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

描述(由申请人提供)：了解抗肿瘤药物的分子作用机制对于增进基础知识和改进癌症治疗具有重要意义。通过定义这些机制，可以发现新的药物靶点；利用肿瘤细胞相对于正常细胞的脆弱性；测试新的药物组合；揭示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，以便确定最有可能受益的患者，并避免那些不太可能受益的患者的治疗和随之而来的不良副作用。微管抑制剂(MIA)包括长春花碱和紫杉烷类化合物，是肿瘤化疗药物中的重要药物。这些药物通常会导致有丝分裂停止，导致纺锤体检查点的持续激活和随后的细胞凋亡。然而，值得注意的是，尽管它们被广泛使用，但对于纺锤体检查点激活后细胞如何死亡的可靠的分子解释尚未出现。微管抑制剂最显著的作用是对抗细胞凋亡蛋白Bcl2和Bclxl的磷酸化。在之前的资金周期中，有研究对我们的主要假设进行了验证，即Bcl2/Bclxl磷酸化是控制抗有丝分裂药物诱导细胞凋亡的关键事件，并由一种新的或未被怀疑的激酶催化。我们在很大程度上成功地完成了目标并验证了我们的假设，令人兴奋的是，我们获得了证据表明CDK1/Cyclin B是MIA诱导的Bcl2/Bclxl磷酸化的激酶。CDK1功能的这种重定向，从正常有丝分裂期间的促增殖到MIA诱导的有丝分裂停止后的促凋亡，在概念和机制上都是有吸引力的。此外，我们还发现，在正常有丝分裂过程中，CDK1部分和短暂地磷酸化了Bcl2/Bclxl。这些结果为CDK1介导的Bcl2/Bclxl磷酸化作为连接有丝分裂停滞和细胞凋亡的功能纽带提供了有力的证据，并提示抗凋亡的Bcl2蛋白可能作为CDK1信号持续时间的感受器。在这项提案中，我们将借鉴这些进展，并提出以下具体目标。特定目的1将检验这样的假设，即：Bcl2/Bclxl磷酸化调节蛋白质：蛋白质相互作用，特别是促凋亡的Bcl2蛋白质的结合/释放，特别是激活剂和/或敏感剂BH3-唯一的蛋白质。在特定的目标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。特异靶3将研究CDK1和抗凋亡的Bcl2蛋白促进细胞凋亡的信号转导机制。具体目标4将使用来自儿童急性淋巴细胞白血病患者的原始细胞来确定CDK1激活在临床相关环境中对长春新碱敏感性的作用。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。 公共卫生相关性： 抗有丝分裂药物作用机制中的bcl2蛋白叙述：抗肿瘤药物广泛应用于癌症治疗。然而，我们并不确切地知道它们是如何发挥作用的，因为在它们对细胞造成初始损伤后，这种损伤是通过复杂的细胞内信号通路来解释和作用的。我们正在研究紫杉醇等药物的损伤感知途径。了解这些途径很重要，因为所获得的知识为适当使用现有制剂提供了基础，也为发现新的制剂和目标提供了理论基础。大多数抗肿瘤药物对正常细胞有毒性，并产生不良副作用。了解这些药物的作用机制对于癌症患者的正确治疗至关重要，特别是在使用联合用药的情况下。

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.