Therapeutics Mcl-1 Stability Determines Mitotic Cell Fate of … · Research Article Mcl-1 Stability Determines Mitotic Cell Fate of Human Multiple Myeloma Tumor Cells Treated with

2046

Published OnlineFirst June 22, 2010; DOI: 10.1158/1535-7163.MCT-10-0033

Research Article Molecular

Cancer

Therapeutics

Mcl-1 Stability Determines Mitotic Cell Fate of HumanMultiple Myeloma Tumor Cells Treated with theKinesin Spindle Protein Inhibitor ARRY-520

Brian J. Tunquist1, Richard D. Woessner2, and Duncan H. Walker1

Abstract

Authors' ABioPharma

Note: SupCancer The

CorresponWalnut Str3861362. E

doi: 10.115

©2010 Am

Mol Canc

Do

Kinesin spindle protein (KSP/Eg5) inhibitors are novel anticancer agents that have thus far shown onlymodest activity in the clinic. Understanding how to identify patients who may be most sensitive to treat-ment is clearly needed to improve the development of these molecules. We studied four multiple myelomacell lines treated with the KSP inhibitor ARRY-520 to identify factors important for initiating apoptosiswhile cells are arrested in mitosis. The majority (three of four) of cell lines underwent mitotic arrest, withapoptosis occurring in mitosis within 24 to 30 hours. The remaining line (NCI H929) is temporally refrac-tory to ARRY-520 treatment, undergoing mitotic slippage and subsequently peaking in apoptotic markersafter 72 hours of treatment, while most cells are in interphase. Interestingly, loss of the antiapoptotic proteinmyeloid cell leukemia 1 (Mcl-1) coincided with mitotic cell death. Stabilization of Mcl-1 resulted in a de-layed onset of apoptosis, whereas enforced downregulation of Mcl-1 increased cell death in response to KSPinhibition. Thus, variation in responses to KSP inhibition is governed by a balance between survival pro-teins and spindle checkpoint integrity. Cells relying on short-lived survival proteins during mitosis are morelikely to undergo apoptosis in response to KSP inhibition. We propose that patients with hematologicmalignancies, which rely on Mcl-1, would therefore be good candidates for treatment with KSP inhibitors.Mol Cancer Ther; 9(7); 2046–56. ©2010 AACR.

Introduction

Antimitotic microtubule inhibitors are among the mostactive and broadly used cancer drugs. These drugs,which include taxanes, epothilones, and Vinca alkaloids,directly perturb microtubule function, resulting in a de-fective mitotic spindle and mitotic arrest. However, asmicrotubules perform critical functions in postmitoticcells, these drugs also exhibit unwanted side effects, in-cluding peripheral neuropathies (1). Thus, inhibitors ofmitosis-specific targets that lack side effects in postmito-tic cells are desired. One such target is kinesin spindleprotein (KSP, Eg5). KSP is a mitosis-specific kinesin es-sential for the assembly of a bipolar spindle and equalsegregation of sister chromatids (2). Inhibition of KSPprevents centrosome separation, resulting in the forma-tion of a monopolar spindle and activation of the mitoticspindle assembly checkpoint (SAC), causing metaphase

ffiliations: 1Translational Oncology and 2Pharmacology, Array, Inc., Boulder, Colorado

plementary material for this article is available at Molecularrapeutics Online (http://mct.aacrjournals.org/).

ding Author: Brian Tunquist, Array BioPharma, Inc., 3200eet, Boulder, CO 80301. Phone: 303-3861512; Fax: 303--mail: [email protected]

8/1535-7163.MCT-10-0033

erican Association for Cancer Research.

er Ther; 9(7) July 2010

on April 6, 2021. ©mct.aacrjournals.org wnloaded from

arrest due to maintenance of cdc2/cyclin B activity (3).Drug-induced accumulation of mitotic cells is tightly as-sociated with cell death (4). ARRY-520 is a selective KSPinhibitor with low nanomolar potency in solid and hema-tologic tumor cell lines (5) and activity in a wide range oftumor xenograft models (6).Mitotic arrest is not permanent, but results in either

death while cells are arrested in mitosis or exit from mi-tosis in the presence of misaligned chromosomes, a pro-cess known as adaptation or mitotic slippage, leading tomultinucleated cells (7). One model for adaptation sug-gests that apoptosis induced by mitotic inhibitors re-quires the activation of the SAC followed by mitoticslippage (8, 9). Presumably, postmitotic cell death resultsfrom a failure of mitosis associated with tetraploidy,which is essential to activate a cell death program in G1to prevent further polyploidization (10). An alternative todeath after mitotic slippage is death during mitotic arrest(11, 12). One hypothesis is that because transcription isabsent in mitosis, yet proteolysis persists, cell fate islargely determined by the rate of depletion of short-livedproteins that have short-lived mRNAs (13). In this model,a decline in antiapoptotic protein levels below a survivalthreshold, before the loss of SAC activity and subsequentdegradation of cyclin B, would be expected to triggerapoptosis during mitotic arrest. Conversely, failure ofthe SAC before depletion of survival signals will resultin adaptation and subsequent cell death in G1. The key

2010 American Association for Cancer Research.

http://mct.aacrjournals.org/

Mcl-1 Determines the Fate of KSP-Inhibited Cells


elements of this hypothesis have recently been verified.Treatment of HeLaM cells with microtubule poisons con-firms the gradual accumulation of apoptotic signals incells that undergo a prolonged mitotic arrest (14). Fur-thermore, it has been shown in multiple cell lines thatcells that typically die directly in mitosis only slowly de-grade cyclin B1, thereby preventing mitotic exit beforedeath (15). By contrast, cells that undergo adaptation pro-gressively destroy cyclin B1, leading to mitotic exit. Thesedata suggest that depending on the differential require-ment for survival protein activity and its rate of degrada-tion, it may be possible to determine whether a tumorwould succumb to mitotic cell death versus postmitoticcell death in the presence of a mitotic inhibitor.Various survival proteins, such as members of the Bcl-2

family or inhibitors of apoptosis proteins (IAP), have beenfound to confer resistance to antimitotic chemotherapy invarious tumor types (16, 17). Of these, the prosurvivalBcl-2 family member myeloid cell leukemia 1 (Mcl-1)stands out as an intriguing candidate for determiningwhether an arrested cell undergoes mitotic cell death.Mcl-1 has an extended PEST region, which is responsiblefor its relatively short half-life (18, 19). In addition, Mcl-1becomes phosphorylated on sites within its PEST regionin response to treatment with phorbol ester, oxidativestress, cytokine withdrawal, and microtubule-damagingagents, which affects its overall stability (20, 21). Lastly,Mcl-1 is predominately expressed in hematopoietic celllineages, including multiple myelomas where it has beenshown to be indispensable (22–25). We have previouslyshown that xenograft tumors of hematologic origin, in-cluding multiple myeloma, are often more responsive todrug treatment with ARRY-520, compared with xenografttumors of nonhematologic origin. Here, we have exam-ined Mcl-1 and other survival proteins in multiple myelo-ma cell lines following treatment with ARRY-520 todetermine whether Mcl-1 plays a role in determining cellfate decisions following KSP inhibition.

