interruption of the ras/mek/erk signaling cascade - blood

NEOPLASIA

Interruption of the Ras/MEK/ERK signaling cascade enhances Chk1inhibitor–induced DNA damage in vitro and in vivo in human multiplemyeloma cellsYun Dai,1 Shuang Chen,1 Xin-Yan Pei,1 Jorge A. Almenara,1 Lora B. Kramer,1 Charis A. Venditti,1 Paul Dent,2,3 andSteven Grant1,2,4

Departments of 1Medicine, 2Pharmacology, 3Radiation Oncology, and 4Biochemistry, Virginia Commonwealth University/Massey Cancer Center, Richmond, VA

The role of the Ras/MEK/ERK pathwaywas examined in relation to DNA damagein human multiple myeloma (MM) cellsexposed to Chk1 inhibitors in vitro and invivo. Exposure of various MM cells tomarginally toxic concentrations of theChk1 inhibitors UCN-01 or Chk1i mod-estly induced DNA damage, accompaniedby Ras and ERK1/2 activation. Interrup-tion of these events by pharmacologic(eg, the farnesyltransferase inhibitorR115777 or the MEK1/2 inhibitorPD184352) or genetic (eg, transfection

with dominant-negative Ras or MEK1shRNA) means induced pronounced DNAdamage, reflected by increased �H2A.Xexpression/foci formation and by cometassay. Increased DNA damage precededextensive apoptosis. Notably, similar phe-nomena were observed in primary CD138�

MM cells. Enforced MEK1/2 activation byB-Raf transfection prevented R115777 butnot PD184352 from inactivating ERK1/2and promoting Chk1 inhibitor–induced�H2A.X expression. Finally, coadministra-tion of R115777 diminished UCN-01–

mediated ERK1/2 activation and mark-edly potentiated �H2A.X expression in aMM xenograft model, associated with astriking increase in tumor cell apoptosisand growth suppression. Such findingssuggest that Ras/MEK/ERK activation op-poses whereas its inhibition dramaticallypromotes Chk1 antagonist–mediated DNAdamage. Together, these findings identifya novel mechanism by which agents tar-geting the Ras/MEK/ERK pathway poten-tiate Chk1 inhibitor lethality in MM. (Blood.2008;112:2439-2449)

Introduction

Checkpoint kinases (ie, Chk1 and Chk2) represent key componentsof the DNA damage checkpoint machinery, which monitors DNAbreaks caused by endogenous/metabolic or environmental geno-toxic insults or by replication stress.1,2 In response to DNA damage,cells activate checkpoint pathways, resulting in cell-cycle arrest,which permits the DNA repair machinery to rectify the damage.Depending on the nature of the DNA lesions and the context inwhich damage occurs, cells either survive and resume cell-cycleprogression through a recovery mechanism when repair is success-ful or are eliminated by apoptosis if repair fails. Thus, checkpointsprovide normal cells with critical surveillance machinery designedto promote genomic integrity and survival. Conversely, checkpointdysfunction contributes to tumorigenesis by permitting cell prolif-eration in the face of genomic instability.3,4 Moreover, checkpointsare activated by numerous chemotherapeutic agents and ionizingradiation.5 This has prompted the development of anticancerstrategies targeting checkpoint machinery.5,6 Among the diversecheckpoint pathway components, Chk1 represents a particularlyattractive target for several reasons, that is, (1) Chk1 is functionallyassociated with all known checkpoints (eg, the G2-M transition,G1, intra-S,5 and, most recently, the mitotic spindle checkpoint7);(2) Chk1 is essential for maintenance of genomic integrity, whereasthe role of Chk2 is conditional3; and (3) for multiple checkpoints,Chk2 function can be mimicked by Chk1, whereas Chk1 cannot bereplaced by a functionally overlapping kinase such as Chk2.3

Chk1 inhibition (eg, by the Chk1 inhibitor UCN-01) results inabrogation of checkpoints induced by DNA-damaging chemo-therapy and radiation, leading to enhanced tumor cell killing.8,9

Given these findings, a major emphasis has been placed on effortsto combine Chk1 inhibitors (eg, UCN-0110 or CHIR-12411) withdiverse DNA-damaging agents. However, an alternative strategy isbased on the concept that transformed cells may be ill-equipped tosurvive simultaneous interruption of both checkpoint machineryand prosurvival signaling. In this context, our group has reportedthat exposure of human leukemia and multiple myeloma (MM)cells to UCN-01 induces pronounced activation of MEK1/2 andERK1/2,12,13 key components of the Ras/Raf/MEK/ERK cascadethat plays a critical role in proliferation and survival of malignantcells.14 Significantly, disruption of ERK1/2 activation by pharmaco-logic MEK1/2 inhibitors,12,13 farnesyltransferase inhibitors (FTIs;eg, L744832)15,16 or HMG-CoA reductase inhibitors (ie, statins)17

results in a dramatic increase in apoptosis of hematopoieticmalignant cells. Together, these findings suggest that activation ofRas/MEK/ERK signaling cascade may represent a compensatoryresponse to Chk1 inhibitor lethality, and that interruption of thisresponse lowers the death threshold.

Although the observation that MEK1/2 inhibitors or FTIsantagonize UCN-01–mediated ERK1/2 activation and potentiatelethality of this agent in various tumor cell types has been welldocumented,12,13,18,19 the mechanism by which interruption of the

Submitted May 23, 2008; accepted June 27, 2008. Prepublished online asBlood First Edition paper, July 9, 2008; DOI 10.1182/blood-2008-05-159392.

The online version of this article contains a data supplement.

