Mcl-1 Stability Determines Mitotic Cell Fate of Human Multiple Myeloma Tumor Cells Treated with the Kinesin Spindle Protein Inhibitor ARRY-520

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 Human Multiple Myeloma Tumor Cells Treated with the Kinesin Spindle Protein Inhibitor ARRY-520

Brian J. Tunquist1, Richard D. Woessner 2, and Duncan H. Walker1

Abstract Kinesin spindle protein (KSP/Eg5) inhibitors are novel anticancer agents that have thus far shown only modest activity in the clinic. Understanding how to identify patients who may be most sensitive to treatment is clearly needed to improve the development of these molecules. We studied four multiple myeloma cell lines treated with the KSP inhibitor ARRY-520 to identify factors important for initiating apoptosis while cells are arrested in mitosis. The majority (three of four) of cell lines underwent mitotic arrest, with apoptosis 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 markers after 72 hours of treatment, while most cells are in interphase. Interestingly, loss of the antiapoptotic protein myeloid cell leukemia 1 (Mcl-1) coincided with mitotic cell death. Stabilization of Mcl-1 resulted in a delayed onset of apoptosis, whereas enforced downregulation of Mcl-1 increased cell death in response to KSP inhibition. Thus, variation in responses to KSP inhibition is governed by a balance between survival proteins and spindle checkpoint integrity. Cells relying on short-lived survival proteins during mitosis are more likely to undergo apoptosis in response to KSP inhibition. We propose that patients with hematologic malignancies, 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 arrest due to maintenance of cdc2/cyclin B activity (3). Drug-induced accumulation of mitotic cells is tightly as- Antimitotic microtubule inhibitors are among the most sociated with cell death (4). ARRY-520 is a selective KSP active and broadly used cancer drugs. These drugs, inhibitor with low nanomolar potency in solid and hema- which include taxanes, epothilones, and Vinca alkaloids, tologic tumor cell lines (5) and activity in a wide range of directly perturb microtubule function, resulting in a de- tumor xenograft models (6). fective mitotic spindle and mitotic arrest. However, as Mitotic arrest is not permanent, but results in either microtubules perform critical functions in postmitotic death while cells are arrested in mitosis or exit from mi- cells, these drugs also exhibit unwanted side effects, in- tosis in the presence of misaligned chromosomes, a pro- cluding peripheral neuropathies (1). Thus, inhibitors of cess known as adaptation or mitotic slippage, leading to mitosis-specific targets that lack side effects in postmito- multinucleated cells (7). One model for adaptation sug- tic cells are desired. One such target is kinesin spindle gests that apoptosis induced by mitotic inhibitors re- protein (KSP, Eg5). KSP is a mitosis-specific kinesin es- quires the activation of the SAC followed by mitotic sential for the assembly of a bipolar spindle and equal slippage (8, 9). Presumably, postmitotic cell death results segregation of sister chromatids (2). Inhibition of KSP from a failure of mitosis associated with tetraploidy, prevents centrosome separation, resulting in the forma- which is essential to activate a cell death program in G1 tion of a monopolar spindle and activation of the mitotic to prevent further polyploidization (10). An alternative to spindle assembly checkpoint (SAC), causing metaphase death after mitotic slippage is death during mitotic arrest (11, 12). One hypothesis is that because transcription is absent in mitosis, yet proteolysis persists, cell fate is Authors' Affiliations: 1Translational Oncology and 2Pharmacology, Array BioPharma, Inc., Boulder, Colorado largely determined by the rate of depletion of short-lived Note: Supplementary material for this article is available at Molecular proteins that have short-lived mRNAs (13). In this model, Cancer Therapeutics Online (http://mct.aacrjournals.org/). a decline in antiapoptotic protein levels below a survival Corresponding Author: Brian Tunquist, Array BioPharma, Inc., 3200 threshold, before the loss of SAC activity and subsequent Walnut Street, Boulder, CO 80301. Phone: 303-3861512; Fax: 303- degradation of cyclin B, would be expected to trigger 3861362. E-mail: [email protected] apoptosis during mitotic arrest. Conversely, failure of doi: 10.1158/1535-7163.MCT-10-0033 the SAC before depletion of survival signals will result ©2010 American Association for Cancer Research. in adaptation and subsequent cell death in G1. The key

