) and, differently from what has been reported in budding yeast (35), centrosomal splitting was normal, thus excluding a major role of the enzymatic activity of MPS1 in centrosomal functions in human cells.
NMS-P715 has the expected mechanism of action in cells. A, time-lapse of YFP-α-tubulin overexpressing U2OS cells. Brightfield and YFP-dependent fluorescent signals of a representative mitotic cell are shown in controls (DMSO) or 1 μmol/L NMS-P715–treated cells. Images were taken every 6 minutes. Scale bar, 10 micrometers/L. B, mitotic length was calculated as the average of 20 mitotic cells treated as described above. C, mitotic index (percentage of mitotic cells) of U2OS cells treated for 20 hours with NMS-P715 (1 μmol/L) or DMSO (control). A total of 900 cells were counted in 3 independent experiments. D, Western blots were obtained as indicated in extracts of nocodazole (Noc.)-arrested mitotic HeLa cells, harvested by shake-off, and then treated for 1 hour as shown. MPS1 was evaluated by Phos-tag gel technology. The ubiquitylated forms of Securin and cyclin B1 are indicated. E, bar graph quantifying pixel intensities of the indicated mitotic proteins at the kinetochore (KT) upon NMS-P715 treatment (1 μmol/L), compared with control treatment (DMSO). Values are normalized to the total kinetochore signal obtained by Crest-specific antibody staining and represent the mean derived from the analysis of at least 5 cells.
MPS1 was previously reported to be heavily phosphorylated during mitosis and a number of autophosphorylation sites were identified (7, 11, 36). To determine a direct in vivo regulation of MPS1 autophosphorylation by NMS-P715, cellular lysates of nocodazole-arrested HeLa cells were analyzed following treatment with the compound. NMS-P715 caused a specific shift in the migration of MPS1. This shift was also observed in the presence of MG132, administered to keep cells in mitosis and thus exclude the possibility that this change was due to mitotic exit (Fig. 2D). Notably, however, NMS-P715 did not cause a complete MPS1 dephosphorylation, which was achieved only in the absence of MG132 upon mitotic exit, indicating that only a subset of MPS1 phosphorylation sites are directly affected by compound treatment. In addition, Securin and cyclin B1 polyubiquitylation was induced, indicating an NMS-P715–dependent activation of the APC/C ubiquitin E3 ligase activity.
As MPS1 kinase has also been implicated in the recruitment of essential SAC components to the kinetochore (8, 9), we investigated the kinetochore localization of different mitotic checkpoint components upon NMS-P715 treatment in the presence of MG132 in nocodazole-arrested HeLa cells. NMS-P715 leads to an almost complete delocalization of MAD1, MAD2, BUB1, and BUB3 (Fig. 2E; Supplementary Fig. S7). Interestingly, MPS1 itself was also reduced, although not to the same extent as the other SAC components. We also observed a delocalization of Borealin, which was not accompanied by a change in Aurora B localization. This confirms a specific role for MPS1 on Borealin, which was recently described as an MPS1 substrate (12). No modulation was observed on CENPB, HEC1, and BUBR1. These data are in full agreement with published data (Supplementary Table S3), the only exception being the marked BUB1 reduction not previously reported. We can exclude the activity of NMS-P715 on BUB1 kinase in vitro (Supplementary Table S1) and an explanation of this observation might be that complete inhibition of MPS1, achieved only by small-molecule inhibitors, is required to cause BUB1 kinetochore disappearance.
NMS-P715 treatment causes SAC inactivation and delocalization of kinetochore components. MAD2, BUBR1, BUB3, and cdc20 have been shown to be components of the mitotic checkpoint complex (MCC) that is required to sustain the mitotic checkpoint activity inhibiting cdc20-dependent activation of APC/C (2). For this reason, MCC complex integrity was evaluated upon NMS-P715 treatment by coimmunoprecipitation (Co-Ip) experiments. Interestingly, in treated cells, cdc20 interaction with MAD2 and BUB3 was lost whereas BUBR1 binding was slightly reduced (Fig. 3A). This was observed not only upon cdc20 Co-Ip but also upon Co-Ip of the APC/C component Cdc27. We also noticed that NMS-P715 causes cdc20 deubiquitylation (Fig. 3B); this indicates that MPS1 inhibition leads to mitotic override affecting both of the two putative SAC quality controls on cdc20: it affects MCC integrity and stability and abrogates cdc20 ubiquitylation allowing it to trigger APC/C activation (37).
