ALK融合基因肺癌Crizotinib获得性耐药机制及其它酪氨酸激酶抑制剂对其作用的研究

ALK-rearranged is a new oncogene in non-small cell lung cancer which was found in 2007 and became a new therapeutic target since then. As an ALK micromolecule inhibitor, Crizotinib represents inspiring effects in the treatment of non-small cell lung cancer with ALK-rearranged , however the resistance always occurs within one year inevitably. Secondary mutations have not been found in most patients with acquired Crizotinib resistance and the mechanism of resistance is unknown. We plan to build up several SCID-mouse models of acquired Crizotinib resistance via the transplantation of primary non-small cell lung cancer tissues with ALK rearranged . We decide to explore the pathways involved in acquired Crizotinib resistance with ALK rearranged to clarify the mechanisms of resistance in the level of post-translational modification of protein applying phosphoproteome and pathway proteomics by PTMScan through camparing the levels of phospholated proteins between the individuals of Crizotinib- sensitive and Crizotinib-resisted SCID mice. Furthermore, we will apply second-generation tyrosine kinase inhibitors in the animal model according to the pathways involved to observe the effects of new drugs dealing with Crizotinib resistance. This study will provide the basis of the clinical application of new drugs against Crizotinib resistance.

ALK融合基因是近年发现的一种新的非小细胞肺癌癌基因，是一种有效的治疗靶点，Crizotinib对其表现出良好的治疗效果，但往往不可避免地在一年之内耐药。大部分Crizotinib获得性耐药的患者未发现ALK融合基因的次级突变，其耐药机制不明。本研究计划建立多个非小细胞肺癌ALK融合基因原代肿瘤移植瘤耐药模型，采用PTMScan技术运用磷酸化蛋白质组学及通道蛋白质组学的方法，通过比对耐药前后标本的磷酸化蛋白质水平的差异，发现并鉴定导致Crizotinib获得性耐药的信号通路，从蛋白质翻译后修饰水平阐明ALK融合基因Crizotinib获得性耐药的耐药机制。同时在Crizotinib获得性耐药模型上，针对耐药的信号通路使用相应的第二代酪氨酸激酶抑制剂，观察新药克服Crizotinib获得性耐药的效果，为临床上使用克服Crizotinib抵抗的新药提供研究基础。

ALK融合基因是近年发现的一种新的非小细胞肺癌癌基因，是一种有效的治疗靶点，Crizotinib 对其表现出良好的治疗效果，但不可避免地在一年之内耐药。大部分 Crizotinib 获得性耐药的患者未发现ALK融合基因的次级突变，其耐药机制不明。本研究收集633例非小细胞肺癌标本，通过western blotting筛查alk阳性标本20例，并经过多重PCR、免疫组化证实及alk直接测序确定其亚型。取其中9例ALK阳性标本接种至免疫缺陷鼠皮下，形成4个原代肿瘤移植瘤模型，其中三个顺利传代保留ALK阳性融合基因并形成耐药移植瘤，能够用于ALK靶向新药的研究开发及非小细胞肺癌ALK融合基因的临床基础研究。取其中一个耐药肿瘤组织送CST进行酪氨酸磷酸化质谱检测分析，通过比对耐药前后标本的磷酸化蛋白质水平的差异，发现PTPN11 Alk GAB2 CDK1 ARHGEF5 SrL MYH2,13 Ckm ACTA2 ANXA2 KISS1R EPHA2 INPPL1 PTPRE Eml1 PTPRA SYNPO CDV3 PRKCD NCK1显著增高，通过genepattern数据库分析信号通路，我们选取P-Tyrosine alk p-alk src p-src met p-met shc p-shc akt p-akt stat3 p-stat3 erk p-erk egfr p-egfr验证了耐药中蛋白水平的变化。同时我们购买了H3122阳性细胞株，经过8个月刺激H3122 crizotinib耐药形成，验证了细胞株内P-Tyrosine alk p-alk src p-src met p-met shc p-shc akt p-akt stat3 p-stat3 erk p-erk egfr p-egfr蛋白水平的变化。通过综合原代移植瘤及细胞株的实验结果，明确了P-Tyrosine alk p-alk p-src met shc p-shc p-akt egfr p-egfr在耐药中的作用，这些位点可作为抗耐药新药研制的阻断通路。