Novel Mps1 Kinase Inhibitors with Potent Antitumor ActivityDirk Kosemund, Ulrich Klar, Detlef...

Small Molecule Therapeutics

Novel Mps1 Kinase Inhibitors with PotentAntitumor ActivityAntje M.Wengner, Gerhard Siemeister, Marcus Koppitz, Volker Schulze,Dirk Kosemund, Ulrich Klar, Detlef Stoeckigt, Roland Neuhaus, Philip Lienau,Benjamin Bader, Stefan Prechtl, Marian Raschke, Anna-Lena Frisk, Oliver von Ahsen,Martin Michels, Bertolt Kreft, Franz von Nussbaum, Michael Brands, Dominik Mumberg,and Karl Ziegelbauer

Abstract

Monopolar spindle 1 (Mps1) has been shown to function as thekey kinase that activates the spindle assembly checkpoint (SAC) tosecure proper distribution of chromosomes to daughter cells.Here, we report the structure and functional characterization oftwo novel selective Mps1 inhibitors, BAY 1161909 and BAY1217389, derived from structurally distinct chemical classes.BAY 1161909 and BAY 1217389 inhibited Mps1 kinase activ-ity with IC50 values below 10 nmol/L while showing anexcellent selectivity profile. In cellular mechanistic assays, bothMps1 inhibitors abrogated nocodazole-induced SAC activityand induced premature exit from mitosis ("mitotic break-through"), resulting in multinuclearity and tumor cell death.Both compounds efficiently inhibited tumor cell proliferationin vitro (IC50 nmol/L range). In vivo, BAY 1161909 and BAY1217389 achieved moderate efficacy in monotherapy in tumor

xenograft studies. However, in line with its unique mode ofaction, when combined with paclitaxel, low doses of Mps1inhibitor reduced paclitaxel-induced mitotic arrest by theweakening of SAC activity. As a result, combination therapystrongly improved efficacy over paclitaxel or Mps1 inhibitormonotreatment at the respective MTDs in a broad range ofxenograft models, including those showing acquired or intrin-sic paclitaxel resistance. Both Mps1 inhibitors showed goodtolerability without adding toxicity to paclitaxel monotherapy.These preclinical findings validate the innovative concept ofSAC abrogation for cancer therapy and justify clinical proof-of-concept studies evaluating the Mps1 inhibitors BAY 1161909and BAY 1217389 in combination with antimitotic cancerdrugs to enhance their efficacy and potentially overcomeresistance. Mol Cancer Ther; 15(4); 583–92. �2016 AACR.

IntroductionCell-cycle deregulation represents one of the classical hallmarks

of cancer, and cell-cycle arrest is the predominant mode of actionof antimitotic cancer drugs (e.g., taxanes and vinca alkaloids).Targeteddisruptionofmitotic cell-cycle checkpoints offers a novelapproach to cancer treatment: driving tumor cells into cell divi-sion despite DNA damage or unattached/misattached chromo-somes, resulting in a lethal degree of DNA damage or aneuploidy.

Monopolar spindle 1 (Mps1), also known as TTK, is a serinethreonine kinase, which ensures proper biorientation of sisterchromatids on themitotic spindle by the activation of the spindleassembly checkpoint (SAC). The SAC controls attachmentsbetween microtubules (MT) and kinetochores (KT) duringprometaphase and blocks transition to anaphase until all chro-mosomes are correctly tensed, attached, and bioriented at themetaphase plate (1). Mps1 therefore plays an important role in

securing proper chromosome alignment, orientation, and segre-gation during mitosis.

Mps1mediates direct activation of SAC components, includingkinetochore null protein 1 (KNL1),mitotic arrest deficient (MAD)1/2, and budding uninhibited by benzimidazole (Bub) 1, 3 andrelated 1 (BubR1) and/or their recruitment to unoccupiedKTs (2–10). Mps1-mediated direct phosphorylation of KNL1 is requiredfor Bub1 and Bub3 kinetochore localization (9, 10). When acti-vated checkpoint proteins are released to the cytosol, they catalyzethe assembly of the mitotic checkpoint complex (MCC), whichinhibits the anaphase-promoting complex (APC)–mediatedpoly-ubiquitination of mitotic proteins, for example, cyclin B andsecurin, thereby delaying anaphase onset until the checkpoint issatisfied (1, 11, 12). Furthermore, Mps1 has been reported to beimplicated in an error correction mechanism that resolves erro-neous KT–MT attachments, which are frequently formed in earlymitosis, by phosphorylation of the chromosome passenger com-plex subunit borealin (13, 14). Accordingly, the inhibition ofMps1 abrogates the SAC, thereby leading to shortened mitosis,chromosome missegregation, aneuploidy or polyploidy, and celldeath (13, 15–17).

Mps1 is expressed during mitosis in proliferating cells. Over-expression of Mps1 is observed in a number of cancer cell linesand tumor types, including breast cancer, anaplastic thyroidcarcinoma, and lung cancers (18–21), correlating with highhistologic grade, tumor aggressiveness, and aneuploidy/CIN inbreast cancer (18). Cancer-associated frameshift mutations,

Bayer Pharma AG, Drug Discovery, Berlin, Germany.

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

Corresponding Author: Antje M. Wengner, Bayer Pharma AG, Muellerstr. 178,Berlin D-13353, Germany. Phone: 004930-4681-5787; Fax: 004930-4681-8069;E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0500

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

www.aacrjournals.org 583

on April 1, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst February 1, 2016; DOI: 10.1158/1535-7163.MCT-15-0500

http://mct.aacrjournals.org/

associated with a premature stop of Mps1 biosynthesis, arefrequently found in gastric and colorectal tumors with microsat-ellite instability (22).

On the basis of these findings, Mps1 has been considered asone among the most promising drug targets for cancer therapy(23). In recent years, several Mps1-inhibitory compounds havebeen identified and explored for their anticancer activity inpreclinical assays (12, 24), although none have entered clinicaldevelopment.

Established antimitotic drugs, such as vinca alkaloids, tax-anes, or epothilones, activate the SAC either by stabilizing ordestabilizing microtubule dynamics, resulting in a mitoticarrest. This arrest prevents separation of sister chromatids toform the two daughter cells. Dependent on the levels of cyclinB1 versus prosurvival proteins prolonged arrest in mitosisforces a cell either into mitotic exit without cytokinesis ("mitot-ic slippage") or into mitotic catastrophe, leading to cell death(25). In contrast, inhibitors of Mps1 induce SAC inactivation,which accelerates progression of cells through mitosis, resultingin chromosomal segregation errors and finally cell death.Silencing of Mps1 leads to the failure of cells to arrest inmitosis in response to antimitotic drugs. Remarkably, thecombination of microtubule-interfering agents and Mps1 inhi-bition even increases chromosomal segregation errors and celldeath (26, 27). Therefore, the combined increase of chromo-somal segregation errors induced by combination of antimi-totics with SAC inhibition constitutes an efficient strategy foreliminating tumor cells. Altogether, the inhibition of Mps1 incombination with microtubule-targeting agents represents avaluable mechanism that is expected to improve therapeuticefficacy of antimitotic drugs.