NMS-P715 affects MCC stability and cdc20 ubiquitylation. A and B, co-IP experiments of extracts of nocodazole-arrested HeLa cells, harvested by shake-off, and then treated for 1 hour as indicated in the presence of nocodazole and MG132. Western blots of total lysates (30 μg) with cdc20 IP in A, or cdc27 IP in B, have been analyzed as indicated. Rabbit IgG IP done in B is a negative control.
In summary, the observed cellular effects indicate that MPS1 is the main target of NMS-P715 in cells.
MPS1 depletion causes chromosomal segregation errors and massive aneuploidy not compatible with cellular survival (Supplementary Fig. S8; refs. 9, 12). Indeed, NMS-P715 treatment also causes SAC inhibition and mitotic acceleration, which ultimately promotes chromosomal misalignment in metaphase and anaphase (Supplementary Fig. S9A and B). As a consequence, chromosome counts after mitotic spreads revealed a dramatic increase in aneuploidy upon treatment with NMS-P715 (Fig. 4A; Supplementary Fig. S9C).
NMS-P715 induces aneuploidy and inhibits tumoral cell proliferation. A, mitotic spread results showing chromosomal number distribution of HCT116 cells treated for 24 hours with NMS-P715 (1 μmol/L) or DMSO (control). Spreads of 68 cells were analyzed. B, DNA content assessed by flow cytometry of A2780 cells treated with DMSO (control) or NMS-P715. C, Western blots were obtained as indicated from extracts of A2780 cells treated with DMSO (Ctr.) or NMS-P715. D, fate profile of U2OS cells exposed to DMSO (control) or NMS-P715 (1 μmol/L). Each lane represents 1 cell followed for 86 hours (5,160 minutes) by time-lapse. E, bar graph quantifying apoptotic induction measured by flow cytometry in HCT116 cells. NMS-P715 (1 μmol/L) was administered with or without aphidicolin. Values are normalized versus control (DMSO). F, colony assay of U2OS cells treated as indicated for 22 hours and grown in compound-free medium for 8 days (washout) or treated continuously for 9 days (prolonged treatment) before staining. Percentages of colony numbers are normalized to control (DMSO). Values are the average of 2 experiments.
To assess the ability of NMS-P715 to promote aneuploidy-mediated cell death, A2780 ovarian cancer cells were treated at different doses for 2, 24, and 72 hours (Fig. 4B and C). Already after 24 hours, NMS-P715 treatment caused a reduction in G1 phase and a flattening in G2/M phase of the cell cycle as measured by flow cytometry. A similar profile was observed upon MPS1 RNAi (Supplementary Fig. S8) and was also confirmed in the colon-derived cell line HCT116 (Supplementary Fig. S10). Aneuploidization, together with the appearance of a sub-G1 population, further increased at 72 hours (Fig. 4B). This was accompanied by histone H3 dephosphorylation, Parp cleavage, and histone H2AX phosphorylation, indicating mitotic exit and confirming subsequent induction of apoptosis (Fig. 4C).
To follow the behavior of individual cells over a long period of treatment, we analyzed U2OS cells upon NMS-P715 administration by time-lapse microscopy and established a fate profile (Fig. 4D; ref. 38). After drug addition, we confirmed that only the mitotic phase was affected. For an average of 39 hours virtually all the cells were able to grow and divide. Faster mitotic division occurred in mitotic cells a few minutes after drug treatment and caused aneuploidy-mediated cell death that starts to be seen after 52 hours of treatment and occurred more frequently after 2 aberrant mitoses and upon a prolonged aneuploid interphase. After 86 hours, almost all the cells died (Supplementary Fig. S11A). In addition, when cells were arrested in early S phase by aphidicolin treatment, we found an almost complete inhibition of NMS-P715–mediated apoptotic induction (Fig. 4E); thus the passage through an aberrant mitosis is critical and essential for induction of cell death. Importantly, colony assay experiments done in U2OS cells treated with NMS-P715 for one entire cell cycle (22 hours) followed by drug washout