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elements of this hypothesis have recently been verified. and Use. Immunocompromised mice were purchased Treatment of HeLaM cells with microtubule poisons con- from Charles River. Tumor growth inhibition included firms the gradual accumulation of apoptotic signals in eight mice per group. Pharmacodynamic analysis in- cells that undergo a prolonged mitotic arrest (14). Fur- cluded three mice per group (average tumor volume thermore, it has been shown in multiple cell lines that of each group ∼165 mm3). cells that typically die directly in mitosis only slowly degrade cyclin B1, thereby preventing mitotic exit before Caspase-3/7 assay death (15). By contrast, cells that undergo adaptation pro- Vehicle or drug-treated cells in growth medium were gressively destroy cyclin B1, leading to mitotic exit. These seeded (10,000 viable cells per well) in duplicate wells data suggest that depending on the differential require- of black-walled 96-well plates and incubated at 37°C ment for survival protein activity and its rate of degrada- in 5% CO2. Caspase-3/7 activity was measured using tion, it may be possible to determine whether a tumor Caspase-Glo 3/7 reagent (Promega) at various time would succumb to mitotic cell death versus postmitotic points after drug addition. Data are reported as the mean cell death in the presence of a mitotic inhibitor. luminescence of drug-treated wells divided by the mean Various survival proteins, such as members of the Bcl-2 luminescence of vehicle control wells. family or inhibitors of apoptosis proteins (IAP), have been found to confer resistance to antimitotic chemotherapy in Cell viability various tumor types (16, 17). Of these, the prosurvival Inhibition of cell proliferation was measured using the Bcl-2 family member myeloid cell leukemia 1 (Mcl-1) Promega CellTiter-Blue assay. Cells were plated in 96- stands out as an intriguing candidate for determining well plates at a density that allowed for logarithmic whether an arrested cell undergoes mitotic cell death. growth over the 72-hour period of the assay. Compound Mcl-1 has an extended PEST region, which is responsible 65 (0.1–50 μmol/L) was added to the cells at a final for its relatively short half-life (18, 19). In addition, Mcl-1 DMSO concentration of 0.5%. Mcl-1 siRNA (10 nmol/L– becomes phosphorylated on sites within its PEST region 1.25 μmol/L) was transfected into the cells as described in response to treatment with phorbol ester, oxidative below. Fluorescent signal was converted to percent of stress, cytokine withdrawal, and microtubule-damaging control relative to high (DMSO-treated control) and low agents, which affects its overall stability (20, 21). Lastly, (fully inhibited control) samples. The EC50 for inhibition Mcl-1 is predominately expressed in hematopoietic cell of viability was determined from the inflection point of a lineages, including multiple myelomas where it has been standard four-parameter logistical curve. shown to be indispensable (22–25). We have previously shown that xenograft tumors of hematologic origin, in- Immunoassays cluding multiple myeloma, are often more responsive to ARRY-520–treated cell lines were harvested at various drug treatment with ARRY-520, compared with xenograft time points after drug addition. Cell lysates were gener- tumors of nonhematologic origin. Here, we have exam- ated by thawing cells in radioimmunoprecipitation assay ined Mcl-1 and other survival proteins in multiple myelo- buffer [50 mmol/L Tris (pH 8), 150 mmol/L NaCl, 1% ma cell lines following treatment with ARRY-520 to NP40, 0.5% deoxycholate, 0.05% SDS, 2 mmol/L EDTA, determine whether Mcl-1 plays a role in determining cell protease inhibitor cocktail set III, EDTA-free, phospha- fate decisions following KSP inhibition. tase inhibitor cocktail set I; Calbiochem]. Mice bearing tumor xenografts were euthanized by CO2 inhalation, and Materials and Methods tumors were harvested and flash frozen in liquid nitro- gen. Tumor lysates were prepared as described previous- Cell lines and culture ly (6). Proteins were resolved on 4% to 12% Tris-glycine RPMI 8226, NCI H929, and U266 cells were obtained gels (NuPage, Invitrogen) and transferred onto nitrocel- from the American Type Culture Collection. JJN3 cells lulose membranes. Membranes were probed with com- were obtained from the Deutsche Sammlung von Mik- mercial primary antibodies: Bcl-2 (BD Biosciences), poly roorganismen und Zellkulturen GmbH. Cells used for ex- (ADP-ribose) polymerase (PARP), XIAP, Bcl-XL (Cell Sig- periments were grown in culture for less than 2 months naling), glyceraldehyde-3-phosphate dehydrogenase under the conditions recommended by the suppliers. (Sigma), phosphorylated histone H3 (Ser10), cyclin B1, Mcl-1, p42/p44 mitogen-activated protein kinase (Santa In vivo xenograft models Cruz Biotechnology), and NOXA (Calbiochem). Blots RPMI 8226 tumor cells, 2 × 107 (tumor growth inhibi- were then probed with secondary antibodies: IRDye tion;Fig.2A)or5×107 (pharmacodynamic analysis; 800–conjugated anti-mouse IgG (Rockland) or Alexa Fluor Fig. 2B–D), in 100 μL of 50% Matrigel were implanted 680 anti-rabbit IgG (Invitrogen), and imaged using the s.c. into female severe combined immunodeficient-beige LiCOR Odyssey infrared imaging system. Lysates from mice as described previously (6). All studies were done cycling NCI H929 cells used for immunoprecipitation in accordance with the Array BioPharma Institutional experiments were prepared as outlined above. Protein Animal Care and Use Committee guidelines and in ac- lysates (300 μg) supplemented with DMSO or 1 μmol/L cordance with the Guide for Laboratory Animal Care compound 65 were subjected to immunoprecipitation

