targeting cdk1 and mek/erk overcomes apoptotic ......cell death and survival targeting cdk1 and...

Cell Death and Survival

Targeting CDK1 and MEK/ERK OvercomesApoptotic Resistance in BRAF-Mutant HumanColorectal CancerPeng Zhang1, Hisato Kawakami1,Weizhen Liu2, Xiangyu Zeng2, Klaus Strebhardt3,Kaixiong Tao2, Shengbing Huang1, and Frank A. Sinicrope1,4

Abstract

TheBRAFV600Emutationoccurs in approximately 8%ofhumancolorectal cancers and is associated with therapeutic resistancethat is due, in part, to reactivation of MEK/ERK signaling cascade.Recently, pathway analysis identified cyclin-dependent kinase 1(CDK1) upregulation in a subset of human BRAFV600E colorectalcancers. Therefore, it was determined whether CDK1 antagonismenhances the efficacy of MEK inhibition in BRAFV600E colorectalcancer cells. BRAFV600E colorectal cancer cell lines expressingCDK1 were sensitized to apoptosis upon siRNA knockdown orsmall-molecule inhibition with RO-3306 (CDK1 inhibitor) ordinaciclib (CDK1, 2, 5, 9 inhibitors). Combination of RO-3306ordinaciclib with cobimetinib (MEK inhibitor) cooperativelyenhanced apoptosis and reduced clonogenic survival versusmonotherapy. Cells isogenic or ectopic for BRAFV600E displayedresistance to CDK1 inhibitors, as did cells with ectopic expressionof constitutively active MEK. CDK1 inhibitors induced a CASP8-dependent apoptosis shown by caspase-8 restoration in deficient

NB7 cells that enhanced dinaciclib-induced CASP3 cleavage. CDKinhibitors suppressed pro-CASP8 phosphorylation at S387, asshown by drug withdrawal, which restored p-S387 and increasedmitosis. In a colorectal cancer xenograft model, dinaciclib pluscobimetinib produced significantly greater tumor growth inhibi-tion in association with a caspase-dependent apoptosis versuseither drug alone. The Cancer Genome Atlas (TCGA) transcrip-tomic dataset revealed overexpression of CDK1 in human colo-rectal cancers versus normal colon. Together, these data establishCDK1 as a novel mediator of apoptosis resistance in BRAFV600E

colorectal cancers whose combined targeting with a MEK/ERKinhibitor represents an effective therapeutic strategy.

Implications: CDK1 is a novel mediator of apoptosis resist-ance in BRAFV600E colorectal cancers whose dual targetingwith a MEK inhibitor may be therapeutically effective.Mol Cancer Res; 16(3); 378–89. �2017 AACR.

IntroductionColorectal cancer is the second leading cause of cancer-related

mortality in the United States (1). BRAFV600E mutations aredetected in 8% of human colorectal cancers where they areassociated with poor prognosis and treatment resistance (2–6).To date, no effective therapeutic options are available for patientswith these tumors. A subset of colorectal cancers with frequentBRAFV600E mutations display the CpG island methylator pheno-type (CIMP) with epigenetic inactivation of theMLH1mismatchrepair gene and p16(Ink4a), a negative regulator of cyclin-depen-dent kinase 1 (CDK1; ref. 7). BRAFV600E results in constitutiveactivation of theMAPKpathway (8, 9). In contrast withmetastaticmelanoma, where BRAF inhibitors produce high rates of initial

tumor response (10, 11), colorectal cancers demonstrate resis-tance to these inhibitors in clinical trials (2). The observedresistance is due, in part, to rebound activation of EGFR thatactivates downstreamMAPKsignalingmediatedbyMEK(12–16).In preclinical models, dual inhibition of BRAF andMEK producesmore potent tumor growth inhibition than didmonotherapy andmarkedly improved efficacy inpatientswithmetastaticmelanomathat led to approval of cobimetinib combined with vemurafenibby the FDA for treatment of this malignancy (17). However, dualinhibition of BRAF and MEK only modestly increased efficacy inpatients with metastatic BRAFV600E colorectal cancer (18), sug-gesting the importance of nonredundant resistance mechanisms.

Recent data suggest that biological subdivisions exist withinBRAFV600E colorectal cancers as these tumors could be sepa-rated by pathway analysis into two subtypes based on geneexpression, one of which shows upregulation of CDK1 (19).CDKs are serine/threonine kinases that regulate the cell cycleby interacting with specific cell-cycle–regulatory cyclins. CDK1is the only essential CDK (20) and functions to promote theG2–M transition and regulates G1 progression and the G1–Stransition (21, 22). Unrestricted cell proliferation, one ofthe hallmarks of malignant tumors, is often driven by altera-tions in CDK activity. Altered CDK expression and/or activityis observed in many human cancers (23, 24). In addition tocell-cycle regulation, CDK1 may regulate apoptosis bycaspase phosphorylation (25), including the inhibitory

1Gastroenterology Research Unit, Mayo Clinic, Rochester, Minnesota.2Department ofGastrointestinal Surgery, UnionHospital, TongjiMedical College,Huazhong University of Science and Technology,Wuhan, China. 3Department ofObstetrics and Gynecology, School of Medicine, Johann Wolfgang Goethe-University, Frankfurt, Germany. 4Mayo Clinic Comprehensive Cancer Center,Rochester, Minnesota.

