Disruption of the anaphase-promoting complex confersresistance to TTK inhibitors in triple-negativebreast cancerK. L. Thua,b, J. Silvestera,b, M. J. Elliotta,b, W. Ba-alawib,c, M. H. Duncana,b, A. C. Eliaa,b, A. S. Merb, P. Smirnovb,c,Z. Safikhanib, B. Haibe-Kainsb,c,d,e, T. W. Maka,b,c,1, and D. W. Cescona,b,f,1

aCampbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada M5G 1L7;bPrincess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada M5G 1L7; cDepartment of Medical Biophysics, University of Toronto,Toronto, ON, Canada M5G 1L7; dDepartment of Computer Science, University of Toronto, Toronto, ON, Canada M5G 1L7; eOntario Institute for CancerResearch, Toronto, ON, Canada M5G 0A3; and fDepartment of Medicine, University of Toronto, Toronto, ON, Canada M5G 1L7

Contributed by T. W. Mak, December 27, 2017 (sent for review November 9, 2017; reviewed by Mark E. Burkard and Sabine Elowe)

TTK protein kinase (TTK), also known as Monopolar spindle 1 (MPS1),is a key regulator of the spindle assembly checkpoint (SAC), whichfunctions to maintain genomic integrity. TTK has emerged as apromising therapeutic target in human cancers, including triple-negative breast cancer (TNBC). Several TTK inhibitors (TTKis) arebeing evaluated in clinical trials, and an understanding ofthe mechanismsmediating TTKi sensitivity and resistance could informthe successful development of this class of agents. We evaluated thecellular effects of the potent clinical TTKi CFI-402257 in TNBC models.CFI-402257 induced apoptosis and potentiated aneuploidy in TNBClines by accelerating progression through mitosis and inducing mitoticsegregation errors. We used genome-wide CRISPR/Cas9 screens inmultiple TNBC cell lines to identify mechanisms of resistance to CFI-402257. Our functional genomic screens identified members of theanaphase-promoting complex/cyclosome (APC/C) complex, whichpromotes mitotic progression following inactivation of the SAC.Several screen candidates were validated to confer resistance to CFI-402257 and other TTKis using CRISPR/Cas9 and siRNA methods. Thesefindings extend the observation that impairment of the APC/C enablescells to tolerate genomic instability caused by SAC inactivation, andsupport the notion that a measure of APC/C function could predictthe response to TTK inhibition. Indeed, an APC/C gene expressionsignature is significantly associated with CFI-402257 response in breastand lung adenocarcinoma cell line panels. This expression signature,along with somatic alterations in genes involved in mitotic progres-sion, represent potential biomarkers that could be evaluated inongoing clinical trials of CFI-402257 or other TTKis.

TTK inhibitor | drug resistance | APC/C | CRISPR/Cas9 | breast cancer

Triple-negative breast cancer (TNBC), characterized by lack ofexpression of estrogen and progesterone receptors or am-

plification of HER2, is recognized as an aggressive disease withpoor outcomes and short survival in the metastatic setting. WhileTNBC is a heterogeneous disease, the majority exhibit high levelsof aneuploidy and a dearth of actionable genetic alterations (e.g.,focal DNA amplifications or activating point mutations that canbe targeted) (1–3). The latter explains in part the current lackof targeted treatment options for this disease, and underscoresthe need for novel treatment strategies. The recurrent somaticchanges that occur in TNBC include nearly ubiquitous TP53mutations, as well as genetic alterations to other tumor sup-pressors including PTEN, RB1, BRCA1 and components of theDNA damage response pathway (1). The loss of these criticalregulators of the cell cycle and genome maintenance contributeto the genomic instability characteristic of TNBC, a hallmarkthat represents a potential therapeutic vulnerability (4, 5).Inhibition of TTK protein kinase (TTK), also known as

monopolar spindle 1 (MPS1), has emerged as a promisingtherapeutic strategy for the treatment of aneuploid tumors, withTNBCs an important focus of clinical development. As a medi-

ator of the spindle assembly checkpoint (SAC), which delaysanaphase until all chromosomes are properly attached to themitotic spindle, TTK has an integral role in maintaining genomicintegrity (6). Because most cancer cells are aneuploid, they areheavily reliant on the SAC to adequately segregate their abnormalkaryotypes during mitosis. This is evidenced by the fact that theSAC is often weakened but rarely completely inactivated in cancercells (7–9). Abrogation of the SAC by TTK inhibition results inintolerable levels of genomic instability that are incompatible withcancer cell survival (10, 11). With several TTK inhibitors (TTKis)currently being evaluated as anticancer therapeutics in clinicaltrials, a more complete understanding of the mechanisms medi-ating TTKi sensitivity and resistance could have a significant im-pact by guiding their successful clinical development.In this study, we aimed to identify cellular mechanisms of

resistance to the clinical TTKi CFI-402257. Importantly, we in-vestigated this question in biologically relevant, aneuploid TNBCcell lines that model one of the principal human malignanciesfor which CFI-402257 is being developed. Using genome-wideCRISPR/Cas9 enrichment screens in three TNBC models, wefound that genetic disruption of anaphase-promoting complex/cyclosome (APC/C) components or other genes involved in mitotic

