Dinaciclib Induces Anaphase Catastrophe in Lung …mct.aacrjournals.org/content/molcanther/15/11/2758.full.pdf · Lung Cancer Cells via Inhibition of Cyclin-Dependent Kinases 1 and

Cancer Biology and Signal Transduction

Dinaciclib Induces Anaphase Catastrophe inLung Cancer Cells via Inhibition of Cyclin-Dependent Kinases 1 and 2AlexeyV.Danilov1,2, ShanhuHu3, BernardoOrr2,4, KristinaGodek2,4, LisaMariaMustachio3,David Sekula3, Xi Liu5, Masanori Kawakami5, Faye M. Johnson5, Duane A. Compton2,4,Sarah J. Freemantle3,4, and Ethan Dmitrovsky2,3,5,6

Abstract

Despite advances in targeted therapy, lung cancer remainsthe most common cause of cancer-related mortality in theUnited States. Chromosomal instability is a prominent featurein lung cancer and, because it rarely occurs in normal cells, itrepresents a potential therapeutic target. Our prior work dis-covered that lung cancer cells undergo anaphase catastrophe inresponse to inhibition of cyclin-dependent kinase 2 (CDK2),followed by apoptosis and reduced growth. In this study, theeffects and mechanisms of the multi-CDK inhibitor dinaciclibon lung cancer cells were investigated. We sought to determinethe specificity of CDK-dependent induction of anaphase catas-trophe. Live cell imaging provided direct evidence that dinaci-clib caused multipolar cell divisions resulting in extensivechromosome missegregation. Genetic knockdown of dinaciclibCDK targets revealed that repression of CDK2 and CDK1, but

not CDK5 or CDK9, triggered anaphase catastrophe in lungcancer cells. Overexpression of CP110, which is a mediator ofCDK2 inhibitor–induced anaphase catastrophe (and a CDK1and 2 phosphorylation substrate), antagonized anaphase catas-trophe and apoptosis following dinaciclib treatment. Consis-tent with our previous findings, acquisition of activated KRASsensitized lung cancer cells to dinaciclib-mediated anaphasecatastrophe and cell death. Combining dinaciclib with themitotic inhibitor taxol augmented anaphase catastrophe induc-tion and reduced cell viability of lung cancer cells. Thus, themulti-CDK inhibitor dinaciclib causes anaphase catastrophe inlung cancer cells and should be investigated as a potentialtherapeutic for wild-type and KRAS-mutant lung cancer, indi-vidually or in combination with taxanes. Mol Cancer Ther; 15(11);2758–66. �2016 AACR.

IntroductionCyclin-dependent kinases (CDK) regulate the cell cycle and are

responsible for its orderly progression (1). Tobecome catalyticallyactive, CDKs associate with specific cyclins during the different

phases of the cell cycle. CDK4/6-cyclin D and CDK2/cyclin Ecomplexes sequentially phosphorylate the retinoblastoma (Rb)protein leading to its inactivation, thus facilitating progressionthrough the G1–S checkpoint (1). CDK1 and its partners cyclins Aand B then ensure G2–M phase transition (1). In cancer cells,diverse genetic and epigenetic events result in overexpression ofcyclins, constitutive activation of CDKs, loss of CDK inhibitors(such as p27 and p16), and mutations of the retinoblastomaprotein (1, 2). These events lead to cell-cycle deregulation andconfer a selective growth advantage to cancer cells. CDK activity isnot restricted to cell-cycle proteins. CDK7/cyclin H and CDK9/cyclin T promote phosphorylation of the carboxy-terminaldomain of RNA polymerase II, facilitating initiation and elonga-tion of RNA transcription, respectively (3, 4). Inhibitors of CDKactivity have been developed and are undergoing evaluation asanticancer treatments. CDK inhibitors that act on CDK1, 2, and 9(5–7) or CDK4/6 (8) have shown preclinical antitumor activities.In mouse knockout studies, there is functional redundancybetween CDK2, 4, and 6 but not CDK1 (9–12). It is not clearwith current CDK inhibitors what is or are the dominant targets ofthese multi-CDK inhibitors.

CDK2 and cyclin E are aberrantly expressed and confer unfa-vorable prognosis in non–small cell lung cancer (NSCLC; refs. 13,14). Our prior work provided direct evidence for the importanceof cyclin E in lung carcinogenesis (15). Transgenic mouse modelswere engineered with surfactant C–targeted cyclin E expression inthe lung (15). This conferred chromosomal instability and caused

1Department of Medicine, Geisel School of Medicine at Dartmouth,Hanover, NewHampshire. 2Norris CottonCancerCenter,Geisel Schoolof Medicine at Dartmouth, Hanover, New Hampshire, and Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire. 3Department ofPharmacology and Toxicology, Geisel School of Medicine at Dart-mouth, Hanover, New Hampshire. 4Department of Biochemistry,Geisel School of Medicine at Dartmouth, Hanover, New Hampshire.5Department of Thoracic/Head and Neck Medical Oncology, TheUniversity of Texas MD Anderson Cancer Center, Houston, Texas.6Department of Cancer Biology,TheUniversity of TexasMDAndersonCancer Center, Houston, Texas.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

A.V. Danilov and S. Hu contributed equally to this work.

