The value of isogenic cell lines in cancer drug discovery: Antitumor activity of WAY-181484, an orally-active, microtubule-binding agent

5924 We have used an isogenic pair of human colon carcinoma cell lines (HCT 116 p21+/+ and p21-/-) to screen compounds for antitumor activity [Waldman et al. (1996), Nature 381, 713-716]. This pair of lines was used to select compounds that preferentially induce apoptosis in the p21-deficient cells, relative to the p21-proficient cells. As a downstream effector of the major tumor suppressor p53, p21 arrests cell cycle progression in response to DNA damage and other environmental stresses, by inhibiting the cyclin-dependent kinases (CDKs). Disruption of this cell cycle checkpoint by deletion of the p21 gene, results in the failure of the cell to arrest, leading to endoreduplication, and ultimately, apoptosis. The role of this checkpoint in modulating chemosensitivity of tumor cells is supported by the observation that the p21-deficient cells show increased sensitivity to a variety of clinically used antineoplastic drugs, compared with the isogenic p21-proficient cells. Compounds active in this assay are, therefore, predicted to target tumor cells that lack one or more checkpoints, a hallmark of cancer. WAY-181484 is the lead compound in the pyrazolopyrimidinyl phenyl amide series, a chemical series identified in this screen. WAY-181484 preferentially inhibits p21-/- cells (IC 50 =120 nM) compared with p21+/+ cells (IC 50 = 5700 nM) producing a selectivity ratio of ∼50. WAY-181484 is microtubule-active agent: it binds tubulin (K d = 4.3 μM) at the colchicine binding site, and inhibits tubulin polymerization in cell-free systems and in intact cells. It shows potent activity against a panel of colon cell lines (IC 50 = 20-60 nM), and inhibits the growth of human tumor xenografts in nude mice when administered orally, once-daily at 20-50 mg/kg. In summary, the identification of WAY-181484, a representative of a novel class of tubulin-binding agents that shows excellent antitumor activity in vitro and in vivo, has validated the use of the p21 isogenic cell-based screen in anticancer drug discovery.