Materials and Methods

Cell lines and cultureRPMI 8226, NCI H929, and U266 cells were obtained

from the American Type Culture Collection. JJN3 cellswere obtained from the Deutsche Sammlung von Mik-roorganismen und Zellkulturen GmbH. Cells used for ex-periments were grown in culture for less than 2 monthsunder the conditions recommended by the suppliers.

In vivo xenograft modelsRPMI 8226 tumor cells, 2 × 107 (tumor growth inhibi-

tion; Fig. 2A) or 5 × 107 (pharmacodynamic analysis;Fig. 2B–D), in 100 μL of 50% Matrigel were implanteds.c. into female severe combined immunodeficient-beigemice as described previously (6). All studies were donein accordance with the Array BioPharma InstitutionalAnimal Care and Use Committee guidelines and in ac-cordance with the Guide for Laboratory Animal Care

www.aacrjournals.org

on April 6, 2021. ©mct.aacrjournals.org Downloaded from

and Use. Immunocompromised mice were purchasedfrom Charles River. Tumor growth inhibition includedeight mice per group. Pharmacodynamic analysis in-cluded three mice per group (average tumor volumeof each group ∼165 mm3).

Caspase-3/7 assayVehicle or drug-treated cells in growth medium were

seeded (10,000 viable cells per well) in duplicate wellsof black-walled 96-well plates and incubated at 37°Cin 5% CO2. Caspase-3/7 activity was measured usingCaspase-Glo 3/7 reagent (Promega) at various timepoints after drug addition. Data are reported as the meanluminescence of drug-treated wells divided by the meanluminescence of vehicle control wells.

Cell viabilityInhibition of cell proliferation was measured using the

Promega CellTiter-Blue assay. Cells were plated in 96-well plates at a density that allowed for logarithmicgrowth over the 72-hour period of the assay. Compound65 (0.1–50 μmol/L) was added to the cells at a finalDMSO concentration of 0.5%. Mcl-1 siRNA (10 nmol/L–1.25 μmol/L) was transfected into the cells as describedbelow. Fluorescent signal was converted to percent ofcontrol relative to high (DMSO-treated control) and low(fully inhibited control) samples. The EC50 for inhibitionof viability was determined from the inflection point of astandard four-parameter logistical curve.

ImmunoassaysARRY-520–treated cell lines were harvested at various

time points after drug addition. Cell lysates were gener-ated by thawing cells in radioimmunoprecipitation assaybuffer [50 mmol/L Tris (pH 8), 150 mmol/L NaCl, 1%NP40, 0.5% deoxycholate, 0.05% SDS, 2 mmol/L EDTA,protease inhibitor cocktail set III, EDTA-free, phospha-tase inhibitor cocktail set I; Calbiochem]. Mice bearing tu-mor xenografts were euthanized by CO2 inhalation, andtumors were harvested and flash frozen in liquid nitro-gen. Tumor lysates were prepared as described previous-ly (6). Proteins were resolved on 4% to 12% Tris-glycinegels (NuPage, Invitrogen) and transferred onto nitrocel-lulose membranes. Membranes were probed with com-mercial primary antibodies: Bcl-2 (BD Biosciences), poly(ADP-ribose) polymerase (PARP), XIAP, Bcl-XL (Cell Sig-naling), glyceraldehyde-3-phosphate dehydrogenase(Sigma), phosphorylated histone H3 (Ser10), cyclin B1,Mcl-1, p42/p44 mitogen-activated protein kinase (SantaCruz Biotechnology), and NOXA (Calbiochem). Blotswere then probed with secondary antibodies: IRDye800–conjugated anti-mouse IgG (Rockland) or Alexa Fluor680 anti-rabbit IgG (Invitrogen), and imaged using theLiCOR Odyssey infrared imaging system. Lysates fromcycling NCI H929 cells used for immunoprecipitationexperiments were prepared as outlined above. Proteinlysates (300 μg) supplemented with DMSO or 1 μmol/Lcompound 65 were subjected to immunoprecipitation

Mol Cancer Ther; 9(7) July 2010 2047



Tunquist et al.

2048


overnight at 4°C using 5 μg of rabbit IgG or Mcl-1 anti-body (Santa Cruz Biotechnology) bound to protein A-Sepharose CL-4B resin (Sigma).

Electroporation of cellsRPMI 8226, JJN3, and U266 cells were diluted to a

final concentration of 1 × 107 cells in 0.4 mL of Opti-MEM (Invitrogen). Cells were mixed with 10 μg ofpZsGreen1-N1 plasmid (Clontech) or pZsGreen1-N1plasmid containing full-length human Mcl-1 cDNA. Cellswere electroporated in a 4-mm cuvette (Bio-Rad Labora-tories) by exponential decay pulse (240 V, 1,070 μF) usingthe Gene Pulser Xcell electroporation system (Bio-RadLaboratories). Cells were immediately placed into growthmedium following electroporation, and dead cells wereremoved 24 hours later by magnetic beads (Dead CellRemoval Kit; Miltenyi Biotec) before being used for drugtreatment.

Transfection of siRNASpecific Accell-modified siRNA duplexes in 1× siRNA

buffer were stored at −20°C: Accell green nontargetingsiRNA, human NOXA siRNA N8 (26), and human Mcl-1siRNA duplex 34 (Dharmacon). Cells were transfected byseeding in fresh Accell delivery medium containing theappropriate siRNA for 24 or 48 hours under normalgrowth conditions.

CompoundsARRY-520 and compound 65 were synthesized at

Array BioPharma. For cell-based assays, ARRY-520 orcompound 65 was added to growth medium, withDMSO used as a vehicle control. DMSO concentrationsnever exceeded 0.5% in the assay.

