The Daoy and D283 medulloblastoma cell lines were purchased from the American Type Culture Collection (ATCC) (Rockville, MD, USA). The ONS-76 medulloblastoma cell line was provided by Dr James T. Rutka at the University of Toronto, Canada, and the UW228 cell line by Dr John Silber at the University of Washington (Seattle, WA, USA). D425 and D458 cell lines were obtained from Dr Darell D. Bigner at Duke University Medical Center (Durham, NC, USA). Cell lines were cultured in Dulbecco' modified Eagle's medium (DMEM) (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA, USA). Cell lines D283, D425 and D458 are all part of the group 3 genomic subgroup. Daoy, ONS-76 and UW228 cluster with the Shh subgroup.

Primary patient samples were obtained from Children's Hospital Colorado and were collected and used in accordance with local and federal human research protection guidelines and Institutional Review Board (IRB) regulations. Informed consent was obtained for all specimens collected. Normal brain tissue was collected from autopsy and purchased from Ambion (Austin, TX, USA), Stratagene (Santa Clara, CA, USA) and Clontech Laboratories, Inc. (Mountain View, CA, USA). NMS-P715 was kindly provided by R. Colombo, Nerviano Medical Sciences (Nerviano, Italy) or were purchased from Calbiochem, EMD Millipore Corp. (Billerica, MA, USA). NMS-P715 was reconstituted in dimethyl sulfoxide (DMSO) and aliquots were stored in a desiccator at −20°C. An equivalent amount of DMSO for the highest concentration of drug was used for each experiment as a vehicle control.

Patient tumor samples were collected at the time of surgery and snap-frozen in liquid nitrogen. Ribonucleic acid was extracted from all samples using an RNeasy kit (Qiagen, Valencia, CA, USA). Samples were evaluated for gene expression using Affymetrix U133 Plus 2.0 GeneChip microarrays as we previously described (9,15).

Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay using CellTiter 96 AQueous One Solution (Promega, Madison, WI, USA). Seventy-two hours after transfection with shTTK1 (shMPS1), 20 µl of MTS reagent was added to the wells already containing 100 µl of media.

For drug treatment, the cells were plated for 24 h before adding 0.5 and 2 µm of NMS-P715. Then, 72 h after the addition of the drug, 30 µl of MTS reagent was added to the wells to make a final volume of 180 µl. Plates were read using a Bio-Tek Synergy 2 plate reader (Bio-Tek, Winooski, VT, USA) every hour for 4 h after the addition of the MTS reagent. Experiments were carried out in triplicate and background absorbance was subtracted from all wells before analysis.

Cell numbers after NMS-P715 treatment (2 µm) were measured using the ViaCount assay (Millipore) on a Guava flow cytometer as per the manufacturer's recommendations. Cells were cultured for 72 h, and collected by centrifugation. The cells were washed, ViaCount reagent was added, and cell counts were measured on the Guava EasyCyte Plus flow cytometer (Millipore).

Daoy, ONS-76 and UW228 medulloblastoma cells were seeded into 6-well plates in triplicate at a density of 500 cells/well in 3 ml medium containing 10% FBS for 24 h. The cells were then treated with DMSO or MPS1 inhibitor, NMS-P715 (500 nm-1 µm) and further cultured for 10 days in a 37°C humidified atmosphere containing 95% air and 5% CO₂. After 10 days of growth, the medium was aspirated, the wells were washed with phosphate-buffered saline (PBS), and cell clones were stained for 15 min with a solution containing 0.5% crystal violet and 25% methanol, followed by 3 rinses with tap water to remove excess dye. The colony numbers were counted using a precise electronic counter (Heathrow Scientific, Vernon Hills, IL, USA) and an inverted microscope with a threshold of 50 cells necessary to constitute a colony.

Protein expression levels were determined by western blotting. Cells were lysed in 1X RIPA buffer (Thermo Scientific, Rockford, IL, USA) containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA), 1 mM sodium vanadate and 0.1 mM sodium molybdate. The lysates were centrifuged for 20 min and the supernatants collected for protein concentration determination by the Bradford reagent (Sigma, St. Louis, MO, USA). Equal amounts of cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and western blot analysis was performed with specific antibodies. Antibodies used for western blot analysis were purchased from the following sources: actin #8H10D10, MPS1#11108 (Abcam, Cambridge, MA, USA). Antibodies for Mps1 and phosphorylated Mps1 were obtained by P.A. Eyers (16). Secondary antibodies conjugated to horseradish-peroxidase were used in conjunction with a chemiluminescent reagent to visualize protein bands.

Flow cytometric analysis was performed to define the cell cycle distribution after NMS-P715 treatment. D425 and D458 cells were seeded into 6-well plates (10⁵ cells/well) and 24 h later were treated with 2 µm NMS-P715. Cells were harvested 72 h later and fixed with chilled 70% ethanol for 24 h. Fixed cells were then washed and stained with propidium iodide-containing cell cycle reagent (Millipore). Flow cytometric analysis was performed on the Guava EasyCyte Plus flow cytometer.

Daoy and UW228 medulloblastoma cells were treated with 2 µm NMS-P715 and allowed to grow in normal culture medium for 72 h. The cell concentration was determined following staining with Guava ViaCount reagent. Equal numbers of cells were then stained using Guava Nexin reagent (Millipore) to detect apoptotic cells. Samples were run on a Guava EasyCyte Plus flow cytometer.