Current address for A.V. Danilov: Knight Cancer Institute, Oregon Health andScience University, Portland, OR 97219; and current address for E. Dmitrovsky:The University of Texas MD Anderson Cancer Center, Houston, TX 77030-4009.

Corresponding Author: Ethan Dmitrovsky, Department of Thoracic/Head andNeck Medical Oncology, The University of Texas MD Anderson Cancer Center,1400 Pressler Street. Unit 1492, Houston, TX 77030. Phone: 713-745-4495; Fax:713-745-1812; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-16-0127

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

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lung cancers to form in mice with tumors recapitulating keyfeatures of human lung carcinogenesis (15). Furthermore, usinglung cancer cells derived from this model, CDK2 inhibition wasfound to trigger anaphase catastrophe, a lethal event where cellswith supernumerary centrosomes segregate chromosomes intomore than 2 daughter cells, resulting in nonviable daughter cells(16–18).In this study, the mechanism of anaphase catastropheinduced by CDK inhibition was further investigated using thenovel multi-CDK inhibitor dinaciclib.

It has been postulated that by simultaneously targetingmultiple CDKs involved in both the cell cycle and transcription,the drug potency would become enhanced (5, 19). Dinaciclibinhibits CDK1, 2, 5, and 9 with an IC50 value of 3, 1, 1, and 4nmol/L, respectively (20). Dinaciclib has exhibited preclinicalactivity in multiple tumors, including pancreatic cancer (6),melanoma (21), and B-cell malignancies (22). In this currentstudy, it is shown that dinaciclib induces anaphase catastrophein lung cancer cells via inhibition of CDK1 and CDK2, but notof CDK5 or CDK9. Activated KRAS mutations sensitized lungcancer cells to dinaciclib-mediated anaphase catastrophe andcell death. Activated KRAS mutations are known to enhanceresistance to chemotherapy including tyrosine kinase inhibitors(23); however, dinaciclib was similarly effective as an antipro-liferative agent in wild-type and mutant KRAS lung cancer celllines. Finally, when combined with the anti-microtubule agentand mitotic inhibitor Taxol, the effects of dinaciclib wereenhanced in NSCLC cell lines. In summary, these data providea strong rationale to study further multi-CDK inhibitors,including dinaciclib, as potential therapeutic agents for lungcancers. This is especially proposed for NSCLC cases harboringactivated KRAS mutations.

Materials and MethodsCell culture and drugs

ED1 murine lung cancer cell line was derived from transgenicmice harboring lung cancers expressing human surfactant C–driven wild-type cyclin E in 2007 (15). Human lung cancer celllines (HOP62, H522, H23, H1299, and H1703) were obtainedfrom ATCC in 2010. Each cell line was briefly cultured and frozenin liquid nitrogen, andonly early passages (<2months of passage)of each cell line were used in these experiments. Cell lines werecultured inRPMI-1640 supplementedwith 10%FBS (Lonza), 100U/mL penicillin, and 100 mg/mL streptomycin at 37�C in 5%CO2

in a humidified incubator. Dinaciclib (SCH727965) was provid-ed by Merck Research Laboratories. Taxol was obtained from LCLaboratories. Dimethyl sulfoxide (10mmol) stock solutions wereprepared for each agent and stored at �20�C.

Cell viability and proliferation assayApoptosis was measured in duplicate using the ApoScreen

Annexin V Apoptosis Kit, as previously described (24). Briefly,cells were trypsinized, washed in PBS, and resuspended in 150 mLof Annexin V binding buffer; 1 mL of Annexin V-PE and 1 mL of 7-AAD (Southern Biotech) were added. Cells were incubated for 15minutes protected from light on ice, followed by flow cytometricanalysis on FACSCalibur (Becton Dickinson). To determine cellproliferative activity, cells were plated in 96-well plates (3,000 perwell in 100 mL, 6wells per sample) and treated with drugs the nextday. After 48hours of culture,MTT (Sigma-Aldrich)was added at afinal concentration of 0.55 mg/mL. Acid isopropanol was added

after 4 hours, and absorbance at 570 nm was measured using anEMax Precision Microplate Reader (Molecular Devices).

