory Ser3 phosphorylation (35, 36). To analyzechanges in cofilin activity, we treated unsynchronized U2OScells with increasing quinolinobenzimidazole concentrations

Figure 3.

CP-673451 and crenolanib inhibit CC ininduced EGFP-PLK4-U2OS cells. A,average percentage of multipolardivisions � SD of induced EGFP-PLK4-U2OS cells within the first 24 hours afterexposure to DMSO, CP-673451 (CP), orcrenolanib (Cre) by time-lapse imaging(n ¼ 2). B, average percentages ofmultipolar telophases in control (�Tet)and induced EGFP-PLK4-U2OS cells(þTet) from two independentexperiments, treated with increasingdrug concentrations for 24 hours(counts/sample � 200). C,representative images of normal bipolar(left), clustered bipolar (middle), andmultipolar (right) metaphases andtelophases in EGFP-PLK4-U2OS cellsstained for Eg5 (green), pericentrin(red), andDNA (blue). Scale bars, 10mm.D, multipolar telophase of an inducedEGFP-PLK4-U2OS cell treated with 1mmol/L CP-673451 (24 hours) andstained for CP110 (green), g-tubulin(red), and DNA (blue). Note that part ofthe green signal could be due to residualEGFP-PLK4. Scale bar, 10mm.E, averagepercentage of multipolar telophases ininduced EGFP-PLK4-U2OS cellsafter PDGFR-b knockdown (72 hours)from two independent experiments(counts/sample � 1,000). Immunoblotshowing PDGFR-b depletion; a-tubulinindicates equal loading.

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and assessed the levels of phosphorylated (inactive) cofilinusing an antibody against phospho-Ser3-cofilin. Both com-pounds induced a concentration-dependent reduction of phos-pho-Ser3-cofilin levels, whereas overall cofilin levels remainedunchanged in both noninduced (Fig. 4B) and induced EGFP-PLK4-U2OS cells carrying CA (data not shown). In addition,we treated several other cancer cell lines with increasing con-centrations of CP-673451. Immunoblot analysis of phospho-Ser3-cofilin clearly showed that cofilin was activated in a dose-dependent manner in all cell lines examined (SupplementaryFig. S3). Next, we analyzed whether cofilin becomes activatedin drug-exposed mitotic cells as well. Mitotic U2OS cellsarrested in metaphase by nocodazole in the presence of CP-673451 were separated from interphase cells by intensiveshaking. As expected, the levels of phospho-Ser3-cofilin werereduced in both CP-673451–treated interphase and mitoticcells as compared with controls (Fig. 4C).

Accumulation of active cofilin during mitosis inhibits CCA previous study has shown that the accumulation of active

cofilin during mitosis strongly affects the orientation of themitotic spindle in HeLa cells due to decreased stability ofthe cortical actin meshwork (37). Accordingly, time-lapsefluorescence microscopy analysis of spindle dynamics in HeLacells stably expressing H2B-mCherry and a-tubulin-EGFPrevealed that CP-673451 markedly affected spindle orienta-tion and caused spindle oscillation. Specifically, treatment

with 2 mmol/L CP-673451 increased the average spindlerotation from 23 degrees to 59 degrees and the averageoscillation distance from 7 to 17 mm (Fig. 5A; SupplementaryMovies S1–S3).

BecauseCP-673451 and crenolanib led to cofilin activation andinhibition of CC, we next addressed whether increased cofilinactivity causes CC inhibition. We increased the levels of activecofilin in dividing EGFP-PLK4-U2OS cells with CA by (i) inhibi-tion of cofilin phosphorylation and (ii) increasing overall cofilinlevels. To inhibit cofilin phosphorylation, we suppressed theactivity of LIM kinases (LIMK) using two highly selective, cell-permeable LIMK inhibitors, LIMKi3 (38) anddamnacanthal (32).Time-lapse microscopy analysis of induced EGFP-PLK4-U2OScells after exposure to LIMKi3 or damnacanthal revealed aconcentration-dependent increase of multipolar divisions(Fig. 5B and C), suggesting that cofilin activation disturbs CC.Next, we examined the effect of cofilin overexpression oninhibition of CC by transiently transfecting induced EGFP-PLK4-U2OS cells with wild-type cofilin (Cof-WT), non-phos-phorylatable cofilin (Cof-S3A), or cofilin containing a phos-phomimetic mutation (Cof-S3E). To increase the number ofmitotic events, cells were synchronized in G1–S-phase by asingle thymidine block and released before transfection. Time-lapse microscopy revealed that overexpression of wild-type andconstitutively active but not inactive cofilin significantlyincreased the frequency of multipolar divisions in comparisonwith cells transfected with empty vector (Fig. 5D). These results