Our recent study suggested that MPS1 is highly expressed in medulloblastoma (9). To further examine the expression of MPS1 in pediatric brain tumors we first analyzed microarray-derived gene expression data from 90 pediatric brain tumors. MPS1 mRNA expression was significantly elevated in high grade pediatric brain tumors including glioblastoma (GBM), medulloblastoma (MED) and ATRT compared to normal brain (Fig. 1A). Notably, lower grade tumors such as pilocytic astrocytoma (PA) expressed much lower amounts of MPS1 mRNA (Fig. 1A). There was no clear correlation between the high MPS1-expressing samples and age, gender or outcomes.

Medulloblastoma consists of four major genomic subgroups [Sonic hedgehog (SHH) signaling, Wnt signaling (WNT), and groups 3 and 4 (7)]. To further elucidate whether there was a correlation between the subgroups of medulloblastoma, we examined expression of MPS1 mRNA in a cohort of 120 recently described medulloblastoma samples (5). MPS1 mRNA was significantly elevated in medulloblastoma compared to adult and fetal cerebellum (Fig. 1B). However, MPS1 expression was not significantly different in any of the medulloblastoma genomic subgroups (Fig. 1B). Similarly, MPS1 protein expression was elevated in the tumor samples from groups 3 and 4 patients compared to normal cerebellum (Fig. 1C). All group 3 and Shh medulloblastoma cell lines commonly used also demonstrated significantly elevated MPS1 protein expression (Fig. 1D). These data suggest that MPS1 may be associated with the oncogenic process in general and is not specific to a particular molecular subgroup of medulloblastoma.

We hypothesized that overexpression of MPS1 drives medulloblastoma cell growth and that inhibition of MPS1 would repress medulloblastoma cell growth. To test this hypothesis we first suppressed MPS1 mRNA in 2-well characterized cell lines using shRNA. In both Daoy and ONS-76 cell lines transfection of shTTK1 (shMPS1) significantly decreased the number of viable cells after 72 h as measured by the MTS assay (Fig. 2A and B). Knockdown of MPS1 was verified by qRT-PCR (43% decrease in MPS1 mRNA compared to sh-control, data not shown).

To further evaluate the impact of MPS1 inhibition we obtained a recently described MPS1 inhibitor NMS-P175 (kind gift from Nervino Medical Sciences). NMS-P715 is an orally bioavailable, potent and selective small-molecule inhibitor of MPS1 kinase activity (14).

NMS-P175 potently inhibited the growth of both Daoy and ONS-76 cells in a dose- and time-dependent manner (Fig. 2C and D). Since both Daoy and ONS-76 cell lines belong to the Shh genomic group we chose to test the impact of chemical inhibition of MPS1 in additional cell lines. We chose 3 cell lines with Myc translocations that are known to be part of the group 3 medulloblastoma genomic signature. The growth of all 3 Myc cell lines (D283, D425 and D458) was also potently suppressed by MPS1 inhibition using NMS-P175 (Fig. 2E).

To evaluate a longer-term impact, we then performed colony formation assays on cells treated with varying concentrations of NMS-P715. We used lower concentrations of NMS-P715 due to the longer-term treatment. Once again, inhibition of MPS1 significantly decreased medulloblastoma cell growth as measured by their ability to form colonies (Fig. 3). Notably, in the ONS-76 cells there was a much more marked inhibition in the colony formation capability (despite treatment with a lower dose) compared to measuring just cell proliferation (92% inhibition vs. 46% inhibition, respectively). These data may reflect the impact of MPS1 on tumor cell self-renewal compared to just mitosis.

We further examined the functional activity of MPS1 inhibition in medulloblastoma cells. Recent evidence suggests that MPS1 phosphorylates a wide range of proteins including itself (16). To verify that inhibition of MPS1 by NMS-P715 inhibited its kinase activity, we evaluated MPS1 autophosphorylation using protein immunoblotting. In both Daoy and UW228 medulloblastoma cells NMS P715 strongly decreased the phosphorylation of serine 821 a key autophosphorylation site on MPS1 (Fig. 4A) (16). The suppression of phosphorylation was dose-dependent for both cell lines (Fig. 4B). Total MPS1 did not change in the Daoy cells, but there was a decrease in total MPS1 protein in the UW228 cells suggesting that pS821 on MPS1 may be important for protein stability.

To determine whether the decreased cell growth upon MPS1 inhibition was due to cell cycle effects, we examined the impact of NMS-P715 on the cell cycle distribution of medulloblastoma cells. In all cells tested, NMS-P715 induced a G2-M arrest as shown in Fig. 5A. These data are consistent with previous studies showing that MPS1 is a critical regulator of mitosis. Moreover, western blot analysis showed that MPS1 also decreased expression of G2-M-associated proteins CDC2 and cyclin B1 (Fig. 5B).

We next examined whether apoptosis was a contributor to medulloblastoma cell growth inhibition by NMS-P715. We measured Annexin V expression on the surface of NMS-P715-treated medulloblastoma cells by flow cytometry. Representative plots are shown for Daoy, UW228, D425 and D458 cells. Annexin V-positive-7-aminoactinomycin D (7-AAD)-negative cells, indicative of early apoptosis, were present at low levels in DMSO control-treated cells. This population increased with increasing doses of NMS-P715 (Fig. 6). In addition, the Annexin V-positive-7-AAD-positive population was significantly enhanced in the NMS-P715-treated cells, indicating increased late apoptosis. The total percentage of apoptosis is quantified in Fig. 6. These data confirm that MPS1 inhibition by NMS-P715 is a strong inducer of apoptosis in medulloblastoma.