Corresponding Author: Frank A. Sinicrope, Mayo Clinic, 200 1st Street SouthWest, Rochester, MN 55905. Phone: 507-266-4338; Fax: 507-255-6318; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-17-0404

�2017 American Association for Cancer Research.

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phosphorylation of caspase-8 shown by the Strebhardt labo-ratory (26). Targeting CDK1 can be achieved by the selectiveCDK1 inhibitor R0-3306 and the nonselective CDK inhibitordinaciclib that is undergoing evaluation in clinical trials.Dinaciclib (formerly SCH727965) is a potent inhibitor ofCDK 1, 2, 5, and 9 with 50% inhibitory concentrations inthe nanomolar range (27). In preclinical studies, dinaciclib hasbeen shown to arrest cell-cycle progression, induce activationof caspase-8, -9 and related apoptosis, and inhibit tumorgrowth in multiple types of cancer (17, 27–30). In addition,early-phase clinical trials have shown that this drug was welltolerated (31) and has significant single-agent activity againstrelapsed and refractory chronic lymphocytic leukemia (31)and multiple myeloma (32), supporting its potential for thetreatment of human malignancies.

In this report, we determined the contribution of CDK1 toapoptosis resistance in BRAF-mutant colorectal cancer cells, andexamined whether CDK antagonism by genetic or pharmacologicmeans can enhance the efficacy of MEK inhibition. We demon-strate that targeting CDK1 is an effective strategy to enhance theefficacy of MEK inhibition in mutant BRAF colorectal cancer celllines and tumor xenografts. The relevance of CDK1 as a thera-peutic target in human colorectal cancers was confirmed in TheCancer Genome Atlas (TCGA) transcriptomic datasets (33).

Materials and MethodsCell culture, drugs, and reagents

BRAF-mutant (HT-29, RKO, VACO432 and WiDr), KRAS-mutant (DLD1, SW620), and BRAF/KRAS wild-type (DiFi)human colorectal cancer cell lines, as well as the melanoma cellline A375, were obtained from the ATCC. Isogenic RKO [A19(BRAFV600E/�/�), T29 (BRAFWT/�/�)] and VACO432 [parental(BRAFV600E/�), VT1 (BRAFWT/�)] human colorectal cancer celllines were obtained from Dr. B. Vogelstein [Genetic ResourcesCore Facility (GRCF), Johns Hopkins University, Baltimore, MD].Neuroblastoma NB7 cell line was used. All cell lines were testedand authenticated by the ATCC using short tandem repeat anal-ysis. Cell lines were also routinely tested forMycoplasma contam-ination every 3monthswith aMycoAlertMycoplasma detection set(Lonza). For isogenicBRAF cells, GRCFuses a short tandem repeatprofiling for authentication. Cellsweremaintained asmonolayersin RPMI medium (Invitrogen, catalog no. 11875) supplementedwith 10% FBS as well as 1% antibiotic–antimycotic (Invitrogen,catalog no. 15240). HEK293T cells, used for lentivirus produc-tion, were grown in high-glucose DMEM (Sigma, catalog no.D5796) with the same supplementation as above.

Cells were treated with cobimetinib (GDC-0973/XL-518;Active Biochem, catalog no. A-1180) at indicated doses and timesalone or combinedwith R0-3306 (Sigma, SML0569) or dinaciclib(Sellekchem, catalog no. S2768) in the presence or absence of acaspase-8 inhibitor, z-IETK-FMK (R&D Systems). Drugs weredissolved in DMSO, prepared as stock solutions, aliquoted, andthen stored at�20�C. Drugs were later diluted in growthmediumat the time of treatment. Carrier-free human recombinant TRAILwas purchased from R&D Systems. For immunoblotting, primaryantibodies includedmouse anti-p16 (BD Biosciences, catalog no.550834), mouse anti-caspase-8 (BD Biosciences, catalog no.551242), and tubulin (Sigma, T4026). A mouse anti-p-caspase-8-s387 was produced in the laboratory of K. Strebhardt. All otherantibodies were purchased from Cell Signaling Technology.

Ectopic gene expression by lentiviral and retroviral deliverypBabePuro3-p16Flag was purchased from Addgene (#24934).

Lentiviral BRAFV600Ewas described previously (34). Lentiviralconstitutive MEK (ERK2–L4A–MEK1 fusion, MEKDD) was gen-erated by subcloning cDNA fragments [pCMV-myc-ERK2-L4A-MEK1 fusion, Addgene #39197] into vector pCDH1-puro-2HA.Production in HEK293T cells and transduction of pseudo-typedlentivirus or retrovirus into target cells were performed utilizing astandard procedure, as described previously (35). A puromycin-resistant pool of cells was produced by eliminating nontrans-duced cells using puromycin (Sigma; catalog no. P8833) at 48hours posttransduction.

