dentifying Anti-cancer Effects and Exploring the Mechanism of an MPS1/TTK Inhibitor in Gastric Cancer

1. Materials

MPS1/TTK inhibitor, compound-9, is a highly selective MPS1 inhibitor based on a 7H pyrrolopyrimidine-5-carbonitrile scaffold. Compound-9 exhibits remarkable selectivity and efficacy, particularly showcasing significant anti-cancer effects in triple-negative breast cancer models [22].

2. Cell lines and cultures

Among the 61 human GC cell lines, 38 of cell lines were established by Song-Dang Institute for Cancer Center and 23 of cell lines were purchased at three different organizations worldwide (American Type Culture Collection, Korean Cell Line Bank and Japanese Collection of Research Bioresources Cell Bank). Cell lines were maintained using their proper complete growth medium. Basically, Eagle’s minimum essential medium, Dulbecco’s modified Eagle medium and Roswell Park Memorial Institute-1640 (RPMI-1640) (Gibco) contained 1% of antibiotics (Lonza) and 5% of fetal bovine serum (Lonza) were consumed for the maintenance of cell lines. Cell lines were grown at 37℃ in 5% CO₂ incubator.

3. Genomic profiling

In-house targeted deep sequencing (CancerMaster), whole exome sequencing (WES) and RNA sequencing data of the 61 GC cell lines were obtained from the genome database of Song-dang Institute for Cancer Research (SICR), Yonsei University College of Medicine [23,24]. TP53, receptor tyrosine kinases (RTKs) (MET, fibroblast growth factor receptor 2, ERBB2, epidermal growth factor receptor), and DNA damage repair (DDR)–related genes were analyzed using CancerMaster [23,25]. MPS1/TTK was analyzed by WES. The mRNA expression levels were measured in transcripts per kilobase million (TPM) without normalization. The GC cell lines were classified according to The Cancer Genome Atlas (TCGA) subgroups based on CancerMaster [23,25]. The 61 GC cell lines were grouped as Epstein-Barr virus (n=2, 3.3%), microsatellite-instable–high (MSI-H; n=4, 6.6%), CIN-likely (n=27, 44.3%), and genomically stable (GS)–likely (n=28, 46.0%) (Fig. 1). In this paper, the CIN and GS types suggested by TCGA are stated as CIN-likely and GS-likely. The 61 GC cell lines used in this study cannot be categorized by the same method as TCGA and we used the gene signature of CIN and GS group presented by TCGA for classification. The tumor mutation burden (TMB) was examined using CancerMaster. The TMB ranged from 2.9 to 63.2, with a median of 10.2. The biological effects of different mutations were assessed using the OncoKB database (https://www.oncokb.org/).

Fig. 1.

Molecular-genomic profiling in 61 gastric cancer (GC) cell lines. Tumor mutation burden (TMB) was counted as the total number of somatic mutations by next-generation sequencing, The Cancer Genome Atlas (TCGA) subtype of 61 GC cell lines and landscape of monopolar spindle 1 (MPS1)/threonine tyrosine kinase (TTK), TP53, receptor tyrosine kinase (RTK)–RAS, phosphoinositide 3-kinase (PI3K), and DNA damage repair–related genes with genetic alterations. CIN, chromosomal instability; EBV, Epstein-Barr virus; GS, genomically stable; HR, homologous recombination; MSI-H, microsatellite-instable–high; NHEJ, non-homologous end joining.

4. Cell viability assay

The cells (8×10³/well) were seeded in 96-well plates overnight at 37℃ and exposed to specific doses of MPS1/TTK inhibitor (0-10 µM) at 37℃ for 72 hours. Cell Counting Kit-8 (Dojindo) solution was added, and the plates were incubated at 37℃ for a further 2 hours. The absorbance was measured at 450 nm and IC₅₀ values were determined using the CalcuSyn software (Biosoft). The cell lines were divided into sensitive, moderate, and resistant groups using the IC₅₀ values. All experiments were repeated three times.

