CK2 Inhibitor CX-4945 Suppresses DNA Repair …...DNA repair mechanisms relevant to cisplatin and...

Preclinical Development

CK2 Inhibitor CX-4945 Suppresses DNA Repair ResponseTriggered by DNA-Targeted Anticancer Drugs andAugments Efficacy: Mechanistic Rationale forDrug Combination Therapy

Adam Siddiqui-Jain1, Joshua Bliesath1, Diwata Macalino1, Mayuko Omori1, Nanni Huser1, Nicole Streiner1,Caroline B. Ho1, Kenna Anderes1, Chris Proffitt1, Sean E. O'Brien1, John K. C. Lim1, Daniel D. Von Hoff2,David M. Ryckman1, William G. Rice1, and Denis Drygin1

AbstractDrug combination therapies are commonly used for the treatment of cancers to increase therapeutic efficacy,

reduce toxicity, anddecrease the incidenceofdrug resistance.Althoughdrugcombination therapieswereoriginally

devised primarily by empirical methods, the increased understanding of drug mechanisms and the pathways

they modulate provides a unique opportunity to design combinations that are based onmechanistic rationale. We

have identified protein kinase CK2 as a promising therapeutic target for combination therapy, because CK2

regulates not just one but many oncogenic pathways and processes that play important roles in drug resistance,

includingDNArepair, epidermal growth factor receptor signaling,PI3K/AKT/mTORsignaling,Hsp90machinery

activity, hypoxia, and interleukin-6 expression. In this article, we show that CX-4945, a clinical stage selective

small molecule inhibitor of CK2, blocks the DNA repair response induced by gemcitabine and cisplatin and

synergizes with these agents in models of ovarian cancer. Mechanistic studies show that the enhanced activity is a

result of inactivation of XRCC1 andMDC1, two mediator/adaptor proteins that are essential for DNA repair and

that require phosphorylation by CK2 for their function. These data position CK2 as a valid pharmacologic target

for intelligent drug combinations and support the evaluation of CX-4945 in combination with gemcitabine and

platinum-based chemotherapeutics in the clinical setting. Mol Cancer Ther; 11(4); 994–1005. �2012 AACR.

IntroductionDNA-targeted chemotherapeutics represent funda-

mental components of modern cancer therapy and arecurrently prescribed for multiple indications. These agentstarget the DNAof tumor cells and can activate one ormoreDNA repair response mechanisms, potentially leadingto the development of drug resistance (1). An emergingstrategy to boost the effectiveness of these drugs is tocombine them with inhibitors of corresponding DNArepair response mechanisms (1, 2). Methods currentlysubject to clinical investigation include inhibitors of the

checkpoint regulators CHK1 and CHK2, inhibitors of thedirect repair enzymeMGMT and inhibitors of PARP, a keymediator of base excision repair (1). Because differentclasses of DNA-targeted anticancer drugs can trigger dis-tinct DNA repair response pathways, it would be advan-tageous to identify and inhibit a target protein to disruptmultiple DNA repair response pathways simultaneously.ProteinkinaseCK2, a constitutivelyactiveserine/threoninekinase that is widely overexpressed in human cancers(3, 4), has recently emerged as a key regulator of the repairof both single and double strand breaks (5–7). Hence, bysimultaneously disruptingmultipleDNArepair pathways,inhibitors of CK2 have the potential to enhance the activityof a wide range of DNA-targeted chemotherapeutics.

Recently validated as an anticancer drug target, CK2regulates a diverse array of prosurvival cellular processes,including epidermal growth factor receptor signaling,PI3K/AKT/mTOR signaling,Hsp90machinery, hypoxia,and interleukin (IL)-6 expression, all ofwhich play impor-tant roles in resistance to various chemotherapeutics(3, 4, 8–11). Furthermore, protein kinase CK2 has emergedas a key participant in DNA repair response, being essen-tial for the surveillance and repair of both single anddouble strand breaks (6). Among the best characterizedof the CK2-dependent DNA repair response substrates

Authors' Affiliations: 1Cylene Pharmaceuticals, SanDiego, California; and2Translational Genomics Research Institute, Phoenix, Arizona

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

The editor-in-chief of Molecular Cancer Therapeutics is a coauthor of thispaper. In keeping with the AACR's Editorial Policy, a member of the AACR'sPublications Committee had the paper reviewed independently of the jour-nal's editorial process and made the decision whether to accept the paper.