Significance

Using functional genomic screens, we have identified resistancemechanisms to the clinical TTK protein kinase inhibitor (TTKi)CFI-402257 in breast cancer. As this and other TTKi are currentlyin clinical trials, understanding determinants of tumor drug re-sponse could permit rational selection of patients for treatment.We found that TTKi resistance is conferred by impairinganaphase-promoting complex/cyclosome (APC/C) function tominimize the lethal effects of mitotic segregation errors. Dis-covery of this mechanism in aneuploid cancer cells builds onprevious reports indicating that weakening the APC/C pro-motes tolerance of chromosomal instability in diploid cells. Ourwork suggests that APC/C functional capacity may serve as aclinically useful biomarker of tumor response to TTKi thatwarrants investigation in ongoing clinical trials.

Author contributions: K.L.T., J.S., M.J.E., W.B.-a., B.H.-K., T.W.M., and D.W.C. designedresearch; K.L.T., J.S., M.J.E., W.B.-a., M.H.D., and A.S.M. performed research; A.S.M., P.S.,and Z.S. contributed new reagents/analytic tools; K.L.T., J.S., M.J.E., W.B.-a., M.H.D.,A.C.E., B.H.-K., T.W.M., and D.W.C. analyzed data; and K.L.T., W.B.-a., B.H.-K., T.W.M.,and D.W.C. wrote the paper.

Reviewers: M.E.B., University of Wisconsin; and S.E., Université Laval.

The authors declare no conflict of interest.

Published under the PNAS license.1To whom correspondence may be addressed. Email: tak.mak@uhnresearch.ca or dave.cescon@uhn.ca.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1719577115/-/DCSupplemental.

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progression confers resistance to CFI-402257 and other TTKis. Ourwork independently validates and extends findings from a previousstudy reporting that APC/C dysfunction promotes diploid cell tol-erability of genomic instability induced by reversine, a chemicalprobe that inhibits TTK (12). Furthermore, we report an APC/Cgene expression signature that is associated with response to CFI-402257 in breast and lung cancer cell line panels. This genetic sig-nature represents a promising biomarker for further developmentand evaluation in ongoing clinical trials, where its application inevaluating APC/C function could inform patient selection or predictdrug response to clinical TTKis.

ResultsCFI-402257 Accelerates Mitosis and Induces Mitotic SegregationErrors and Apoptosis in TNBC. To study the cellular effects ofCFI-402257 in TNBC, we selected three commonly used cellline models: MDA-MB-231, MDA-MB-468, and MDA-MB-436.Each line is reportedly aneuploid and contains a TP53 mutation(13), characteristic of clinical TNBC. The SAC functions toprevent anaphase onset until all chromosomes are sufficientlyattached to the mitotic spindle, thereby ensuring proper chro-mosome segregation during mitosis (6). TTK inhibition causesSAC inactivation and premature onset of anaphase with im-properly segregated chromosomes. To assess the effects of TTKinhibition on mitotic timing, live-cell microscopy was used tomeasure the time from nuclear envelope breakdown (NEBD) toonset of anaphase. CFI-402257 treatment (150 nM) significantlyreduced mitotic timing by twofold to threefold in all three celllines (Fig. 1A). As expected, scoring of mitotic cells identifiedsignificantly more mitotic errors (e.g., lagging chromosomes,anaphase bridges, and multipolar divisions) in CFI-402257–treated compared with DMSO control-treated cells (Fig. 1Band Fig. S1). We next assessed whether treatment with CFI-402257 potentiated aneuploidy using propidium iodide (PI)staining to measure DNA content. While 72 h of low-dose CFI-402257 (100 nM) had a modest effect on DNA content, a higherdose (400 nM) reproducibly increased the fraction of cellswith >4n content in all three lines (Fig. 1C). Finally, we de-termined that aneuploidy induced by 72 h of treatment was as-sociated with induction of apoptosis (Fig. 1D). Taken together,these cellular effects in TNBC are consistent with TTK inhibition-driven abrogation of the SAC, which accelerates mitotic progressionand induces mitotic errors, aneuploidy, and apoptosis, consistentwith reports for other TTKis (12, 14–17).