Figure 4.

CP-673451 and crenolanib disturb actinorganization associated with cofilinactivation both in interphase andmitotic cells. A, representative imagesof compound-induced disorganizationof the actin network in phalloidin-TRITC–stained U2OS cells aftercompound addition (3 hours).� , magnified view of a rectangular inset.Scale bar, 20 mm. B, immunoblotshowing a decrease of phospho-Ser3-cofilin levels in unsynchronized U2OScells after exposure to CP-673451 orcrenolanib (3 hours), in comparisonwith DMSO. C, CP-673451 decreasesphospho-Ser3-cofilin levels in bothinterphase (I) and mitotic (M) cells.U2OS cells were synchronized withnocodazole in the presence of 1 mmol/LCP-673451 (CP) or DMSO (D). Phospho-Thr927-Eg5 positivity characterizes themitotic fraction. � , longer exposure.






demonstrate that increased amounts of active cofilin in U2OScells with amplified centrosomes perturb CC.

CP-673451- and crenolanib-induced cofilin activation ismediated by SSHs

The putative mechanisms of cofilin activation upon treatmentwith CP-673451 or crenolanib include drug-induced inhibitionof LIMK and/or the activation of SSHs (35). Because insufficientactivity of LIMK leads to the accumulation of active cofilin (39,40), we first analyzed the phosphorylation status of LIMK1 andLIMK2 in U2OS cells after exposure to increasing concentrationsof CP-673451 or crenolanib. Immunoblot analysis using a phos-pho-LIMK1/2 antibody revealed that the levels of phosphorylatedLIMK did not decrease, suggesting that both compounds do notinhibit kinase activity. In fact, LIMK phosphorylation appeared toincrease after compound addition both in interphase (Fig. 6A)and mitotic cells (Fig. 4C). To exclude direct inhibition of LIMK,independent from its phosphorylation status, LIMK1 expressedin kidney HEK293T cells was immunoprecipitated and subjectedto an in vitro kinase assay in the presence of CP-673451, usingHis6-cofilin as a substrate. Autoradiography of incorporated 32P

revealed that exposure to CP-673451 had no effect on LIMK1activity (Fig. 6B), indicating that impaired kinase activity is notresponsible for the decrease in cofilin phosphorylation.

In contrast, cofilin activation might be triggered by increasedSSH activity. Indeed, we observed that transient overexpression ofGFP-tagged SSH1 in U2OS cells decreased phospho-cofilin tosimilar levels as exposure to CP-673451 or crenolanib (Fig. 6C).To examine the involvement of SSH in drug-induced cofilinactivation, we depleted SSH isoforms 1, 2, or 3 from U2OS cellsand monitored cofilin activation after exposure to both quinoli-nobenzimidazoles. RNAi-mediated depletion of SSH1 and SSH2partially rescued drug-induced inhibition of cofilin phosphory-lation (Fig. 6D). Importantly, SSH2 depletion had the mostpronounced effect, while knockdown of SSH3 failed to rescuecofilin activation. Next, we investigated whether SSH depletioncan also rescue drug-induced CC inhibition. We depleted eachSSH isoform in induced EGFP-PLK4-U2OS cells with CA andassessed CC by time-lapse microscopy during the first day fol-lowing addition of the compounds. Despite relatively low knock-down efficiencies (Supplementary Fig. S6), silencing of SSH1 andSSH2 partially rescued induction of multipolar cell divisions by

Figure 5.

