the mitotic kinesin kif11 is a driver of invasion ...cancer the mitotic kinesin kif11 is a driver of...

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The mitotic kinesin KIF11 is a driver of invasion,proliferation, and self-renewal in glioblastomaMonica Venere,1,2 Craig Horbinski,3 James F. Crish,1 Xun Jin,2 Amit Vasanji,4 Jennifer Major,1

Amy C. Burrows,1 Cathleen Chang,2 John Prokop,2 Quilian Wu,2 Peter A. Sims,5,6 Peter Canoll,7

Matthew K. Summers,1,8 Steven S. Rosenfeld,1,8* Jeremy N. Rich2,8*

The proliferative and invasive nature of malignant cancers drives lethality. In glioblastoma, these two processes arepresumedmutually exclusive and hence termed “go or grow.”We identified amolecular target that shuttles betweenthese disparate cellular processes—themolecular motor KIF11. Inhibition of KIF11 with a highly specific small-moleculeinhibitor stopped the growth of the more treatment-resistant glioblastoma tumor-initiating cells (TICs, or cancer stemcells) as well as non-TICs and impeded tumor initiation and self-renewal of the TIC population. Targeting KIF11 also hitthe other arm of the “go or grow” cell fate decision by reducing glioma cell invasion. Administration of a KIF11 inhibitorto mice bearing orthotopic glioblastoma prolonged their survival. In its role as a shared molecular regulator of cellgrowth and motility across intratumoral heterogeneity, KIF11 is a compelling therapeutic target for glioblastoma.

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INTRODUCTION

The prognosis for patients afflicted with glioblastoma (GBM) has re-mained grim despite decades of translational and clinical investigation.Several features contribute to the malignant phenotype of this disease.GBM has a high proliferative capacity that is supported by a highly pro-angiogenic microenvironment (1). In addition, although GBM rarelymetastasizes outside the central nervous system (CNS), it is capable ofwidely disseminating within the brain—a feature that severely limitsthe efficacy of surgery and radiotherapy (2, 3). Each of these features isaugmented in a subset of GBM cells that have stem cell–like propertiesand are referred to as GBM tumor-initiating cells (TICs). TICs are re-sistant to radiotherapy and alkylating chemotherapy, drive angiogen-esis, and are highly invasive (4). These features have led to efforts inmultiple laboratories to find points of vulnerability for the TIC pop-ulation. However, under some circumstances, the non-TIC subpopu-lation can assume TIC properties (5, 6). This implies that effective GBMtherapy will require use of either two classes of drugs—one to targetTICs and another to target the non-TIC population—or one class thattargets both populations. A target relevant to both cell populationswould be expected to play several essential roles in maintaining theGBM phenotype. First, it would drive mitosis to support tumor cellproliferation. Second, it would be needed for cell motility, which un-derlies tumor cell dispersion. Finally, it would be beneficial to blocksuch a target with highly specific, high-affinity small-molecule inhibitors.

Mitosis and cell motility require the microtubule-based cytoskele-ton, and these cellular physiologies are important not only for GBMbut also for a number of other highly aggressive malignancies. Severalclasses of drugs that inhibit microtubule dynamics, including thetaxanes, Vinca alkaloids, and epothilones, have been used successfullyin treating hematologic and solid malignancies (7). However, the

1Department of Cancer Biology, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.2Department of Stem Cell Biology and Regenerative Medicine, Cleveland Clinic Founda-tion, Cleveland, OH 44195, USA. 3Department of Pathology and Laboratory, MedicineUniversity of Kentucky College of Medicine, Lexington, KY 40506, USA. 4ImageIQ Inc.Cleveland, OH 44128, USA. 5Department of Systems Biology, Columbia University MedicalCenter, New York, NY 10032, USA. 6Department of Biochemistry and Molecular Biophysics,Columbia University Medical Center, New York, NY 10032, USA. 7Department of Pathologyand Cell Biology, Columbia University Medical Center, New York, NY 10032, USA. 8CaseComprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA.*Corresponding author. E-mail: [email protected] (S.S.R.); [email protected] (J.N.R.)

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microtubule-based cytoskeleton is crucial for CNS function, includingaxonal transport, and neurotoxicity is the dose-limiting side effect ofmany of these drugs (8). This has spurred efforts to identify and targetmicrotubule-associated proteins (MAPs) whose inhibition would blockmitosis without producing neurotoxicity. One class of MAPs that ap-pear to satisfy these requirements are a group of molecular motors, themitotic kinesins, that orchestrate a number of steps in the mitotic pro-cess, including chromosome congression, formation of the mitoticspindle, kinetochore microtubule dynamics, and cytokinesis (9). High-ly specific small-molecule inhibitors directed against several of thesehave been developed and evaluated both in preclinical models andin clinical trials (10), and as expected, these drugs have not producedthe neurotoxicity seen with microtubule poisons. Furthermore, an in-hibitor of one of these, KIF11 (also known as EG5 or kinesin-5), isbeing tested in multiple phase 2 trials in recurrent multiple myelomawith plans for a phase 3 trial in the near future (11).

KIF11 is a plus end–directed kinesin required for formation of thebipolar spindle in metaphase, where it opposes the action of minus end–directed molecular motors (12). It is the target for more than 20 high-affinity, specific small-molecule inhibitors that all bind to the samestructural motif in the catalytic domain (13). Suppression of KIF11 func-tion results in prolongedmitotic arrest, leading to cell death inmitosis or toinappropriate progression throughmitosis that is subsequently followed bycell death (14). KIF11 appears to have nonmitotic functions as well. It hasbeen shown to regulate axonal branching and growth cone motility and,more recently, was shown to be involved in cell motility (15–20). Althoughthe importance of KIF11 in the malignant behavior of GBM cells has notbeen explored, the evidence that it is required formitotic progression aswellas for cellmotility suggests that itmay play an especially important role.Wetherefore sought to determinewhetherKIF11 is indeed an essential driver ofboth the proliferative and invasive behaviors that are characteristic of GBM.