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Ras/MEK/ERK pathway potentiates the lethality of Chk1 inhibi-tors remains to be fully elucidated. Recently, it has been found thatChk1 inhibition by either Chk1 inhibitors (eg, UCN-01 andCEP-3891) or Chk1 siRNA leads to formation of single-strandedDNA (ssDNA) and induction of DNA strand breaks20 (ie, mani-fested by increased expression of the phosphorylated form of theatypical histone H2A.X, referred to as �H2A.X9). Interestingly,ERK1/2 signaling has been implicated in attenuation of DNAdamage through positive regulation of DNA repair mechanism.21

Such findings raise the possibility that interruption of Ras/MEK/ERK signaling may promote Chk1 inhibitor–mediated DNA dam-age, leading to enhanced lethality. To explore this possibility, wehave examined the effects of the Ras/MEK/ERK pathway on Chk1inhibitor–mediated DNA damage in MM cells. We report here forthe first time that disruption of Ras/MEK/ERK signaling cascade,by either pharmacological agents (eg, the FTI R115777 or theMEK1/2 inhibitor PD184352) or genetic approaches (eg, dominant-negative S17N Ras or MEK1 shRNA) results in markedly in-creased DNA damage prior to massive apoptosis in MM cells.Conversely, enforced MEK1/2 activation by B-Raf attenuates theability of R115777 but not PD184352 to promote Chk1 inhibitor–related DNA damage and lethality. Moreover, in an in vivo murinexenograft model of MM cells, coadministration of R115777diminishes UCN-01–induced ERK1/2 activation, markedly in-creases �H2A.X expression and foci formation, strikingly inducesapoptosis, and suppresses tumor growth. These findings arguestrongly for a functional role of Ras/MEK/ERK signaling in theregulation of Chk1 inhibitor–mediated DNA damage both in vitroand in vivo. Moreover, they provide a novel mechanistic basis forunderstanding interactions between agents targeting the DNAdamage checkpoint (eg, Chk1 inhibitors) and prosurvival signalingpathways (eg, farnesyltransferase or MEK1/2 inhibitors).

Methods

Cells and reagents

Human MM U266 (wild-type Ras), RPMI8226 (activated K-Ras), H929(activated N-Ras),22 MM.1S, and MM.1R cells were maintained asreported previously.13 All experiments used logarithmically growing cells(3-5 � 105 cells/mL). Bone marrow samples were obtained with informedconsent from 2 patients with MM undergoing routine diagnostic aspirationwith IRB approval from Virginia Commonwealth University. Informedconsent was provided in accordance with the Declaration of Helsinki.CD138� myeloma cells were isolated as previously reported.13,16 UCN-01was provided by Cancer Therapy Evaluation Program (CTEP), NationalCancer Institute (NCI, Bethesda, MD). 4-(2-Phenyl)-9-hydroxypyrrolo[3,4-c]carbazole-1,3-(2H,6H)-dione (designated Chk1i throughout), a specificWee1/Chk1 inhibitor (IC50 toward Chk1 vs Wee1 � 2.06-fold, determinedby an in vitro assay),23 and PD184352 were purchased from Calbiochem(San Diego, CA) and Upstate Biotechnology (Lake Placid, NY), respec-tively. R115777 was provided by Johnson & Johnson Pharmaceuticals (LaJolla, CA) via CTEP. Agents were dissolved in DMSO and stored at �20°C.In all experiments, the final concentration of DMSO did not exceed 0.1%.

Plasmids and stable transfection

cDNAs for human H-Ras S17N mutant (dominant negative), full-lengthB-Raf, as well as MEK1 shRNA (pKD-MEK1-v2; GenBank no.NM 00275524) and its negative control (pKD-NegCon-v1) were obtainedfrom Upstate Biotechnology. U266 cells were transfected with theseconstructs using Amaxa Nucleofector (Amaxa, Cologne, Germany) as perthe manufacturer’s instructions. Clones were selected by G418.

Flow cytometry

Apoptosis was evaluated by annexin V–FITC or 7AAD staining and flowcytometry as described previously.17 To analyze DNA damage, cells werestained with Alexa Fluor 488–conjugated phospho–histone H2A.X (Ser139)rabbit monoclonal antibody (Cell Signaling, Beverly, MA) as per themanufacturer’s protocol, followed by flow cytometry to determine thepercentage of cells with relatively increased �H2A.X expression. Theresults for each condition were normalized to values for cells stained withrabbit IgG (Southern Biotech, Birmingham, AL) as the primary antibody.

Comet assay

Single-cell gel electrophoresis assays were performed to assess both single-and double-stranded DNA breaks in cells using a Comet Assay Kit(Trevigen, Gaithersburg, MD) as per the manufacturer’s instructions.Images were captured using fluorescence microscopy at 20�/0.50. Tailmoment was determined25 using TriTek CometScore v1.5, a free softwareprogram downloaded from http://www.tritekcorp.com.

Western blot

Samples from whole-cell pellets were prepared and 30 �g protein for eachcondition was subjected to Western blot following previously describedprocedures.12 Blots were reprobed with antibodies against �-actin or�-tubulin to ensure equal loading and transfer of proteins. The followingprimary antibodies were used: phospho-p44/42 MAPK (ERK1/2, Thr202/Tyr204) and p44/42 MAPK (Cell Signaling); H-Ras, B-Raf, and MEK1(Santa Cruz, Santa Cruz, CA); phospho–histone H2A.X (Ser139), and Ras(clone RAS10) that recognizes H-, K-, and N-Ras (Upstate Biotechnology);and PARP (Biomol, Plymouth Meeting, PA).

Ras activation assay

Ras activity was detected using a Ras Activation Assay Kit (UpstateBiotechnology) as described previously17 using 400 �g protein for eachcondition. Ras activity was reflected by the amount of Ras-GTP pulleddown by Raf-1 RBD (Ras-binding domain).

Membrane distribution of Ras

Following treatment, cells were lysed and membrane proteins fractionedusing Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit(Pierce, Rockford, IL) to assess membrane localization of Ras as per themanufacturer’s instructions. Samples (5 �g protein for each condition) ofmembrane fractions were diluted 5-fold to prevent band distortion andsubjected to Western blot analysis using Ras antibody (clone RAS10).

