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

紫杉烷类是临床肿瘤学中最常用的化疗药物之一，并且是治疗肿瘤的主要药物。 胃癌然而，尽管它们用于一线和二线治疗，但患者通常表现出内在抵抗力 导致边际效益。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;我们发现弥漫组织学亚型的GC患者没有从DTX治疗中获益，这表明， 弥漫性GC可能对紫杉烷类具有内在抗性。迄今为止，尽管紫杉烷类在肿瘤学中广泛使用，但其分子基础 临床紫杉烷耐药的机制仍不清楚。使用GC内在耐药的临床前模型，我们发现了一种新的 微管（MT）加末端结合蛋白CLIP-170的更快迁移同种型，下文称为CLIP-170 S，其被富集 在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加- 末端跟踪蛋白（+TIPs），在生长的MT的远端积累，将MT末端连接到各种细胞结构 和调节MT动力学。质谱蛋白质组学和5 'RACE显示CLIP-170 S缺失了前150个氨基酸， 包括正确的+TIP定位所需的第一个Cap-Gly（结构域）。共聚焦显微镜实验表明， CLIP-170 S从MT末端到MT晶格的定位错误。使用荧光技术对天然细胞骨架进行活细胞成像- 标记的紫杉醇（Flutax）显示Flutax从表达CLIP-170 S的细胞中的MT的解离速率显著更快， 表明与MT的短暂相互作用。紫杉烷与MT的结合是一个两步过程。首先，紫杉烷类化合物与MT外膜结合， 它们通过与MT孔处的低亲和力结合位点相互作用而粘附到表面，然后，它们被内化到MT腔中， 结合到它们的高亲和力管腔结合位点。我们的数据使用化学探针的外表面和内表面的具体 MT孔显示CLIP-170 S表达与紫杉烷对MT的结合亲和力降低相关。稳定爆震- CLIP-170 S的KD完全逆转紫杉烷耐药性（约300倍），而经典CLIP-170的KD没有逆转紫杉烷耐药性。 因此，提示CLIP-170 S表达与紫杉烷耐药性之间存在因果关系。一起 这些数据使我们提出了一个模型，其中CLIP-170 S阻断MT孔，削弱紫杉烷与MT外表面的结合，抑制MT的生长。 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（Bayesian Analysis to Identify Drug Interaction Targets，贝叶斯分析识别药物相互作用靶点），可准确识别靶蛋白 用于孤儿药或小分子药物。BANDIT确定伊马替尼是一种预计在紫杉烷耐药GC细胞中具有活性的药物。 实验验证表明，伊马替尼不仅能够完全逆转紫杉烷耐药性，而且通过抑制 特别是CLIP-170 S的表达。我们的中心假设是CLIP-170 S由于缺少第一个N端CAP-GLY， 基序从+TIP错误定位到MT晶格，阻塞MT孔并阻断紫杉烷获得其高亲和力 管腔药物结合位点。计算模型预测，伊马替尼将逆转紫杉烷耐药性。实验 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 这种作用模式与伊马替尼，并协同紫杉烷类提供了一个新的针对性的治疗策略，为GC患者。

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.