Immunoblot analysesCells were lysed in RIPA buffer [20 mmol/L Tris, 150 mmol/L

NaCl, 1% NP-40, 1 mmol/L NaF, 1 mmol/L Na3PO4, 1 mmol/LNaVO3, 1 mmol/L EDTA, 1 mmol/L EGTA, supplemented withprotease inhibitor cocktail (Roche) and 1mmol/L phenyymethyl-sulfonyl fluoride (PMSF)]. Proteins were analyzed by immuno-blotting, as described (24). The following antibodies were used:cleaved PARP, survivin, CDK1, CDK2, phospho-RbS780 (CellSignaling); phospho-RbT821 (Life Technologies); Rb (C-15),CP110 (N-14), K-Ras (F234; Santa Cruz Biotechnology); phos-pho-RNA polymerase II ser-2 (H5) and ser-5 (H14), total RNApolymerase (8WG16, all from Covance); b-actin (Sigma), andhorseradish peroxidase–conjugated secondary anti-mouse andanti-rabbit antibodies (Bio-Rad).

siRNA-mediated gene silencing and transfectionsThe siRNA-mediated gene silencing in lung cancer cells was

performed using Lipofectamine 2000 Plus (Life Technologies).The siRNA oligonucleotides targeting CDK1 (CDK1.1, sensestrand 50-GGAACUUCGUCAUCCAAAUAUAGTC-30, CDK1.2sense strand 50-GACUAACUAUGGAAGAUUAUACCAA-30),CDK2 (CDK2.1, sense strand 50-ACAAGAGCGAGAGGUAUA-CUGCGTT-30, CDK2.2 sense strand 50-GCCACAAUGUUUAU-AAAGGCCAAAT-30), CDK5 (CDK5.1, sense strand 50-GCCAGA-CUAUAAGCCCUAUCCGATG-30 and CDK5.2, sense strand 50-GCGUAUCUCAGCAGAAGAGGCCCTG-30) were synthesized byIntegratedDNATechnologies andCDK9 (CDK9.1 sense strand 50-GGUGCUGAUGGAAAACGAG-30, CDK9.2 sense strand 50-GGA-GAAUUUUACUGUGUUU-30) was from Ambion/Life Technolo-gies. For enforced expression, pCDEF3-CP110 plasmid wasobtained from Dr. Brian D. Dynlacht (25), pcDNA-CDK1 andpcDNA-CDK2 plasmids and vector control (pcDNA) were pur-chased from Addgene. Lung cancer cells were transfected usingXtreme Gene 9 transfection reagent according to the manufac-turer's protocol (Roche).

RT-PCR assaysTotal RNA from cells was isolated using the RNeasy Mini Kit

(Qiagen). The cDNA was synthesized from 500 ng RNA using theiScript cDNA Synthesis Kit (Bio-Rad). qRT-PCR assays were per-formed in aC1000ThermalCycler (Bio-Rad) usingUniversal PCRMaster Mix according to the manufacturer's protocol (AppliedBiosystems), with template cDNA and gene-specific probes. Thefollowing TaqMan probes were used: CDK1: Hs00938777_m1;CDK2: Hs01548894_m1; CDK9: Hs00977896_g1. Amplificationof the sequence of interest was compared with a reference probe(RPS18, #4308329; all from Life Technologies). All samples wereanalyzed in duplicate. We used the comparative Ct method forrelative quantitation (2�DDCt, where DDCt ¼ DCt_P � DCt_K; P ¼probe and K ¼ reference sample). For CDK5 mRNA quantifica-tion, total RNA was isolated from cells using the RNA Easy Kit(Invitrogen). Reverse transcription (RT) was done using the HighCapacity cDNA Reverse Transcription Kit (Applied Biosystems)with a Peltier Thermal Cycler (MJ Research). Quantitative real-time PCR assays were done using SYBR Green PCR Master Mix(Applied Biosystems) and the 7500 Fast Real time PCR System(Applied Biosystems) for quantitation. RT-PCR assays wereconducted following the manufacturer's protocol (Applied

Dinaciclib Inhibits CDK1/2-Inducing Anaphase Catastrophe

www.aacrjournals.org Mol Cancer Ther; 15(11) November 2016 2759




Biosystems). Three replicate experiments were done. Primerssequences were human glyceraldehyde-3-phosphate dehydroge-nase (GAPDH) forward 50-ACGTGTCAGTCAGTGGTGGACCT-30

and reverse 50-GTCCACCACCCTGTTGCTG-30 and human CDK5forward 50-TCTGTGACCAGGACCTGA-30 and reverse 50- GCA-CATTGCGGCTATGAC-30 sequences.

