佛波酯与三氧化二砷通过调控自噬在靶向清除慢性粒细胞白血病干细胞中的作用及机制研究

The previous evidence strongly suggest that the combination of imatinib with autophagy inhibitors may result in targeting eradication of leukemic stem cells and that arsenic trioxide (ATO) may target and ultimately eradicate leukemic stem cells through arsenic-induced autophagic degradation of BCR-ABL oncoprotein in the treatment of chronic myeloid leukemia (CML). In this study, the imatinib-resistant CML leukemic stem cells will be obtained as the object of the research firstly. After these, by flow cytometry, immunofluorescence, western blotting, transmission electron microscopy analysis, co-immunoprecipitation analysis, RNA interference and so on, we plan to clear two outstanding questions: (i)Whether 12-0-tetradecanoylphorbol-13-ac-etate (TPA) could enhance the therapeutic effects of imatinib in the targeting elimination imatinib-resistant CML leukemic stem cells in vitro. At the same time, the mechanisms of synergistic interactions between TPA and imatinib need to be studied; (ii) Whether ATO can target and ultimately eradicate imatinib-resistant CML leukemic stem cells in vitro through ATO-induced autophagic machinery and the mechanisms underlying induction of autophagy need to be studied. In vivo study, we plan to use an established NOD/SCID mouse model of CML to examine the efficiency of sustained molecular response with ATO maintenance after induction therapy with imatinib plus TPA in patients with CML. In conclusion, the present study was designed to create a novel treatment with ATO enables discontinuation of imatinib in most patients after prior imatinib/TPA combination therapy which would appear to completely eradicate imatinib-resistant CML stem cells through regulation of autophagy in different stages of CML treatment.

有证据提示：抑制自噬极可能会增强伊马替尼（Imatinib）靶向清除慢性粒细胞白血病干细胞（CML干细胞）的药效；通过诱导自噬,三氧化二砷（ATO) 极可能会通过靶向降解BCR-ABL癌蛋白进而杀灭CML干细胞。本研究拟以对Imatinib天然耐药的CML干细胞为研究对象，利用免疫荧光、透射电镜、免疫共沉淀、RNA干扰等实验方法，体外探讨：①氟波酯（TPA）通过抑制自噬而增强Imatinib靶向清除CML干细胞的有效性及分子机制；②ATO通过诱导自噬、靶向清除CML干细胞的有效性及分子机制。借助NOD/SCID小鼠CML模型，体内探讨采用TPA + Imatinib诱导治疗CML后再继以单药ATO维持分子学缓解的有效性。本研究拟在CML治疗的不同阶段，借助TPA与ATO对自噬的不同调控作用，力求达到彻底清除CML干细胞之目的，初步探讨能适时终止Imatinib治疗的可行性。

慢性粒细胞白血病（CML）患者体内处于细胞周期静止期、BCR-ABL+的CML白血病干细胞，很难被酪氨酸激酶靶向抑制剂伊马替尼（Imatinib）彻底清除，这些在CML患者体内残留的极少量的CML白血病干细胞，必然会成为疾病复发及产生耐药的突变白血病细胞的潜在根源。因此，无限期服用imatinib仍然是当前CML治疗的标准推荐模式。但对于持续接受imatinib治疗的CML患者，继发耐药、安全性与耐受性、依从性及患者和社保系统的经济负担等，都是我们血液学工作者无法回避的严峻问题。因此，用何种方法有效清除天然耐药的CML白血病干细胞，从而使绝大多数CML患者能适时中断imatinib治疗并长期无病生存，无疑是目前血液界亟需解决的课题之一。本研究以对Imatinib耐药的Flk1+CD31-CD34- CML白血病干细胞为研究对象，利用流式细胞术、免疫荧光、透射电镜、免疫共沉淀、RNA干扰等实验方法，体内外探讨了佛波酯(TPA)与三氧化二砷（亚砷酸，ATO)通过调控自噬在靶向清除CML白血病干细胞中的作用及分子机制。结果提示：①TPA通过抑制自噬而发挥出增强Imatinib靶向清除CML白血病干细胞的作用；②ATO通过诱导自噬而发挥出靶向降解BCR-ABL癌蛋白并进而杀灭CML白血病干细胞的作用；③以NOD/SCID小鼠CML模型为研究工具，借助TPA与ATO所发挥的抑制或诱导自噬作用，发现采用TPA联合Imatinib诱导治疗NOD/SCID小鼠CML后，再续以单药ATO维持，可显著延长分子学缓解的持续时间；④以CML患者为研究对象，发现采用TPA联合Imatinib诱导治疗CML患者后再续以单药ATO维持，极有可能使CML患者能适时终止Imatinib治疗并长期无病生存。综上，本研究为探索Imatinib联合其他药物彻底清除耐药CML干细胞提供了新的切入点，为使CML患者能适时终止服用Imatinib探索了新思路。