Animal studies

Animal studies were approved by the American Association for Accredita-tion of Laboratory Animal Care, and performed in accordance with currentregulations and standards of the US Department of Agriculture, the USDepartment of Health and Human Services, and the National Institutes ofHealth. Female athymic NCr-nu/nu mice were purchased from The JacksonLaboratory (Bar Harbor, ME), and inoculated subcutaneously with 107

MM.1S cells into the right rear flank. Treatment was administratedintraperitoneally daily after tumors reached a volume of approximately100 mm3. R115777 was freshly prepared in 2% B-cyclodextrin in 0.1 NHCl for a final dose of 25 mg/kg. UCN-01 in DMSO was diluted in 2%(wt/vol) Na citrate (pH 3.5) for a final dose of 0.5 mg/kg. Control animalswere injected with equal volume of vehicle to that administered withR115777 and/or UCN-01. Tumors were measured every 2 days indepen-dently by 3 operators. Tumor volumes were calculated from the formula(L � W2)/2, where L and W represent longest and shortest lengths of thetumor, respectively.

Immunohistochemistry, immunofluorescence, and TUNELstaining

For animal studies, tumors were removed 30 minutes after the final dose.Tissue cryostat sections were prepared for immunohistochemical staining

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for phospho-ERK and Ki67, using phospho-p44/42 MAPK (Thr202/Tyr204) rabbit monoclonal antibody (20G11, IHC preferred; Cell Signal-ing) or Ki67 antibody (Oncogene, San Diego CA), respectively, as per theinstructions of the manufacturer. To assess apoptosis, tumor sections werestained for terminal deoxynucleotidyl transferase-mediated dUTP-biotinnick-end labeling (TUNEL) using In Situ Cell Death Detective Kit(fluorescein; Roche, Penzberg, Germany) as per the manufacturer’s instruc-tions. To monitor �H2A.X foci formation, samples were stained with AlexaFluor 488–conjugated �H2A.X antibodies as per the manufacturer’sinstructions. Images were captured with Olympus BX40 fluorescencemicroscope (Olympus, Center Valley, PA) and a CE digital camera (AlphaInnotech, San Leandro, CA) with RS Image software (Roper ScientificPhotometrics, Tucson, AZ).

Statistical analysis

For analysis of apoptosis, values represent the means plus or minus SD forat least 3 separate experiments performed in triplicate. Significance ofdifferences between experimental variables was determined using the Student ttest. Analysis of synergism was performed using median dose effectanalysis using the Calcusyn software program (Biosoft, Ferguson, MO).17

Results

The FTI R115777 or the MEK1/2 inhibitor PD184352 disruptsRas3ERK1/2 signaling in human MM cells exposed to Chk1inhibitors, accompanied by increased �H2A.X expression andlethality

Previous studies indicated that exposure to UCN-01 inducedERK1/2 phosphorylation/activation in human leukemia and MMcells,12,13 and very recently, triggered Ras activation in MM cells.17

Consistent with those findings, exposure of U266 and RPMI8226cells to marginally toxic concentrations of UCN-01 (ie, 100-200 nM; 14% or 7% cell death in U266 [48 hours] or RPMI8226[24 hours] cells, respectively) or Chk1i (1-2 �M; 19% or 8%cell death in U266 [48 hours] or RPMI8226 [24 hours] cells,respectively) resulted in a clear increase in membrane localizationand activation of Ras, accompanied by marked ERK1/2 phosphor-ylation (Figure 1A). In vitro loading with GTP�S rather than GDPin untreated cell lysates for each cell line resulted in a strikingincrease in the amount of active Ras-GTP pulled down by Raf-1RBD-agarose beads (Figure S1A, available on the Blood website;see the Supplemental Materials link at the top of the online article),verifying specificity of the Ras activation assay. Similar resultswere obtained in MM.1S and its dexamethasone-resistant counter-part MM.1R (Figure S1B). These results indicate that pharmaco-logic inhibition of Chk1 triggers activation of Ras and ERK1/2 invarious human MM cell lines, and also suggest that this representsa generalized phenomenon involving Chk1 inhibitors, rather thanbeing restricted to the prototype UCN-01.

In view of earlier evidence that treatment with Chk1 inhibitorspromotes DNA breaks, manifested by phosphorylation of histoneH2A.X on Ser139 residues (designated �H2A.X),20 the possibilitythat interruption of ERK1/2 activation by pharmacological agentsmight affect Chk1 inhibitor–induced DNA damage was examined.Cotreatment with either the FTI R115777 or the MEK1/2 inhibitorPD184352 largely attenuated ERK1/2 phosphorylation inRPMI8226 or U266 cells exposed to UCN-01 or Chk1i (Figure1B,C). Notably, exposure to UCN-01 or Chk1i induced a modestbut discernible increase in �H2A.X, whereas coadministration ofeither R115777 or PD184352 dramatically increased Chk1 inhibitor–induced �H2A.X expression. Furthermore, both R115777 andPD184352 significantly potentiated apoptosis in MM cells exposed

to either Chk1 inhibitor (Figure 1D; P .01 in each case,compared with treatment with each Chk1 inhibitor individually), aphenomenon confirmed by increased PARP cleavage (Figure1B,C). Similar results were obtained in MM.1S and MM.1R cells(Figure S1C,D), as well as H929 cells (Figure S1E,F). Finally,isobologram analysis revealed combination index values less than0.4 (�H2A.X expression) or less than 0.2 (cell death, manifested by7AAD�), indicating a highly synergistic interaction (Figure 1E).Together, these findings raise the possibility that simultaneousinterference with compensatory Ras3ERK1/2 activation enhancesChk1 inhibitor–induced DNA damage, and that these events maycontribute to synergism between Chk1 inhibitors and agentstargeting the Ras/MEK/ERK pathway.