RESULTS

Mitotic kinesins, including KIF11, are up-regulated in GBMBecause cell proliferation depends on mitotic kinesins, we sought to char-acterize their expression in human GBM. We queried the expressionof 17 kinesins in a data set generated by RNA-seq of MRI (magnetic

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resonance imaging)–localized biopsies of human GBM (21). These in-cluded samples from the highly cellular, contrast-enhancing (CE) coreand the nonenhancing (NE) margins of GBM, which harbor the in-filtrating glioma cells that often escape surgery and give rise to recur-rence. Previous analysis of this data set showed that GBM subtypes, asdefined by the Verhaak classifier, differed in the expression of genesassociated with proliferation (21, 22). We used DESeq to perform dif-ferential expression analysis and determine which kinesins are signif-icantly increased or decreased in each GBM subtype compared tononneoplastic brain samples (Fig. 1A). This analysis revealed thatmost mitotic kinesins, including KIF11, for which clinically relevantsmall-molecule inhibitors exist, were elevated in the CE samples acrosssubtypes, with the highest expression in the proneural subtype. Thispattern was even more pronounced when examining the samplesfrom the NE margins of GBM. In an effort to understand the potentialimpact of KIF11 inhibition on nonneoplastic cells as well as tumorcells, we calculated the Spearman correlation between KIF11 andtwo sets of cell type–specific genes: those expressed by TICs and oligo-dendrocyte progenitor-like (OPC-like) proneural glioma cells andthose expressed by the major lineages found in nonneoplastic braintissue, including astrocytes, microglia, oligodendrocytes, and neurons.Results show that KIF11 expression is highly correlated with markersof TICs and OPC/proneural glioma cells in both the CE core and NEmargins of GBM (Fig. 1B). KIF11 expression does not correlate withmarkers of oligodendrocytes or neurons despite the abundance of thesecell types in the margins. This is important because the infiltrating tumorcells that reside in the margins are the target for postsurgical chemo-therapy. These findings suggest that a KIF11 inhibitor such as ispinesibwould selectively target tumor cells and not neurons or oligodendrocytes.To understand the genes most strongly correlated with KIF11 expressionin the CE core and NE margin, we performed an unsupervised gene ontol-ogy analysis (iPAGE), which showed highly enriched ontologies associatedwith mitotic cell cycle and DNA replication genes (fig. S1). Together, theseresults suggest that mitotic kinesins in general, and KIF11 in particular,are expressed in proliferating and migrating cells in glioma.

We also used quantitative polymerase chain reaction (PCR) to com-pare three matched sets of TICs and non-TICs from patient-derivedxenografts that span the spectrum of GBM subtypes (3691 proneural,08-387 classical, and 3565 mesenchymal) and examine the differentialexpression of a group of mitotic regulators (fig. S2). Of these, onlyKIF11 was consistently elevated above twofold (Fig. 1C). We prospec-tively isolated TICs and non-TICs so that mRNA could be acutelygenerated with minimal exposure of the cells to tissue culture conditionsand potential changes in expression profiles. The glycosylated cell sur-face epitope CD133 was used to separate TICs from non-TICs. TICswere functionally validated for their ability to self-renew and form tu-mors in immunocompromised mice (23–25). These data establish up-regulation of mitotic kinesins as a hallmark of GBM and implicateKIF11 as a putative therapeutic target in GBM. Furthermore, althoughKIF11 inhibitors have been extensively studied clinically, they havenever been examined in GBM (13). This motivated us to examine theroles of KIF11 in the GBM phenotype to determine whether it wouldbe a compelling therapeutic target for treatment of this disease.

Attenuated protein turnover contributes to increased KIF11concentration in TICsHaving established KIF11 as a potential target in GBM, we thenwanted to further explore the differences in the expression of KIF11

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between TICs and non-TICs. We isolated matched TICs and non-TICsfrom patient-derived xenografts and evaluated KIF11 concentrations byWestern blot. TICs and non-TICs were grown under the same cultureconditions for all experiments. We have previously used this approach

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and CE regions of human GBM surgical specimens. (B) Correlation of celltype–specific marker genes with KIF11 expression. (C) Quantitative PCR wasperformed for the listed mitotic regulators on RNA isolated from TICs andmatched non-TICs from three patient-derived xenografts.

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to maintain the TIC or non-TIC pheno-types while also providing mitogenic sig-nals that produce similar cell cycle profilesand proliferation rates (fig. S3A) (26, 27).In all three TIC/non-TIC pairs tested,KIF11 protein concentrations were higherin the TICs (Fig. 2A). To ensure that theTIC and non-TIC phenotypes were main-tained after sorting, we probed for OLIG2,a marker for TICs, and for glial fibrillaryacidic protein (GFAP), a marker for moredifferentiated non-TICs. We found a high-er concentration of OLIG2 protein in theTICs and, conversely, a higher concentra-tion of GFAP in the non-TICs (Fig. 2A).

In nontransformed cells, KIF11 expres-sion fluctuates throughout the cell cycle andis regulated by targeted protein destructionby means of ubiquitination (28, 29). Theanaphase-promoting complex/cyclosome(APC/C) is an E3 ubiquitin ligase that is acentral mediator of the ubiquitin-mediateddegradation of mitotic proteins. APC/Csubstrate specificity is dictated by its asso-ciation with the substrate adaptors Cdc20(APC/CCdc20, active in early mitosis) orCdh1 (APC/CCdh1, active in late mitosisand during G1). KIF11 is ubiquitinatedand degraded in an APC/CCdh1-dependentmanner to reduce its concentration in G1(28, 29). To determine whether KIF11turnover differs between TICs and non-TICs and whether this contributes to thedifferences in protein concentration, weused a double-thymidine block to arrestTICs and matched non-TICs from twohuman GBM xenografts. The cells werethen released from the arrest, sampleswere harvested for protein lysates every2 hours over a 10-hour time course forspecimen 3691 and over a 24-hour timecourse for specimen 08-387, and the re-sulting lysates were probed for KIF11 byWestern blot. Flow cytometry was per-
formed to validate the degree of cell synchronization (fig. S3A). WhereasKIF11 concentration in non-TICs rose in G2/M and then fell oncemitosis was complete, as previously reported for nontransformed cells(29), TICs maintained a high concentration of KIF11 throughout thecell cycle (Fig. 2B and fig. S3B). Because APC/CCdh1 targets KIF11 fordestruction, this finding suggests that there is a defect in APC/CCdh1
activity in TICs. To confirm this, we examined the concentration of anadditional APC/CCdh1 target, CDC20, in TICs and non-TICs. We foundthat, as in the case of KIF11, the concentration of CDC20 failed todrop in G1 in TICs (fig. S3C). These results imply that APC/CCdh1,a central component in cell cycle regulation and tumor suppression, isdysfunctional in TICs (30). To gain additional insight into this postu-late, we treated TICs and non-TICs with nocodazole to arrest cells atthe start of M phase. Mitotically arrested cells were then released from

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the nocodazole block. Four hours later, at a time when the cells werein late M/G1 and when APC/CCdh1 should be active, cell extracts weregenerated. The activity of APC/CCdh1 was directly tested by intro-ducing hemagglutinin (HA)–tagged securin, an APC/CCdh1 substrate,into the lysates. The concentration of HA-securin was evaluated overtime by Western blot to monitor the extent of protein turnover duringthe time course between TICs and non-TICs. HA-securin exhibitedgreater stability in extracts of G1 TICs, indicating that the activityof APC/CCdh1 is attenuated in TICs compared to non-TICs (Fig. 2, Cand D). These results indicate that chronic expression of central cellcycle regulators such as KIF11 and CDC20 is a key feature in TICsand establishes attenuated ubiquitin-mediated proteolysis as a con-tributing factor to their differential protein expression between TICsand non-TICs.

