Microtubule (MT) Interfering Agents (MTAs): Mechanisms of Action and Resistance

微管 (MT) 干扰剂 (MTA)：作用和耐药机制

• 批准号: 7965477
• 负责人: Antonio Fojo
• 金额: $ 66.58万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965477
• 关键词: Acetylation; Affect; Aftercare; Antineoplastic Agents; Area; Binding; Biological; Biological Assay; Biological Factors; Biopsy; Cell Death; Cell Line; Cell Nucleus; Cell Survival; Cell physiology; Cells; Chemical Structure; Chemicals; Chemotherapy-Oncologic Procedure; Cisplatin; Clinic; Clinical; Clinical Data; Clinical Oncology; Clinical Trials; Complex; Conduct Clinical Trials; Cytoplasm; Cytoskeleton; DNA Damage; Data; Development; Disease; Dose; Drug Delivery Systems; Drug resistance; Dynein ATPase; Epothilone B; Epothilone B Analogue; Eukaryotic Cell; Family; Future; Gene Targeting; Glucocorticoid Receptor; Goals; Growth; HTI-286; Head and Neck Cancer; Hodgkin Disease; Human; Interphase; Interphase Cell; Intracellular Transport; Ixabepilone; Kidney; Kinesin; Knowledge; Laboratories; Lead; Learning; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Malignant neoplasm of lung; Malignant neoplasm of ovary; Malignant neoplasm of prostate; Malignant neoplasm of thyroid; Measurement; Mediating; Methodology; Methods; Microtubules; Mitotic spindle; Modeling; Modification; Monitor; Motor; Multiple Myeloma; Mutation; New Agents; Non-Hodgkin' s Lymphoma; Nuclear; Nuclear Translocation; Paclitaxel; Patients; Pharmaceutical Preparations; Pharmacodynamics; Phase II Clinical Trials; Post-Translational Protein Processing; Process; Proteins; Randomized Clinical Trials; Recurrence; Renal Cell Carcinoma; Research; Resistance; Role; Sampling; Signal Transduction; Stabilizing Agents; System; TP53 gene; Therapeutic; Thinking; Time; Transcriptional Regulation; Travel; Treatment Protocols; Tubulin; Vinca Alkaloids; Vincristine; Work; alpha Tubulin; base; cancer cell; cell motility; chemotherapy; drug development; hemiasterlin; interest; kidney cell; macromolecule; malignant breast neoplasm; malignant stomach neoplasm; neoplastic cell; novel; polymerization; preclinical study; resistance mechanism; response; sarcoma; success; trafficking; tumor

The cytoskeleton of eukaryotic cells participates in various cellular functions such as motility, secretion, signaling and proliferation. Microtubules (MTs) are an integral part of the cytoskeleton. Among anti-cancer agents, drugs targeting tubulin or MTs are among the most, if not the most, effective class of agents. The list of compounds that bind to tubulin or the MTs is large and continues to expand. The overwhelming majority are natural products, and their chemical structures are remarkably diverse. The vinca alkaloids were introduced in the 1950's, and although they were useful in a wide range of malignancies, interest in developing new agents targeting MTs gradually declined, until the introduction of Taxol. Arguably the most effective agent since cisplatin, the remarkable activity of Taxol stimulated interest in tubulin and MTs as targets for chemotherapy. The clinical success of Taxol has led to a wealth of new scientific knowledge, reinforced the importance of the tubulin/MT system as a target for cancer chemotherapy and spurred efforts to identify novel tubulin-active agents. In the field of MT targeting agents (MTAs), our current research goals are to (1) to increase our understanding of how MTAs interact with tubulin and lead to cell death; (2) understand the mechanisms of resistance to MTAs; and (3) develop assays to monitor the pharmacodynamics of MTAs in patients. In the clinic, we continue to conduct trials examining MTAs. Given our success in identifying mutations in paclitaxel- and epothilone-resistant cells, and encouraged by the information accumulated and the lessons learned, we began selections with HTI-286 a synthetic hemiasterlin in development at that time by Wyeth-Ayerst. As we were investigating the hemiasterlin resistant cell lines, we were also conducting a phase II clinical trial with