a novel eg5 inhibitor (ly2523355) causes mitotic arrest ... · kinesin eg5 represents an attractive...

Small Molecule Therapeutics

A Novel Eg5 Inhibitor (LY2523355) CausesMitotic Arrest and Apoptosis in Cancer Cells andShows Potent Antitumor Activity in XenograftTumor ModelsXiang S. Ye1, Li Fan1, Robert D. Van Horn1, Ryuichiro Nakai2, Yoshihisa Ohta3,Shiro Akinaga4, Chikara Murakata3, Yoshinori Yamashita2, Tinggui Yin1,Kelly M. Credille1, Gregory P. Donoho1, Farhana F. Merzoug1, Heng Li1,Amit Aggarwal1, Kerry Blanchard1, and Eric H.Westin1

Abstract

Intervention of cancer cell mitosis by antitubulin drugs isamong the most effective cancer chemotherapies. However, anti-tubulin drugs have dose-limiting side effects due to importantfunctions of microtubules in resting normal cells and are oftenrendered ineffective by rapid emergence of resistance. Antimitoticagents with different mechanisms of action and improved safetyprofiles are needed as new treatment options. Mitosis-specifickinesin Eg5 represents an attractive anticancer target for discov-ering such new antimitotic agents, because Eg5 is essentialonly in mitotic progression and has no roles in resting, non-dividing cells. Here, we show that a novel selective Eg5 inhibitor,

LY2523355, has broad target-mediated anticancer activity in vitroand in vivo. LY2523355 arrests cancer cells at mitosis and causesrapid cell death that requires sustained spindle-assembly check-point (SAC) activation with a required threshold concentration.In vivo efficacy of LY2523355 is highly dose/schedule-dependent,achieving complete remission in a number of xenograft tumormodels, including patient-derived xenograft (PDX) tumor mod-els. We further establish that histone-H3 phosphorylationof tumor and proliferating skin cells is a promising pharmaco-dynamic biomarker for in vivo anticancer activity of LY2523355.Mol Cancer Ther; 14(11); 2463–72. �2015 AACR.

IntroductionDuring G2–M progression, duplicated centrosomes are sepa-

rated by themicrotubule-based bipolar mitotic spindle through acomplex process involving chromosomes, mitotic kinases, andmicrotubule-associated proteins (1–3). The bipolar mitotic spin-dle orchestrates the segregation of replicated chromosomes accu-rately and equally into two daughter cells to maintain genomestability during cell division. Antimitotic drugs such as antitubu-lins, including taxanes and vincas, are among the most effectivecancer therapies in current clinical use (4). However, these anti-tubulins also have severe side effects as they disrupt the normalmicrotubule functions in resting cells, including neurons, whichmay lead to neuropathic disorders (5–7). Furthermore, emerging

resistance to these antimitotic agents renders them virtuallyineffective (8).

Eg5, a member of BIMC family of kinesins, is a plus-enddirected homotetrameric kinesin (1, 9, 10). It plays a vital rolein chromosome segregation and bipolar mitotic spindle orga-nization and elongation by sliding antiparallel microtubulesaway from each other (11–14). Its function in bipolar spindleformation was found to be evolutionarily conserved (14–16).Eg5 is tightly regulated by mitotic kinases and cell-cycle check-point controls (14, 15, 17). Overexpression of Eg5 in trans-genic mice caused mitotic defects leading to genome instabilityand tumorigenesis (18, 19). Unlike microtubules that arepresent throughout the cell cycle and have important biologicfunctions in many nondividing cells, Eg5 is expressed andactive only during mitosis (20), thus making it an attractivetarget for discovering new antimitotic drugs with a differentmode of action and an anticipated improved safety profile(5, 21). The discovery of small-molecule Eg5 inhibitors spurredstrong interest in their development as anticancer agents(6, 22–24).

Inhibition of Eg5with smallmolecules disruptsmitotic spindleformation inducing spindle assembly checkpoint (SAC)-mediat-ed mitotic arrest (25) and apoptosis in cancer cells, includingtaxol-resistant cancer cells (22, 25–27). Recent studies have dem-onstrated that both activation of SAC and subsequent mitoticslippage are required for apoptosis induction by the Eg5 inhibi-tors (25, 28). However, how activation of SAC and mitoticslippage are linked to induction of apoptosis is still not wellunderstood.

1Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indi-ana. 2Drug Discovery Research Laboratories, Kyowa Hakko Kirin Co.Ltd., Shizuoka, Japan. 3Medicinal Chemistry Research Laboratories,Kyowa Hakko Kirin Co. Ltd., Shizuoka, Japan. 4Clinical DevelopmentDepartment 1, Development Division, Kyowa Hakko Kirin Co. Ltd.,Shizuoka, Japan.

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

Current address for H. Li: CrownBio, Taicang, Jiansu, China.

Corresponding Author: Xiang S. Ye, Lilly Corporate Center, Eli Lilly and Com-pany, Indianapolis, IN 46285. Phone: 317-277-1467; Fax: 317-277-3652; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0241

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

www.aacrjournals.org 2463

on April 6, 2021. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst August 24, 2015; DOI: 10.1158/1535-7163.MCT-15-0241

http://mct.aacrjournals.org/

Eg5 inhibitors generally show excellent anticancer activities in arange of xenograft tumor models (21). However, clinical experi-enceswith several Eg5 inhibitors that enteredphase I and II studieshave been rather disappointing, resulting in termination of theirclinical studies (6). The lack of clinically meaningful activities forthese Eg5 inhibitors may be a result of their poor pharmacoki-netics or dose selection, which was not optimized on the basis ofan understanding of pharmacokinetic and pharmacodynamicrelationships. Moreover, early Eg5 inhibitors were developedfollowing a dosing paradigm established for antitubulins. Thefact that cell-type variation and the length of time in sustainedmitotic arrest contribute to differences in induction of apoptosisby Eg5 inhibitors (25, 29–31) reinforce the need for careful doseand schedule selection.

We describe here the discovery and characterization ofLY2523355, a selective allosteric inhibitor of human Eg5, derivedthrough an extensive structure–activity relationship programfrom a precursor compound identified by a phenotype-basedscreening formitotic arrest of HCT116 cells (32).We demonstratethat inhibition of Eg5 in cancer cells by LY2523355 causes rapidapoptosis following SAC-dependent mitotic arrest. We furthershow that abrogation of SAC by inactivation of cyclin-dependentkinase 1 (CDK1) or knockdown of mitotic arrest deficient2 (Mad2) causes rapid exit from mitosis and antagonizesinduction of apoptosis by LY2523355. LY2523355 shows high-ly schedule-dependent, broad-spectrum anticancer activityagainst tumor models, including patient-derived xenograft(PDX) models. We establish that histone H3 phosphorylationof tumor proliferating skin cells is a promising pharmacody-namic marker for LY2523355 activity.