5. Western blotting

Cells were lysed in the M-PER mammalian protein extraction reagent (Pierce) containing phosphatase inhibitor (Sigma) and protease inhibitor (Roche). Protein concentrations were determined by the Bradford Assay (Bio-Rad). Proteins were separated by 8%-15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Non-specific antibody binding was reduced by blocking process with 5% skim milk at 1hrs incubation. Membranes were incubated overnight at 4℃ with following primary antibodies: cyclinB1 (sc-245, Santa Cruz Biotech), phospho-histone H2A.X (γ-H2AX) (Ser139) (9718S, Cell Signaling), p21^WAF1 (OP64, Calbiochem), BUBR1 (612503, BD Biosciences), phospho-BUBR1 (612503, BD Biosciences), MPS1/TTK (35-9100, Invitrogen), and α-tubulin (T6199, Sigma) After 6 times of wash, membranes were incubated for 2 hours at room temperature with horseradish peroxidase–conjugated secondary antibody. anti-mouse (1706516, Bio-Rad) or anti-rabbit (1721019, Bio-Rad) antibodies were used as secondary antibodies. Immunoblots were developed using LumiFlash Ultima Chemiluminescent Substrate (Visual Protein) and visualized by ChemiDoc XRS+system (Bio-Rad). Data were normalized to α-tubulin, and protein intensity was semi-quantified using the ImageJ software (NIH). All experiments were repeated three times and quantification was performed with ImageJ using α-tubulin as loading control.

6. Cell cycle analysis

Cells (1×10⁶/well) were seeded in 60-mm cell culture dishes overnight at 37°C, treated with 0.05, 0.5, and 1 µM MPS1/TTK inhibitor for 12, 24, 48, and 72 hours and harvested with 0.25% trypsin/EDTA (Gibco), before being fixed with cold 75% ethanol at −20℃ for at least 24 hours. Next, cells were washed twice with cold phosphate buffered saline (PBS), and stained with propidium iodide/RNase Staining Buffer (BD Pharmingen) for 20 minutes in the dark at 20℃. Cell cycle distribution was analyzed using a BD LSRII (BD Pharmingen) and the Flow Jo software (Tristar). All experiments were repeated three times.

7. Apoptosis assay

Cells (1×10⁶/well) were seeded in 60-mm cell culture dishes and incubated for 24 hours. Cells were then treated with drugs for 12 and 24 hours and the supernatant (containing floating death cells) and adherent cells were collected together. The collected cells were washed twice with PBS, then centrifuged. The pellet was resuspended in 100 µL of AnnexinV binding buffer and stained using BD AnnexinV/FITC apoptosis kit (BD Biosciences). Apoptotic cells were measured using flow cytometry. All experiments were repeated three times.

8. Statistical analysis

All assays were performed independently at least three times unless indicated. Mean with standard error mean was represented in each graph. IBM SPSS Statistics for Windows ver. 27.0 software (IBM Corp.) was used for statistical analysis including correlation analysis, paired, non-paired t tests, correlation coefficient, and chi-square test.

1. Molecular-genomic profiling of 61 GC cell lines

We profiled the status of MPS1/TTK, TP53, and DDR-related genes (Fig. 1). Frameshift mutation at coding mononucleotide repeats (cMNR) in the MPS1/TTK gene were observed in four of the 61 cell lines (6.6%) using WES, all of which belonged to the MSI-H subgroup. These cMNR mutations are known to result in the production of truncated MPS1/TTK proteins and occur at a high frequency in microsatellite-instable (MSI) colorectal cancer and GC [26]. The mRNA level of MPS1/TTK is expressed at low levels (0.2-153 TPM). However, the mRNA and protein expression levels of MPS1/TTK showed a moderate correlation in 61 GC cell lines (R²=0.1801, p < 0.001).

According to in-house deep sequencing, 46 GC cell lines (75%) containing TP53 alterations (32 missense mutations, 11 truncating mutations, or 4 splicing variants) were discovered. Moreover, we also checked any alterations of DDR-related gene changes which are based on 35 genes that involved in two mechanisms of double-strand break repair, homologous recombination and non-homologous end joining (Fig. 1) [27]. As a result, we confirmed 17 GC cell lines (28%) had DDR alterations (Table 1). Based on 61 GC cell lines genomic profiling, we performed experiments to identify anti-cancer effect and explore the mechanisms of MPS1/TTK inhibitor in the GC cell lines.

Table 1.