Transfection of siRNACDK1 and ERK1/2 siRNA were purchased from Cell Signaling

Technology (catalog no. 3500, 6560); AllStars Negative ControlsiRNAwas obtained fromQiagen (catalog no. SI03650318). Cellswere seeded 1 day before transfection in medium without anti-biotics to allow 30%–50% confluence at transfection. Lipofecta-mine RNAi Max (Invitrogen, catalog no. 13778150) and siRNAwere each diluted in Opti-MEMmedium (Invitrogen). The result-ing two solutions were then mixed and incubated to enablecomplex formation. Cells were then transfected by adding theRNAi–Lipofectamine complex dropwise to medium to achieve asiRNA concentration of 100 nmol/L. Cells were then incubated at37�C and knockdown efficiency was examined at 48 hoursposttransfection.

Apoptosis assayApoptosis was analyzed by Annexin Vþ staining and quan-

tified by flow cytometry as described previously (36). Briefly,cells were treated with the study drugs for a prespecifiedduration. After drug treatment, TrypLE Express Enzyme withoutphenol red (Invitrogen) was used to detach adherent cells thatwere then combined with floating cells. The cell pellet fromcentrifugation was rinsed three times in cold PBS. Then, cellswere resuspended in 1� Annexin V binding solution (BDBiosciences, catalog no. 556454) and stained with Annexin Vconjugated with FITC (BD Biosciences, catalog no. 556419).The proportion of Annexin V–labeled cells was quantified byflow cytometry.

ImmunoblottingProtein samples were prepared in a lysis buffer [5 mmol/L

MgCl2, 137 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1%CHAPS, 10 mmol/L HEPES (pH 7.5)] supplemented with aprotease inhibitor cocktail and a phosphatase inhibitor cocktail2 (both from Sigma), normalized using NanoDropmeasurement(NanoDrop Technologies) or Bio-Rad protein assay (catalog no.500-0006). Samples were denatured in LDS sample buffer (Invi-trogen) supplemented with 2-Mercaptoethanol (Bio-Rad) andthen loaded onto 10% or 14% SDS-PAGE gels followed byelectrophoretic transfer onto a polyvinylidene difluoride mem-brane (Bio-Rad). The membrane was blocked with 0.2% I-Block(Applied Biosystems) in PBS-T (PBS containing 0.1% Tween 20)and incubated with the primary antibodies in PBS-T containing0.2% I-Block overnight at 4�Cor at room temperature for 3 hours.The membranes were then incubated with a secondary antibodyin PBS-T containing 0.2% I-Block conjugated to alkaline phos-phatase, and then developed with CDP-Star substrate (AppliedBiosystems).

Dual Targeting CDK1 and MEK in BRAF-Mutant Colorectal Cancer

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Clongenic assayCells were seeded into 6-well plates at a density of 200 cells per

well. After attachment, the cells were treated with drugs for12 hours. Drugs were removed by replacing with fresh growthmedium. After incubation for 8–10 days, cells grown as colonieswere visualized by fixation in 10%methanol/10% acetic acid andstaining with 0.5% crystal violet in 10% methanol. Each condi-tionwas performed in triplicate. Colony areawas computed usingthe ImageJ plugin ColonyArea (37).

Gene expression analysisTCGA RNA-Seq and associated somatic mutation data (in VCF

format) together with the metadata for 478 human colorectalcancers and 41 normal colonic tissue samples (33) were down-loaded using GDC data portal (https://gdc.cancer.gov/access-data/gdc-data-portal). The VCF file was utilized to classifyRNA-Seq samples from human colorectal cancers as being mutat-ed for BRAF or KRAS or wild-type for both genes. Log transfor-mation of normalized gene expression in Fragments Per Kilobaseof transcript per Million mapped reads (FPKM) was performed.In the colorectal cancers, a Pearson correlation coefficient wascomputed between expression of CDK1 and p16(INK4a) genes.The R ggplot2 package was utilized for data plotting (38).

Colorectal cancer tumor xenograft modelFive-week-old male BALB/c mice were purchased from Beijing

HFK Bioscience and received sterilized food and acidified waterdaily under pathogen-free conditions. RKO colorectal cancer cellswere grown in vitro and then 106 cells (in 0.1 mL Hanks balancedsolution) were injected subcutaneously into the left dorsalflanks of each mouse. When mean tumor volume reached100–150 mm3, mice were randomly divided into four groups:vehicle (n ¼ 8), dinaciclib (n ¼ 8), cobimetinib (n ¼ 8), andcombination of dinaciclib plus cobimetinib (n ¼ 8). Dinaciclibwas administered as 40 mg/kg (2% DMSO/30% PEG 300/ddH2O) by intraperitoneal injection 3� per week for 3 weeks.Cobimetinib was administered by oral gavage at 15 mg/kg (5%DMSO/30% PEG 300/5% Tween 80/ddH2O) 3� per week for 3weeks. Tumor volume and body weight were measured 3� perweek postimplantation. Tumor volumewas determined using thefollowing formula: length � width2 � 0.5.

After 3weeks of treatment,micewere euthanized and xenografttumor tissues were immediately harvested. Tissues were dividedinto those snap frozen with subsequent protein extraction forimmunoblotting, and those fixed in 10% neutral buffered for-malin and embedded in paraffin for IHC. Five-micron-thicksections were cut and used for IHC staining performed according

to the manufacturer's instructions. For IHC, primary antibodiesused included rabbit anti-cleaved caspase-8, -3, and anti-Ki-67 (allfrom Cell Signaling Technology). All animal experiments wereperformed in accordance with guidelines of the Animal CareFacility of the Huazhong University of Science and Technology(Wuhan, China).

