Functional characterization of CFI-402257, a potent and selective Mps1/TTK inhibitor, for the treatment of cancer

Jacqueline M. Masona, Xin Weia, Graham C. Fletchera, Reza Kiarasha, Richard Brokxa, Richard Hodgsona, Irina Beletskayaa, Mark R. Braya, and Tak W. Makb,1

aThe Campbell Family Institute Therapeutics Group at Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada M5G 1L7; and bThe Campbell Family Institute for Breast Cancer Research at Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada M5G 2M9

Contributed by Tak W. Mak, February 13, 2017 (sent for review January 6, 2017; reviewed by Sabine Elowe and Kari Wisinski)

Loss of cell-cycle control is a hallmark of human cancer. Cell-cycle correction (6–8). Inhibition of Mps1 activity causes cells to checkpoints are essential for maintaining genome integrity and bal- prematurely exit with unattached , resulting ancedgrowthanddivision.Theyarespecifically deregulated in cancer in severe missegregation, aneuploidy, and ulti- cells and contain regulators that represent potential therapeutic targets. mately cell death (4, 9–12). Mps1 is expressed during mitosis in Monopolar spindle 1 (Mps1; also known as TTK kinase) is a core proliferating cells. Overexpression of Mps1 is observed in several component of the spindle assembly checkpoint (SAC), a genome-sur- human tumors, and correlates with worse prognosis (13–19). veillance mechanism that is important for cell survival, and has Further, Mps1 has been implicated in facilitating an aneuploidy- emerged as a candidate target for anticancer therapy. Here, we report tolerant state in cancer cells (13). Together, these findings pointed to Mps1 as a rational target for anticancer therapy. the cellular and antitumor effects of CFI-402257, a potent (Mps1 Ki = We describe here the cellular and antitumor effects of CFI- 0.09 ± 0.02 nM; cellular Mps1 EC50 = 6.5 ± 0.5 nM), highly selective, and orally active small-molecule inhibitor of Mps1 that was identified 402257, a potent and selective Mps1 kinase inhibitor. CFI-402257 through a drug-discovery program. Human cancer cells treated with shows strong antineoplastic activity on a broad panel of human cancer-derived cell lines, and causes effects consistent with de- CFI-402257 exhibit effects consistent with Mps1 kinase inhibition, spe- – CELL BIOLOGY cifically SAC inactivation, leading to chromosome missegregation, an- pletion or inhibition of Mps1 (4, 9 12). Oral administration of CFI- euploidy, and ultimately cell death. Oral administration of CFI-402257 402257 as a single agent to mice bearing human cancer xenografts results in inhibition of tumor growth at doses that are well-tolerated. in monotherapy or in combination with an anti-programmed cell death Notably, the combination of CFI-402257 and an anti-programmed 1 (PD-1) antibody in mouse models of human cancer results in inhibi- cell death 1 (PD-1) antibody causes tumor regression and increases tion of tumor growth at doses that are well-tolerated. Our findings survival in a murine colon cancer model that mimics the human provide a rationale for the clinical evaluation of CFI-402257 in patients disease, suggesting that this might be a clinically viable approach. with solid tumors. Results Mps1/TTK | inhibitor | CFI-402257 | cancer CFI-402257 Is a Selective Inhibitor of Mps1. Mps1 was identified as a potential therapeutic target through a systematic approach that ammalian is tightly regulated by the activation combined RNAi screening with -expression analysis in hu- Mand inactivation of that regulate progression through man breast cancers and cell lines, which previously yielded polo- the phases of the . To ensure that only healthy cells pro- like kinase 4 as an anticancer drug target (20). Reduction of liferate, cell-cycle checkpoints have evolved that play key roles in genome maintenance under various stress conditions and even Significance during an unperturbed cell cycle. These checkpoints are specifically deregulated in cancer cells, and contain regulators that represent At present, -targeting agents are the most important potential targets for the design of cancer cell-selective therapies (1). antimitotic used in the clinic. However, there is an urgent The spindle