Abstract 1459: Combined inhibition of Wee1 and PARP1 sensitizes pancreatic cancer cells to radiation

Abstract

Abstract PARP1 [poly (ADP-ribose) polymerase-1] inhibitors are maximally effective in cells with HRR (homologous recombination repair) defects, a concept known as synthetic lethality. We have previously shown that inhibition of HRR and the G2 checkpoint by small molecule inhibitors of Chk1 (checkpoint kinase 1), can induce synthetic lethality by increasing PARP1 inhibitor-mediated radiosensitization selectively in tumor cells and preferentially in tumor cells with p53 mutations. In order to begin to determine whether other inhibitors of the DNA damage response might induce synthetic lethality in combination with PARP1 inhibition and radiation, we evaluated MK-1775, a Wee1 kinase inhibitor. Wee1 regulates the G2 checkpoint by phosphorylation of Cdk1, thus preventing entry into mitosis in the presence of DNA damage, while its influence on HRR is not clear. To determine whether inhibition of Wee1 might synergistically radiosensitize in combination with PARP1 inhibition, we treated pancreatic cancer cells with MK-1775 (100-200nM) and the PARP1 inhibitor, olaparib (1uM) and assessed radiosensitization by clonogenic survival. We found that the combination of MK-1775 with olaparib induced marked radiosensitization in MiaPaCa-2 (RER: 1.9) and AsPC-1 (RER: 1.7) pancreatic cancer cells, which was greater than the radiosensitization produced by either agent alone. In order to determine the mechanism(s) by which Wee1 and PARP1 inhibitors interact to produce radiosensitization, we investigated the G2 checkpoint as well as HRR. In response to radiation, MK-1775 abrogated the G2 checkpoint while olaparib treatment resulted in increased G2 accumulation which was associated with increased γH2AX, suggesting the presence of unrepaired DNA double strand breaks. Abrogation of the olaparib+radiation-mediated G2 checkpoint, by MK-1775 resulted in even greater DNA damage as assessed by γH2AX expression. Given the efficacy of PARP1 inhibitors in HRR defective cells, we also investigated the effect of MK-1775 on HRR. We found that MK-1775 inhibited homology-directed repair of an I-SceI endonuclease-induced DNA double strand break in MiaPaCa-2 cells (Control 5.2% versus MK-1775 3.3%, P<0.05) as well as impaired Rad51 focus formation in response to radiation. Taken together, these results suggest that inhibition of HRR and the G2 checkpoint by inhibition of Wee1 can induce synthetic lethality with PARP1 inhibitors in combination with radiation by increasing unrepaired DNA double strand breaks. These studies support the continued investigation of combined molecularly targeted agents such as Wee1 and PARP1 inhibitors with radiation, to induce synthetic lethality selectively in tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1459. doi:1538-7445.AM2012-1459