R115777 or PD184352 promotes DNA damage in MM cellsexposed to Chk1 inhibitors, a phenomenon that occurs prior toinduction of extensive apoptosis

Although �H2A.X expression is primarily induced by DNAdouble-stranded breaks (DSBs),26 DNA fragmentation during apo-ptosis may also lead to H2A.X phosphorylation,27 includingphosphorylation on Ser139.28 To address this issue, 2 sensitiveassays were used to determine whether increased �H2A.X expres-sion is indeed related to enhanced DNA damage in the presentsettings. Notably, consistent with observations in established MMcell lines (ie, U266; Figure S2A), immunofluorescence analysisusing Alexa Fluor 488–conjugated �H2A.X antibodies revealedthat coadministration of Chk1 inhibitors (ie, UCN-01 or Chk1i)with R115777 markedly increased the number of nuclear foci, aphenomenon reflecting sustained colocalization of �H2A.X withdiverse DNA damage mediator/repair factors at or near DNA breaksites,29-31 in primary CD138� MM cells (Figure 2A), but not inCD138� bone marrow cells (Figure S2B). Similar phenomena wereobserved when PD184352 was used instead of R115777 (data notshown). In addition, flow cytometric assays demonstrated thatincreased �H2A.X expression occurred in primary CD138� MMcells as early as 6 hours following coexposure to Chk1 inhibitorswith R115777 or PD184352 (Figure S3A), but this phenomenonwas not observed in CD138� bone marrow cells even after a24-hour exposure (Figure S3B). Moreover, after drug treatment(24 hours), U266 cells were analyzed by comet assay, an estab-lished method for evaluating both single- and double-strandedDNA breaks.32 In this assay, denatured, broken DNA fragmentsmigrate out of the cell under the influence of an electric field,producing a comet tail, whereas undamaged DNA migrates moreslowly and remains within the confines of the nucleus.33 As shownin Figure 2B, coadministration of R115777 or PD184352 witheither UCN-01 or Chk1i induced a striking increase in the numberof comet-positive cells compared with treatment with the agentsindividually. Notably, increased �H2A.X foci formation and theappearance of DNA comet tails in U266 cells coexposed to Chk1inhibitors with R115777 or PD184352 occurred substantiallybefore the induction of massive apoptosis (ie, 24 hours vs48 hours). As shown in Figure 2C, quantification of data indicatedthat coadministration (24 hours) of R115777 or PD184352 witheither UCN-01 or Chk1i induced approximately a 50% to 75%increase in comet tail moment, whereas only a modest increase inapoptosis (eg, � 25% annexin V� cells) occurred at this exposureinterval. Together, these results confirm that coadministration ofeither R115777 or PD184352 markedly potentiates DNA damagein MM cells exposed to Chk1 inhibitors, and indicate that this eventprecedes induction of extensive apoptosis.

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Dominant-negative Ras (S17N) prevents ERK1/2 activation andsensitizes MM cells to induction of �H2A.X expression andlethality mediated by Chk1 inhibitors

To gain further insights into the functional role of Ras3ERK1/2signaling in Chk1 inhibitor–mediated DNA damage, U226 cellswere stably transfected with a dominant-negative form (S17N) ofH-Ras34,35 (Figure 3A top panel). As shown in Figure 3A (bottompanel), ectopic expression of S17N Ras substantially blocked Rasactivation induced by UCN-01. Moreover, exposure to Chk1inhibitors (ie, UCN-01 or Chk1i) failed to induce ERK1/2 phosphor-ylation in these cells (Figure 3A,B). Significantly, these eventswere accompanied by a pronounced increase in �H2A.X expres-sion and PARP cleavage compared with EV controls. Consistentwith these findings, cells expressing S17N Ras were significantly

more sensitive to apoptosis induced by both Chk1 inhibitors thantheir EV counterparts (Figure 3C,D; P .05 or P .01). Collec-tively, these findings indicate that disruption of Ras signaling playsa significant functional role in potentiating Chk1 inhibitor–inducedDNA damage and lethality.

MEK1 down-regulation by shRNA significantly enhances Chk1inhibitor–mediated �H2A.X expression

An shRNA approach targeting MEK1, a key mediator forRas3ERK1/2 signaling,36 was then used to further examinefunctional relationships between MEK/ERK signaling and DNAdamage. As shown in Figure 4, stable transfection of U266 cellswith a construct encoding MEK1 shRNA resulted in marked

Figure 1. R115777 or PD184352 potentiates Chk1 inhibitor–induced �H2A.X expression and lethality in human MM cells in association with diminishedRas3ERK1/2 signaling. (A) U266 and RPMI8226 cells were exposed for 24 hours to UCN-01 (UCN, top panels) or Chk1i (bottom panels), after which Ras activation assaysand Western blot analysis (WB) were performed to monitor Ras activation status and ERK1/2 phosphorylation, respectively. Ras activity was reflected by amount of Ras-GTPpulled down by Raf-1 RBD. Alternatively, membrane fractions were separated and subjected to WB. In parallel, the percentage of cell death (7AAD�) was assessed by flowcytometry to determine the toxicity of UCN-01 or Chk1i at the indicated concentrations in U266 (48h) or RPMI8226 cells (24h). (B,C) Cells were exposed to UCN-01(RPMI8226, 100 nM for 24 hours; U266, 150 nM for 48 hours) or Chk1i (RPMI8226, 1 �M for 24 hours; U266, 2 �M for 48 hours) in the absence or the presence of either 5 �MR115777 (R115) or 5 �M PD184352 (PD184), after which cells were lysed and subjected to WB for phosphorylation of ERK1/2 and H2A.X (�H2A.X), as well as PARPdegradation. For panels A through C, results of a representative experiment are shown; 2 additional studies yielded equivalent results. CF indicates cleavage fragment.(D) Alternatively, the percentage of apoptotic cells was determined by annexin V–FITC staining and flow cytometry. Veh indicates vehicle. Results represent the means plus orminus SD for 3 separate experiments performed in triplicate. ** indicates significantly greater than values for those treated with Chk1 inhibitors alone (P .01). (E) U266 cellswere treated with a range of R115777 and UCN-01 concentrations alone or in combination for 24 hours (for �H2A.X staining) or 48 hours (for 7AAD staining) at a fixed ratio(R115777/UCN-01, 50:1). At the end of this period, the percentage of cells with relative increases in �H2A.X expression (ie, drug treatment vs untreated controls, left panel) or7AAD� cells (right panel) was determined by flow cytometry, respectively. Median dose effect analysis was used to characterize the nature of the interaction. Two additionalstudies yielded equivalent results.