assembly checkpoint (SAC; also known as the mi- need for the discovery of new approaches to more effectively tar- totic checkpoint) is a signaling cascade that prevents chromosome get tumor cells with less toxicity. Emerging strategies for anticancer missegregation by arresting the cell cycle in mitosis until all chro- mosomes are properly attached to the mitotic spindle (2). This ar- therapy include exploiting cell-cycle checkpoint vulnerabilities and rest is achieved by inhibiting the -promoting complex/ genomic instability in cancer cells. The spindle assembly checkpoint cyclosome (APC/C), an E3 ubiquitin that is essential for (SAC) is important for cell survival, and its inactivation generates mitotic progression. Normal cells have a robust SAC in which one lethal genomic instability in cancer cells. Inhibition of SAC signaling or more unattached chromosomes can produce a signal strong through targeting of monopolar spindle 1 (Mps1) has provided an enough to inhibit all cellular APC/C activity and thereby block indication of the feasibility of such an approach. We report here the progression to anaphase. By contrast, many cancer cells have a cellular and antitumor effects of CFI-402257, a potent and specific weakened, but not absent, SAC response that may cause aneuploidy small-molecule inhibitor of Mps1. CFI-402257 is currently in a phase I and drive malignancy. However, more severe disabling of SAC clinical trial (ClinicalTrials.gov ID: NCT02792465). checkpoint signaling generates a level of chromosome instability that exceeds the adaption capacity of cancer cells and is a potential Author contributions: J.M.M., X.W., G.C.F., R.B., R.H., M.R.B., and T.W.M. designed research; anticancer strategy. Of note, nontransformed cells appear to be less J.M.M., X.W., G.C.F., R.K., R.B., R.H., and I.B. performed research; J.M.M., X.W., G.C.F., R.K., R.B., sensitive than cancer cells to death caused by SAC inhibition (3, 4). R.H., I.B., M.R.B., and T.W.M. analyzed data; and J.M.M., M.R.B., and T.W.M. wrote the paper. The core SAC kinase, monopolar spindle 1 (Mps1; also known as Reviewers: S.E., Université Laval; and K.W., University of Wisconsin School of Medicine TTK protein kinase), is a candidate target for this approach. and Public Health. Mps1 is an essential, dual-specificity kinase (5) that functions as an The authors declare no conflict of interest. important guardian of the fidelity of chromosome segregation. It is Freely available online through the PNAS open access option. critical for the recruitment of SAC proteins to unattached kineto- 1To whom correspondence should be addressed. Email: [email protected]. chores, mitotic checkpoint complex formation, and thus APC/C in- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. hibition. Mps1 is also required for chromosome alignment and error 1073/pnas.1700234114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1700234114 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Mps1 resulted in a loss of cell viability in 8 of 14 cell lines (Fig. S1), Cellular Effects of CFI-402257. Mps1 contributes to the alignment of and Mps1 mRNA was found to be overexpressed in 38% of all breast chromosomes on the metaphase plate, and is a core component of tumors and in 85% of triple-negative/basal-like (TNBC) tumors (Fig. the SAC, a surveillance mechanism that ensures mitotic fidelity and S2A) (21), the subtype considered to be the most genomically un- genome stability. Depletion or inhibition of Mps1 causes cells to exit stable class of breast cancer (22). Of note, reduction of Mps1 mitosis with unattached chromosomes, resulting in severe chromo- inhibited the cell viability of all four basal breast cancer cell lines and some missegregation, aneuploidy, and cell death (4, 9–12). CFI- both immortalized basal breast cell lines examined. Immunoblot 402257 inhibited the activation of the SAC with an IC50 value of 64 ± analysis was used to compare Mps1 levels in breast cancer cell lines to 5 nM as monitored in an assay in which the disappearance of histone those in primary human mammary epithelial cells derived from H3 serine 10 phosphorylation was assessed in HCT116 human colon normal breast tissue samples. Mps1 levels were