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Fig. 2. KIF11 is increased in TICs because of attenuated protein turnover. (A) Whole-cell lysates frommatched TICs and non-TICs from three patient-derived xenografts were probed for KIF11, OLIG2, and
GFAP. (B) TICs and non-TICs from xenograft specimen 08-387 were synchronized at G1/S using a double-thymidine block. After release, whole-cell lysates were made every 2 hours over a 24-hour time course.Asynchronous (A) lysates were also harvested. Resulting lysates were probed for KIF11. (C) TICs and non-TICs from xenograft specimen 3691 were synchronized at M phase using a nocodazole block. Lysates weremade 4 hours after release at late M and early G1, and APC/CCdh1 activity was evaluated using exogenousHA-securin as a substrate. Samples for immunoblot analysis were taken every 30 min over a 90-min timecourse and probed for HA. For all immunoblots, b-actin served as a loading control. The molecular weight(MW) of resulting bands is given in kilodaltons. (D) Quantification of band intensity, normalized to actin.




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KIF11 inhibition targets TICsand compromises their abilityto self-renewTo examine the importance of KIF11 inGBM pathophysiology, we used ispinesib,a cell-permeable and highly specific small-molecule inhibitor to KIF11 (31). We firstdetermined the dose-response relationshipsfor cytotoxicity versus ispinesib concentra-tion for matched TICs and non-TICs fromfive independent xenograft specimens after72 hours of ispinesib exposure. The meaneffective concentration (EC50) of ispine-sib for these five TIC samples (1.15 ±0.35 nM) was slightly but significantly lowerthan that for the matched non-TICs fromthe same tumor (1.79 ± 0.22 nM, P = 0.0085)(Fig. 3A). We next exposed TICs and non-TICs for 96 hours to 3 nM ispinesib andused flow cytometry to measure the sub-G1population, a surrogate marker for apoptosis.TICs demonstrated a higher sub-G1 frac-tion than non-TICs in three xenograftspecimens over 4 days of observation (Fig.3B). To confirm these findings, we usedan adenosine triphosphate (ATP)–based

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TICs from patient-derived xenografts (n = 5,with three technical replicates per specimen)were exposed to increasing concentrationsof ispinesib (0 to 32 nM) for 72 hours, fol-lowed by analysis of cell viability using anATP-based assay. Shown are means ± SD;P = 0.0085, two-tailed t test. (B) Matched TICsand non-TICs from three patient-derived xe-nografts were evaluated for the percentageof sub-G1 (apoptotic) cells over a 4-day timecourse after a single exposure to vehicle [di-methyl sulfoxide (DMSO)] or 3 nM ispinesibat day 0. (C) Matched TICs and non-TICs fromtwo patient-derived xenografts were moni-tored for cell viability with an ATP-based assayover a 5-day time course after a single expo-sure to vehicle (DMSO) or ispinesib. Shownare means ± SD; n = 3 biological replicates(with three technical replicates per biologicalreplicate); P < 0.01 or P < 0.0001, one-wayanalysis of variance (ANOVA) with a Bonferroniposttest. (D) TICs were isolated from xeno-grafts 3565, 3691, and 08-387 (three biolog-ical replicates for each xenograft) and platedfrom 50 down to 1 cell per well. Wells werescored 10 days later for the presence of atumorsphere. Representative results fromthe third xenograft harvested for each spec-imen are shown.
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viability assay to monitor the impact of exposure to 3 nM ispinesibover a 5-day time course for two matched TICs and non-TICs. Bothpopulations demonstrated sensitivity to KIF11 inhibition, with theTICs showing a slightly increased sensitivity over the non-TICs at ear-lier time points (Fig. 3C). These data indicate that although TICs andnon-TICs may have slightly different sensitivities to ispinesib, the pro-liferation of both cell populations is effectively inhibited by this drug.

We also examined the effect of transientKIF11 inhibition onTIC self-renewal as measured by the ability of a single cell to form a tumorsphere(32). We sorted TICs from three different patient-derived xenografts(three independent tumors per specimen) and allowed them to recoverovernight before pretreatment for 18hourswith 3nM ispinesib or vehiclecontrol. Cells were then released from drug inhibition and a single-cellsuspension was prepared by flow cytometry. The cells were then plated at arange of 1 to 50 cells perwell and scored for tumorsphere formation 10dayslater. In all specimens tested, stem cell frequencywas compromised in TICspreexposed to ispinesib (P=1.37×10−23 for 3565,P=1.12×10−16 for 3691,and P = 9.96 × 10−26 for 08-387) (Fig. 3D and fig. S4). Together, these datasupport KIF11 as a robust target that not only compromises the viabilityof both TICs and non-TICs but also affects TIC self-renewal.