Statistical analysisApoptosis data in cell culture experiments derived from

Annexin V data, clonogenic survival assays, and tumor volumesinmurine xenograft models were expressed asmean� SD. All cellculture experiments were performed in triplicate. Data wereanalyzed using the Student t test (two-tailed). A P value of<0.05 was considered statistically significant. Methods used toanalyze gene expression data in human tissue samples from theTCGA datasets are described above.

ResultsDual inhibitionofCDK1andMEKenhances cell death inBRAF-mutant colorectal cancer cells

Colorectal cancers with mutant BRAF show constitutive acti-vation of the MAPK pathway that promotes unrestricted cellproliferation (8). Rebound MAPK activation has been observedin cancer cells with BRAFV600E and is believed to represent a keymechanism of treatment resistance (12–16). In a panel of colo-rectal cancer cells with BRAFV600E, we observed that treatmentwith the MEK inhibitor cobimetinib can more potently suppressp-ERK and induce BIM and PARP cleavage compared with treat-ment with the BRAF inhibitor vemurafenib (Fig. 1A). Further-more, we previously found that cobimetinib produced moresustained inhibition of downstream pERK activity than didthe varamefinib, although it did not induce significant apoptosisin these colorectal cancer cells (34). Cobimetinib inducedthe proapoptotic BH3-only BIM protein, as has been shown forother MEK/ERK inhibitors (39), whose induction was due tosuppression of ERK-mediated phosphorylation that blocks pro-teasome-mediated BIM degradation, as previously shown by ourlaboratory (40).

Recent data indicate thatmany BRAF-mutant colorectal cancersshow cell-cycle dysregulation with upregulation of CDK1 (19).We detected abundant CDK1 expression in multiple humancolorectal cancer cell lines, including those with BRAFV600E

(Fig. 1A). To investigate whether CDK1 can confer apoptosisresistance, we determined whether CDK1 inhibition can enhancecobimetinib-induced apoptosis in BRAFV600E colorectal cancercells. To test this hypothesis, we performed gene knockdown of

Figure 1.Inhibition of CDK1 enhances cell death induction by cobimetinib in BRAF-mutant colorectal cancer cells. A, A panel of BRAF mutant colorectal cancer cellsand A375 melanoma cells were treated with cobimetinib (Cobi) or vemurafenib for 48 hours and expression of pERK1/2 and apoptosis-related proteins weredetermined by immunoblotting. Basal levels of CDK1 expression were examined in colorectal cancer cell lines with mutant or wild-type BRAF or KRAS genes.B,RKO andHT29 cells were transiently transfected with siRNA against CDK1 versus nontargeting control siRNA. After treatment with cobimetinib for 24 or 48 hours,immunoblotting using designated antibodies or Annexin V labeling was performed, respectively; �, P < 0.05. In HT29 and WiDr colorectal cancer cellswith siRNA knockdown of ERK1/2, expression of CDK1, pERK1/2 and ERK1/2 was detected by immunoblotting. Tubulin was probed as a loading control. C, RKO andHT29 cell lines were treated with cobimetinib, R0-3306 (CDK1 inhibitor), or their combination for 24 hours. Cell lysates were collected and subjected toimmunoblotting for pERK1/2, ERK1/2, CDK1, pH2Ax, and cleaved caspase-3 or PARP. Cells were treated with same drugs for 48 hours and apoptosis was analyzed byAnnexin V labeling followed by quantification using flow cytometry; �� , P < 0.002. D, Cells were seeded in 6-well plates at a density of 200 cells per welland allowed to attach overnight. Cells were then treated with designated drugs for 12 hours followed by replacement of media and continued incubationfor 8–10 days. Colonies were stained and a percentage of colony area was quantified (see Materials and Methods). Results represent mean � SD fromtriplicate experiments (� , P < 0.02; �� , P < 0.005).





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CDK1 by siRNA that was shown to increase cobimetinib-inducedcleavage of PARP, caspase-3, and double-strand DNA damage(pH2Ax) concurrentwith enhancedAnnexinV labeling comparedwith control siRNA cells (Fig. 1B). Addition of the selective CDK1inhibitor RO-3306 to cobimetinib was shown to enhance apo-ptosis induction shown by cleavage of PARP, caspase-3, and anincrease in pH2Ax that was accompanied by increased Annexin Vlabeling in RKO and HT29 cell lines (Fig. 1C). The effect of thedrug combination on long-term cell viability was examined byclonogenic survival assay. The combination of R0-3306 andcobimetinib suppressed colony formation to a greater extentcompared with either drug alone (Fig. 1D). Interestingly, cobi-metinib suppressedCDK1 expression (Fig. 1B andC) thatwas dueto MEK/ERK inhibition shown clearly in WiDr cells with knock-down of ERK (Fig. 1B). Together, these results indicate that CDK1inhibition can significantly enhance cobimetinib-induced apo-ptosis in BRAF-mutant colorectal cancer cell lines.

