ALK阳性的间变大细胞淋巴瘤对克唑替尼的耐药机理及逆转研究

Anaplastic lymphoma kinase (ALK) gene was generating the NPM-ALK oncoprotein. In the oncoprotein NPM-ALK, the tyrosine kinase activity of ALK is constitutively activated via its dimerization mediated through the NPM oligomerization domain. NPM-ALK activates and deregulates a host of cellular signaling pathways， leading to uncontrollable cell growth and malignant transformations. Moreover, fusions between other genes and ALK were also identified in several ALK "addict" malignant diseases, including echinoderm microtubule-associated protein-like 4 (EML4)-ALK in a small subset of non-small-cell lung cancer (NSCLC) cells, the Ran-binding protein 2 (RANBP2)-ALK or tropomyosin-3 and -4 (TPM-3 and -4)-ALKin inflammatory myofibroblastic tumors. Increased population of patients may potentially benefit from the treatment of the ALK inhibition. Crizotinib functions as ALK kinase inhibitor. It has been approved by FDA for the treatment of ALK+ NSCLC patients in US, and is undergoing with clinical trials in the treatment of anaplastic large cell lymphoma. Some promising results have been shown in the clinical trials of using crizotinib treatments of advanced NSCLC patients. However, several groups had reported the crizotinib resistance in the treatments of NSCLC patients. The crizotinib resistant mechanisms involved in using crizotinib for the treatments of ALK+ALCL cells are still largely unknown. In this study, we used two ALK+ALCL cell lines underwent long term crizotinib treatment with gradually increasing doses to set up a research model for mimicing the drug resistant development process in clinic. We found that high NPM-ALK expression level was uniformly predominant in the crizotinib resistant Karpas 299 and SUP-M2 cell lines. In those resistant cells, we will study ALK gene,NPM-ALK protein and phospho-NPM-ALK protein expression and the change of downstream target gene AKT and Twist1;HSP90 and IGF signal pathway inhibition alone or combination will reverse the crizotinib resistance.so the hypothesis is that ALK gene over expression maybe contributing to the resistance of cancer cells to the treatments of kinase inhibitors,ALK protein up-regulation via IGF-IR signal pathway or/and the less degradation of ALK protein via proteasome pathway.our data suggest that up-regulation of the NPM-ALK oncoprotein contrubite to the resistance to Crizotinib. Further biological analysis and evaluations can be performed using those cell lines, animal model and patients' samples to study the mechanisms of crizotinib resistance in ALK+ ALCL.we will develop more biomarker to predict the effectiveness for clinic.

间变大细胞淋巴瘤基因（Anaplastic Lymphoma Kinase, ALK）的异位与点突变等成为该类肿瘤的驱动基因。并被确定为此类癌症的治疗靶点。ALK抑制剂克唑替尼对ALK+的肿瘤细胞有抑制作用，但疗效受到肿瘤细胞获得性耐药的限制。文献报告的耐药机理可能为继发耐药突变、旁路激活、基因扩增等，而在间变大细胞淋巴瘤（ALCL）主要为继发性突变引起，我们前期用已建立的ALK+的ALCL耐药细胞系为模型，发现耐药细胞系中ALK基因、蛋白及磷酸化水平明显升高；IGF-I、AKT及Twist1明显升高； HSP90抑制剂能够增加耐药细胞对克唑替尼的敏感性，并未发现继发性耐药性突变。我们推测ALK过表达是引起耐药的主要原因之一，可能通过IGF-IR信号通路的激活或通过蛋白酶体途径降解减少引起。本研究为明确ALK+的ALCL对克唑替尼的耐药机理，建立疗效预测指标，指导临床治疗提供科学依据。

携带NPM-ALK融合基因的ALK阳性间变性大细胞淋巴瘤（ALCL）对ALK / MET双重抑制剂克唑替尼敏感。然而，在最初的明显治疗反应后，癌细胞最终对克唑替尼产生耐药性，但在NPM-ALK阳性的ALCL中克唑替尼耐药的机制尚不清楚。.目前，克唑替尼耐药的多种机制已被证实，包括ALK扩增，ALK激酶结构域突变和不同旁路信号传导途径的激活。因而，确定耐药机制并寻找克服ALCL克唑替尼耐药的策略具有重要意义。.为了研究ALCL细胞对克唑替尼耐药的机制，我们通过将敏感的NPM-ALK阳性ALCL细胞系逐渐暴露于较高浓度的克唑替尼的方法建立了克唑替尼耐药的模型。我们发现在400nM耐药克隆中存在NPM-ALK G1269A突变以及IGF-1R通路的激活。然后我们通过极限稀释的方法建立了G1269A突变的耐药细胞亚系，其对第二代ALK抑制剂AP26113敏感。 IGF-1R抑制剂PPP对这些亚克隆没有影响，并且不改变对第一代和第二代ALK TKI的敏感性。但IGF-1R抑制剂在400nM克唑替尼耐药细胞中增加对克唑替尼和AP26113的敏感性。因此，我们证明，IGF-1R通路参与了ALK TKIs的耐药，并且联合ALK / IGF1R抑制剂能够提高治疗疗效。