Corresponding Author: Adam Siddiqui-Jain, Cylene Pharmaceuticals,5820 Nancy Ridge Dr. Ste 200, San Diego, CA 92121. Phone: 858-875-5100; Fax: 858-875-5101; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0613

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 11(4) April 2012994

on May 26, 2020. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst January 20, 2012; DOI: 10.1158/1535-7163.MCT-11-0613

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are the mediator/adaptor proteins XRCC1 and MDC1,which are essential components of the single strand break(SSB) and double strand break (DSB) repair machinery,respectively (Fig. 1).XRCC1 is a key mediator of SSB repair, which includes

both the base excision repair and nucleotide excisionrepair (NER) mechanisms (12). It exists in a tight complexwith DNA ligase IIIa, which serves to religate brokenDNA single strands following the processing of damagedbases/nucleotides. XRCC1 is continuously phosphorylat-ed by CK2 (13), an event that is required for its interactionwith 2 proteins, aprataxin and polynucleotide kinase(PNK), which participate in DNA end-processing beforeligation (Fig. 1). In addition, phosphorylation of XRCC1by CK2 may also be required to maintain stability of theXRCC1–ligase IIIa complex itself (5, 14, 15).MDC1 is a key mediator of homologous recombina-

tion (HR) DSB repair and is the principle binding partnerof g-H2AX that is anchored to DNA at sites of DSBs(16–18). Once bound to g-H2AX, MDC1 recruits a keymultiprotein complex "MRN" that is required for DSBrepair signaling. This interaction is dependent on thephosphorylation of MDC1 at multiple acidophilic sitesby CK2 (16–18). The functional consequences of MRNcomplex binding to MDC1 include activation of both theS-phase and G2-M checkpoints following the treatmentof cells with ionizing radiation (19–21) and amplificationof ATM signaling (Fig. 1; ref. 22). In addition, the phos-phorylation of MDC1 by CK2 also promotes binding ofaprataxin, implicatingMDC1/CK2 in direct HR repair (6).Following the discovery of CX-4945, a first-in-class

clinical stage inhibitor of CK2 (23, 24), we sought toinvestigate whether the pharmacologic targeting ofCK2-dependent DNA repair response functions couldpotentiate the ability of DNA-targeted chemotherapeu-tic agents to kill tumor cells. For this purpose we

selected cisplatin (or carboplatin) and gemcitabine inthe context of ovarian cancer, in which both drugs areused in combination chemotherapy (25). The primarymechanism involved in the repair of DNA–platinumadducts is NER, in which XRRC1/ligase IIIa complexplays a prominent role in religating the broken DNAstrand (Fig. 1; refs. 12, 26). During DNA replication,unrepaired platinum adducts can stall the replicationfork, triggering ATR-mediated repair. Likewise, thetriphosphorylated form of gemcitabine is incorporatedinto DNA during DNA replication, also causing repli-cation forks to stall. Failure to repair either cisplatin- orgemcitabine-induced stalled replication forks leads toreplication fork collapse (DSBs), triggering the accumu-lation of CK2-phosphorylated MDC1 (bound tog-H2AX), amplified ATM signaling, and repair by HR(Fig. 1; refs. 27–30). Thus, inhibition of CK2 couldpotentially synergize with cisplatin by disruptingXRCC1-dependent NER (SSB repair) and with cisplatinand gemcitabine by disrupting MDC1-mediated HRrepair (DSB repair).

Materials and MethodsMaterials

CX-4945 [5-(3-chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic acid)] was synthesized byCylene Pharmaceuticals (>99%pure by high-performanceliquid chromatography assay). Cisplatin, gemcitabine,and carboplatin were purchased from Sigma-Aldrich(Fig. 2).

Cell cultureA2780 and SKOV-3 human ovarian carcinoma cell lines

were purchased fromAmerican TissueCultureCollection(ATCC) and used within 6 months with weekly monitor-ing for growth rates and morphology consistency. ATCC

Figure 1. Reported roles of CK2 inDNA repair mechanisms relevant tocisplatin and gemcitabine.

CX-4945 Enhances Activity of Gemcitabine and Cisplatin

www.aacrjournals.org Mol Cancer Ther; 11(4) April 2012 995




carries out authentication testing of the cell lines usingDNA profiling and cytogenetic analysis. Cell lines werecultured according to the suppliers’ recommendations.

ImmunoprecipitationUntreated or CX-4945–treated (10 mmol/L, 24 hours)

cells were washed twice with PBS and lysed in 1X radio-immunoprecipitation assay Buffer supplemented withphenylmethylsulfonylfluoride and Protease Inhibitor Set1 (EMD Chemicals). Samples were sonicated on ice andcentrifuged at 14,000� g for 10minutes at 4�C. Proteinwasquantitated using the Bradford protein assay. Ten micro-grams of anti-MDC1 antibody (Bethyl Laboratories) wasadded to the cell lysate (2 mg) and 100 mL of 20% protein Asuspension. The immunoprecipitation reactions were rot-ated overnight at 4�C. The samples were centrifuged andthe resulting pelletswerewashed 3 timeswith 500 mL cold-cell lysis buffer. Samples were analyzed by Western blot.The antibody for the CK2 substrate consensus sequence(S�/T�D/EXD/E) was purchased from Cell Signaling.