In a previous work, we have presented Mps1 kinase inhibitorsfrom two structural distinct chemical classes (28). Derived fromthese structures, we have developed two novel, potent, and highlyselectiveMps1 kinase inhibitors, BAY1161909andBAY1217389.Here, we present, for the first time, their chemical structuresand pharmacologic in vitro and in vivo profiles. Both compoundsare currently in phase I clinical trials (NCT02138812 andNCT02366949).

Materials and MethodsChemicals

BAY 1161909, (2R)-2-(4-fluorophenyl)-N-[4-(2-{[2-methoxy-4-(methylsulfonyl)phenyl]amino}[1,2,4]triazolo[1,5-a]pyridin-6-yl)phenyl]propanamide, and BAY 1217389, N-cyclopropyl-4-{6-(2,3-difluoro-4-methoxyphenoxy)-8-[(3,3,3-trifluoropropyl)amino]imidazo[1,2-b]pyridazin-3-yl}-2-methylbenzamide, weresynthesized at Bayer Pharma AG. Paclitaxel was purchased fromBristol-Myers Squibb, and cisplatin from Sigma-Aldrich.

Kinase assayThe inhibition of recombinant human Mps1 by BAY 1161909

or BAY 1217389 was assessed in TR-FRET–based in vitro kinaseassays via phosphorylation of a biotinylated peptide (biotin-Ahx-PWDPDDADITEILG-NH2). Kinase and test compound were pre-incubated for 15 minutes before enzyme reaction was started bythe addition of substrate and ATP upon 10 mmol/L. For furtherdetails, see Supplementary Materials and Methods.

Kinase selectivity profilingBAY 1161909 and BAY 1217389 were counterscreened against

a panel of kinases using theMillipore Kinase orDiscoveRx profilerscreen (29). BAY 1161909 was initially tested at 1 mmol/L in theDiscoveRx kinase panel, followed by KD determination for 11kinases (Supplementary Table S1). BAY 1161909 was tested at 10mmol/L in the Millipore kinase panel, followed by retesting at 1and 0.1 mmol/L and IC50 determination for JNK1alpha, JNK2al-pha, and JNK3 (Supplementary Table S1). BAY 1217389 wasinitially tested at 1, 0.1, and 0.01 mmol/L in the DiscoveRx kinasepanel (Supplementary Table S2).

Cell lines and cell cultureTumor cell lines were obtained either from the ATCC or from

the German Collection of Microorganisms and Cell Cultures.HeLa-MaTu and HeLa-MaTu-ADR cells were obtained from EpoGmbH. Authentication of all human cell lines used was per-formed at the German Collection of Microorganisms and CellCultures. Cells were propagated under the suggested growthconditions in a humidified 37�C incubator.

Cell proliferation assayCells were seeded into 96-well plates at densities ranging from

1,000 to 5,000 cells per well in the appropriate medium supple-mented with 10% FCS. After 24 hours, cells were treated inquadruplicates with serial dilutions of compounds. After further96 hours, adherent cells were fixed with glutaraldehyde andstained with crystal violet. IC50 values were calculated by meansof a 4-parameter fit using the company's own software.

Cell-based mechanistic assaysSAC assay. HeLa (ATCC CCL-2) cells were plated at a density of1,000 cells per well in a 1,536-well microtiter plate in 2 mL cellculture medium and incubated overnight at 37�C. G2–M arrestwas initiated by adding 0.1 mg/mL nocodazole for 24 hours. Cellswere then treated for 4 hours at 37�C in the presence of testcompounds (0 mmol/L, 0.005–10 mmol/L) solubilized in DMSO0.5% (v/v), fixed with 4% (v/v) paraformaldehyde (PFA), per-meabilized with 0.5% (v/v) Triton X-100, and blocked with 1%(v/v) BSA in PBS. After washing with PBS, the phosphorylation of

Table 1. Mean pharmacokinetic parameters of BAY 1161909 and BAY 1217389 after single administration in preclinical species

Intravenous (i.v.) administration Oral (p.o.) administrationSpecies Gender Dose (mg/kg) t1/2 (h) Vss (L/kg) CLblood (L/h/kg) Gender Dose (mg/kg) Tmax (h) Cmax (mg/L) F (%)

BAY 1161909Mouse M 0.5 4.9 3.4 0.69 F 1.0 4.0 120 59Rat M 0.5 14 2.4 0.19 M 0.5 4.0 110 49

BAY 1217389Mouse F 1.0 4.1 2.7 0.82 F 1.0 7.0 74 37Rat M 0.5 7.8 2.2 0.54 M 0.5 1.5 177 73

NOTE: intravenous administration as bolus.Abbreviations: M, male; F, female.

Wengner et al.

Mol Cancer Ther; 15(4) April 2016 Molecular Cancer Therapeutics584




histone H3 at serine 10 was labeled by antibody detection(Upstate Biotechnology, cat# 16-222). Nuclei were marked withHoechst 33342 (Life Technologies, cat# H1399). After washing,cells were covered with PBS and stored at 4�C until imageacquisition. Images for high-throughput microscopy wereacquiredwith aPerkinElmerOperaHigh-Content Analysis reader.Images were analyzed with image analysis software MetaXpressfrom Molecular Devices utilizing the Mitotic Index ApplicationModule. Assay data were further analyzed by 4-parameter Hillequation using Genedata's Assay Analyzer and Condoseosoftware.

Multinuclearity assay.U-2OS (osteosarcomaATCC:HTB-96) cellswere plated at a density of 2,500 cells per well in a 384-wellmicrotiter plate in 20 mL cell culture medium and incubatedovernight at 37�C. BAY 1161909 or BAY 1217389 was added ata final concentration of 100 nmol/L in triplicates. Cells weretreated for 0, 24, 48, and 72 hours at 37�C in the presence oftest compounds. Thereafter, cells were fixed with 4% (v/v) PFA,permeabilized with 0.5% (v/v) Triton X-100, and blocked with0.5% (v/v) BSA in PBS. a-Tubulin structures were detected byantibody labeling (Abcam, cat# ab7291). After blockingwith goatIgG (Jackson ImmunoResearch, cat# 005-000-003), secondaryantibodies (Life Technologies, cat# A-11017) were applied in theblocking solution. Cells were washed with PBS, and nuclei weremarked with Hoechst 33342 (Life Technologies, cat# H1399).Finally, cells were washed and covered with PBS and stored at 4�Cuntil image acquisition. Images were acquired with a PerkinElmerOpera High-Content Analysis reader.

