Abstract Pixantrone is a new non-cardiotoxic aza-anthracenedione anticancer drug structurally related to anthracyclines and anthracenediones such as doxorubicin and mitoxantrone. Pixantrone is approved in the EU for the treatment of relapsed or refractory aggressive B-cell non-Hodgkin lymphoma. This study was undertaken to investigate both the mechanisms of its anticancer activity and its lack of cardiotoxicity. The reduced cardiotoxicity of pixantrone compared to doxorubicin may be due to the fact that it did not bind iron(II) or iron(III). Thus, unlike doxorubicin it would not be able to induce iron-based oxidative stress in the heart. Pixantrone did, however, form a weak complex with Cu(II). Pixantrone was 10-fold less damaging to neonatal rat myocytes than doxorubicin as measured by LDH release. As measured by EPR spectrometry pixantrone generated about two-fold higher levels of its semiquinone free radical than doxorubicin in the hypoxanthine-xanthine oxidase reducing system. Conversely, in intact K562 cells under nitrogen, doxorubicin produced much more semiquinone radical than pixantrone. The reduced myocyte toxicity and semiquinone-forming ability in K562 cells compared to doxorubicin may be due low cell accumulation of dicationic pixantrone. Multidrug resistant Pgp- (ABCB1) overexpressing MDCK/MDR cells were 40-fold cross resistant to pixantrone, compared to parental cells. Pixantrone bound to DNA much more strongly than doxorubicin as determined in a DNA melting point assay. Pixantrone targeted topoisomerase II as evidenced by its ability to inhibit kDNA decatenation, to produce linear double-stranded DNA in a pBR322 DNA cleavage assay, and to form covalent topoisomerase II-DNA complexes in a cellular ICE assay. Pixantrone was 3-fold cross-resistant to K562-derived etoposide-resistant cells. DNA double strand breaks produced by pixantrone assessed in a cellular γH2AX assay were also characteristic of a topoisomerase II-targeted drug. These results support the conclusion that pixantrone displays characteristics that are consistent with targeting topoisomerase II. Support: CIHR; a Canada Research Chair in Drug Development; R01 CA090787. Citation Format: Brian B. Hasinoff, Xing Wu, Daywin Patel, Jack C. Yalowich. The non-cardiotoxic anticancer drug pixantrone targets topoisomerase II. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1659. doi:10.1158/1538-7445.AM2015-1659