BI-2536 is a potent inhibitor of PLK1 (Polo-like kinase 1, IC50 ~0.83 nM) and BRD4 (IC50 ~37 nM). It also inhibits PLK2 and PLK3 with IC50 of 3.5 nM and 9.0 nM, respectively. BI-2536 treatment ranging from 10 nM to 100 nM leads to the blocking of the recruitment of γ-tubulin and phosphorylation of Apc6 at mitotic centrosomes, inhibition of cohesin release from chromosome arms, induction of monopolar spindles, and other Plk1 dependent processes. BI-2536 inhibits the growth of a panel of 32 human cancer cell lines with EC50 of 2-25 nM. It also displaces BRD4 from chromatin and suppresses c-Myc expression. The combination of inhibitory activities on independent kinase and bromodomain oncogenic pathways exemplifies a new strategy for rational single-agent polypharmacological targeting.