MPS1/TTK inhibitor sensitivity group’s distribution by genomic profiling

2. Anti-proliferative effects of the MPS1/TTK inhibitor in 61 GC cell lines

Next, we evaluated the effects of the MPS1/TTK inhibitor in 61 GC cell lines. We confirmed cell line viability to check the sensitivity of the MPS1/TTK inhibitor. Moreover, we classified the cell lines according to their IC₅₀ values, ranged from 0.002 µM to ~10 µM, with a median value of 1.80 and a mean value of 4.15 (Fig. 2A, S1 Fig.). Cell lines were classified into three groups: sensitive (IC₅₀ ≥ 0.1 µM), moderate (0.1 µM < IC₅₀ < 10 µM), and resistant (IC₅₀ ≥ 10 µM). As a result, the viability of 61 cell lines treated with the MPS1/TTK showed that 34% (n=21) of the cell lines were sensitive, 38% (n=23) were moderate, and 28% (n=17) were resistant to the inhibitor (Table 1).

Fig. 2.

Monopolar spindle 1 (MPS1)/threonine tyrosine kinase (TTK) inhibitor sensitivity in 61 gastric cancer cell lines. (A) At the top panel, Waterfall plots representing IC₅₀ (µM) values for each cell lines determined using the CalcuSyn software (color-coded as per the legend: sensitive, blue [0.1 µM ≤ IC₅₀]; moderate, red [0.1 µM < IC₅₀ < 10 µM]; resistant, green [IC₅₀ ≥ 10 µM]). At the middle panel, Protein expression of MPS1/TTK was determined by western blotting with α-tubulin as loading control and compared to YCC-25. Quantification of each protein levels is indicated on the band above. At the bottom panel, mRNA expression was analyzed using RNA sequencing data and is color-coded according to the heatmap legend. (B) Correlation between MPS1/TTK mRNA expression level (transcripts per kilobase million [TPM]) and MPS1/TTK protein expression. (C) Association between MPS1/TTK inhibitor sensitivity and MPS1/TTK mRNA expression level (TPM). (D) Association between MPS1/TTK inhibitor sensitivity MPS1/TTK protein expression (unpaired t test with Welch’s correction between sensitive and resistant group). (E) Dose-response curves for the 61 gastric cancer (GC) cell lines according to The Cancer Genome Atlas subtype. MPS1/TTK inhibitor cytotoxicity was evaluated using Cell Counting Kit-8 assays and is expressed as the mean percentage cell viability according to The Cancer Genome Atlas subtype (color-coded as per the legend: Epstein-Barr virus [EBV], blue; microsatelliteinstable–high [MSI-H], red; genomically stable (GS)–likely, orange; chromosomal instability [CIN]–like, green). (F) Dose-response curves for the 61 GC cell lines according to TP53 status (color-coded as per the legend: TP53_wild type [WT], red; TP53_mutant, blue). The statical tests performed with chi-square test and data are expressed as mean±standard error of mean.

3. Association between molecular-genomic profiling and sensitivity to the MPS1/TTK inhibitor

Next, we compared the sensitivity of 61 GC cell lines to MPS1/TTK inhibitor with the previous molecular-genomic profiling data in Fig. 1. There was no correlation observed between IC₅₀ values and MPS1/TTK mRNA or protein expressions not like inhibitor treatment (Fig. 2B-D). Next, we compared sensitivity to the MPS1/TTK inhibitor according to the TCGA subtype (Fig. 2E). Of the four cell lines in the MSI-H group, three were sensitive (75%), and one was moderately sensitive (25%). Of the 27 cell lines in the CIN-likely group, 13 cell lines were sensitive (48.2%), five cell lines were moderate (18.5%), and nine cell lines were resistant (33.3%). Of the 28 cell lines in the GS-likely group, four cell lines were sensitive (14.8%), 16 cell lines were moderate (57.1%), and eight cell lines were resistant (28.6%). Intriguingly, MSI-H group was highly sensitive to MPS1/TTK inhibitor. This result consistent with previous reports of high sensitivity to the MPS1/TTK inhibitor in MSI colorectal cancers [26].

The GS-likely group tended to be moderate-to-resistant, whereas the CIN-likely group was extremely sensitive or resistant to the MPS1/TTK inhibitor. The CIN-likely group was more sensitive than the GS-likely group (chi-squared p=0.0051) (Table 1). Based on this, we investigated the differences in cell lines that were sensitive or resistant to the MPS1/TTK inhibitor in the CIN-likely group.