Chromosome stability assayCells were fixed in 10% formalin, stained with anti-a-tubulin–

specific antibody (Sigma Aldrich), and independently mountedwith Pro-Long Gold antifade reagent supplemented with 40,6-diamidino-2-phenylindole (DAPI; Life Technologies). Fluores-cent images were captured with an F-view II monochrome camera(Olympus, U-CMAD3) mounted on an Olympus BX51 micro-scope. Total anaphase cellswere counted and those that contained3 or more spindle poles were scored as multipolar.

Live cell imagingHOP62 and H1299 cells were plated on glass coverslips in

complete RPMI media and treated with 25 to 50 nmol/L dina-ciclib or vehicle control for 24 hours before live cell imaging. Forlive cell imaging, cells were placed in a modified chamber andmaintained at 37�C in complete RPMImedia and25 to 50nmol/Ldinaciclib. Differential interference contrast (DIC) images wereacquiredwith aNikon Eclipse Timicroscope and anAndor cooledCCD camera using a 60� 1.4 NA oil immersion objective. Elevenz-axis optical sections of 1 mm were acquired at 10-minute inter-vals for a total of 25 hours (H1299) and 13 hours 40 minutes(HOP62). Images were processed using Nikon Elements andAdobe Photoshop software. DIC images represent the most in-focus single focal plane at each time point.

Generation of stable KRAS-expressing transfectantsEither pCGN KRAS12V,188L expressing (Addgene) or an empty

vector were transfected into ED1 cells concomitantly with pPURplasmid (Clontech) using Lipofectamine 2000 as per the manu-facturer's protocol. KRAS-transfected pools were selected withpuromycin beginning 24 hours after transfection. Engineeredexpression of the KRAS was confirmed by immunoblotting.

High-throughput proliferation assaysLogarithmically growing cells were seeded at optimized den-

sities for each line into 384-well tissue culture plates in triplicate.Proliferation assayswere performed using theCellTiter-Glo Lumi-nescent Assay (Promega) following 72-hour exposure to serial-fold drug dilutions. IC50 and IC70 values were estimated using thedrexplorer R package.

Statistical analysisResults of individual experiments were analyzed using paired

and unpaired Student t test, Fisher exact test, nonparametricMann–Whitney test, and Spearman r. Statistical analyses werecompleted using the GraphPad Prism 6.07 software package(GraphPad Software, Inc.). All tests were 2-sided, and data wereconsidered to be statistically significant when P < 0.05

ResultsDinaciclib induces multipolar cell division in lung cancer cells

Our previous work reported that pharmacologic inhibition ofCDK2 with seliciclib led to anaphase catastrophe in murine and

human lung cancer cell lines (16, 26, 27). The effect of the multi-CDK inhibitor dinaciclib in inducing apoptosis and anaphasecatastrophe in lung cancer cells was investigated. Apoptosis wasinduced in a dose-dependent manner in ED-1 cells as comparedwith vehicle-treated cells (Fig. 1A). Apoptosis induction wasaccompanied by anaphase catastrophe induction (Fig. 1B; Sup-plementary Fig. S1). At dinaciclib doses of 100 nmol/L or higher,the mitotic index was lower likely due to inhibition of transcrip-tion via CDK9 inhibition (data not shown).

Using real-time live cell imaging techniques, we followed thefate of individual human lung cancer cells undergoing chromo-some missegregation induced by dinaciclib treatment. As shownin Fig. 1C (videos are provided in Supplementary Fig. S2), H1299andHOP62 lung cancer cell lines underwentmultipolar anaphasefollowed bymultipolar cell divisionswhen treatedwith dinaciclib(25 and 50 nmol/L). While this event was shown to be incom-patible with survival (18), apoptosis of daughter cells of multi-polar cell divisions may be delayed by 12 to 72 hours uponcompletion of a multipolar division or occur following oneadditionalmitotic division (18). Decreased cellmetabolic activityasmeasuredby theMTTassaywasdetected in lung cancer cell linesfollowing 24 hours of dinaciclib treatment (Fig. 1D).

Anaphase catastrophe is mediated via CDK1 and CDK2inhibition and CP110

As dinaciclib potently inhibits CDK1, CDK5, and CDK9, inaddition to CDK2, in vitro (20), the consequences of ablation ofthe individual CDKs on anaphase catastrophe induction wereassessed in lung cancer cells. Knockdown of individual CDKs wasconfirmed by RT-PCR assays (Fig. 2A). Knockdown of CDK2resulted in multipolar anaphases in H1299 and HOP62 cells,confirming our earlier observations (Fig. 2B). Notably, knock-down of CDK1 also led to an increase in multipolar anaphases inboth cell lines (Fig. 2B). Finally, knockdown of CDK5 or CDK9did not have an appreciable effect on anaphase catastropheinduction in lung cancer cells (Fig. 2B). We postulated from thesefindings that forced expression of CDK1 or CDK2 would protectlung cancer cells from inducing anaphase catastrophe after dina-ciclib induction. However, increased levels of CDKs in vitro aloneled to an increased frequency of multipolar anaphases and sub-sequent addition of dinaciclib had no appreciable effect, com-plicating experimental interpretation (Supplementary Fig. S3).

