Mechanism of Action of TOP2-Directed Anticancer Drugs

描述（申请人提供）：本申请的长期目标是了解拓扑异构酶II（Top2）靶向药物的作用机制。Top2靶向药物如依托泊苷（VP-16）、阿霉素和米托蒽醌是临床上最有效和最广泛使用的抗癌药物之一。尽管具有令人印象深刻的抗肿瘤活性，但已知Top2靶向药物会引起严重的副作用，如t-AML（治疗相关的急性髓性白血病）。依托泊苷诱导的t-AML通常与混合谱系白血病基因（MLL）和许多MLL伴侣基因的平衡易位相关，导致MLL融合蛋白的表达。有大量证据表明，这些易位主要是依托泊苷诱导的人MLL基因断裂点簇区域（BCR）内DNA切割的结果。Top2和CAD（一种凋亡核酸酶）均参与依托泊苷诱导的MLL BCR内DNA切割。我们最近的研究表明，Top2的两种同工酶Top21和Top22可能在Top2靶向药物的抗肿瘤活性和副作用中发挥不同的作用。Top21靶向被认为有助于依托泊苷的抗肿瘤活性，而Top22靶向依托泊苷的作用尚不清楚。我们的初步研究表明，依托泊苷靶向Top22触发转录依赖性，蛋白酶体介导的Top22降解（Top22下调），导致暴露的Top22隐藏的DNA双链断裂。此外，Top22主要负责依托泊苷诱导的DNA序列重排和皮肤癌发生。在目前的提案中，我们计划测试的假设，Top22同工酶是主要负责依托泊苷诱导的MLL易位和t-AML。此外，我们计划阐明依托泊苷诱导的MLL易位的分子机制，通过确定Top22同工酶，蛋白酶体和CAD使用细胞培养和动物模型的作用。具体目的是（1）确定Top22同工酶在依托泊苷诱导的MLL易位和t-AML中的作用，（2）确定蛋白酶体和凋亡核酸酶CAD是否参与依托泊苷诱导的MLL易位和t-AML，以及（3）阐明依托泊苷诱导的Top22下调的分子机制。成功完成拟议的研究不仅将促进我们对Top2药物诱导的t-AML的分子基础的理解，而且还为开发Top2同工酶特异性抗癌药物以及更有效的基于Top2的癌症治疗的新策略提供理论基础。公共卫生相关性：Top22在MLL易位中的作用的成功证明将为开发Top21同工酶特异性抗癌药物提供必要的理论基础，这些药物有望表现出降低的毒副作用（例如t-AML）。此外，阐明依托泊苷诱导的MLL易位的分子基础也可以导致开发新的策略来改善目前基于Top2的治疗。例如，ICRF-187（用于特异性下调Top22）、硼替佐米（用于抑制蛋白酶体）或半胱天冬酶抑制剂（用于阻断CAD活化）可用于减少与当前基于Top2的疗法相关的毒副作用。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.