Genome-Wide CRISPR/Cas9 Screen Reveals APC/C Impairment ConfersResistance to CFI-402257. To understand mediators of CFI-402257 response, we used a functional genomics approach. StableCas9-expressing lines were generated for each model and used toconduct genome-wide CRISPR screens with the Toronto HumanKnockout Pooled Library (18). We used a positive enrichmentapproach to select gene knockouts that confer resistance to CFI-402257. Cells were continuously cultured in media containingCFI-402257 or DMSO vehicle control. Three different concen-trations of CFI-402257 were attempted for each cell line. Of thenine screens attempted, six were successful, as evidenced by theemergence of a drug-resistant cell population (one in MDA-MB-468, two in MDA-MB-436, and three in MDA-MB-231), andthree were unsuccessful (i.e., no drug resistant cell populationemerged because drug concentrations were too high). Screenswere ended once a drug-resistant population had clearly emergedfollowing the initial lagging period where CFI-402257 impairedsurvival and proliferation of the pooled cells (Fig. 2A). Impor-tantly, cells transduced with an sgRNA targeting LacZ were cul-tured with CFI-402257 in parallel to ensure that cell deathoccurred at the concentrations used for the screens. Targetedsequencing of sgRNA inserts in baseline, DMSO-treated, anddrug-resistant cell populations were evaluated using the MAGeCK

algorithm to identify sgRNAs significantly enriched in the resistantpopulation (19). Comparison of the CRISPR library representationat the beginning and end of the screens indicated that represen-tation was reduced in the final CFI-402257–resistant population, asexpected (Fig. S2A).Single guide RNAs enriched in the final drug-resistant pop-

ulation are those that target genes whose inactivation promotesresistance to CFI-402257. To identify the most robust candidates,

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Fig. 1. CFI-402257 induces mitotic errors and leads to cell death. (A) Live-cellimaging was used to measure mitotic timing. Cells were synchronized withdouble-thymidine block and released into DMSO or CFI-402257 for at least 4 hbefore time-lapse imaging. Each dot represents a single cell, and at least100 cells were counted per experiment. (B) Classification of mitoses in treatedcells. Mitoses observed were scored as normal (N) or as abnormal ifthey exhibited segregation errors, including lagging chromosomes (LC),endoreduplication (ER), anaphase bridges (AB), or multipolar divisions (MP).(C) DNA content analysis of treated cells. Cells were synchronized as above andreleased into DMSO or CFI-402257 for 72 h. Live cells were stained with PI andanalyzed by flow cytometry. (Inset) Numbers indicate the percentage of cellsexhibiting >4n DNA content. (D) Assessment of apoptosis induction by CFI-402257. Cells were collected and costained with Annexin-V and PI to de-termine the percentage of cells undergoing apoptosis after 72 h of treatment. Pvalues indicate significance for two-tailed Student’s t tests (mitotic timing andapoptosis) and χ2 tests (mitotic errors, normal vs. abnormal). All statistics werecalculated using GraphPad Prism software. *P < 0.05; **P < 0.01; ***P < 0.001;ns, not significant. Error bars indicate mean ± SD.

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we trimmed each screen’s list of candidate genes to only those withan enrichment P value < 0.05, and then compared these lists acrossthe six screens. This stringent analysis revealed 15 genes that weresignificantly enriched in at least one screen per cell line and in atleast four of the six screens conducted (Table 1 and Fig. S2B).Assessment of these candidates using Enrichr analysis identifiedthe APC/C as the most significantly enriched cellular componentfor two different Gene Ontology databases (GO and Jensen) (20, 21)(Fig. 2B and Table S1). ANAPC13 and ANAPC15 are both compo-nents of the APC/C itself, while MAD2L1BP, better known asp31(comet), is a negative regulator of the SAC through its antago-nism of the mitotic checkpoint complex (22). In light of the identifi-cation of APC/C components in our top hits, we examined the sgRNAlists and identified other APC/C components in individual cell lines,including ANAPC4, ANAPC5, CDC16, CDC20, and CDC23 inMDA-MB-468; ANAPC4, CDC20, and CDC16 in MDA-MB-436;and ANAPC5, ANAPC10, and CDC27 in MDA-MB-231. Taken to-gether, our functional genomics approach revealed numerous com-ponents of the complex responsible for anaphase initiation followingSAC inactivation, implicating a delay in anaphase onset and mitoticprogression as a mechanism mediating resistance to CFI-402257,and thus a potentially important determinant of drug response.

Inactivation of ANAPC4, ANAPC13, and MAD2L1BP Confers Resistanceto Multiple TTKis. We chose to further investigate the mitoticcheckpoint complex antagonist MAD2L1BP and the APC/Ccomponent ANAPC13 identified in our CFI-402257 screen asmediators of TTKi resistance. We also investigated ANAPC4,which was previously described to be involved in diploid celltolerance of chromosomal instability in an siRNA screen (TableS2) (12). To confirm that these candidate genes enable TNBCresistance to CFI-402257, we disrupted them using CRISPR/Cas9 editing with sgRNAs identified in our screens and withsiRNA as an orthogonal method. Knockdowns and genome editswere confirmed by quantitative PCR (qPCR), Western blotanalysis (when sufficient antibodies were available), or se-quencing (Fig. 3, Figs. S3 and S4, and Table S3). FollowingCRISPR editing or siRNA knockdown, we conducted colonysurvival assays to determine the effects of gene manipulation onsensitivity to CFI-402257. Both of these methods confirmed thatANAPC4, ANAPC13, and MAD2L1BP mediate CFI-402257 re-sponse in MDA-MB-231 cells, as genetic interference with these