KIF11 is required for the motility and morphogenesis of TICsand affects microtubule polymerizationKIF11 has been considered to be an essential component in mitosis,but there have been a number of reports suggesting that it also hasroles outside of the mitotic cycle, including in the regulation of cellmotility, axonal branching, and angiogenesis (15–20, 33, 34). A po-tential caveat to the interpretation of some of these studies, however,is that cell motility ceases during mitosis, when both the microtubule-and actin-based cytoskeletons are recruited to form mitotically impor-tant structures, including the spindle and the cytokinetic ring. Hence,studying a putative extramitotic role for KIF11 in cell motility or mor-

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phogenesis requires ensuring that any effects of KIF11 inhibition onthese processes occur in cells that are outside of mitosis. To accom-plish this, we treated TICs with a double-thymidine block to arrestthem at the G1/S boundary. Cells were then released from the arrestand allowed to reenter the cell cycle in a synchronized manner. Theircell cycle state was monitored by flow cytometry to determine whenthe cells were enriched in G1 (fig. S5). About 12 hours after release fromthe thymidine block, when the cells had fully recovered from the arrestand had begun to enter G1 (fig. S5), they were plated in a 3-mm Trans-well assay in the presence of increasing concentrations of ispinesib(Fig. 4A). After 8 hours, while the cells were still largely outside of

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Fig. 4. KIF11 inhibition blocks glioma cell motility and brain invasion.(A) Schematic of Transwell assay. TICs from patient-derived xenograft
08-387 were enriched within interphase using a thymidine arrest and re-lease paradigm. Interphase-enriched cells were plated on a Matrigel-coatedTranswell (125,000 per well) and given 8 hours to migrate in the presenceof increasing concentrations of ispinesib (0 to 200 nM) before fixation andstaining of the nuclei with DAPI. (B) Resulting membranes were scored forthe movement of nuclei through the Transwell membrane. Shown aremeans ± SD; n = 3 biological replicates (with three technical replicatesper biological replicate). (C) The Transwell assay was run as above withvehicle (DMSO) or 200 nM ispinesib, with data represented as the numberof migrated cells per field. Shown are means ± SD; n = 3 biological repli-cates (with three technical replicates per biological replicate); P = 0.0009,unpaired t test. (D) Schematic of slice culture assay. A PDGF–IRES (internalribosomal entry site)–GFP retrovirus was used to generate tumors in a ratglioma model. Resulting tumor-bearing brains were isolated to generateslice cultures. GFP-positive tumor cells were monitored by time-lapse videomicroscopy over a 10-hour time course. (E) Wind rose plots of tumor celldispersion (55 cells per group). Individual cell tracks from time-lapse mi-croscopy were plotted to a common origin to generate wind rose plotsfor displaying the dispersion of tumor cells in the presence of DMSO vehi-cle (left, black) or 200 nM ispinesib (right, red). (F) MSD versus time for ve-hicle (black) and 200 nM ispinesib (red) conditions. MSD was calculated for55 cells each from control and ispinesib conditions for each time intervalover 9.5 hours of observation. Black and red curves depict MSD (±1 SD)versus time, and superimposed white curves depict fitting of the data toa persistent random walk model using a nonlinear least-squares regression.




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G2/M, they were fixed and their nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for visualization and quantification of cells thathad migrated through the membrane. Ispinesib blocked Transwell mi-gration under these conditions with an EC50 of 67.2 nM (Fig. 4B). Inthe presence of 200 nM ispinesib, about sixfold fewer cells migratedthrough the Transwell compared to vehicle control (Fig. 4C). We nextexamined the effect of ispinesib on glioma dispersion in brain tissueusing a rodent GBM model produced by intracranially injecting abicistronic retrovirus encoding for platelet-derived growth factor (PDGF)and green fluorescent protein (GFP) in 3-day-old rat pups (Fig. 4D).As we have previously shown, diffusely infiltrating tumors with the his-tological features of GBM develop robustly in this rodent model within10 days after injection (35). The retrovirus-infected cells are highly mi-gratory and proliferative and express the OPC/proneural markers OLIG2,PDGF receptor a (PDGFRa), and NG2 (35). Furthermore, althoughTICs are thought to represent a relatively small subpopulation, SOX2and OLIG2 are seen in the vast majority of cells in proneural GBM andrepresent the predominant proliferating population in these tumors(21, 36). We generated 300-mm-thick sections from the tumor-bearingportion of the brain and monitored the effect of ispinesib (200 nM) orvehicle (DMSO) on the migration of the fluorescent tumor cells usingtime-lapse microscopy. Slices were counterstained with rhodamine-labeled isolectin B4 to fluorescently label tumor-associated microglialcells and allow for visualization of the interface between tumor and thesurrounding nonneoplastic, microglial-rich tissue. Individual greenfluorescent tumor cells (n = 50) from the treatment and control groupsfrom six separate recordings were tracked over the course of 9.5 hours.Representative videos of ispinesib and vehicle-treated brain slices areshown in movies S1 and S2. Cell tracks were transposed to a commonorigin to generate wind rose plots for ispinesib- and vehicle-treatedsamples (Fig. 4E). These demonstrate a reduction in tumor dispersion,as illustrated by a plot of mean square displacement (MSD) versustime (Fig. 4F). Data were fit to a persistent random walk model(37), which relates MSD to time by the following equation:

MSD ¼ 2S2P • ½t − Pð1 − expð−t=PÞÞ�where S is cell velocity and P is the persistence time (average length oftime a cell moves in a single direction). This analysis reveals that ispinesibreduces cell velocity by about twofold (21.9 ± 0.1 mm/hour for controlversus 11.3 ± 0.2 mm/hour for ispinesib treatment) but has little effect onpersistence time (1.9 ± 0.02 hours for control versus 2.3 ± 0.1 hours forispinesib treatment). BecauseMSDvaries as the square of velocity, a twofoldreduction in the latter corresponds to a fourfold reduction in the former.

Microtubules play important roles in cell motility. Several mitotickinesins, including KIF11, are known to affect tubulin polymerizationat the dynamic, plus end, which in interphase cells is located near thecell periphery at the leading edge (15–17). This implies that KIF11 in-hibitors, such as ispinesib, might inhibit cell motility by altering micro-tubule lengthening at the leading edge of migrating cells. To test thishypothesis, we first seeded TICs enriched in interphase via cell synchro-nization (fig. S5) at a low density in the presence of vehicle or 200 nMispinesib and monitored the kinetics of cytoplasmic process formationin these cells. Process formation in TICs was appreciably attenuated bydrug treatment (P < 0.0001) in a time-dependent manner (Fig. 5, A andB). Next, we examined the effect of ispinesib on microtubule content ininterphase-enriched TICs (fig. S5). We plated TICs in a wound assay toinduce cell polarization toward the cell-free zone. Cells were treated witheither vehicle or 200 nM ispinesib for 6 hours, fixed, permeabilized, and

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stained with anti-tubulin primary antibody followed by a secondary an-tibody conjugated to Alexa Fluor 488. Vehicle-treated cells at the woundedge displayed organized arrays of microtubules, but ispinesib treatmentreduced their appearance, and few cells had developed processes (Fig.5C). We measured the intensity of the Alexa Fluor 488 signal with con-focal microscopy, and this revealed that ispinesib treatment significantly(P < 0.0001) decreased polymerized tubulin in the TICs (Fig. 5D). Thesefindings highlightKIF11 as amediator of theTIC invasive phenotype thatcan be attenuated with small-molecule inhibition.