down-regulation of MEK1 expression, accompanied by a pro-nounced reduction in basal levels of phospho-ERK1/2. Moreover,this approach dramatically diminished ERK1/2 phosphorylationinduced by either UCN-01 or Chk1i, accompanied by morepronounced �H2A.X expression as well as PARP cleavage follow-ing exposure to either Chk1 inhibitor, compared with controlcounterparts transfected with a construct encoding a scrambledsequence (Figure 4A,B). Furthermore, MEK1 knockdown signifi-

cantly sensitized U266 cells to Chk1 inhibitor lethality comparedwith controls (Figure 4C,D; P .05 or P .01). In conjunctionwith the preceding results, these findings argue strongly that activationof the Ras/MEK/ERK signaling cascade limits DNA damage inducedby Chk1 inhibitors, and that blockade of these compensatory responsesby targeting either Ras (eg, by FTIs or dominant-negative mutant Ras)or MEK (eg, by MEK1/2 inhibitors or MEK1 siRNA) results indramatic potentiation of DNA damage and lethality.

Figure 2. Cotreatment with R115777 and Chk1 inhibitors induces a pronounced increase in �H2A.X foci formation and DNA breaks. (A) Primary CD138� cells wereisolated from the bone marrow of a patient (no. 1) with MM. Cells were then either untreated or exposed (16 hours) to 150 nM UCN-01 (UCN) or 2 �M Chk1i in the absence orthe presence of 5 �M R115777 (R115). After treatment, cells were harvested and stained with Alexa Fluor (AF) 488–conjugated phospho-H2A.X (Ser139) antibody forimmunocytochemical analysis. Images were captured at 60�/1.40 oil. (B) U266 cells were treated with 150 nM UCN-01 or 2 �M Chk1i with or without 5 �M R115777 or 5 �MPD184352 for 24 hours, after which a comet assay was performed to assess DNA breaks. As control, U266 cells were treated with 100 �M hydrogen peroxide for 20 minutes.(C) Tail moment was calculated as the percentage of DNA in the tail and the distance between the means of head and tail distributions. Mean tail moment was determined bymeasuring at least 100 cells per sample. Results represent the means plus or minus SD for 3 separate experiments. In parallel, the percentage of annexin V� cells wasdetermined by flow cytometry. V indicates vehicle. Results represent the means plus or minus SD for 3 separate experiments performed in triplicate.





Enforced activation of MEK1/2 by ectopic B-Raf expressionprevents R115777 but not PD184352 from potentiating Chk1inhibitor–mediated �H2A.X expression and lethality

Attempts were made to determine whether enforced MEK1/2activation at an intermediate level of the Ras/Raf/MEK/ERKsignaling cascade (eg, by ectopic B-Raf expression) might differen-tially affect the ability of FTIs versus MEK1/2 inhibitors topotentiate Chk1 inhibitor–mediated DNA damage and lethality. Tothis end, U266 cells were stably transfected with a constructencoding full-length B-Raf. As anticipated, ectopic expression ofB-Raf led to a marked increase in basal levels of phosphorylatedMEK1/2 (Figure 5A inset) and ERK1/2 (Figure 5B).37,38 Notably,enforced activation of MEK1/2 by B-Raf substantially diminishedthe capacity of the FTI R115777, but not the MEK1/2 inhibitorPD184352, to block ERK1/2 phosphorylation and to potentiate�H2A.X expression induced by either Chk1 inhibitor (Figure 5B).Moreover, these cells also displayed significant resistance to thelethality of the Chk1 inhibitor/R115777 (P .01 in each case) butnot the Chk1 inhibitor/PD184352 (P .05 in each case) regimen,compared with EV control cells (Figure 5A). Together, thesefindings suggest that FTIs target signaling events upstream of Rafto attenuate ERK1/2 activation and to promote DNA damage andlethality in MM cells exposed to Chk1 inhibitors, whereas MEK1/2inhibitors act at a more distal site in the Ras/Raf/MEK/ERK

cascade. These observations also provide further support for thenotion that interruption of the Ras/Raf/MEK/ERK signaling path-way (eg, by FTIs and MEK1/2 inhibitors) potentiates Chk1inhibitor–mediated DNA damage and lethality.

Coadministration of R115777 with UCN-01 attenuates ERK1/2activation and increases �H2A.X expression/foci formation invivo in a MM xenograft model, accompanied by strikinginduction of apoptosis and suppression of tumor growth

Finally, attempts were made to determine whether the preceding invitro findings would be operative in vivo. To address this issue,athymic nude mice were inoculated in the flank with humanmyeloma MM.1S cells, and after tumors were measurable, treateddaily intraperitoneally with either vehicle, UCN-01 (0.5 mg/kg),R115777 (25 mg/kg), or both R115777 and UCN-01 for 12 days.As shown in Figure 6, combined treatment with UCN-01 andR115777 resulted in a dramatic suppression of tumor growthduring (Figure 6A) and following (Figure 6B) treatment, whereasagents administrated individually exerted only modest inhibitoryeffects. Similar interactions were observed in an RPMI8226bioluminescence xenograft model (Figure S4). Moreover, immuno-histochemical analysis of tumor sections excised 30 minutes afterthe final drug doses revealed that UCN-01 administered aloneresulted in a clear increase in phospho-ERK1/2 expression in

