MZH29治疗FLT3-ITD突变型急性髓性白血病的作用机制研究

发布时间：2018-04-10 11:57

本文选题：络氨酸激酶抑制剂 + 白血病　；参考：《北京协和医学院》2017年博士论文

【摘要】：急性髓性白血病(Acute Myeloid Leukemia,AML)中约有三分之一的患者是由于FLT3酪氨酸激酶的突变引起的,其中内部串联型(Internal Tandem Duplication,ITD)突变患者预后极差。以FLT3为靶点用于治疗FLT3突变型AML的药物开发是当前研究的热点之一。AC220(Quizartinib)是目前已知的十分具有开发潜力的FLT3抑制剂之一,并处于临床Ⅲ期研究阶段。但包括AC220在内的许多FLT3抑制剂,尤其是TypeⅡ的FLT3抑制剂,所面临的主要问题是患者一般服用3个月后会发生进一步的点突变而产生耐药性。因此,继续寻找具有FLT3抑制活性且对耐药突变有效的化合物,并详细阐述药物作用机制是当前亟需解决的问题,也是本研究的主要目的。本课题前期通过对c-MET激酶抑制剂LS177进行了 46种络氨酸激酶抑制作用的体外筛选实验,发现LS177同时具有FLT3激酶抑制活性,但随后发现LS177对FLT3-ITD突变细胞系MV4-11的抑制活性显著弱于阳性对照药AC220。经过对一系列化合物的筛选,最终设计合成并筛选出4-苯氧基喹啉衍生物MZH29。MZH29对体外表达的FLT3-WT和FLT3-ITD激酶具有强的抑制活性,半数有效抑制浓度(IC50)分别为9 nM和7 nM,活性强度与AC220相同;MZH29同时对FLT3-WT依赖性的AML细胞系和FLT3-ITD突变型细胞系具有抑制作用,在FLT3-ITD突变型白血病细胞系上的IC50均低于1nM。在促凋亡作用实验中,MZH29同时对FLT3-WT依赖性的AML细胞系和FLT3突变型细胞系具有显著的促凋亡作用,主要通过促进凋亡蛋白Cleaved Caspase 3和Cleaved PARP的表达上调。为了明确MZH29对耐药突变细胞株的抑制作用情况,我们体外构建了各种耐药突变细胞系,结果表明MZH29对耐药突变型细胞系,包括FLT3-ITD-F691L,FLT3-D835H/Y/V和FLT3-K663Q突变细胞系的生长均具有很强的抑制作用,且明显优于阳性对照药AC220,在FLT3-K663Q和FLT3-ITD-F691L突变细胞上的抑制活性强度分别是AC220的47倍和40倍。在信号通路方面,MZH29主要通过抑制FLT3及其下游的STAT5和ERK的磷酸化而抑制细胞的增殖,MZH29对FLT3-ITD-F691L和FLT3-ITD-D835Y耐药突变细胞系的FLT3及其下游磷酸化信号通路仍然具有抑制作用,表明MZH29能够用于治疗FLT3耐药突变型的AML。我们通过Docking的方法计算了 MZH29与FLT3的作用方式,MZH29能够结合于非活性状态FLT3的激酶结构域,并发现MZH29与FLT3-F691L耐药突变仍能形成稳定的结合构象,解释了 MZH29对耐药突变有效的作用机制,同时也暗示着MZH29可能属于TypeⅡ的FLT3抑制。体内实验中,通过构建皮下种植瘤和血液瘤两种模型证实了 MZH29的药物活性。口服MZH29 10 mg/kg可以完全抑制小鼠皮下移植瘤生长和显著延长血液种植瘤模型小鼠的生存期,且具有较低的体内毒性。进一步的蛋白质组学研究发现,与AC220相比较,MZH29作用后对整体蛋白组的表达水平影响较小,二者调控的蛋白主要富集在转录调节活性蛋白和DNA结合蛋白,以及重要的细胞周期蛋白。总之,本研究证实MZH29具有作为治疗FLT3突变型急性髓性白血病的新型抑制剂的开发潜力。
[Abstract]:Acute myeloid leukemia (Acute Myeloid, Leukemia, AML) in about 1/3 of patients is due to a mutation of FLT3 tyrosine kinase, including internal tandem type (Internal Tandem Duplication, ITD) mutations in patients with poor prognosis. FLT3 targeted for drug development for the treatment of FLT3 mutant AML is currently one of the hotspots in the Research of.AC220 (Quizartinib) is currently known is the potential inhibitors of FLT3, and in the stage of clinical stage III. But many FLT3 inhibitors including AC220, especially FLT3 inhibitors of Type II, the main problems are generally taking patients after 3 months will further point mutation of drug resistance. Therefore, continue to search with FLT3 inhibitory activity and effective compound mutation on resistance, and expounds the mechanism of drug action is needed to solve the current problems, this study is the The main purpose of this paper. By the early on the c-MET kinase inhibitor LS177 for 46 inhibition of tyrosine kinase in vitro screening experiments, LS177 has found in inhibition of FLT3 kinase activity, but then found that LS177 inhibitory activity of the FLT3-ITD mutant cell line MV4-11 was