found to be signifi- carcinoma cells treated with the (Fig. 1C). cantly higher in breast cancer cells relative to their normal tissue This phosphorylation event in histone H3 is a posttranslational modi- counterparts and in basal breast cancer cell lines relative to luminal fication that identifies mitotic cells (prophase to anaphase), and is not a breast cancer cell lines (Fig. S2B). result of Mps1 activity. To examine the effect of CFI-402257 on the On the basis of these studies, we initiated a drug discovery fidelity of chromosome segregation, HCT116 cells were treated with program that culminated in the discovery of CFI-402257 as a the inhibitor, and the number of chromosome segregation errors potent and selective small-molecule inhibitor of Mps1 (Fig. 1A) present in mitotic cells was determined by immunostaining for cen- (23). In an assay using recombinant human Mps1, CFI-402257 tromeres, α- to visualize the mitotic spindle, and DAPI. Treat- inhibited Mps1 with an IC50 value of 1.2 ± 0.4 nM, and was ATP ment with 200 nM CFI-402257 caused a massive increase in competitive with a Ki value of 0.09 ± 0.02 nM. Consistent with chromosome missegregations relative to DMSO control cells (Fig. 2A). these results, CFI-402257 inhibited autophosphorylation of Mps1 SAC inactivation and gross chromosome segregation errors caused at threonine 12/serine 15 with an EC50 value of 6.5 ± 0.5 nM in by CFI-402257 had a major impact on the cell-cycle progression and cells exogenously expressing human Mps1 (Fig. 1B). Mps1 thre- survival of human cancer cells. CFI-402257 induced a dose-dependent onine 12/serine 15 autophosphorylation occurs as a consequence dysregulation of the cell cycle, resulting in an increase in the frequency of kinase activation, and thus enables identification of active Mps1 of cells exhibiting an aneuploid DNA content (Fig. 2B). Massive an- in cells (24). Immunoblotting of endogenous phosphorylated euploidy was observed with 100 nM CFI-402257, and significant poly- Mps1 in cell lysates was technically challenging; therefore, cells ploidy was not observed with CFI-402257 concentrations as high as exogenously expressing Mps1 were used to determine the EC50 3,000 nM as a result of the potency and selectivity of the compound. value for inhibition of Mps1 autophosphorylation by CFI-402257. The ability of CFI-402257 to cause aneuploidy in cancer cells was To evaluate selectivity, CFI-402257 was profiled against a panel confirmed by metaphase chromosome spread assay (Fig. 2C). CFI- of 265 recombinant protein and lipid and a panel of 98 402257–induced aneuploidy was accompanied by a progressive accu- nonkinase targets, including G protein-coupled receptors, nuclear mulation of apoptotic cells that were detectable as early as 16 h receptors, membrane channels, and . Of the 265 kinases following treatment (Fig. 2D). The effects described here for tested, none showed >50% inhibition when incubated with 1 μM CFI-402257 are in accordance with cellular depletion of Mps1 CFI-402257, and no significant inhibitory activity was observed protein or inhibition of Mps1 with other compounds (4, 9–12). against , Polo-like kinase, or Cyclin-dependent kinase Thus, CFI-402257 inhibits Mps1 in human cancer cells and family members. Only 3 of 98 nonkinase targets were inhibited modulates the physiological role of the kinase. ≥50% with 10 μM CFI-402257 (Table S1). Overall, these results We evaluated the effect of CFI-402257 on growth inhibition (i.e., indicate that CFI-402257 selectively and potently inhibits Mps1 ki- IC50) in a panel of human cancer-derived cell lines covering various nase activity in cells with minimal off-target effects. cancer histologies after 5 d. CFI-402257 exerted potent growth in- hibitory activity across the vast majority of cell lines with a median IC50 value of 15 nM (range, 1 to >1,500nM;Fig.3andTable S2). Notably, no single gene mutation in several proteins relevant to A C oncogenesis (e.g., APC, BRAF, CDKN2A, PIK3CA, RAS and TP53; Sanger Institute Catalog Of Somatic Mutations In Cancer 1.5 database, cancer.sanger.ac.uk/cosmic/) or cell doubling time was correlated with statistical significance with the resistance/sensitivity 1.0 of the cell lines to CFI-402257.

