Abstract
Chemotherapeutic DNA topoisomerase II (top2) poisons (i.e. agents that stabilize the top2 covalent complex and have the overall effect of increasing cleavage complexes) have been implicated in the treatment complication of leukemia characterized by balanced translocations, among which are translocations of the NUP98 gene at chromosome band 11p15. NUP98, which encodes a 98-kd docking protein in the nuclear pore complex, is disrupted by translocations in de novo and chemotherapy-related leukemias and has ~15 partner genes encoding proteins of diverse function. There have been no studies to delineate the mechanism whereby top2 poisons result in DNA damage leading to NUP98 translocations. Previously we described the TOP1 gene at chromosome band 20q11 as a recurring partner gene of NUP98 in 2 cases of treatment-related MDS (Ahuja 2000). The first patient was treated 9 years before for ALL with therapy including doxorubicin (ADR), daunorubicin and etoposide (VP). The second patient received VP and ADR in therapy for non-Hodgkin’s lymphoma and MDS was diagnosed 14 months after the primary cancer. In both cases, the NUP98 breakpoints were in intron 13 and the TOP1 breakpoints were in intron 7, though at distinct locations (Ahuja 2000). Sequencing of the breakpoint junctions on both derivative chromosomes showed near-precise recombinations relative to the germline sequences, indicating that the translocation breakpoints were at the sites of damage. Here, to interrogate whether drugs to which the patients were exposed stimulated formation of top2 cleavage complexes at these sites of damage, DNA substrates from germline NUP98 and TOP1 sequences were utilized in top2 in vitro cleavage assays, which entail trapping the cleavage complexes and mapping the cleavage sites in the sequence. ADR was examined over a range of concentrations because of its known effects of cleavage stimualtion at low concentrations and, at high concentrations, top2 catalytic inhibition due to intercalation. Etoposide and its catechol and quinone metabolites stimulated strong, heat stable top2 cleavage at several sites proximal to the NUP98 and TOP1 translocation breakpoints. At low concentration (~0.01μM) ADR also enhanced cleavage in a NUP98 substrate at several sites near the translocation breakpoint while dose-dependent cleavage diminution occurred at higher concentrations. Dose-response experiments with ADR are in progress using TOP1 substrates. Prior models based on the coincidence of in vitro top2 cleavage sites with cloned translocation breakpoints in MLL and partner genes or in PML and RARA and feasibility of resolution of the cleavage sites to form the respective breakpoint junctions have suggested drug-stimulated top2 cleavage as the relevant damage mechanism in other translocations (Whitmarsh 2003; Mistry 2003). The results of the present study support a model in which the chromosomal breakage leading to chemotherapy-related NUP98-TOP1 translocations is a consequence of drug-stimulated top2 cleavage also. Dose-dependent dual effects of ADR as a poison and a catalytic inhibitor of top2 function with respect to the sequence of a specific leukemia-associated gene have implications for its role in the genesis of translocations.
Identification of potent catalytic inhibitors of human DNA topoisomerase II by structure-based virtual screening