We also examined the ability of the CDK inhibitor dinaciclib toenhance apoptosis induction by cobimetinib in BRAFV600E colo-rectal cancer cells. The combination of dinaciclib with cobimeti-nib enhanced caspase-3 cleavage, induced pH2Ax, and increasedthe proportion of RKO cells undergoing apoptosis in a dose-dependent manner (Fig. 2A). While cobimetinib or dinaciclibmonotherapy reduced colony formation, the drug combinationdid so to a greater extent in both RKO and HT29 cell lines.Specifically, the colony area formed by cells treated with the drugcombination was reduced by approximately 50% compared witheither drug alone (Fig. 2B and C).

Mutant BRAF–mediated MEK/ERK activation attenuatesapoptosis induction by CDK inhibitors

Dual inhibition of CDK1 and MEK/ERK signaling coopera-tively enhanced apoptosis induction (Figs. 1 and 2), suggestingthat these two pathways act interdependently to conferapoptosis resistance. Ectopic expression of mutant BRAF orconstitutively active MEK each increased MEK/ERK signalingindicated by increased pERK expression, and attenuated dina-ciclib-induced DNA damage (pH2Ax), cleavage of caspase-3and PARP (Fig. 3A), and apoptosis shown by Annexin Vlabeling (Fig. 3B). Using isogenic cells that differ only innumber of mutant BRAF alleles, we found that RKO cells withone (A19) or two copies (RKO parental) of mutant BRAF (vs.WT allele only, T29) conferred resistance to R0-3306, as shownby attenuation of pH2Ax and cleaved caspase-3 and PARP (Fig.3C). Together, these data support the strategy of dual targetingof CDK1 and MEK/ERK pathways to overcome BRAF-mediatedapoptosis resistance.

Caspase-8 mediates apoptosis induced by inhibition of CDK1and MEK/ERK signaling

Caspase-8 is a key cell death regulator and procaspase-8 isknown to be regulated during the cell cycle through the inhibitoryaction of CDK1/cyclin B1 (41). We determined whether inhibi-tion of MEK/ERK signaling can activate caspase-8. Cobimetinibtreatment inhibited pERK expression that was associated with anincrease in caspase-8 cleavage and pH2Ax expression inHT29 andWiDr colorectal cancer cell lines (Fig. 4A). Similar promotion ofcaspase-8 cleavage andDNAdamagewere found inuntreated cellswith ERK1/2 siRNA (Fig. 4A), which also suppressed CDK1(Fig. 1B). Given that CDK1 was shown to phosphorylate cas-pase-8 at S387 (p-C8-S387; ref. 41), we tested the ability of CDK1

inhibitors to reduce this inhibitory phosphorylation event.Because the basal level of p-C8-S837 is difficult for detection, weperformed drug withdrawal experiments. Withdrawal of R0-3306or dinaciclib was each shown to upregulate p-C8-S387 expressionthat was accompanied by restoration of mitosis (shown bypH3S10; Fig. 4B). These findings suggest that CDK1 inhibitorscan induce apoptosis by reducing caspase-8 phosphorylation. Theability of caspase-8 to mediate dinaciclib-induced apoptosis wasconfirmed by ectopic caspase-8 expression in caspase-8–deficientNB7 cells that was shown to increase both dinaciclib- and TRAIL-induced caspase-3 and PARP cleavage (Fig. 4C). Further supportfor the dependence of apoptosis on caspase-8 activation wasachieved using the caspase-8 inhibitor, Z-IETD-FMK, that atten-uated apoptosis (Annexin Vþ cells) induced by dinaciclib �cobimetinib (Fig. 4C). Similarly, Z-IETD-FMK reduced DNAdamage, cleavage of caspase-8 and -3, and attenuated AnnexinVþ labeling induced by the combination of R0-3306 plus cobi-metinib in colorectal cancer cells (Fig. 4D).

Combination of dinaciclib and cobimetinib enhance tumorgrowth inhibition in vivo

To evaluate the efficacy of our combinatorial strategy in vivo, wegenerated murine xenograft models using the BRAF-mutant RKOcolorectal cancer cell line. Treatment of mice with intraperitonealdinaciclib or oral cobimetinibwas shown to significantly suppresstumor growth compared with vehicle. Furthermore, the drugcombinationwas shown to inhibit tumor growth to a significantlygreater extent compared with dinaciclib or cobimetinib (Fig. 5Aand B). Drugs were well tolerated by the mice as indicated by thelack of significant changes in body weight of the mice in eachgroup during the experimental period. Tumor growth inhibitionin the xenograft model was accompanied by induction of apo-ptosis thatwas enhanced by the drug combination comparedwithmonotherapy, as shown in tumor tissues analyzed by immuno-blotting and IHC. Specifically, we observed an increase in cleavedcaspase-8, -3, and PARP as well as increased pH2AX proteins intumors treated with dinaciclib plus cobimentib compared withsingle drugs as shownby immunoblotting (Fig. 5C). Furthermore,IHC staining of tumor xenografts demonstrated cleavage of cas-pase-8 and -3 and inhibition of the cell proliferationmarker Ki-67inmice treated with the drug combination compared withmono-therapy (Fig. 5D). Together, these results confirm our in vitrofindings and suggest that cooperative apoptosis induction is a keymechanism of the antitumor effect of dual CDK1 and MEKinhibition.