genes led to increased TNBC resistance to TTK inhibition (Fig.3 A–C). Furthermore, we found that resistance conferred byknockdown of these genes was associated with reduced apoptosis,dampened aneuploidy induction, and elongated mitotic timing withCFI-402257 treatment in MDA-MB-231 cells (Fig. 3 D–I). Despitethe high rate of mitotic errors in basal conditions (Fig. 1), we foundan increase in the number of normal mitoses when knockdowncells were treated with CFI-402257 (Fig. 3 G–I). Similar effectswere observed in MDA-MB-436 cells (Fig. S3).We next asked whether genetic manipulation of ANAPC4,

ANAPC13, and MAD2L1BP affected sensitivity to additional pub-lished selective TTKis, including MPI-0479605 (17), NMS-P715(23) and Mps-Bay2a (15). Compared with CFI-402257, theseexhibited similar effects on TNBC viability in sulforhodamine B(SRB) dose–response assays, although their potency was lower(Fig. 4A). Consistent with our results for CFI-402257, CRISPR/Cas9and siRNA-mediated knockdown of ANAPC4, ANAPC13, andMAD2L1BP also conferred resistance to these TTKis, although withvariable penetrance across cell lines and the genes manipulated atthe concentrations tested (Fig. 4 B and C and Fig. S4).

Response to CFI-402257 Is Associated with Reduced APC/C GeneExpression Signature in Breast and Lung Cancer Cell Lines. Finally,we sought to investigate whether the biological mechanismrevealed by our functional genomics screens could be useful toidentify a biomarker correlate of intrinsic CFI-402257 response.Drug response profiles were generated for a panel of 52 breastcancer cell lines for which gene expression profiles were avail-able (Fig. 5A and Table S4). We hypothesized that cancers withlow expression of APC/C components or MAD2L1BP would berelatively resistant to CFI-402257. To test this, we evaluated theassociation between a gene set comprising 16 APC/C genes andMAD2L1BP (Fig. 5B), and CFI-402257 response using gene setenrichment analysis (GSEA) (24). We found that the APC/C-MAD2L1BP gene set was significantly associated with the in vitroresponse to CFI-402257 (Fig. 5C). Interestingly, among breastcancer subtypes, the APC/C-MAD2L1BP gene set associationwith CFI-402257 response was most significant in TNBC models(Fig. 5D). Assessment of the gene set in an independent panel of20 lung adenocarcinoma cell lines confirmed the association(Fig. S5A). We then evaluated an APC/C-MAD2L1BP genesignature defined as the mean expression of the 16 APC/C genesand MAD2L1BP (Fig. 5B), and found a significant associationbetween this metagene and CFI-402257 response in breast can-cer cell lines (Fig. S5B). Furthermore, of the 17 genes composingthe APC/C-MAD2L1BP gene set, we found that a metagene

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Table 1. Top 15 candidate genes from CRISPR/Cas9 screens

Gene Number of screens

ANAPC13 6ANKS1A 5ETS1 5LRTM1 5MAD2L1BP 5PLA2G16 5ANAPC15 4BID 4CMIP 4ENPP5 4GPSM3 4KCNH8 4LACE1 4SERPINA7 4VSIG1 4

Bold indicates candidate genes validated in this study.

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consisting of only ANAPC4 and CDC20 was most strongly as-sociated with CFI-402257 response (Fig. 5E).To address the potential utility of the APC/C metagene as a

clinical correlate of CFI-402257 response, we investigated the var-iability in the two-gene metagene score across various tumor typesusing public gene expression data from The Cancer Genome Atlas(TCGA). This showed substantial variability within breast and othertumors, and importantly, revealed numerous outliers with very lowscores that could represent tumors with intrinsic resistance to CFI-402257 (Fig. 5F). The variation we observed in APC/C metagenescores prompted us to assess the frequency of APC/C complexgenetic disruption in clinical tumors. A previous study reported thatpoint mutations in APC/C complex components occur in up to 23%of nearly 8,000 tumors in the TCGA pan-cancer dataset (12). Ourfocused analysis of TCGA breast cancers considering somatic DNAalterations with potential loss of function consequences (i.e., pointmutations and homozygous deletions) identified these in 4% ofprimary tumors (17/482), while down-regulation of gene expressionwas apparent in 46% of cases (222/482) (Fig. S6). Thus, clinicalcancers exhibit measurable differences in markers of APC/C func-tion, which are predicted to be associated with TTKi responsebased on our functional and correlative studies.