0.5 histone H3 Toxicity Studies of CFI-402257. The tolerability and pharmacoki- EC 50 netic properties of CFI-402257 were initially evaluated in mice. 0.0 -10 -9 -8 -7 -6 To determine a tolerable dose range for in vivo efficacy studies, Phospho-histone H3 (S10)/ Phospho-histone H3 log [CFI-402257] mice were given CFI-402257 orally for 21 d, with doses ranging B from 4.5 to 6.5 mg/kg. The upper dose level [i.e., maximum Mps1 1.0 tolerated dose (MTD)] for once-daily (QD) administration of CFI-402257 was between 6 and 6.5 mg/kg, defined as the highest dose 0.8 that did not cause >10% loss in body weight or induce overt toxicity nM CFI-402257 0.6 DMSO 50 100 400 mock 1 10 25 200 (i.e., signs of ill health or abnormal behaviors, including hunched phospho-Mps1 0.4 posture, poor grooming, urine stains, diarrhea, or dehydration) in (T12/S15) 0.2 accordance with institutional humane endpoint guidelines. EC50 Mps1 0.0 Comprehensive toxicologic evaluation of CFI-402257 adminis- -10 -9 -8 -7 -6 tered to rats and dogs has been completed. In summary, dose-lim-

Phospho-Mps1 (T12/S15)/Mps1 log [CFI-402257] iting toxicities were found to be reversible gastrointestinal effects and myelosuppression, which are consistent with the proposed mecha- Fig. 1. CFI-402257 is a selective inhibitor of Mps1. (A) The chemical structure of CFI-402257. (B) HCT116 cells transfected with full-length human Mps1 were treated nism of action of the molecule (Table S3). The toxicological profile with nocodazole for 17 h. Cells were pretreated with MG132 for 30 min before of CFI-402257 supports its clinical development to determine its treatment with CFI-402257 or DMSO for 4 h. Lysates were analyzed by immunoblot potential as an effective treatment in oncology. analysis with antibodies against phospho-Mps1 (T12/S15) and Mps1. (C)HCT116 cells were treated with nocodazole for 17 h before treatment with CFI-402257 or In Vivo Characterization of the Anticancer Potential of CFI-402257. Phar- DMSO for 4 h. Lysates were analyzed by immunoblot analysis with antibodies macokinetic parameters were determined by treating mice with single against phospho-histone H3 (S10) and histone H3. Band intensities were quantified, oral doses of CFI-402257 (Fig. S3). Plasma was obtained at specified

and EC50 values were calculated by using GraphPad Prism software. time points, and concentrations of CFI-402257 were determined by

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Fig. 2. Mechanism of action studies for CFI-402257. (A) CFI-402257 causes aberrant chromosomal segregation in cancer cells. HCT116 cells were synchronized by using a double thymidine block, released after 6 h, and treated with 200 nM CFI-402257 or DMSO for 2 h. (Left) Representative mitotic figures of CFI-402257– or DMSO- treated cells. Staining of is in green, α-tubulin in red, and DAPI in blue. (Scale bar: 5 μm.) (Right) Graph of the mean percentage of anaphase and telophase cells with and without lagging chromosomes from three independent experiments, with a total of 100 cells examined per treatment condition.Mean percentage of lagging chromosomes ± SEM: DMSO, 11 ± 3%, CFI-402257, 79 ± 6% (P = 0.0002, Student’s t test). (B) CFI-402257 induces aneuploidy with no significant polyploidy in cancer cells. HCT116 cells were treated with CFI-402257 or DMSO for 2 d before staining with propidium iodide and analysis of DNA content by flow cytometry. Percentages of 8N and 16N cells are normalized to the DMSO control. Aneuploidy is defined as having an abnormal numbers of chromosomes. Polyploidy is defined as having an 8N DNA population greater than or equal to 15% after normalization to the DMSO control. Data are representative of three independent experiments. (C) CFI-402257 causes aneuploidy in cancer cells. (Top) Graph of the chromosome counts on metaphase spreads from HCT116 cells treated with 200 nM CFI-402257 or DMSO for 28 h. Data are representative of three independent experiments, with a total of 98 spreads examined per treatment condition. (Bottom) Representative images of metaphase spreads from CFI-402257– or DMSO-treated cells. Chromosomes were stained with DAPI. (D) HCT116 cells were treated for the indicated amounts of time with 0, 50, or 100 nM CFI-402257. Lysates were analyzed by immunoblot analysis with antibodies against cleaved PARP and GAPDH.

