A novel, short isoform of the +TIP microtubule (MT) binding protein CLIP170 confers taxane resistance by obstructing the MT pore.

TIP 微管 (MT) 结合蛋白 CLIP170 的一种新型短亚型通过阻塞 MT 孔而赋予紫杉烷抗性。

• 批准号: 9918278
• 负责人: Olivier Elemento
• 金额: $ 50.42万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-19 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9918278
• 关键词: Affinity; Amino Acids; Bayesian Analysis; Binding; Binding Proteins; Binding Sites; Biological Markers; Biopsy; CLIP-170 gene; Cancer Patient; Cancer cell line; Cells; Cellular Structures; Chemicals; Clinical; Clinical Oncology; Clinical Trials; Combined Modality Therapy; Computer Analysis; Computer Models; Confocal Microscopy; Cytoskeleton; Data; Diffuse; Diffuse gastric cancer; Dissociation; Distal; Down-Regulation; Drug Binding Site; Drug Interactions; Drug resistance; Exhibits; Family; Genes; Glycine; Goals; Histologic; Imatinib; Impairment; International; Kinetics; Label; Length; Link; Mediating; Microtubules; Modeling; Molecular; Neoplasm Circulating Cells; Oncology; Orphan Drugs; Paclitaxel; Pathway interactions; Patients; Pharmaceutical Preparations; Plus End of the Microtubule; Pre-Clinical Model; Process; Progression-Free Survivals; Property; Protein Isoforms; Proteins; Proteomics; Receptor Protein-Tyrosine Kinases; Resistance; Role; Sampling; Signal Pathway; Signal Transduction; Surface; Testing; Transmembrane Transport; Tumor Bank; Tyrosine Kinase Inhibitor; Validation; Variant; base; cancer cell; chemical genetics; chemotherapy; clinical predictors; computerized tools; disorder control; docetaxel; drug discovery; drug sensitivity; experimental study; genetic approach; knock-down; live cell imaging; malignant stomach neoplasm; new therapeutic target; novel; novel therapeutics; overexpression; precision medicine; predictive modeling; prospective; response; small molecule; taxane; transcriptome sequencing; tumor

The taxanes are amongst the most commonly used chemotherapy drugs in clinical oncology, and are a mainstay of treatment in gastric cancer. However, despite their use in both first and second line therapy, patients commonly exhibit intrinsic resistance resulting in marginal benefit. A post hoc analysis of taxane therapy in an international clinical trial (TAX-325) confirmed this observation; we found that GC patients with diffuse histological subtype did not benefit from DTX therapy, suggesting that diffuse GC is may be intrinsically resistant to taxanes. To date, despite the wide use of taxanes in oncology, the molecular underpinnings of clinical taxane resistance remain poorly elucidated. Using preclinical models of GC intrinsic resistance, we identified a novel faster-migrating isoform of the microtubule (MT) plus-end binding protein CLIP-170, hereafter CLIP-170S, which was enriched in GC cell lines with intrinsic taxane resistance. The canonical full-length CLIP-170 protein belongs to the family of MT plus- end-tracking proteins (+TIPs) which accumulate at the distal ends of growing MTs, linking MT ends to various cell structures and regulating MT dynamics. Mass-spec proteomics and 5’RACE revealed that CLIP-170S was missing the first 150 amino acids, including the first Cap-Gly (domain, required for proper +TIP localization. Confocal microscopy experiments showed that CLIP-170S was miss-localized from the MT ends to the MT lattice. Live-cell imaging of native cytoskeletons using fluorescently- labeled paclitaxel (Flutax) revealed significantly faster dissociation rates of Flutax from MTs in the cells expressing CLIP-170S, indicating transient interaction with MTs. Taxane binding to MTs is a two-step process. First, taxanes bind to the MT outer surface by interacting with their low affinity binding site at the MT pore, then, they get internalized to the MT lumen where they bind to their high affinity luminal binding site. Our data using chemical probes specific for the outer and inner surface of the MT pore showed that CLIP-170S expression was associated with decreased binding affinity of taxanes for MTs. Stable knock- down (KD) of CLIP-170S reversed completely taxane resistance (~300-fold)— while KD of the canonical CLIP-170 had no effect on drug activity— thereby, suggesting a cause-effect relationship between CLIP-170S expression and taxane resistance. Together these data led us propose a model where CLIP-170S blocks the MT-pore, impairs taxane binding to the MT outer surface inhibiting taxane access to the high-affinity luminal binding site resulting in drug resistance. We have developed a drug discovery platform, BANDIT (Bayesian Analysis to Identify Drug Interaction Targets), which allows for accurate identification of target proteins for orphan drugs or small molecules. BANDIT identified Imatinib as a drug predicted to be active in taxane-resistant GC cells. Experimental validation showed that Imatinib not only was able to completely reverse taxane resistance, but it did so by inhibiting specifically the expression of CLIP-170S. Our central hypothesis is that CLIP-170S, by lacking the first N-terminus CAP-GLY motif is miss-localized from the +TIP to the MT lattice, obstructing the MT-pore and blocking taxane access to its high affinity luminal drug binding-site. Computational modeling predicted that Imatinib would reverse taxane resistance. Experimental validation of this prediction led us further hypothesize that additional clinically used tyrosine kinase inhibitors (TKIs), may share this mode of action with Imatinib, and synergize with taxanes providing a new targeted therapeutic strategy for GC patients.