Materials and MethodsCancer cell lines, reagents, and treatments

Cancer cell lines (A2780, A549, ARH-77, COLO-205, Calu-6,DU 145, HCT-116, HT-29, Hep 3B2.1-7, LN-18, LN-229, M059K,MDA-MB-231, NAMALWA, NCI-H460, NIH:OVCAR-3, RPMI8226, SK-BR-3, SK-OV-3, SW620, U-87 MG, HeLa) used for invitro anticancer and mechanism studies at Eli Lilly and Companywere all obtained from ATCC between September 2005 to July2007, unless otherwise stated. The cell lines were authenticated byshort tandem repeat (STR) assay and compared against existingreference standards at RADIL (now IDEXX Bioresearch) and weremaintained in media per ATCC instructions and as describedpreviously (33). Sources of the cancer cell lines used for the in vivoxenograft tumor model studies at Kyowa Hakko Kirin as sum-marized in Table 1 were as follows: HCT-116 cells were obtainedin 1998, NCI-H460 cells in 1999, NCI-H929 cells in 2001, andColo-205, Calu-6, SK-OV-3 cells in 2002 from ATCC; EBC-1 cellswere obtained from JCRB Cell Bank in 2002; KPL-4 and KMS-11cells were obtained from Kawasaki Medical University in 1997and 2001, respectively; PC-14 and PC-14/CDDP cells wereobtained from National Cancer Center Hospital in 2001 and2002, respectively; A2780 cells were obtained from JapaneseFoundation for Cancer Research in 2002; RMG-1 cells wereobtained from Ishiwata Hospital in 1999 and MX-1 cells wereobtained from Central Institute for Experimental Animals in1998. These cancer cell lines at Kyowa Hakko Kirin were notauthenticated and were maintained as previously described (32).

Mouse anti-BubR1 antibody was purchased from Becton Dick-inson Transduction Labs. Mouse anti-a-tubulin, actin, and anti-

Mad2 antibodies were from Sigma-Aldrich. Rabbit anti-pericen-trin antibody was from Abcam. PARP1, Mcl1, Bcl2, p38, and anti-cleaved PARP (Asp214) antibodies were from Cell SignalingTechnologies. Rabbit anti-phospho-histone H3 (Ser10) andphospho-histone H2A.X (Ser139) and cyclinB1 antibodies werepurchased from Millipore. Mouse anti-GAPDH antibody wasfrom Meridian Life Science Inc.. Mad2 ON-TARGETplus SMARTpool siRNA and DharmaFECT1 transfection reagent were pur-chased from Dharmacon. Propidium iodide was purchased fromInvitrogen. Paclitaxel, docetaxel, CPT-11, vinorelbine, and cis-platin were from Sigma Aldrich. RO3306 was from Santa CruzBiotechnology. The Eg5 inhibitor LY2523355 was synthesizedinternally.

RNAi, compound handling and treatment, immunoblot-ting, flow cytometry, and immunofluorescence microscopywere performed as previously described (32, 33). Assays forATPase activity of kinesins and in vitro microtubule polymer-ization and depolymerization were also done as previouslydescribed (32).

Mitotic index and apoptosis measurementCancer cells were plated in poly-D-lysine coated 96-well plates

(BD cat. no. 356640) and incubated overnight. Cells were thentreated with indicated concentrations of LY2523355 for varioustime periods. Cells were then fixed with 3.7% formaldehyde inPBS for 45minutes or 1�Preferfixative solution for 30minutes, atroom temperature, and permeabilized with cold methanol for 10minutes and then 0.2% Triton X-100 in PBS for 10 minutes. Cellswere washed three timeswith PBS. Cells were then incubatedwith100 mg/mLDNase-free RNase and 10 mg/mL of propidium iodide

Table 1. Summary of the sensitive and insensitive tumors to LY2523355

Number of sensitivetumors (%)

Number ofinsensitive

Type of tumor Regresseda Suppressedb tumorsc (%)

In xenograft tumor models with established cancer cell linesd

Colorectal 2/2 0/2 0/2Non–small cell lung 3/5 2/5 0/5Ovarian 1/3 1/3 1/3Breast 1/2 0/2 1/2Multiple myeloma 2/2 0/2 0/2Overall 9/14 (64) 3/14 (21) 2/14 (14)

In PDX tumor modelsNon–small cell lung 7/12 4/12 1/12Small cell lung 3/4 1/4 0/4Colorectal 1/4 2/4 1/4Gastric 1/3 2/3 0/3Melanoma 1/5 2/5 2/5Pancreatic 0/1 1/1 0/1Breast 1/1 0/1 0/1Brain 0/1 0/1 1/1Pleuramesothelioma 0/1 1/1 0/1Testis 1/1 0/1 0/1Bladder 1/1 0/1 0/1Ovarian 0/1 0/1 1/1Sarcoma 0/1 0/1 1/1Liver 0/1 1/1 0/1Overall 16/37 (43) 14/37 (38) 7/37 (20)

aV/V0 min < 1.bT/C min < 0.5.cT/C min ¼ 0.5–1.dThe cancer cell lines used are: Colo-205, HCT-116, EBC-1, Calu-6, NCI-H460, PC-14, PC-14/CDDP, A2780, SK-OV-3, RMG-1, KPL-4, MX-1, KMS-11, NCI-H929.

Ye et al.

Mol Cancer Ther; 14(11) November 2015 Molecular Cancer Therapeutics2464




for 1 hour and scanned with Acumen for mitotic index (MI)measurement based on DNA condensation as percentage ofcells with condensed DNA. For MI based on histone H3phosphorylation or apoptosis analysis, cells were incubatedovernight at 4�C with anti-phospho-histone H3 antibody oranti-phospho-histone H2AX at 1:1,000 dilution with 5%bovine serum albumin (BSA) in PBS, respectively. Cells werewashed three times with 0.2% Triton-X 100 in PBS and incu-bated with Alexa 488 secondary antibody (1:1,000 in PBS–2%BSA, Invitrogen) for 60 minutes at room temperature. Cellswere washed three times again with PBS and stained for 15minutes in PBS containing 10 mg/mL propidium iodide (Molec-ular Probes, Invitrogen) and 100 mg/mL RNaseA (MolecularProbes, Invitrogen). The stained cells were scanned with anAcumen Explorer eX3 microplate cytometer (TTP LabTech). Theresults were expressed as percentage of cells positive for phos-pho-histone H3-Ser10 or phospho-histone H2AX.