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overnight at 4°C using 5 μg of rabbit IgG or Mcl-1 anti- NCI H929 cell line also displayed a gradual increase in body (Santa Cruz Biotechnology) bound to protein A- caspase-3/7 activity over time, the peak in activity oc- Sepharose CL-4B resin (Sigma). curred much later (72–80 hours). As a secondary method of verifying apoptosis, and to Electroporation of cells characterize drug responses at a molecular level, we per- RPMI 8226, JJN3, and U266 cells were diluted to a formed immunoblotting for survival and apoptotic pro- final concentration of 1 × 107 cells in 0.4 mL of Opti- teins, and/or their posttranslational modification sites, MEM (Invitrogen). Cells were mixed with 10 μgof from all four multiple myeloma cell lines. All cell lines pZsGreen1-N1 plasmid (Clontech) or pZsGreen1-N1 treated with 10 nmol/L ARRY-520 exhibited rapid induc- plasmid containing full-length human Mcl-1 cDNA. Cells tion of mitotic arrest as judged by both increased phos- were electroporated in a 4-mm cuvette (Bio-Rad Labora- phorylation of histone H3 (pHH3) and accumulation of tories) by exponential decay pulse (240 V, 1,070 μF) using cyclin B1 (see Fig. 1B and C; Supplementary Fig. S2). Mi- the Gene Pulser Xcell electroporation system (Bio-Rad totic arrest caused by ARRY-520 was also confirmed by Laboratories). Cells were immediately placed into growth flow cytometry as all four cell lines exhibited an in- medium following electroporation, and dead cells were creased amount of 4 N cells after 24 hours of ARRY-520 removed 24 hours later by magnetic beads (Dead Cell treatment (Supplementary Fig. S1B). PARP is one of the Removal Kit; Miltenyi Biotec) before being used for drug main targets of caspase-3 (29), and its cleavage has been treatment. used as a marker for cells undergoing apoptosis (30). Us- ing the JJN3 cell line as a representative for cell lines that Transfection of siRNA undergo rapid onset of apoptosis, it was found that sig- Specific Accell-modified siRNA duplexes in 1× siRNA nificant PARP cleavage occurred within 24 hours of buffer were stored at −20°C: Accell green nontargeting ARRY-520 treatment, consistent with the peak in caspase siRNA, human NOXA siRNA N8 (26), and human Mcl-1 activation in this cell line (Fig. 1A) and correlated with siRNA duplex 34 (Dharmacon). Cells were transfected by maximal mitotic arrest (Fig. 1B). However, whereas the seeding in fresh Accell delivery medium containing the NCI H929 cell line exhibited a slight increase in the levels appropriate siRNA for 24 or 48 hours under normal of cleaved-PARP during mitotic arrest, the majority of growth conditions. PARP cleavage was seen after 72 hours of treatment, once the cells had exited mitosis (Fig. 1C). Compounds ARRY-520 and compound 65 were synthesized at Loss of Mcl-1 correlates with mitotic cell death Array BioPharma. For cell-based assays, ARRY-520 or In an effort to identify molecular activities that have a compound 65 was added to growth medium, with role in regulating mitotic cell death in these cells, the le- DMSO used as a vehicle control. DMSO concentrations vels of key antiapoptotic proteins were measured at var- never exceeded 0.5% in the assay. ious times following exposure to ARRY-520. In all cell lines tested, levels of Bcl-2 and Bcl-XL remained relative- Results ly stable following treatment with ARRY-520 (see Fig. 1B and C; Supplementary Fig. S2). It has been suggested that The onset of apoptosis varies among cell lines although the abundance of XIAP is not correlated with treated with ARRY-520 cell death, the degradation of XIAP in solid tumor lines We have previously shown that multiple myeloma tu- is associated with onset of cell death in cells treated with mor xenografts are highly sensitive to ARRY-520 in vivo a KSP inhibitor (28) or other chemotherapeutics (31). Al- (6). Thus, we decided to investigate the molecular me- though XIAP degradation indeed occurred following chanisms responsible for mitotic cell death in four differ- treatment with ARRY-520 in myeloma lines, the overall ent multiple myeloma cell lines (RPMI 8226, JJN3, U266, loss of XIAP protein was relatively modest and inconsis- and NCI H929). Most studies report that cell death result- tent between cell lines, suggesting that, at least in these ing from prolonged mitotic arrest is executed by proteo- cell lines, other proteins play a role in signaling cell lytic caspase enzymes (9, 11, 15, 27, 28). The kinetics of death. This is compatible with the finding that cell death apoptosis induction in these four cell lines was investi- caused by ARRY-520 in leukemia cell lines also did not gated by measuring the activation of caspase-3 and depend on XIAP (5). By contrast, we observed that the caspase-7 at various time points following drug addition. degradation of Mcl-1 coincided closely with PARP clea- Cells seeded into identical 96-well plates were treated vage in JJN3 cells (Fig. 1B) and other cell lines that with either 10 nmol/L ARRY-520 (∼5-fold above the cel- underwent rapid apoptosis in response to ARRY-520 lular IC50 for these lines; Supplementary Fig. S1A) or an (Supplementary Fig. S2). This effect was not seen in equivalent volume of vehicle (DMSO). Three of the cell immunoblots of the NCI H929 cell line exposed to lines (RPMI 8226, JJN3, and U266) exposed to ARRY- ARRY-520 (Fig. 1C), suggesting that Mcl-1 destabiliza- 520 exhibited a rapid onset of apoptosis as defined by a tion, promoted during prolonged mitotic arrest, may con- peak in caspase-3/7 activity that occurred ∼24 hours af- tribute to initiation of cell death. Interestingly, cells that ter compound treatment (Fig. 1A). However, whereas the have slipped from mitosis may not be as dependent on