Figure 3. Ectopic expression of dominant-negative Ras (S17N) prevents ERK1/2 activation and enhances �H2A.X expression following exposure to Chk1inhibitors. (A,B) U266 cells were stably transfected with S17N H-Ras or its empty vector (EV), and ectopic expression of mutant protein was detected by WB (A, top panels).Cells were then exposed to UCN-01 or Chk1i for 24 hours, after which Ras activation assay and WB were performed to monitor Ras activity (A, bottom panel) and expression ofphospho-ERK1/2 and �H2A.X, as well as PARP cleavage. The results of representative experiments are shown; 2 additional studies yielded equivalent results. CF indicatescleavage fragment. (C,D) Cells were incubated with UCN-01 or Chk1i for 24 hours and 48 hours, after which percentage of annexin V–FITC–positive cells was determined byflow cytometry. The results represent the means plus or minus SD for 3 separate experiments performed in triplicate. *P .05; **P .01.





nuclei of tumor cells (arrows), an event dramatically diminished bycoadministration of R115777 (Figure 7A top panels). In addition,combined treatment with UCN-01 and R115777 dramaticallyreduced expression of the cell proliferation marker Ki67 in tumorcells, as well as diminished infiltration of tumor cells into thesurrounding muscle (M) tissue, whereas the agents administratedindividually had little or no effect (Figure 7A middle panels).Moreover, coadministration of R115777 with UCN-01 resulted in astriking induction of apoptosis in tumor cells, manifested byTUNEL positivity, whereas the agents administered individuallyhad minimal effects (Figure 7A bottom panels). Notably, treatmentwith UCN-01 alone resulted in only a modest increase in �H2A.Xexpression (reflected by increased fluorescent intensity of �H2A.X)as well as foci formation (Figure 7B arrows), whereas R115777administered alone had no discernible effect. However, thesephenomena were markedly potentiated when UCN-01 was coadmin-istered with R115777 (Figure 7B). Collectively, these findingssuggest that disruption of Ras3ERK1/2 signaling (eg, by the FTIR115777) may also contribute to promotion of DNA damage andlethality induced by Chk1 inhibitors (eg, UCN-01) in vivo.

Discussion

Chk1 plays a critical role in maintaining genomic integrity byregulating DNA damage–associated checkpoint responses, andrepresents an attractive therapeutic target.5 UCN-01, a first-generation Chk1 inhibitor, potently blocks Chk1 function at low,submicromolar concentrations,10 an action that can promote DNAdamage by itself20 or in combination with DNA-damagingagents.39-41 As UCN-01 exerts pleiotropic actions, including inhibi-tion of PKC, CDKs, and PDK1 in addition to Chk1,10 and becauseUCN-01’s therapeutic effectiveness is limited by human alpha(1)-acid glycoprotein binding,42 efforts to develop newer generation,more specific Chk1 inhibitors are currently under way.43,44 Thesehave focused primarily on disrupting checkpoint responses inducedby DNA-damaging agents and ionizing radiation.5,6,43 However,UCN-01 also triggers compensatory activation of the prosurvivalRas/MEK/ERK cascade in various tumor cell types, includinghuman MM cells.13,17 Significantly, interruption of the latterpathway (eg, by MEK1/2 inhibitors,12,13 as well as FTIs15,16 and

Figure 4. MEK1 knockdown by shRNA blocksERK1/2 activation and sensitizes MM cells to �H2A.Xexpression and lethality induced by Chk1 inhibi-tors. (A,B) U266 cells were stably transfected withconstructs encoding MEK1 shRNA or a scrambledsequence as a control, and exhibited down-regulationof MEK1 expression by WB (top panels). Cells werethen incubated with UCN-01 or Chk1i for 24 hours, afterwhich WB analysis was performed to detect expressionof phospho-ERK1/2 and �H2A.X, as well as PARPdegradation. Blots were subsequently stripped andreprobed for expression of �-tubulin to ensure equiva-lent loading and transfer of protein. Additional 2 studiesyielded equivalent results. CF indicates cleavage frag-ment. (C,D) Alternatively, after 24-hour and 48-hourtreatment, flow cytometry was performed to monitorapoptosis (annexin V–FITC staining). Results repre-sent the means plus or minus SD for 3 separateexperiments performed in triplicate. *P .05; **P .01; NS indicates no significance (P .05).

Figure 5. Enforced activation of MEK1/2 by B-Rafdisables R115777 but not PD184352 to block ERK1/2activation and to potentiate �H2A.X expressionmediated by Chk1 inhibitors. (A) U266 cells werestably transfected with a construct encoding full-lengthB-Raf, and WB was performed to monitor B-Raf expres-sion as well as resulting MEK1/2 phosphorylation(inset). Blots were subsequently stripped and reprobedusing �-actin antibodies to ensure equivalent loadingand transfer of proteins. Cells were then exposed to150 nM UCN-01 or 2 �M Chk1i with or without 5 �MPD184352 or 5 �M R115777 for 48 hours, after whichthe percentage of annexin V� cells was determined byflow cytometry. The results represent the means plus orminus SD for 3 separate experiments performed intriplicate. **P .01; NS indicates no significance(P .05). (B) Alternatively, after 24-hour exposure todrugs, WB was performed to monitor expression ofphospho-ERK1/2 and �H2A.X. Results are representa-tive of 3 separate experiments.





statins17) dramatically increases UCN-01 lethality in human leuke-mia and MM cells. Although this phenomenon may stem from thepleiotropic actions of UCN-01, the present results demonstrate thata more selective Chk1 inhibitor (Chk1i)23 also triggered activationof both Ras and ERK1/2 in MM cells. Moreover, coadministrationof either FTIs or MEK1/2 inhibitors dramatically potentiated Chk1ilethality in MM cells. Very recently, similar phenomena have beenobserved in the case of AZD7762,45 another specific and clinicallyrelevant Chk1 inhibitor.44 Collectively, such findings argue that Ras/MEK/ERK pathway activation is not restricted to the prototypical Chk1inhibitor UCN-01, but in all likelihood represent a more generalizedresponse of tumor cells exposed to agents targeting Chk1.