Results

The onset of apoptosis varies among cell linestreated with ARRY-520We have previously shown that multiple myeloma tu-

mor xenografts are highly sensitive to ARRY-520 in vivo(6). Thus, we decided to investigate the molecular me-chanisms responsible for mitotic cell death in four differ-ent multiple myeloma cell lines (RPMI 8226, JJN3, U266,and NCI H929). Most studies report that cell death result-ing from prolonged mitotic arrest is executed by proteo-lytic caspase enzymes (9, 11, 15, 27, 28). The kinetics ofapoptosis induction in these four cell lines was investi-gated by measuring the activation of caspase-3 andcaspase-7 at various time points following drug addition.Cells seeded into identical 96-well plates were treatedwith either 10 nmol/L ARRY-520 (∼5-fold above the cel-lular IC50 for these lines; Supplementary Fig. S1A) or anequivalent volume of vehicle (DMSO). Three of the celllines (RPMI 8226, JJN3, and U266) exposed to ARRY-520 exhibited a rapid onset of apoptosis as defined by apeak in caspase-3/7 activity that occurred ∼24 hours af-ter compound treatment (Fig. 1A). However, whereas the

Mol Cancer Ther; 9(7) July 2010


NCI H929 cell line also displayed a gradual increase incaspase-3/7 activity over time, the peak in activity oc-curred much later (72–80 hours).As a secondary method of verifying apoptosis, and to

characterize drug responses at a molecular level, we per-formed immunoblotting for survival and apoptotic pro-teins, and/or their posttranslational modification sites,from all four multiple myeloma cell lines. All cell linestreated with 10 nmol/L ARRY-520 exhibited rapid induc-tion of mitotic arrest as judged by both increased phos-phorylation of histone H3 (pHH3) and accumulation ofcyclin B1 (see Fig. 1B and C; Supplementary Fig. S2). Mi-totic arrest caused by ARRY-520 was also confirmed byflow cytometry as all four cell lines exhibited an in-creased amount of 4 N cells after 24 hours of ARRY-520treatment (Supplementary Fig. S1B). PARP is one of themain targets of caspase-3 (29), and its cleavage has beenused as a marker for cells undergoing apoptosis (30). Us-ing the JJN3 cell line as a representative for cell lines thatundergo rapid onset of apoptosis, it was found that sig-nificant PARP cleavage occurred within 24 hours ofARRY-520 treatment, consistent with the peak in caspaseactivation in this cell line (Fig. 1A) and correlated withmaximal mitotic arrest (Fig. 1B). However, whereas theNCI H929 cell line exhibited a slight increase in the levelsof cleaved-PARP during mitotic arrest, the majority ofPARP cleavage was seen after 72 hours of treatment, oncethe cells had exited mitosis (Fig. 1C).

Loss of Mcl-1 correlates with mitotic cell deathIn an effort to identify molecular activities that have a

role in regulating mitotic cell death in these cells, the le-vels of key antiapoptotic proteins were measured at var-ious times following exposure to ARRY-520. In all celllines tested, levels of Bcl-2 and Bcl-XL remained relative-ly stable following treatment with ARRY-520 (see Fig. 1Band C; Supplementary Fig. S2). It has been suggested thatalthough the abundance of XIAP is not correlated withcell death, the degradation of XIAP in solid tumor linesis associated with onset of cell death in cells treated witha KSP inhibitor (28) or other chemotherapeutics (31). Al-though XIAP degradation indeed occurred followingtreatment with ARRY-520 in myeloma lines, the overallloss of XIAP protein was relatively modest and inconsis-tent between cell lines, suggesting that, at least in thesecell lines, other proteins play a role in signaling celldeath. This is compatible with the finding that cell deathcaused by ARRY-520 in leukemia cell lines also did notdepend on XIAP (5). By contrast, we observed that thedegradation of Mcl-1 coincided closely with PARP clea-vage in JJN3 cells (Fig. 1B) and other cell lines thatunderwent rapid apoptosis in response to ARRY-520(Supplementary Fig. S2). This effect was not seen inimmunoblots of the NCI H929 cell line exposed toARRY-520 (Fig. 1C), suggesting that Mcl-1 destabiliza-tion, promoted during prolonged mitotic arrest, may con-tribute to initiation of cell death. Interestingly, cells thathave slipped from mitosis may not be as dependent on

Molecular Cancer Therapeutics





Mcl-1 for survival during interphase as the levels of Mcl-1seem to remain stable in the postmitotic time points takenfrom NCI H929 cells (Fig. 1C), despite the fact that thesecells eventually undergo apoptosis as measured by PARPcleavage and caspase activation.RPMI 8226 tumor xenografts were particularly sensi-

tive to low doses of ARRY-520 (12.5 mg/kg) adminis-tered on days 1 and 2 (Fig. 2A). Immunoblot analysisof tumor lysates following ARRY-520 treatment showedsimilar effects on Mcl-1 stability and induction of apopto-sis during mitotic arrest as seen in RPMI 8226 cells trea-ted in vitro (Fig. 2B–D). Mcl-1 levels were significantlydecreased after only 24 hours of treatment with the ma-jority of Mcl-1 expression in the tumor lost by 48 hours,corresponding with an increase in cleaved PARP at thesetime points. In total, these experiments suggest that Mcl-1



may be a critical factor important for multiple myelomacell survival during prolonged mitotic arrest. Subsequentexperiments test the validity of this hypothesis in multi-ple myeloma cell lines.

Increased expression or stabilization of Mcl-1delays apoptosisOur data suggest that Mcl-1 degradation in cells ar-

rested in mitosis by ARRY-520 is a key step in the onsetof apoptosis. To verify this, we stabilized Mcl-1 proteinin cells through both direct overexpression of recombi-nant Mcl-1 protein and by knockdown of the Mcl-1regulatory protein NOXA. In the first instance, we an-alyzed the effect of overexpression of Mcl1-ZsGreen1fusion protein on the three cell lines that underwent arapid apoptosis in mitosis following treatment with

Figure 1. Caspase-3/7 activation in multiple myeloma cell lines treated with ARRY-520 correlates with Mcl-1 degradation. A, multiple myeloma cell lineswere seeded in the presence of 10 nmol/L ARRY-520 at the 0-h time point. Caspase-3/7 activity was measured at the indicated times and reportedas the fold-increase above control (DMSO)–treated cells for that time point. B, immunoblot analysis of JJN3 cells in response to 10 nmol/L ARRY-520 at11 time points starting with drug addition (0 h) to 48 hours following drug addition. C, NCI H929 cell reaction to 10 nmol/L ARRY-520. Note the longerduration of time points (80 h total) for this cell line. Full-length and cleaved forms of PARP are indicated by arrows. pHH3, phosphorylated histone H3(Ser10). The mean of at least three experiments is shown.




