The long-term goal of this application is to understand the mechanism of action of topoisomerase II (Top2)-targeting drugs. Top2-targeting drugs such as etoposide (VP-16), doxorubicin and mitoxantrone are among the most effective and widely used anticancer drugs in the clinic. Despite their impressive antitumor activities, Top2-targeting drugs are known to cause serious side effects such as t-AML (therapy-related acute myeloid leukemia). Etoposide-induced t-AML is frequently associated with balanced translocations of the mixed lineage leukemia gene (MLL) and many MLL partner genes, resulting in the expression of MLL fusion proteins. There has been substantial evidence that these translocations are primarily the consequence of etoposide-induced DNA cleavage(s) within the breakpoint cluster region (BCR) of the human MLL gene. Both Top2 and CAD (an apoptotic nuclease) have been shown to be involved in etoposide-induced DNA cleavage(s) within the MLL BCR. Our recent studies have suggested that the two Top2 isozymes, Top21 and Top22 may play different roles in the antitumor activity and side effects of Top2-targeting drugs. Top21 targeting has been suggested to contribute to the antitumor activity of etoposide while the role of Top22 targeting by etoposide is unclear. Our preliminary studies have demonstrated that Top22 targeting by etoposide triggers transcription-dependent, proteasome-mediated degradation of Top22 (Top22 down-regulation), resulting in the exposure of Top22-concealed DNA double-strand breaks. In addition, Top22 is primarily responsible for etoposide-induced DNA sequence rearrangements and skin carcinogenesis. In the current proposal, we plan to test the hypothesis that the Top22 isozyme is primarily responsible for etoposide-induced MLL translocations and t-AML. In addition, we plan to elucidate the molecular mechanism for etoposide-induced MLL translocations by determining the roles of Top22 isozyme, proteasome and CAD using both cell culture and animal models.
The specific aims are (1) to determine the role of the Top22 isozyme in etoposide-induced MLL translocations and t-AML, (2) to determine whether proteasome and the apoptotic nuclease CAD are involved in etoposide-induced MLL translocations and t-AML, and (3) to elucidate the molecular mechanism for etoposide-induced Top22 down-regulation. Successful completion of the proposed studies will not only advance our understanding of the molecular basis for Top2 drug-induced t-AML but also provide a theoretical foundation for developing Top2 isozyme-specific anticancer drugs, as well as new strategies, for more efficacious Top2-based cancer therapy.
The successful demonstration of the role of Top22 in MLL translocations will provide the necessary theoretical foundation for developing Top21 isozyme-specific anticancer drugs which are expected to exhibit reduced toxic side effects (e.g. t-AML). In addition, elucidation of the molecular basis for etoposide-induced MLL translocations can also lead to development of new strategies for improving current Top2-based therapy. For example, ICRF-187 (for specific down-regulation of Top22), bortezomib (for inhibition of proteasome) or caspase inhibitors (for blocking CAD activation) could be employed to reduce the toxic side effects associated with current Top2-based therapy.
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