Pharmacokinetic investigationsPharmacokinetic studies were performed in male Wistar rats

and female CD1 or NMRI nu/nu mice. For i.v. studies in rats andmice, BAY 1161909 was solubilized in 1% DMSO, 99% plasma;for p.o. studies in rats in 50%polyethylene glycol (PEG) 400, 10%ethanol, 40%water, and for p.o. studies inmice in 75% PEG 400,5% ethanol, and 25% solutol. BAY 1217389 was solubilized in50% PEG 400, 10% ethanol, and 40% water for intravenous andp.o. dosing in rats and mice. In pharmacokinetic studies, plasmasamples were collected after 2, 5, 15, 30, 45 minutes, 1, 2, 4, 6, 8,and 24 hours after intravenous application and after 8, 15, 30, 45minutes, 1, 2, 4, 6, 8, and 24 hours after p.o. administration andprecipitated with ice-cold acetonitrile (1:5). Supernatants wereanalyzed via LC/MS-MS. Pharmacokinetic parameters were esti-mated from the plasma concentration data, e.g., using the log-linear trapezoidal rule for AUC estimation. Maximal plasmaconcentrations (Cmax) and time thereof (Tmax) were taken directlyfrom the concentration time profiles.

Animal efficacy studiesHousing and handling of animals was in strict compliancewith

European andGermanGuidelines for LaboratoryAnimalWelfare.For tumor xenograft studies, female athymic NMRI nu/nu mice(Taconic), 50 days old, average body weight 20 to 22 g, wereused after an acclimatization period of 14 days. Feedingand drinking was ad libitum 24 hours per day. Human tumorcells derived from exponentially growing cell cultures were resus-pended for A2780cis, NCI-H1299, and SUM-149 modelsin 100% Matrigel (BD Biosciences) to a final concentration of2� 107, 3� 107, or 5� 107 cells/mL, respectively. Subcutaneous

implants of 0.1mL of 2� 106 A2780cis, 3� 106NCI-H1299, or 5� 106 SUM-149 cells were inoculated into the inguinal region ofathymic mice. Tumor fragments of patient tumor explants MAXF1384 or LU384, obtained from serial passage in nude mice, werecut into fragments of 4 to 5 mm diameter and transplantedsubcutaneously into the flank of athymic mice. Tumor area(product of the longest diameter and its perpendicular),measuredwith a caliper, and body weight were determined two to threetimes a week. Tumor growth inhibition is presented as treat-ment/control ratio (T/C) calculated with tumor areas at the endof the study. Animal body weight was used as a measure fortreatment-related toxicity. Body weight loss > 20% was dedi-cated as toxic. When tumors reached a size of approximately 20to 40 mm2, depending on growth of the tumor model, animalswere randomized to treatment and control groups (8–10 mice/group) and treated p.o. with vehicle (70% PEG 400, 5 %ethanol, and 25% Solutol), BAY 1161909, BAY 1217389,and/or paclitaxel, as indicated in Tables and Figure legends.In combination treatment groups, Mps1 inhibitor and pacli-taxel were applied at the same day within a time frame of1 hour. The treatment of each animal was based on individualbody weight. Animals were euthanized according to theGerman Animal Welfare Guidelines. Data were expressed asmeans � SD. Statistical analysis included one-way ANOVA,and differences to the control were compared versus controlgroup by pair-wise comparison procedure using the SigmaStatsoftware.

In vivo mode of action studiesFor analysis of polyploidy and multinuclearity induction

in vivo, A2780cis tumor–bearing female NMRI nude mice (seeabove) were treated with paclitaxel (intravenously once with24 mg/kg), BAY 1161909 (p.o. twice daily for 2 days with 2.5mg/kg), and in combination with paclitaxel (i.v. once 24 mg/kg)and BAY1161909 (p.o. twice daily for 2 days 1mg/kg). Treatmentfor all groups started at a tumor size of 60 mm2 at day 14 aftertumor cell inoculation. Tumor samples were prepared 4 and 8hours after first BAY 1161909 application at treatment day 1, aswell as 4, 8, and24hours afterfirst applicationof BAY1161909ontreatment day 2. At each time point, 3 animals per treatmentgroup were analyzed. Tumors were used for histologic examina-tion after paraffin embedding and hematoxylin and eosinstaining.

For determination of phosphorylated KNL1 or total KNL1 intumor tissue, A2780cis tumor–bearing female NMRI nude mice(see above) were treated with vehicle, BAY 1217389 alone p.o. for2 days with 1, 2, 4, or 8 mg/kg (day 1 twice daily, day 2 once),paclitaxel alone intravenously once 24 mg/kg, and in combina-tion with paclitaxel (i.v. once 24 mg/kg) and BAY 1217389 (p.ofor 2 days with 1, 2, or 4 mg/kg, day 1 twice daily, day 2 once).Treatment for all groups started at a tumor size of 60 mm2 at day15 after tumor cell inoculation. Tumor samples were prepared 36hours after the first treatment at day 1. Five animals per treatmentgroup were analyzed. Tumors were used for histologic examina-tion after paraffin embedding and pKNL1 staining.

Peripheral blood was collected in K-EDTA tubes for the deter-mination of relative reticulocyte counts (%RET) andmicronuclei-containing reticulocytes counts (%MN-RET) using theMicroFlowPlus Kit (mouse blood) from Litron Laboratories and the FlowCytometer BDFACSCanto II (BDBiosciences). Sample collection,

Mps1 Kinase Inhibitors BAY 1161909 and BAY 1217389

www.aacrjournals.org Mol Cancer Ther; 15(4) April 2016 585




processing, and analysis were performed according to the man-ufacturer's instructions (version 130712).

Antibodies and IHCAn antibody directed against phosphothreonine 875 in KNL1

protein was produced by immunization of rabbits with a phos-pho-KNL1 peptide as described previously (9). Sera with highestphospho-KNL1 specific titer were used for IHC. Paraffin-embed-ded tumor sections were stained using the rabbit anti-phospho-threonine 875 KNL1 antibody (1:400) inDAKO antibody diluent(S3022) and anti-rabbit Envision secondary antibody (DAKOK4010). Stained slides were scanned using a Mirax Slide Scannerand evaluated for positive pixels above background in threeindependent fields per slide. Total KNL1 peptide was detectedwith the primary rabbit anti-human CASC5 antibody (Sigma-Aldrich HPA026624) in a 1:400 dilution. The development andquantification of KNL1 staining was performed accordingly topKNL1.