Comet assaySKOV-3 cells (1� 105 cells) were combinedwithmolten

LMAgarose (Trevigen) at a ratio of 1:10 (v/v) and wereimmediately pipetted onto CometSlide (Trevigen). Slideswere incubated at 4�C in the dark for 10 minutes, thenimmersed in prechilled Lysis Buffer and incubated at 4�Cfor 30 minutes. Slides were immersed in AlkalineUnwinding Solution, pH > 13 (200 mmol/L NaOH, 1mmol/L EDTA) for 20 minutes at room temperature inthe dark. Electrophoresis was done at 21 V for 30 minutesusing Alkaline Electrophoresis solution (200 mmol/LNaOH, 1 mmol/L EDTA). The slides were washed twicein water for 5 minutes and once in 70% EtOH for 5minutes, then dried overnight and visualized by micros-copy. Under these conditions the formation of "comet tail"is indicative of SSDs, DBSs, and/or active excision repairof DNA cross-links.

Cell-cycle analysisCell cycle was analyzed by flow cytometry as described

elsewhere (23). Data were analyzed with FlowJo software(TreeStar, Inc.).

Cell viability assayA2780 and SKOV-3 cells were seeded at a density of 750

and 1,000 cells per well, respectively, and treated 24 hourslater according to the preaddition or postaddition sched-ule (Preaddition schedule: 4-hour treatmentwithCX-4945followed by 24-hour treatment with cisplatin/gemcita-bine. Postaddition schedule: 24-hour treatment with cis-platin/gemcitabine followed 8-hour treatment with CX-4945). Following completion of the treatment schedule,the media was replaced and the cells were cultured at37�C for the remainder of 96 hours. Cell viability wasanalyzed by CyQuant assay (Invitrogen).

In vivo efficacy studiesFemale immunocompromised mice CrTac:Ncr-Foxn1nu

(5–7 weeks old) were obtained from Taconic Farms.Animals were maintained under clean room conditionsin sterile filter top cages. Animals received sterile rodentchow and water ad libitum. All procedures were con-ducted in accordance with the Institute for LaboratoryAnimal Research Guide: The Care and Use of LaboratoryAnimals. Xenografts were initiated by subcutaneousinjection of A2780 cells into the right hind flank regionof each mouse. When tumors reached a designated vol-ume of 100 to 150 mm3, animals were randomized anddivided into groups of 9 to 10 mice per group. CX-4945was administered by oral gavage twice daily at 75 or100 mg/kg as indicated. Cisplatin (5 mg/kg) and carbo-platin (75 mg/kg) were administered via intraperitonealinjection once a week. Gemcitabine (60 mg/kg) wasadministered via intraperitoneal injection according toa once every 3 days � 4 schedule. Tumor volumes andbody weights were measured twice weekly. The lengthand width of the tumor were measured with calipersand the volume calculated using the following formula:tumor volume ¼ (length � width2)/2. Mean percenttumor growth inhibition (TGI) values were calculatedon the final day of the study for drug-treated comparedwith vehicle-treated mice and were calculated as 100 �{1-[(TreatedFinal day � TreatedDay 1)/(ControlFinal day �ControlDay 1)]}. Time-to-endpoint (TTE) is defined as themedian time taken for the tumors to reach a median of2,000 mm3.

Pharmacodynamic studiesMice bearing A2780 tumors (300–400 mm3) were

randomized and divided into 3 groups of 5 mice. Fol-lowing a single dose of gemcitabine, CX-4945 wasadministered by oral gavage at 3.5 and 15.5 hours.Animals were euthanized 24 hours after administrationof gemcitabine; tumors were resected, lysed, and result-ing protein extracts were subjected to Western blotanalysis.

Figure 2. Structures of CX-4945 (A), gemcitabine (B), cisplatin (C), andcarboplatin (D).

Siddiqui-Jain et al.

Mol Cancer Ther; 11(4) April 2012 Molecular Cancer Therapeutics996




ResultsCX-4945 enhances the activity of cisplatin andgemcitabine in cell-based antiproliferativeexperimentsGemcitabine and cisplatin are commonly used to treat

patients with ovarian cancers (25). Because CK2 isinvolved in multiple processes that regulate the sensitiv-ity of cancer cells to such DNA-targeted anticancer drugs(10, 13, 16), we asked whether combining CX-4945 withgemcitabine or cisplatin can improve the effectiveness ofthese chemotherapeutics. As p53 status is known to playan important role in the sensitivity of ovarian cancer cellsto DNA-targeted agents (31) and because of the highfrequency of p53 mutations in ovarian cancer (32), weused both p53 WT A2780 cells and p53 null SKOV-3 cellsfor our mechanistic studies. To ensure inhibition of CK2

signaling throughout the experiment, cells were pre-treated with CX-4945 4 hours before addition of eithergemcitabine or cisplatin, and the presence of CX-4945wasmaintained throughout the treatment period.Under theseconditions (Fig. 3A), CX-4945 promoted synergistic anti-proliferative effects in both cell lines (antiproliferativeactivity 8% to 17% greater than Bliss additivity; ref. 33).