Cofilin activation inhibits CC. A, spindlerotation (left, angle variation betweeninitial metaphase plate and anaphase)and oscillation (right, cumulative traveldistance of the metaphase plate center)induced by 2 mmol/L CP-673451 asquantified by fluorescence time-lapseimaging of H2B-mCherry-a-tubulin-EGFP-HeLa cells. Bars, averages. Cellswere synchronized in G1–S-phase(thymidine, 18 hours), drug orDMSOwereadded 3 hours after release, and imagingstarted 6 hours after release (nangle¼ 100;nmovement¼ 70; �� , P <0.0001).B andC,average percentages � SD of multipolardivisions from two independentexperiments in induced EGFP-PLK4-U2OScells after exposure to the indicatedconcentrations of LIMKi3 (B) ordamnacanthal (C), determined by time-lapse microscopy over the first24 hours after drug addition.Immunoblots showing the respectivephospho-Ser3-cofilin levels are shown inthe corresponding Supplementary Fig.S4A and S4B. D, average percentages ofmultipolar divisions in induced EGFP-PLK4-U2OS cells, transiently transfectedwith the indicated constructs,determined by time-lapse microscopyover the first 12 hours after transfection(� , P < 0.05; �� , P < 0.01; n ¼ 5;Supplementary Fig. S5A and S5B).

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both drugs (Fig. 6E). Again, SSH2 knockdown had the strongesteffect and decreased the percentage of multipolar divisionsinduced by CP-673451 and crenolanib by 35% and 43%, respec-tively, whereas depletion of SSH3 had no effect. These resultscorrelate with cofilin activation observed in U2OS cells undersimilar conditions (Fig. 6D), emphasizing the negative effect ofcofilin activation on CC. It can be concluded that CP-673451 andcrenolanib-induced cofilin activation is mediated by slingshotphosphatases 1 and 2.

CP-673451 and crenolanib activate PI3K/Akt and MEK/ERKsignaling under physiologic conditions

Earlier studies have demonstrated that isoforms of PI3K playan important role in mediating extracellular signals leading tothe activation of SSH1 and SSH2, resulting in cofilin activationand actin cytoskeleton rearrangement (41, 42). As direct SSHactivation by CP-673451 was not observed (Supplementary Fig.S7), we next examined whether PI3K signaling is required forCP-673451–induced cofilin activation by SSHs. We preincu-bated U2OS cells with the PI3Ka inhibitor BYL719 before theaddition of CP-673451. Immunoblot analysis showed thatcofilin phosphorylation was partially rescued by BYL719 (Fig.7A). Similar results were obtained by preincubating cells withthe pan-PI3K inhibitor wortmannin, but not the PI3Kd-specificinhibitor CAL-101 (data not shown). Importantly, pretreat-ment of induced EGFP-PLK4-U2OS cells with BYL719 alsopartially rescued CP-673451–induced multipolar divisions intime-lapse microscopy experiments (Fig. 7B). These results

suggest that SSH-mediated cofilin activation by quinolinoben-zimidazoles may be mediated by PI3K. However, both com-pounds are known to potently inhibit PDGFR-b, and severalstudies have demonstrated their inhibitory effects on PDGFR-bdownstream signaling (33, 34, 43, 44). To assess the effects ofselective PDGFR-b inhibition on cofilin activation, we depletedPDGFR-b by RNAi and found levels of phospho-cofilin to beunaltered (Fig. 7C). In addition to our finding that PDGFR-bdepletion had no effect on CC (Fig. 3E), we conclude that CP-673451- and crenolanib-induced cofilin activation is indepen-dent of PDGFR-b.

To gain further insights into signaling alterations caused byboth quinolinobenzimidazoles, we next analyzed the impact ofthese compounds on Akt and MEK, the main signaling branchesdownstream of several RTKs, in different cancer cell lines. Undernormal growth conditions, CP-673451 unexpectedly elevated thelevels of phospho-Akt and phospho-MEK in almost all cell lineswithin 3 hours of exposure (Supplementary Fig. S8A). AlthoughAkt phosphorylation was not increased in U2OS cells at that timepoint, CP-673451 treatment led to a significant increase in phos-pho-Akt levels at 24 hours in a dose-dependent manner (Supple-mentary Fig. S8B). In conclusion, both compounds stimulate Aktand MEK in cultured cells.