Next, we examined TP53 status to identify its influence on the response to the MPS1/TTK inhibitor (Fig. 2F). The TP53^WT group was more sensitive than the TP53^MUT group (chi-squared p < 0.001) (Table 1). This suggests that deleterious CIN induced by the MPS1/TTK inhibitor triggers detection by p53, leading to cell death in p53-competent cells [20]. Interestingly, TP53^WT and CIN-likely GC cell lines were sensitive to the MPS1/TTK inhibitor, whereas TP53^MUT and CIN-likely GC lines were sensitive or resistant (Table 1). From these results, MPS1/TTK resistant and sensitive cell lines based on TP53 status in the CIN-likely group were selected for further studies.

4. Effect of the MPS1/TTK inhibitor on cell-cycle progression

We investigated the effects of MPS1/TTK inhibitor treatment on cell-cycle progression and genomic instability using flow cytometry on YCC-47 (IC₅₀=0.02 µM, sensitive, TP53^WT), YCC-30 (IC₅₀=0.07 µM, sensitive, TP53^MUT), and YCC-28 (IC₅₀ > 10 µM, resistant, TP53^MUT) cell lines (Fig. 3).

Fig. 3.

Effect of the monopolar spindle 1 (MPS1)/threonine tyrosine kinase (TTK) inhibitor on cell-cycle progression. Effect of the MPS1/TTK inhibitor on cell-cycle progression in YCC-47 (TP53^WT, sensitive) cell line (A), YCC-30 (TP53^MUT, sensitive) cell line (B), and YCC-28 (TP53^MUT, resistant) cell line (C). At the upper panel, histograms of cell cycle analysis after treatment with the MPS1/TTK inhibitor were represented (The y-axis represents cell counts, and the x-axis represents DNA modal contents). At the lower panel, cell cycle distribution bar graph (The y-axis represents the percentage of cell population, and the x-axis represents the conditions) was represented.

After 24 hours of treatment, all three cell lines showed a reduction in the percentage of cells in the G0/G1 phase, with an increase in the G2/M phase and aneuploidy or polyploidy (> 4N) (Fig. 3A-C). However, this increase was most pronounced in YCC-47, the most sensitive cell line (Fig. 3A). After 48 hours of treatment, the YCC-47 cell line had less aneuploid or polyploid (> 4N ploidy) than before 24 hours of treatment, with a significant increase in the sub-G1 phase (Fig. 3A). In contrast, YCC-28 cells remained mostly in the G2-M phase and still developed aneuploidy and polyploidy (> 4N), similar to the 24-hour treatment (Fig. 3C). The YCC-30 cell line showed a significant increase in 8N ploidy compared to the other two cell lines (Fig. 3B). After 72 hours of treatment, the YCC-47 cell line showed concentration-dependent cell cycle collapse, with an increase in the sub-G1 phase (Fig. 3A). In YCC-30, the majority of cells had shifted towards 8N polyploidy and sub-G1 phase, whereas YCC-28 continued to exhibit resistance, with most cells remaining in G2/M and showing no increase in subG1 (Fig. 3B and C).

These results suggest that MPS1/TTK inhibition leads to rapid apoptosis in sensitive cell lines like YCC-47, whereas resistant lines like YCC-28 can tolerate aneuploidy and polyploidy, contributing to their drug resistance. YCC-30 shows a delayed response, eventually leading to cell death through polyploidy.

5. Changes in molecular factors and cell death induced by the MPS1/TTK inhibitor

To explore further mechanisms of the MPS1/TTK inhibitor to the YCC-47, YCC-30, and YCC-28 cell lines, we checked protein expression of MPS1/TTK, cyclin B1, γ-H2AX, and p21 after 24- or 48-hours treatment at concentrations of 0.05, 0.5, or 1 (Fig. 4).

Fig. 4.

