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推进基础知识和改善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;利用肿瘤细胞与正常细胞的脆弱点;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使用这些信息来更准确地预测临床反应，以便可以识别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伴随的不良副作用。微管抑制剂（MIA）包括长春花生物碱和紫杉烷类，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停滞，导致纺锤体检查点的持续激活和随后的凋亡性细胞死亡。然而，值得注意的是，尽管它们被广泛使用，但关于纺锤体检查点激活后细胞如何死亡的可靠分子解释尚未出现。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。在上一个资助周期中，进行了研究来测试我们的主要假设，即Bcl-2/Bcl-xL磷酸化是控制抗有丝分裂药物诱导凋亡的关键事件，并且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，获得的证据表明，CDK 1/细胞周期蛋白B作为负责MIA诱导的Bcl-2/Bcl-XL磷酸化的激酶。CDK 1功能的这种重定向，从正常有丝分裂期间的促增殖到MIA诱导的有丝分裂停滞后的促凋亡，在概念上和机制上是有吸引力的。此外，我们发现，在正常的有丝分裂过程中，CDK 1部分和瞬时磷酸化Bcl-2/Bcl-xL。结果提供了令人信服的证据，CDK 1介导的Bcl-2/Bcl-XL磷酸化作为一个功能链接耦合有丝分裂阻滞和凋亡，并建议的可能性，抗凋亡Bcl-2蛋白作为传感器的CDK 1信号持续时间。在本提案中，我们将借鉴这些进展，并提出以下具体目标。具体目标1将检验Bcl-2/Bcl-xL磷酸化调节蛋白质：蛋白质相互作用的假设，特别是促凋亡Bcl-2蛋白的结合/释放，尤其是激活剂和/或敏化剂仅BH 3蛋白。在具体目标2中，我们将根据CDK 1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。具体目标3将研究CDK 1和抗凋亡Bcl-2蛋白的促凋亡信号传导机制。具体目标4将使用来自急性淋巴细胞白血病儿科患者的原始细胞，在临床相关环境中确定CDK 1活化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多的新见解。 公共卫生相关性： Bcl-2蛋白在抗有丝分裂药物作用机制中的作用叙述：抗肿瘤药物广泛用于癌症治疗。然而，我们并不确切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，这种损伤会通过复杂的细胞内信号通路被解释和作用。我们正在研究这些药物如紫杉醇的损伤传感途径。了解这些途径很重要，因为所获得的知识为适当使用现有药物提供了基础，并为发现新药物和靶点提供了理论基础。大多数抗肿瘤剂对正常细胞是有毒的，并产生不希望的副作用。了解这些药物的作用机制对于癌症患者的适当管理至关重要，特别是当使用药物组合时。

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.