Because crenolanib acts as a type I tyrosine kinase inhibitor(TKI) and binds preferentially to phosphorylated RTKs (45,46), we reasoned that CP-673451 and crenolanib would inhibitRTK signaling only when receptors are in their active confor-mation. To validate this hypothesis, we assessed Akt and MEK

Figure 6.

CP-673451- and crenolanib-induced cofilin activation is mediated by SSH1 and SSH2. A, analysis of LIMK1/LIMK2 phosphorylation (Thr508/Thr505) in U2OS cellsexposed to indicated drug concentrations or DMSO for 3 hours. B, LIMK1 kinase activity assay. Ectopically expressed Myc-hLIMK1 was immunoprecipitated fromHEK293T cells and kinase activity was analyzed in vitro, by comparing the amounts of incorporated 32P into His6-cofilin. CP-673451 was added to the assaybuffer at the indicated concentrations. LIMK1-D460A–inactive mutant (DA) and damnacanthal were used as controls. Whole-cell lysates show overexpression ofMyc-hLIMK1 variants. C, phospho-Ser3-cofilin levels in U2OS cells transiently transfected with empty vector (GFP) or GFP-SSH1L (24 hours) and treated with DMSO(D), 2 mmol/L CP-673451 (CP), or crenolanib (Cre; 3 hours). D, partial rescue of drug-induced cofilin activation by knockdown of SSH1 and SSH2. U2OS cellswere transfected with RNAi pools against SSH1, SSH2, SSH3, or control (72 hours) and exposed to 2 mmol/L compound (3 hours). Silencing was validated byqPCR (Supplementary Fig. S6) and for SSH1L by immunoblotting as well (top). Relative levels of phospho-Ser3-cofilin are indicated for each sample. E, averagepercentages � SD of multipolar divisions in induced EGFP-PLK4-U2OS cells, depleted for the indicated SSH isoform (72 hours) and treated with DMSO or1 mmol/L compound. Quantification was done by time-lapse microscopy during the first 24 hours after drug addition (� , P < 0.05; �� , P < 0.01; �� , P ¼ 0.001;�� , P < 0.001).






phosphorylation (as indicators of PDGFR-b downstream sig-naling) in PDGF-BB–stimulated and nonstimulated U2OScells pretreated with either CP-673451 or crenolanib. Asexpected, stimulation of PDGFR-b strongly enhanced Aktand MEK phosphorylation and activated cofilin (Fig. 7D).Preincubation with CP-563451 and crenolanib attenuatedPDGFR-BB–induced Akt and MEK activation, demonstratingtheir inhibitory role on PDGFR-b signaling. In contrast, expo-sure of nonstimulated, serum-starvedU2OScells toCP-673451orcrenolanib increased Akt and MEK phosphorylation. This con-firms that the inhibitory capability of bothmolecules depends onthe RTK activation state. Finally, we tested whether PDGFR-b wasrequired for drug-induced activation of downstream signaling innonstimulated cells. Exposure of PDGFR-b–depleted U2OS cellsto CP-673451 and crenolanib still resulted in Akt and MEKphosphorylation (Supplementary Fig. S8C), indicating that otherkinases are involved in this signaling.

DiscussionBecause CC is regarded as a promising target for cancer

treatment, several studies have focused on the characterization

of this mechanism, the discovery of new druggable targetproteins, and the identification of small-molecule inhibitors.To date, most cell-based assays have utilized high-contentmicroscopic imaging (13, 14, 21, 47). However, these screensdelivered little information on direct cellular cytotoxicityand thus therapeutic potential because their readouts wereconfined to metaphase multipolarity induction. In this study,we employed a screening concept to identify small-moleculeinhibitors of CC based on differential viabilities of inducedversus noninduced isogenic EGFP-PLK4-U2OS cells. Highlevels of CA and robust CC in these cells allowed for theidentification of small molecules, which selectively interferewith the mechanisms of CC.