Changes in molecular factors and cell death induced by the monopolar spindle 1 (MPS1)/threonine tyrosine kinase (TTK) inhibitor. Protein expression of MPS1/TTK, cyclin B1, γ-H2AX, and p21 after 24- or 48-hour treatment with 0.05, 0.5, or 1 µM of the MPS1/TTK inhibitor in YCC-47 cell line (A), YCC-30 cell line (B), and YCC-28 cell line (C). Quantification of each western blot data is represented on YCC-47 cell line (D), YCC-30 cell line (E), and YCC-28 cell line (F). α-tubulin was used as a loading control for each experiment. All the statical test was compared to control group (blue) with the Wilcoxon matched-pairs signed rank test. Data are expressed as mean±standard error or mean; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

First, MPS1/TTK expression levels decreased in YCC-47 and YCC-30 cells in a time- and concentration-dependent manner (Fig. 4A, B, D, and E). However, in YCC-28 cells, MPS1/TTK levels remained unchanged across all concentrations (Fig. 4C and F), indicating resistance to the inhibitor in this cell line. Second, the cyclin B1, a substrate of APC/C, exhibited different patterns of regulation across the cell lines. YCC-30 cells showed a significant reduction in cyclin B1 after 24 hours of treatment at 1 µM (Fig. 4B and E). In contrast, YCC-47 (Fig. 4A and D) and YCC-28 (Fig. 4C and F) cell lines demonstrated increased cyclin B1 levels after the same treatment. By 48 hours, cyclin B1 levels continued to decrease in YCC-47 cell line (Fig. 4A and D), while YCC-28 cell line (Fig. 4C and F) exhibited a concentration-dependent decline in cyclin B1 expression. Third, γ-H2AX, a marker of DNA damage, showed an increase in both YCC-47 and YCC-30 cells after treatment with the MPS1/TTK inhibitor (Fig. 4A, B, D, and E). This suggests that DNA damage accumulation was induced by MPS1/TTK inhibition in the more sensitive cell lines. At last, p21 expression was increased in the TP53^WT YCC-47 (Fig. 4A and D) cell line upon treatment with the MPS1/TTK inhibitor. However, this increase was not observed in the TP53^MUT cell lines, YCC-30 and YCC-28 (Fig. 4B, C, E, and F).

These results suggest that YCC-47 and YCC-30 cells undergo significant DNA damage accumulation in response to MPS1/TTK inhibition, as evidenced by increased γ-H2AX levels and cell cycle arrest at the G2/M phase. In contrast, YCC-28 cells exhibited resistance to the inhibitor, with no significant changes in DNA damage markers or cell cycle progression, further indicating their resistance to MPS1/TTK inhibition.

6. Apoptosis induced by the MPS1/TTK inhibitor

The effect of the MPS1/TTK inhibitor on the apoptosis was determined by apoptosis assay (Fig. 5). Percentage of apoptotic cells increased significantly in the YCC-47 (Fig. 5A and D) or YCC-30 (Fig. 5B and E) cell lines in a concentration- and time-dependent manner, whereas YCC-28 (Fig. 5C and F) cell line does not show any increase. Additionally, the YCC-47 cell line exhibited a high proportion of apoptotic cells, while the YCC-30 cell line had a high proportion of necrotic cells, suggesting that cell death occurred through different mechanisms in these two cell lines.

Fig. 5.

Apoptosis induced by the monopolar spindle 1 (MPS1)/threonine tyrosine kinase (TTK) inhibitor. Representative flow cytometry plots obtained using annexin V–FITC/propidium iodide (PI) staining for apoptosis analysis in YCC-47 cell line (A), YCC-30 cell line (B), and YCC-28 cell line (C) (The y-axis represents % of apoptotic or necrotic cells, and the x-axis represents MPS1/TTK inhibitor concentrations). Quantification of each apoptosis analysis data is represented on YCC-47 cell line (D), YCC-30 cell line (E), and YCC-28 cell line (F) (The y-axis represents PI, and the x-axis represents annexin V–FITC). All the statical test was compared to ctr group with the Wilcoxon matched-pairs signed rank test. Data are expressed as mean±standard error or mean; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

These results suggest that DNA damage accumulated in a time- and concentration-dependent manner in YCC-47 and YCC-30 cell lines, leading to DNA damage stress-induced cell death. Additionally, the post-mitotic checkpoint was activated in the YCC-47 cell line upon MPS1/TTK inhibitor treatment, but not in YCC-30 cells, suggesting that cell death occurred through different mechanisms in the YCC-47 and YCC-30 cell lines.

