Bcl-2 Proteins in Mechanism of Anti-mitotic Drug Action

描述（由申请人提供）：了解抗肿瘤药物作用的分子机制对于推进基础知识和改善癌症治疗具有重要意义。通过确定这些机制，可以发现新的药物靶点；利用肿瘤细胞与正常细胞的易损点；新型药物组合试验；揭示了药物敏感性的决定因素。因此，一个主要的长期目标是利用这些信息更准确地预测临床反应，从而确定最有可能受益的患者，并避免对那些不太可能受益的患者进行治疗和随之而来的不良副作用。微管抑制剂（MIAs）包括长春花生物碱和紫杉烷是癌症化疗药物库中的重要药物。这些药物通常诱导有丝分裂阻滞，导致纺锤体检查点的持续激活和随后的凋亡细胞死亡。然而，值得注意的是，尽管它们被广泛使用，一个可靠的分子解释如何细胞死亡后纺锤体检查点激活尚未出现。微管抑制剂引起的最突出的作用是抗凋亡蛋白Bcl-2和Bcl-xL的磷酸化。在之前的资助周期中，我们进行了研究以验证我们的主要假设，即Bcl-2/Bcl-xL磷酸化是抗有丝分裂药物诱导细胞凋亡的关键事件，并由一种新的或未知的激酶催化。我们成功地在很大程度上完成了目标并验证了我们的假设，令人兴奋的是，我们获得了CDK1/cyclin B作为mia诱导的Bcl-2/Bcl-xL磷酸化的激酶的证据。这种CDK1功能的重定向，从正常有丝分裂时的促增殖到mia诱导的有丝分裂停止后的促凋亡，在概念上和机制上都很有吸引力。此外，我们发现CDK1在正常有丝分裂过程中部分和短暂地磷酸化Bcl-2/Bcl-xL。结果提供了令人信服的证据，表明CDK1介导的Bcl-2/Bcl-xL磷酸化是偶联有丝分裂停止和凋亡的功能纽带，并提示抗凋亡的Bcl-2蛋白可能作为CDK1信号持续时间的传感器。在本建议中，我们将借鉴这些进展，提出以下具体目标。特异性目的1将验证Bcl-2/Bcl-xL磷酸化调节蛋白：蛋白相互作用的假设，特别是促凋亡Bcl-2蛋白的结合/释放，特别是激活剂和/或致敏剂BH3-only蛋白。在Specific Aim 2中，我们将基于CDK1介导Mcl-1降解的初步数据，研究Mcl-1磷酸化在mia诱导的细胞凋亡中的作用。特异性目的3将探讨CDK1和抗凋亡蛋白Bcl-2的促凋亡信号传导机制。特异性Aim 4将使用来自急性淋巴细胞白血病儿童患者的细胞，在临床相关环境中确定CDK1激活在长春新碱敏感性中的作用。在这些实验完成后，我们希望对这类重要的抗癌药物的分子作用机制有相当大的新认识。

DESCRIPTION (provided by applicant): Understanding the molecular mechanisms of action of antitumor agents is significant for advancing fundamental knowledge and for improvement in cancer treatment. By defining these mechanisms, new drug targets can be uncovered; points of vulnerability in tumor cells versus normal cells exploited; novel drug combinations tested; and determinants of drug sensitivity revealed. Thus a major long-term goal is to use this information to more accurately predict clinical response, such that patients most likely to benefit can be identified, and treatment and attendant adverse side-effects avoided for those unlikely to benefit. Microtubule inhibiting agents (MIAs) including the vinca alkaloids and taxanes are important drugs in the arsenal of cancer chemotherapeutics. These drugs typically induce mitotic arrest leading to sustained activation of the spindle checkpoint and subsequent apoptotic cell death. Remarkably however, despite their widespread use, a solid molecular explanation of how cells die after spindle checkpoint activation has yet to emerge. The most prominent effect elicited by microtubule inhibitors is the phosphorylation of the antiapoptotic proteins, Bcl-2 and Bcl-xL. In the previous funding cycle, studies were undertaken to test our main hypothesis, that Bcl-2/Bcl-xL phosphorylation is a key event controlling apoptosis induction by anti-mitotic drugs and is catalyzed by a novel or unsuspected kinase. We were successful in largely completing the aims and validating our hypothesis, and excitingly, obtained evidence implicating CDK1/cyclin B as the kinase responsible for MIA-induced Bcl-2/Bcl-xL phosphorylation. Such a redirection in CDK1 function, from pro- proliferative during normal mitosis to pro-apoptotic after MIA-induced mitotic arrest, is conceptually and mechanistically appealing. Further, we showed that CDK1 partially and transiently phosphorylates Bcl-2/Bcl-xL during normal mitosis. The results provide compelling evidence that CDK1-mediated Bcl-2/Bcl-xL phosphorylation acts as a functional link coupling mitotic arrest and apoptosis, and suggest the possibility that anti-apoptotic Bcl-2 proteins act as sensors for CDK1 signal duration. In this proposal we will draw on these advances and propose the following Specific Aims. Specific Aim 1 will test the hypothesis that Bcl-2/Bcl-xL phosphorylation regulates protein:protein interaction, in particular the binding/release of pro-apoptotic Bcl-2 proteins and especially activator and/or sensitizer BH3-only proteins. In Specific Aim 2, we will examine the role of Mcl-1 phosphorylation in MIA-induced apoptosis, based on preliminary data that CDK1 mediates Mcl-1 degradation. Specific Aim 3 will investigate the mechanisms of pro-apoptotic signaling by CDK1 and anti- apoptotic Bcl-2 proteins. Specific Aim 4 will use blasts derived from pediatric patients with acute lymphoblastic leukemia to establish the role of CDK1 activation in vincristine sensitivity in a clinically relevant setting. At the completion of these experiments, we hope to have gained considerable new insight into the molecular mechanisms of action of this important class of cancer drugs. PUBLIC HEALTH RELEVANCE: Bcl-2 Proteins in Mechanism of Anti-mitotic Drug Action Narrative: Anti-tumor drugs are widely used in cancer treatment. However, we do not know exactly how they function, because after they cause initial damage to a cell, the damage is interpreted and acted upon through complex intracellular signaling pathways. We are investigating these damage sensing pathways for drugs such as Taxol. Understanding these pathways is important because the knowledge gained provides a basis for the appropriate use of existing agents and a theoretical basis for the discovery of new agents and targets. Most anti-tumor agents are toxic to normal cells and produce undesirable side-effects. Knowledge of the mechanism of these drugs is vital for the proper management of cancer patients especially when drug combinations are used.