EphB4/RhoA通路参与慢性髓细胞白血病对于二代酪氨酸激酶抑制剂耐药与阻断机制研究

It was a major question to study the mechanisms of drug resistance to tyrosine kinase inhibitor. EphB4/RhoA may be a new marker of IM resistance and homoharringtonine gained more treatment advantages than IM alone by blocking EphB4/RhoA pathways in CML cell lines. However it did still not identify the role of EphB4/RhoA and homoharringtonine in the second generation tyrosine kinase inhibitor. In the study, we monitored the expression of EphB4/RhoA protein and mRNA by follow-up the patients with advanced phases of chronic myeloid leukemia (CML) and explored the relations between EphB4/RhoA with nilotinib. We further established cell lines from a typical patient with CML at the time of first diagnosis and relapsed phase. Nilotinib-resistant cell line was generated and knockdown of EphB4 was established by RNA interference. Activated EphB4/RhoA was detected in EphB4-modulated CML cell lines. Meanwhile the CML xenograft models were also established by SCID nude mice. The influence of EphB4/RhoA resistance to nilotinib was verified by SCID models. In summary, we hope to make a theoretical explanation and value for the efficacy of targeted therapy and reversing the resistance to the second generation tyrosine kinase inhibitor in the treatment of myeloid leukemia.

耐药机制是慢性髓系白血病靶向治疗的最主要问题。研究已发现EphB4/RhoA是一条介导伊马替尼耐药的新信号通路，高三尖杉酯碱具有阻断EphB4/RhoA的作用。但EphB4/RhoA和高三尖杉酯碱在二代酪氨酸激酶抑制剂耐药中的角色尚不清楚。本课题随访监测对尼洛替尼耐药的慢性髓系白血病进展期患者EphB4、RhoA的蛋白和mRNA表达情况，初步探讨其与尼洛替尼耐药的关系；采集典型病例的白血病标本进行体外培养，诱导尼洛替尼耐药株，通过RNA干扰技术调变EphB4表达水平，并测定调变EphB4表达的人白血病，对尼洛替尼耐药性的变化，检测EphB4/RhoA通路表达水平对尼洛替尼耐药性变化的影响；利用SCID裸鼠，建立白血病模型，验证EphB4/RhoA对尼洛替尼耐药的影响。通过本实验研究，为克服二代酪氨酸激酶抑制剂耐药性，寻找更有效的靶标奠定基础，提供重要的理论意义和应用价值。

目的 为明确EphB4在进展期人慢性粒细胞白血病（CML）中对达沙替尼（DAS）耐药的作用和机制，我们以CML患者和K562细胞株为的对象，探索EphB4调控的基质微环境通路。方法 分别从5名CML受试者初诊时（A1-E1）和进展期耐药状态下（A2-E2）获得肝素化骨髓标本。梯度浓度诱导K562-W细胞产生高度耐DAS的K562-R细胞株。利用RNA干扰技术，通过K562-R细胞株构建稳定低表达EphB4的K562-R-EphB4-sh细胞株。将K562-W，K562-R和K562-R-EphB4-sh细胞株（108）分别注入BALB/C雌性裸鼠皮下成瘤。结果 EphB4 mRNA/蛋白水平在DAS耐药的人A2-E2细胞中过表达，在初诊状态下的人A1–E1细胞中低表达。和K562-R细胞相比，EphB4和磷酸化-Rac1/Cdc42蛋白在K562-R-EphB4-sh细胞株中显著低水平表达（P<0.01）。K562–R细胞株对DAS高度耐药性（IC50 10.54±0.67 µg/ml），但K562-R-EphB4-sh细胞株对DAS敏感性显著增强（IC50 1.02±0.1 µg/ml, P<0.01）。配体EphrinB2-Fc刺激下，K562-R-EphB4-sh 细胞株中，EphB4/磷酸化-RhoA/MCL-1蛋白水平较前显著增高（P<0.01），对DAS耐受性也随之增强（4.18±0.30µg/ml）。和K562-R移植瘤裸鼠相比，K562-R-EphB4-sh移植瘤裸鼠对DAS治疗反应较好。移植瘤组织中蛋白测定结果和细胞水平相近，EphB4/RhoA/ROCK1/PTEN/MCL-1在K562-R移植瘤中过表达，但在K562-R-EphB4-sh移植瘤中表达下降（P<0.01）。结论 进展期CML中，存在一个新的DAS耐药独特作用机制，就是EphrinB2-Fc 配体刺激EphB4过表达后，进一步活化RhoA/ROCK1/PTEN/MCL-1凋亡通路，产生对DAS的耐受。