Tunquist et al.

2050


ARRY-520. If multiple myeloma cells depend on Mcl-1for mitotic survival, we hypothesized that overexpressionof Mcl1-ZsGreen1 would delay apoptosis induction inthese cells. Indeed, overexpression of Mcl1-ZsGreen1caused an ∼24 hours (2-fold) delay in the onset of peakcaspase-3/7 activity (Fig. 3A) and PARP cleavage(Fig. 3B and C) in the JJN3 cell line following exposureto ARRY-520. Similar results were also obtained in theRPMI 8226 and U266 cell lines (Supplementary Figs. S3and S4, respectively). Although endogenous Mcl-1 andMcl1-ZsGreen1 were still degraded, the excess Mcl1-ZsGreen1 seemed to compensate for the loss of thenative protein, thus delaying the onset of apoptosis inthese cells.It is possible that overexpression of a survival protein

such as Mcl-1 could induce survival signals indepen-dent of a role in mitotic cell death responses. For thisreason, we chose to investigate whether we could sta-



bilize Mcl-1 protein by an alternative mechanism thatdoes not directly alter physiologic levels of Mcl-1. Thesmall, BH3-only protein NOXA functions as a specificantagonist for Mcl-1 binding to proapoptotic Bcl-2 fam-ily members, such as Bim and Bak (32–34). Binding ofNOXA to Mcl-1 prohibits the formation of Mcl-1/Bakor Mcl-1/Bim dimers, thus increasing the pool of freeBak and Bim capable of promoting mitochondrial outermembrane permeabilization. In addition, NOXA bind-ing to Mcl-1 has been suggested to promote Mcl-1 ubi-quitination and subsequent degradation by theproteasome (Fig. 4A; ref. 34). Such a role for NOXAhas been verified in multiple myeloma cell lines treatedwith a wide range of chemotherapeutics (32, 33, 35, 36).We were interested in whether the rapid decline ofMcl-1 protein in ARRY-520–arrested RPMI 8226, JJN3,or U266 cell lines was mediated through NOXA. To testthis, we used NOXA-specific siRNA to diminish NOXA

Figure 2. RPMI 8226 in vivo tumor growth inhibition correlates with Mcl-1 degradation and apoptosis. Saline vehicle or 12.5 mg/kg ARRY-520 wereadministered i.p. to mice bearing RPMI 8226 tumor xenografts at 0 and 24 h. A, tumor growth was analyzed by measuring tumor volume on the indicateddays. Tumors were excised at the indicated times, and lysates were blotted for Mcl-1 (B), pHH3 (C), or PARP (D). Band intensities were normalized tototal Erk (p42/p44 mitogen-activated protein kinase) for each sample, then averages (three mice per time point) and percentages of control were calculated.Data in D are the ratio of Erk-normalized cleaved PARP to Erk-normalized full-length PARP.






protein levels before ARRY-520 treatment. Transfectionof cells with NOXA siRNA 48 hours before ARRY-520treatment resulted in significant knockdown of NOXAthroughout the time course of ARRY-520 treatment asdetermined by immunoblotting (Fig. 4D). Caspase-3/7activity measured in JJN3 cells transfected with controlsiRNA behaved very similarly to what was seen inFigs. 1 and 2, whereas cells containing reduced NOXAexhibited a significant 24-hour delay in the onset of celldeath caused by ARRY-520 (Fig. 4B). Similar resultswere observed following knockdown of NOXA inRPMI 8226 and U266 cell lines (Supplementary Figs.S5 and S6). NOXA knockdown correlated with greaterstabilization of Mcl-1 protein 24 hours after ARRY-520treatment, and a delay in detectable levels of cleaved-PARP (Fig. 4D) compared with control-transfected cells(Fig. 4C). Taken together, these results suggest thatMcl-1 is capable of maintaining survival signals in mul-tiple myeloma cell lines arrested in mitosis by ARRY-520, and that only once Mcl-1 levels decrease below athreshold level for survival can cell death pathwaysbecome active.



Reduced Mcl-1 protein or function enhances celldeath caused by KSP inhibition in NCI H929 cellsThe observation that increased Mcl-1 protein can post-

pone cell death in M phase in most cell lines tested sug-gests that perhaps the NCI H929 multiple myeloma cellline could be made to undergo an early cell death inmitosis by lowering Mcl-1 activity. We reduced Mcl-1protein function by two separate methods: the use ofMcl-1–specific siRNA and treating with a small-moleculeMcl-1 inhibitor. As the loss of Mcl-1 alone is sufficient toinduce cell death in multiple myeloma cell lines (22, 25),we established conditions that resulted in knockdown ofMcl-1 without incurring substantial cell death on its own.We detected measurable Mcl-1 knockdown 24 hoursposttransfection using Mcl-1 siRNA concentrations >15nmol/L (Fig. 5A), and further determined cellular EC50values for cell viability of 20 and 14 nmol/L, at 24 and48 hours posttransfection, respectively (Fig. 5B). Usingan Mcl-1 siRNA concentration above its EC50 value(25 nmol/L), we found that addition of 10 nmol/LARRY-520 to NCI H929 cells 24 hours posttransfectionenhanced both the magnitude and time to initiation of

Figure 3. Mcl-1 overexpression delays cell death in ARRY-520 sensitive cells. A, caspase-3/7 activity measurements were carried out in emptyvector– and Mcl1-ZsGreen1–transfected JJN3 cells treated with 10 nmol/L ARRY-520. Data are reported as the fold increase above empty vector orMcl1-ZsGreen1–transfected JJN3 cells treated with DMSO. The mean of three experiments is shown; error bars, SD. Immunoblot analysis of proteinsfrom JJN3 cell lysates following transfection with empty vector (B) or Mcl1-ZsGreen1 vector (C) treated with 10 nmol/L ARRY-520.




Tunquist et al.