Next, expression and phosphorylation of known targets ofCDK1, 2, 5, and 9 were investigated after dinaciclib treatmentsat varying concentrations in lung cancer cells. As expected,decreased phosphorylation of RNA polymerase II in serine 2position in H1299 cells upon exposure to dinaciclib was detectedlikely due to CDK9 inhibition (Fig. 2C). Downregulation ofsurvivin and decreased Rb phosphorylation at residue T821 (Fig.2C) provide evidence for CDK1 and CDK2 inhibition, respec-tively (28, 29). At the same time, RbS780 phosphorylation wasunchanged, indicating that cyclin D and its partners CDK4 andCDK6 were not inhibited by dinaciclib. Dinaciclib also inhibitsCDK5, but as CDK5 function is most prominent in neuronaltissue (30) and knockdown of CDK5 did not induce anaphasecatastrophe in lung cancer cells (Fig. 2B), we did not investigate itsdownstream substrates phosphorylation status.

Our recent work reported that CDK2 inhibition–induced ana-phase catastrophe is mediated through the centrosomal proteinCP110 (26, 27). CP110 is a substrate for both CDK1 and CDK2kinases and is involved in centrosome duplication and separation

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Mol Cancer Ther; 15(11) November 2016 Molecular Cancer Therapeutics2760




(25, 31). Engineered expression of CP110 reduced the frequencyof multipolar anaphases in H1299 and HOP62 cells treated withdinaciclib (Fig. 2D). In contrast, CP110 did not prevent inductionof aberrant anaphases byTaxol, amicrotubule-targeting agent thatis not known to inhibit CDK activity (Fig. 2D). Together, thesedata indicate that dinaciclib induces anaphase catastrophethrough inhibition of both CDK1 and CDK2 and that this ismediated, at least in part, by CP110.

Activated KRAS mutations can sensitize lung cancer cells todinaciclib

Our previous studies reported that activated KRASmutationssensitized lung cancer cells to the CDK2 inhibitor seliciclib(16, 27). This provided a rationale to study whether activatedKRAS mutations also sensitized lung cancer cells to dinaciclib.Three lung cancer cell lines with KRAS mutations (H1299,HOP62, and H23) and 2 without (H522 and H1703) werescreened for NRAS, HRAS, or KRAS mutations within codons12, 13, and 61 using Sanger sequencing, as previously described(32). In aggregate, activated KRAS-mutant lung cancer cell lines

exhibited a higher susceptibility to dinaciclib-induced apopto-sis than wild-type KRAS-expressing lines (Fig. 3A). Concurrent-ly, such cell lines demonstrated higher frequency of multipolaranaphases (Fig. 3B). To further study the biologic effects ofactivated KRAS mutations with minimal cell line genetic back-ground differences in vitro, lentiviral-mediated mutant KRASexpression in ED-1 cells was achieved. Expression of oncogenicKRAS was confirmed by immunoblot analyses (Fig. 3C, bot-tom). ED1 cells that expressed mutant KRAS exhibited a sig-nificant increase in apoptosis induction when treated withdinaciclib compared with mock-transduced cells (Fig. 3C).Increased apoptosis rate was accompanied by significantlyhigher frequency of multipolar anaphases in these cells (Fig.3D). Therefore, the acquisition of activated KRAS increasessusceptibility to dinaciclib-induced anaphase catastrophe andapoptosis in lung cancer cells.

To extend the analysis of lung cancer cell lines, a high-through-put system was used to test the dinaciclib sensitivity of 108 lungcancer cell lines with known KRASmutation status for cell growthusing the CellTiterGlo assay. IC50 and IC70 concentrations were

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Dinaciclib induces anaphasecatastrophe in lung cancer cells.A, ED1cellswere incubatedwith the indicatedconcentrations of dinaciclib or vehiclecontrol for a period of 10 or 24 hours.Cells were trypsinized and theproportion of Annexin Vþ cells wasmeasured by flow cytometry (induplicate). Data are themean� SEof 3independent experiments. B, ED1 cellswere incubated with the indicatedconcentrations of dinaciclib or vehiclecontrol for 24 hours. Cells wereimmunostained with a-tubulin andcounterstained with DAPI. Totalanaphases were counted (�100) andthe proportion of multipolaranaphases is shown. Data are themean� SE of 3 independent experiments.C,H1299 (top) andHOP62 (lower) cellswere treated with 25 or 50 nmol/Ldinaciclib and imaged at the indicatedtime points as described in Materialsand Methods. White asterisksrepresent 3 daughter cells generatedfrom dinaciclib-induced multipolarmitoses. D, cells were incubated withthe indicated concentrations ofdinaciclib or vehicle control for 24hours, followed by a washout. Cellularproliferation was determined usingan MTT assay after 72 hours. Data arethe mean � SE of 3 independentexperiments performed inquadruplicate.