Results of previous reports indicate that Chk1 inhibitors,including UCN-01, not only enhance genotoxic agent-mediatedDNA damage,39-41,46 but also induce DNA breaks by themselves,manifested by increased phosphorylation of histone H2A.X atSerine 139 (�H2A.X).20 Thus, induction of DNA damage by Chk1inhibitors may contribute to their antitumor activity. On the otherhand, earlier findings suggested that Ras/MEK/ERK pathwayactivation represents a cytoprotective response of transformed cellsto Chk1 inhibitors such as UCN-01.13,16 In this context, recentevidence that Raf/MEK/ERK signaling is critical to DNA damageresponses and DNA repair21 prompted us to examine whetherdisruption of the Ras/Raf/MEK/ERK signaling cascade mightenhance Chk1 inhibitor–induced DNA damage. Notably, coadmin-istration of the FTI R115777 or the MEK1/2 inhibitor PD184352dramatically increased DNA damage, reflected by both increased�H2A.X expression/foci formation and DNA comet tail appear-ance, in UCN-01– or Chk1i-treated cells. Significantly, theseevents closely correlated with blockade of Chk1 inhibitor–inducedERK1/2 activation by FTIs or MEK1/2 inhibitors, and occurredprior to massive apoptosis. It is tempting to speculate that even inthe absence of exogenous DNA damage stimuli (eg, DNA damag-ing agents or radiation), Chk1 plays an important role in protecting

cells from DNA breaks or stresses occurring during DNA replica-tion. Indeed, interruption of Chk1 function (ie, by Chk1 siRNA orChk1 inhibitors) induces DNA breaks, possibly a consequence ofaberrant events related to increased initiation and/or elongation ofDNA replication.20,41 The finding that interruption of Ras3ERK1/2signaling dramatically promotes Chk1 inhibitor–mediated DNAdamage in MM cells has not, to the best of our knowledge, beenpreviously described, and may provide new insights into the basisfor synergism between these agents.

The mechanism(s) responsible for potentiation of Chk1 inhibi-tor lethality by Ras/MEK/ERK pathway disruption may reflectmultiple interrelated events. For example, ERK1/2 activationexerts cytoprotective actions through posttranslational modifica-tion of several components of the apoptotic pathway (eg, phosphor-ylation/inactivation of caspase-947) or proapoptotic Bcl-2 familymembers (eg, Bad48). Notably, interruption of the Ras/ERK path-way by pharmacologic or genetic approaches (ie, cells expressingS17N Ras) increased Chk1 inhibitor–mediated cdc2 activation(Y.D. and S.G., unpublished results, May 2008), an event associ-ated with apoptosis.47 In addition, the ability of MEK1/2 inhibitorsto potentiate UCN-01 lethality in MM cells has very recently beenlinked to diminished BimEL phosphorylation and degradation.49

Interestingly, MEK1/2 inhibitors administered alone at concentra-tions that substantially increase Bim expression are minimallylethal to MM cells.49 This suggests that Bim up-regulation by itselfmay be insufficient to trigger apoptosis, but instead lowers thelethal threshold for other events, such as DNA damage and/or cdc2activation. Studies designed to test this hypothesis are under way.

The present findings argue strongly that the Ras/Raf/MEK/ERKpathway plays an important functional role in regulating Chk1inhibitor–related DNA damage in MM cells. For example, ectopicexpression of dominant-negative Ras (S17N) or MEK1 knockdownby shRNA largely recapitulated the capacity of R115777 andPD184352 to prevent Chk1 inhibitor–mediated ERK1/2 activation,

Figure 6. Cotreatment with R115777 and UCN-01results in marked tumor growth suppression in amurine xenograft model of MM.1S cells. (A,B) Nudemice were inoculated subcutaneously with 107 MM.1Scells into the right rear flank. After tumors were measur-able, 25 mg/kg R115777 with or without 0.5 mg/kgUCN-01 were administrated intraperitoneally daily for12 days. Tumor size was monitored every 2 daysduring drug treatment (days 1-12; panel A) and for anadditional 12 days after cessation of drug treatment(days 13-24; panel B). Mean tumor volumes are shown(n � 9 per group).





thereby sensitizing MM cells to DNA damage and lethality. Suchfindings provide genetic evidence that both Ras and MEK1 arerequired for Chk1 inhibitor–mediated ERK1/2 activation andattenuation of DNA damage. Moreover, enforced MEK1/2 activa-tion by B-Raf substantially diminished the ability of FTIs to blockChk1 inhibitor–induced ERK1/2 activation and protected MM cellsfrom R115777/Chk1 inhibitor–induced DNA damage and lethality.In marked contrast, PD184352, by targeting MEK1/2, which actsdirectly downstream of Raf,36 circumvented the protective effectsof ectopic B-Raf expression. Together, such findings suggest thatactivation of Ras/Raf/MEK/ERK signaling represents a self-protective response limiting DNA damage in Chk1 inhibitor–treated cells. Currently, the mechanism(s) by which Ras/MEK/ERK pathway interruption potentiates DNA damage by Chk1inhibitors or by other genotoxic stimuli remains to be determined.In this context, it has been observed that Chk1 inhibition by eitherChk1 inhibitors or Chk1 siRNA promotes DNA breaks through anATR-dependent mechanism.20 Moreover, Chk1 is required for therepair of DNA double-stranded breaks.50,51 In addition, the Raf/MEK/ERK pathway has been linked to DNA repair through anATM-dependent process.21 Thus, one plausible explanation for thepresent findings is that interference with the Ras/Raf/MEK/ERKsignaling cascade disrupts DNA repair processes, thereby potentiat-ing DNA damage induced by Chk1 inhibitors. Whatever the basisfor this interaction, the observation that disruption of Ras3ERK1/2signaling (eg, by R115777) dramatically potentiated UCN-01–mediated DNA damage and apoptosis in murine xenograft MMmodels argues that such mechanisms do not simply reflect in vitrophenomena, but are also operative in vivo.