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Figure 1. Caspase-3/7 activation in multiple myeloma cell lines treated with ARRY-520 correlates with Mcl-1 degradation. A, multiple myeloma cell lines were 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 reported as 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 at 11 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 longer duration 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.

Mcl-1 for survival during interphase as the levels of Mcl-1 may be a critical factor important for multiple myeloma seem to remain stable in the postmitotic time points taken cell survival during prolonged mitotic arrest. Subsequent from NCI H929 cells (Fig. 1C), despite the fact that these experiments test the validity of this hypothesis in multi- cells eventually undergo apoptosis as measured by PARP ple myeloma cell lines. cleavage and caspase activation. RPMI 8226 tumor xenografts were particularly sensi- Increased expression or stabilization of Mcl-1 tive to low doses of ARRY-520 (12.5 mg/kg) adminis- delays apoptosis tered on days 1 and 2 (Fig. 2A). Immunoblot analysis Our data suggest that Mcl-1 degradation in cells ar- of tumor lysates following ARRY-520 treatment showed rested in mitosis by ARRY-520 is a key step in the onset similar effects on Mcl-1 stability and induction of apopto- of apoptosis. To verify this, we stabilized Mcl-1 protein sis during mitotic arrest as seen in RPMI 8226 cells trea- in cells through both direct overexpression of recombi- ted in vitro (Fig. 2B–D). Mcl-1 levels were significantly nant Mcl-1 protein and by knockdown of the Mcl-1 decreased after only 24 hours of treatment with the ma- regulatory protein NOXA. In the first instance, we an- jority of Mcl-1 expression in the tumor lost by 48 hours, alyzed the effect of overexpression of Mcl1-ZsGreen1 corresponding with an increase in cleaved PARP at these fusion protein on the three cell lines that underwent a time points. In total, these experiments suggest that Mcl-1 rapid apoptosis in mitosis following treatment with