Caspase-3/7 activity after 48-hour treatment was measuredusing Apo-ONE Homogeneous Caspase-3/7 Assay (cat. no.G7791, Promega) and cell viability after 72-hour treatmentwas measured using CellTiter-Glo Luminescent Cell ViabilityAssay (cat. no. G7573, Promega), following the manufacturer'sinstruction.

Animal models and in vivo pharmacologyPrimary human-tumor xenograft models were established and

maintained in nudemice as previously described (34). Antitumorefficacy in subcutaneous xenograft tumor-bearing mice with 10mice per treatment group from either established cancer cell linesor fragments of human tumor explants was evaluated as tumorvolume by serial caliper measurements and was calculated asdescribed previously (32, 34). The p388 syngeneic tumor modelwas developed for high-content imaging analysis using femaleBDF1 mice, weighing 20 to 23 g, which were purchased fromCharles River and acclimated in-house for one week before theiruse in experiments. p388 murine lymphocytic leukemia cells,obtained from the NCI in 2006, were authenticated by STR assayas described above and maintained in RPMI1640 medium con-taining 10% FBS. For inoculation, the cells were washed withserum-free medium three times and 1.25 million cells wereimplanted by intraperitoneal injection into mice. On day 5 afterimplantation, mice were treated with LY2523355, via eitherintravenous bolus or intravenous infusion at appropriate dosesand durations. Mice were euthanized and the ascitic (intraperi-toneal) fluid containing the p388 tumor cells was drawn andanalyzed by acumen, flow cytometry, and TUNEL assays forphospho-histoneH3, G2–M, and apoptosis. For pharmacokineticstudy, we collected the blood samples via cardiac puncture andgenerated plasma with EDTA and determined LY2523355 expo-sure in the plasma.

All in vivo studies were reviewed by Ethical Committees of EliLilly, Kyowa Hakko, and Oncotest, respectively, and were per-formed in accordance with the guidelines stated in the Interna-tional Guiding Principles for Biomedical Research InvolvingAnimals developed by the Council for InternationalOrganizationof Medical Sciences.

ImmunohistochemistryXenograft tumors and mouse skin (ears) were fixed in 10%

neutral buffered formalin for IHC studies. The sections weretrimmed, routinely processed, and embedded in paraffin. Three

micrometer sections were immunohistochemically labeled forhistone H3-serine 10 phosphorylation using a rabbit polyclonalprimary antibody (Upstate, Millipore) without antigen retrievaland with 3,30-diaminobenzidine as chromagen. Sections werecounterstained with hematoxylin.

ResultsThreshold concentration effect on cell-cycle–dependentmitoticarrest by LY2523355

Biochemical studies established that LY2523355 is a potentand selective inhibitor of the human Eg5 (Fig. 1A and Supple-mentary Fig. S1). Cell-cycle analysis showed that LY2523355caused a dose-dependent increase of cells with 4N DNA content(Supplementary Fig. S2A). Immunofluorescence microscopy fur-ther revealed that LY2523355 arrested proliferating normal andcancerous human cells inmitosiswithmonopolar spindles in vitro(Supplementary Fig. S2B and S2C). Interestingly, inactivation ofEg5 by LY2523355 also inhibited Eg5 localization to the mitoticspindle, suggesting spindle localization of Eg5 is an active processand requires its functional activity (Supplementary Fig. S2C).

LY2523355-treated HCT116 cells also showed a dose-depen-dent increase in MI based on phospho-histone H3 or DNAcondensation (Fig. 1B). Furthermore, there was a time-dependentincrease in MI in LY2523355-treated HCT116 cells reaching amaximum at 12 hours (Fig. 1C). Importantly, LY2523355 at 10nmol/L was sufficient to cause maximal mitotic arrest at all thetime points and concentrations higher than 10 nmol/L had noadditional effects either on the timing or themagnitude ofmitoticarrest. Mitotic block by LY2523355 was closely correlated with itsantiproliferation activity in cell culture as demonstrated by com-plete inhibition of HeLa cell growth at approximately 10 nmol/L(Supplementary Fig. S2D). Indeed, LY2523355 showed potent,broad-spectrum anticancer activity in vitro at low concentrations(IC50 values ranging from 0.4 nmol/L to14 nmol/L) when testedagainst a panel of 21 cancer cell lines,whichwas further confirmedin proliferation assays with the NCI-60 panel (SupplementaryTables S1 and S2).

LY2523355 kills cancer cells as a result of mitotic arrestLY2523355 at 10 nmol/L or above causes maximal mitotic

arrest with monopolar spindle (Fig. 1B and Supplementary Fig.S2B and S2C). A substantial sub-G1 fraction of cells was observed48 hours after LY2523355 treatment (Supplementary Fig. S2A),indicating that cell death occurred. To establish the causal rela-tionship between mitotic arrest and cell death, real-time mea-surement of HCT116 cell growth after LY2523355 treatment andsubsequent withdrawal was performed. LY2523355 caused max-imal mitotic arrest and cell death 12 to 24 hours after additionand cells treated for less than 12hours were able to resume growthafter withdrawal of LY2523355 (Fig. 1D). The data thus stronglysuggest that cell killing by LY2523355 is linked to cell-cycle arrestatmitosis. Furthermore, it was observed that LY2523355-inducedmitotic arrest and apoptosis were closely associated with eachother in a time- and dose-dependent manner (Fig. 1C andSupplementary Fig. S2E).

A role for SAC activation in cancer cell killing by LY2523355DNA damage and cell death in LY2523355-treated HeLa cells,

as indicated by histoneH2AX phosphorylation and cleaved PARP(Fig. 2A), occurred in mitotic cells but not in interphase cells

Effect of the Eg5 Inhibitor LY2523355 in Cancer

www.aacrjournals.org Mol Cancer Ther; 14(11) November 2015 2465




(adherent). This confirmed that cell killing by LY2523355actually occurred during mitotic arrest. Mitotic arrest was indi-cated by phosphorylation of histone H3 and the spindlecheckpoint kinase, budding uninhibited by benomyl Related-1 (BubR1). Immunofluorescence microscopy of mitotic cellsfurther revealed that mitotic cells had condensed DNA, phos-phorylated histone H3, and also phosphorylated histone H2AX(Supplementary Fig. S3A).