With this screening approach, we identified CP-673451 andcrenolanib (CP-868596), two molecules with similar chemicalstructures and proven antitumor activity, as inhibitors of CC. Atclinically relevant concentrations (48), both compounds effec-tively inducedmultipolar cell divisions and consequent cell deathin EGFP-PLK4-U2OS cells with CA as well as in a variety of cancercell lines harboring different degrees of spontaneous CA. Impor-tantly, drug-induced multipolarity was restricted to cells withsupernumerary centrosomes and did not lead to the formation

Figure 7.

CP-673451 and crenolanib stimulate Akt and MEK signaling under physiologic conditions. A, immunoblot analysis of phospho-Ser3-cofilin levels in U2OS cellspretreated with BYL719 for 2 hours, followed by addition of 2 mmol/L CP-673451 (CP) or DMSO (D) for 3 hours. Phospho-Ser473-Akt levels indicate PI3K inhibition.MCM7 shows equal loading. B, average percentage � SD of multipolar cell divisions of induced EGFP-PLK4-U2OS cells, pretreated with BYL719 (2 hours) andexposed to 1 mmol/L CP-673451 in the continuous presence of BYL719. Quantificationwas done by time-lapsemicroscopy during the first 24 hours after the additionof CP-673451 (�� , P <0.001; n¼ 3).C, immunoblot showing the effects of RNAi-mediated PDGFR-b silencing (72 hours) in U2OS cells on phospho-Ser3-cofilin levels.Akt andMEK phosphorylationwas detected using phospho-Ser473-Akt and phospho-Ser217/221-MEK1/2 antibodies.D, immunoblot comparing downstreamMEK1/2-Ser217/221-, Akt-Ser473-, and cofilin-Ser3-phosphorylation between PDGF-BB–stimulated and nonstimulated U2OS cells in the presence or absence ofcompounds. Starved U2OS cells were preincubated with DMSO (D), 2 mmol/L CP-673451 (CP), or crenolanib (Cre) for 2 hours before PDGF-BB addition.

Konotop et al.





of acentrosomal spindle poles as seen with the other inhibitors ofCC (21–23, 47).

Previous studies have shown that CC is inhibited upon inter-ference with spindle pole integrity, microtubule–kinetochoreattachment, SAC activation, or cortical actin cytoskeleton(13, 14, 17). The absence of acentriolar spindle poles in cellstreatedwithCP-673451 and crenolanib suggests that spindle poleintegrity is not affected by these compounds. As both CP-673451and crenolanib prolonged the average duration ofmitosis but didnot induce mitotic arrest, SAC inactivation and interference withmicrotubule–kinetochore attachment is unlikely.

We show here that CP-673451 and crenolanib affect the orga-nization of cortical actin filaments by the activation of cofilin.Cofilin is one of the key regulators of actin remodeling in responseto external stimuli; it promotes severing and dissociation of F-actin filaments and increases the cellular pool of G-actin for newfilament growth (36). Cofilin activity is negatively regulated bySer3 phosphorylation, mediated by LIM-domain kinases (LIMK1and LIMK2) and related testicular kinases TESK1 and TESK2.Cofilin dephosphorylation ismainly regulated by slingshot phos-phatases SSH1, SSH2, and SSH3 (35).

We demonstrate that cofilin activation in EGFP-PLK4-U2OScells with CA inhibits CC. A previous study showed that elevatedlevels of active cofilin strongly affect spindle orientation andpositioning in dividing HeLa cells with regular centrosome con-tent (37).We observed similar effects upon exposure of HeLa cellsto CP-673451 and crenolanib. In cells with extra centrosomes,normal actin and actin-based contractility has been shown topromote bipolar spindle assembly and suppress spindle multi-polarity (13). In accordance with our results, an independentstudy identified LIMK2 and TESK1 as important regulators of CC(15).

