CD20膜转运障碍介导B淋巴细胞瘤对美罗华耐药的机制研究

Lymphoma incidence has been one of the top 10 tumors, and B-NHL is the most common and threatening subclass. When combined rituximab(R), a molecular target monoclonal antibody, with CHOP chemotherapy regimen, though the curative effect becomes more satisfying, the drug resistance of R is still the biggest obstacle for treatment, which is worthy of a further study of the resistance mechanism. The therapeutic target of R is the membrane protein CD20. In the previous studies, the applicant found that in R resistant cells, the expression level of CD20 only decreased on the cell surface but not in total protein level and the proportion of cancer stem cells(CSC) increased significantly. After a deep research, the applicant found an obvious up-regulation of SOX2 on protein level. As an important CSC transcription factor, SOX2 may negatively regulate the transcription of Golgi protein PLPP3, that caused the blockage of CD20 transportation to cellular membrane, resulting in the reduction of CD20 distribution on the cell surface, which ultimately led to R resistance. This project is based on the level of molecular, cellular, animal and clinical by means of proteomics, biochemistry, molecular biology, immunology and others. It intends to study the molecular mechanism of R drug resistant formation, discover the prediction markers of the curative effect in B-NHL treatment using R-CHOP, explore the mechanism of SOX2 up-regulation in R resistant cells, identify the targets that can reverse R drug resistance and extend a new thinking for novel clinical tumor treatment.

淋巴瘤发病率已进入十大肿瘤之列，其中B-NHL发病率最高，威胁也最大。通过联用分子靶向单抗药物美罗华显著提高了原CHOP化疗方案的疗效，但美罗华耐药仍然是最大的治疗困扰因素，其耐药机制也有待深入研究。美罗华的治疗靶点为膜蛋白CD20，申请人前期研究发现，美罗华耐药细胞中CD20仅膜蛋白水平下降而总蛋白水平没有变化，同时肿瘤干细胞的比例明显增加。深入研究还发现耐药细胞中，重要干细胞转录因子SOX2蛋白水平明显升高，且很可能通过负性调控高尔基体蛋白PLPP3的转录，引起CD20的膜转运障碍，造成美罗华的靶分子膜水平下降，最终导致耐药。本课题拟通过蛋白质组学、生化、分子生物学等多种手段，在分子、细胞、动物和临床整体水平，多角度多层面多途径进一步研究美罗华耐药形成的分子机制，筛选预测耐药标志物，探讨R耐药中SOX2上调表达的分子机制，鉴定逆转耐药的药物靶点，为临床肿瘤预测和治疗提供新思路和新方案。

R-CHOP疗法是目前DLBCL的一线治疗方案，然而将近三分之一的患者最终将对此疗法产生耐药，而目前对于R-CHOP耐药的DLBCL患者行之有效的治疗方案十分有限，因此，因此亟需深入研究R-CHOP耐药产生的机制，并制定新的治疗策略。我们制备了两株（GCB和ABC亚型）的R-CHOP耐药的DLBCL细胞株，并通过连续移植成瘤评估、干细胞相关指标（CD34、CD133）检测、侧群（SP）细胞检测以及ALDH1检测在体内体外评估耐药细胞的干细胞特性。通过RNA-seq技术揭示耐药细胞的表达谱特征，同时通过IHC检测对配对的耐药前后的DLBCL患者临床样本对关键差异分子进行验证。我们研究发现，在耐药DLBCL细胞中由于PI3K/AKT信号的过度活化导致干细胞关键转录因子SOX2第118位磷酸化，进而抑制了SOX2的泛素化降解，从而使用肿瘤干细胞在耐药细胞中富集。我们进一步发现在耐药细胞中BCR信号，FAK信号，趋化因子信号以及FGFR1/2信号调控PI3K/AKT的活化，而抑制SOX2下游靶点CDK6，FGFR1/2可使耐药细胞恢复对R-CHOP的敏感性。更重要的是R-CHOP与PI3K抑制剂联用后可使肿瘤干细胞转化为对R-CHOP敏感的分化肿瘤细胞，从而完全抑制耐药细胞的生长。PI3K/AKT/SOX2信号轴在R-CHOP耐药形成中发挥着重要作用，而本研究提出的靶向肿瘤干细胞的促分化治疗策略可为将来临床上使用PI3K/AKT抑制剂联合R-CHOP治疗复发难治型DLBCL提供理论依据。