Results and DiscussionKinase inhibition profile and cellular mechanism of actionstudies of two novel Mps1 inhibitors

In biochemical assays, BAY 1161909 and BAY 1217389, twonovel Mps1 kinase inhibitors from different structural classes(Fig. 1), inhibited Mps1 kinase activity, with IC50 values of0.34 � 0.09 nmol/L and 0.63 � 0.27 nmol/L, respectively.

Both compounds showed high selectivity against other kinases.When assayed against the Millipore panel (230 kinases), BAY1161909 inhibited only two kinases, JNK2 and JNK3, more than50% at a concentration of 1 mmol/L and no other kinase at100 nmol/L (Supplementary Table S1). BAY 1217389 wastested against the DiscoveRx kinase panel (395 kinases) andwas found to bind to PDGFRb (3,000 nmol/L), whereas BAY 1217389 was found to inhibit cellproliferation with a median IC50 of 6.7 nmol/L (range 3 to >300nmol/L). Sensitive and rather insensitive cell lines were identified,and overall, both compounds showed a very similar sensitivitypattern based on the comparisonof relative IC50s (ratio of cell lineIC50 vs. median IC50; Supplementary Table S4). Attempts tocorrelate cell line sensitivity or insensitivity towards Mps1 inhi-bition with gene mutations (Sanger) did not lead to the identi-fication of single-genemutationswith statistical significance (datanot shown). Even the comparison of the two pairs of isogenicTP53WTandTP53 knockoutHCT116 andRKOcell lines revealedonly slightly lower IC50s for the cell lines with a TP53–WTbackground, which is in line with our previous findings (28).

Taken together, BAY 1161909 and BAY 1217389 represent twopotent Mps1 inhibitors with high-kinase selectivity, specific cel-lular on-target activity, and antiproliferative potency. Althoughcell lines with high and low sensitivity towards Mps1 inhibitionwere identified, no single-gene mutation could be correlated,indicating that multiple genes may contribute to cellular sensi-tivity to SAC inhibition or that other local factors at the kineto-chore, such as the presence of phosphatases (10), may determinethe cellular response to Mps1 inhibition.

In vivo pharmacokinetic parametersPharmacokinetic parameters were determined in mouse and

rat. Following intravenous administration of BAY 1161909 asbolus of 0.5 mg/kg to male CD1 mouse and 0.5 mg/kg to male

NN

NNH

SOOCH3

OCH3

NH

F

CH3

O

Chiral

BAY 1161909 BAY 1217389

Figure 1.Structure of novel Mps1 inhibitors.Chemical structure of BAY 1161909,(2R)-2-(4-fluorophenyl)-N-[4-(2-{[2-methoxy-4-(methylsulfonyl)phenyl]amino} [1,2,4]triazolo[1,5-a]pyridin-6-yl)phenyl]propanamide,and BAY 1217389, N-cyclopropyl-4-{6-(2,3-difluoro-4-methoxyphenoxy)-8-[(3,3,3-trifluoropropyl)amino]imidazo[1,2-b]pyridazin-3-yl}-2-methylbenzamide.

Wengner et al.





Wistar rat, the compound exhibited low blood clearance. Thevolume of distribution (Vss) was high in both species andterminal half-lives were long. After oral administration of 1mg/kg to female NMRI mouse and 0.5 mg/kg to male Wistarrat, peak plasma levels were reached after 4 hours. The oralbioavailability was moderate in mouse and rat (Table 1). BAY1217389 was administered intravenously as bolus of 1.0 and0.5 mg/kg to female CD1 mouse and male Wistar rat, respec-tively. Blood clearance was found to be low in the testedspecies. Vss was high and terminal half-lives were long. BAY1217389 was administered orally to female NMRI mouse (1mg/kg) and male Wistar rat (0.5 mg/kg). Peak plasma concen-trations were observed between 1.5 and 7 hours. Oral bioavail-ability was high in rat and moderate in mouse (Table 1).

Our data demonstrate that we have identified two novel Mps1kinase inhibitors with a favorable pharmacokinetic profile, sup-porting further development for clinical application.

In vivo efficacy of Mps1 inhibitors in monotherapyTherapeutic efficacy and tolerability of theMps1 inhibitors BAY

1161909 or BAY1217389was investigated inmonotherapy usingthe A2780cis human cisplatin-resistant ovarian tumor xenograftmodel. Athymic mice bearing established A2780cis xenografttumors were treated with MTDs of BAY 1161909 or BAY1217389 in an intermittent 2 days on/5 days off dosing schedulein comparison with cisplatin. As expected, cisplatin treatmentresulted in weak antitumor efficacy in A2780cis tumors (T/C0.75). BAY 1161909 showed moderate antitumor efficacy (T/C0.43; Fig. 2A)but achieved statistically significant improvement oftumor growth inhibition compared with vehicle control andcisplatin treatment at overall good tolerability (Fig. 2B). Compa-rable antitumor efficacy was achieved for BAY 1217389 (T/C of0.53) at acceptable tolerability (Fig. 2C and D).

Our data demonstrate that monotreatment with Mps1 inhibi-tors BAY 1161909 or BAY 1217389 can achieve moderate

240

Vehicle

A C

DB

BAY 1161909 2.5 mg/kg 2QD 2 on/5 off p.o.

Vehicle

BAY 1217389 5 mg/kg 2QD 2 on/5 off p.o.; tox 1/8

Vehicle

BAY 1217389 5 mg/kg 2QD 2 on/5 off p.o.;animal death 1/8

Cisplatin 6 mg/kg once Q14D i.p.

Vehicle

BAY 1161909 2.5 mg/kg 2QD 2 on/5 off p.o.

Cisplatin 6 mg/kg once Q14D i.p.

T/C

0.75

0.43*T/C

0.53*

200

160

120

Tum

or a

rea

(mm

2 + S

D)

80

40

30

20

40

10

0

-10

-20

30

20

40

10

0

-10

-20

05 8

*Statistically significantvs. vehicle P < 0.05

11

Days after tumor inoculation

Days after tumor inoculation Days after tumor inoculation

14 17 20 23

5 8 11 14 17 20 23

240

280

200

160

120

Tum

or a

rea

(mm

2 + S

D)

Bod

y w

eigh

t cha

nge

(%)

Bod

y w

eigh

t cha

nge

(%)

80

40

3 60

9

*Statistically significantvs. vehicle P < 0.05

12


15 18 21 24

3 6 9 12 15 18 21 24

Figure 2.Response of A2780cis human ovarian xenograft tumors to treatment with BAY 1161909 and BAY 1217389 monotherapy. A2780cis human ovarian tumor cellswere implanted subcutaneously into nude mice on day 0. Treatment was started when tumors had reached a size of approximately 30 mm2. BAY 1161909was administered orally (p.o.) upon 2.5 mg/kg twice daily (2QD) for 2 days on/5 days off (2 on/5 off). Cisplatin was administered intraperitoneally (i.p.) upon6mg/kg every 2weeks (Q14D). BAY 1217389was administered orally (p.o.) upon 5mg/kg twice daily (2QD) for 2 dayson/5 daysoff. Tumor growthwasmonitoredbydetermination of the tumor area using caliper measurement three times weekly. A and C, time course of tumor growth. B and D, time course of animal bodyweight change.






antitumor efficacy at MTDs, as it has been presented for otherMps1 inhibitors before (2).