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ARRY-520. If multiple myeloma cells depend on Mcl-1 bilize Mcl-1 protein by an alternative mechanism that for mitotic survival, we hypothesized that overexpression does not directly alter physiologic levels of Mcl-1. The of Mcl1-ZsGreen1 would delay apoptosis induction in small, BH3-only protein NOXA functions as a specific these cells. Indeed, overexpression of Mcl1-ZsGreen1 antagonist for Mcl-1 binding to proapoptotic Bcl-2 fam- caused an ∼24 hours (2-fold) delay in the onset of peak ily members, such as Bim and Bak (32–34). Binding of caspase-3/7 activity (Fig. 3A) and PARP cleavage NOXA to Mcl-1 prohibits the formation of Mcl-1/Bak (Fig. 3B and C) in the JJN3 cell line following exposure or Mcl-1/Bim dimers, thus increasing the pool of free to ARRY-520. Similar results were also obtained in the Bak and Bim capable of promoting mitochondrial outer RPMI 8226 and U266 cell lines (Supplementary Figs. S3 membrane permeabilization. In addition, NOXA bind- and S4, respectively). Although endogenous Mcl-1 and ing to Mcl-1 has been suggested to promote Mcl-1 ubi- Mcl1-ZsGreen1 were still degraded, the excess Mcl1- quitination and subsequent degradation by the ZsGreen1 seemed to compensate for the loss of the proteasome (Fig. 4A; ref. 34). Such a role for NOXA native protein, thus delaying the onset of apoptosis in has been verified in multiple myeloma cell lines treated these cells. with a wide range of chemotherapeutics (32, 33, 35, 36). It is possible that overexpression of a survival protein We were interested in whether the rapid decline of such as Mcl-1 could induce survival signals indepen- Mcl-1 protein in ARRY-520–arrested RPMI 8226, JJN3, dent of a role in mitotic cell death responses. For this or U266 cell lines was mediated through NOXA. To test reason, we chose to investigate whether we could sta- this, 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 were administered 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 indicated days. Tumors were excised at the indicated times, and lysates were blotted for Mcl-1 (B), pHH3 (C), or PARP (D). Band intensities were normalized to total 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.

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Figure 3. Mcl-1 overexpression delays cell death in ARRY-520 sensitive cells. A, caspase-3/7 activity measurements were carried out in empty vector– and Mcl1-ZsGreen1–transfected JJN3 cells treated with 10 nmol/L ARRY-520. Data are reported as the fold increase above empty vector or Mcl1-ZsGreen1–transfected JJN3 cells treated with DMSO. The mean of three experiments is shown; error bars, SD. Immunoblot analysis of proteins from JJN3 cell lysates following transfection with empty vector (B) or Mcl1-ZsGreen1 vector (C) treated with 10 nmol/L ARRY-520. protein levels before ARRY-520 treatment. Transfection Reduced Mcl-1 protein or function enhances cell of cells with NOXA siRNA 48 hours before ARRY-520 death caused by KSP inhibition in NCI H929 cells treatment resulted in significant knockdown of NOXA The observation that increased Mcl-1 protein can post- throughout the time course of ARRY-520 treatment as pone cell death in M phase in most cell lines tested sug- determined by immunoblotting (Fig. 4D). Caspase-3/7 gests that perhaps the NCI H929 multiple myeloma cell activity measured in JJN3 cells transfected with control line could be made to undergo an early cell death in siRNA behaved very similarly to what was seen in mitosis by lowering Mcl-1 activity. We reduced Mcl-1 Figs. 1 and 2, whereas cells containing reduced NOXA protein function by two separate methods: the use of exhibited a significant 24-hour delay in the onset of cell Mcl-1–specific siRNA and treating with a small-molecule death caused by ARRY-520 (Fig. 4B). Similar results Mcl-1 inhibitor. As the loss of Mcl-1 alone is sufficient to were observed following knockdown of NOXA in induce cell death in multiple myeloma cell lines (22, 25), RPMI 8226 and U266 cell lines (Supplementary Figs. we established conditions that resulted in knockdown of S5 and S6). NOXA knockdown correlated with greater Mcl-1 without incurring substantial cell death on its own. stabilization of Mcl-1 protein 24 hours after ARRY-520 We detected measurable Mcl-1 knockdown 24 hours treatment, and a delay in detectable levels of cleaved- posttransfection using Mcl-1 siRNA concentrations >15 PARP (Fig. 4D) compared with control-transfected cells nmol/L (Fig. 5A), and further determined cellular EC50 (Fig. 4C). Taken together, these results suggest that values for cell viability of 20 and 14 nmol/L, at 24 and Mcl-1 is capable of maintaining survival signals in mul- 48 hours posttransfection, respectively (Fig. 5B). Using tiple myeloma cell lines arrested in mitosis by ARRY- an Mcl-1 siRNA concentration above its EC50 value 520, and that only once Mcl-1 levels decrease below a (25 nmol/L), we found that addition of 10 nmol/L threshold level for survival can cell death pathways ARRY-520 to NCI H929 cells 24 hours posttransfection become active. enhanced both the magnitude and time to initiation of

