Abstract 2746: Combined inhibition of Wee1 and Poly(ADP Ribose)Polymerase 1/2 synergistically inhibits acute leukemia cells by enhancing DNA damage and inducing apoptosis in vitro and in vivo
Authors: Garcia Tamara B. , Snedeker Jonathan C. , Porter Christopher C. 
Published: July 2016

Introduction: The goal of this study was to test the hypothesis that pharmacologic inhibition of Wee1 can sensitize acute leukemia cells to Parp1/2 inhibition by inducing defects in homologous recombination.

Methods: Human cell lines MV4;11 and Molm-13 (AML), Jurkat (T-ALL), and Reh (B-ALL) were treated with various concentrations of a Wee1 inhibitor (AZD1775) and a Parp1/2 inhibitor (olaparib) and counted 72 hours later by propidium iodide exclusion and flow cytometry In some experiments, cells were split into fresh media to recover for an additional 72 hours. Combination Index (CI) values were calculated by the method of Chou and Talalay. Apoptosis was measured using Annexin V/7AAD and flow cytometry. Western blots examining γH2AX as well as comet assays were used to measure DNA damage induction. Inhibitory phosphorylation of BRCA2, a critical protein in the homologous recombination pathway, was examined by Western blot. Finally, wild type Bl6 mice were injected with a murine AML cell line (Mll-Enl+FLT3-ITD+) and treated daily with olaparib and/or AZD1775. Leukemia progression was determined by measuring luciferase activity biweekly via In Vivo Imaging Systems (IVIS).

Results: Combined inhibition of Wee1 and Parp1/2 was synergistic, as measured by cell numbers at 72 hours, in all 4 cell lines tested, with CI values ranging from 0.3 to 0.9. When cells were allowed to recover after treatment, those treated by single agents were able to continue proliferating. However, those treated with the combination did not recover as well or at all, indicating greatly impaired proliferative capacity. Combined inhibition of Wee1 and Parp1/2 also resulted in a significant increase in apoptosis greater than either drug alone. Comet assays and western blots for γH2AX confirmed that the combination of Wee1 and Parp1/2 resulted in more DNA damage than either drug alone. Western blots demonstrated increased inhibitory phosphorylation of BRCA2 in cells treated with AZD1775 suggesting impaired homologous recombination upon Wee1 inhibition. In preliminary in vivo studies, mice with AML treated with olaparib and AZD1775 displayed prolonged survival compared to mice treated with single agents.

Conclusion: Combined inhibition of Wee1 and Parp1/2 results in greater inhibition of AML, T-ALL, and B-ALL cell proliferation, DNA damage and apoptosis than either drug alone. Inhibition of Wee1 resulted in increased BRCA2 inhibitory phosphorylation, suggesting that this increased sensitivity to PARP1/2 inhibition could be due to impairment of the homologous recombination pathway. Preliminary studies indicate that combined inhibition of Wee1 and Parp1/2 enhances survival in a murine AML model. These preliminary studies raise the possibility of rational combinations of targeted agents for leukemia in patients for whom conventional chemotherapeutics may not be well tolerated.

Citation Format: Tamara B. Garcia, Jonathan C. Snedeker, Christopher C. Porter. Combined inhibition of Wee1 and Poly(ADP Ribose)Polymerase 1/2 synergistically inhibits acute leukemia cells by enhancing DNA damage and inducing apoptosis in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2746.