Abstract 3507: Facilitating pixantrone-induced DNA damage and subsequent cell kill through formaldehyde activation and improvements in drug design

Abstract

Abstract Pixantrone is a novel aza-anthracenedione originally developed to improve the therapeutic profile of mitoxantrone, a DNA-interactive agent currently used in the clinical management of a range of haematological malignancies and solid tumours. Like mitoxantrone, pixantrone interacts with DNA via intercalation and stimulates topoisomerase II-mediated DNA cleavage, presumably by stabilising the cleavable complex. Despite the drug's ability to stimulate DNA cleavage via topoisomerase II impairment, this form of DNA damage does not directly correlate with drug cytotoxicity, suggesting that pixantrone may be operating by a distinct, currently undefined mechanism of cell kill. A novel form of pixantrone-DNA interaction has recently emerged in which the drug can be efficiently activated by formaldehyde to yield covalent drug-DNA adducts. In cellular systems, pixantrone exhibits a mild synergistic relationship with the formaldehyde-releasing prodrug AN-9, indicating a favourable role for formaldehyde in mediating pixantrone-induced cell kill. A variety of methods including in vitro transcription and mass spectrometry have established that formaldehyde-activated pixantrone is a monofunctional DNA alkylator that binds selectively to CpG and CpA dinucleotides via the exocyclic N2 amino group of guanine within DNA. Crucially, formaldehyde provides the methylene bridge that covalently links DNA with a single drug side-chain. A major functional limitation of the monoadduct is its poor intrinsic stability (t1/2 = 75 min). It was subsequently rationalised that an increase in drug side-chain length may permit the drug to completely bridge the two strands of duplex DNA, much like an interstrand crosslink. Accordingly, new anthracenediones were synthesised that incorporated symmetrical side chains of the nature -NH-(CH2)n-NH2 (where n = 2—5). An in vitro crosslinking assay demonstrated that side-chain extension generally conferred a remarkable increase in the temporal stability of formaldehyde-activated anthracenedione-DNA adducts. A clear and direct relationship existed between side-chain length and drug-DNA adduct half-life with MX5 (n = 5) exhibiting exceptional stability (t1/2 > 2 days). Significantly, the enhanced drug-DNA adduct stability was reflected in breast adenocarcinoma MCF-7 cells where derivatives with longer side-chains were synergistic in combination with the formaldehyde-releasing prodrug AN-9. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3507.