significantly weaker than the positive control drug AC220. after the screening of a series of compounds the final design, synthesized and screened 4- phenoxy quinoline derivatives MZH29.MZH29 on the in vitro expression of FLT3-WT and FLT3-ITD kinase has strong inhibiting activity, the median effective inhibitory concentration (IC50) were 9 nM and 7 nM, the strength of activity similar to AC220; MZH29 and FLT3-WT dependent AML cell lines and FLT3-ITD mutant cells the Department has inhibitory effect on FLT3-ITD mutant leukemia cell line IC50 were lower than that of 1nM. in the apoptosis experiments, MZH29 dependent AML cells to FLT3-WT And FLT3 mutant cells with apoptosis significantly, mainly through up regulation of apoptosis protein Cleaved Caspase 3 and Cleaved PARP expression. In order to suppress the effect of MZH29 on the clear mutant cell lines in vitro, we construct a variety of mutant cell lines, the results showed that MZH29 of resistant mutant cell lines, including FLT3-ITD-F691L FLT3-D835H/Y/V, FLT3-K663Q mutation and cell growth has a strong inhibitory effect, and significantly better than the positive control drug in AC220, FLT3-K663Q and FLT3-ITD-F691L mutation on the cell inhibitory activity intensity were 47 times and 40 times of the AC220. In the MZH29 signaling pathway, mainly through inhibition of FLT3 and its downstream STAT5 and ERK phosphorylation and inhibition of cell proliferation, FLT3 phosphorylation and downstream signal pathway of FLT3-ITD-F691L cell lines MZH29 and FLT3-ITD-D835Y mutant still Inhibitory effect of MZH29 that can be used for the treatment of FLT3 resistant mutant AML. we through the Docking method to calculate the interaction between MZH29 and FLT3, MZH29 kinase domain can bind to the non active state of FLT3, and found that MZH29 and FLT3-F691L mutant still can form stable binding conformations, explains the mechanism of effective MZH29 the mutant, also implies that MZH29 may belong to Type II FLT3 inhibition. In vivo, by constructing the confirmed drug activity MZH29 subcutaneous tumor and blood tumor model. Two kinds of oral MZH29 10 mg/kg can completely inhibit the growth and prolong the blood tumor model in mice subcutaneous xenograft survival. In vivo and has low toxicity. Further proteomics study found that compared with AC220, has little effect on expression level of whole protein group after MZH29, two The proteins regulated mainly are transcriptional regulation of active protein and DNA binding protein, and important cyclin. In conclusion, this study confirms that MZH29 has potential as a new inhibitor for the treatment of FLT3 mutant acute myeloid leukemia.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R733.71

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