Abstract 1759: MK-1775, a novel Wee1 kinase inhibitor, radiosensitizes p53-defective non-small cell lung cancer (NSCLC) cells

Abstract

Abstract Wee1 is a tyrosine kinase involved in the S>G2 and G2>M cell cycle transitions. Following DNA damage, Wee1 is phosphorylated by upstream kinases and stabilized. Stabilized Wee1, in turn, phosphorylates and inhibits CDC2, preventing the cells from entering mitosis before repairing their damaged DNA. Abrogation of Wee1 activity, either by decreasing expression using siRNA, or by inhibiting protein activity with selective small molecule inhibitors, sensitizes tumor cells to DNA-damaging agents. This sensitization preferentially takes place in a p53 null or mutant context. We have investigated the ability of a novel, small molecule inhibitor of Wee1, MK-1775, to radiosensitize NSCLC cells treated in vitro. Four NSCLC cell lines were used in the study: A549 and H460 with wild-type p53 status and H1299 and Calu6 with null or mutant p53 status, respectively. A 24 hr pre-irradiation treatment with 200 nM MK-1775 suppressed phosphorylation of CDC2 in both A549 and H1299 cells, however, this treatment did not produce a radiosensitizing effect in either line. An 18 hr post-irradiation treatment slightly radiosensitized the H1299 line but not the A549 line. The optimal treatment of a 1 hr pre-irradiation followed by an 18 hr post-irradiation treatment substantially radiosensitized the H1299 and Calu6 lines, yielding dose-enhancement factors (DEFs) at 10% survival of 1.39 and 1.47 respectively, but did not sensitize the A549 or H460 cells, DEFs of 1.0. To determine the mechanism responsible for the radiosensitizing effect of MK-1775, cells were treated with nocodazole following a dose of 4 Gy and analyzed for phospho-histone H3 as an indication of mitotic index after 4 hrs. Unirradiated cells accumulated in mitosis during the 4 hrs in nocodazole and this accumulation was substantially enhanced by MK-1775 in both p53 wild type A549 and p53 null H1299 cells suggesting that Wee1 inhibition accelerates unirradiated cells into mitosis prematurely. Neither A549 nor H1299 cells, irradiated with 4 Gy, accumulated in mitosis during the 4 hr nocodazole treatment in the absence of MK-1775 indicating a robust G2 block for both lines. However, H1299 cells irradiated with 4 Gy and treated with MK-1775 accumulated in mitosis to a level higher than the unirradiated cells treated with MK-1775 alone. This enhanced entry of irradiated cells into mitosis was not evident to nearly the same degree in the A549 cells. Thus, we conclude that the Wee1 inhibitor, MK-1775, accelerates cells harboring DNA-damage prematurely into mitosis in a p53-dependent manner correlating with its radiosensitizing effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1759.