A time course study of multiple mitotic and apoptotic cellularbiomarkers showed that mitotic arrest clearly preceded apoptosis(Fig. 2B). LY2523355 treatment caused a dramatic increase inphosphorylation of histone H3, B-cell lymphoma 2 (Bcl2), andBubR1, and accumulation of cyclin B from4 to 16 hours (Fig. 2B).After peaking at 16 hours, phosphorylation of histone H3, Bcl2,and BubR1, and protein level of cyclin B began to decreasesubstantially and no cyclin B was detectable 48 hours after

LY2523355 treatment. Interestingly, the level of BubR1 proteinalso markedly decreased 24 hours after LY2523355 treatment. Incontrast, apoptosis, indicated byPARP1 cleavage and adecrease inthe level of antiapoptotic proteins Bcl2 and Mcl1, occurred 16hours after LY2523355 treatment and continued up to 48 hours(Fig. 2B). Mcl1 appeared to be most sensitive to LY2523355treatment; its protein level decreased appreciably at 8 hours andcompletely disappeared 24 hours after treatment (Fig. 2B). Themarked decrease in multiple mitotic markers may indicate amitotic slippage after a sustained LY2523355-mediated mitoticarrest.

CDK1 kinase activity is required to maintain activation of SACduring mitosis (35). Therefore, to investigate a potential role ofSAC in apoptotic cell death caused by LY2523355, we used aselective CDK1 inhibitor, RO3306, to abrogate SAC activationand force cells to exit mitosis rapidly. Upon addition of RO3306

C

BA

Concentration (nmol/L)

Mito

tic in

dex

(%)

100

80

60

40

20

00.1 1 10 10,0001,000100 100,000

0

20

40

60

80

100

109 121 13397857361483624120

% C

onflu

ence

Time (h)

Control3 h6 h12 h24 h48 h

0

20

40

60

80

100

48241263DMSO

% C

ell c

ycle

Time (h)

G1%

S%

G2–M%

D

Concentration (log µmol/L)

−4 −3 −2 −1 0 10

1020304050607080

3 h1 h

6 h12 h18 h24 hM

itotic

inde

x (%

)

Figure 1.LY2523355 kills cancer cells during mitotic arrest. A, the chemical structure of LY2523355. B, dose-dependent mitotic arrest of HCT116 cells by LY2523355. MI wasdetermined by fluorescent microscopy based on DNA condensation 18 hours after LY2523355 addition. C, mitotic block by LY2523355 is time dependentwith a threshold concentration for maximal activity. MI was determined using Acumen high content technology. D, cell-cycle arrest at G2–M and cell killing byLY2523355 were closely correlated from 12 to 24 hours after LY2523355 addition. HCT116 cells were treated with 25 nmol/L LY2523355 for various time intervalsand then LY2523355 was removed from the growth medium. Cell-cycle profiles of the treated HCT116 cells were analyzed (top graph) and the ability of thetreated cells to grow after LY2523355 removal was measured in real time using Incucyte (bottom graph).

Ye et al.





tomitoticHeLa cells 12hours after pretreatmentwith LY2523355,cells resumed flat interphase morphology, reattached to the platequickly, and exited from mitosis. Consistent with rapid mitoticexit, dephosphorylation of histone H3 occurred rapidly uponaddition of RO3306 (Fig. 2C). Addition of RO3306 stronglyinhibited increase in both PARP1 cleavage and phospho-histoneH2AX normally associated with LY2523355 treatment (Fig. 2C).Interestingly, while rapid dephosphorylation of BubR1 (mobilityshift) indicates SAC abrogation by RO3306 as expected, BubR1degradation was also prevented. Taken together, the data supportan important role of activated SAC in cancer cell killing by Eg5inhibitors.

To independently confirm the role of SAC in cell killing byLY2523355, we silenced SAC protein Mad2 by siRNA. Ascompared with the parental cells, cells with silenced Mad2failed to be arrested at mitosis and consequently had noactivated caspase activity, indicating that SAC has an essentialrole in mitotic arrest and subsequent cell killing by LY2523355(Supplementary Fig. S3B–S3D).

Schedule-dependent antitumor activity in vivo by LY2523355As shown in Fig. 3A, the in vivo antitumor activity of LY2523355

was highly schedule-dependent, although all treatment groupsreceived the same total amount of the compound. Strong anti-tumor efficacy was observed when LY2523355 was administeredwith a time interval of

LY2523355 caused a dramatic increase in cancer cells immuno-positive for histone H3 phosphorylation and that had a cellularphenotype consistent withmonopolar spindles, indicatingmitot-ic arrest of cancer cells (Fig. 4B). Quantitative analysis of histoneH3phosphorylation showed a correlationwith antitumor activityin a dose-dependent manner (Fig. 4C). It is worth noting that atefficacious doses, the increase in histoneH3 phosphorylationwasfollowed by cell death on Day 2 after dosing (Fig. 4B). Further-more, increased cell death was observed after each successivedosing and by Day 8, very few abnormal tumor cells remained,providing a cellular mechanism for marked tumor shrinkage byLY2523355.

To further correlate cancer-cell mitotic arrest with antitumoractivity in vivo, we examined the effect of LY2523355 in tworesistant and two sensitive PDX tumor models (SupplementaryFig. S4A). Immunohistochemistry showed that each tumor had adistinct tissue structure and a low level of phospho-histone H3-positive cells (Supplementary Fig. S4B,a). The number of cellspositive for phospho-histone H3 and cell death increased dra-matically in the two sensitive tumor models, whereas in the tworesistant tumor models there was only a slight increase, if any(Supplementary Fig. S4B, b and c, and 4C). Moreover, IHCanalysis of skin tissues observed a marked increase in phos-pho-histone H3-positive proliferating skin cells in all treatedanimals (Supplementary Fig. S4D). Taken together, the datademonstrated that the antitumor activity of LY2523355 is linkedto Eg5 inhibition in cancer cells andmitotic arrest of proliferating

skin cells may serve as a promising surrogate pharmacodynamicbiomarker for dose and schedule assessments.

Therapeutic threshold concentration and efficacy byLY2523355

To understand the relationship between therapeutic thresh-old concentration, duration for which the threshold must bemaintained for cell-cycle–dependent mitotic arrest, and sub-sequent cell killing, we used the p388 ascites tumor modelwhich, being a liquid tumor, is amenable to high-throughputquantitative cell-cycle analysis as compared with immunohis-tochemistry in solid tumors. Cell-cycle analysis showed a clearthreshold dose between 5 to 10 mg/kg for maximal G2–Marrest of cancer cells (Fig. 5A). Indeed, the 5-mg/kg dosemarkedly extended survival of mice bearing p388 tumors andhigher doses did not proportionately increase survival benefit(Fig. 5B).