In separate experiments using postaddition of CX-4945,we carried out combination studies using a schedulewherein CX-4945 was added 24 hours after treatment withgemcitabine or cisplatin and then maintained in combina-tion for only 8 hours. Under these conditions, the chemo-therapeutic agents have sufficient time to cause DNAstrand breaks before the addition of CX-4945, whereasthe presence of CX-4945 for only 8 hours contributesminimal single-agent antiproliferative activity. Using this

Figure 3. Synergisticantiproliferative effects of combiningCX-4945 with either cisplatin orgemcitabine in A2780 and SKOV-3cells. The additivity line is equivalentto the theoretical combinedantiproliferative effects of 2 drugs Aand B [%A þ%B(100 � %A)/100] inwhich %A and %B are thepercentage of cells killed by drug Aand B, respectively, at givenconcentrations. In A2780 cells,3 mmol/L CX-4945, 300 nmol/Lcisplatin (Cis), and 3 nmol/Lgemcitabine (Gem) were used. InSKOV-3 cells, 10 mmol/L CX-4945,1 mmol/L cisplatin (Cis), and10 nmol/L gemcitabine (Gem) wereused. A, CX-4945 preadditionschedule. B, CX-4945 postadditionschedule. C, inhibition of replicationrecovery for A2780 cells treated with10 nmol/L gemcitabine and10 mmol/L CX-4945.






schedule (Fig. 3B), CX-4945 significantly enhanced theantiproliferative effects of gemcitabine and cisplatin (anti-proliferative activity 23%–38% higher than Bliss additi-vity). These data were consistent with an enhancementof antiproliferative activity by CX-4945 as a consequenceof inhibiting DNA repair response mechanisms.

Stalledor collapsed replication forksproducedbyDNA-targeted drugs are known to cause cancer cells to arrest inS-phase (34). Indeed, we showed that SKOV-3 cells and, toa greater extent, A2780 cells can respond to gemcitabine orcisplatin treatment by accumulating in S-phase (data notshown). To determine the effects of CX-4945 on chemo-therapeutically induced cell-cycle arrest, we evaluated theeffects of gemcitabine alone or the combination of gemci-tabine with CX-4945 in A2780 cells. After 28 hours, A2780cells treated with gemcitabine alone reached the maximalS-phase arrest and then began recovery from S-phaseand progressed to G2-M by 36 hours. However, the com-

bination of CX-4945 with gemcitabine delayed replicationrecovery (Fig. 3C),whereasCX-4945 alone producedG2-Marrest, as previously described (24). These data suggestedthat by inhibiting DNA repair response in ovarian cancercells, the combination with CX-4945 inhibits replicationrecovery and increases cancer cell death induced byDNA-targeted drugs.

CX-4945 decreases XRCC1 and MDC1phosphorylation and prevents DNA repair responsein combination with cisplatin and gemcitabine

To further define the mechanistic processes underlyingthe synergistic antiproliferative activity, we askedwheth-er CX-4945 alone or in combination with cisplatin orgemcitabine could reduce the phosphorylation of theDNA repair mediator/adaptor proteins XRCC1 andMDC1, thus inhibiting DNA repair response. Treatmentof A2780 or SKOV-3 cells with CX-4945 led to a significant

Figure 4. Western blot analysis ofXRCC1 and MDC1 from CX-4945combined with cisplatin (A) orgemcitabine (B) in A2780 or SKOV-3 cells. Cells were treated withgemcitabine (10 nmol/L) orcisplatin (3 mmol/L) for 24 hoursfollowed by CX-4945 (10 mmol/L)and the cells lysed 8 hours after theaddition of CX-4945; quantifiedphospho-XRRC1 levels(normalized to total XRCC1 levels).C, levels of phospho-MDC1 in CX-4945 (10 mmol/L, 24 hours) treatedA2780 and SKOV-3 cells byimmunoprecipitation.






