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

紫杉烷是临床肿瘤学中最常用的化疗药物之一，也是治疗癌症的主要药物。 胃癌。然而，尽管在一线和二线治疗中都使用了它们，患者通常表现出内在的抵抗力。 从而产生边际效益。一项国际临床试验(TAX-325)中对紫杉烷疗法的特殊分析证实了这一点。 观察：我们发现弥漫组织学亚型的GC患者并没有从DTX治疗中受益，这表明 弥漫性GC IS可能对紫杉烷类药物具有内在抗药性。到目前为止，尽管紫杉烷在肿瘤学中被广泛使用，但分子基础 临床上紫杉烷耐药的原因仍不清楚。利用GC内在耐药的临床前模型，我们发现了一种新的 迁移速度更快的微管(MT)+末端结合蛋白CLIP-170的异构体，以下简称CLIP-170，它被浓缩 在具有内在紫杉烷耐药的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(+TIPS)，聚集在生长的MT末端，将MT末端连接到各种细胞结构 调节MT动态。质谱蛋白质组学和5‘RACE显示，CLIP-170缺失前150个氨基酸， 包括第一个Cap-Gly(结构域，正确定位+TIP所需。共聚焦显微镜实验表明 Clip-170从MT末端到MT晶格未定位。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(福尔泰素)在表达CLIP-170的细胞中显示出显著更快的福尔泰素从MTS的解离速率， 指示与MTS的瞬时交互。紫杉烷与MTS结合是一个分两步进行的过程。首先，紫杉烷与MT外层结合 通过与它们在MT孔处的低亲和力结合部位相互作用，它们被内化到MT管腔，在那里它们 结合到它们的高亲和力的管腔结合部位。我们的数据使用了化学探针，专门针对外表面和内表面 MT孔显示，CLIP-170S的表达与紫杉烷与MTS的结合亲和力降低有关。稳定的敲门声- CLIP-170的向下(Kd)完全逆转了紫杉烷的耐药性(~300倍)，而标准CLIP-170的Kd没有 对药物活性的影响-因此，提示CLIP-170表达与紫杉烷耐药性之间存在因果关系。同舟共济 这些数据使我们提出了一个模型，即CLIP-170阻断MT-孔，削弱紫杉烷与MT外表面的结合抑制 紫杉烷进入高亲和力的管腔结合部位，导致耐药。我们已经开发了一个药物发现平台， BANDIT(识别药物相互作用靶点的贝叶斯分析)，允许准确识别目标蛋白质 用于孤儿药物或小分子药物。班迪特确认伊马替尼是一种预计在紫杉烷耐药GC细胞中有效的药物。 实验验证表明，伊马替尼不仅能够完全逆转紫杉烷耐药性，而且它通过抑制 特别是CLIP-170的表达。我们的中心假设是，由于缺少第一个N末端CAP-Gly，CLIP-170 Motif从+尖端到MT晶格的位置缺失，阻碍了MT-孔，并阻止紫杉烷获得其高亲和力 鲁米那药物结合部位。计算模型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(TKIs)可能与 与伊马替尼的这种作用模式，以及与紫杉烷的协同作用，为GC患者提供了一种新的靶向治疗策略。

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.