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apoptosis as measured by activation of caspase-3 and BH3-only proteins. Consistent with this, compound 65 μ caspase-7 (Fig. 5C). This was significantly different than induced rapid cell death (IC50 2 mol/L at 48 hours; the amount of cell death caused by 25 nmol/L Mcl-1 Fig. 6C) in NCI H929 cells consistent with its Mcl-1 inhi- siRNA in combination with vehicle (DMSO), or through bitory activity. Treatment of NCI H929 cells with 10 nmol/L transfection of 25 nmol/L control siRNA in combination ARRY-520, followed 8 hours later with the addition of with 10 nmol/L ARRY-520 (Fig. 5C). Thus, although NCI 2 μmol/L compound 65, resulted in a significant increase H929 cells depend on Mcl-1 for survival, it is hypothe- in apoptosis by 24 hours compared with either ARRY-520 sized that they either lack a mechanism sufficient for or compound 65 alone (Fig. 6D), similar to the effect of triggering its mitotic degradation or have developed a Mcl-1 knockdown (Fig. 5C). These results are consistent mechanism to promote its stabilization during a pro- with a role for Mcl-1 functioning to prevent apoptosis longed M-phase arrest caused by ARRY-520. during mitotic arrest of NCI H929 cells. As an alternative method of modulating Mcl-1 function, we used a selective, small-molecule inhibitor of Discussion Mcl-1. Compound 65 (Fig. 6A) has been reported to disrupt the binding of interacting proteins, such as The inhibition of mitosis-specific drug targets, such as NOXA, to the hydrophobic pocket of Mcl-1 (37). To KSP, is expected to yield improvements in clinical effica- confirm the reported activity, we show that coimmuno- cy and safety profiles compared with current microtu- precipitation of BH3-only proteins NOXA and Bim bule-targeted chemotherapeutics. To date, however, with Mcl-1 is prevented in the presence of 1 μmol/L KSP inhibitors have shown only sporadic clinical activity compound 65 (Fig. 6B), suggesting that compound 65 in a subset of tumor types. Thus, an unresolved challenge can disrupt the association of Mcl-1 with proapoptotic is 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 in Mcl-1 regulation and promotion of cell death. NOXA is important for sequestering Mcl-1 from proapoptotic factors, such as Bak. Further, NOXA-bound Mcl-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. The mean of three independent experiments is shown; error bars, SD. Western blot analysis of control (C) and NOXA (D) siRNA-transfected JJN3 cells. Cells were immunoblotted for the indicated proteins before transfection (−48 h time point) and at various time points following 10 nmol/L ARRY-520 addition.

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Figure 5. Knockdown of Mcl-1 confers increased ARRY-520 sensitivity to NCI H929 cells. A, Mcl-1 siRNA dose titration 24 h posttransfection. Antibodies used for immunoblotting and siRNA concentrations (nmol/L) are indicated. B, cell viability of NCI H929 cells 24 and 48 h following transfection with indicated concentrations of Mcl-1 siRNA. Data are reported as the percentage of viable Mcl-1 siRNA-transfected cells compared with control siRNA-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.