To further demonstrate the relationship between thresholdexposure and cell-cycle arrest, we performed a series of infusionstudies with a p388 tumor model. Following a 72-hour infusion,wedetermined steady-state exposure in theplasma andquantifiedphospho-histone H3-positive cancer cells in the ascites fluid byhigh-content imaging and also in skin biopsies by immunohis-tochemistry. As shown in Table 2, Fig. 5C and Supplementary Fig.S5A and S5B, LY2523355 at the threshold exposure level of about10 ng/mL, as established in vitro, achieved maximal mitotic blockin both cancer cells and proliferating skin cells. TUNEL staining

A

B

Control

q4d×3 9.6 mg/kg

q1d×3 9.6 mg/kg

q2d×3 9.6 mg/kg

q7d×3 9.6 mg/kg

Single 9.6 mg/kg

Tum

or v

olum

e (V

/V0)

Tum

or v

olum

e (V

/V0)

Days after dosing Days after dosing

Bod

y w

eigh

t cha

nge

(g)

0.01

0.1

1

10

100

35302520151050

Colo-205

−8

−6

−4

−2

0

2

4

35302520151050

1/5 dead

Days after dosing35302520151050

Tum

or v

olum

e (V

/V0)

0.1

1

10

100A2780

Control

LY2523355 (30 mg/kg, q4d x3)Paclitaxel(30 mg/kg, q4d x3)Docetaxel(15 mg/kg, q4d x3)CPT-11(67 mg/kg, q4d x3) Vinorelbine(16 mg/kg, qdx1)Cisplatin(11 mg/kg, qdx 1)

ECB-1

25201510500.01

0.1

1

10

100

Days after dosing

Figure 3.Anticancer efficacy of LY2523355in xenograft tumor models.A, demonstration of schedule-dependent efficacy by LY2523355 inColo205 tumor models. When tumorsreached an average size of 150 mm3,LY2523355 was administered at 9.6mg/kg to the tumor-bearingmice with5 mice per treatment group by 1-hourintravenous infusion with variousdosing schedules for a total of 3 dosingcycles ranging from daily dosing (q1d)to once every 7 days (q7d). Tumor sizeand mouse body weight weremeasured every few days for morethan a month after the start of dosing.LY2523355, when dosed once every 4days, caused marked tumor shrinkagewith minimal toxicity seen as body-weight loss. B, comparison ofanticancer efficacy by LY2523355 withother chemotherapeutic agents inpreclinical xenograft tumor models.Mice bearing non–small cell lung (ECB-1; 160 mm3) or ovarian (A2780)xenograft tumors of an average size of220 mm3 were treated with theanticancer agents at their maximumtolerated doses and schedulesindicated. Tumor size of ECB1 andA2780 xenografts was measuredevery few days for up to 24 and 28days, respectively, after the start ofdosing.

Ye et al.





showed that many treated p388 tumor cells were also apoptotic(Supplementary Fig. S5A).

Taken together, both in vitro and in vivo data clearly indicate thatefficacy of LY2523355 is highly dosing schedule-dependent andthat histone H3 phosphorylation of proliferating skin cells is apromising surrogate pharmacodynamic biomarker for dose andschedule evaluation of LY2523355 to study its anticancer activity.

DiscussionThis article describes in vitro and in vivo studies to charac-

terize the anticancer activity of a novel selective Eg5 inhibitorLY2523355. LY2523355 showed broad-spectrum antiprolifera-tive activity against cancer cell lines. In preclinical xenografttumor models, LY2523355 also showed a broad-spectrumanticancer activity with complete remission in some. We fur-ther demonstrated that in vivo efficacy of LY2523355 is highlydependent on threshold therapeutic concentration and dosingschedule. We also showed that increase in histone-H3 phos-phorylation of proliferating skin cells is quantitatively corre-lated with anticancer activity of LY2523355, thus offering apromising surrogate pharmacodynamic biomarker for the eval-uation of LY2523355 anticancer activity.

Our extensive cellular studies showed that the anticancer activ-ity by LY2523355 is mediated through cell cycle–dependentmitotic arrest and requires sustained activation of SAC.LY2523355 activates SAC as a result of monopolar spindle for-

mation, leading to mitotic arrest. Spindle defects such as mono-polar spindles activates SAC to delay exit from mitosis by pre-venting the proteolysis of cyclin B, which is mediated by ana-phase-promoting complex (APC), until spindle defects are cor-rected (35). However, Cancer cells often escape from SAC-mediated mitotic arrest via a poorly defined process known asmitotic slippage.Mitotic slippage has been shown to be caused bygradual degradationof cyclin B in the presence of a functional SAC(36, 37). The time course study with HeLa cells showed that cellsthat were arrested at mitosis began to escape from the mitoticarrest 16 hours after LY2523355 treatment, as indicated by thedecrease of multiple mitotic biochemical markers. Interestingly,we observed that, in addition to cyclin B degradation, mitoticslippage is also associated with BubR1 degradation. Perhapsdegradation of BubR1, an essential regulatory component of SAC,is part of themechanism responsible formitotic slippage. Analysisofmultiplemitotic and apoptotic markers show that induction ofapoptosis by LY2523355 clearly followed mitotic slippage. Ourdata are consistent with the previous report that both activation ofSAC and mitotic slippage are required for rapid cancer cell killing(25). This is further supported by the compound wash-outexperiment that showed that killing of cancer cells by LY2523355requires 12 hours or more and coincides with sustained mitoticarrest. Furthermore, phosphorylation of Bcl2 and rapid degrada-tion of Mcl1 observed in cells mitotically arrested by LY2523355may also contribute to induction of apoptosis. Indeed, recentstudies that shows CDK1-mediated phosphorylation of Bcl-xL/

0 1 2 3 4 5 6 7 8 9 10 11 120

200

400

600

800

1,000 Saline1.1 mg/kg3.3 mg/kg10 mg/kg30 mg/kg

Day 1, 30 mg/kgSaline

Day 2, 30 mg/kg

B

Day 8, 30 mg/kgTu

mor

vol

ume

(mm

3 )

Days after dosing

A

0 2 4 6 8 10 120

10

20

30

40

50

60C

Days after dosing

Saline1.1 mg/kg3.3 mg/kg10 mg/kg30 mg/kg

Pho

spho

-his

tone

H3+

ce

lls (%

)

Figure 4.Anticancer activity of LY2523355 isassociated with mitotic arrest andsubsequent cell death in Colo205xenograft tumors. LY2523355 wasadministered by intravenous bolus atthe doses indicated for 3 cycles (redarrow) of every 4 days and anticanceractivity was assessed by measuringtumor volume (A), and phosphorylationof histone H3 was analyzed byimmunostaining (B). Insert is anenlargement of a phospho-histone H3-positive cell to show a phenotypeconsistent with monopolar spindle andarrows point to dead cells on day 2 afterthe first dose. C, tumor tissues from 2mice were harvested, fixed, andprocessed for phospho-histone H3immunostaining and positive cells werequantified at various times after eachdosing.






