Interactions of pixantrone with apurinic/apyrimidinic sites in DNA.

Pixantrone and mitoxantrone are structurally related anticancer drugs which have been shown to generate covalent conjugates at apurinic/apyrimidinic (AP) sites in DNA. Mitoxantrone binding to AP sites induces DNA strand cleavage and inhibits the endonuclease activity of human AP endonuclease 1 (APE1). Here, pixantrone was demonstrated to have similar properties, but relative to mitoxantrone, it was significantly less potent in both DNA incision and APE1 inhibition. Consistent with these observations, pixantrone had ~ 15-fold lower affinity for DNA containing an AP site analogue, tetrahydrofuran, as measured by a Thiazole Orange (ThO) displacement assay.

Interaction of mitoxantrone with abasic sites - DNA strand cleavage and inhibition of apurinic/apyrimidinic endonuclease 1, APE1.

Mitoxantrone (1,4-dihydroxy-5,8-bis[2-(2-hydroxyethylamino)ethylamino]-anthracene-9,10-dione) is a clinically-relevant synthetic anthracenedione that functions as a topoisomerase II poison by trapping DNA double-strand break intermediates. Mitoxantrone binds to DNA via both stacking interactions with DNA bases and hydrogen bonding with the sugar-phosphate backbone. It has been shown that mitoxantrone inhibits apurinic/apyrimidinic (AP) endonuclease 1 (APE1)-catalyzed incision of DNA containing a tetrahydrofuran (THF) moiety and more recently, that mitoxantrone forms Schiff base conjugates at AP sites in DNA. In this study, mitoxantrone-mediated inhibition of APE1 at THF sites was shown to be consistent with preferential binding to, and thermal stabilization of DNA containing a THF site as compared to non-damaged DNA. Investigations into the properties of mitoxantrone at AP and 3' α,ß-unsaturated aldehyde sites demonstrated that in addition to being a potent inhibitor of APE1 at these biologically-relevant substrates (∼ 0.5 µM IC50 on AP site-containing DNA), mitoxantrone also incised AP site-containing DNA by catalyzing ß- and ß/δ-elimination reactions. The efficiency of these reactions to generate the 3' α,ß-unsaturated aldehyde and 3' phosphate products was modulated by DNA structure. Although these cell-free reactions revealed that mitoxantrone can generate 3' phosphates, cells lacking polynucleotide kinase phosphatase did not show increased sensitivity to mitoxantrone treatment. Consistent with its ability to inhibit APE1 activity on DNAs containing either an AP site or a 3' α,ß-unsaturated aldehyde, combined exposures to clinically-relevant concentrations of mitoxantrone and a small molecule APE1 inhibitor revealed additive cytotoxicity. These data suggest that in a cellular context, mitoxantrone may interfere with APE1 DNA repair functions.

The distinct roles of NEIL1 and XPA in limiting aflatoxin B1-induced mutagenesis in mice.

Dietary exposure to aflatoxin B1 (AFB1) is a risk factor for the development of hepatocellular carcinomas (HCCs). Following metabolic activation, AFB1 reacts with guanines to form covalent DNA adducts, which induce high-frequency G > T transversions. The molecular signature associated with these mutational events aligns with the single base substitution signature 24 (SBS24) in the Catalog of Somatic Mutations in Cancer (COSMIC) database. Deficiencies in either base excision repair (BER) due to the absence of Nei-like DNA glycosylase 1 (NEIL1) or nucleotide excision repair (NER) due to the absence of xeroderma complementation group A protein (XPA) contribute to HCCs in murine models. In the current study, ultra-low error duplex sequencing was used to characterize mutational profiles in liver DNAs of NEIL1-deficient, XPA-deficient, and DNA repair-proficient mice following neonatal injection of 1 mg/kg AFB1. Analyses of AFB1-induced mutations showed high cosine similarity to SBS24, regardless of repair proficiency status. The absence of NEIL1 resulted in an approximately 30% increase in the frequency of mutations, with distribution suggesting preferential NEIL1-dependent repair of AFB1 lesions in open chromatin regions. A trend of increased mutagenesis was also observed in the absence of XPA. Consistent with the role of XPA in transcription-coupled repair, mutational profiles in XPA-deficient mice showed disruption of the transcriptional bias in mutations associated with SBS24. Implications: Our findings define the roles of DNA repair pathways in AFB1-induced mutagenesis and carcinogenesis in murine models, with these findings having implications in human health for those with BER and NER deficiencies.

Frequencies and spectra of aflatoxin B1-induced mutations in liver genomes of NEIL1-deficient mice as revealed by duplex sequencing.

Increased risk for the development of hepatocellular carcinoma (HCC) is driven by a number of etiological factors including hepatitis viral infection and dietary exposures to foods contaminated with aflatoxin-producing molds. Intracellular metabolic activation of aflatoxin B1 (AFB1) to a reactive epoxide generates highly mutagenic AFB1-Fapy-dG adducts. Previously, we demonstrated that repair of AFB1-Fapy-dG adducts can be initiated by the DNA glycosylase NEIL1 and that male Neil1-/- mice were significantly more susceptible to AFB1-induced HCC relative to wild-type mice. To investigate the mechanisms underlying this enhanced carcinogenesis, WT and Neil1-/- mice were challenged with a single, 4 mg/kg dose of AFB1 and frequencies and spectra of mutations were analyzed in liver DNAs 2.5 months post-injection using duplex sequencing. The analyses of DNAs from AFB1-challenged mice revealed highly elevated mutation frequencies in the nuclear genomes of both males and females, but not the mitochondrial genomes. In both WT and Neil1-/- mice, mutation spectra were highly similar to the AFB1-specific COSMIC signature SBS24. Relative to wild-type, the NEIL1 deficiency increased AFB1-induced mutagenesis with concomitant elevated HCCs in male Neil1-/- mice. Our data establish a critical role of NEIL1 in limiting AFB1-induced mutagenesis and ultimately carcinogenesis.