Efficacy of Mps1 inhibitors in combination with paclitaxelAs antimitotics/antitubulins lead to mitotic checkpoint activa-

tion, checkpoint overrun by Mps1 kinase inhibition shouldimpact antitumor activity in a combination treatment setting(Figs. 3, 4, and 5; Supplementary Tables S5 and S6). On the basisof our mechanistic in vitro findings supported by shRNA knock-down experiments in combination with low doses of paclitaxelshowing synergistic effects (31), we tested the ability of ourMps1 inhibitors to inhibit tumor growth in combination withpaclitaxel.

The response of human triple-negative breast cancer xenografttumors (no expression of Her2/neu, progesterone receptor, andestrogen receptor) to treatment with Mps1 inhibitor in combi-nation with paclitaxel was tested in tumor models that developpaclitaxel insensitivity during continued paclitaxel treatment,the cell line derived model SUM-149, and the patient-derivedtumor model MAXF 1384 (Fig. 3). In the SUM-149 xenograftmodel, the effect of combination treatment of BAY 1217389 andpaclitaxel was studied. BAY 1217389 was applied upon dosing

with 30% of MTD in a twice-daily intermittent (2 days on/5 daysoff) dosing schedule in combination with paclitaxel upon itsrespective MTD. A clear tumor growth delay was observed uponcombination of paclitaxel and BAY 1217389 after 48 treatmentdays, achieving statistically significant improvement over pacli-taxel monotherapy efficacy at acceptable tolerability (Fig. 3A). Inthe patient-derived triple-negative breast cancer xenograft modelMAXF 1384, BAY 1161909 was applied upon dosing with 80% ofMTD in a twice-daily intermittent (2 days on/5 days off) dosingschedule in combinationwith paclitaxel upon its respectiveMTD.After 42 treatment days, statistically significant improvement overpaclitaxel monotherapy efficacy was achieved in the paclitaxel/BAY 1161909 combination treatment group, inducing completetumor remission (Fig. 3C).

The activity of Mps1 inhibitors in combination with paclitaxelwas further tested in human lung carcinoma (NSCLC) xenograftmodels, the cell line–derived model NCI-H1299, which acquirespaclitaxel insensitivity during continued paclitaxel treatment,and the intrinsically taxane resistant patient-derived modelLU387 (Fig. 4). In the NCI-H1299 NSCLC xenograft model, theeffect of combination treatment of BAY 1217389 and paclitaxelwas tested. BAY 1217389 was applied upon dosing with 60% of

180 SUM-149 MAXF 1384350

300

30

DB

A C

20

10

0

250

200

150

100

50

0-4 0 4 8 12 16 20 24 28 32 36 40 44 48

-4

-10

-20

30

20

10

0

-10

-200 4 8 12 16 20 24 28 32 36 40 44 48

Days after randomization

Days after randomizationDays after tumor inoculation


160

140

120

100

80

60

Tum

or a

rea

(mm

2 + S

D)

Tum

or a

rea

(mm

2 + S

D)

40

20

010 15 20 25 30 35 40 45 50 55 60 65

10 15 20 25 30 35 40 45 50 55 60 65

*Statistically significantvs. paclitaxel P < 0.001


Bod

y w

eigh

t cha

nge

(%)

Bod

y w

eigh

t cha

nge

(%)

Vehicle

Paclitaxel 20 mg/kg QD 1 on/6 off

BAY 1217389 1.5 mg/kg 2QD 2 on/5 off

BAY 1217389 1.5 mg/kg 2QD 2 on/5 off + Paclitaxel 20 mg/kg QD 1 on/6 off

Vehicle


BAY 1161909 2 mg/kg Q2D 2 on/5 off

BAY 1161909 2 mg/kg Q2D 2 on/5 off + Paclitaxel 15 mg/kg QD 1 on/6 off

Vehicle

Paclitaxel 15 mg/kg QD 1 on/6 offBAY 1161909 2 mg/kg Q2D 2 on/5 off

BAY 1161909 2 mg/kg Q2D 2 on/5 off + Paclitaxel 15 mg/kg QD 1 on/6 off

Vehicle


BAY 1217389 1.5 mg/kg 2QD 2 on/5 off

BAY 1217389 1.5 mg/kg 2QD 2 on/5 off + Paclitaxel 20 mg/kg QD 1 on/6 off

Figure 3.Response of human triple-negative breast cancer xenograft tumors to treatment with BAY 1217389 or BAY 1161909 in combination with paclitaxel. SUM-149human breast cancer cells were implanted subcutaneously into nude mice on day 0. Treatment was started on day 14 when tumors had reached a size ofapproximately 26 mm2. MAXF 1384 patient-derived tumor fragments were transplanted subcutaneously into nude mice. Treatment was started whentumors had reached a size of approximately 40 mm2. BAY 1217389 was administered orally (p.o.) upon 1.5 mg/kg twice daily (2QD) for 2 days on/5 days off(2 on/5 off) in monotherapy and in combination with paclitaxel. BAY 1161909 was administered orally (p.o.) upon 2 mg/kg twice daily for 2 days on/5 days off inmonotherapy and in combinationwith paclitaxel. Paclitaxel was administered intravenously (i.v.) upon 20mg/kg or 15mg/kg once daily (QD) once per week (1 on/6off) in monotherapy and combination therapy. Tumor growth was monitored by determination of the tumor area using caliper measurement three times weekly.A and C, time course of tumor growth. B and D, time course of animal body weight change.

Wengner et al.





MTD in the twice-daily intermittent (2 days on/5 days off) dosingschedule. Paclitaxel was applied upon itsMTD. After 36 treatmentdays, statistically significant improvement over paclitaxel mono-therapy efficacy was achieved by paclitaxel/BAY 1217389 com-bination (Fig. 4A). BAY1217389was slightly overdosedwith 60%MTD in combination treatments as transiently critical bodyweight loss (>10%) and toxicity occurred (Fig. 4B). The intrinsi-cally taxane-resistant patient-derived lung carcinoma xenograftmodel LU387 was used to study the effect of BAY 1161909/paclitaxel combination therapy. BAY 1161909 was applied upondosing with doses 40% of MTD in the intermittent (2 days on/5days off) dosing schedule in combinationwith paclitaxel, appliedupon its respective MTD. The combination therapy of paclitaxeland BAY 1161909 achieved statistically significant reduction oftumor size compared with vehicle-treated control andmonother-apy groups, inducing clear tumor growth delay at good tolera-bility of treatments (Fig. 4C and D).