Despite encouraging preclinical results, the clinical promise ofFTIs remains to be fulfilled,14,52 and the lack of correlation between

FTI responsiveness and Ras mutational status raises the possibilityof alternative targets (eg, Rho proteins53). In the present study,genetic disruption of Ras function (eg, by dominant-negative S17NRas) largely prevented Chk1 inhibitor–induced ERK1/2 activationand sensitized MM cells to these agents. Moreover, ectopicexpression of B-Raf largely prevented R115777 from blockingERK1/2 activation and enhancing DNA damage in MM cellsexposed to Chk1 inhibitors. Such genetic evidence indicates thatChk1 inhibitors induce ERK1/2 activation via a Ras-dependentmechanism, and that FTIs act through interruption of the Ras/Raf/MEK/ERK cascade to promote Chk1 inhibitor–mediated DNAdamage and lethality. In this context, members of the Ras familyare frequently mutated/activated in human cancers,14,54 includingMM (ie, 40%, increasing to 70% at relapse).55,56 A question ariseswhether Ras mutational status might influence MM cell responsesto strategies combining Chk1 inhibitors with agents targeting theRas3ERK1/2 signaling cascade, particularly inhibitors of up-stream (eg, FTIs) versus downstream (eg, MEK1/2 inhibitors)events. However, the observations that the R115777/ and PD184352/Chk1 inhibitor regimens displayed similar activity toward MMcells with disparate Ras status, for example, RPMI8226 (activatedK-Ras), U266 (wild-type Ras), and H929 (activated N-Ras),22

argue against this possibility.In summary, the present findings suggest that activation of

the Ras3ERK1/2 signaling cascade plays an important cytopro-tective role in limiting Chk1 inhibitor–mediated DNA damagein MM cells. They also indicate that interruption of this pathwayby FTIs or MEK1/2 inhibitors enhances Chk1 inhibitor lethalitythrough a novel mechanism (ie, potentiation of DNA damage).Previous studies implicating the MEK/ERK pathway in regulat-ing checkpoint responses have largely focused on DNA damage–

Figure 7. R115777 diminishes UCN-01–inducedERK1/2 activation in association with an increasein �H2A.X expression/foci formation and a dra-matic induction of apoptosis induced by UCN-01 invivo. (A) Following the experiments described in Figure6, tumors were excised after the final dose, andsubjected to immunohistochemistry for ERK1/2 phos-phorylation and Ki67 expression, as well as TUNELstaining. indicates nuclear staining of phospho-ERK1/2 in tumor cells. M indicates muscle. (B) Alterna-tively, tumor tissue sections were stained with AlexaFluor 488–conjugated �H2A.X antibodies to monitor�H2A.X expression and nuclear foci formation. Arrowsindicate cells with �H2A.X foci. Results are representa-tive of 3 separate sets of tumor sections.





related stimuli (eg, DNA-damaging agents or ionizing radia-tion).21,57,58 However, it is noteworthy that even absent suchnoxious stimuli, interruption of Chk1 function (eg, by Chk1inhibitors or Chk1 siRNA) induces DNA breaks.20 The presentresults suggest that Ras3ERK1/2 activation provides a cellulardefense permitting cells to survive the lethal consequences ofChk1 inhibition (eg, DNA damage), possibly by promotingDNA repair. Such findings provide a mechanistic basis foran alternative Chk1 inhibitor–based strategy that targets thisdefensive response. Finally, defective cell- cycle checkpointscharacteristic of transformed cells3 may render them uniquelysusceptible to such a strategy. In this context, the frequency ofgenetic abnormalities involving cell-cycle regulatory proteins inMM,59,60 as well as the genetic instability of such cells,60,61 maymake the use of checkpoint antagonists particularly attractive inMM. Analogously, the Ras/MEK/ERK signaling cascade is akey pathway implicated in MM cell survival signaling mediatedby IL-6,62,63 IGF-1,64 and stromal cells,65 and represents apromising candidate for therapeutic intervention.66,67 Impor-tantly, simultaneous disruption of Chk1 and the Ras/Raf/MEK/ERK pathway (eg, by either FTIs or MEK1/2 inhibitors) hasdemonstrated selective toxicity toward neoplastic cells, includ-ing primary MM cells, in vitro.13,15,16 The in vivo activity of theR115777/UCN-01 regimen, as well as the preferential inductionof �H2A.X in primary CD138� MM cells versus CD138�

normal cells, further supports this notion. Aside from providinginsights into novel mechanisms (ie, potentiation of DNAdamage) underlying interactions between agents targeting the

checkpoint machinery and those blocking the Ras3ERK signal-ing cascade, such findings could have therapeutic implications.

Acknowledgments

This work was supported by awards CA63753, CA93738,CA100866, CA88906, and CA72955 from the National Institutesof Health (NIH, Bethesda, MD), a Translational Research Awardfrom the Leukemia & Lymphoma Society of America (New York,NY; 6045-03), an award from the Department of Defense (Washing-ton, DC; DAMD-17-03-1-0209), an award from the V Foundation(Cary, NC), and the Universal Professorship (P.D.).

Authorship

Contribution: Y.D. designed and performed the research, ana-lyzed the data, and wrote the paper; S.C. designed andperformed the research, and analyzed the data; X.-Y.P., J.A.A.,L.B.K., and C.A.V. helped to perform the research; P.D. helpedto design the research; and S.G. designed the research, analyzeddata, and wrote the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Steven Grant, Division of Hematology/Oncology, Virginia Commonwealth University/Medical College ofVirginia, MCV Station Box 230, Richmond VA, 23298; e-mail:[email protected].

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online July 9, 2008 originally publisheddoi:10.1182/blood-2008-05-159392

2008 112: 2439-2449

Dent and Steven GrantYun Dai, Shuang Chen, Xin-Yan Pei, Jorge A. Almenara, Lora B. Kramer, Charis A. Venditti, Paul myeloma cells

induced DNA damage in vitro and in vivo in human multiple−inhibitor Interruption of the Ras/MEK/ERK signaling cascade enhances Chk1

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