Abstract A77: Inhibition of TTK promotes chromosome segregation defects in a manner distinct from inhibitors of Aurora or PLK mitotic kinases
Authors: Williams Brandi L. , Roth Bruce L. , Cassiano Jared , McKinnon Rena S. , Brown Brita , Hoarau Christophe , Saunders Michael , Kumar D. Vijay 
Published: May 2012

Background: TTK, also known as Mps1, is a dual-specificity kinase that is essential for the proper amphitelic attachment of chromosomes to the mitotic spindle during mitosis and for preventing anaphase progression until the chromosomes are properly attached. We evaluated the effect of MPI-0479605, a potent and selective inhibitor of TTK, on chromosome segregation and cell cycle progression during an unperturbed mitosis and compared it to the effects of an Aurora or PLK kinase inhibitor.

Materials and Methods: The TTK, Aurora, and PLK inhibitors used include, MPI-0479605 (TTK IC50 ∼ 4 nM), VX-680 (Aurora IC50 ∼5 nM) and a thiophene benzimidazole (PLK1 and PLK3 IC50 ∼5 nM). For image analysis, cells were fixed and stained with Hoechst dye and anti-β-tubulin and anti-pericentrin antibodies. Images were taken on a BD Pathway high content imaging system. For cell cycle analysis, cells were fixed, stained with propidium iodide and analyzed by FACS. Alternatively, live cells were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) and analyzed by FACS. BrdU incorporation was measured using a FITC BrdU Flow kit (BD Pharmingen).

Results: In asynchronously growing cells, treatment with TTK inhibitor caused defects in chromosome congression at the metaphase plate. This resulted in lagging chromosomes during anaphase and a large increase in the percentage of cells with micronuclei during interphase. These micronuclei stained positive for centromeres, suggesting the presence of whole chromosomes. Aurora inhibitor induced the formation of multipolar spindles and caused severe defects in chromosome alignment at the metaphase plate. This resulted in micronuclei formation as well as multi-nucleated cells. The PLK inhibitor caused a monopolar spindle phenotype, consistent with the requirement for PLK1 to facilitate centrosome maturation. In asynchronously growing cells, treatment with TTK inhibitor had no obvious cell cycle arrest phenotype but induced a significant increase in the apoptotic sub-G1 population of cells. If cells were synchronized at the G1/S border and then released into drug, the majority of cells arrested after one cell division in response to inhibitors of all three mitotic kinases. Cells treated with TTK inhibitor were diploid, whereas cells treated with Aurora inhibitor became tetraploid. PLK inhibitor caused a metaphase arrest followed by apoptotic cell death. Finally, cells treated with either TTK or PLK inhibitor, but not Aurora inhibitor, induced a strong caspase response.

Conclusions: Inhibitors of TTK, Aurora or PLK produce distinct phenotypic effects on chromosome segregation and cell cycle progression. Inhibition of TTK abrogates the spindle assembly checkpoint during an unperturbed mitosis. This allows anaphase progression in the presence of misattached chromosomes and leads to chromosome segregation defects and aneuploidy. Subsequent activation of a post-mitotic checkpoint in G1 or S phase results in apoptosis. In contrast, an Aurora inhibitor causes defects in mitotic spindle formation, chromosome segregation, and cytokinesis, resulting in tetraploidy. PLK inhibitor causes metaphase arrest followed by mitotic catastrophe. The phenotypic differences demonstrated here provide evidence that TTK inhibition may provide a novel anti-mitotic mechanism for treating cancer.

Citation Information: Cancer Res 2009;69(23 Suppl):A77.