NK314, a topoisomerase II inhibitor that specifically targets the alpha isoform

Abstract

Topoisomerase II (Top2) is a ubiquitous nuclear enzyme that relieves torsional stress in chromosomal DNA during various cellular processes. Agents that target Top2, involving etoposide, doxorubicin, and mitoxantrone, are among the most effective anticancer drugs used in the clinic. Mammalian cells possess two genetically distinct Top2 isoforms, both of which are the target of these agents. Top2alpha is essential for cell proliferation and is highly expressed in vigorously growing cells, whereas Top2beta is nonessential for growth and has recently been implicated in treatment-associated secondary malignancies, highlighting the validity of a Top2alpha-specific drug for future cancer treatment; however, no such agent has been hitherto reported. Here we show that NK314, a novel synthetic benzo[c]phenanthridine alkaloid, targets Top2alpha and not Top2beta in vivo. Unlike other Top2 inhibitors, NK314 induces Top2-DNA complexes and double-strand breaks (DSBs) in an alpha isoform-specific manner. Heterozygous disruption of the human TOP2alpha gene confers increased NK314 resistance, whereas TOP2beta homozygous knock-out cells display increased NK314 sensitivity, indicating that the alpha isoform is the cellular target. We further show that the absence of Top2beta does not alleviate NK314 hypersensitivity of cells deficient in non-homologous end-joining, a critical pathway for repairing Top2-mediated DSBs. Our results indicate that NK314 acts as a Top2alpha-specific poison in mammalian cells, with excellent potential as an efficacious and safe chemotherapeutic agent. We also suggest that a series of human knock-out cell lines are useful in assessing DNA damage and repair induced by potential topoisomerase-targeting agents.

FIGURE 1.

NK314 induces Top2 complexes and DSBs in a manner highly specific to theα isoform. A, PFGE analysis of Nalm-6 genomic DNA from untreated cells (None) or cells treated with NK314 (1 μm) or etoposide (100 μm) for 1 h. The cells were preincubated with (+) or without (–)10 μm ICRF-193 for 30 min. B, Western blot analysis for Top2 of Nalm-6 and HeLa cells using 7B9 antibody (31). Ku70 served as a loading control. C and D, ICE bioassay for detection of Top2 complex formation. Indicated amounts of DNA from drug-treated Nalm-6 (C) or HeLa (D) cells were blotted to polyvinylidene difluoride or nitrocellulose membrane. Top2 protein covalently bound to DNA was detected with Top2α- or Top2β-specific antibody. E, TARDIS analysis in MEF cells treated with 100 μm etoposide or 5 μm NK314 for 90 min. Total Top2 complexes (α + β) were detected with antibody 4882, whereas Top2β complexes (β) were detected with antibody 18513β. Mean fluorescence values were calculated for each treatment and normalized to the mean value for etoposide-treated MEFs. The data shown are from three to six independent experiments. F, effect of NK314 on Top2-mediated DNA cleavage. Supercoiled plasmid DNA was incubated with Top2α or Top2β or without enzyme (–Top2) in the absence (–solvent) or presence of 5% DMSO (None), NK314 (25–400 μm), or etoposide (100–400 μm). N, nicked circular DNA; L, linear DNA; S, supercoiled DNA; and R, relaxed DNA. G, inhibitory effect of NK314 on Top2 decatenation activity. Kinetoplast DNA was incubated with 2 units of purified Top2α or Top2β in the absence (5% DMSO; 0 μm) or presence of NK314 (3–100 μm).

FIGURE 2.

siRNA-mediated Top2α knockdown leads to increased NK314 resistance. A, Western blot analysis for Top2 in siRNA-transfected HeLa cells. Whole cell extract was prepared from mock-transfected cells (lane 1) or cells transfected with Top2α siRNA (lane 2), Top2β siRNA (lane 3), or control siRNA (lane 4). Actin served as a loading control. B, NK314 sensitivity of siRNA-transfected cells. Data are the mean ± S.D. of three independent assays. Where absent, error bars fall within symbols.

FIGURE 3.