BMS-247550 (ixabepilone) in patients with renal cell carcinoma. BMS-247550 is an epothilone B analogue and MT-stabilizing agent. As a part of this ongoing trial, we were attempting to obtain tumor biopsies before therapy and after the fifth dose of BMS-247550. The original goal had been to examine the pharmacodynamics of BMS-247550 by quantitating the degree of tubulin polymerization before and after drug administration. This measurement would allow us to establish whether BMS-247550 had stabilized MTs in the tumor cells. However, we also considered alternate methods to demonstrate MT stabilization. Encouraged by the results in our hemiasterlin resistant cells where tubulin acetylation and detyrosination had been correlated with MT stabilization, we chose to investigate these chemical modifications in the patient samples. As a first step we demonstrated that the levels of detyrosinated (glu-terminated) and acetylated alpha-tubulin correlated well with MT stabilization induced by BMS-247550 in cultured renal and ovarian cancer cells, suggesting these modifications could be used to monitor MT-stabilization. More importantly, in examining the patient samples we found that after treatment with BMS-247550, the levels of glu-terminated and/or acetylated alpha-tubulin increased 2- to 100-fold in 8 out of 8 serial tumor biopsies. These data indicate BMS-247550 reached the tumors and engaged the MT target, leading to MT stabilization consistent with its ability to avert Pgp and reach its target. We conclude that glu-terminated and/or acetylated alpha-tubulin levels are simple and reliable markers for the pharmacodynamic effects of BMS-247550. We believe that assessing post-translationally modified tubulin levels may provide a simple and reliable assay of the pharmacodynamic effects of other MTAs. Puzzled by the recurrent observation that our paclitaxel and epothilone resistant cell lines had acquired mutations in p53, we set out to determine if p53 could interact with tubulin in a meaningful way. We found that both wt and mt p53 associate with MTs and this interaction is lost following treatment with MT-depolymerizing drugs. Furthermore we showed that p53 accumulates in the nucleus following DNA damage only in cells with a functional MT network. Pre-treatment with either vincristine or paclitaxel reduced nuclear accumulation of p53, indicating nuclear translocation of p53 requires a functional MT network. In most cells, MTs are organized with their 'minus ends' near the nucleus and their 'plus ends' towards the cell periphery. MT-based intracellular transport is mediated via the kinesins, plus-end directed MT motors, and the dyneins, minus-end directed MT motors. Both families of MT-motor proteins require ATP to move along MTs with their cargoes. We have demonstrated the dynein family mediates transport of p53 to the nucleus and more recently we have been able to show that p53 oligomerizes prior to association with dynein and that this association then occurs in the cytoplasm. Only then does the p53-dynein complex associate with microtubules and travel to the nucleus. The residues in p53 involved in this have been identified as the residues important in the oligomerization of p53, so that mutations at these residues not only impairs p53 oligomerization and hence its ability to trans-activate its target genes, but also interferes with the trafficking of p53 to the nucleus - in effect a double hit impairing the trans-activation of target genes. The association of p53 with cellular MTs may be important in several ways. First, this may provide a mechanistic basis to regulate p53 subcellular localization. Second, our findings suggest p53 is an indirect target for MT-active agents. In this regard, the demonstration that MT active drugs may affect p53 levels and activate p53 dependent checkpoints could be explained by our findings. Third, by binding MTs, p53 is brought in close proximity to other cellular proteins. Moreover, MTs could provide a reservoir for p53. This hypothesis is