Abstract
Introduction: Successful treatment of AML remains dependent upon cytotoxic chemotherapy. However, traditional regimens are not well tolerated by older patients who are at highest risk of disease, and salvage rates after relapse are low, necessitating novel therapeutic strategies. Our groups identified Wee1 as a potential therapeutic target in AML, particularly in the context of concomitant treatment with cytarabine (Tibes et al, Blood, 2012; Porter et al, Leukemia, 2012). Wee1 inhibits CDK1&2 via phosphorylation thereby stalling cell cycle progression. One consequence of Wee1 inhibition/CDK1 activation is impairment of DNA repair via homologous recombination (Krajewska et al, Oncogene, 2013). Cells in which HR is impaired are dependent upon Parp1/2 function, and HR deficient cells are particularly sensitive to Parp1/2 inhibition. Therefore, we hypothesized that combined Wee1 and Parp1/2 inhibition may result in greater inhibition of AML cell proliferation and survival than either alone.
Methods: Human AML cell lines, MV4-11 and Molm-13, and a mouse AML that expresses MLL-ENL/FLT3-ITD were cultured with various concentrations of a Wee1 inhibitor (AZ1775) 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 72 more 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 were used to confirm inhibition of CDK1/2 phosphorylation and to measure DNA damage induction (gamma-H2AX).
Results: Combined inhibition of Wee1 and Parp1/2 was synergistic, as measured by cell numbers at 72 hours, in all 3 cell lines tested, with combination index 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. Western blots for gamma-H2AX confirmed that the combination of Wee1 and Parp1/2 resulted in more DNA damage than either drug alone.
Discussion: Combined inhibition of Wee1 and Parp1/2 results in greater inhibition of AML cell proliferation, DNA damage and apoptosis than either drug alone. Future studies will include experiments with primary patient samples, as well as in vivo trials combining Wee1 inhibition with Parp1/2 inhibition. These preliminary studies raise the possibility of rational combinations of targeted agents for leukemia in those for whom conventional chemotherapeutics may not be well tolerated.
Disclosures
No relevant conflicts of interest to declare.
Innovative Analyses Support a Role for DNA Damage and an Aberrant Cell Cycle in Myelodysplastic Syndrome Pathogenesis