calculated for each cell line. IC50 values ranged from 0.05 to1.4 mmol/L and these data are shown in Supplementary Fig. S4.Fromourprevious study, the average IC50 values for seliciclibwerecloser to 15 mmol/L for lung cancer cell lines (16). This indicatesthe increased potency of dinaciclib as compared with seliciclib.While acquisition of mutant KRAS sensitized cells to dinaciclib-mediated apoptosis, the cell line analysis indicated wild-type andmutant KRAS cell lines were similarly sensitive to dinaciclibtreatment.

Dinaciclib cooperates with taxol to induce anaphasecatastrophe

Taxanes aremicrotubule-targeting agents that induce apoptosisthrough multiple mechanisms including mitotic catastrophe(33). Our prior work indicated that combinations of taxanes withseliciclib, a CDK2 inhibitor, enhanced effects of apoptosis andanaphase catastrophe induction in lung cancer cells as compared

with single-agent treatment (16). Given this, effects of combiningdinaciclib with Taxol in inducing anaphase catastrophe wereinvestigated in a panel of lung cancer cells.

As shown in Fig. 4A, treatment of Taxol alone at the 1 and 5nmol/L dosages did not induce a substantial reduction in cellgrowth in H23 and HOP62 lung cancer cell lines. Similarly,dinaciclib had no appreciable effect on this outcome when usedat low dosages (5 and 10 nmol/L). In contrast, concurrent treat-ment with the low dosages of Taxol and dinaciclib resulted in asignificant (P < 0.05) decrease in lung cancer cell metabolicactivity (Fig. 4A).

The effect of combining Taxol with dinaciclib in anaphasecatastrophe induction was investigated. As shown in Fig. 4B,H23 cells showed at least a 2-fold increase in multipolar ana-phases when treated with Taxol combined with dinaciclib ascompared with single-agent treatments. A less marked but statis-tically significant increase in anaphase catastrophe induction was

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Dinaciclib mediates anaphasecatastrophe via CDK1/2 inhibition.A and B, H1299 and HOP62 cells weretransfected with CDK1-, CDK2-,CDK5-, or CDK9-specific siRNA orcontrol siRNA. The mRNAknockdown was confirmed byRT-PCR assays (A) in H1299 cells andthe proportion of multipolaranaphases was analyzed 48 hourslater (B). C, H1299 cells wereincubated with or without theindicated concentrations of dinaciclibfor 10 or 24 hours. Whole-cell lysateswere subjected to immunoblotting. Arepresentative immunoblot of 3independent experiments is shown.Data are the mean � SE of 3independent experiments performedin duplicate. D, H1299 and HOP62cells were transfected with CP110-expressing vector or insert-lessvector control for 24 hours andincubated with the indicatedconcentrations of dinaciclib orvehicle control for 10 hours. CP110overexpression was confirmed byimmunoblotting (right, H1299 cellsshown). Cells were immunostainedwith a-tubulin and counterstainedwith DAPI. Total anaphases werecounted, and the proportion ofmultipolar anaphases is displayed inthis figure. Data are themean� SE of3 independent experimentsperformed in duplicates.

Danilov et al.





also observed in the Hop62 cells. Analysis of these data revealedthat the combination of dinaciclib with taxol was additive forboth H23 and Hop62 cells. Taken together, dinaciclib cooperateswith a microtubule-stabilizing agent to deregulate anaphase andrestrict growth of lung cancer cells.

DiscussionIt was estimated more than 225,000 were diagnosed with and

nearly 160,000 patients succumbed to lung cancer in 2015,accounting for the most cancer-related deaths in both men andwomen (34). The 5-year survival rate for lung cancer is less than17%, and this drives the development of innovative therapeuticapproaches for lung cancer (34).

Lung cancer cells exhibit substantial chromosomal instability(35). Many cases exhibit aneuploidy and also have structuralcytogenetic abnormalities, such as translocations, deletions, andgene copy number gains, leading to decreased function of tumorsuppressors and enhanced activity of oncogenes. Chromosomalinstability correlates with poor prognosis in lung cancer and othersolid tumors, indicating that increasing genetic diversity contri-butes to enhanced tumor survival and chemoresistance (36).However, because of the propensity for lung cancers to be genet-

ically unstable, this represents a potential tumor-specific thera-peutic target.