KIF11 inhibition affects TICs in vivoOur results so far show that targeting KIF11 blocks both invasion andproliferation in both TICs and non-TICs, and therefore suggest thatKIF11 may be a very compelling therapeutic target. We therefore nextexamined how ispinesib affects the defining features of TICs—tumorinitiation and propagation. We started with a flank tumor model whereGBM cells (100,000 per mouse) were injected into NSG mice. When tu-mors reached 0.12 cm3, mice were randomized into either vehicle (DMSO)or ispinesib (10 mg/kg) treatment groups, with drug or vehicle givendaily for seven consecutive days via intraperitoneal injection. Flank tumorvolume was measured daily. Two hours after the last administration, tu-mors were harvested, and their weight and volume were measured, atwhich point we observed that ispinesib treatment significantly reducedboth (Fig. 6, A and B; P < 0.0001 at day 7). We then evaluated the ex-cised tumors by immunocytochemistry for the marker SOX2, which isexpressed both by TICs and by transformed OPCs. Ispinesib-treated tu-mors had no SOX2-positive cells (Fig. 6C). These findings support in vivocell killing of GBM cells by ispinesib, including the TIC subpopulation.

Tumor initiation is a defining characteristic of TICs. To determinewhether ispinesib treatment alters tumor initiation and survival in or-thotopic xenograft models, we pretreated TICs in vitro for 18 hourswith either 3 nM ispinesib or an equal volume of vehicle (DMSO).Cells were then intracranially implanted at 2500 or 25,000 cells permouse (n = 10 mice per group for vehicle and n = 7 for ispinesib).Mice were monitored daily for weight loss and neurological signs in-dicative of brain tumor development. TICs that had been treated withispinesib were impaired in the ability to form secondary tumors, fur-ther confirming a compromised stem cell phenotype (Fig. 6D). At thefirst sign of neurologic impairment within the vehicle group, mice werealso harvested from the ispinesib group to evaluate tumor burden.Representative hematoxylin and eosin (H&E)–stained brains fromthese mice indicate tumor burden only within the vehicle group, sup-porting a delay in tumor development for drug-treated TICs (Fig. 6D).In an effort to evaluate the impact of in vivo exposure to ispinesib ontumor initiation, we treated mice with established subcutaneous tumorsfor 3 days with ispinesib (10 mg/kg) or vehicle. The tumors were thendissociated, and FACS (fluorescence-activated cell sorting)–isolatedCD133-positive TICs from each group were intracranially injected at1000 or 10,000 cells per mouse (n = 10 mice per group). In line within vitro exposure to ispinesib, in vivo targeting of TICs also reducedtheir tumor-initiating capability (Fig. 6E). Altogether, these data indicatethat TICs, which resist alkylating chemotherapy and radiation therapy,are highly sensitive to a KIF11 inhibitor in vivo.

High KIF11 portends poor patient prognosis, and targetingimproves survival in a preclinical modelTo evaluate the efficacy of KIF11 inhibition in a clinically relevant sys-tem, we used a TIC-derived orthotopic xenograft model to examine





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the effect of systemically delivered ispinesibon survival. We first confirmed that ispinesibreaches an intracranial tumor by adminis-tering a single dose (10 mg/kg) to an ortho-topic tumor-bearing mouse and harvestingthe tumor 5 hours later. The tumor wassectioned and probed for tubulin to eval-uate for the presence of monoastral spin-dles, a histological hallmark of KIF11inhibition (38). The ispinesib-treated tumorcontained numerous cells with monoas-tral spindles (Fig. 7A), which promptedus to evaluate the effect of systemicallyadministered ispinesib on survival in anorthotopic GBM model, in which 10,000luciferase-expressing TICs were injectedintracranially in each of a group of 20NSG mice. After 7 days, mice were ran-domized into two groups of 10, with onegroup receiving vehicle (DMSO) and theother receiving ispinesib (10 mg/kg, ad-ministered every 4 days for six doses),both administered by intraperitoneal in-jection. We monitored each cohort forweight loss and neurological signs indica-tive of brain tumor development and/ordrug toxicity (fig. S6). Mice treated withispinesib demonstrated a significant surviv-al advantage over those treated with vehicle

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formation was monitored for 6 hours bytime-lapse microscopy in the presence ofvehicle (DMSO) or ispinesib (200 nM) in08-387 TICs plated on Geltrex and enrichedin interphase. Representative images ofthe two treatment groups are shown at the6-hour time point. Cell bodies are masked inred, and cell processes are marked in yellow.Scale bars, 10 mm. (B) Process length wasmeasured over the time course. Shown aremeans ± SD; n > 500 cells per condition fromthree biological replicates; P < 0.0001, two-wayANOVA with a Bonferroni posttest. (C) Rep-resentative images from a modified scratchwound assay used to drive formation of aleading cellular process in 08-387 TICs en-riched in interphase. Just before woundformation, medium was changed to that con-taining vehicle (DMSO) or ispinesib (200 nM).Six hours later, cells were fixed and processedfor immunofluorescence to b-tubulin. (D)Anti-tubulin fluorescence signal from con-focal images of the primary cell layer adja-cent to the wound was quantified for bothtreatment groups and presented as signalover area. Horizontal bar represents the meanand error bars represent SD; n > 40 cells percondition; P < 0.0001, unpaired t test.




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(P < 0.001), with a median survival of 36 daysversus 24 days for the DMSO vehicle cohort(Fig. 7B). These data demonstrate that KIF11inhibition is effective in a preclinical model,where it appears to prolong tumor latencyand survival.