decrease in the phosphorylation of XRCC1 at multipleCK2-specific sites (Fig. 4A and B). As specific antibodiesfor theCK2phosphorylation sites onMDC1were unavail-able, we probed immunoprecipitated MDC1 from A2780and SKOV-3 cells treated with CX-4945 using an anti-body designed to bind to phospho-peptides with the CK2substrate consensus sequence. Treatment of either celltype with CX-4945 led to significant reductions in MDC1phosphorylation (Fig. 4C).To determine whether decreased XRCC1 and MDC1

phosphorylation prevented DNA repair after treatment

with gemcitabine or cisplatin, we used the alkaline cometassay to monitor the production of DNA strand breaks(35). At concentrations at which neither CX-4945 norgemcitabine or cisplatin caused significant comet forma-tion inA2780 cells, the combination ofCX-4945with eithergemcitabine or cisplatin produced prominent tails. Suchtails are indicative of SSBs, DSBs, and/or active excisionrepair of DNA cross-links. This shows that CX-4945 pre-vented DNA repair after gemcitabine or cisplatin treat-ment (Fig. 5A). Addition of the pan-caspase inhibitorzVAD-FMK did not reduce tail formation, indicating that

Figure 5. A, comet assay showingincreased DNA strand breaksresulting from the combination ofCX-4945 (10 mmol/L) withgemcitabine (100 nmol/L) or cisplatin(10 mmol/L), with and without ZVAD(100 mmol/L). Western blot analysisof g�H2AX and CHK1/2 from CX-4945 combined with cisplatin (B) orgemcitabine (C) in A2780 or SKOV-3cells. Cells were treated withgemcitabine (10 nmol/L) or cisplatin(3 mmol/L) for 24 hours followed byCX-4945 (10 mmol/L) and the cellslysed8hours after the addition ofCX-4945; quantified phospho-CHK1/2levels normalized to total CHK levels.Quantified g-H2AX levels are shownin Supplementary Fig S4.






the observed DNA strand breaks were not secondary tothe induction of apoptosis. To confirm these findings, wemonitored changes in the levels of g-H2AX, a widelyestablished marker of DNA strand breaks (36). Combin-ing CX-4945 with either gemcitabine or cisplatin in eitherA2780 or SKOV-3 cells increased levels of g-H2AX com-paredwith either agent used alone, confirming that levelsof DNA strand breaks were increased in the cancer cells(Fig. 5B and C and Supplementary Fig. S1).

Mechanistic data with CX-4945 presented thus far indi-cated that CK2, XRCC1, and MDC1, all act in a coordi-nated and essential fashion to facilitate the DNA repairresponse that is triggered by cisplatin or gemcitabinetreatment. This contention is corroborated by studies inwhich we used short interfering RNA to knockdown CK2

(both the a and a’ isoforms simultaneously), XRCC1 orMDC1 in SKOV-3 cells. Knockdown of XRCC1, MDC1, orsimultaneous knockdown of CK2a/a’ significantlyincreased the levels of g-H2AX produced by either cis-platin or gemcitabine treatment alone (SupplementaryFig. S2). These data confirmed the relevance of CK2,XRCC1, and MDC1 in mediating gemcitabine or cisplat-in-induced DNA repair response and highlight the utilityof CK2 as a drug target to prevent the DNA repairresponse triggered by such chemotherapeutic agents.

The presence of DNA strand breaks would be expectedto trigger the activation of ATR and ATM, 2 kinases thatplay prominent roles in DNA repair response signaling,which in turn phosphorylate CHK1 and CHK2, respec-tively, halting cell-cycle progression to allow DNA repair

Figure 6. A, increased caspase-3/7 activation in ovarian cancercells treated with indicatedconcentrations of CX-4945 andcisplatin or gemcitabine. B,cleavedPARP levelsmeasured inovarian cancer cells treatedwith CX-4945 (10 mmol/L) andcisplatin (3 mmol/L) orgemcitabine (10 nmol/L).Quantified cleaved PARP levelsare shown in Supplementary FigS4. C, DAPI staining of SKOV-3cells treated with cisplatin(10 mmol/L), gemcitabine(100 nmol/L), and/or CX-4945(10 mmol/L).






Figure 7. A2780 ovarian cancer xenograft efficacy studies. Mean tumor volumes, median times to endpoint, and changes in body weights are presented.A,CX-4945wasdosed75mg/kgorally twicedaily for 21days; cisplatinwasdosed5mg/kg i.p. onceevery 7days for 21days.B,CX-4945wasdosed75mg/kgorally twice daily for 21 days; carboplatin was dosed 75 mg/kg i.p. once every 7 days for 21 days. C, CX-4945 was dosed 100 mg/kg orally twicedaily for 21 days; gemcitabinewas dosed 60mg/kg i.p. once every 3days for 10days (days 1, 4, 7, and 10). D, CX-4945was dosed 100mg/kg orally twice daily(days 2, 5, 8, and 11); gemcitabine was dosed 30 mg/kg i.p. once every 3 days (days 1, 4, 7, and 10). Combination treatments used combined single-agentschedules. E, analysis of cleaved PARP levels in A2780 tumor cells subjected to CX-4945 and gemcitabine alone or in combination.