Bcl2 to be a functional link between mitotic arrest and apoptosis(38), and that suppression of Mcl1 sensitizes cancer cells to thetreatment of anticancer agents, including an Eg5 inhibitor (ARRY-520), in human multiple myeloma (39–41).

Consistent with Eg5 biology andmode of action of LY2523355as discussed above, we demonstrated that anticancer activity ofLY2523355 is highly schedule-dependent and has an optimalthreshold exposure level and duration to achieve significantanticancer activity. Furthermore, the in vivo anticancer activity insensitive xenograft tumor models but not resistant tumor models

is associated with first mitotic arrest and then apoptosis in cancercells very similarly as observed in vitro, indicating that LY2523355has the same mechanism of anticancer activity in vivo as estab-lished in vitro in cell-based studies.

The salient finding in this study on characterization ofLY2523355 anticancer activity in vitro and in vivo is that durationof threshold exposure is extremely important to anticancer activityby selective Eg5 inhibitors. This finding has significant implica-tions for a successful effort in the discovery and developmentefforts not only on selective inhibitors of Eg5, but also on selective

0

20

40

60

80

100

30 mpk10 mpk5 mpk3 mpkVehicle

% o

f Cel

ls a

t G2–

MA

B

C

Con

trol

LY25

2335

5

Vehicle5 mg/kg15 mg/kg

30 mg/kg

Figure 5.Efficacy and therapeutic thresholdconcentration of LY2523355 in p388 ascitestumor model. A, cell-cycle analysis by flowcytometry of p388 tumor cells from ascitesfluid 18 hours after dosing by intravenousbolus. B, survival extension by LY2523355.Note that LY2523355 achieved maximalcell-cycle arrest and significant survivalextension benefit at 5 mg/kg. C, changes inphospho-histone H3-positive epidermalcells in the ear skin of vehicle- andLY2523355-treated mice. The top panelshows no phospho-histone H3-positivekeratinocytes in a 1 mm length of epidermisfrom the ear of a mouse that receivedvehicle. In the bottom panel, multiplephospho-histone H3-positive mitotic cellsare present in the same length of epidermisin a mouse that received LY2523355 (bar,100 mm).

Ye et al.





inhibitors of Plk1 and aurora kinases as novel new antimitoticcancer drugs. Our data on LY2523355 convincingly showed thatcompounds targeting these mitotic regulators must have goodpharmacokinetic properties that give rise to sustained duration ofthreshold exposure through careful dose/dosing schedule selec-tion to achieve robust anticancer efficacy. Thus, dosing schedulesneed to be optimized for each compound in accordancewith theirpotency and pharmacokinetic properties to differentially killsensitive cancer cells while sparing or minimizing toxicity to,proliferating normal cells. This also provides a good biologicexplanation why clinical studies on early generation Eg5 inhibi-tors, such as SB-715992, that followed the development paradigmof antitubulin drugs showed little clinical activity (6).

Microtubules as target for antitubulins are always presentthroughout the cell cycle of dividing cancer cells. In contrast, Eg5is only expressed briefly duringmitosis. Furthermore, cancer cell–cycle progression is asynchronous and mitosis is completed veryrapidly. Consequently, mitotic cells in tumor tissues are alwaysonly a small fraction of the total cancer cell population at anygiven time. Therefore, Eg5 inhibitorsmust be available at or abovethe threshold concentration relative to their potency for anadequate duration to achieve sustained mitotic arrest of mostdividing cancer cells as they asynchronously progress intomitosis.The length of this required duration of threshold exposure wouldalso depend on the cell-cycle time of proliferating cancer cells inindividual tumors. The importance of sustained duration ofthreshold exposure of drugs that target cell-cycle regulators nowhas been borne out by promising clinical results of a second-generation Plk1 inhibitor volasertib (BI6727),which showed veryencouraging anticancer activity (42). The reason for clinical anti-cancer activity of volasertib, in contrast to the first-generation Plk1inhibitor BI2536, which has similar potency on Plk1, is likely dueto the fact that volasertib has a large volume of distribution and along half-life in humans of up to 110 hours. Such sustainedexposure conceivably is able to causemitotic arrest of proliferatingcancer cells as they progress into mitosis and also sustained SACactivation for induction of apoptosis (42, 43).

Eg5mitotic kinesin, although overexpressed in various cancers,is not a selective cancer target, per se, and plays an essential role incell proliferation of both cancer and normal cells. It is thusanticipated that the primary dose-limiting toxicity of an Eg5inhibitor is likely to be myelosupression and gastrointestinaltoxicity. As discussed above, unlike traditional antimitotic che-motherapies, treatment at a MTD may not be the best option forapplication of targeted Eg5 inhibitors. Rather,what is important isthe determination of a dose and dosing schedule that achieves thedesired duration of exposure to cause sustainedmitotic arrest andsubsequent mitotic slippage of cancer cells while keeping target-mediated toxicity manageable. Histone H3 phosphorylation(pHH3), which closely correlates with its antitumor activity, isa promising pharmacodynamic biomarker that has the potentialto guide dose and schedule selection for optimal cancer treatmentof Eg5 inhibitors with improved safety profiles and new antican-cer mechanisms as compared with antitubulins.

Disclosure of Potential Conflicts of InterestT. Yin is a scientist Eli Lilly and Company. G.P. Donoho has ownership

interest (including patents) in Eli Lilly and Co. A. Aggarwal has ownershipinterest (including patents) in Eli Lilly. E.H. Westin has ownership interest(including patents) in Eli Lilly. No potential conflicts of interest were disclosedby the other authors.

