Abstract 4481: Myeloperoxidase as a determinant for activity of etoposide (VP-16) and other phenolic and non-phenolic anticancer agents: implications for drug-induced leukemogenesis.
Authors: Yalowich Jack C. , Skwarska Anna , Rabovsky Andrew J. , Zhao Yuan , Phelps Mitch A. , Day Billy W. , Kanagasabai Ragu 
Published: August 2014

Abstract
Clinical utilization of the phenolic anticancer agent etoposide (VP-16) is limited in selected malignancies due to its ability to induce acute myeloid leukemias causally linked to MLL gene rearrangements. Previously, we demonstrated that myeloperoxidase (MPO) found in myeloid precursors converts VP-16 to its phenoxyl radical [Mol. Pharm. 79: 479-87, 2011], which can redox cycle leading to enhanced DNA topoisomerase II (topo II)-mediated strand cleavage, and resultant MLL translocations. In the present study, we utilized MPO shRNA in myeloid leukemia HL-60 cells to further examine MPO dependency for VP-16-induced: 1) inhibition/poisoning of topo II isoforms; 2) DNA damage; 3) formation of reactive oxygen species; 4) generation of a VP-16 ortho-quinone GSH adduct; 5) antiproliferative/cytotoxic activity. In MPO knockdown cells, mature MPO expression was reduced to 6% and enzyme activity was reduced to 18% of the level found in shRNA controls. Topo II alpha and beta, and DNA topoisomerase I (topo I) levels were similar in MPO knockdown and control cells. VP-16 (0-100 μM) induced a progressive increase in DNA double strand breaks in MPO+ HL-60 cells which was attenuated in MPO-knockdowns. Direct VP-16-induced topo II alpha and beta complexes with genomic DNA were reduced in MPO knockdown cells compared to shRNA controls. Using 3’-(p-hydroxyphenyl) fluorescein (HPF), pro-oxidant activity of VP-16 was demonstrated in MPO+ HL-60 cells which converted to anti-oxidant effects in MPO knockdowns. Mass spectrometric analysis indicated that the level of inactive VP-16 ortho-quinone GSH adducts in MPO knockdown cells was reduced to 33% of that in control cells. Paradoxically, cytotoxicity of VP-16 and additional topo II poisons mAMSA, and mitoxantrone was decreased in MPO+ cells compared to MPO knockdown cells. Podophyllotoxin, the non-phenolic natural product related to VP-16, and camptothecin were equally cytotoxic in MPO knockdown and control cells. Hydroquinone and the dietary flavonol quercetin were more cytotoxic in MPO+ cells compared to knockdowns. Together results suggest that MPO conversion of VP-16 to a pro-oxidant, which leads to genotoxicity/carcinogenicity, may simultaneously protect cells from cytotoxicity, in part due to subsequent production of inactive GSH conjugates. The consequences of MPO activity on mAMSA and mitoxantrone metabolism, topo II poisoning, and MLL gene rearrangements are currently under investigation as are the redox-related activities of dietary flavonoids dependent on MPO activity. Support: NIH CA090787.
Citation Format: Jack C. Yalowich, Anna Skwarska, Andrew J. Rabovsky, Yuan Zhao, Mitch A. Phelps, Billy W. Day, Ragu Kanagasabai. Myeloperoxidase as a determinant for activity of etoposide (VP-16) and other phenolic and non-phenolic anticancer agents: implications for drug-induced leukemogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4481. doi:10.1158/1538-7445.AM2013-4481