Radiosensitization of p53 mutant cells by PD0166285, a novel G(2) checkpoint abrogator

The lack of functional p53 in many cancer cells offers a therapeutic target for treatment. Cells lacking p53 would not be anticipated to demonstrate a G(1) checkpoint and would depend on the G(2) checkpoint to permit DNA repa ir prior to undergoing mitosis. We hypothesized that the G(2) checkpoint ab rogator could preferentially kill p53-inactive cancer cells by removing the only checkpoint that protects these cells from premature mitosis in respon se to DNA damage. Because Wee1 kinase is crucial in maintaining G2 arrest t hrough its inhibitory phosphorylation of Cdc2, we developed a high-throughp ut mass screening assay and used it to screen chemical library for Wee1 inh ibitors. A pyridopyrimidine class of molecule, PD0166285 was identified tha t inhibited Wee1 at a nanomolar concentration. At the cellular level, 0.5 m um PD0166285 dramatically inhibits irradiation-induced Cdc2 phosphorylation at the Tyr-15 and Thr-14 in seven of seven cancer cell lines tested. PD016 6285 abrogates irradiation-induced G. arrest as shown by both biochemical m arkers and fluorescence-activated cell sorter analysis and significantly in creases mitotic cell populations. Biologically, PD0166285 acts as a radiose nsitizer to sensitize cells to radiation-induced cell death with a sensitiv ity enhancement ratio of 1.23 as shown by standard clonogenic assay. This r adiosensitizing activity is p53 dependent with a higher efficacy in p53-ina ctive cells. Thus, G(2) checkpoint abrogators represent a novel class of an ticancer drugs that enhance cell killing of conventional cancer therapy thr ough the induction of premature mitosis.