Authors' ContributionsConception and design: X.S. Ye, R. Nakai, Y. Ohta, Y. Yamashita, G.P. Donoho,K. Blanchard, E.H. WestinDevelopment of methodology: L. Fan, R. Nakai, C. Murakata, Y. Yamashita,T. Yin, H. Li, A. AggarwalAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Fan, R.D.V. Horn, R. Nakai, T. Yin, K.M. Credille,F.F. Merzoug, H. Li, A. AggarwalAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Nakai, K.M. Credille, F.F. Merzoug, A. Aggarwal,K. Blanchard, E.H. WestinWriting, review, and/or revision of the manuscript: X.S. Ye, L. Fan, R. Nakai,T. Yin, K.M. Credille, G.P. Donoho, F.F. Merzoug, E.H. WestinAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R. Nakai, Y. Ohta, C. MurakataStudy supervision: X.S. Ye, R. Nakai, S. Akinaga, C. Murakata, Y. Yamashita,G.P. Donoho, F.F. Merzoug, E.H. Westin

AcknowledgmentsThe authors thank Dr. Richard Gaynor for his interest and support, Dr.

Christoph Reinhard for having LY2523355 tested against the gastric cancer cellline panel, Mrs. Michele Dowless for performing Cellomic assays for p388tumor cells using the ascites tumor model, the scientists at Oncotest, Germany,for testing LY2523355 in PDX models, and Dr. Chunping Yu for carefullyreading the manuscript.

Grant SupportThis work was supported by KyowaHakko Karin and Eli Lilly and Company.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedMarch 27, 2015; revised August 13, 2015; accepted August 17, 2015;published OnlineFirst August 24, 2015.

References1. Sawin KE, LeGuellec K, Philippe M, Mitchison TJ. Mitotic spindle organi-

zation by aplus-end-directedmicrotubulemotor.Nature 1992;359:540–3.2. Mishima M, Pavicic V, Gr€uneberg U, Nigg EA, Glotzer M. Cell cycle

regulation of central spindle assembly. Nature 2004;430:908–13.

3. TanenbaumME, Medema RH. Mechanisms of centrosome separation andbipolar mitotic spindle assembly. Dev Cell 2010;19:797–806.

4. Dumontet C, Jordan MA. Microtubule-binding agents: a dynamic field ofcancer therapeutics. Nat Rev Drug Discov 2010;9:790–803.

Table 2. Cell-cycle arrest and steady-state exposure in p388 tumor model afterLY2523355 infusion

Total dose(mg/kg)

Infusion rate(mg/kg�h) % pHH3

Cells positivefor pHH3 per unitlength of epidermis

Plasma(ng/mL)

Vehicle Vehicle 5.07 0.1830 0.416 28.37 7.87 94.6815 0.208 28.73 5.18 56.1610 0.139 20.38 7.75 39.677.5 0.104 20.14 7.86 15.225a 0.069 17.24 11.16 19.603.74a 0.052 22.28 6.09 7.791.22a 0.017 15.81 3.86 4.370.65 0.009 17.70 0.34 1.520.29 0.004 5.70 0.50 1.770.14 0.002 3.98 0.19 1.13aDose within the threshold efficacious dose range.






5. Huszar D, Theoclitou ME, Skolnik J, Herbst R. Kinesin motor proteins astargets for cancer therapy. Cancer Metastasis Rev 2009;28:197–208.

6. Jackson JR, Patrick DR,DarMM,Huang PS. Targeted anti-mitotic therapies:can we improve on tubulin agents? Nat Rev Cancer 2007;7:107–17.

7. Lobert S. Neurotoxicity in cancer chemotherapy: vinca alkaloids. Crit CareNurse 1997;17:71–9.

8. Orr GA, Verdier-Pinard P, McDaid H, Horwitz SB. Mechanisms of Taxolresistance related to microtubules. Oncogene 2003;22:7280–95.

9. Kashina AS, Rogers GC, Scholey JM. The bimC family of kinesins: essentialbipolar mitotic motors driving centrosome separation. Biochim BiophysActa 1997;1357:257–71.

10. Kim AJ, Endow SA. A kinesin family tree. J Cell Sci 2000;113:3681–2.11. Kapitein LC, Peterman EJ, Kwok BH, Kim JH, Kapoor TM, Schmidt CF. The

bipolar mitotic kinesin Eg5moves on both microtubules that it crosslinks.Nature 2005;435:114–8.

12. Valentine MT, Fordyce PM, Krzysiak TC, Gilbert SP, Block SM. Individualdimmers of the mitotic kinesin motor Eg5 step processively and supportsubstantial loads in vitro. Nat Cell Biol 2006;8:470–6.

13. Kashina AS, Baskin RJ, Cole DG, Wedaman KP, Saxton WM, Scholey JM. Abipolar kinesin. Nature 1996;379:270–2.

14. Sawin KE, Mitchison TJ. Mutations in the kinesin-like protein Eg5 disrupt-ing localization to the mitotic spindle. Proc Natl Acad Sci U S A 1995;92:4289–93.

15. Blangy A, Lane HA, d'H�erin P, Harper M, Kress M, Nigg EA. Phosphory-lation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Cell1995;83:1159–69.

16. Dagenbach EM, Endow SA. A new kinesin tree. J Cell Sci 2004;117:3–7.17. Cahu J, Olichon A, Hentrich C, Schek H, Drinjakovic J, Zhang C, et al.

Phosphorylation by Cdk1 increases the binding of Eg5 to microtubules invitro and in Xenopus egg extract spindles. PLoS One 2008;3:e3936.

18. Liu M,Wang X, Yang Y, Li D, Ren H, Zhu Q, et al. Ectopic expression of themicrotubule-dependent motor protein Eg5 promotes pancreatic tumour-igenesis. J Pathol 2010;221:221–8.

19. Castillo A,MorseHC3rd,Godfrey VL,NaeemR, JusticeMJ.Overexpressionof Eg5 causes genomic instability and tumor formation inmice. Cancer Res2007;67:10138–47.

20. Uzbekov R, Prigent C, Arlot-Bonnemains Y. Cell cycle analysis and syn-chronizationof theXenopus laevis XL2 cell line: studyof the kinesin relatedprotein XlEg5. Microsc Res Tech 1999;45:31–42.

21. Sakowicz R, Finer JT, Beraud C, Crompton A, Lewis E, Fritsch A, et al.Antitumor activity of a kinesin inhibitor. Cancer Res 2004;64:3276–80.

22. Mayer TU, Kapoor TM, Haggarty SJ, King RW, Schreiber SL, Mitchison TJ.Small molecule inhibitor of mitotic spindle bipolarity identified in aphenotype-based screen. Science 1999;286:971–4.

23. LiuM, YuH,HuoL, Liu J, LiM, Zhou J. Validating themitotic kinesin Eg5 asa therapeutic target in pancreatic cancer cells and tumor xenografts using aspecific inhibitor. Biochem Pharmacol 2008;76:169–78.