2052


apoptosis as measured by activation of caspase-3 andcaspase-7 (Fig. 5C). This was significantly different thanthe amount of cell death caused by 25 nmol/L Mcl-1siRNA in combination with vehicle (DMSO), or throughtransfection of 25 nmol/L control siRNA in combinationwith 10 nmol/L ARRY-520 (Fig. 5C). Thus, although NCIH929 cells depend on Mcl-1 for survival, it is hypothe-sized that they either lack a mechanism sufficient fortriggering its mitotic degradation or have developed amechanism to promote its stabilization during a pro-longed M-phase arrest caused by ARRY-520.As an alternative method of modulating Mcl-1 func-

tion, we used a selective, small-molecule inhibitor ofMcl-1. Compound 65 (Fig. 6A) has been reported todisrupt the binding of interacting proteins, such asNOXA, to the hydrophobic pocket of Mcl-1 (37). Toconfirm the reported activity, we show that coimmuno-precipitation of BH3-only proteins NOXA and Bimwith Mcl-1 is prevented in the presence of 1 μmol/Lcompound 65 (Fig. 6B), suggesting that compound 65can disrupt the association of Mcl-1 with proapoptotic



BH3-only proteins. Consistent with this, compound 65induced rapid cell death (IC50 2 μmol/L at 48 hours;Fig. 6C) in NCI H929 cells consistent with its Mcl-1 inhi-bitory activity. Treatment of NCI H929 cells with 10 nmol/LARRY-520, followed 8 hours later with the addition of2 μmol/L compound 65, resulted in a significant increasein apoptosis by 24 hours compared with either ARRY-520or compound 65 alone (Fig. 6D), similar to the effect ofMcl-1 knockdown (Fig. 5C). These results are consistentwith a role for Mcl-1 functioning to prevent apoptosisduring mitotic arrest of NCI H929 cells.

Discussion

The inhibition of mitosis-specific drug targets, such asKSP, is expected to yield improvements in clinical effica-cy and safety profiles compared with current microtu-bule-targeted chemotherapeutics. To date, however,KSP inhibitors have shown only sporadic clinical activityin a subset of tumor types. Thus, an unresolved challengeis to determine which tumors or patients are most likely

Figure 4. Knockdown of NOXA increases the duration of mitotic survival and stability of Mcl-1 in JJN3 cells treated with ARRY-520. A, role for NOXA inMcl-1 regulation and promotion of cell death. NOXA is important for sequestering Mcl-1 from proapoptotic factors, such as Bak. Further, NOXA-boundMcl-1 is targeted for degradation. B, caspase-3/7 activity in control or NOXA siRNA-transfected JJN3 cells following addition of 10 nmol/L ARRY-520. Themean of three independent experiments is shown; error bars, SD. Western blot analysis of control (C) and NOXA (D) siRNA-transfected JJN3 cells. Cellswere immunoblotted for the indicated proteins before transfection (−48 h time point) and at various time points following 10 nmol/L ARRY-520 addition.






to be sensitive to compounds such as ARRY-520. Suchtranslational knowledge would enable the design of bet-ter clinical trials and patient selection strategies. Previousefforts to identify the molecular sources of variation insensitivity to apoptosis have suggested that the balancebetween apoptotic signals and the integrity of the SACare key events in determining the onset of cell death inresponse to mitotic inhibitors (13–15, 27, 28, 38). To ourknowledge, ours is the first study to successfully identifya single survival protein, Mcl-1, as a determinant control-ling the decision to undergo apoptosis during mitotic ar-rest in multiple myeloma cell lines.

Survival protein reliance determines cell fateWe have observed that of the four myeloma cell lines

tested, three undergo a rapid onset of apoptosis, commit-ting to cell death within 24 hours of exposure to ARRY-520, while still arrested in mitosis. By contrast, NCIH929 cells showed a significantly delayed apoptosis fol-lowing KSP inhibition compared with the other threemultiple myeloma cell lines (Fig. 1). The majority ofH929 cells seemed to undergo SAC failure and subse-quent cell death after 72 hours when the cells were in in-terphase (Fig. 2B). Western analysis failed to show asignificant difference in mitotic duration between theselines (Fig. 2; Supplementary Fig. S2), suggesting that thisdissimilarity could not be due solely to a SAC defect. Ofnote, although Mcl-1 levels declined rapidly in cells thatdied in mitosis, Mcl-1 levels remained stable in NCI H929



throughout the time course of the experiment, suggestingthat Mcl-1 stability enabled survival of ARRY-520–arrested cells until SAC failure and mitotic exit. Compa-rable results have recently been reported wherebydecreasing Mcl-1 expression in various tumor cell lines,including multiple myeloma cell lines, led to the sensitiza-tion of cells previously resistant to CDK inhibition (39).These data support a model wherein the fate of a cell onceit has become arrested in mitosis is controlled by two in-dependent clocks: the cell-specific rate of decline in sur-vival signals below a threshold for triggering cell deathand the maintenance of mitotic arrest as determined bythe intrinsic rate of cyclin B1 degradation. In some tumorcells, as exemplified by JJN3, RPMI 8226, and U266, theloss of Mcl-1 during a KSP inhibitor–induced mitotic arrestis sufficient to promote the onset of apoptosis. Conversely,tumor cells that rely onMcl-1 for survival, but are less capa-ble of inducing Mcl-1 degradation during mitotic arrest,such as NCI H929, may exhibit a delayed onset of apopto-sis and require far longer exposure to induce cell death.

Therapeutic importance of Mcl-1 in determiningcell fateThe importance of survival proteins in determining cell

fate in response to KSP inhibition has been shown byothers. In a population-based analysis similar to whathas been presented here, Shi et al. characterized the re-sponse of several solid tumor cell lines to three differentantimitotic drugs (28). Although these cells all responded

Figure 5. Knockdown of Mcl-1 confers increased ARRY-520 sensitivity to NCI H929 cells. A, Mcl-1 siRNA dose titration 24 h posttransfection. Antibodiesused for immunoblotting and siRNA concentrations (nmol/L) are indicated. B, cell viability of NCI H929 cells 24 and 48 h following transfection withindicated concentrations of Mcl-1 siRNA. Data are reported as the percentage of viable Mcl-1 siRNA-transfected cells compared with controlsiRNA-transfected cells at each concentration. POC, percent of control. C, NCI H929 cells were transfected with 25 nmol/L of control or Mcl-1 siRNA.Twenty-four hours after siRNA transfection, DMSO or 10 nmol/L ARRY-520 was added and caspase-3/7 activity was measured at various time points.Time refers to the time since DMSO or ARRY-520 addition. Data are the mean of three independent experiments; error bars, SD.




Tunquist et al.