7. Differential cell cycle recovery after SAC activation in YCC-47 and YCC-28 cell lines following MPS1/TTK inhibition

We next examined the impact of the MPS1/TTK inhibitor on the SAC activity in TP53^WT (YCC-47) and TP53^MUT (YCC-28) cells lines. SAC activity was induced by treating cells with nocodazole, a MT depolymerizer, which arrests cells in the G2/M phase by generating unattached kinetochores (Fig. 6).

Fig. 6.

Comparisons of inhibition of monopolar spindle 1 (MPS1)/threonine tyrosine kinase (TTK) function and spindle assembly checkpoint activity induced by the MPS1/TTK inhibitor. Effect of the MPS1/TTK inhibitor on cell-cycle progression in YCC-47 cell line (A) and YCC-28 cell line (B). At the left panel, histograms of cell cycle analysis were represented (The y-axis represents cell counts, and the x-axis represents DNA modal contents). At the right panel, cell cycle distribution bar graph were represented (The y-axis represents the percentage of cell population, and the x-axis represents the conditions). All the statical test was compared to media group with the Wilcoxon matched-pairs signed rank test.

Analysis of cell cycle distribution showed that both YCC-47 (Fig. 6A) and YCC-28 (Fig. 6B) cells were arrested in the G2/M phase upon nocodazole treatment. Notably, YCC-47 cells displayed a higher proportion of polyploid cells (> 4N) when arrested with nocodazole compared to YCC-28 cells. Following a 4-hour release from nocodazole, the G2/M arrest was reduced in YCC-47 cells, leading to increased aneuploidy and polyploidy, with some cells recovering to the G1 phase (Fig. 6A). In contrast, YCC-28 cells remained largely arrested in the G2/M phase, with limited polyploidy, and the recovery of these cells into the G1 phase was also observed (Fig. 6B). When treated with the MPS1/TTK inhibitor, YCC-47 cells failed to recover to the G1 phase, showing a significant increase in the sub-G1 population, indicating cell death (Fig. 6A). In contrast, YCC-28 cells did not exhibit an increase in the sub-G1 population and remained in the G2/M phase, suggesting resistance to the inhibitor’s effects (Fig. 6B).

These results suggest that the MPS1/TTK inhibitor disrupted proper cell cycle recovery in YCC-47 cells following SAC activation, leading to cell death. In contrast, YCC-28 cells were able to proceed through the cell cycle and exhibited resistance to cell death induced by MPS1/TTK inhibition.

8. Changes in molecular factors after SAC activation in YCC-47 and YCC-28 cell lines following MPS1/TTK inhibition

In the previous paragraph, YCC-47 and YCC-28 cell lines were treated with nocodazole to induce SAC activation by arresting the cells in the G2/M phase. After release from nocodazole, we assessed molecular changes, including the expression levels of MPS1/TTK, cyclin B1, and phosphorylated or non-phosphorylated BUBR1, one of the downstream molecules of MPS1/TTK, in response to MPS1/TTK inhibitor (Fig. 7).

Fig. 7.

Changes in molecular factors after spindle assembly checkpoint activation in YCC-47 and YCC-28 cell lines following monopolar spindle 1 (MPS1)/threonine tyrosine kinase (TTK) inhibitor treatment. After nocodazole-induced G2-M arrest, protein expression of MPS1/TTK, cyclin B1, and phospho- or non-phosphorylated BUBR1 treatment with 0.05, 0.5, or 1 µM of the MPS1/TTK inhibitor in YCC-47 cell line (A) or YCC-28 cell line (B). Quantification of each western blot data is represented on YCC-47 (C) or YCC-28 (D). α-tubulin was used as a loading control for each experiment. All the statical test was compared to Media group with the Wilcoxon matched-pairs signed rank test. Data are expressed as mean±standard error of mean; ^*p < 0.05, ^**p < 0.01.

The expression levels of cyclin B1 and phosphorylated BUBR1 decreased with increasing concentration of the MPS1/TTK inhibitor in YCC-47 cell line (Fig. 7A and C). In contrast, cyclin B1 and phosphorylated BUBR1 expression levels increased in the YCC-28 (Fig. 7B and D) cell line, and then decreased after treatment with MPS1/TTK inhibitor concentrations up to 0.5 µM.

These results show that the MPS1/TTK inhibitor inhibited SAC activity in the YCC-47 cell line, resulting in mitotic catastrophe and cell death. However, SAC activity was maintained in the YCC-28 cell line.