Taken together, we show here for the first time that the Mps1inhibitors BAY 1161909 and BAY 1217389 exhibit strongcooperativity with taxanes in acquired or intrinsically taxane-resistant tumor models of triple-negative breast cancer and lung

carcinoma. Paclitaxel monotherapy efficacy upon dosing at therespective MTD could be significantly improved in combina-tion with sub-MTD doses of Mps1 inhibitors at overall goodtolerability. Therefore, we could validate the hypothesis thatMps1 inhibitors in combination with antitubulins, such astaxanes, can enhance their efficacy and potentially overcomeresistance. This opens the opportunity to improve overallsurvival of taxane-resistant patients in indications where tax-anes are used as standard of care, such as breast, lung or ovariancancer.

In vivo mode of action of Mps1 inhibitor monotherapy andcombination with paclitaxel

To demonstrate that in vivo efficacy is mediated by the antic-ipated mode of action of Mps1 inhibition, different in vivomechanistic assays were applied.

The induction of cellular polyploidy and multinuclearitywas analyzed in A2780cis ovarian xenograft tissue after treatmentwith Mps1 inhibitor in monotherapy or combination with pac-litaxel at doses that have shown in vivo antitumor efficacy. BAY1161909 monotreated A2780cis tumors showed induction of a

NCI-H1299 LU387350

300

CA

DB

250

200

150

100

50

0-4 0 4 8 12 16 20 24 28 32 36 40 44 48

-4-20

-10

0

10

20

30

40

-206 10 14 18 22 26 30 34 38 42 42

-10

0

10

20

30

0 4 8 12 16 20 24 28 32 36 40 44 48

Days after randomizationDays after tumor inoculation

Days after randomizationDays after inoculation

Tum

or a

rea

(mm

2 + S

D)


180

160

140

120

100

80

60

40

20

06 10 14 18 22 26 30 34 38 42 46

Tum

or a

rea

(mm

2 + S

D)

Bod

y w

eigh

t cha

nge

(%)

Bod

y w

eigh

t cha

nge

(%)

Vehicle


BAY 1217389 3 mg/kg 2QD 2 on/5 off

BAY 1217389 3 mg/kg 2QD 2 on/5 off +Paclitaxel 20 mg/kg QD 1 on/6 off

Vehicle



BAY 1161909 1 mg/kg 2QD 2 on/5 off +Paclitaxel 20 mg/kg QD 1on/6 off

VehiclePaclitaxel 20 mg/kg QD 1 on/6 offBAY 1161909 1 mg/kg 2QD 2 on/5 off

BAY 1161909 1 mg/kg 2QD 2 on/5 off + Paclitaxel 20 mg/kg QD 1 on/6 off

Vehicle



BAY 1217389 3 mg/kg 2QD 2 on/5 off + Paclitaxel 20 mg/kg QD 1 on/6 off


Figure 4.Response of human NSCLC xenograft tumors to treatment with BAY 1217389 or BAY 1161909 in combination with paclitaxel. NCI-H1299 human NSCLC tumorcells were implanted subcutaneously into nude mice on day 0. Treatment was started on day 8 when tumors had reached a size of approximately 30 mm2.LU387 patient-derived tumor fragmentswere transplanted subcutaneously into nudemice. Treatmentwas startedwhen tumors had reached a size of approximately40 mm2. BAY 1217389 was administered orally (p.o.) upon 3 mg/kg twice daily (2QD) for 2 days on/5 days off (2 on/5 off) in monotherapy and in combinationwith paclitaxel. BAY 1161909 was administered orally (p.o.) upon 1 mg/kg twice daily for 2 days on/5 days off in monotherapy and in combination with paclitaxel.Paclitaxel was administered intravenously (i.v.) upon 20 mg/kg once daily (QD) once per week (1 on/6 off) in monotherapy and combination therapy. Tumorgrowth was monitored by determination of the tumor area using caliper measurement two to three times weekly. A and C, time course of tumor growth.B and D, time course of animal body weight change.






pleomorphic phenotype when compared with vehicle-treatedcontrol tumors, including multinuclearity, whereas paclitaxeltreatment alone induced atypical mitoses, as well as increasednecrosis and apoptosis. Combination treatment with paclitaxeland BAY 1161909 induced atypical mitoses as well as increasedpleomorphism and multinucleated tumor cells in A2780cisovarian tumors, demonstrating the expected mode of actionof Mps1 inhibition (Supplementary Fig. S2). Incorrect distri-bution of chromosomal material leads in some cell types, e.g.,erythrocytes or reticulocytes, to the formation of cytoplasmicbodies of chromosome fragments or whole chromosomes, socalled micronuclei. Therefore, peripheral blood reticulocytes of

Mps1 inhibitor–treated mice were analyzed. A clear increase ofthe fraction of micronuclei-harboring reticulocytes 3 days afterBAY 1161909 treatment at doses �1 mg/kg twice daily for 2days corresponding to �40% monotherapy MTD could bedetermined. Total reticulocyte counts dropped at doses �2mg/kg twice daily for 2 days (�80% monotherapy MTD;Supplementary Fig. S3). In addition, the effect of Mps1 inhib-itor alone or in combination with paclitaxel on KNL1 Thr-875phosphorylation has been determined in A2780cis ovariantumor xenografts. Monotherapy of Mps1 inhibitor BAY1217389 dosed upon 4 mg/kg (80% of efficacious dose inmonotherapy) led to 80% reduction of basal pKNL1; 2 mg/kg

pKNL1 in A2780Cis tumors in nude mice

pKNL1 in A2780Cis tumors in nude mice

1.2A B

C D

% p

KN

L1-p

ositi

ve p

ixel

s in

tum

or IH

C(m

edia

n)%

pK

NL1

-pos

itive

pix

els

in tu

mor

IHC

(med

ian)

1.0

0.8

0.6

0.4

0.2

0.0

12

10

8

6

4

2

0

Vehicle 1 mg/kg 4 mg/kg 8 mg/kg2 mg/kgBAY 1217389

Vehicle Paclitaxel

1 mg/kgBAY 1217389

+Paclitaxel

2 mg/kgBAY 1217389

+Paclitaxel

4 mg/kgBAY 1217389

+Paclitaxel

BAY 12173892 mg/kg

+ Paclitaxel

BAY 12173892 mg/kg

Paclitaxel

Vehicle

Vehicle

Figure 5.Effect of BAY 1217389 in monotherapy and combination therapy with paclitaxel on phosphorylation of KNL1 in A2780Cis tumors in nude mice. Untreated femalenude mice were implanted with 2 � 106 A2780Cis cells (in 100% Matrigel) subcutaneously. Animals were randomized to treatment groups when tumorsreached a mean tumor area of 52 mm2 (n ¼ 3 per group). Animals were treated with vehicle, BAY 1217389 in monotherapy at the indicated doses for 2 days(day 1 twice daily and day 2 once p.o.), once with paclitaxel (24 mg/kg once i.v.) on day 1, or once with paclitaxel (24 mg/kg once i.v.) and BAY 1217389 at theindicated doses (day 1 twice daily and day 2 once p.o.). On day 2, 36 hours after first application, animals were sacrificed und tumors isolated. The amount(%) of phosphorylated KNL1 (pKNL1) in tumors was determined by immunohistochemical staining (IHC). A and C show quantification of pKNL1 signals inA2780Cis tumors. B and D show representative pKNL1 tumor sections. The blue bar, 100 mm.