DiscussionSeveral TTKis, including CFI-402257, are currently being testedin early-phase clinical trials to characterize their safety andexplore their antitumor activity as cancer therapeutics (e.g.,

NCT02792465, NCT02138812, NCT02366949; EudraCT no.2014–002023-10) (25) as monotherapy or in combination withtaxane chemotherapy. For several of the agents under investigation,breast cancer (and TNBC in particular) is a primary indication ofinterest. Patient stratification is becoming increasingly important forthe development of novel therapies, in order to improve theprobability of success in the clinic. This effort can benefit signifi-cantly from an understanding of mechanisms of sensitivity and re-sistance characterized in nonclinical settings (26). Previous reportshave identified gatekeeper mutations in the TTK kinase domainas potential mechanisms of acquired resistance to TTKi (anal-ogous to those observed in many clinically approved kinase in-hibitors) (27), or have suggested somatic mutations and alterationsassociated with response (28), but the relevance of these to in-trinsic TNBC response are uncertain.Our approach, using functional genomic screens followed

by validation and correlative analyses in TNBC models, wasdesigned to identify biologically relevant processes or pathwaysthat could be linked to drug response for the clinical TTKi CFI-402257. The identification of the APC/C as a central complexmediating sensitivity to CFI-402257 is directly relevant to therationale for TTK inhibition in TNBC, whereby the charac-teristic genomic instability of these tumors was identified as atherapeutic vulnerability that can be exploited by TTK-targetingagents (11). Inhibition of TTK in these tumors will cause syntheticlethality by abrogating the SAC and consequently increase aneuploidyto intolerable levels that lead to cancer cell death (29).

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Fig. 3. Inhibition of genes regulating mitotic progression promotes resistance of MDA-MB-231 to CFI-402257. (A) Colony survival assay for MDA-MB-231 cellstransfected with siRNAs targeting ANAPC4, ANAPC13, and MAD2L1BP and treated with DMSO or CFI-402257. Colonies surviving 10–14 d of treatment werestained with SRB, solubilized, and quantified by spectrophotometry. Survival is illustrated as the proportion of drug-treated colonies relative to DMSO-treated(control) colonies. Dotted lines indicate colony growth in siNTC control cells. A representative assay is shown. (B and C) siRNA knockdown efficiencies weredetermined by qRT-PCR [error bars indicate maximum/minimum relative quantification (RQ) values] and Western blot analysis. (D) Quantitation of apoptosisinduction in siRNA-transfected cells treated with DMSO or CFI-402257 for 72 h. (E and F) DNA content analysis of siRNA-transfected cells treated with DMSO orCFI-402257 at 100 nM or 400 nM doses for 72 h. (G–I) Mitotic timing and error analysis of siRNA-transfected cells treated with 150 nM CFI-402257. Error barsindicate mean ± SD. P values indicate significance for two-tailed Student’s t tests (apoptosis, DNA content, and mitotic timing) or χ2 tests (mitotic errors, normalvs. abnormal). All statistics were calculated using GraphPad Prism software. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. Error bars indicate mean ± SD.

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Our study carried out in TNBC cell lines has revealed multiplecomponents of the APC/C, which promotes mitotic progressioninto anaphase, as well as additional genes involved in initiatingmitotic exit. These models harbor TP53 mutations and exhibitaneuploidy, characteristic features of clinical TNBCs, supportingthe clinical relevance of our findings. Another group has recentlyreported their investigation of diploid cell tolerance to chro-mosomal instability using reversine, a chemical probe that in-hibits TTK, to model this phenomenon (12). Sansregret et al.conducted a 4-day siRNA screen in immortalized nonmalignant,diploid retinal-pigment epithelial (RPE1) cells, and validatedcandidate genes in RPE1 and HCT116, a near-diploid coloncancer cell line. These authors reported that APC/C dysfunctionenables these cells to tolerate excessive chromosomal instabilityinduced by reversine treatment (12). Interestingly, this siRNA-screen identified both overlapping and nonoverlapping candi-dates compared with our screen. Among other factors, this mayreflect the differing ploidy states or genetic backgrounds of themodels studied or the use of CRISPR/Cas9 vs. siRNA systems(Table S3). For instance, TP53 was identified in the Sansregretscreen, but not in our screen, where the TNBC models alreadyharbor TP53 mutations, like nearly all TNBC tumors. Althoughwe did not identify ANAPC1 and UBE2C, the APC/C compo-nents ANAPC13, ANAPC15, and CDC20 were uniquely identifiedin our screens, as were other genes implicated in progression to

anaphase or mitotic exit: MAD2L1BP/p31(comet), DYNC1LI1,DYNC1LI2, TRIP13, and RNF8 (30–34). Importantly, siRNAknockdown of ANAPC15 and MAD2L1BP, two of the top candi-dates identified in our screens, have been shown to delay mitoticprogression in HeLa and RPE1 cells (35–37) providing mechanisticinsight for their association with TTKi resistance and corroboratingour mechanistic studies. The shared finding of the APC/C as acentral mediator of resistance to TTK inhibition, despite the dif-ferences in approach and cellular contexts, lends strong support tothe biological importance of these discoveries.Also relevant to our findings, Wild et al. (14) studied the