to progress (1). In all combinations tested, addition ofCX-4945 increased phosphorylation of CHK2 when com-pared with the cytotoxic agent used alone (Fig. 5B andC). The effects on CHK1 phosphorylation were mixed,particularly in A2780 cells, where significant reductionsof total CHK1 levels were also observed. Taken together,our data showed that inhibition of CK2 by CX-4945prevents activation of MDC1 and XRCC1 proteins andconsequently suppresses the ability of cancer cells torepair DNA strand breaks caused by treatment withgemcitabine or cisplatin.

The combination of CX-4945 with cisplatin orgemcitabine results in apoptosis in p53 WT A2780cells andmitotic catastrophe inp53nullSKOV-3cells

It has previously been reported that cisplatin-treatedovarian cancer cells undergo distinct modes of cell deaththat are dependent on the status of p53 (34). Therefore,we examined the mode of cell death of A2780 and SKOV-3 cells treated with CX-4945 and gemcitabine or cisplatin.First, we analyzed the change in levels of effector cas-pase-3/7 activity. In p53WTA2780 cells, the combinationof CX-4945 with either cisplatin or gemcitabine resultedin a significant increase in caspase-3/7 activity comparedwith either agent used alone (Fig. 6A). In p53 null SKOV-3 cells, the same combinations did not produce signifi-cant increases in caspase-3/7 activity. Consistent withthese observations, in p53 WT A2780 cells a significantincrease in cleaved PARP was seen upon combinationtreatment, confirming elevated apoptosis, whereas in p53null SKOV-3 cells the levels of cleaved PARP wereundetectable (Fig. 6B and Supplementary Fig. S1).

Because p53 null SKOV-3 cells were previously shownto be susceptible to mitotic catastrophe in response tocisplatin treatment (34), we wanted to test whether thecombination of CX-4945 with cisplatin would lead tothe same outcome. For this purpose we monitorednuclear morphology changes following drug treatmentof SKOV-3 cells by 40,6-diamidino-2-phenylindole(DAPI) staining (Fig. 6C). The nuclear morphology ofSKOV-3 cells treated with cisplatin for up to 72 hourswas unchanged. Combination treatment of cisplatinwith CX-4945 for 72 hours produced enlarged multi-nucleated cells, a phenotypic response previously char-acterized as cisplatin-induced mitotic catastrophe(34, 37, 38). Similar results were also seen when CX-4945 was combined with gemcitabine in SKOV-3 cells(Fig. 6C).

CX-4945 synergizes with cisplatin, carboplatin, andgemcitabine in xenograft models of ovarian cancer

To determine whether the synergy observed betweenCX-4945 and cisplatin or gemcitabine in vitro could betranslated into increased antitumor efficacy in vivo, wetested these combinations in a xenograft model. For thispurpose we selected the A2780 model because it isaggressively tumorigenic (TTE approximately 15 days),and it allowed us to investigate cleaved PARP as a

pharmacodynamic biomarker of combination activityin tumors. CX-4945 (dosed orally at 75 mg/kg twicedaily) or cisplatin (dosed intraperitoneal at 5 mg/kgonce every 7 days) had minimal antitumor effects assingle agents in this model. However, combining CX-4945 with cisplatin produced robust TGI and extendedTTE to 30 days (Fig. 7A). Because cisplatin is known tocause negative effects on the body weights of treatedanimals, we combined CX-4945 with carboplatin, acisplatin analog with a reduced side-effect profile thatis widely used in the treatment of ovarian cancer.Neither agent was effective when administered alone,whereas the combination resulted in robust TGI andextended TTE to 34 days. The body weight effects forcombination were comparable with those produced bysingle agents (Fig. 7B). In combination with gemcita-bine, CX-4945 improved the therapeutic benefit byincreasing TGI to 99% and extending TTE to 39 days,while being well tolerated (Fig. 7C).

Our in vitro antiproliferative studies suggested thattransient exposure to CX-4945 was sufficient to producesynergy with gemcitabine or cisplatin. Therefore, weconducted an additional A2780 xenograft study whereinCX-4945 was dosed only on 4 occasions, each dose given24 hours after administration of gemcitabine (Fig. 7D).This dosing schedule delivered significantly longer TTEthan gemcitabine alone (51 vs. 37 days). Moreover, theenhanced efficacy using this dosing regimen provides invivo support that CX-4945 augments the antitumor effectsof chemotherapeutic agents as a consequence of inhibitingDNA repair response mechanisms.