24. Yan Y, Sardana V, Xu B, Homnick C, Halczenko W, Buser CA, et al.Inhibition of a mitotic motor protein: where, how, and conformationalconsequences. J Mol Biol 2004;335:547–54.

25. TaoW, South VJ, Zhang Y, Davide JP, Farrell L, Kohl NE, et al. Induction ofapoptosis by an inhibitor of the mitotic kinesin KSP requires both acti-vation of the spindle assembly checkpoint and mitotic slippage. CancerCell 2005;8:49–59.

26. Marcus AI, Peters U, Thomas SL, Garrett S, Zelnak A, Kapoor TM, et al.Mitotic kinesin inhibitors induce mitotic arrest and cell death in Taxol-resistant and -sensitive cancer cells. J Biol Chem 2005;280:11569–77.

27. Kapoor TM, Mayer TU, Coughlin ML, Mitchison TJ. Probing spindleassembly mechanisms with monastrol, a small molecule inhibitor of themitotic kinesin, Eg5. J Cell Biol 2000;150:975–88.

28. Riffell JL, Zimmerman C, Khong A, McHardy LM, Roberge M. Effects ofchemical manipulation of mitotic arrest and slippage on cancer cellsurvival and proliferation. Cell Cycle 2009;8:3025–38.

29. Bekier ME, Fischbach R, Lee J, Taylor WR. Length of mitotic arrest inducedby microtubule-stabilizing drugs determines cell death after mitotic exit.Mol Cancer Ther 2009;8:1646–54.

30. Dalton WB, Yang VW. Role of prolonged mitotic checkpoint activation inthe formation and treatment of cancer. Future Oncol 2009;5:1363–70.

31. Shi J, Orth JD, Mitchison T. Cell type variation in responses to antimitoticdrugs that target microtubules and kinesin-5. Cancer Res 2008;68:3269–76.

32. Nakai R, Iida S, Takahashi T, Tsujita T, Okamoto S, Takada C, et al. K858, anovel inhibitor of mitotic kinesin Eg5 and antitumor agent, induces celldeath in cancer cells. Cancer Res 2009;69:3901–9.

33. Van Horn RD, Chu S, Fan L, Yin T, Du J, Beckmann R, et al. Cdk1 activity isrequired for mitotic activation of aurora A during G2/M transition ofhuman cells. J Biol Chem 2010;285:21849–57.

34. Fiebig HH, Maier A, Burger AM. Clonogenic assay with established humantumor xenografts: correlation of in vitro to in vivo activity as a basis foranticancer drug discovery. Eur J Cancer 2004;40:802–20.

35. Yu H. Regulation of APC-Cdc20 by the spindle checkpoint. Curr Opin CellBiol 2002;14:706–14.

36. Brito DA, Rieder CL. Mitotic checkpoint slippage in humans occurs viacyclin B destruction in the presence of an active checkpoint. Curr Biol2006;16:1194–200.

37. BritoDA, Yang Z, Rieder CL.Microtubules donot promotemitotic slippagewhen the spindle assembly checkpoint cannot be satisfied. J Cell Biol2008;182:623–9.

38. TerranoDT, Upreti M, Chambers TC. Cyclin-dependent kinase 1-mediatedBcl-xL/Bcl-2 phosphorylation acts as a functional link coupling mitoticarrest and apoptosis. Mol Cell Biol 2010;30:640–56.

39. Lestini BJ, Goldsmith KC, Fluchel MN, Liu X, Chen NL, Goyal B, et al. Mcl1downregulation sensitizes neuroblastoma to cytotoxic chemotherapy andsmall molecule Bcl2-family antagonists. Cancer Biol Ther 2009;8:1587–95.

40. Tunquist BJ, Woessner RD, Walker DH. Mcl-1 stability determines mitoticcell fate of human multiple myeloma tumor cells treated with the kinesinspindle protein inhibitor ARRY-520. Mol Cancer Ther 2010;9:2046–56

41. Wertz IE, Kusam S, Lam C, Okamoto T, Sandoval W, Anderson DJ, et al.Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 andFBW7. Nature 2011;471:110–4.

42. Gil T, Schoffski P, AwadaA, Bartholomus S, Selleslach J, TatonM, et al. Finalanalysis of phase I single dose-escalation study of the novel polo-likekinase inhibitor BI 6727 in patients with advanced solid tumors. J ClinOncol 28:15s. 2010 [suppl; abstr 3061]

43. Rudolph D, Steegmaier M, Hoffman M, Grauert M, Baum A, Quant C,et al. BI 6727, a Polo-like kinase inhibitor with improved pharmaco-lokinetic profile and broad anticancer activity. Clin Cancer Res 2009;15:3094–102.


Ye et al.




2015;14:2463-2472. Published OnlineFirst August 24, 2015.Mol Cancer Ther Xiang S. Ye, Li Fan, Robert D. Van Horn, et al. Xenograft Tumor ModelsApoptosis in Cancer Cells and Shows Potent Antitumor Activity in A Novel Eg5 Inhibitor (LY2523355) Causes Mitotic Arrest and

Updated version

10.1158/1535-7163.MCT-15-0241doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2015/08/22/1535-7163.MCT-15-0241.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/14/11/2463.full#ref-list-1

This article cites 43 articles, 16 of which you can access for free at:

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

Subscriptions

Reprints and

[email protected]

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

Permissions

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

.http://mct.aacrjournals.org/content/14/11/2463To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-15-0241http://mct.aacrjournals.org/content/suppl/2015/08/22/1535-7163.MCT-15-0241.DC1http://mct.aacrjournals.org/content/14/11/2463.full#ref-list-1http://mct.aacrjournals.org/cgi/alertsmailto:[email protected]://mct.aacrjournals.org/content/14/11/2463http://mct.aacrjournals.org/

/ColorImageDict > /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /CropGrayImages false /GrayImageMinResolution 200 /GrayImageMinResolutionPolicy /Warning /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /CropMonoImages false /MonoImageMinResolution 600 /MonoImageMinResolutionPolicy /Warning /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 900 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False

/CreateJDFFile false /Description > /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ > /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MarksOffset 18 /MarksWeight 0.250000 /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /NA /PageMarksFile /RomanDefault /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /LeaveUntagged /UseDocumentBleed false >> > ]>> setdistillerparams> setpagedevice

a novel eg5 inhibitor (ly2523355) causes mitotic arrest ... · kinesin eg5 represents an attractive...

Documents