consistent with the substantial amount of p53 bound to MTs and the large capacity of MTs for p53 storage. This is evidenced by the ability of MTs to bind the higher levels of mt p53, and the increased levels of wt p53, following DNA damage. Most importantly, our data indicate the p53/MT association is important for p53 nuclear accumulation. As p53 exerts many of its effects by transcriptional regulation, translocation to nuclear targets is critical for biological responses. Our data showing that disruption of a functional MT-network prior to DNA damage results in impaired trans-activation of p53-target genes further supports a role of MTs in p53 intracellular trafficking. As we go forward we plan to focus on several aspects of this work. We plan to further examine post-translational modifications as surrogates for MT stability. While MTAs have been successfully developed without clinical evidence of MT engagement by drug, we believe a simple, sensitive, and reliable assay to monitor the pharmacodynamic effect of these agents would be of value in their future development. In addition, our work has revealed that MTs facilitate intracellular trafficking and nuclear accumulation of several proteins and are in the process of clarifying how they interact with microtubules. The ultimate goal is to determine whether interphase microtubules are effective drug targets. Current thinking favors interference with the mitotic spindle as the principal effect of MTAs, however, we believe that interfering with the interphase spindle may also be very important. In the clinic we have been involved in the conduct of clinical trials with a novel epothilone B an [summary truncated at 7800 characters]

真核细胞的细胞骨架参与多种细胞功能，如运动、分泌、信号传导和增殖。微管（MTs）是细胞骨架的组成部分。在抗癌药物中，靶向微管蛋白或MTs的药物即使不是最有效的一类药物，也是最有效的一类药物。与微管蛋白或mt结合的化合物清单很大，并且还在继续扩大。绝大多数是天然产物，它们的化学结构非常多样。长春花生物碱在20世纪50年代被引入，尽管它们在许多恶性肿瘤中都很有用，但开发针对mt的新药物的兴趣逐渐下降，直到紫杉醇的引入。紫杉醇可以说是继顺铂之后最有效的药物，其显著的活性激发了人们对微管蛋白和mt作为化疗靶点的兴趣。紫杉醇的临床成功带来了丰富的新科学知识，加强了微管蛋白/MT系统作为癌症化疗靶点的重要性，并刺激了鉴定新型微管蛋白活性药物的努力。在MT靶向药物（mta）领域，我们目前的研究目标是：(1)增加我们对mta如何与微管蛋白相互作用并导致细胞死亡的理解；(2)了解mta耐药机制；(3)开发检测mta在患者体内的药效学。在临床中，我们继续进行mta试验。鉴于我们成功地鉴定了紫杉醇和埃泊替龙耐药细胞的突变，并受到积累的信息和经验教训的鼓舞，我们开始使用HTI-286进行选择，这是当时惠氏公司正在开发的一种合成半氯司他林。当我们在研究hemiasterlin耐药细胞系时，我们也在肾细胞癌患者中进行了BMS-247550 （ixabepilone）的II期临床试验。BMS-247550是一种艾替隆B类似物和mt稳定剂。作为这项正在进行的试验的一部分，我们试图在治疗前和第五剂BMS-247550后获得肿瘤活检。最初的目的是通过定量给药前后的微管蛋白聚合程度来检查BMS-247550的药效学。这一测量将使我们能够确定BMS-247550是否稳定了肿瘤细胞中的mt。然而，我们也考虑了其他方法来证明MT稳定。在我们的半司他林耐药细胞中，微管蛋白乙酰化和去酪氨酸化与MT稳定相关，我们受到结果的鼓舞，选择在患者样本中研究这些化学修饰。作为第一步，我们证明了在培养的肾癌和卵巢癌细胞中，去酪化（葡聚糖终止）和乙酰化α -微管蛋白的水平与BMS-247550诱导的MT稳定密切相关，表明这些修饰可用于监测MT稳定。更重要的是，在检查患者样本时，我们发现在接受BMS-247550治疗后，在8个系列肿瘤活检中，8个中葡聚糖终止和/或乙酰化的α -微管蛋白水平增加了2- 100倍。这些数据表明BMS-247550到达肿瘤并与MT靶标结合，导致MT稳定，与其避免Pgp的能力一致，并达到其靶标。我们得出结论，葡萄糖终止和/或乙酰化的α -微管蛋白水平是BMS-247550药效学效应的简单可靠的标志物。我们认为，评估翻译后修饰的微管蛋白水平可能为其他mta的药效学效应提供一种简单可靠的分析方法。反复观察到我们的紫杉醇和埃泊霉素耐药细胞系在p53中获得突变，这让我们感到困惑，我们开始确定p53是否可以以一种有意义的方式与微管蛋白相互作用。我们发现wt和mt p53都与mt相关联，并且这种相互作用在mt解聚药物治疗后消失。此外，我们发现p53仅在具有功能MT网络的细胞中在DNA损伤后在细胞核中积累。用长春新碱或紫杉醇预处理都能减少p53的核积累，这表明p53的核易位需要一个功能性的MT网络。在大多数细胞中，mt的“负端”靠近细胞核，“正端”靠近细胞外周。以MT为基础的细胞内运输是通过运动蛋白（正端定向MT马达）和动力蛋白（负端定向MT马达）介导的。MT-motor蛋白的两个家族都需要ATP和它们的货物一起沿着mt移动。我们已经证明动力蛋白家族介导p53到细胞核的运输，最近我们已经能够证明p53在与动力蛋白结合之前寡聚化，这种结合随后发生在细胞质中。只有这样p53-动力蛋白复合物才会与微管结合并进入细胞核。与此相关的p53残基已被确定为p53寡聚化过程中重要的残基，因此这些残基的突变不仅会损害p53寡聚化，从而影响其反式激活靶基因的能力，还会干扰p53向细胞核的运输——实际上是双重打击，损害了靶基因的反式激活。p53与细胞mt的关联可能在几个方面很重要。首先，这可能为调控p53亚细胞定位提供了机制基础。其次，我们的研究结果表明p53是mt活性药物的间接靶点。在这方面，MT活性药物可能影响p53水平并激活p53依赖检查点的证明可以用我们的发现来解释。第三，通过结合MTs， p53与其他细胞蛋白紧密结合。此外，mt可以提供p53的储存库。这一假设与大量p53结合到MTs上以及MTs储存p53的大容量相一致。DNA损伤后，mt结合高水平mt p53的能力和wt p53水平的增加证明了这一点。最重要的是，我们的数据表明p53/MT关联对p53核积累很重要。由于p53通过转录调控发挥其许多作用，因此向核靶点的易位对生物反应至关重要。我们的数据显示，在DNA损伤之前，功能性mt网络的破坏会导致p53靶基因的反式激活受损，这进一步支持了mt在p53细胞内运输中的作用。在我们前进的过程中，我们计划把重点放在这项工作的几个方面。我们计划进一步研究翻译后修饰作为MT稳定性的替代品。虽然mta在没有药物参与的临床证据的情况下已经成功开发，但我们相信，一种简单、敏感、可靠的方法来监测这些药物的药效学效果，将对它们未来的开发有价值。此外，我们的工作还揭示了mt促进细胞内运输和几种蛋白质的核积累，并正在阐明它们如何与微管相互作用。最终目的是确定间期微管是否是有效的药物靶点。目前认为mta的主要作用是干扰有丝分裂纺锤体，然而，我们认为干扰间期纺锤体也可能是非常重要的。在临床中，我们参与了一种新型艾泊霉素B的临床试验[摘要截短为7800个字符]