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.

紫杉烷类药物是临床肿瘤学中最常用的化疗药物之一，是治疗癌症的主要药物。 然而，尽管它们用于一线和二线治疗，但患者通常表现出内在耐药性。 一项国际临床试验 (TAX-325) 中紫杉烷治疗的事后分析证实了这一点。 观察；我们发现具有弥漫性组织学亚型的GC患者没有从DTX治疗中获益，这表明 迄今为止，尽管紫杉烷类药物在肿瘤学中得到广泛应用，但弥漫性GC可能对紫杉烷类药物具有内在耐药性。 临床紫杉烷耐药性的机制仍不清楚。利用 GC 内在耐药性的临床前模型，我们发现了一种新的耐药性。 微管 (MT) 正端结合蛋白 CLIP-170（以下简称 CLIP-170S）的快速迁移亚型，已富集 具有内在紫杉烷抗性的 GC 细胞系中，典型的全长 CLIP-170 蛋白属于 MT plus- 家族。 末端跟踪蛋白 (+TIP) 在生长中的 MT 远端积累，将 MT 末端与各种细胞结构连接起来 并调节 MT 动力学和 5’RACE 揭示 CLIP-170S 缺失前 150 个氨基酸， 包括第一个 Cap-Gly（结构域，正确 +TIP 定位所需的。共焦显微镜实验表明 使用荧光法对天然细胞骨架进行活细胞成像，CLIP-170S 从 MT 末端错误定位。 标记的紫杉醇 (Flutax) 显示在表达 CLIP-170S 的细胞中 Flutax 与 MT 的解离速率明显加快， 表明紫杉烷与 MT 的短暂相互作用是一个两步过程，首先，紫杉烷与 MT 外层结合。 通过与 MT 孔处的低亲和力结合位点相互作用，它们进入表面，然后，它们被内化到 MT 腔，在那里它们 我们的数据使用特定于外腔和内表面的化学探针结合到它们的高亲和力结合位点。 MT 孔显示 CLIP-170S 表达与紫杉烷类与 MT 的结合亲和力降低有关。 CLIP-170S 的 KD 下降（KD）完全逆转了紫杉烷耐药性（约 300 倍），而经典 CLIP-170 的 KD 没有 对药物活性的影响——从而表明 CLIP-170S 表达与紫杉烷耐药性之间存在因果关系。 这些数据使我们提出了一个模型，其中 CLIP-170S 阻断 MT 孔，损害紫杉烷与 MT 外表面的结合，抑制 紫杉烷进入高亲和力的管腔结合位点导致耐药性我们开发了一个药物发现平台， BANDIT（识别药物相互作用目标的贝叶斯分析），可准确识别目标蛋白 BANDIT 确定伊马替尼是一种预计对紫杉烷耐药的 GC 细胞具有活性的药物。 实验验证表明，伊马替尼不仅能够完全逆转紫杉烷耐药性，而且是通过抑制 特别是 CLIP-170S 的表达，我们的中心假设是 CLIP-170S 缺乏第一个 N 末端 CAP-GLY。 基序从 +TIP 错误定位到 MT 晶格，阻碍了 MT 孔并阻止紫杉烷进入其高亲和力 Luminal 药物结合位点预测伊马替尼将逆转紫杉烷耐药性。 这一预测的验证使我们进一步发现临床上使用的其他酪氨酸激酶抑制剂 (TKI) 可能会分享 这种作用方式与伊马替尼，以及与紫杉烷类药物的协同作用，为GC患者提供了新的靶向治疗策略。