We previously demonstrated that either genetic or pharmaco-logic (seliciclib) inhibition of cyclin E–driven CDK2 activityinduces anaphase catastrophe and cell death (16, 27). In thiscurrent work, it is demonstrated that dinaciclib, a novel inhibitorof CDK1/2/5/9, like seliciclib, induces multipolar anaphases inlung cancer cells, leading to apoptosis. It is established here thattreatmentwith dinaciclib leads tomultipolar cell divisions in lungcancer cells, which is incompatible with viable progeny (18) andaccounts for lung cancer apoptosis observed in these studies.

Dinaciclib inhibits both CDK1 and CDK2 with high affinity inin vitro kinase assays (1 and3nmol/L, respectively; ref. 20).Hence,the individual contribution of these kinases to the induction ofanaphase catastrophe may not be elucidated with dinaciclibtreatment alone. In an effort to confirm the role of CDK2 indinaciclib-mediated induction of anaphase catastrophe, geneticknockdown of individual CDKs was achieved in human lungcancer cells. Surprisingly, it was found that, like CDK2 antago-nism, siRNA-mediated knockdownofCDK1 resulted in inductionof anaphase catastrophe in the studied lung cancer cells. In fact,incidence ofmultipolar anaphases occurredwith similar or higherfrequency in response to CDK1 manipulation, compared with

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00 10 25 50

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35P = 0.001

P = 0.003

10 25 50

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Figure 3.

Ras mutations sensitize lung cancercells to dinaciclib. A, RasWT (H522,H1703) and RasMUT (H1299, HOP62,H23) human lung cancer cells wereincubated with the indicatedconcentrations of dinaciclib or vehiclecontrol for 12 hours. Cells weretrypsinized, and the proportion ofAnnexin Vþ cells was measured byflow cytometry in duplicate. B, cellswere scored for multipolar anaphasesafter a 6-hour incubation withdinaciclib. C, activated mutant KRASor vector control–transduced ED1 cellswere incubated with the indicatedconcentrations of dinaciclib or vehiclecontrol for 24 hours. Cells weretrypsinized, and the proportion ofAnnexin Vþ cells was measured byflow cytometry (in duplicates). KRASprotein expression was confirmed byimmunoblotting, whereas theseexperiments were conducted(bottom). D, activated mutant KRASor vector control–transduced ED1 cellswere scored for multipolar anaphasesafter 10-hour incubation withdinaciclib. A–D, data are the mean �SE of at least 3 independentexperiments.






that of CDK2. This finding is in contrast to our previous studieswhere anaphase catastrophe was not observed in response togenetic knockdown of CDK1 (16). The potential reasons are: (i)higher efficiency of CDK1 suppressionwas achieved in the currentwork and (ii) earlier experiments were performed in ED1 cells,which are addicted to cyclin E expression andmaybe therefore lessresponsive to manipulations of CDK1.

CDK1 is amitotic kinase active during G2–Mtransition and hasmultiple phosphorylation targets, many of which are also targetsof CDK2–cyclin complexes (37). We previously reported that thecentrosomal protein CP110 mediates CDK2 inhibition–inducedanaphase catastrophe, as CP110 overexpression partially com-pensates for loss of CDK2 activity (26, 27). As CP110 is alsoregulated byCDK1 (25), it is possible that loss of CDK1-mediatedphosphorylation of CP110 results in enhanced susceptibility toanaphase catastrophe. Consistent with this, engineered overex-

pression of CP110 was found to confer at least partial protectionfrom dinaciclib-induced anaphase catastrophe. Of the uniqueCDK1 targets, survivin is an important mitotic regulator with adistinct antiapoptotic function in cancer (29). CDK1-mediatedphosphorylation prevents survivin degradation, and thus inhibi-tion of CDK1 rapidly downregulates its expression (30). Inter-estingly, interference with survivin function results in formationof abnormal mitotic spindle and multipolar mitoses, similar tothose observed in our study (38). Here, we demonstrated thatdinaciclib treatment results in downregulation of survivin in lungcancer cells, and this could account for the development ofmultipolar anaphases. Thus, it is likely that anaphase catastropheis induced through severalmechanismsdownstreamofCDK1and2 inhibition.

A phase II trial with dinaciclib as a single agent in patients withNSCLC did not report objective responses (39). Yet, as for other

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MTT activity, fold compared to untreated control MTT activity, fold compared to untreated control

% Multipolar anaphases % Multipolar anaphases

**

** *

*

*

* *

B

A

*, P < 0.05; **, P < 0.01

Figure 4.

