Mps1 Inhibition Preferentially Induces DNA Damage in Tetraploid Cancer Cells

Abstract

Background: Tetraploidy, a state of whole-genome duplication, is frequently observed in human cancers and confers resistance to conventional DNA-damaging therapies. The spindle assembly checkpoint (SAC) kinase Mps1 (TTK) is essential for mitotic fidelity and has emerged as a promising anticancer target. However, whether Mps1 inhibition differentially induces DNA damage in tetraploid versus diploid cancer cells, and whether this response engages the p53 pathway, remains unknown.

 Methods: Using isogenic diploid and tetraploid RKO colon carcinoma cells, we assessed the effects of pharmacological (Reversine) and genetic (siRNA) Mps1 inhibition on cell viability, clonogenic potential, and DNA damage induction. DNA double-strand breaks (DSBs) were quantified by γH2AX immunofluorescence and western blot analysis of γH2AX and phosphorylated CHK2 (T68). The involvement of the p53/p21 axis was examined using immunoblot and functional validation in p53-proficient and p53-deficient isogenic HCT116 cells.

 Results: Tetraploid RKO cells exhibited significantly greater resistance to bleomycin, cisplatin, etoposide, and camptothecin compared to diploid cells (p < 0.001). Conversely, Mps1 inhibition or depletion preferentially compromised tetraploid cell viability and clonogenic potential. Mechanistically, Mps1 knockdown induced a marked increase in γH2AX foci and pCHK2 (T68) specifically in tetraploid cells, indicative of a robust double-strand break (DSB) response. Notably, this occurred without significant changes in p53 or p21 expression, and p53-deficient tetraploid cells remained sensitive to Mps1 targeting, suggesting a p53-independent mechanism.

 Conclusion: Mps1 inhibition preferentially induces enhanced DNA damage in tetraploid cancer cells via a p53-independent pathway. These new findings position Mps1 as a promising therapeutic target for tetraploid-rich tumors and potentially including those with p53 mutations.