AbstractPlasticity delineates cancer subtypes with more or less favourable outcomes. In breast cancer, the subtype triple-negative lacks expression of major differentiation markers, e.g., estrogen receptor α (ERα), and its high cellular plasticity results in greater aggressiveness and poorer prognosis than other subtypes. Whether plasticity itself represents a potential vulnerability of cancer cells is not clear. However, we show here that cancer cell plasticity can be exploited to differentiate triple-negative breast cancer (TNBC). Using a high-throughput imaging-based reporter drug screen with 9 501 compounds, we have identified three polo-like kinase 1 (PLK1) inhibitors as major inducers of ERα protein expression and downstream activity in TNBC cells. PLK1 inhibition upregulates a cell differentiation program characterized by increased DNA damage, mitotic arrest, and ultimately cell death. Furthermore, cells surviving PLK1 inhibition have decreased tumorigenic potential, and targeting PLK1 in already established tumours reduces tumour growth both in cell line- and patient-derived xenograft models. In addition, the upregulation of genes upon PLK1 inhibition correlates with their expression in normal breast tissue and with better overall survival in breast cancer patients. Our results indicate that differentiation therapy based on PLK1 inhibition is a potential alternative strategy to treat TNBC.