Dual TTK/PLK1 inhibition combined with G-CSF treatment synergistically suppresses metastatic progression in triple-negative breast cancer

Threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1), critical regulators of the spindle assembly checkpoint (SAC) required for faithful mitotic progression, are frequently overexpressed and dysregulated in human cancers. In recent years, strategies targeting the SAC, including TTK and/or PLK1, have emerged as promising therapeutic approaches in cancer treatment. However, these SAC inhibitors are known to induce adverse effects, including neutropenia. Granulocyte-colony stimulating factor (G-CSF) is widely used to prevent anti-cancer therapy-induced neutropenia, but growing evidence suggests that G-CSF may also promote tumor progression. Here, we demonstrate the broad anti-cancer activity of a novel dual TTK/PLK1 inhibitor, BAL0891, and its functional link to the G-CSF/G-CSF receptor (G-CSFR) axis in metastatic breast cancer. In patients with breast cancer, both TTK and PLK1 were markedly upregulated in triple-negative breast cancer (TNBC), which exhibits aggressive behavior, compared with other subtypes, and their expression levels were positively correlated among TNBC patients. The expression of G-CSFR was upregulated in breast tumors compared with normal tissues and was further elevated in metastatic tumors. In multiple human and murine TNBC cell lines, BAL0891 exerted potent anti-proliferative activity, accompanied by G2/M cell-cycle arrest and apoptosis. BAL0891 showed markedly higher potency in TNBC cells (GI50 ≈ 50─90 nM) than in immortalized MCF10A and ER+ MCF7 cells GI50 > 700 nM). In addition, BAL0891 treatment suppressed the migratory and invasive capacities of metastatic TNBC cells, with a concomitant reduction in vimentin expression, an epithelial-mesenchymal transition (EMT) marker. When comparing the effects of BAL0891 alone and in combination with G-CSF on TNBC progression, G-CSF treatment did not alter the anti-proliferative or apoptotic responses to BAL0891 in TNBC cells harboring moderate to high G-CSFR levels. However, combined BAL0891 and G-CSF treatment further potentiated BAL0891-mediated suppression of migration and invasion in these cells. Taken together, these findings suggest that dual TTK/PLK1 inhibition in combination with G-CSF may represent a valuable therapeutic strategy to suppress metastatic TNBC while potentially reducing the risk of neutropenia.