impact of deletion of the E2 ubiquitin-conjugating enzymes,UBE2C and UBE2S, on APC/C function in HCT116 cells. TheseE2s are used by the APC/C to ubiquitinate mitotic proteins, in-cluding CCNB1 and securin, whose degradation is required formitotic exit (22). Concordant with our findings, Wild et al. (14)showed that UBE2C and UBE2S deletion weakened APC/Cfunction and elongated NEBD to anaphase time, rendering cellsinsensitive to reversine or deletion of the spindle assemblycheckpoint gene, MAD2. Collectively, these data support thehypothesis that prolonging anaphase onset provides time forcancer cells to avoid otherwise lethal mitotic segregation errorsinduced by TTK inhibition. Our study provides independentsupport of these findings, but does so in multiple aneuploidcancer models, which is the clinically relevant disease being

Fig. 4. Delaying anaphase confers resistance to multiple TTKis. (A) Response of MDA-MB-231, MDA-MB-436, and MDA-MB-468 to various TTKis. Dose–responsecurves were generated using SRB assays with nine-point serial drug dilutions. For each drug dose, cell viability is plotted as the proportion of viability observed inDMSO-treated control cells. Curves were plotted with GraphPad Prism software, with error bars indicating SD. (B) Colony survival assays for MDA-MB-231 cellstransfected with siRNAs targeting ANAPC4, ANAPC13, andMAD2L1BP. (C) Quantitation of colony survival assays after solubilizing SRB. Colony survival is plotted asthe proportion of drug-treated colonies relative to DMSO-treated (control) colonies. Error bars indicate mean ± SD. Dotted lines indicate colony growth in siNTCcontrol cells. Representative experiments are shown.

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targeted by TTKi in development. Moreover, we demonstratedthat this mechanism confers resistance not only to CFI-402257, aclinical TTKi, but also several other selective TTKis.To extend these mechanism-based discoveries toward predictors

of TTKi response that could be applied clinically, we pursued agene expression-based approach, with the rationale that gene ex-pression might capture various alterations affecting the mitotic exitpathway, and supported by the observation that gene expressionpredictors are often the strongest predictors of cancer dependencies(38). To do so, we assembled a metagene expression signaturebased on the biological findings from our functional genomicscreens. The metagene comprised 16 APC/C complex componentsand MAD2L1BP, another governor of anaphase progression iden-tified by our screens. Analysis of this metagene in both breast andlung cancer cell lines revealed a significant association with re-sponse to CFI-402257: models with low APC/C metagene scores(i.e., low expression of APC/C and MAD2L1BP genes) exhibitedrelative resistance to TTK inhibition, consistent with our CRISPR/Cas9 screen findings. Subsequent analyses revealed that a two-genesignature of CDC20 and ANAPC4 expression alone was even morestrongly associated with CFI-402257 response in breast cancer celllines. Interestingly, we observed the strongest TTKi resistancephenotypes with ANAPC4 depletion in our validation studies.To investigate the potential clinical utility of the APC/C

metagene for stratifying or selecting patients for TTKi therapy,we assessed the reduced metagene signature in 11 different tu-mor types from TCGA’s pan-cancer dataset. We observed sub-stantial variability in metagene scores both across and within

different tumor types, and identified outliers with very low scoresin multiple tumor types. These markers could potentially indicatepatients with intrinsic resistance to CFI-402257. Characterizationof these APC/C low cancers may reveal alternative vulnerabilitiesthat could be exploited (29). Assessment of the APC/C metagene,other biomarkers of APC/C functional capacity, or somatic al-terations in components of the APC/C pathway in ongoing clinicaltrials will determine the clinical significance of our findings. Ifvalidated in the clinic, these discoveries could have an importantimpact on the successful development of TTKis, such as CFI-402257, as novel cancer therapeutics for TNBC and other cancers.

MethodsCell Lines. The breast cancer cell line (39) and the lung adenocarcinoma cellline (40) panels were generous gifts from Drs. Benjamin Neel and AdiGazdar, respectively. Cas9 was introduced into MDA-MB-231, MDA-MB-468, and MDA-MB-436 using lenti-Cas9-blast (52962; Addgene). For MDA-MB-231 and MDA-MB-468, cells stably expressing Cas9 were subcloned toselect lines with efficient Cas9 editing activity, evaluated by transduction ofcells with sgRNAs targeting essential genes followed by assessment of cellviability. A nonclonal Cas9 expressing population of MDA-MB-436 cells wasused for the screens.