这些紫杉烷是临床肿瘤学中最常用的化学疗法药物之一，是治疗中的主要治疗方法 胃癌。然而，多皮特在第一线和第二线治疗中使用，患者通常会暴露于固有阻力 导致边际收益。国际临床试验中紫杉烷治疗的事后分析（税务-325）证实了这一点 观察；我们发现具有弥漫性组织学亚型的GC患者没有受益于DTX疗法，这表明 弥漫性GC可能对紫杉烷具有本质上的抗性。迄今为止，dospite在肿瘤学中广泛使用了分子的基础 临床紫杉烷耐药性的阐明仍然很差。使用GC内在电阻的临床前模型，我们确定了一种新颖的 微管（MT）加末端结合蛋白夹170，Herelafter Clip-170S的更快迁移同工型，该蛋白 在具有内在紫杉烷耐药性的GC细胞系中。规范的全长夹170蛋白属于MT Plus-家族 末端跟踪蛋白（+尖端）积聚在生长MT的远端，将MT末端连接到各种细胞结构 并调节MT动力学。质谱蛋白质组学和5'race表明，夹170s缺少第一个150个氨基酸， 包括第一个帽盖（域，适当 +尖端定位所需的域）。共聚焦显微镜实验表明 从MT的末端到MT晶格，夹170s被遗忘了。使用荧光的天然细胞骨架的活细胞成像 - 标记的紫杉醇（Flutax）显示，在表达夹夹170S的细胞中，Flutax的分解速率明显更快， 指示与MTS的瞬时相互作用。紫杉烷与MTS的结合是两个步骤的过程。首先，紫杉虫与MT外部结合 通过与MT孔的低亲和力结合位点相互作用，它们将其内部化为MT腔内 结合其高亲和力腔结合位点。我们使用针对外表面和内表面的化学问题的数据 MT孔表明，Clip-170S的表达与MTS的结合亲和力降低有关。稳定的敲门 夹170的下降（kd）完全逆转了完全紫杉烷的耐药性（〜300倍），而规范夹的KD则没有 对药物活性的影响 - 从而表明夹170年代表达与紫​​杉烷耐药性之间存在因果关系。一起 这些数据导致我们提出了一个模型，其中clip-170阻断了MT孔，会损害紫杉烷与MT外表面的结合 紫杉烷进入高亲和力的管腔结合位点，导致耐药性。我们已经开发了一个药物发现平台， 强盗（贝叶斯分析以识别药物相互作用目标），这允许准确鉴定靶蛋白 用于孤儿药或小分子。匪徒将伊马替尼确定为一种药物，被预测在抗紫杉烷的GC细胞中活跃。 实验验证表明，伊马替尼不仅能够完全逆转紫杉烷的耐药性，而且通过抑制它来做到这一点 特别是夹170S的表达。我们的核心假设是，夹子170s缺少第一个N末端帽盖。 主题从 +尖端到MT晶格被遗忘，阻塞了MT孔和阻塞紫杉烷的高亲和力 腔内药物结合位置。计算建模预测，伊马替尼将逆转紫杉烷耐药性。实验 对这一预测的验证使我们进一步假设，其他临床使用的酪氨酸激酶抑制剂（TKI）可能共享 与伊马替尼的这种行动方式，并与紫杉烷协同作用，为GC患者提供了新的针对性治疗策略。