2054


similarly to a defective mitotic spindle, the response ofthe apoptotic machinery was variable between cell lines.Loss of the survival protein XIAP correlated with the on-set of apoptosis. Although supporting the hypothesisthat cell fate decisions in response to KSP inhibition aredetermined in part by modulation of survival proteins,the loss of XIAP has not yet been directly shown as beingresponsible for cell death. Here, we clearly show a rolefor Mcl-1 degradation in determining cell fate in responseto KSP inhibition in several myeloma cell lines. We be-lieve that these data can inform the identification of tu-mors likely to be most sensitive to KSP inhibitorsclinically. KSP inhibitors are typically given over short,infusional schedules and therefore have defined periodsof therapeutic exposure, with reported clinical half-livestypically ranging from 28 to 50 hours (40). Thus, wehypothesize that cells that rely on more stable survivalproteins, such as Bcl-2 or Bcl-XL, may be temporally re-fractory to KSP inhibition: By maintaining survival sig-



nals during mitotic arrest, these cells may be able tosurvive transient mitotic inhibition until drug levels dropbelow therapeutic levels (at which time a normal spindlecan be reassembled and cell cycle progression can com-mence). In contrast, cells that depend on Mcl-1 and rap-idly degrade it during mitotic arrest are more likely to beresponsive to the drug because apoptosis can be initiatedwithin the window of exposure to the drug.The importance of Mcl-1 in determining apoptosis in

mitosis in hematologic cell lines makes particular sense,given the fact that hematologic cells typically rely on thisshort-lived survival protein. Thus, we suggest that hema-tologic malignancies, such as multiple myeloma, leuke-mia, and lymphoma, are particularly good candidatesto investigate the clinical activity of KSP inhibitors. Ofnote, the KSP inhibitor SB-743921 has shown clinicalactivity in Hodgkin's and non–Hodgkin's lymphomas,although a relationship between Mcl-1 and responsehas not been investigated (41). Many solid tumor cell

Mole

2010 American Association

Figure 6. Inhibition of Mcl-1binding to NOXA in NCI H929 cellsconfers increased sensitivity toARRY-520. A, chemicalstructure of compound 65.B, coimmunoprecipitation of NOXAwith endogenous Mcl-1 is blockedin the presence of 1 μmol/Lcompound 65 in NCI H929 cellextracts. Rabbit IgG was used as acontrol for immunoprecipitation.C, cell viability of NCI H929 cellsexposed to the indicatedconcentrations of compound 65 for24 or 48 h. Data are reported as thepercentage of viable NCI H929cells following compound 65treatment compared with DMSOcontrol–treated NCI H929 cells.D, NCI H929 cells treated withDMSO or 2 μmol/L compound 65were analyzed for caspase-3/7activity at various time pointsfollowing the addition of DMSO or10 nmol/L ARRY-520. Total DMSOconcentration of the control didnot exceed 0.5% (v/v). Foldcaspase-3/7 activity is reported asthe mean of three separateexperiments; error bars, SD.

cular Cancer Therapeutics

for Cancer Research.




lines do not express or depend on Mcl-1. Here, it is pos-sible that other mechanisms, such as degradation of XIAP(28), may determine cell fate following KSP inhibitortreatment. Alternatively, it has been reported that subsetsof nonhematologic tumor types, such as non–small celllung carcinomas, melanomas, and biliary cancers, maygain the ability to express Mcl-1 during tumorigenesis(42–44), and these Mcl-1–expressing solid tumors mayalso be expected to be sensitive to shorter drug exposuresof ARRY-520. These findings would be expected to havesignificant value in selecting potentially sensitive patientpopulations in solid tumor indications.

Temporal resistance to ARRY-520: predictive valueof Mcl-1 regulation by NOXAThe fact that NCI H929 cells rely on Mcl-1 but are un-

able to significantly degrade Mcl-1 during mitotic arrest,and therefore avoid mitotic cell death, indicates that Mcl-1stabilization may represent a mechanism of resistance toKSP inhibitors. One possible mechanism for Mcl-1 stabi-lization in the NCI H929 cell line may be mediatedthrough the proapoptotic BH3-only protein NOXA.NOXA antagonizes the survival function of Mcl-1 andhas been found to be sufficient to promote its degrada-tion by the proteasome (34, 45, 46). Interestingly, NOXAprotein levels are lowest in the NCI H929 cell line, whichmay help explain why Mcl-1 levels are more stable dur-ing a prolonged mitotic arrest (Supplementary Fig. S7).Reducing NOXA levels in the other three cell lines de-layed Mcl-1 degradation and prolonged M-phase arrestand survival (Fig. 4; Supplementary Figs. S5 and S6).Thus, tumor cells with higher expression of NOXA pro-tein may be more sensitive to antimitotics, such as ARRY-520, by promoting the rapid degradation of Mcl-1 by theproteasome during mitotic arrest. As Mcl-1 has also beenreported to be cleaved by active caspases (47), we hy-pothesize that the NOXA-driven decline in Mcl-1 levelsbelow a death threshold would trigger apoptosis and a



further decline in Mcl-1 protein levels to cement thecommitment to apoptosis. Interestingly, a recent ge-nome-wide siRNA screen of HeLa cells exposed to KSPinhibitor identified NOXA (also known as PMAIP1)knockdown as a potential suppressor of cell death causedby low concentrations of K5I (48). Further investigationof the role of NOXA in modulating cell fate in responseto KSP inhibition is needed to confirm these hypotheses.In summary, our data suggest that tumors that express

and rely on Mcl-1 for survival may be more sensitive tothe KSP inhibitor ARRY-520. However, this observation isdependent on the ability of the tumor to degrade Mcl-1during mitotic arrest. Mcl-1 is a key antiapoptotic proteinin a wide variety of hematologic tumor types, such asmultiple myeloma (22, 25), leukemia (49), and lymphoma(50). These data therefore support further investigation ofARRY-520 in hematologic malignancies, which rely onMcl-1 for survival. To test this hypothesis clinically,ARRY-520 is being explored in clinical trials in both acutemyelogenous leukemia and multiple myeloma.

Disclosure of Potential Conflicts of Interest

The authors are employees and shareholders of Array BioPharma, Inc.,Boulder, CO, USA. This company has patent rights on some reagentsused in this study.

Acknowledgments

We thank all those at Array BioPharma who have contributed to thedevelopment of ARRY-520; Heidi Simmons, Dana Longo, Shelley Allen,Christine Lemieux, Jennifer Garrus, Stefan Gross, Jim Winkler, EliWallace, and Kevin Koch for helpful discussions and advice; and D.Ross Camidge, University of Colorado Denver, for the critical review ofthe manuscript.

