Abstract C203: Inhibition of TTK activates the p53 pathway

• Published in: Molecular cancer therapeutics 2009-12, Vol.8 (12_Supplement), p.C203-C203
• Main Authors: Williams, Brandi L., Rogers, Aaron, Cassiano, Jared, Tardif, Keith D., Roth, Bruce L., Saunders, Michael, Hoarau, Christophe, Kumar, D. Vijay
• Format: Article
• Language: English
• ISSN: 1535-7163; 1538-8514
• DOI: 10.1158/1535-7163.TARG-09-C203

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. Given the role of TTK in genome integrity, we evaluated the effect of MPI-0479605, a potent and selective inhibitor of TTK, on the p53 pathway. Materials and Methods: 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. For Western blot analysis, cell lysates were run on SDS-polyacrylamide gels and proteins were transferred to PVDF membrane. Membranes were probed with the indicated antibodies. Results: Inhibition of TTK induced severe defects in chromosome segregation, as shown by the presence of lagging chromosomes and/or anaphase bridges during mitosis and the formation of micronuclei at interphase. Anaphase bridges may result in DNA double-strand breaks. Cell cycle analysis showed that a subpopulation of treated cells exhibited a tetraploid phenotype. Because either DNA damage or tetraploidy may induce the p53 pathway, we examined whether p53 was induced by treatment with MPI-0479605. Expression of p53 was induced in a dose- and time-dependent manner in response to MPI-0479605, with maximal levels occurring between 24 and 48 hours of treatment. Induction of p53 correlated with an increase in p21 expression and a decrease in survivin protein levels, indicating that p53 is transcriptionally active. Using phosphospecific antibodies, we determined that p53 is phosphorylated on Ser15 in response to MPI-0479605 treatment but not Thr18, Ser20, Ser37 or Ser46. Phosphorylation of γH2AX at Ser139, which is a marker for the presence of double-strand DNA breaks, was induced by treatment with MPI-0479605 in a time-dependent manner similar to p53 induction. Phosphorylation of p53 on Ser15 and H2AX on Ser139 can be mediated by the DNA damage checkpoint protein ATM. ATM also phosphorylates Chk2 on Thr68 and induces its activity, but, treatment with MPI-0479605 did not induce an increase in Chk2 Thr68 phosphorylation. Conclusions: Treatment of cells with the small molecule TTK inhibitor, MPI-0479605, activates p53. This may be due to chromosome segregation defects, which cause subsequent DNA doubl