To investigate the potential of cleaved PARP as a phar-macodynamic biomarker, we treated mice implantedwith A2780 xenografts to a single dose of gemcitabine(60 mg/kg i.p.), followed by 2 subsequent doses of CX-4945 (75 mg/kg orally) at 3.5 and 15.5 hours after admin-istration of gemcitabine. Twenty four hours followinggemcitabine addition, the tumors were resected, lysed,and the resulting protein extracts were analyzed for levelsof cleaved PARP usingWestern hybridization (Fig. 7E). Aclear increase in cleaved PARP levels was observed intumors from mice treated with CX-4945 and gemcitabinein combination compared with either drug used alone,confirming that the antitumor effect of combining the 2drugs resulted in increased apoptosis inA2780 xenograftsin vivo.

DiscussionDNA-targeted chemotherapeutics are commonly used

as single agents or in combination for the treatment ofvarious types of cancer. Although these drugs are knownfor their robust initial efficacy, they are often limited bytoxicity and inherent or acquired resistance (39). One ofthe mechanisms driving such resistance is DNA repairresponse, which limits the ability of DNA-targeted che-motherapeutics to kill cancer cells (1). Several approachesaimed at combining DNA-targeted chemotherapeutics






with inhibitors of DNA repair response are being inves-tigated in the clinic. Themajority of these inhibitors targethighly specific DNA repair response pathways; therefore,there is considerable need for DNA repair response inhi-bitors that target a broader spectrum of DNA repairresponse and potentially offer combinability with a great-er number of anticancer chemotherapies. CK2, with itsnewly recognized role in the genomic surveillance andrepair of both single and double strand breaks and itsestablished overexpression in cancer cells, ideally fulfillsthese criteria.We showed that the presence of the CK2 inhibitor CX-

4945 strongly enhances the antiproliferative activity ofgemcitabine or cisplatin in ovarian cancer cells (Fig. 3Aand B). By varying dosing schedules we showed thatrelatively short exposure of cells already treated withgemcitabine or cisplatin to CX-4945 was sufficient toproduce synergy. Cell-cycle analysis established thatCX-4945 prevented the replication recovery of cells trea-ted with gemcitabine, indicating that the inhibition ofDNA repair response is the primary mechanism behindthe observed synergy (Fig. 3C).We show that inhibiting CK2 with CX-4945 decreased

the phosphorylation of XRCC1 andMDC1 at CK2-specificphosphorylation sites (Fig. 3A), which prevented efficientDNA repair response in cisplatin- or gemcitabine-treatedovarian cancer cells, as judged by tail formation in thecomet assay and increased H2AX phosphorylation (Fig.5A–C). The combination treatments also resulted inamplification of DNA repair signaling, as evident fromthe increased levels of phospho-CHK2. Interestingly,changes in the phosphorylation of CHK1 were cell linedependent, with relative levels of phospho-CHK1 beingsomewhat higher for the combination in SKOV-3 cells, butlower in A2780 cells, when compared with the effects ofsingle agents (Fig. 5B and C). This can potentially beexplained by the reduction of total CHK1 levels producedby both gemcitabine or cisplatin and combination treat-ments with CX-4945, which may be linked to the previ-ously described caspase-mediated cleavage of CHK1 thatcan occur during apoptosis (40, 41). It is also possible thatCK2 inhibition may directly regulate CHK1 stability inA2780 cells, as CHK1 is a client protein of HSP90 co-chaperone CDC37, which is known to depend on phos-phorylation by CK2 for its activity (8).Using short interfering RNA knockdowns, we showed

that CK2a/a’, MDC1, and XRCC1 proteinswere essentialfor efficient DNA repair response resulting from cisplatinor gemcitabine treatment. Although XRCC1 has beenimplicated in cisplatin DNA repair response in HepG2cells (42), the role of XRCC1 in repairing gemcitabine-induced DNA repair response has not previously beendescribed. One possible explanation is that XRCC1 hasbeen shown to bind to RAD51, implicating XRCC1 in HRrepair (43). An alternative explanation derives from theemerging role of XRCC1 in the coordination of DNArepair and replication during S-phase, mediated by itsinteraction with the p58 subunit of DNA Pol a-primase

(44). Thismodelwould involveXRCC1 in the stabilizationand repair of stalled replication forks created by gemci-tabine, although the putative role of CK2 in this processremains to be investigated (Fig. 1).