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 01/12/2010; revised 04/14/2010; accepted 04/30/2010;published OnlineFirst 06/22/2010.

References

1. Scripture CD, Figg WD, Sparreboom A. Peripheral neuropathy in-

duced by paclitaxel: recent insights and future perspectives. CurrNeuropharmacol 2006;4:165–72.

2. Blangy A, Lane HA, d'Herin P, Harper M, Kress M, Nigg EA. Phos-phorylation by p34cdc2 regulates spindle association of human Eg5,a kinesin-related motor essential for bipolar spindle formation in vivo.Cell 1995;83:1159–69.

3. Stern BM, Murray AW. Lack of tension at kinetochores activatesthe spindle checkpoint in budding yeast. Curr Biol 2001;11:1462–7.

4. Milross CG, Mason KA, Hunter NR, Chung WK, Peters LJ, Milas L.Relationship of mitotic arrest and apoptosis to antitumor effect ofpaclitaxel. J Natl Cancer Inst 1996;88:1308–14.

5. Carter BZ, Mak DH, Woessner R, et al. Inhibition of KSP by ARRY-520 induces cell cycle block and cell death via the mitochondrialpathway in AML cells. Leukemia 2009;23:1755–62.

6. Woessner R, Tunquist B, Lemieux C, et al. ARRY-520, a novel KSPinhibitor with potent activity in hematological and taxanes resistanttumor models. Anticancer Res 2009;29:4373–80.

7. Rieder CL, Maiato H. Stuck in division or passing through: what hap-

pens when cells cannot satisfy the spindle assembly checkpoint?Dev Cell 2004;7:637–51.

8. Kienitz A, Vogel C, Morales I, Muller R, Bastians H. Partial downre-gulation of Mad1 causes spindle checkpoint inactivation and aneu-ploidy, but does not confer resistance towards Taxol. Oncogene2004;24:4301–10.

9. TaoW, South VJ, Zhang Y, et al. Induction of apoptosis by an inhibitorof the mitotic kinesin KSP requires both activation of the spindle as-sembly checkpoint and mitotic slippage. Cancer Cell 2005;8:49–59.

10. Vogel C, Kienitz A, Hofmann I, Muller R, Bastians H. Crosstalk of themitotic spindle assembly checkpoint with p53 to prevent polyploidy.Oncogene 2004;23:6845–53.

11. Panvichian R, Orth K, DayML, Day KC, Pilat MJ, Pienta KJ. Paclitaxel-associated multimininucleation is permitted by the inhibition ofcaspase activation: a potential early step in drug resistance. CancerRes 1998;58:4667–72.

12. Sherwood SW, Sheridan JP, Schimke RT. Induction of apoptosis bythe anti-tubulin drug colcemid: relationship of mitotic checkpointcontrol to the induction of apoptosis in HeLa S3 cells. Exp CellRes 1994;215:373–9.




Tunquist et al.

2056


13. Blagosklonny MV. Mitotic arrest and cell fate: why and how mitoticinhibition of transcription drives mutually exclusive events. Cell Cycle2007;6:70–4.

14. Bekier ME, Fischbach R, Lee J, Taylor WR. Length of mitotic arrestinduced by microtubule-stabilizing drugs determines cell death aftermitotic exit. Mol Cancer Ther 2009;8:1646–54.

15. Gascoigne KE, Taylor SS. Cancer cells display profound intra- andinterline variation following prolonged exposure to antimitotic drugs.Cancer Cell 2008;14:111–22.

16. Danson S, Dean E, Dive C, Ranson M. IAPs as a target for anticancertherapy. Curr Cancer Drug Targets 2007;7:785–94.

17. Kang MH, Reynolds CP. Bcl-2 inhibitors: targeting mitochondrialapoptotic pathways in cancer therapy. Clin Cancer Res 2009;15:1126–32.

18. Kozopas KM, Yang T, Buchan HL, Zhou P, Craig RW. MCL1, a geneexpressed in programmed myeloid cell differentiation, has sequencesimilarity to BCL2. Proc Natl Acad Sci U S A 1993;90:3516–20.

19. Warr MR, Shore GC. Unique biology of Mcl-1:therapeutic opportuni-ties in cancer. Curr Mol Med 2008;8:138–47.

20. Domina AM, Vrana JA, Gregory MA, Hann SR, Craig RW. MCL1 isphosphorylated in the PEST region and stabilized upon ERK activa-tion in viable cells, and at additional sites with cytotoxic okadaic acidor Taxol. Oncogene 2004;23:5301–15.

21. Maurer U, Charvet C, Wagman AS, Dejardin E, Green DR. Glycogensynthase kinase-3 regulates mitochondrial outer membrane permea-bilization and apoptosis by destabilization of MCL-1. Mol Cell 2006;21:749–60.

22. Derenne S, Monia B, Dean NM, et al. Antisense strategy shows thatMcl-1 rather than Bcl-2 or Bcl-x(L) is an essential survival protein ofhuman myeloma cells. Blood 2002;100:194–9.

23. Opferman JT, Letai A, Beard C, Sorcinelli MD, Ong CC, KorsmeyerSJ. Development and maintenance of B and T lymphocytes requiresantiapoptotic MCL-1. Nature 2003;426:671–6.

24. Opferman JT, Iwasaki H, Ong CC, et al. Obligate role of anti-apopto-tic MCL-1 in the survival of hematopoietic stem cells. Science 2005;307:1101–4.

25. Zhang B, Gojo I, Fenton RG. Myeloid cell factor-1 is a critical survivalfactor for multiple myeloma. Blood 2002;99:1885–93.

26. Smit LA, Hallaert DY, Spijker R, et al. Differential NOXA/Mcl-1 bal-ance in peripheral versus lymph node chronic lymphocytic leukemiacells correlates with survival capacity. Blood 2007;109:1660–8.

27. Brito DA, Rieder CL. The ability to survive mitosis in the presence ofmicrotubule poisons differs significantly between human nontrans-fromed (RPE-1) and cancer (U2OS, HeLa) cells. Cell Motil Cytoskel-eton 2009;66:437–47.

28. Shi J, Orth JD, Mitchison T. Cell type variation in responses to anti-mitotic drugs that target microtubules and kinesin-5. Cancer Res2008;68:3269–76.

29. Tewari M, Quan LT, O'Rourke K, et al. Yama/CPP32 β, a mamma-lian homolog of CED-3, is a CrmA-inhibitable protease that cleavesthe death substrate poly(ADP-ribose) polymerase. Cell 1995;81:801–9.