CRISPR/Cas9 Screens. The Toronto Human Knockout pooled library (TKO) was agift from Dr. Jason Moffat (1000000069; Addgene) (18). Cas9-expressing celllines were transduced with the TKO library at low multiplicity of infection toensure single viral integrations per cell with 200× library coverage. Follow-ing puromycin selection, library infected cells were expanded for 7–10 d.Genomic DNA (gDNA) was harvested to determine baseline library repre-sentation and cells were plated at densities to maintain 200× library

A B

ANAPC1ANAPC2ANAPC4ANAPC5ANAPC7ANAPC10ANAPC11ANAPC13ANAPC15

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APC/C-MAD2L1BP gene set

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ANAPC4/CDC20 Metagene Score

CFI-402257 response associationwith 2-gene metagene

ρ: 0.693, p-value = 1.3 x 10 -8

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ANAPC4/CDC20 Metagene Score AcrossVarious TCGA Tumour Types

APC/C-MAD2L1BP gene set: 52 Breast Cancer Cell LinesGenes ranked by association with CFI-402257 response

Enrichment Score: 0.715, p-value = 4.3 x 10-4

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APC/C-MAD2L1BP gene set: 26 TNBC Cell LinesGenes ranked by association with CFI-402257 response

Enrichment Score: 0.711, p-value = 8.2 x 10 -4

0 10000 20000 40000Rank

30000 50000

Fig. 5. The APC/C gene signature is associated with CFI-402257 response in vitro. (A) Distribution of CFI-402257 sensitivity across 52 breast cancer cell lines. TheAAC was calculated from dose–response assays and used as a metric of cell line drug sensitivity. The P value for an ANOVA comparing AACs across breast cancersubtypes is indicated. (B) List of 17 genes composing the APC/C-MAD2L1BP gene set investigated. The genes composing the two-gene metagene (below) areindicated in bold. (C and D) GSEA of APC/C-MAD2L1BP gene set association with CFI-402257 response in all breast cancer cell lines (n = 52) (C), and only in TNBClines (n = 26) (D). (E) Correlation between the two-gene metagene and CFI-402257 response in 52 breast cancer cell lines. The metagene score was calculated asthe mean expression of the genes for each sample. Pearson correlation coefficients and P values are indicated. (F) Violin plots displaying the distribution andprobability density of the two-gene metagene scores across various TCGA tumor types. Only tumor types with 500 or more patients were assessed.

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coverage at the onset of CFI-402257 or DMSO (vehicle) treatments. For eachcell line, three doses ranging from IC60–IC90 concentrations were attempted.Cas9 lines transduced with sgLacZ were used as a negative control to ensurethat screen drug doses resulted in cell death. During the screens, cells werecultured as usual and counted at each passage to monitor cell doublings for30–50 d. At the end of the screen, gDNA was extracted from CFI-402257–treated cells and doubling-matched DMSO controls, and together withbaseline gDNA, was subjected to targeted sequencing of the sgRNA locus.Enriched sgRNAs were identified using the MAGeCK algorithm (19), andGene Ontology analyses were conducted using Enrichr (20, 21).

Candidate Gene Validation Studies. To validate candidate genes, MDA-MB-231 and MDA-MB-436 were transduced with lentiCRISPR-V2 (LCV2) encodingCas9 and the candidate gene-targeting sgRNAs identified as most significantlyenriched in our screens. sgRNA sequences were as follows: ANAPC4, CCTGCAG-CATCTAGTCCAAG; ANAPC13, CCTGAACCTGAACAAGACAA; MAD2L1BP, ACTT-GAGACAAGCTCTACGC; and GFP (negative control), GGGGCGAGGAGCTGTT-CACCG. Editing of candidate genes in LCV2 lines was confirmed byTA-cloning and sequencing of the sgRNA-target sites. As an orthogonalapproach, we conducted siRNA knockdowns to confirm their effects onCFI-402257 response using ON-TARGETplus SMARTpools (Dharmacon).Lipofectamine 3000 (Thermo Fisher Scientific) was used to deliver 10 nMsiRNA or nontargeting control (siNTC) to cells. Knockdown efficiencieswere determined using qPCR and Western blot analysis (anti-ANAPC4,A301-176A, Bethyl Laboratories; anti-MAD2L1BP, sc-134381, Santa CruzBiotechnology) at 48–72 h posttransfection.

Drug Response Assays. Response of cell lines to TTKi (CFI-402257, MPI-0479605,NMS-P715, andMps-Bay2a)was evaluated using colony survival and SRBassays.For colony assays, cellswere seeded sparsely and treatedwithDMSOor TTKi for10–14 d, and then fixed and stained with SRB. For quantification, SRB wassolubilized with 10 mM Tris·HCl, and absorbance was quantified on a spec-trophotometer. For SRB dose–response assays, cells were seeded in 96-wellplates and treated with serial drug dilutions. After 5 d of treatment, cells werefixed, stained with SRB, and solubilized, and absorbance was quantified on aspectrophotometer. CFI-402257 was synthesized as described previously (10),and MPI-0479605, NMS-P715, and Mps-Bay2a were synthesized by theCampbell Family Institute for Breast Cancer Research.