Wengner et al.




led to 60% of basal pKNL1 level in A2780Cis tumors (Fig. 5Aand B). Treatment with paclitaxel strongly induced the pKNL1signal in A2780cis tumor tissue (Fig. 5C and D). Remarkably,cotreatment with Mps1 inhibitor BAY 1217389 was able tocompletely suppress paclitaxel-induced pKNL1 at an efficaciouscombination dose of BAY 1217389 (Fig. 5C and D). Nochanges of total KNL1 level were detectable (SupplementaryFig. S4), indicating a specific effect of Mps1 inhibitors on KNL1phosphorylation.

These results demonstrate that Mps1 inhibitor treatment pre-vents correct distribution of chromosomal material during celldivision in tumors as well as peripheral blood reticulocytes.Furthermore, KNL1 phosphorylation might serve as a direct readout of target engagement in tumors. Overall, our data suggest thatan early pharmacodynamic effect of Mps1 inhibition in mono-therapy or paclitaxel combination could be determined bymicro-nuclei induction in peripheral blood reticulocytes or by moni-toring the phosphorylation status of KNL1.

ConclusionAntitubulin–based chemotherapy is used as standard of care in

many cancer patients across a variety of tumor types, such asbreast, lung, or ovarian cancers. Resistance to antitubulins is acommon drawback in cancer chemotherapy (32). Alternativetreatment options are often not available, whereby antimito-tics/antitubulins are still the only treatment option for patientswith aggressive cancer types. Therefore, we have evaluated wheth-er efficacy of antitubulin reagents (taxanes) can be improved bycombination treatment with Mps1 inhibitors in line with theirmechanisms of action.

Our findings validate the innovative concept of SAC abro-gation and justify clinical proof-of-concept studies evaluatingMps1 inhibitors BAY 1161909 and BAY 1217389 in combina-tion with antimitotic cancer drugs to enhance their efficacy andpotentially overcome resistance. BAY 1161909 and BAY1217389 are currently in phase I clinical trials (NCT02138812and NCT02366949). To our knowledge, these are the firstclinical trials of Mps1 kinase inhibitors.

Disclosure of Potential Conflicts of InterestAll authors are employees of Bayer Pharma AG. A.M. Wenger, P. Lienau,

B. Bader, G. Siemeister, D. Kosemund, and U. Klar have ownership interests(including patents) in Bayer Pharma AG. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: A.M. Wengner, G. Siemeister, M. Koppitz, V. Schulze,D. Kosemund, U. Klar, B. Bader, M. Michels, B. Kreft, F. von Nussbaum,M. Brands, D. MumbergDevelopment of methodology: A.M. Wengner, G. Siemeister, V. Schulze,D. Kosemund, U. Klar, B. Bader, S. Prechtl, O. von AhsenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.M. Wengner, G. Siemeister, D. Stoeckigt, R. Neu-haus, P. Lienau, B. Bader, S. Prechtl, M. Raschke, A.-L. Frisk, O. von AhsenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.M. Wengner, G. Siemeister, D. Kosemund, U. Klar,D. Stoeckigt, R. Neuhaus, P. Lienau, B. Bader, S. Prechtl, M. Raschke, A.-L. Frisk,O. von Ahsen, M. Michels, F. von NussbaumWriting, review, and/or revision of the manuscript: A.M. Wengner, G. Sieme-ister, M. Koppitz, V. Schulze, D. Kosemund, U. Klar, D. Stoeckigt, R. Neuhaus,P. Lienau, B. Bader, S. Prechtl, M. Raschke, A.-L. Frisk, O. von Ahsen, M.Michels,B. Kreft, F. von Nussbaum, M. Brands, D. Mumberg, K. ZiegelbauerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.M. Wengner, G. Siemeister, M. Koppitz,V. Schulze, D. Kosemund, U. Klar, D. Stoeckigt, R. Neuhaus, P. Lienau, B. Bader,S. Prechtl, M. Raschke, A.-L. Frisk, O. von Ahsen, M. Michels, B. Kreft,D. Mumberg, K. ZiegelbauerStudy supervision: A.M. Wengner, P. Lienau, M. Brands, K. ZiegelbauerOther (invention and synthesis of candidate compound): M. KoppitzOther (design and chemical synthesis of novel Mps1 kinase inhibitors):V. SchulzeOther (medicinal chemistry): F. von Nussbaum

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 June 17, 2015; revised December 28, 2015; accepted January 4,2016; published OnlineFirst February 1, 2016.

References1. Musacchio A, Salmon ED. The spindle-assembly checkpoint in space and

time. Nat Rev Mol Cell Biol 2007;8:379–93.2. Colombo R, Caldarelli M, Mennecozzi M, Giorgini ML, Sola F, Cappella P,

et al. Targeting the mitotic checkpoint for cancer therapy with NMS-P715,an inhibitor of MPS1 kinase. Cancer Res 2010;70:10255–64.

3. Hewitt L, Tighe A, Santaguida S, White AM, Jones CD, Musacchio A, et al.Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to theMad1-C-Mad2 core complex. J Cell Biol 2010;190:25–34.

4. Kwiatkowski N, Jelluma N, Filippakopoulos P, Soundararajan M, ManakMS, Kwon M, et al. Small-molecule kinase inhibitors provide insight intoMps1 cell cycle function. Nat Chem Biol 2010;6:359–68.

5. London N, Ceto S, Ranish JA, Biggins S. Phosphoregulation of Spc105 byMps1 and PP1 regulates Bub1 localization to kinetochores. Curr Biol2012;22:900–6.

6. Maciejowski J, George KA, Terret ME, Zhang C, Shokat KM, Jallepalli PV.Mps1 directs the assembly of Cdc20 inhibitory complexes during inter-phase and mitosis to control M phase timing and spindle checkpointsignaling. J Cell Biol 2010;190:89–100.

