TNK2 Inhibitor (R)−9bMS Causes Polyploidization Through Mitotic Failure by Targeting Aurora B

Abstract

TNK2 is a ubiquitously expressed nonreceptor-type tyrosine kinase. TNK2 participates in tumorigenesis, and TNK2 activation has been found in various cancers; therefore, TNK2 is a promising target for cancer chemotherapy. While the TNK2 inhibitor XMD16-5 is highly selective, it inhibits cytokinesis at higher concentrations by targeting Aurora B kinase, a key enzyme for cell division. Cytokinesis failure frequently generates polyploid cells, and the surviving polyploid cells risk leading to cancer development and malignant progression via chromosome instability. In this study, to investigate the possibility that (R)−9bMS, a TNK2 inhibitor structurally related to XMD16-5, drives malignant progression by inducing abnormal cell division, we examined its effects on cell division, Aurora B autophosphorylation, and colony formation. Cell count results showed a reduction in the number of A431, HeLa S3, HCT116, and MCF7 cells upon TNK2 inhibitor treatment. Microscopic observation indicated the formation of multinucleated and nucleus-enlarged cells. An increase in DNA content was confirmed with flow cytometry, which was underpinned by an increased number of centrosomes. Time-lapse imaging revealed mitotic failure, such as mitotic slippage and cytokinesis failure, as a cause of polyploidization. Of note, TNK2 knockdown significantly increased multinucleated cells, but the effect was quite weak, suggesting that TNK2 inhibition may only partially contribute to mitotic failure and polyploidization. Expectedly, Aurora B phosphorylation was reduced by (R)−9bMS like XMD16-5, but not by TNK2 knockdown. Collectively, TNK2 inhibitors (R)−9bMS and XMD16-5 induce polyploidization via mitotic failure caused by the inhibition of Aurora B kinase rather than TNK2. Notably, (R)−9bMS treatment promoted anchorage-independent colony formation, a hallmark of cancer. Our findings suggest that (R)−9bMS at a high concentration risks promoting cancer development or malignant progression. Therefore, caution should be used when using TNK2 inhibitors for cancers where TNK2 activation is not the transforming mutation and higher concentrations of TNK2 inhibitors are required to slow proliferation.