We probed a well-annotated tissue micro-array from normal subjects and glioma patientsand found that KIF11 protein expression pos-itively correlates withWHO grade (P = 0.0001;Fig. 7C) and, in the case of grade III andgrade IV glioma, with patient survival (P =0.047; Fig. 7D and table S1). Within GBM(WHO grade IV), KIF11 protein expressionwas markedly elevated over normal brain, withthe specificity of the KIF11 antibody vali-dated by localization to the spindles of mitot-ically active cells (Fig. 7, E and F). These data

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Fig. 6. KIF11 inhibition targets TICs in vivoand compromises tumor initiation. (A) Mice
were injected in the flank with 100,000 3691GBM cells. When tumors reached about 0.12 cm3,mice were randomized into one of two treat-ment groups: vehicle only (DMSO; n = 5) orispinesib (10 mg/kg; n = 5), with daily admin-istration over a 7-day time course. Tumor vol-ume was measured daily. Shown are means ±SD; P < 0.01, P < 0.001, or P < 0.0001, one-wayANOVA with a Bonferroni posttest. (B) Tumorswere removed at day 7 from both groups tocalculate the final weight. Shown are means ±SD. (C) Tumors isolated on day 7 were processedfor immunofluorescence with the stem cellmarker SOX2 (green), and nuclei were counter-stained with DAPI (blue). Scale bars, 20 mm.(D) TICs from patient-derived xenograft 3691were pretreated in vitro for 18 hours with vehi-cle or 3 nM ispinesib. Viable cells (2500 or25,000) were intracranially (IC) injected (DMSO,n = 10; ispinesib, n = 7). Mice were monitoredfor signs indicative of brain tumor development,at which time they were sacrificed. Three micefrom each group were harvested at the time offirst indication of neurological signs among thegroups, which occurred in the vehicle group.Representative H&E images are shown. Whitearrowheads indicate tumor. P = 0.012 for the2500 cohort and P = 0.020 for the 25,000 cohort(log-rank test). (E) Mice were injected in the flankwith 100,000 3691 GBM cells. When tumorsreached 0.2 to 0.6 cm3, mice were randomizedinto one of two treatment groups: vehicle only(DMSO; n = 5) or ispinesib (10 mg/kg; n = 5),with daily administration over a 3-day time course.Tumors were harvested and viable TICs wereisolated. Viable cells (1000 or 10,000) were intra-cranially injected into mice, which were thenmonitored for signs indicative of brain tumor de-velopment, at which time they were sacrificed.P = 0.0019 for the 1000 cell cohort and P =0.021 for the 10,000 cell cohort (log-rank test).




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establish that KIF11 is consistently up-regulated in GBM at the protein level,and they implicate KIF11 as a putativetherapeutic target.

DISCUSSION

Proliferation and dispersion are hall-marks of the malignant phenotype, andboth contribute to the grim prognosis thatcharacterizes GBM (2). Thus, an ideal ther-apeutic target would be a cellular compo-nent that drives both proliferation anddispersion in both TICs and non-TICs.The cytoskeleton is an essential compo-nent for both mitosis, which drives tumorproliferation, and cell motility, whichdrives invasion. Microtubules play impor-tant roles in cell motility, including mem-brane vesicle transport, signal transduction,

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Fig. 7. KIF11 informs patient prognosis,and targeting improves survival in a pre-
clinical model. (A) A mouse bearing an or-thotopic tumor was injected with a singledose of ispinesib (10 mg/kg). The tumor wasisolated 5 hours later and processed for im-munofluorescence to b-tubulin with nucleicounterstained with DAPI. Arrowheads indi-cate cells with monoastral spindles. a′ and a″represent enlarged regions of interest. Scalebars, 10 mm. (B) TICs (10,000) from patient-derived xenograft 3691 modified to expressluciferase were intracranially implanted intomice. Seven days later, when positive lumi-nescence signal indicated tumor burden, micewere randomized into one of two treatmentgroups: vehicle only (DMSO; n = 10) or ispinesib(10 mg/kg; n = 10), administered every 4 daysfor six doses. Arrows indicate day of vehicleor drug administration. Mice were monitoredfor signs indicative of brain tumor develop-ment, at which time they were sacrificed togenerate a Kaplan-Meier survival curve. P <0.001, log-rank test. (C) KIF11 protein expres-sion was scored on a tissue microarray con-taining normal brain control (n = 9) andpatient specimens from WHO grade II (n =25), grade III (n = 22), and grade IV (n = 47)gliomas. P = 0.0001, one-way ANOVA withpost hoc Tukey’s test. (D) Kaplan-Meier curvesof patients with grade III or grade IV gliomasstratified by low or high KIF11 expression(anti-KIF11 score). P = 0.047, log-rank test.(E) Representative H&E and immunohisto-chemistry (IHC) for KIF11 in normal brain anda GBM specimen. Scale bars, 20 mm. (F) Repre-sentative signal for KIF11 within the mitoticspindle of GBM cells. Scale bars, 10 mm.




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and stabilization and cross-linking of actin microfilaments, and theirrole in generating the mitotic spindle is well known (39). They can betargeted with a variety of clinical grade drugs that block microtubuledynamics, including the Vinca alkaloids and taxanes. These drugs havebeen very effective in treating a variety of malignancies (7). However,microtubules are essential for CNS function, which means that ther-apeutic concentrations of these microtubule inhibitors in the CNSwould be expected to produce severe neurotoxicity, a common issuefor the peripheral nervous system after systemic administration ofthese drugs (8).

These considerations have served as the rationale for developingsmall-molecule inhibitors directed at the mitotic kinesins that arelargely expressed in mitosis and are involved in forming the mitoticspindle (10). Such targets would be expected to be markedly up-regulatedin a mitotically active CNS tumor, such as GBM, compared to therelatively postmitotic brain. The most intensively investigated ofthese mitotic motors is KIF11, a member of the kinesin-5 family,for which well over 20 inhibitors have been synthesized (13). Unlikethe Vinca alkaloids and taxanes, KIF11 inhibitors are not neurotoxic—a point of direct relevance for a possible GBM therapy (10). However,clinical trials with some of these drugs, none of which were used inGBM, have been disappointing (40). These inhibitors have a shorthalf-life and have largely been used in tumors with a low proliferationindex. This mismatch between pharmacokinetics and cell cycle ki-netics means that it is unlikely that adequate concentrations of drugcan be sustained long enough to kill large numbers of tumor cells whenthey are most vulnerable—in the mitotic cycle. However, more recentclinical trials with other KIF11 inhibitors that have a long half-life(ARRY-520) or can be given by oral metronomic dosing (4SC-205)have been much more encouraging, particularly when combined withother drugs that synergize (41, 42). Unlike many other solid tumors,GBM has a high proliferation index, and it rarely spreads outside theCNS, so optimizing regional delivery of a KIF11 inhibitor to a largelypostmitotic tissue, such as the brain, should improve efficacy and re-duce toxicity. Consequently, we began our investigation of KIF11 byexamining its expression in GBM at both the RNA and protein levels.Our annotated tissue microarray data clearly demonstrated that KIF11protein expression correlates with both glioma grade and patient out-come. This finding is complemented by our DEseq analysis, whichshows that mitotic kinesins are most highly expressed in the proneuralsubtype of GBM, and this pattern is most pronounced in samples takenfrom the NE margins of the tumor. KIF11 expression is highly cor-related with genes that are expressed in TIC and OPC-like proneuralglioma cells. Although TICs are thought to represent a relatively smallsubpopulation, SOX2 and OLIG2 are highly expressed by OPC-likeglioma cells, which account for the vast majority of cells in proneuralGBM and represent the predominant proliferating population in thesetumors (21, 36). Similarly, an unsupervised gene ontology analysis(iPAGE) showed that KIF11 expression is most highly correlated withgenes associated with mitotic cell cycle and DNA replication. Theseresults show that KIF11 expression is increased in proliferating popu-lations in GBM, both in the CE core and in the NE margins, whichharbor the infiltrating glioma cells that give rise to recurrence.

