[CANCER RESEARCH 61, 8211– 8217, November 15, 2001] Radiosensitization of p53 Mutant Cells by PD0166285, a Novel G 2 Checkpoint Abrogator Yuli Wang, 1 Jun Li, Robert N. Booher, Alan Kraker, Theodore Lawrence, Wilbur R. Leopold, and Yi Sun Departments of Cancer Molecular Sciences [Y. W., J. L., Y. S.] and Cancer Pharmacology [A. K., W. R. L.], Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, Michigan 48105; Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan 48109 [J. L., T. L.]; and Onyx Pharmaceutical, Inc., Richmond, California 94806 [R. N. B.] ABSTRACT The lack of functional p53 in many cancer cells offers a therapeutic target for treatment. Cells lacking p53 would not be anticipated to dem- onstrate 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 mole- cule, PD0166285 was identified that inhibited Wee1 at a nanomolar con- centration. At the cellular level, 0.5 M 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 fluores- cence-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. INTRODUCTION The driving force for G23 M progression is the Cdc2/cyclin B1 protein complex (1–3). In addition to association with cyclin B1, Cdc2 is also subjected to both positive and negative phosphorylation con- trols. Thr-161 phosphorylation, catalyzed by cyclin-dependent kinase activating kinase, is required for Cdc2 kinase activity (1). On the other hand, Thr-14 and Tyr-15 phosphorylations on Cdc2 inhibit its kinase activity. Wee1 is the major kinase phosphorylating Cdc2 on Tyr-15 (2– 4), therefore inhibiting Cdc2 activity. At the onset of mitosis, the inhibitory phosphates are removed by a dual-specific phosphatase Cdc25C, leading to activation of Cdc2 kinase. Thus, Cdc25 phospha- tase plays a key role in normal cell cycle progression between G 2 and M phases. DNA damage can cause Cdc25 inactivation, resulting in a G 2 arrest and allowing the damaged DNA to be repaired. The mech- anism of Cdc25 inactivation is through its phosphorylation on Ser- 215, catalyzed by Chk1/Chk2 or C-TAK1 kinases (5–7). This phos- phorylation creates a binding site for 14-3-3. The interaction between Cdc25 and 14-3-3 results in nuclear exporting of Cdc25 and its cytoplasmic accumulation (8). The upstream kinase that activates Chk2 is ATM, 2 which can be activated by DNA damage (9 –12). Therefore, DNA damage activates a G 2 checkpoint by activation of ATM/ATR, followed by Chk1/2 activation and Cdc25 and Cdc2 inactivation. We hypothesized that a strategy could be developed that would permit us to exploit the G 2 checkpoint to obtain a therapeutic index in the treatment of cancers lacking a G 1 checkpoint. The lack of a G 1 checkpoint is common in 50% of cancers containing p53 mutations. In this strategy, normal cells arrest in G 1 after DNA damage from irradiation (or chemotherapy), whereas cancer cells with a defective G 1 checkpoint would progress through S-phase and into G 2 . There- fore, abrogation of G 2 checkpoint will be more detrimental to cancer than normal cells. In an effort to search for specific G 2 checkpoint abrogators, Wee1 kinase was selected as a anticancer target for following reasons: (a) Wee1 is a negative regulator of Cdc2 kinase activity, and expression of Cdc2AF mutant, a mutant that cannot be phosphorylated by Wee1 and Myt1 kinases, caused premature mitosis (13–15); (b) Wee1 was down-regulated in p53-positive cells after DNA damage (16); (c) Wee1 was degraded in Fas-ligand induced apoptosis, involving caspase-dependent activation of Cdc2 (17); and (d) Wee1 overexpression rescues apoptosis (14, 18). In this report, we show the identification of a Wee1 kinase inhibitor PD0166285 with an IC 50 at nanomolar concentration. Interestingly, the compound also has nanomolar IC 50 for Myt1 kinase. PD0166285 inhibits Cdc2 phospho- rylation on both Tyr-15 and Thr-14 in vivo in seven tumor cell lines tested. It also abrogates radiation-induced G 2 arrest as measured by biochemical markers and mitotic index. Furthermore, PD0166285 sensitizes radiation-induced cell killing in p53 mutant HT29 cells and in the E6-transfected, p53-null ovarian cancer cell line PA-1 but to a lesser extent in p53 wild-type PA-1 cells. Our observations support the concept that abrogation of G 2 checkpoint potentiates cancer cells, particularly those with a functional inactive p53 to DNA damage- induced cell killing. Therefore, the G 2 checkpoint abrogator presents a new class of anticancer drug functioning as a radiosensitizer. MATERIALS AND METHODS Compounds. PD0166285 was synthesized at Pfizer Global Research and Development. UCN-01 was obtained from National Cancer Institute. Caffeine was purchased from Sigma Chemical Co. (St. Louis, MO). Cell Culture. Ovarian carcinoma PA-1 cells, transfected with the vector control (PA-1/neo, p53wt) or E6 (PA-1/E6, p53-null) were kindly provided by Dr. El Deiry (University of Pennsylvania, Philadelphia, PA) and cultured with Basic Medium Eagle with 10% FBS. All other cell lines are from American Tissue Culture Collection. HT29 (human colon carcinoma cell line with p53 mutation) and HeLa (human cervical cancer cell line with p53 wt/hpv) cells were cultured in high glucose DMEM with 10% FBS. HCT8 (human colon carcinoma cell line with wild-type p53 status) cells were cultured in RPMI 1640 with 10% FBS. HCT116 (human colon carcinoma cell line with wild- type p53) cells were cultured in McCoy’s 5a medium with 10% FBS. DLD-1 (human colon carcinoma cell line with mutant p53) cells were cultured in Eagle’s MEM with 10% FBS. H460 (human lung carcinoma cell line with wild-type p53) cells were cultured in RPMI 1640 with sodium pyruvate and 10% FBS. C26 (mouse colon carcinoma cell line with wild-type p53) 3 cells were cultured in DMEM/F12 and 10% FBS. All cell culture media were from Life Technologies, Inc. Received 6/19/01; accepted 9/13/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Pfizer Global Research and Development, Ann Arbor Laboratory, 2800 Plymouth Road, Ann Arbor, MI. Phone: (734) 622-3885; Fax: (734) 622-5668; E-mail: Yuli.Wang@Pfizer.com. 2 The abbreviations used are: ATM, ataxia telangiecstasia mutated; PD0166285, 6- aryl-pyrido[2,3-d] pyrimidine; UCN-01, 7-hydroxy staurosporine; FBS, fetal bovine se- rum; FACS, fluorescence-activated cell sorter; SER, sensitizing enhancement ratio. 3 Y. Sun et al., unpublished observation. 8211