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应用最广泛和最有效的抗癌药物。尽管TOP2靶向药物具有令人印象深刻的抗肿瘤活性，但已知会导致严重的副作用，如t-AML(与治疗相关的急性髓系白血病)。依托泊苷诱导的t-AML常常与混合系白血病基因(MLL)及其伴侣基因的平衡易位有关，导致MLL融合蛋白的表达。已有大量证据表明，这些易位主要是依托泊苷诱导的人类MLL基因断点簇区(BCR)内的DNA切割(S)的结果。TOP2和CAD(一种凋亡核酸酶)都被证明参与了依托泊苷诱导的MLL BCR内DNA的切割(S)。我们最近的研究表明，TOP2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靶向与依托泊苷的抗肿瘤活性有关，而依托泊苷靶向Top22的作用尚不清楚。我们的初步研究表明，依托泊苷靶向Top22会触发蛋白酶体介导的转录依赖的Top22降解(Top22下调)，导致Top22隐藏的DNA双链断裂的暴露。此外，Top22主要负责依托泊苷诱导的DNA序列重排和皮肤癌的发生。在目前的方案中，我们计划检验Top22同工酶主要负责依托泊苷诱导的MLL易位和t-AML的假设。此外，我们计划通过细胞培养和动物模型来确定Top22同工酶、蛋白酶体和CAD的作用，从而阐明依托泊苷诱导MLL易位的分子机制。其具体目的是(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是否参与了依托泊苷诱导的MLL易位和t-AML，以及(3)阐明依托泊苷诱导Top22下调的分子机制。这些研究的成功完成不仅将促进我们对TOP2药物诱导t-AML的分子基础的了解，而且将为开发TOP2同工酶特异性抗癌药物以及更有效的基于TOP2的癌症治疗提供新的策略。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，这些药物有望显示出较少的毒副作用(例如t-AML)。此外，阐明依托泊苷诱导MLL易位的分子基础也有助于开发新的策略来改进目前基于TOP2的治疗。例如，ICRF-187(用于特异性下调Top22)、Bortezomib(用于抑制蛋白酶体)或caspase抑制剂(用于阻断CAD激活)可用于减少与当前基于TOP2的治疗相关的毒副作用。

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.