liquid chromatography (LC)-tandem MS (LC/MS/MS). CFI-402257 PDX model of high-grade serous ovarian cancer (Fig. 4D). CFI- was absorbed rapidly after oral administration, and exposure [in 402257 was well-tolerated at the doses examined as measured by terms of area under the curve (AUC) and maximum plasma little to no decrease in body weight (<10% change) and normal concentrations (Cmax)] was dose-dependent and approximately behavior. Together, these results indicate that CFI-402257 can in- dose-linear to 55 mg/kg. At the repeat-dose MTD of 6.5 mg/kg, hibit the growth of a range of tumor types and may be effective in the Cmax was 1,070 ng/mL and the AUC0–24h was 3,170 ng·h/mL. a clinical setting. The elimination t1/2 ranged between 2 and 3 h. These data dem- To determine the pharmacodynamics of CFI-402257 in vivo, onstrate that CFI-402257 has a favorable pharmacokinetic profile. phospho-histone H3 serine 10-positive cells were counted in the CFI-402257 has demonstrated efficacy as a monotherapy in MDA-MB-231 breast tumor xenografts treated with the daily dose cancer cell line and patient-derived xenograft (PDX) models across MTD of 6 mg/kg for 3 d or a large acute dose of 35 mg/kg twice daily various indications, and representative studies are described here. (BID) for five doses (Fig. 5). Relative to vehicle controls, a decrease CFI-402257 given orally QD showed dose-dependent activity in in phospho-histone H3 serine 10-positive cells per square millimeter mice with established tumors from xenografted MDA-MB-231 of tumor tissue was measured in CFI-402257–treated tumors (40 human TNBC cells [5 mg/kg CFI-402257, tumor growth inhibition phospho-histone H3-positive cells per square millimeter with 6 mg/kg (TGI) = 74%, P = 0.02; 6 mg/kg CFI-402257, TGI = 89%, P = CFI-402257 QD × 3 treatment, and 29 phospho-histone H3-positive 0.004; Fig. 4A]. Similar results were obtained in tumors from xen- cells per square millimeter with 35 mg/kg CFI-402257 BID × 5 ografted MDA-MB-468 human TNBC cells in mice (5 mg/kg CFI- treatment vs. 70 phospho-histone H3-positive cells per square 402257, TGI = 75%, P = 0.003; 6 mg/kg CFI-402257, TGI = 94%, millimeter with vehicle control treatment). Thus, CFI-402257 P = 0.001; Fig. 4B). CFI-402257 also demonstrated antitumor ac- reduces the mitotic index in vivo, consistent with inhibition of tivity in a platinum-resistant PDX model of high-grade serous Mps1 in vivo. ovarian cancer (6.5 mg/kg CFI-402257, TGI = 61%, P = 0.04; CFI-402257 induces genomic instability and apoptotic cell death, 75 mg/kg , TGI = 97%, P = 0.03; Fig. 4C). Further, CFI- and therefore could promote tumor immunity. To explore the po- 402257 was found to be similarly efficacious in a platinum-sensitive tential to combine Mps1 inhibitors with immune checkpoint

Mason et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 10000 Mouse plasma concentration–time profiles indicate that, follow- ) ing oral administration of efficacious doses of CFI-402257 in mice, nM ( 1000 plasma levels of CFI-402257 were sustained and remained above 50 the EC50 value for half-maximal inhibition of cellular Mps1 auto- 100 phosphorylation and the median growth-inhibition IC50 value for greater than 12 h. Moreover, CFI-402257 monotherapy demon- strated dose-dependent antitumor activity. Analysis of xenograft 10 tumors from mice treated with an efficacious dose of CFI-402257

Growth inhibition IC inhibition Growth showed pharmacodynamic effects consistent with inhibition of 1 Mps1. Together, these results provide evidence that the antitumor activity of CFI-402257 results from its in vivo inhibition of Mps1. PC-3 A549 HT29 SW48 T-47D CAL51

SKBr-3 Mps1 inhibitors BAY 1161909 (ClinicalTrials.gov ID: BT-474 BxPC-3 DU-154 PANC-1 HCT116 HCC202 -MB-361 NCI-H23 SK-OV-3 SK-OV-3 HCC1419 HCC1937 OVCAR-3 NCT02138812), BAY 1217389 (ClinicalTrials.gov ID: – MDA-MB-231 MDA-MB-468 MDA-MB-436 MDA MDA-MB-330 NCT02366949), and S 81694 (EudraCT no.: 2014 002023-10) are currently in phase I clinical trials. As has been presented for CFI- Fig. 3. CFI-402257 inhibits human cancer cell growth. After 5 d of treatment, cell growth was determined by measuring total protein content by sulforhodamine 402257, these inhibitors have cellular on-target activity and have significant tumor growth-inhibitory activity in preclinical models (27, B (SRB) assay, and growth-inhibition IC50 values were calculated by using GraphPad Prism software. 28). However, comparison of the reported pharmacological prop- erties and preclinical data for these inhibitors reveals differences. CFI-402257 shows higher kinase selectivity compared with BAY inhibitors, immunocompetent BALB/cJ mice were inoculated 1161909, BAY 1217389, or S 81694. Although additional activities with syngeneic CT26 mouse colon carcinoma cells and then treated could provide enhanced efficacy in particular settings, they may also with CFI-402257 alone and in combination with an anti–PD-1 an- result in reduced tolerance. Specifically, the toxicities observed for tibody (Fig. 6). Tumors in the vehicle-treated control arm grew this class of drug targets (i.e., myelosuppression and gastrointestinal rapidly, and the average size was >1,500 mm3 by day 11 of treat- disturbances) suggest that the balance between efficacy and toler- ment. Although there was tumor growth delay in the anti–PD-1 ability may not be one that allows additional activities to contribute antibody- and the CFI-402257–treated single-agent arms, there to an antitumor effect. These inhibitors have been evaluated by were no instances in which complete regression was observed. In the using different dosing schedules and routes of delivery. Daily oral combination anti–PD-1 antibody and CFI-402257–treated arm, however, two of the eight tumors completely regressed. Very similar results were also seen in a duplicate experiment (Fig. S4), again with complete regression (two of eight tumors) only seen in the combi- AB 500 Vehicle 350 Vehicle nation arm. In the former experiment, the two animals in CFI-402257, 5 mg/kg PO, QD CFI-402257, 5 mg/kg PO, QD 300 ) CFI-402257, 6 mg/kg PO, QD ) CFI-402257, 6 mg/kg PO, QD 3 which complete regression had occurred were rechallenged by 400 3 inoculation with CT26 cells on day 31. Tumors did not grow in 250 either mouse, indicating that immunity to the CT26 cells had 300 200 been generated. 200 150 Discussion 100

