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Radiosensitization of p53 mutant cells by PD0166285, a novel G(2) checkpoint abrogator
Y Wang1, J Li, R N Booher
1Department of Cancer Molecular Sciences, Pfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, USA. Yuli.Wang@Pfizer.com
Abstract:
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 microM 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.
Insights
Targeting cancer cells lacking p53 with G(2) checkpoint abrogators offers a novel therapeutic strategy. PD0166285, a Wee1 inhibitor, preferentially kills these cells by inducing premature mitosis, enhancing radiosensitivity.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Many cancers lack functional p53, a tumor suppressor protein.
- p53-inactive cells rely on the G(2) cell cycle checkpoint for DNA repair before mitosis.
- Abrogating the G(2) checkpoint could selectively eliminate p53-inactive cancer cells.
Purpose of the Study:
- To identify inhibitors of Wee1 kinase, a key regulator of the G(2) checkpoint.
- To evaluate the potential of G(2) checkpoint abrogators as a targeted cancer therapy.
- To assess the radiosensitizing effects of Wee1 inhibitors in p53-inactive cancer cells.
Main Methods:
- High-throughput mass screening assay to identify Wee1 inhibitors.
- Chemical library screening leading to the identification of PD0166285 (a pyridopyrimidine).
- Cell-based assays to assess G(2) arrest, Cdc2 phosphorylation, and radiosensitivity.
Main Results:
- PD0166285 inhibited Wee1 kinase at nanomolar concentrations.
- PD0166285 abrogated irradiation-induced G(2) arrest and increased mitotic cell populations.
- PD0166285 demonstrated radiosensitizing activity, with enhanced efficacy in p53-inactive cells.
Conclusions:
- G(2) checkpoint abrogators, like PD0166285, represent a novel class of anticancer drugs.
- These agents enhance cancer cell killing by inducing premature mitosis.
- Targeting the G(2) checkpoint offers a promising strategy for treating p53-inactive cancers.