CDK1 is upregulated in human colorectal cancersA schematic diagram shows the proposed mechanism by

which CDK1 inhibition can antagonize inhibitory caspase-8phosphorylation to promote apoptosis that is attenuated byBRAFV600Ein human colorectal cancer cells. Combined inhibitionof CDK1 and MEK/ERK were shown to cooperatively enhanceapoptosis induction in BRAFV600Ecolorectal cancer cells (Fig. 6A).

To establish CDK1 as a relevant therapeutic target in humancolorectal cancers with mutant BRAF, we used public RNA-Seqdatasets from TCGA to examine CDK1 gene expression. CDK1mRNA was found to be significantly overexpressed in humancolorectal cancer versus normal colonic tissue (Fig. 6B). As shownby normalized and log-transformed FPKM (log-FPKM], we foundthat themean log-RPKMwas approximately 3 in tumor comparedwith approximately 1.5 in normal colonic tissue. Similar levels

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of CDK1 expression were found among colorectal cancers withmutant versus wild-type copies of BRAF or KRAS genes (Fig. 6C).CDK1 expression has been shown to be negatively regulated byp16 at the posttranscriptional level by interaction of miR-410 ormiR-650 with CDK1-30UTR (42). p16(INK4a), which blocks cell-cycle progression from G1 to S phase, is epigenetically silencedalong withMLH1 in colorectal cancers with CIMP (7). Consistentwith this finding, we observed a negative correlation between theexpression of p16(INK4a) and CDK1 (r, �0.19, P < 0.001) inTCGA datasets (Fig. 6D). To confirm this regulation, we ectopi-

cally expressed retroviral p16(INK4a) into RKO human colorectalcancer cells and observed reduced CDK1 protein expression (Fig.6E). Consistent with our analysis of TCGA datasets, we found thatneither ectopic BRAFV600E nor presence of mutant BRAFV600E

alleles in isogenic RKO cells altered CDK1 expression, althoughpERK and the transcription factor MAFG were upregulated bymutant BRAF (Fig. 6E) consistent with published reports (7).MAFG is a transcriptional repressor that has been identified as akey factor required for MLH1 silencing and CIMP in colorectalcancers harboring BRAFV600E (7).

Figure 2.

Dinaciclib enhances cobimetinib-induced cell death in BRAF-mutant colorectal cancer cells. A, RKO cells were treated with increasing doses ofdinaciclib (Dina) alone or combined with cobimetinib for 24 hours. Immunoblotting was performed for detection of PARP and caspase-3 cleavageas well as DNA double-strand breaks using pH2Ax. Treatment-induced apoptosis was analyzed by Annexin V labeling; �� , P < 0.003. B and C, Clonogenicsurvival was determined in RKO and HT-29 cells that were treated with dinaciclib, cobimetnib, or their combination for 12 hours. Colony percent areawas then quantified and results are shown as mean � SD from triplicate experiments; �� , P < 0.006.






DiscussionCombined treatment with inhibitors of both BRAF and MEK

onlymodestly improved response rates in patients with colorectalcancer (18), suggesting the need to target other pathways. Recentpathway analysis identified two subtypes of BRAFV600E humancolorectal cancers, one of which shows prominent cell-cycledysregulation (19). Using colorectal cancer cells lines withBRAFV600E that express CDK1, we made the novel observationthat CDK1 can confer apoptosis resistance and that antagonismof

CDK1 by genetic or pharmacologic means can significantly andcooperatively enhance apoptosis induction by cobimetinib thatwas also shown in long-term clonogenic survival assays. Interest-ingly, cobimetinib suppressed CDK1 expression due to its abilityto inhibitMEK/ERK signaling as shown by ERK1/2 knockdown bysiRNA. Dual inhibition of CDK1 and BRAF-mediated MEK/ERKsignaling were required for effective tumor cell death given thatcells with isogenic BRAFV600E versus wild-type alleles conferredresistance to cobimetinib-induced DNA damage and apoptosisin a gene dose-dependent manner. Ectopic BRAFV600E or

Figure 3.

BRAFV600E or constitutively active MEK/ERK confer resistance to apoptosis induction by CDK inhibitors. A, BRAFV600E or a fusion of MEK1–ERK2(ERK2-L4A-MEK1) versus empty vector was ectopically expressed in VACO432 VT1 cells. Cells were exposed to dinaciclib or vehicle for 24 hours and thenprobed for cleavage of caspase-3 and PARP as well as pH2Ax by immunoblotting. B, Cells were treated with vehicle or dinaciclib at the indicated doses (24 hours)and apoptosis induction was examined by Annexin V labeling. C, Isogenic RKO colorectal cancer cell lines that differ in number of mutant BRAF alleles[parental (BRAFV600E/V600E/WT), A19 (BRAFV600E/�/�), and T29 (BRAFwt/�/�)] were treated with RO-3306 versus vehicle for 48 hours. Cell lysates wereprepared and then probed with antibodies against CDK1, pERK1/2, ERK1/2, pH2Ax, and cleaved PARP and caspase-3 by immunoblotting.

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Figure 4.