30. Oliver FJ, de la Rubia G, Rolli V, Ruiz-Ruiz MC, de Murcia G, MurciaJM. Importance of poly(ADP-ribose) polymerase and its cleavage inapoptosis. Lesson from an uncleavable mutant. J Biol Chem 1998;273:33533–9.

31. Park SJ, Wu CH, Gordon JD, Zhong X, Emami A, Safa AR. Taxol in-duces caspase-10 dependent apoptosis. J Biol Chem 2004;279:51057–67.

32. Gomez-Bougie P, Wuilleme-Toumi S, Menoret E, et al. Noxa up-regulation and Mcl-1 cleavage are associated to apoptosis induc-



tion by bortezomib in multiple myeloma. Cancer Res 2007;67:5418–24.

33. Qin JZ, Stennett L, Bacon P, et al. p53-independent NOXA inductionovercomes apoptotic resistance of malignant melanomas. Mol Can-cer Ther 2004;3:895–902.

34. Willis SN, Chen L, Dewson G, et al. Proapoptotic Bak is sequesteredby Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only pro-teins. Genes Dev 2005;19:1294–305.

35. Moore HE, Davenport EL, Smith EM, et al. Aminopeptidase inhibitionas a targeted treatment strategy in myeloma. Mol Cancer Ther 2009;8:762–70.

36. Nefedova Y, Sullivan DM, Bolick SC, Dalton WS, Gabrilovich DI. In-hibition of Notch signaling induces apoptosis of myeloma cells andenhances sensitivity to chemotherapy. Blood 2008;111:2220–9.

37. Nikolovska-Coleska Z, Qiu S, Long J, et al. Discovery and character-ization of new small-molecule inhibitors of Mcl-1 by high-throughputscreening [abstract]. Proc Am Assoc Cancer Res 2008, Abstractnumber 4148.

38. Orth JD, Tang Y, Shi J, et al. Quantitative live imaging of cancer andnormal cells treated with kinesin-5 inhibitors indicates significant dif-ferences in phenotypic responses and cell fate. Mol Cancer Ther2008;7:3480–9.

39. Eguchi T, Itadani H, Shimomura T, Kasanishi N, Hirai H, Kotani H.Expression levels of p18INK4C modify the cellular efficacy of cy-clin-dependent kinase inhibitors via regulation of Mcl-1 expressionin tumor cell lines. Mol Cancer Ther 2009;8:1460–72.

40. Schmidt M, Bastians H. Mitotic drug targets and the development ofnovel anti-mitotic anticancer drugs. Drug Resist Update 2007;10:162–81.

41. Gerecitano JF, Van Deventer O'ConnorO, Hainsworth H, Leonard J,Afanasayev J. A phase I/II trial of the kinesin spindle protein (KSP)inhibitor SB-743921 dosed q14d without and with prophylactic G-CSF in non-Hodgkin lymphoma (NHL) or Hodgkin lymphoma (HL).J Clin Oncol 2009;27:15s, (suppl; abstr 8578).

42. Chetoui N, Sylla K, Gagnon-Houde JV, Alcaide-Loridan C, CharronD, Al-Daccak R. Down-regulation of mcl-1 by small interfering RNAsensitizes resistant melanoma cells to fas-mediated apoptosis. MolCancer Res 2008;6:42–52.

43. Okaro AC, Deery AR, Hutchins RR, Davidson BR. The expression ofantiapoptotic proteins Bcl-2, Bcl-X(L), and Mcl-1 in benign, dysplas-tic, and malignant biliary epithelium. J Clin Pathol 2001;54:927–32.

44. Song L, Coppola D, Livingston S, Cress D, Haura EB. Mcl-1 regu-lates survival and sensitivity to diverse apoptotic stimuli in humannon-small cell lung cancer cells. Cancer Biol Ther 2005;4:267–76.

45. Letai AG. Diagnosing and exploiting cancer's addiction to blocks inapoptosis. Nat Rev Cancer 2008;8:121–32.

46. Nijhawan D, Fang M, Traer E, et al. Elimination of Mcl-1 is requiredfor the initiation of apoptosis following ultraviolet irradiation. GenesDev 2003;17:1475–86.

47. Herrant M, Jacquel A, Marchetti S, et al. Cleavage of Mcl-1 bycaspases impaired its ability to counteract Bim-induced apoptosis.Oncogene 2004;23:7863–73.

48. Tsui M, Xie T, Orth JD, et al. An intermittent live cell imaging screenfor siRNA enhancers and suppressors of a kinesin-5 inhibitor. PLoSONE 2009;4:e7339.

49. Ji M, Li J, Yu H, et al. Simultaneous targeting of MCL1 and ABCB1 asa novel strategy to overcome drug resistance in human leukaemia. BrJ Haematol 2009;145:648–56.

50. Zhou P, Levy NB, Xie H, et al. MCL1 transgenic mice exhibit a highincidence of B-cell lymphoma manifested as a spectrum of histologicsubtypes. Blood 2001;97:3902–9.




2010;9:2046-2056. Published OnlineFirst June 22, 2010.Mol Cancer Ther Brian J. Tunquist, Richard D. Woessner and Duncan H. Walker Inhibitor ARRY-520Myeloma Tumor Cells Treated with the Kinesin Spindle Protein Mcl-1 Stability Determines Mitotic Cell Fate of Human Multiple

Updated version

10.1158/1535-7163.MCT-10-0033doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2010/06/22/1535-7163.MCT-10-0033.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/9/7/2046.full#ref-list-1

This article cites 49 articles, 23 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/9/7/2046.full#related-urls

This article has been cited by 15 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/9/7/2046To request permission to re-use all or part of this article, use this link

on April 6, 2021. © 2010 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-10-0033http://mct.aacrjournals.org/content/suppl/2010/06/22/1535-7163.MCT-10-0033.DC1http://mct.aacrjournals.org/content/9/7/2046.full#ref-list-1http://mct.aacrjournals.org/content/9/7/2046.full#related-urlshttp://mct.aacrjournals.org/cgi/alertsmailto:[email protected]://mct.aacrjournals.org/content/9/7/2046http://mct.aacrjournals.org/

Documents

Therapeutics Mcl-1 Stability Determines Mitotic Cell Fate of … · Research Article Mcl-1 Stability Determines Mitotic Cell Fate of Human Multiple Myeloma Tumor Cells Treated with