We report for the first time the use of a selective small-molecule inhibitor of DNA repair to potentiate topoisomerase II (topo II) poisons, identifying DNA-dependent protein kinase (DNA-PK) as a potential target for leukemia therapy. Topo II poisons form cleavable complexes that are processed to DNA double-strand breaks (DSBs). DNA-PK mediates nonhomologous end joining (NHEJ). Inhibition of this DSB repair pathway may sensitize cells to topo II poisons. We investigated the effects of a novel DNA-PK inhibitor, NU7026 (2-(morpholin-4-yl)-benzo[h]chomen-4-one), on the response to topo II poisons using K562 leukemia cells. NU7026 (10 μM) potentiated the growth inhibition of idarubicin, daunorubicin, doxorubicin, etoposide, amsacrine (mAMSA), and mitroxantrone with potentiation factors at 50% growth inhibition ranging from approximately 19 for mAMSA to approximately 2 for idarubicin (potentiation of etoposide was confirmed by clonogenic assay). In contrast, NU7026 did not potentiate camptothecin or cytosine arabinoside (araC). NU7026 did not affect the levels of etoposide-induced topo IIα or β cleavable complexes. NU7026 alone had no effect on cell cycle distribution, but etoposide-induced accumulation in G2/M was increased by NU7026. A concentration-dependent increase in etoposide-induced DSB levels was increased by NU7026. The mechanism of NU7026 potentiation of topo II poisons involves inhibition of NHEJ and a G2/M checkpoint arrest. (Blood. 2004;103:4659-4665)

Type II topoisomerases are required for the management of DNA superhelicity and chromosome segregation, and serve as frontline targets for a variety of small-molecule therapeutics. To better understand how these enzymes act in both contexts, we determined the 2.9-Å-resolution structure of the DNA cleavage core of human topoisomerase IIα (TOP2A) bound to a doubly nicked, 30-bp duplex oligonucleotide. In accord with prior biochemical and structural studies, TOP2A significantly bends its DNA substrate using a bipartite, nucleolytic center formed at an N-terminal dimerization interface of the cleavage core. However, the protein also adopts a global conformation in which the second of its two inter-protomer contact points, one at the C-terminus, has separated. This finding, together with comparative structural analyses, reveals that the principal site of DNA engagement undergoes highly quantized conformational transitions between distinct binding, cleavage, and drug-inhibited states that correlate with the control of subunit-subunit interactions. Additional consideration of our TOP2A model in light of an etoposide-inhibited complex of human topoisomerase IIβ (TOP2B) suggests possible modification points for developing paralog-specific inhibitors to overcome the tendency of topoisomerase II-targeting chemotherapeutics to generate secondary malignancies.

The histone variant histone H2A.X comprises up to 25% of the H2A complement in mammalian cells. It is rapidly phosphorylated following exposure of cells to double-strand break (DSB) inducing agents such as ionising radiation. Within minutes of DSB generation, H2AX molecules are phosphorylated in large chromatin domains flanking DNA double-strand breaks (DSBs); these domains can be observed by immunofluorescence microscopy and are termed γH2AX foci. H2AX phosphorylation is believed to have a role mounting an efficient cellular response to DNA damage. Theoretical considerations suggest an essentially random chromosomal distribution of X-ray induced DSBs, and experimental evidence does not consistently indicate otherwise. However, we observed an apparently uneven distribution of γH2AX foci following X-irradiation with regions of the nucleus devoid of foci. Using immunofluorescence microscopy, we show that focal phosphorylation of histone H2AX occurs preferentially in euchromatic regions of the genome following X-irradiation. H2AX phosphorylation has also been demonstrated previously to occur at stalled replication forks induced by UV radiation or exposure to agents such as hydroxyurea. In this study, treatment of S-phase cells with hydroxyurea lead to efficient H2AX phosphorylation in both euchromatin and heterochromatin at times when these chromatin compartments were undergoing replication. This suggests a block to H2AX phosphorylation in heterochromatin that is at least partially relieved by ongoing DNA replication. We discus a number of possible mechanisms that could account for the observed pattern of H2AX phosphorylation. Since γH2AX is regarded as forming a platform for the recruitment or retention of other DNA repair and signaling molecules, these findings imply that the processing of DSBs in heterochromatin differs from that in euchromatic regions. The differential responses of heterochromatic and euchromatic compartments of the genome to DSBs will have implications for understanding the processes of DNA repair in relation to nuclear and chromatin organization.