Our results indicate that CP-673451 and crenolanib stimulatephosphatase activity of SSH1 and SSH2 to decrease cofilin phos-phorylation. SSH1 and SSH2 are known to be activated byexternal factors that involve production of PI(3,4,5)P3 (41, 42).Accordingly, we foundPI3K inhibition byBYL719orwortmanninto partially rescue CP-673451/crenolanib-induced cofilin activa-tion, suggesting that both drugs activate cofilin, at least in part,through PI3Ka stimulation.

It is important to note that CP-673451 has been described to bea highly selective ATP-competitive inhibitor of PDGFR-b (33).Similarly, crenolanib is a potent TKI with strongest affinity forPDGF-a and -b receptors and FLT3 (34). Stimulation of severaldifferent RTKs and G protein–coupled receptors, for example,insulin receptor, formyl peptide receptor 1, and PDGFR-b, pro-motes cofilin activity via activation of SSH1/2 to generate rapidturnover of actin filaments in different cell types (42, 49–51).Accordingly, although PDGFR-b depletion did not affect cofilinregulation, we corroborate that stimulation of U2OS cells withPDGF-BB decreases overall cofilin phosphorylation. As CP-673451 and crenolanib stimulated cofilin activation in all testedcell lines in a concentration-dependent manner, we conclude thatthis effect is not mediated by RTK/PDGFR-b inhibition.

Our data suggest that the downstream inhibitory effect of thesecompounds is dependent on the activation state of PDGFR-b.Although CP-673451 and crenolanib attenuated PDGF-BB–induced Akt and MEK activation, in the absence PDGF-BB stim-ulation, they enhanced downstream Akt and MEK pathway sig-naling in almost all cell lines tested. These observations may beexplained by the fact that crenolanib behaves as a type I TKI andtherefore preferentially binds to RTKs in their active conformation(45). Its affinity toward active FLT3 is more than 10-fold higherthan toward inactive FLT3, and for ABL1, phosphorylationincreases drug affinity by 7-fold (46). To the best of our knowl-edge, no data concerning this matter are available for CP-673451.

In summary, we present a novel high-throughput screeningconcept for the identification of small molecules that inhibit CC.By applying this strategy, we have identified CP-673451 andcrenolanib as inhibitors of CC with increased cytotoxicity forcells with CA. Both compounds induce multipolar cell divisionand subsequent cell death by cofilin-mediated disruption of thecortical actin cytoskeleton, reemphasizing the importance ofcortical actin for CC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: G. Konotop, A. Kr€amer, M.S. RaabDevelopment of methodology: G. Konotop, M. Boutros, A. Kr€amer, M.S. RaabAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Konotop, E. Bausch, T. Nagai, A. Turchinovich,K. Mizuno, A. Kr€amerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Konotop, E. Bausch, N. Becker, A. Benner,K. Mizuno, A. Kr€amer, M.S. RaabWriting, review, and/or revision of the manuscript: G. Konotop, N. Becker,A. Benner, A. Kr€amer, M.S. RaabAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Kr€amerStudy supervision: A. Kr€amer, M.S. Raab

AcknowledgmentsWe thank Barbara Schmitt and Thilo Miersch for excellent technical assis-

tance and advice during the small-molecule screen. We acknowledge BiancaKraft for the 3Flag-STIL-HCT116 cells and Marion Schmidt-Zachmann for theNO66 antibody.

Grant SupportThis study was supported by the Max-Eder program of the German Cancer

Aid (Deutsche Krebshilfe; awarded to M.S. Raab) and a German ResearchFoundation (DFG) grant (KR 1981/4-1 to A. Kr€amer).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 8, 2016; revised July 15, 2016; accepted August 21, 2016;published OnlineFirst September 13, 2016.

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Konotop et al.




2016;76:6690-6700. Published OnlineFirst September 13, 2016.Cancer Res Gleb Konotop, Elena Bausch, Tomoaki Nagai, et al. DestabilizationSlingshot-Mediated Cofilin Activation and Actin Cortex Pharmacological Inhibition of Centrosome Clustering by

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