Live-Cell, Time-Lapse Microscopy. Cells were synchronized with double thymi-dine block, plated in Eppendorf chamber slides, and released into 167 nM siR-DNA stain (Cytoskeleton) and CFI-402257 at 150 nM or DMSO for a minimum of4 h before imaging. Cells were held in a humidified Chamlide stage incubatorkept at 37 °C and 5% CO2 (Live Cell Instrument). Time-lapse images werecaptured using Volocity 6.3 software (Quorum Technologies) on a Yoko-gawa spinning disk confocal microscope (Quorum Technologies) equippedwith a Hamamatsu ImageEM EM-CCD camera at 20× magnification every4 min for 20–28 h. The time from NEBD to anaphase was recorded foreach dividing cell. For all dividing cells, mitoses were scored as normal orabnormal (i.e., endoreduplication, lagging chromosomes, anaphase bridge,or multipolar divisions).

Flow Cytometry. Induction of aneuploidy and apoptosis after 72 h of treat-ment with CFI-402257 were measured by PI and annexin-V (AnV) combinedwith PI staining, respectively. To assess drug-induced apoptosis, cells were

collected following treatment, fixed, and stainedwith AnV-FITC at 2.25 μg/mL(BioLegend) and PI at 10 μg/mL (Sigma-Aldrich), and measured on a BDFACSCanto II flow cytometer. For ploidy analysis, viable cells were fixed withethanol, stained with PI (10 μg/mL), and measured on a BD FACSCanto II flowcytometer. FlowJo software was used to quantify the proportion of AnV+PI−

and AnV+PI+ cells as a readout of apoptosis, and to determine the fraction ofcells with 2n, 4n, or >4n DNA content based on PI staining.

Pharmacogenomic Analyses. CFI-402257 dose–response curves were generatedfor a panel of 52 breast cancer cell lines and 20 lung adenocarcinoma cell lines.The PharmacoGx pipeline was used to generate drug responsemetrics for eachcell line, including area above the drug dose–response curve (AAC) (41–45).Drug response data were integrated with publicly available gene expressionprofiles to evaluate the association between cell line CFI-402257 sensitivity(AAC) and a gene set comprising 16 APC/C genes and MAD2L1BP using GSEA(24). All genes in the genome were ranked according to their univariate as-sociation with CFI-402257 response and entered into GSEA. GSEA was run withthe 17-gene APC/C-MAD2L1BP gene list submitted as a gene set for testingenrichment compared with one million random permutations of the rankedgene list. Metagene scores were defined as the mean expression of the genescomposing them (e.g., 17 genes composing the APC/C-MAD2L1BP gene set, orANAPC4 and CDC20 in the two-gene metagene). Breast cancer cell line geneexpression profiles were obtained from Marcotte et al. (39), and lung ade-nocarcinoma cell line gene expression profiles were obtained from the CancerCell Line Encyclopedia (CCLE) (46). All gene expression profiles were reproc-essed from raw data files using the Kallisto pipeline (47). TCGA gene expres-sion profiles were obtained from University of California Santa Cruz Xenabrowser (xena.ucsc.edu), and genetic analyses were conducted using the Na-ture 2012 breast cancer cohort in cBioPortal (48, 49).

Research Reproducibility. The genomic data used in this study are publiclyavailable through our PharmacoGx platform. CCLE raw data are available athttps://portals.broadinstitute.org/ccle/. The raw RNA-seq data for the breastcancer cell line panel are available from the National Center for Bio-technology Information’s Gene Expression Omnibus (accession no. GSE73526).Our code and documentation are open-source and publicly available throughthe GitHub repository (https://github.com/bhklab/). A detailed tutorial de-scribing how to run our pipeline and reproduce our analysis results is availablein the GitHub repository.

ACKNOWLEDGMENTS. We thank Dr. Troy Ketela and members of theT.W.M. laboratory and Pelletier laboratory for experimental discussions,Dr. Jacqueline Mason and the CFIBCR Therapeutics group for providing drugsand scientific input, Drs. Benjamin Neel and Adi Gazdar for sharing cancer celllines, The Cancer Genome Atlas for data access, and the Advanced OpticalMicroscopy Facility for technical support. This work was supported by the TerryFox Research Institute, Canadian Institutes of Health Research, the PrincessMargaret Cancer Foundation, and Stand Up To Cancer Canada–CanadianBreast Cancer Foundation Breast Cancer Dream Team Research Funding, withsupplemental support of the Ontario Institute for Cancer Research throughfunding provided by the Government of Ontario (Funding Award SU2C-AACR-DT-18-15). Stand Up To Cancer Canada is a program of the EntertainmentIndustry Foundation Canada. Research funding is administered by the AmericanAssociation for Cancer Research International–Canada, the Scientific Partner ofSU2C Canada.

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