The role of p53 in the cellular and clinical response tochemotherapies reflects the dual nature of p53 as anactivator of both DNA repair and apoptosis (45). In thecontext of ovarian cancer, p53mutations have been shownto confer resistance to cisplatin in vitro (46) and alsocorrelate with resistance to platinum-based chemothera-py and poor prognosis in the clinic (31). Although inantiproliferative experiments the combination of CX-4945 with gemcitabine or cisplatin was synergistic in bothA2780 and SKOV-3 cells, the fate of these cells was foundto be different, with p53 WT A2780 cells readily under-going increased apoptosis, whereas p53 null SKOV-3 cellsshowed evidence of mitotic catastrophe. These data sug-gest that even though different cell death pathways areactivated, the ability of CX-4945 to augment the antipro-liferative activity of gemcitabine or cisplatin seems not todepend on the status of p53, suggesting that such combi-nations can be used successfully in a broad spectrum ofpatients with ovarian cancer, a disease characterized by ahigh frequency of p53 mutation (32).

In vivo studies with mice bearing A2780 xenograftsconfirmed that the combination of CX-4945with cisplatin,carboplatin, or gemcitabine increased the antitumor effi-cacy compared with the efficacy observed with singleagents. We also showed that administration of CX-4945on an intermittent schedule, that is, 24 hours after eachdose of gemcitabine could enhance the efficacy of gemci-tabine in mice. These findings further illustrate that CX-4945 prevents CK2 from activating DNA repair responsemechanisms, thereby preventing replication recovery.Finally, we showed that the increased antitumor activityof the CX-4945/gemcitabine combination correlates withenhanced apoptosis by measuring cleaved PARP as apharmacodynamic biomarker in xenograft tumors.

CK2 potentially regulates multiple functions withinDNA repair response. However, a clearly prevailingmechanism is to facilitate the binding of signaling mole-cules andDNA end-processing factors to noncatalyticallyactive mediator/adaptor proteins involved in both SSBRand DSBR. Remarkably, mirroring its function withXRCC1, phosphorylation by CK2 promotes the bindingof both aprataxin and PNK to XRCC4, the mediator/adaptor binding partner of DNA ligase IV (7, 15). BecauseXRCC4/Ligase IV complex is an essential component ofnonhomologous end-joining repair (NHEJ) of DSBs, theprimary repair pathway triggered by ionizing radiation, itis possible that CK2 inhibition may synergize with radio-therapy in addition to the large number of DNA-targetedanticancer drugs that trigger SSBR, HR, and NHEJ (Sup-plementary Fig. S3).

Along with the DNA repair response, CK2 positivelyregulates an extensive list of additional cellular processesthat are also established effectors of sensitivity to DNA-targeted chemotherapeutics as well as other anticancer






drugs, including PI3K/AKT/mTOR signaling, NF-kBtranscription, Hsp90 machinery activity, hypoxia, inhibi-tion of apoptosis, and IL-6 expression (3, 8–11). Indeed,sensitization of pancreatic cancer cells to gemcitabineand head and neck cancer cells to cisplatin by geneticmanipulation of CK2 expression has been attributedto resulting suppression of Akt and NF-kB activity,respectively (47, 48). Thus it is possible that in additionto the suppression of DNA repair response, CX-4945enhances activity of gemcitabine and cisplatin by modu-lating PI3K/Akt and NF-kB signaling. Our data com-bined with previous reports showing enhancement oftherapeutic activity by suppression of CK2 (e.g., ref. 49)shows that CK2 represents a promising pharmacologictarget that can be explored in combination therapy withmultiple anticancer therapeutics.

Here we have described how targeting CK2 withCX-4945 can inhibit multiple DNA repair responsemechanisms by blocking phosphorylation of XRCC1 andMDC1 and synergize with the DNA-targeted anticancer

drugs cisplatin and gemcitabine in ovarian cancer cells.These data provide a strong rationale for combining CX-4945 with gemcitabine and platinum-based chemother-apeutics in clinical trials for ovarian and possibly othercancers.

Disclosure of Potential Conflicts of InterestAll authors are affiliated with Cylene Pharmaceuticals. A. Siddiqui-

Jain, K. Anderes, S.E. O’Brien, D.M. Ryckman, W.G. Rice, D. Drygin, J.Bliesath, C.B. Ho, J.K.C. Lim, and C. Proffitt have ownership interest(including patents) in Cylene Pharmaceuticals. D.D. Von Hoff is a con-sultant/on advisory board of Cylene Pharmaceuticals.

Grant SupportThis work received support from Cylene Pharmaceuticals.

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 August 9, 2011; revised December 5, 2011; accepted January 9,2012; published OnlineFirst January 20, 2012.

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2012;11:994-1005. Published OnlineFirst January 20, 2012.Mol Cancer Ther Adam Siddiqui-Jain, Joshua Bliesath, Diwata Macalino, et al. Mechanistic Rationale for Drug Combination Therapyby DNA-Targeted Anticancer Drugs and Augments Efficacy: CK2 Inhibitor CX-4945 Suppresses DNA Repair Response Triggered

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