Tumor volume (mm 100 The primary function of the SAC is to prevent chromosome mis- Tumor volume (mm 50 segregation and the generation of aneuploid or polyploid cells. Thus, 0 0 the SAC ensures healthy proliferation and precise division in cells, and 0246810121416182022 02468101214161820 Day Day targeting this checkpoint could provide new therapeutic opportunities C D for killing cancer cells. Differently from antimitotic/antitubulin ther- 750 Vehicle 1000 Vehicle apy, which arrests cell proliferation, inactivation of the SAC would CFI-402257, 6.5 mg/kg PO, QD CFI-402257, 6.5 mg/kg PO, QD Carboplatin, 75 mg/kg IP, QWX2 Carboplatin, 75 mg/kg IP, QWX2 ) )

lead to an accelerated mitosis and the consequent generation of lethal 3 3 750 genomic instability in cancer cells. The strong requirement for Mps1 500 for the SAC has made it a candidate for this therapeutic approach, and has generated intense interest in developing small-molecule in- 500 hibitors against this kinase (4, 9–12, 25, 26). 250 We report here the functional characterization of CFI-402257, 250 Tumor volume (mm a potent and orally active inhibitor of Mps1 that has entered a Tumor volume (mm phase I clinical trial (ClinicalTrials.gov ID: NCT02792465). CFI- 0 0 02468101214161820 02468101214161820 402257 is highly selective toward Mps1 relative to other protein Day Day and lipid kinases, and did not inhibit several kinases that are known for their role in mitosis. CFI-402257 pharmacologically re- Fig. 4. In vivo characterization of the anticancer potential of CFI-402257 on capitulates previous findings obtained upon Mps1 deletion or with human xenograft tumors. (A) CD-1 Nude mice with established MDA-MB-231 other Mps1-specific inhibitors (4, 9–12). Specifically, CFI-402257 xenografts were treated for 22 d (n = 7). CFI-402257 5 mg/kg orally QD vs. vehicle, treatment potently inhibited Mps1 kinase activity in cancer cells, and TGI = 74%, P = 0.02; CFI-402257 6 mg/kg orally (PO) QD vs. vehicle, TGI = 89%, P = causes cancer cells to exit mitosis with missegregated chromosomes, 0.004. (B) CD-1 nude mice with established MDA-MB-468 xenografts were treated which results in severe aneuploidy and finally cell death. Cancer cell for 21 d (n = 7). CFI-402257 5 mg/kg orally QD vs. vehicle, TGI = 75%, P = 0.003; = = line profiling demonstrated that CFI-402257 has broad and strong CFI-402257 6 mg/kg orally QD vs. vehicle, TGI 94%, P 0.001. (C)NODscid antiproliferative activity. Although cell lines with high and low sen- gamma (NSG) mice with established human platinum-resistant ovarian tumor xenografts were treated for 21 d (n = 6). CFI-402257 6.5 mg/kg orally QD vs. ve- sitivity toward CFI-402257 treatment were identified, no single gene hicle, TGI = 61%, P = 0.04; carboplatin 75 mg/kg i.p. weekly × 2(QWX2)vs.vehicle, implicated via mutation in human cancer could be clearly correlated. TGI = 97%, P = 0.03. (D) NSG mice with established human platinum-sensi- This suggests that multiple and/or -specific factors tive ovarian tumor xenografts were treated for 21 d (n = 6). CFI-402257 6.5 may determine the cellular sensitivity to Mps1 inhibitors. Further mg/kg orally QD vs. vehicle, TGI = 66%, P = 0.11; carboplatin 75 mg/kg i.p. investigation is ongoing to identify candidate predictive biomarkers QWX2 vs. vehicle, TGI = 124%, P = 0.02. Data are represented as mean ± SEM for CFI-402257 and to understand which particular class of malig- (for figure clarity, only positive error bars are shown). P values were calculated by nancies might benefit from Mps1-targeted agents. using Student’s t test.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1700234114 Mason et al. Downloaded by guest on September 30, 2021 p=0.002 clinical activity. Here, we show that the combination of CFI- 402257 and an anti–PD-1 antibody causes tumor regression and p=0.009 80 increases the survival in a murine colon cancer model. The reason for this response is likely complex and under investigation. 70