The TIC subpopulation contains the most treatment-resistant sub-set of cells that constitute a GBM. We found that KIF11 expression isup-regulated in TICs compared to non-TICs at both the RNA and pro-tein levels. Although increased transcription might account for some ofthis increased KIF11 expression, our data clearly demonstrated that

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increased KIF11 in TICs also reflects a defect in protein degradationat the level of the APC/CCdh1. The delayed kinetics of KIF11 proteinturnover likely overlaps with cell cycle–regulated transcription of KIF11at the G1/S phase transition, maintaining KIF11 at a chronically higherconcentration in TICs. Furthermore, failed protein turnover of KIF11by APC/CCdh1 has been reported to impair clustering of supernumer-ary centrosomes, a common feature of cancer cells (28). Thus, TICsmight maintain their viability by balancing genomic instability causedby the reported persistent DNA damage and/or by the loss of APC/CCdh1, a key regulator of genomic stability, with altered cell cycle reg-ulation (23, 27, 30). Additionally, KIF11 has been reported to play arole in polypeptide synthesis in interphase, and the differential expres-sion of KIF11 between TICs and non-TICs could alter the kinetics andfidelity of protein translation (43).

Both TICs and non-TICs are exquisitely sensitive to nanomolarconcentrations of ispinesib, although our results support increasedsensitivity for TICs. These data could indicate an increased depen-dence on KIF11 for TICs and/or an increased sensitivity to perturba-tion of mitosis. The latter hypothesis is supported by our own dataregarding altered APC/CCdh1 activity as well as by recent findings thatTIC viability is compromised when other key mitotic regulators, suchas CDC20 and BUBR1, are targeted (44–46). Beyond viability, the keystem cell phenotypes of self-renewal and tumor initiation are abrogatedby KIF11 inhibition. Furthermore, systemic administration of ispinesibto mice bearing orthotopic, TIC-derived GBMs produces the expectedphenotype of monopolar spindles, as well as a prolongation in survival,implying that sufficient amounts of drug reach the intracranial tumorsto exert a therapeutic effect. Together, our results indicate that ispinesibhas a broad-spectrum antiproliferative activity against the major sub-populations that constitute a GBM. Although it is well appreciated thatthe TIC subpopulation can generate non-TICs, recent evidence suggeststhat under at least some conditions, the reverse is also true, implyingthat there is dynamic interchange between the TIC and non-TIC pop-ulations (5, 6). This finding suggests that therapies that are only activeagainst TICs may ultimately fail, if the remaining non-TICs can repop-ulate the TIC niche. Thus, our results with KIF11 provide an example ofa cellular component whose inhibition targets both TICs and non-TICs,and which may therefore prevent the inevitable recurrence that is seenin humans afflicted with GBM.

Inhibition of KIF11 induces axonal lengthening and branching,suggesting that this motor acts as a “brake” on microtubule slidingand regulates microtubule entry into neuronal dendrites (16, 17). In hu-man foreskin fibroblasts, inhibition of KIF11 blocks cell motility andmembrane ruffling on a two-dimensional surface when nonmusclemyosin II is inhibited (47), suggesting that KIF11 can act as an “ac-cessory” motor in driving cell motility in the absence of myosin II.These earlier studies motivated us to determine whether ispinesibalso affects morphology and cell motility in TICs. An in vitro Trans-well assay demonstrated that TIC invasion could be abolished by ispinesibunder conditions where we could rule out any appreciable effect froma block in G2/M, when cells normally become immobile. We furtherconfirmed that ispinesib also reduced GBM dispersion in brain tissueusing an ex vivo slice assay. Fitting to a persistent random walk modelrevealed that ispinesib reduces tumor cell velocity and not directionalpersistence. Of the eight residues in KIF11 that interact with ispinesib,all are conserved in the KIF11 sequences from human, rat, and mouse,making it highly likely that the effects of ispinesib that we see in the ratex vivo slice assay reflect direct inhibition of KIF11 (31). Associated

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with this block in dispersion is a change in tubulin polymerization andcell polarization with ispinesib. A number of studies have shown thatmicrotubules contribute to cell motility by regulating actin polymeriza-tion, transporting vesicles to the leading edge, and controlling the local-ization of focal adhesions (48). Furthermore, there also is evidence thatKIF11 can enhance elongation of tubulin protofilaments at the dy-namic, plus end, acting in essence as a tubulin polymerase (49).

We note that the EC50 for ispinesib inhibition of Transwell migra-tion by TICs is 30- to 50-fold larger than that for cytotoxicity. Webelieve that this difference is not likely due to an off-target effect ofthe drug for two reasons. First, the viability dose response experimentsinvolved prolonged exposure to ispinesib for 72 hours, whereas expo-sure in the Transwell assay was for only 8 hours. Prolonged exposurecould allow for cumulative toxic effects that would not necessarily beevident by briefer exposure times. Second, the antimitotic effects ofispinesib are on the mitotic spindle—a structure in which KIF11 gen-erates sustained force to oppose cytoplasmic dynein and non-claretdisjunctional (ncd). Thus, even a small degree of KIF11 inhibitionwould cause an imbalance of forces within the spindle, implying thata low dose of inhibitor over a sustained period may be sufficient toproduce mitotic arrest. By contrast, if KIF11 acted more as a supple-mentary driver of cell motility, it might be expected that higher dosesof ispinesib would be needed to detect an antimigratory phenotype.

The power of short hairpin RNA (shRNA)/small interfering RNA(siRNA) transfection is well established, but their use in determiningwhether KIF11 has a separate role in cell motility is problematic. Geneknockdown with these tools generally requires several days, and dur-ing this time, it would be expected that the gradual loss of KIF11would lead to a gradual mitotic block at the G2/M boundary, whencells normally become nonmotile. Interrogating the role of KIF11 intumor invasion requires instead a way of rapidly blocking its functionintracellularly and under controlled conditions. Ispinesib is freely cell-permeable, and we have shown that it binds to and inhibits KIF11 withinseveral seconds (31). The high specificity and affinity of this drug forits target and its rapid onset of action mean that interrogating the roleof KIF11 in cell motility can be most directly accomplished throughthe use of highly specific small-molecule inhibitors, such as ispinesib.