to be sensitive to compounds such as ARRY-520. Such throughout the time course of the experiment, suggesting translational knowledge would enable the design of bet- that Mcl-1 stability enabled survival of ARRY-520– ter clinical trials and patient selection strategies. Previous arrested cells until SAC failure and mitotic exit. Compa- efforts to identify the molecular sources of variation in rable results have recently been reported whereby sensitivity to apoptosis have suggested that the balance decreasing Mcl-1 expression in various tumor cell lines, between apoptotic signals and the integrity of the SAC including multiple myeloma cell lines, led to the sensitiza- are key events in determining the onset of cell death in tion of cells previously resistant to CDK inhibition (39). response to mitotic inhibitors (13–15, 27, 28, 38). To our These data support a model wherein the fate of a cell once knowledge, ours is the first study to successfully identify it has become arrested in mitosis is controlled by two in- a single survival protein, Mcl-1, as a determinant control- dependent clocks: the cell-specific rate of decline in sur- ling the decision to undergo apoptosis during mitotic ar- vival signals below a threshold for triggering cell death rest in multiple myeloma cell lines. and the maintenance of mitotic arrest as determined by the intrinsic rate of cyclin B1 degradation. In some tumor Survival protein reliance determines cell fate cells, as exemplified by JJN3, RPMI 8226, and U266, the We have observed that of the four myeloma cell lines loss of Mcl-1 during a KSP inhibitor–induced mitotic arrest tested, three undergo a rapid onset of apoptosis, commit- is sufficient to promote the onset of apoptosis. Conversely, ting to cell death within 24 hours of exposure to ARRY- tumor cells that rely on Mcl-1 for survival, but are less capa- 520, while still arrested in mitosis. By contrast, NCI ble of inducing Mcl-1 degradation during mitotic arrest, H929 cells showed a significantly delayed apoptosis fol- such as NCI H929, may exhibit a delayed onset of apopto- lowing KSP inhibition compared with the other three sis and require far longer exposure to induce cell death. multiple myeloma cell lines (Fig. 1). The majority of H929 cells seemed to undergo SAC failure and subse- Therapeutic importance of Mcl-1 in determining quent cell death after 72 hours when the cells were in in- cell fate terphase (Fig. 2B). Western analysis failed to show a The importance of survival proteins in determining cell significant difference in mitotic duration between these fate in response to KSP inhibition has been shown by lines (Fig. 2; Supplementary Fig. S2), suggesting that this others. In a population-based analysis similar to what dissimilarity could not be due solely to a SAC defect. Of has been presented here, Shi et al. characterized the re- note, although Mcl-1 levels declined rapidly in cells that sponse of several solid tumor cell lines to three different died in mitosis, Mcl-1 levels remained stable in NCI H929 antimitotic drugs (28). Although these cells all responded

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similarly to a defective mitotic spindle, the response of nals during mitotic arrest, these cells may be able to the apoptotic machinery was variable between cell lines. survive transient mitotic inhibition until drug levels drop Loss of the survival protein XIAP correlated with the on- below therapeutic levels (at which time a normal spindle set of apoptosis. Although supporting the hypothesis can be reassembled and cell cycle progression can com- that cell fate decisions in response to KSP inhibition are mence). In contrast, cells that depend on Mcl-1 and rap- determined in part by modulation of survival proteins, idly degrade it during mitotic arrest are more likely to be the loss of XIAP has not yet been directly shown as being responsive to the drug because apoptosis can be initiated responsible for cell death. Here, we clearly show a role within the window of exposure to the drug. for Mcl-1 degradation in determining cell fate in response The importance of Mcl-1 in determining apoptosis in to KSP inhibition in several myeloma cell lines. We be- mitosis in hematologic cell lines makes particular sense, lieve that these data can inform the identification of tu- given the fact that hematologic cells typically rely on this mors likely to be most sensitive to KSP inhibitors short-lived survival protein. Thus, we suggest that hema- clinically. KSP inhibitors are typically given over short, tologic malignancies, such as multiple myeloma, leuke- infusional schedules and therefore have defined periods mia, and lymphoma, are particularly good candidates of therapeutic exposure, with reported clinical half-lives to investigate the clinical activity of KSP inhibitors. Of typically ranging from 28 to 50 hours (40). Thus, we note, the KSP inhibitor SB-743921 has shown clinical hypothesize that cells that rely on more stable survival activity in Hodgkin's and non–Hodgkin's lymphomas, proteins, such as Bcl-2 or Bcl-XL, may be temporally re- although a relationship between Mcl-1 and response fractory to KSP inhibition: By maintaining survival sig- has not been investigated (41). Many solid tumor cell

Figure 6. Inhibition of Mcl-1 binding to NOXA in NCI H929 cells confers increased sensitivity to ARRY-520. A, chemical structure of compound 65. B, coimmunoprecipitation of NOXA with endogenous Mcl-1 is blocked in the presence of 1 μmol/L compound 65 in NCI H929 cell extracts. Rabbit IgG was used as a control for immunoprecipitation. C, cell viability of NCI H929 cells exposed to the indicated concentrations of compound 65 for 24 or 48 h. Data are reported as the percentage of viable NCI H929 cells following compound 65 treatment compared with DMSO control–treated NCI H929 cells. D, NCI H929 cells treated with DMSO or 2 μmol/L compound 65 were analyzed for caspase-3/7 activity at various time points following the addition of DMSO or 10 nmol/L ARRY-520. Total DMSO concentration of the control did not exceed 0.5% (v/v). Fold caspase-3/7 activity is reported as the mean of three separate experiments; error bars, SD.

