Radiosensitization of p53 mutant cells by PD0166285, a novel G2 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 repair prior to undergoing mitosis. We hypothesized that the G 2 checkpoint abrogator could preferentially kill p53-inactive cancer cells by removing the only checkpoint that protects these cells from premature mitosis in response to DNA damage. Because Wee1 kinase is crucial in maintaining G 2 arrest through its inhibitory phosphorylation of Cdc2, we developed a high-throughput mass screening assay and used it to screen chemical library for Wee1 inhibitors. A pyridopyrimidine class of molecule, PD0166285 was identified that inhibited Wee1 at a nanomolar concentration. At the cellular level, 0.5 μm PD0166285 dramatically inhibits irradiation-induced Cdc2 phosphorylation at the Tyr-15 and Thr-14 in seven of seven cancer cell lines tested. PD0166285 abrogates irradiation-induced G 2 arrest as shown by both biochemical markers and fluorescence-activated cell sorter analysis and significantly increases mitotic cell populations. Biologically, PD0166285 acts as a radiosensitizer to sensitize cells to radiation-induced cell death with a sensitivity enhancement ratio of 1.23 as shown by standard clonogenic assay. This radiosensitizing activity is p53 dependent with a higher efficacy in p53-inactive cells. Thus, G 2 checkpoint abrogators represent a novel class of anticancer drugs that enhance cell killing of conventional cancer therapy through the induction of premature mitosis.