One major limitation to this study is that our preclinical model didnot allow for interrogation of the impact of KIF11 inhibition on inva-sion in an in vivo system. This limitation was caused by the studybeing designed to assess the impact on survival; hence, the time be-tween the last administration of ispinesib and the end-point of themice was too great to evaluate this factor. Despite this limitation, weshow that ispinesib targets both TICs and non-TICs and inhibits boththe proliferative and invasive phenotypes that characterize GBM. An-other limitation is balancing efficacy while avoiding the dose-limitingbone marrow suppression seen with systemic administration. This raisesthe possibility of administering KIF11 inhibitors intracerebrally withtechnologies that are being used clinically, such as convection-enhanceddelivery (CED), where prolonged intracerebral CED could be achievedwith a subcutaneous implantable infusion pump (50). These consid-erations imply that KIF11 inhibitors may be relevant therapies forGBM, particularly when combined with existing treatments.

MATERIALS AND METHODS

Detailed materials and methods can be found in the SupplementaryMaterials.

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SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/7/304/304ra143/DC1Materials and MethodsFig. S1. iPAGE gene ontology analysis of the Spearman correlation for KIF11 in the NE and CEregions.Fig. S2. Subtype analysis for patient-derived xenograft specimens.Fig. S3. Synchronization of TICs and non-TICs.Fig. S4. Stem cell frequency after KIF11 inhibition.Fig. S5. Representative cell synchronization histograms.Fig. S6. Weights of mice during the orthotopic preclinical study.Table S1. Patient data correlating with the Kaplan-Meier survival curves.Movie S1. Representative movie from an untreated organotypic slice culture from a rodentglioma model.Movie S2. Representative movie from an ispinesib-treated organotypic slice culture from arodent glioma model.References (51–53)

REFERENCES AND NOTES

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3. D. R. Smith, J. M. Hardman, K. M. Earle, Metastasizing neuroectodermal tumors of the centralnervous system. J. Neurosurg. 31, 50–58 (1969).

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Acknowledgments: We appreciate the flow cytometry assistance by C. Shemo, S. O’Bryant,and P. Barrett; the imaging assistance by J. Drazba, E. Diskin, and C. Rogers; the biostatisticalconsultation with J. Bena; and the animal support provided by the Lerner Research InstituteBRU. We also thank B. Slone from the Kentucky Cancer Registry for outcome data and D. Napier forhistotechnical support. Funding: We wish to acknowledge support from the following fundingsources: the NIH (NS073610 to S.S.R. and P.C.; CA172986 to S.S.R.; NS066955 to P.C.; EB016071 toP.A.S.; K08CA155764 to C.H.; GM108743 and GM112895 to M.K.S.; and CA154130, CA169117,CA171652, NS087913, and NS089272 to J.N.R.), the Research Programs Committees of ClevelandClinic (to J.N.R.), the James S. McDonnell Foundation (J.N.R.), the American Brain Tumor Associ-ation Basic Research Fellowship (to M.V.), the Ohio Cancer Research Award (to M.K.S.), and theBrain Tumor Ecology Collaborative funding from the James S. McDonnell Foundation (to P.C.and P.A.S.). The University of Kentucky Biospecimen and Tissue Procurement Shared ResourceFacility is supported by the Markey Cancer Center (P30CA177558). Author contributions: M.V.designed, performed, and analyzed experiments and wrote the manuscript; C.H. performedand analyzed the tissue microarray and edited the manuscript. J.F.C. designed and performedslice culture experiments and edited the manuscript; A.V. analyzed the slice culture experiments;J.M., X.J., A.C.B., C.C., J.P., and Q.W. performed experiments and contributed to acquisition of data;P.A.S. and P.C. performed bioinformatics analysis and data interpretation and edited the man-uscript; M.K.S. designed and performed the APC/C assays, analyzed data, and edited the man-uscript; S.S.R. and J.N.R. coordinated the project, analyzed experiments, and wrote themanuscript. Competing interests: M.V., S.S.R., and J.N.R. are co-inventors on a filed patententitled “Mitotic kinesin EG5 inhibiting anticancer agents”; U.S. Patent Application Serial No.14/678,310. S.S.R. is a paid consultant for the Mayo Clinic Comprehensive Cancer Center. Allother authors declare that they have no competing interests. Data and materials availability:RNA-seq data are available from the Gene Expression Omnibus under accession no. GSE59612.

Submitted 29 May 2015Accepted 5 August 2015Published 9 September 201510.1126/scitranslmed.aac6762

Citation: M. Venere, C. Horbinski, J. F. Crish, X. Jin, A. Vasanji, J. Major, A. C. Burrows, C. Chang,J. Prokop, Q. Wu, P. A. Sims, P. Canoll, M. K. Summers, S. S. Rosenfeld, J. N. Rich, The mitotickinesin KIF11 is a driver of invasion, proliferation, and self-renewal in glioblastoma. Sci. Transl.Med. 7, 304ra143 (2015).



glioblastomaThe mitotic kinesin KIF11 is a driver of invasion, proliferation, and self-renewal in

N. RichChang, John Prokop, Quilian Wu, Peter A. Sims, Peter Canoll, Matthew K. Summers, Steven S. Rosenfeld and Jeremy Monica Venere, Craig Horbinski, James F. Crish, Xun Jin, Amit Vasanji, Jennifer Major, Amy C. Burrows, Cathleen

DOI: 10.1126/scitranslmed.aac6762, 304ra143304ra143.7Sci Transl Med

therapeutic target for treating glioblastoma in patients.findings, together with the availability of a KIF11 inhibitor that's safe for human use, suggest KIF11 as a viable proliferation and invasion of glioblastoma cells and to prolong survival in mouse models of this disease. Thesecalled KIF11 plays a role in both. By inhibiting KIF11 with a small molecule, the authors were able to block the

show that a molecular motoret al.tissue. Although these are usually thought to be independent processes, Venere mechanisms that contribute to its lethality are proliferation of the tumor cells and their invasion into normal brain

Glioblastoma is a common and aggressive brain tumor, which is very difficult to treat. Two of theShutting off cancer's motor

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