2 One possible explanation may lie in the genomic instability and 60 in tumor cells caused by CFI-402257–induced Mps1 50 inhibition. These events might lead to the creation of new immu- 40 nogenic epitopes, which are able to induce cytotoxic T lymphocyte activity against tumor cells. Further studies of immune-checkpoint 30 inhibitors are merited to support their use in combination treat- 20 ment with Mps1 inhibitors. positive nuclei per mm positive nuclei Phospho-histone H3 (S10) Phospho-histone H3 10 In summary, CFI-402257 is a potential small-molecule in- 0 hibitor for the treatment of cancer with a mechanism of action Vehicle 6 mg/kg 35 mg/kg that is consistent with potent and selective inhibition of Mps1. CFI-402257 CFI-402257 QDx3 BIDx5 CFI-402257 has been approved for human use by Health Canada, and a phase I clinical trial has been initiated. Fig. 5. In vivo effect of CFI-402257 on human xenograft tumors. C.B.-17 severe combined immunodeficiency (SCID) mice with established MDA-MB-231 xe- Methods nografts were treated with CFI-402257 6 mg/kg orally QD or vehicle for 3 d (n = Compounds and Plasmids. CFI-402257 was synthesized as described previously 3) or CFI-402257 35 mg/kg orally BID for 5 d (n = 3); mice were killed and tumor (23). The hydrochloride or bisphosphate hemihydrate salt forms of CFI- ± tissue was removed 4 h after the final dose. Graph shows the mean SEM of 402257 were used in all studies. Thymidine, nocodazole, and MG132 were the number of phospho-histone H3 (S10)-positive nuclei per square millimeter obtained from Sigma. Rat IgG2a anti–PD-1 antibody (clone RMP1-14; cat. no. 2 of tumor tissue, with 14.4 mm of tumor tissue analyzed for the vehicle control BE0146) and rat IgG2a isotype control (clone 2A3; cat. no. BE0089) were 2 – tumors, 14.5 mm of tumor tissue analyzed for the 6 mg/kg CFI-402257 treated obtained from Bio X Cell. Carboplatin was obtained from Teva Canada. 2 – tumors, and 32.7 mm of tumor tissue analyzed for the 35 mg/kg CFI-402257 The expression construct encoding full-length human Mps1 was obtained ’ treated tumors. P values were calculated by using Student s t test. from Origene.