7. Santaguida S, Tighe A, D'Alise AM, Taylor SS, Musacchio A. Dissecting therole of MPS1 in chromosome biorientation and the spindle checkpointthrough the small molecule inhibitor reversine. J Cell Biol 2010;190:73–87.

8. Sliedrecht T, Zhang C, Shokat KM, Kops GJ. Chemical genetic inhibition ofMps1 in stable human cell lines reveals novel aspects of Mps1 function inmitosis. PLoS One 2010;5:e10251.

9. Yamagishi Y, YangCH, TannoY,WatanabeY. MPS1/Mph1phosphorylatesthe kinetochore protein KNL1/Spc7 to recruit SAC components. Nat CellBiol 2012;14, 746–52.

10. Nijenhuis W, Vallardi G, Teixeira A, Kops GJPL. Negative feedback atkinetochores underlies a responsive spindle checkpoint signal. Nat CellBiol 2014;16:1257–64.

11. Weaver BA, Cleveland DW. Decoding the links between mitosis, cancer,and chemotherapy: the mitotic checkpoint, adaptation, and cell death.Cancer Cell 2005;8:7–12.

12. Liu X, Winey M. The MPS1 family of protein kinases. Annu Rev Biochem2012;81:561–85.

13. Jelluma N, Brenkman AB, van den Broek NJ, Cruijsen CW, van Osch MH,Lens SM, et al. Mps1 phosphorylates Borealin to control Aurora B activityand chromosome alignment. Cell 2008;132:233–46.

14. Nezi L, Musacchio A. Sister chromatid tension and the spindle assemblycheckpoint. Curr Opin Cell Biol 2009;21:785–95.

15. Hardwick KG, Weiss E, Luca FC, Winey M, Murray AW. Activation of thebudding yeast spindle assembly checkpoint without mitotic spindle dis-ruption. Science 1996;273:953–6.






16. Liang H, LimHH, Venkitaraman A, Surana U. Cdk1 promotes kinetochorebi-orientation and regulates Cdc20 expression during recovery from spin-dle checkpoint arrest. EMBO J 2012;31:403–16.

17. TigheA, StaplesO, Taylor S. Mps1 kinase activity restrains anaphase duringan unperturbed mitosis and targets Mad2 to kinetochores. J Cell Biol2008;181:893–901.

18. Daniel J, Coulter J, Woo JH, Wilsbach K, Gabrielson E. High levels of theMps1 checkpoint protein are protective of aneuploidy inbreast cancer cells.Proc Natl Acad Sci U S A 2011;108:5384–9.

19. Landi MT, Dracheva T, Rotunno M, Figueroa JD, Liu H, Dasgupta A,et al. Gene expression signature of cigarette smoking and its role inlung adenocarcinoma development and survival. PLoS One 2008;3:e1651.

20. Salvatore G, Nappi TC, Salerno P, Jiang Y, Garbi C, Ugolini C, et al. A cellproliferation and chromosomal instability signature in anaplastic thyroidcarcinoma. Cancer Res 2007;67:10148–58.

21. YuanB, XuY,Woo JH,Wang Y, Bae YK, YoonDS, et al. Increased expressionof mitotic checkpoint genes in breast cancer cells with chromosomalinstability. Clin Cancer Res 2006;12:405–10.

22. Ahn CH, Kim YR, Kim SS, YooNJ, Lee SH. Mutational analysis of TTK genein gastric and colorectal cancers with microsatellite instability. Cancer ResTreat 2009;41:224–8.

23. Manchado E, Guillamot M, Malumbres M. Killing cells by targetingmitosis. Cell Death Differ 2012;19:369–77.

24. Kusakabe K, Ide N, Daigo Y, Itoh T, Yamamoto T, HashizumeH, et al. Discovery of imidazo[1,2-b]pyridazine derivatives: selec-

tive and orally available Mps1 (TTK) kinase inhibitors exhibit-ing remarkable antiproliferative activity. J Med Chem 2015;58:1760–75.

25. Topham C, Taylor S. Mitosis and apoptosis: how is the balance set? CurrOpin Cell Biol 2013;25:1–6

26. Abrieu A, Magnaghi-Jaulin L, Kahana JA, Peter M, Castro A, Vigneron S,et al. Mps1 is a kinetochore-associated kinase essential for the vertebratemitotic checkpoint. Cell 2001;106:83–93.

27. Stucke VM, Sillj�e HH, Arnaud L, Nigg EA. HumanMps1 kinase is requiredfor the spindle assembly checkpoint but not for centrosome duplication.EMBO J 2002;21:1723–32.

28. Jema�a M, Galluzzi L, Kepp O, Brands M, Boemer U, Koppitz M, et al.Characterization of novel MPS1 inhibitors with preclinical anticanceractivity. Cell Death Differ 2013;11:1532–45.

29. FabianMA, Biggs WH, Treiber DK, Atteridge CE, Azimioara MD, BenedettiMG, et al. A small molecule-kinase interaction map for clinical kinaseinhibitors. Nat Biotechnol 2005;23:329–36.

30. Schmidt M, Budirahardja Y, Klompmaker R, Medema RH. Ablation of thespindle assembly checkpoint by a compound targeting Mps1. EMBO Rep2005;6:866–72.

31. Janssen A, Kops GJ, Medema RH. Elevating the frequency of chromosomemis-segregation as a strategy to kill tumor cells. Proc Natl Acad Sci U S A2009;106:19.

32. Gonzalez-Angulo AM, Morales-Vasquez F, Hortobagyi GN. Overview ofresistance to systemic therapy in patients with breast cancer. Adv Exp MedBiol 2007;608:1–22.


Wengner et al.




2016;15:583-592. Published OnlineFirst February 1, 2016.Mol Cancer Ther Antje M. Wengner, Gerhard Siemeister, Marcus Koppitz, et al. Novel Mps1 Kinase Inhibitors with Potent Antitumor Activity

Updated version

10.1158/1535-7163.MCT-15-0500doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2016/01/30/1535-7163.MCT-15-0500.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/15/4/583.full#ref-list-1

This article cites 32 articles, 12 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/15/4/583.full#related-urls

This article has been cited by 13 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]

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/15/4/583To 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-15-0500http://mct.aacrjournals.org/content/suppl/2016/01/30/1535-7163.MCT-15-0500.DC1http://mct.aacrjournals.org/content/15/4/583.full#ref-list-1http://mct.aacrjournals.org/content/15/4/583.full#related-urlshttp://mct.aacrjournals.org/cgi/alertsmailto:[email protected]://mct.aacrjournals.org/content/15/4/583http://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

Novel Mps1 Kinase Inhibitors with Potent Antitumor ActivityDirk Kosemund, Ulrich Klar, Detlef...

Documents

Transcript of Novel Mps1 Kinase Inhibitors with Potent Antitumor ActivityDirk Kosemund, Ulrich Klar, Detlef...