Bcl-2家族蛋白在慢性粒细胞白血病Bcr/Abl非依赖型耐药中的机制研究

The tyrosine kinase inhibitor Gleevec has undoubtedly revolutionized the therapy of chronic myeloid leukaemia (CML).However, relapse and drug resistance is increasingly more serious in CML therapy. A clearcut molecular mechanism or molecular player in BCR-ABL-independent resistance to Gleevec has not yet evolved. In this study, a variety of specific inhibitors of Bcl-2 family proteins, including the small molecules inhibitor of phosphorylation Bcl-2 developed by ourselves is used as a research tool. These small molecules play a direct role in screening and induction of Bcr/Abl-independent TKI resistant CML cells. We also use these tools to study the function of Bcl-2 family proteins, especially the relationship between the combination and dissociation of phosphorylated Bcl-2 and its pro-apoptotic partner and the drug resistance of CML. This study will reveal the key role of Bcl-2 phosphorylation in drug resistance. We will also study the crosstalk between Bcl-2 family proteins and kinase pathway. Besides Bcr/Abl, we will found the new kinase pathway involved in the development of CML. This study will explain the Bcr / Abl-independent molecular mechanisms of TKI resistance and provide new therapeutic targets to overcome this resistance. We will also provide the therapeutic strategies of combination therapy, in order to truly improve the cure rate of TKIs in CML.

酪氨酸激酶抑制剂在慢性粒细胞白血病(CML)治疗中的复发和耐药问题日益凸显。但是，Bcr/Abl非依赖的TKIs耐药机制，目前还没有系统的研究和确切的结论，更没有有效的逆转耐药方案。本研究以多种特异性的Bcl-2家族蛋白抑制剂，包括我们自主研发的磷酸化Bcl-2蛋白的抑制剂作为小分子研究工具，直接作用于筛选和诱导的Bcr/Abl非依赖型TKI耐药的CML细胞，研究Bcl-2家族蛋白，尤其是磷酸化Bcl-2蛋白与促凋亡伴侣蛋白的二聚体的形成和解离与CML耐药和逆转耐药的关系，揭示Bcl-2蛋白磷酸化在这种耐药中的关键作用和意义；发现参与Bcl-2磷酸化的、Bcr/Abl之外的激酶通路，在理解Bcr/Abl非依赖型TKI耐药的分子机制的同时，为克服耐药提供新的治疗靶点和联合用药的治疗策略，以期提高TKIs对CML的治愈率。

本研究以多种本课题组自主研发特异性的Bcl-2家族蛋白抑制剂作为小分子研究工具，以筛选和诱导的Bcr/Abl非依赖型多种激酶抑制剂耐药的慢性粒细胞白血病细胞系，和临床采集的三代激酶抑制剂原发耐药和获得性耐药的慢性粒细胞白血病患者的外周血癌细胞为对象，直接研究了Bcr/Abl非依赖型TKI耐药的分子机制。本研究构建了新的Bcl-2家族蛋白凋亡网络数学模型，整合了Bcl-2家族蛋白在表达水平和后修饰水平后对凋亡网络的影响，在原有的Bcl-2凋亡调控模型基础上，定量分析和补充了磷酸化修饰的Bcl-2（pBcl-2）与促凋亡蛋白二聚体形成、解离的动力学参数，完善了网络拓扑结构对凋亡输出信号的调控模型。该模型结合实验验证了pBcl-2对肿瘤细胞凋亡网络的重塑，提出了更精准的分子标志物，用于预测Bcl-2抑制剂与化疗药物联合治疗包括白血病的多种肿瘤的治疗方案，更大程度地克服耐药。尤其是，本研究通过化学蛋白质组学研究方法，发现了Bcl-2家族新成员：Tubulin和Hsp70，可能是Bcr/Abl非依赖TKIs耐药的白血病的新的治疗靶点。