Dinaciclib cooperates with taxol to inhibit growth and induce anaphase catastrophe in lung cancer cells. A and B, H23 and HOP62 cells were incubated withthe indicated concentrations of dinaciclib, taxol, and combination or vehicle control. Cell proliferationwas determinedusing anMTTassayafter 48hours of incubationwith the indicated drugs, in quadruplicate (A). Also, H23 cells were subjected to Annexin Vþ cell measurements by flow cytometry after 10 hours ofincubation with the indicated drugs (B). H23 and HOP62 cells were incubated with taxol and dinaciclib alone or the drugs combined for 10 hours. Cells were fixed,total anaphases were counted, and the proportion of multipolar anaphases is presented in this figure. Data are the mean � SE of 3 independent experiments.

Danilov et al.





targeted agents, the distinct genetic characteristics of the tumorshould be used to guide therapeutic decision making. Mutationsin the RAS oncogene are present in up to 30% of lung cancers,typically occurring in smokers, and this confers poor prognosis(40). Point mutations in codon 12 of KRAS with G!T transver-sion are among the most common KRAS alterations. Here, wedemonstrate that acquisition of mutant KRAS enhanced sensitiv-ity toward dinaciclib treatments. Human lung cancer cells withmutations in KRAS and murine lung cancer cells geneticallymodified to express activated KRAS demonstrated increased dina-ciclib-mediated anaphase catastrophe and apoptosis. High-throughput analysis of 108 human lung cancer cells did notindicate a significant difference in sensitivity between KRASwild-type or mutant cells. However, KRAS-mutant cancers arefrequently resistant to chemotherapeutic agents that are effectivefor KRAS wild-type cancers (23).

Finally, we observed that dinaciclib cooperates with Taxol, amicrotubule-stabilizing agent, to restrict growth of the lung cancercells. While Taxol alone did not appreciably affect cell growth (atthe studied concentrations), a marked enhancement in the fre-quency of multipolar anaphases was detected in its combinationwith dinaciclib and was accompanied by a reduction in cellviability. We demonstrated a similar additive effect previouslywith seliciclib (16), suggesting that sensitization to anaphasecatastrophe may be one of the underlying mechanisms account-ing for cooperation between the CDK inhibitors and taxanes, 2distinct classes of drugs, which affect cellular progression throughmitosis.

In summary, here we report that dinaciclib disrupts anaphaseprogression and restricts growth of lung cancer cells. Our studyindicates a need to further investigate dinaciclib and other CDKinhibitors as a potential therapeutic approach in lung cancerwith activating RAS mutations, either alone or in combinationwith taxanes.

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

Authors' ContributionsConception and design: A.V. Danilov, S. Hu, E. DmitrovskyDevelopment of methodology: A.V. Danilov, S. Hu, E. DmitrovskyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.V. Danilov, S. Hu, B. Orr, K. Godek, L.M.Mustachio,D. Sekula, M. Kawakami, F.M. Johnson, E. DmitrovskyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.V. Danilov, S. Hu, B. Orr, K. Godek, L.M. Musta-chio, X. Liu, M. Kawakami, S.J. Freemantle, E. DmitrovskyWriting, review, and/or revisionof themanuscript:A.V.Danilov, S.Hu, B.Orr,K. Godek, M. Kawakami, D.A. Compton, S.J. Freemantle, E. DmitrovskyAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.V. Danilov, X. Liu, E. DmitrovskyStudy supervision: D.A. Compton, E. Dmitrovsky

Grant SupportThis study was supported by the National Cancer Institute grants R01-

CA087546 (E. Dmitrovsky and S.J. Freemantle) and R01-CA190722 (E. Dmi-trovsky and S.J. Freemantle), the Samuel Waxman Cancer Research FoundationAward (E. Dmitrovsky and D.A. Compton), by an American Cancer SocietyClinical Research Professorship (E. Dmitrovsky), by a UT-STARS Award (E.Dmitrovsky), by a grant fromUniting Against Lung Cancer with Mary Jo's Fundto Fight Cancer (S.J. Freemantle), and by an American Cancer Society Postdoc-toral Fellowship (K.G Godek). Support to A.V. Danilov was provided by aNational Cancer Institute new faculty award (3P30CA023108-31S4) to theNorris Cotton Cancer Center.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 3, 2016; revised July 28, 2016; accepted August 10, 2016;published OnlineFirst August 22, 2016.

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2016;15:2758-2766. Published OnlineFirst August 22, 2016.Mol Cancer Ther Alexey V. Danilov, Shanhu Hu, Bernardo Orr, et al. Inhibition of Cyclin-Dependent Kinases 1 and 2Dinaciclib Induces Anaphase Catastrophe in Lung Cancer Cells via

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