Caspase-8 mediates apoptosis induced by inhibition of CDK1 and MEK/ERK signaling. A, MEK/ERK signaling was suppressed in HT29 and WiDr colorectalcancer cells by increasing doses of cobimetinib for 24 hours or by siRNA knockdown of ERK1/2 (48 hours). Detection of pERK1/2, ERK1/2, cleaved caspase-8,or pH2Ax was performed by immunoblotting. B, RKO cells were treated with RO-3306 (5 mmol/L) or dinaciclib (12.5 nmol/L) for 16 hours. The drug was thenwithdrawn and cells allowed to incubate in drug-free media for indicated times (0–3 hours). Phosphorylation of procaspase-8 at S387(p-C8-S387) andphosphorylation of the mitotic marker histone H3 at Ser10 (pH3S10) were then probed by immunoblotting. Caspase-8–deficient NB7 cells were used as anegative control for procaspase-8.C,Caspase-8was reconstituted in caspase-8–deficient NB7 cells by ectopic expression. NB7 cells were then treatedwith TRAIL ordinaciblib for 24 hours and probed for cleavage of caspase-8, caspase-3, PARP, and expression of pH2Ax. Apoptosis induction by cobimetinib � dinaciclibversus vehicle was analyzed by Annexin V staining in the presence or absence of the caspase-8 inhibitor, z-IETD-fmk (�� , P < 0.002). D, HT29 and WiDr cells weretreated with vehicle, cobimetinib (5 mmol/L), RO-3306 (5 mmol/L), or their combination for 48 hours in the presence/absence of z-IETD-fmk. Expression ofdesignated proteins was examined in cell lysates by immunoblotting. Annexin V staining was performed to determine the effect of RO-3306 plus cobimetinibon apoptosis in the presence or absence of z-IETD-fmk (�� , P < 0.002).






Figure 5.

Effect of cobimetinib plus dinaciclib on tumor growth in the RKO colorectal cancer xenograft model. A, Isolated tumor tissue size (left) and tumor volumemeasurements (right) are shown at termination of treatment (day 20) for each drug and vehicle control (n ¼ 8 mice; � , P < 0.05). B, Tumor volume growthcurve for mice treated with study drugs versus vehicle over time (days; � , P < 0.05). C, Immunoblot of representative tissue lysates prepared from freshtumor tissues for each experimental group were analyzed for cleavage of caspase-8, caspase-3, PARP, and expression of p-ERK1/2, total ERK1/2 and pH2Ax.GAPDH served as protein loading control. D, Representative images of tumor tissues from drug- versus vehicle-treated mice were immunostained forcleaved caspase-8, caspase-3, Ki-67, and hematoxylin and eosin (H&E).


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constitutively activeMEK were also shown to confer resistance toapoptosis induction by CDK inhibitors. These in vitro data dem-onstrate that a combinatorial strategy that targets both CDK1 andMEK signaling can overcome apoptosis resistance and, thereby,

achieve tumor cell death in BRAF-mutant colorectal cancer cells.Todemonstrate the clinical relevance of this strategy,we generateda murine tumor xenograft model of BRAFV600E colorectal cancerwhereby the combination of dinaciclib and cobimetinib were

Figure 6.

CDK1 mRNA expression in human colorectal cancers versus normal colonic tissue. A, Schematic diagram of proposed mechanism by which CDK1 promotesinhibitory caspase-8 phosphorylation and together with BRAFV600E signaling, confers resistance to apoptosis that can be reversed by combined inhibitionof CDK1 and MEK/ERK. B,CDK1 gene expression data were extracted from TCGA RNA-Seq datasets for human colorectal cancers (N ¼ 478) andcomparedwith expression levels in normal colonic tissues (N¼41);P¼ 8.61925E�18.C,Colorectal cancer tissue sampleswere categorizedbased onmutantBRAF (N¼ 49), mutant KRAS (N ¼ 177) or wild-type (WT) copies of both genes (N ¼ 225) using associated metadata. CDK1 expression was then compared amongthese three colorectal cancer subgroups; P > 0.6. D, Association of CDK1 and p16(INK4a) expression were analyzed by linear regression and a Pearsoncorrelation coefficient was computed (r ¼ �0.19, P ¼ 3.548e-05). E, CDK1 expression was compared in RKO cells with ectopic retroviral p16 expressionversus empty vector. CDK1 expression was also compared in VACO432 VT1 [BRAFWT/�] cells with ectopic BRAFV600E versus empty vector, and in parental(BRAFV600E/V600E/WT) or isogenic RKO cells with a mutant [A19 (BRAFV600E/�/�)] or wild-type BRAF allele [T29 (BRAFwt/�/�)]. In these cells, MAFG and pERKexpression were also probed.






shown to significantly suppress tumor growth to a greater extentthan did either drug alone. Consistent with our in vitro findings,the drug combination induced a caspase-dependent apoptosis,including caspase-8 cleavage, in tumor xenografts that wasenhanced compared with monotherapy. The relevance of CDK1in the clinical behavior of human colorectal cancers has beenshown by the finding that a high CDK1 nuclear to cytoplasmicexpression ratiowas associatedwith poor overall survival andwasan independent risk factor for outcome (43). Further support forthe use of dinaciclib in the treatment of refractory solid tumors,including those with mutant KRAS, derives from the finding thatits combination with the pan-AKT inhibitor MK-2206 can strong-ly suppress tumor growth in murine orthotopic and patient-derived xenograft models of human pancreatic cancer (44).