Targeted disruption of the human TOP2α gene. A, scheme for TOP2α gene targeting. The targeting vector was designed to replace exons 5 to 7 with a hygromycin-resistance (Hyg^r) gene. Triangles represent loxP sequences. DT-A, a gene encoding diphtheria toxin A fragment. B, Southern blot analysis of EcoRI-digested genomic DNA from wild-type (+/+) and TOP2α heterozygous (+/–) cells using the probe shown in A. C, Western blot analysis for Top2 in mutant cell lines. Whole cell extract from 1 × 10⁵ cells was loaded on a 7.5% SDS-polyacrylamide gel. Levels of expression were quantified using an image analyzer. Ku70 served as a loading control. D, growth curves of wild-type and mutant cell lines. Data are the mean ± S.D. of three independent experiments. Where absent, error bars fall within symbols.

FIGURE 4.

Targeted disruption of the human TOP2β gene. A, scheme for TOP2β gene targeting. The targeting vector was designed to replace exons 9 to 11 with a hygromycin-resistance (Hyg^r) or puromycin-resistance (Puro^r) gene. Symbols are as described in the legend to Fig. 3. B, Southern blot analysis of KpnI/NcoI-digested genomic DNA from wild-type (+/+) and TOP2β heterozygous (+/–) and homozygous (–/–) cells using the probe shown in A. C, Western blot analysis for Top2 in mutant cell lines. Whole cell extract from 1 × 10⁵ cells was loaded on a 7.5% SDS-polyacrylamide gel. Levels of expression were quantified using an image analyzer. Ku70 and Actin served as loading controls. D, growth curves of wild-type and mutant cell lines. Data are the mean ± S.D. of three independent experiments. Where absent, error bars fall within symbols.

FIGURE 5.

NK314, unlike other Top2 inhibitors, specifically targets the α isoform. A–G, sensitivities of wild-type, TOP2α^+/–, and TOP2β^–/– cells to topoisomerase inhibitors: NK314 (A), etoposide (B), doxorubicin (C), amsacrine (D), mitoxantrone (E), XK469 (F), and camptothecin (G). Data are the mean ± S.D. of three independent experiments. Where absent, error bars fall within symbols. H, PFGE analysis of Nalm-6 genomic DNA from untreated cells (None) or cells treated with NK314 (1 μm) or etoposide (30 μm) for 1 h. WT, wild-type; α, TOP2α^+/–; and β, TOP2β^–/– cells.

FIGURE 6.

Loss of Top2β does not affect NK314 sensitivity in MEFs. Clonogenic survival assays were performed using NK314-treated Top2β^+/+ (WT) and Top2β^–/– MEFs. Data are the mean ± S.D. of three independent experiments. Where absent, error bars fall within symbols.

FIGURE 7.

NHEJ-deficient human cells are hypersensitive to NK314, although less prominent than to other Top2 inhibitors. A–C, sensitivities of wild-type, RAD54^–/–, LIG4^–/–, and LIG4^–/–RAD54^–/– cells to NK314 (A), etoposide (B), and doxorubicin (C). D and E, sensitivities of wild-type, TOP2β^–/–, LIG4^–/–, and LIG4^–/–TOP2β^–/– cells to NK314 (D) and etoposide (E). F and G, sensitivities of wild-type, TOP2α^+/–, LIG4^–/–, and LIG4^–/–TOP2α^+/– cells to NK314 (F) and etoposide (G). In all assays, cells were allowed for colony formation in agarose medium containing the indicated concentrations of drugs. Data are the mean ± S.D. of three to six independent experiments. Where absent, error bars fall within symbols. H, summary of sensitivity assays shown in A and B. I, summary of sensitivity assays shown in D–G.

FIGURE 8.

NK314 cytotoxicity is less dependent on exposure time than etoposide cytotoxicity. A and B, sensitivities of wild-type, RAD54^–/–, LIG4^–/–, and LIG4^–/–RAD54^–/– cells to NK314 (A) and etoposide (B). Sensitivity was measured by treating cells with Top2 inhibitor for 1 h, followed by colony formation in drug-free agarose medium. Data are the mean ± S.D. of three independent experiments. Where absent, error bars fall within symbols. C, summary of sensitivity assays shown in A and B. D, inhibitory effect of NK314 and etoposide on proliferation of various cancer cell lines. Cell proliferation was measured by using CellTiter-Glo (for Nalm-6) or methylene blue staining (for other cell lines). Cells were cultured for 72 h (for Nalm-6, 96 h) in the presence of Top2 inhibitor (long exposure, L), or for 1 h in the presence of Top2 inhibitor, followed by culture in drug-free medium for 71 h (for Nalm-6, 96 h) (short exposure, S). Exposure time dependence of the growth inhibitory effect of NK314 or etoposide was determined by dividing IC₅₀ (S) by IC₅₀ (L).