2054 Mol Cancer Ther; 9(7) July 2010 Molecular Cancer Therapeutics

Mcl-1 Determines the Fate of KSP-Inhibited Cells

lines do not express or depend on Mcl-1. Here, it is pos- further decline in Mcl-1 protein levels to cement the sible that other mechanisms, such as degradation of XIAP commitment to apoptosis. Interestingly, a recent ge- (28), may determine cell fate following KSP inhibitor nome-wide siRNA screen of HeLa cells exposed to KSP treatment. Alternatively, it has been reported that subsets inhibitor identified NOXA (also known as PMAIP1) of nonhematologic tumor types, such as non–small cell knockdown as a potential suppressor of cell death caused lung carcinomas, melanomas, and biliary cancers, may by low concentrations of K5I (48). Further investigation gain the ability to express Mcl-1 during tumorigenesis of the role of NOXA in modulating cell fate in response (42–44), and these Mcl-1–expressing solid tumors may to KSP inhibition is needed to confirm these hypotheses. also be expected to be sensitive to shorter drug exposures In summary, our data suggest that tumors that express of ARRY-520. These findings would be expected to have and rely on Mcl-1 for survival may be more sensitive to significant value in selecting potentially sensitive patient the KSP inhibitor ARRY-520. However, this observation is populations in solid tumor indications. dependent on the ability of the tumor to degrade Mcl-1 during mitotic arrest. Mcl-1 is a key antiapoptotic protein Temporal resistance to ARRY-520: predictive value in a wide variety of hematologic tumor types, such as of Mcl-1 regulation by NOXA multiple myeloma (22, 25), leukemia (49), and lymphoma The fact that NCI H929 cells rely on Mcl-1 but are un- (50). These data therefore support further investigation of able to significantly degrade Mcl-1 during mitotic arrest, ARRY-520 in hematologic malignancies, which rely on and therefore avoid mitotic cell death, indicates that Mcl-1 Mcl-1 for survival. To test this hypothesis clinically, stabilization may represent a mechanism of resistance to ARRY-520 is being explored in clinical trials in both acute KSP inhibitors. One possible mechanism for Mcl-1 stabi- myelogenous leukemia and multiple myeloma. lization in the NCI H929 cell line may be mediated through the proapoptotic BH3-only protein NOXA. Disclosure of Potential Conflicts of Interest NOXA antagonizes the survival function of Mcl-1 and has been found to be sufficient to promote its degrada- The authors are employees and shareholders of Array BioPharma, Inc., Boulder, CO, USA. This company has patent rights on some reagents tion by the proteasome (34, 45, 46). Interestingly, NOXA used in this study. protein levels are lowest in the NCI H929 cell line, which may help explain why Mcl-1 levels are more stable dur- Acknowledgments ing a prolonged mitotic arrest (Supplementary Fig. S7). Reducing NOXA levels in the other three cell lines de- We thank all those at Array BioPharma who have contributed to the layed Mcl-1 degradation and prolonged M-phase arrest development of ARRY-520; Heidi Simmons, Dana Longo, Shelley Allen, Christine Lemieux, Jennifer Garrus, Stefan Gross, Jim Winkler, Eli and survival (Fig. 4; Supplementary Figs. S5 and S6). Wallace, and Kevin Koch for helpful discussions and advice; and D. Thus, tumor cells with higher expression of NOXA pro- Ross Camidge, University of Colorado Denver, for the critical review of the manuscript. tein may be more sensitive to antimitotics, such as ARRY- The costs of publication of this article were defrayed in part by the 520, by promoting the rapid degradation of Mcl-1 by the payment of page charges. This article must therefore be hereby marked proteasome during mitotic arrest. As Mcl-1 has also been advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. reported to be cleaved by active caspases (47), we hypothesize that the NOXA-driven decline in Mcl-1 levels Received 01/12/2010; revised 04/14/2010; accepted 04/30/2010; below a death threshold would trigger apoptosis and a published OnlineFirst 06/22/2010.

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2056 Mol Cancer Ther; 9(7) July 2010 Molecular Cancer Therapeutics

Mcl-1 Stability Determines Mitotic Cell Fate of Human Multiple Myeloma Tumor Cells Treated with the Kinesin Spindle Protein Inhibitor ARRY-520

Brian J. Tunquist, Richard D. Woessner and Duncan H. Walker

Mol Cancer Ther 2010;9:2046-2056. Published OnlineFirst June 22, 2010.

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