Animals. C.B.-17 SCID and NSG mice were obtained from the animal colony at dosing has been used for CFI-402257, intermittent oral dosing for the Ontario Cancer Institute of the University Health Network (Toronto). CD- CELL BIOLOGY BAY 1161909 and BAY 1217389, and intermittent i.v. dosing for S 1 Nude mice were obtained from Charles River Laboratories. BALB/cJ mice 81694. Notably, the preclinical studies for these inhibitors have were obtained from The Jackson Laboratory. Six- to eight-week-old female shown that both daily and intermittent dosing schedules are effi- animals were used for the studies and were allowed unrestricted access to cacious and generally well-tolerated, giving confidence that an food and water. All animal procedures were approved by the institutional efficacious and tolerated schedule can be found in patients. animal care and use committee of the University Health Network (Toronto). Preclinical efficacy results for BAY 1161909 and BAY 1217389 showed moderate activity as monotherapies, but strong coopera- Xenograft Tumor Growth. The PDXs were generated from platinum-resistant or tivity with , and this combination has advanced to the clinic platinum-sensitive high-grade serous ovarian cancers that were obtained from the (discussed later). CFI-402257 and S 81694 showed single-agent University Health Network Tissue Bank with patient consent and research ethics activity in preclinical studies, and have entered clinical testing in board approval (protocol 06–0903T) (34). Xenografts were established by injection × 6 monotherapy. The relative importance of these factors and other of 1 10 H2K-depleted tumor cells in 1:1 HBSS:growth factor-reduced Matrigel subtle differences between these drug candidates, and others that (BD Biosciences) into mammary fat pad of female NSG mice. Animals were ran- may emerge, as well as the genetic makeup of the tumor, will be domized, and treatments were initiated when tumor volumes reached 100−170 mm3. Cancer cell line xenografts were established by injecting established by ongoing clinical studies. s.c. 1 × 107 tumor cells into the flank of female mice. Animals were randomized, Mechanism-based combination strategies are being explored for Mps1 inhibitors in an effort to expand their therapeutic utility and delay the onset of resistance. Mps1 inhibitors are mitosis-promoting agents that, when used with antitubulin agents, 2000 Control 2000 Anti-PD-1, 150 µg, Day 0, 3, 6, 10 IP ) ) 3 which perturb proper chromosome alignment, are expected to 3 increase chromosome segregation errors above the threshold re- 1500 1500 quired to kill cancer cells. In support of this hypothesis, Mps1 inhibitors have been shown to sensitize cancer cells to 1000 1000 treatment (29), and this approach is being tested clinically (27). 500 500 Furthermore, the combination of Mps1 and cyclin-dependent Tumor volume (mm Tumor volume (mm kinase 4/6 inhibition has been investigated as a strategy to in- 0 0 crease the therapeutic window for Mps1 inhibitors (30). Cdk4/6 0 10 20 30 40 50 0 10 20 30 40 50 inhibitors trigger G1 cell cycle arrest in Rb1-competent cells, and Day Day thus might improve the tolerability and efficacy of Mps1 inhibi- 2000 CFI-402257, 6 mg/kg PO, QD 2000 CFI-402257 + Anti-PD-1 ) )

tors. Early results support this hypothesis, and suggest that the 3 3 combination may prove particularly effective in Rb1-dysregu- 1500 1500 lated cancers. The rationale for another combination approach for Mps1 inhibitors may lie in the proteotoxic or metabolic stress 1000 1000 caused by the chromosome imbalance in aneuploid cancer cells (31). Here, treatment of cancer cells with Mps1 inhibitors that 500 500 Tumor volume (mm Tumor volume (mm increase aneuploidy could synergize with ones that target the 0 0 proteotoxic or metabolic stress induced by aneuploidy. Whether 0 10 20 30 40 50 0 10 20 30 40 50 aneuploidy-induced stress pathway inhibitors might synergize Day Day with Mps1 inhibitors remains to be determined. , Fig. 6. CFI-402257 in combination with anti–PD-1 antibodies induces complete targeted therapies, and radiotherapy can promote tumor im- regressions in the syngeneic CT26 model. When CT26 tumors reached an average munity by inducing immunogenic cell death (32, 33). Combi- target size of ∼60 mm3, Balb/cJ mice were treated with four doses of anti–PD-1 nations of these cancer therapies with immune-checkpoint antibody (150 μg on days 0, 3, 6, and 10) or 21 doses of CFI-402257 6 mg/kg orally inhibitors are being explored to achieve additive or synergistic (PO) QD. The size of each individual tumor within each treatment arm is plotted.

Mason et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 and treatments were initiated when tumor volumes reached 80−100 mm3.To initiation of treatment. In cases in which tumor regression occurred, percentage

treat an established tumor, CFI-402257 and the vehicle [10% (vol/vol) NMP/40% of tumor regression was defined as 100 × [1 − (TVf,treated/TVi,treated)]. At the (vol/vol) PEG-300/50% (vol/vol) water for the hydrochloride form, or 80–90% (vol/ completion of the study, the mice were killed by an overdose, and vol) PEG-300 or PEG-400/20–10% (vol/vol) water for the bisphosphate hemi- tumor tissue was removed for further studies. hydrate form] were administered by oral gavage, and carboplatin, anti–PD-1 antibody, and isotype control were administered by i.p. injection to mice as ACKNOWLEDGMENTS. The authors thank Benjamin Neel for providing the described earlier. Animal weights were monitored daily, and tumor volume was ovarian cancer PDX models and members of The Campbell Family Institute measured three times per week. Tumor volume (in cubic millimeters) was de- for Breast Cancer Research for helpful discussions. This work was supported fined as length × width2/2. Percentage of TGI was defined as 100 × [1 − by the Princess Margaret Cancer Foundation, the Canadian Institutes of (TVf,treated − TVi,treated)/(TVf,control − TVi,control)], where TVf is the average tumor Health Research, Genome Canada, and the California Institute for volume at the end of study and TVi is the average tumor volume at the Regenerative Medicine.

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