Abstract 826: Myeloperoxidase (MPO) dependency for DNA damage, genotoxicity and cytotoxicity induced by etoposide (VP-16): Implications for therapy-induced second malignancies

Abstract

Abstract Use of the phenolic anticancer agent etoposide (VP-16), which targets DNA topoisomerase II (topo II), can lead to an increased incidence of acute myeloid leukemia linked to rearrangements in the MLL gene. Previous work (Mol. Pharm. 79: 479-87, 2011) demonstrated that MPO, found in myeloid progenitor cells, oxidized VP-16 to its phenoxyl radical form and led to enhanced DNA topo II-mediated strand cleavage through redox cycling resulting in MLL translocations. In the present study, we utilized MPO shRNA in myeloid leukemia HL-60 cells to further examine MPO dependency for VP-16-, VP-16 catechol (VP-OH)-, and VP-16 ortho-quinone (VP-oQ)-induced: 1) DNA strand breaks; 2) oxidative DNA damage; 3) caspase 3 activation; 4) cytotoxicity. Using Comet assays, VP-16 induced a concentration-dependent increase in DNA strand breaks which was attenuated in MPO knockdown cells, as well as in cells incubated with succinylacetone (SA), a heme synthesis inhibitor which depletes cells of active MPO. Similar MPO dependency was demonstrated using VP-OH, and parthenolide (PTL), a sesquiterpene lactone, whose activity is known to be dependent on MPO. In contrast, DNA damage induced by VP-oQ, a fully oxidized VP-16 metabolite, and by the topo I inhibitor camptothecin (CPT), was independent of MPO. VP-16 and VP-OH also induced DNA abasic sites in MPO-replete HL-60 cells. In MPO depleted cells, VP-16 diminished oxidative DNA damage to levels below those seen in controls. Paradoxically, caspase 3 activation induced by VP-16 and VP-OH was decreased in MPO-replete compared to MPO-depleted cells. By comparison, PTL-induced caspase 3 activation was MPO dependent and CPT-induced caspase 3 activation was MPO independent. Cytotoxicity (trypan blue exclusion) induced by VP-16 and VP-OH was inversely related to, while that of PTL corresponded to, MPO levels and activity. VP-oQ, CPT, and podophyllotoxin (PDT) were equally cytotoxic regardless of MPO levels or activity. Clonogenic assays revealed similar paradoxes where VP-16 cytotoxicity was diminished in MPO-replete compared to MPO knockdown cells. In contrast, the effects of VP-oQ, CPT, and PDT on colony formation were not influenced by expression of MPO. Mass spectrometric analysis demonstrated MPO-dependent production of VP-oQ GSH adducts. The production of these inactive adducts were reduced in MPO depleted cells accounting, in part, for the unexpected increase in VP-16 cytotoxicity in MPO knockdown cells. Together, results strongly suggest that MPO-catalyzed oxidation of VP-16 to redox active species leads to enhanced genotoxic events linked to the known leukemogenic action of this anticancer agent. Future studies will be directed toward use of nutritional antioxidants to diminish formation of genotoxic radical species of VP-16 in MPO-containing myeloid precursors as a strategy to prevent drug-induced second malignancies. Citation Format: Ragu Kanagasabai, Jason Goodspeed, Soumendra Krishna Karmahapatra, Alex Klausing, Anna Skwarska, Michael Darby, Yuan Zhao, Jiang Wang, Mitchell A. Phelps, Jack C. Yalowich. Myeloperoxidase (MPO) dependency for DNA damage, genotoxicity and cytotoxicity induced by etoposide (VP-16): Implications for therapy-induced second malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 826. doi:10.1158/1538-7445.AM2014-826