抑制PREX2逆转胃肠道间质瘤对酪氨酸激酶抑制剂耐药的研究

Tyrosine Kinase Inhibitors(TKIs) have made a great success in Gastrointestinal stromal tumor (GIST) target thrapy, however, most patients become resistant after TKIs treatment, some of them with unknown mechanisms. We established an Imatinib resistant cell line after treating GIST 882 with Imatinib in the dose of IC50 10nM, it's resistant to Imatinib without any secondary mutations and other known resistant mechanisms had not been found, named GIST882IM whose morphology and biological behavior were different from the parental cells. We found PTEN loss in GIST882IM by SNP analysis, this PTEN loss led to abnormal pAKT hyperactvation, which caused TKIs resistance eventually. This phenomenon was also confirmed in patients' samples, we further identified PREX2 loss by SNP in GIST882. According to those important findings,we are going to either over-express PTEN PREX2 or knockdown those genes with shRNA hairpins in KIT/PDGFRA dependent GIST, investigate the possibility of enhencing target therapy effect by targeting PERX2 by downregulating the downstream of KIT/PDGFRA in the PI3K/AKT pathway, thus suppress the growth of GIST cells. We are going to design a small compound for inhibiting PREX2, combine this inhibitor with TKIs for the treatment in TKIs-resistant GIST, preclinically validate and evulate this new stratege of the treatment in vitro cell lines and in vivo nude mice modle. Hopefully this study will provide a new path for GIST treatment in the clinic.

酪氨酸激酶抑制剂（TKIs）治疗胃肠道间质瘤取得了巨大的成功，然而，绝大多数病人治疗后耐药，部分机制未明。本课题通过建立伊马替尼耐药株，通过单核苷酸多态性（SNP）首次发现伊马替尼耐药株的PTEN基因缺失，诱导pAKT异常激活而致胃肠道间质瘤对TKIs耐药，这种现象同样在临床标本得到了验证。进一步SNP筛选发现原代细胞株中PREX2基因缺失，而PREX2正是PTEN的上游调控分子。拟利用这些重要发现，研究PTEN缺失导致TKIs耐药的机制，深入探讨其负调控因子PREX2生物学功能，拟靶向抑制PREX2，从而调控该肿瘤生长主要信号通路PI3K/AKT的下游分子，抑制肿瘤细胞生长。设计小分子药物阻滞PREX2蛋白表达联合TKIs治疗，进行体外细胞试验和体内裸鼠模型等临床前验证，评估PREX2抑制剂与TKIs联用在胃肠道间质瘤的治疗效果。为逆转TKIs耐药的临床治疗提供新思路。

酪氨酸激酶抑制剂（TKIs）治疗胃肠间质瘤(GIST)取得了巨大的成功，然而最终大部分病人会发生耐药。本课题首先大样本调查了中国GIST临床病理因素与治疗现状，对大样本患者回顾性分析发现，Ki67对高危的GIST患者预后影响显著，找到了“极高危”的新判断标准。伊马替尼（IM）作为GIST的一线治疗药物，疗效显著，大部分GIST患者能够从IM的治疗中获益， 首先对血药浓度检测的方法进行了改进, 基于液相色谱-串联质谱法定量测定人血浆中IM及其代谢物n-demethyl-imatinib（NDI） 。 结果发现，NR1I2 rs3814057的基因多态性与IM和NDI的需要浓度具有潜在的相关性（P<0.1）我们还在关注了患者在服用IM期间出现的毒副反应，IM诱导的眼科的副作用，包括结膜出血和眶周水肿， 探讨药物靶点和膜转运蛋白的基因多态性对这些副作用的影响.课题组采用磷酸RTK和immunoblot RTK技术的分析，我们发现胰岛素受体（IR）对伊马替尼耐药细胞株 表达充分，但不在伊马替尼敏感细胞 中表达。在耐药细胞中，治疗药物 linsitinib特异性抑制剂抑制IR，及中间和下游的信号通路AKT、MAPK和S6，然而 在伊马替尼敏感的细胞 效应最小 。 我们研究结果显示，KIT抑制剂与IR抑制的联合治疗是对伊马替尼耐药的GIST有效的治疗策略 。基于此的耐药机制，我们研究了新药HQP135 具有全球自主知识产权的高效的口服的多靶点酪氨酸激酶抑制剂，HQP1351有效克服了上市药物对c-Kit激酶A-loop突变体诱导的格列卫耐药型GIST无效的缺点。体内、体外实验中，HQP-1351单药对c-KIT突变的GIST细胞、 异种移植肿瘤表现显著的剂量相关的生长抑制作用。