Themechanism by which CDK1 inhibitors can induce apoptosisis unknown; however, it has been shown that procaspase-8 isphosphorylated by CDK1/cyclin B1 on Ser-387 in cancer cell lines(41). Our data demonstrate that caspase-8 is a key mediator of celldeath induction by the CDK1 inhibitors RO-3306 or dinaciclib.Caspase-8 is a key effector of cell death, particularly through thedeath receptor (DR)–mediated apoptotic pathway (45), although itcan be activated independent of DRs as shown for paclitaxel (46).Although inhibition of caspase-8 attenuated apoptosis in responseto combined targeting of CDK1 and MEK/ERK, this partial effectsuggests that additionalmechanisms besides caspase-8 signaling arelikely to contribute. Our finding that withdrawal of R0-3306 ordinaciclib restored procaspase-8 phosphorylation at S387 is consis-tent with the ability of CDK1 to inhibit apoptosis by phosphory-lation of procaspase-8. This event occurred in association withincreased cell mitosis that is known to be regulated by CDK1(47). Confirmation of the ability of caspase-8 to mediate dinaci-blib-induced apoptosis was shown by reexpression of caspase-8 incaspase-8–deficient NB7 cells. Moreover, treatment of colon cancerxenografts with the combination of dinaciclib and cobimetinibpromoted caspase-8 cleavage. Together, these data indicate theimportanceof caspase-8activation inmediatingcell death inductionby CDK1 antagonists alone or in combination with cobimetinib.

We found that CDK1 was frequently overexpressed in humancolorectal cancers relative to normal colonic tissues from TCGAdatasets that was not limited to the BRAF-mutant subtype. How-ever, ectopic BRAFV600E conferred resistance to apoptosis inducedby CDK1 inhibitors that was further shown to be gene dose-dependent in BRAFV600E isogenic cells. These data indicate thatCDK1 is a therapeutic target in human colorectal cancer and thesusceptibility to CDK1 inhibition is regulated by mutant BRAF,which supports the rationale for the combinatorial strategyof targeting CDK1 and BRAF-mediated MEK/ERK signaling inBRAF-mutant colorectal cancer. Given that KRAS-mutant colo-rectal cancers also show aberrant activation of MEK/ERK signal-ing, the drug combination holds promise in this tumor subtype.We found a negative correlation between expression ofCDK1 andp16(Ink4a) in TCGA datasets. CDK1 is known to be negatively

regulated by p16 (42), and we confirmed that ectopic p16 cansuppress CDK1 expression inBRAF-mutant colorectal cancer cells.Ectopic BRAFV600E did not increase CDK1 expression but was ableto upregulate MAFG, a transcriptional repressor and target ofCIMP that is frequently detected in colorectal cancers withmutantBRAF and deficient DNA mismatch repair (7).

In conclusion, we identify CDK1 as a novel mediator ofapoptosis resistance inBRAF-mutant colorectal cancer cells whoseantagonism by CDK1 inhibitors, including dinaciclib, was shownto significantly enhance the efficacy of cobimetinib both in vitroand in vivo that was shown to be mediated, in part, by caspase-8activation.Mutant BRAF is a negative regulator of susceptibility toCDK1 inhibitors that supports combined inhibition of CDK1-and BRAFV600E-mediated MEK/ERK signaling in this tumor sub-type. Frequent CDK1 overexpression in human colorectal cancerssuggests its relevance as a therapeutic target. Taken together, ourdata support a novel combinatorial strategy to inhibit CDK1 andMEK for the treatment of BRAF-mutant colorectal cancer.

Disclosure of Potential Conflicts of InterestF.A. Sinicrope is a consultant/advisory board member for Hoffmann La

Roche. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: P. Zhang, H. Kawakami, S. Huang, F.A. SinicropeDevelopment of methodology: P. Zhang, H. Kawakami, W. Liu, K. Strebhardt,S. Huang, F.A. SinicropeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Zhang, W. Liu, X. Zeng, K. Tao, S. Huang,F.A. SinicropeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. Zhang, H. Kawakami, W. Liu, K. Tao, S. Huang,F.A. SinicropeWriting, review, and/or revision of the manuscript: P. Zhang, H. Kawakami,K. Strebhardt, S. Huang, F.A. SinicropeAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. Kawakami, S. Huang, F.A. SinicropeStudy supervision: S. Huang, F.A. Sinicrope

AcknowledgmentsThis study was supported, in part, by National Cancer Institute grant R01

CA210509(toF.A.Sinicrope).Additional supportwasobtainedfromtheNationalNatural Science Foundation of China (#81572413 to K.T, #81702386; toP. Zhang). P. Zhang was supported by the Scientific Research Training Programfor Young Talents of Wuhan Union Hospital, PRC; his current address is Depart-ment of Gastrointestinal Surgery, Union Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan, China.

The authors express their gratitude to Mr. Matthew A. Bockol for down-loading TCGA data.

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 July 25, 2017; revised October 13, 2017; accepted November 14,2017; published OnlineFirst December 12, 2017.

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2018;16:378-389. Published OnlineFirst December 12, 2017.Mol Cancer Res Peng Zhang, Hisato Kawakami, Weizhen Liu, et al. BRAF-Mutant Human Colorectal CancerTargeting CDK1 and MEK/ERK Overcomes Apoptotic Resistance in

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