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新型Bcr-Abl/Src激酶抑制剂FB2抗伊马替尼耐药慢性髓系白血病作用及机制研究
【作者】 袁霞；
【作者基本信息】 北京协和医学院 ， 药理学， 2010， 博士
【副题名】K562/Imatinib获得性耐药细胞的耐药机制研究
【摘要】 慢性髓系白血病(CML)是一种起源于造血干细胞的恶性克隆性疾病,其特征性遗传学标志是由染色体易位形成费城(Ph)染色体。由易位染色体产生的Bcr-Abl融合基因编码产生分子量为210kD的Bcr-Abl融合蛋白,最初在造血干细胞中表达,促进骨髓及淋巴样细胞的分化,具有异常增高的蛋白酪氨酸激酶活性,被认为是CML发病的始发因素。传统的CML的治疗采用羟基脲、马利兰、甲异靛及干扰素等药物疗效欠佳且副作用大。随着对Bcr-Abl蛋白活性的深入研究,使其成为CML治疗的重要的分子靶点。伊马替尼(Imatinib)作为小分子Bcr-Abl酪氨酸激酶抑制剂以其显著的疗效及毒副作用低的特点,成为目前CML治疗的一线用药。但随着临床用药的增加Imatinib耐药问题逐渐突显。另外,Imatinib不能根除残留的白血病干细胞及原始祖细胞,成为疾病复发的危险因素。因此寻找新靶点或多靶点药物成为CML研究的热点。临床上常见的获得性Imatinib耐药机制主要分为两类：Bcr-Abl依赖型及Bcr-Abl非依赖型。Bcr-Abl依赖型耐药中主要原因为Bcr-Abl基因发生位点突变以及扩增引起的表达增加。另外,从Immatnib耐药的患者中分离的细胞系中发现Src激酶家族(SFK)成员中Lyn及Hck过表达,提示SFK涉及Bcr-Abl非依赖型Imatinib耐药。由于Abl与SFK成员中具有明显的序列同源性及活性中心结构相似性。因此,在对多种不同化学结构的Src激酶抑制剂的研究中包括Bosutinib、Dasatinib及INNO-0406等,发现这些化合物能够较Imatinib更有效地抑制Bcr-Abl激酶活性,并且对多种位点突变的Bcr-Abl有效。FB2是一种新型的噻唑类衍生物,前期研究已经证实其可有效地抑制Imatinib敏感及无Bcr-Abl点突变的耐药K562细胞系。本研究将观察FB2对Imatinib耐药的具有Abl激酶点突变的Ba/F3 P210 (WT、Y253F及T315I)白血病细胞的作用及机制,并建立四种不同的动物模型Ph+的K562及体外建立的Imatinib耐药株K562/G7.0 NOD/SCID小鼠模型,Ba/F3 P210 (WT及Y253)白血病小鼠模型,以观察FB2对不同耐药机制动物模型中位生存期的影响。基于FB2是在Dasatinib的结构基础上优化改造,推测其与Abl结合不会像Imatinib对蛋白结构要求严格,因此本实验首先纯化野生型(WT)及具有突变位点(Y253F及T315I)的Abl激酶区,利用Histone2B作为Abl激酶的底物,结果显示FB2在纳摩尔水平能够有效地抑制Abl (WT及Y253F)激酶活性,而对T315I突变的Abl无效。同时进一步检测了FB2对Src及Lyn的体外激酶活性抑制作用,结果显示其对激酶活性的ICso值分别为1.37nmol/L和2.83nmol/L此后,采用Ba/F3 P210细胞系包括野生型及两株具有突变位点Y253F及T315I的细胞观察FB2对细胞增殖的影响。MTT结果显示,FB2明显抑制Ba/F3 P210 WT及Y253F细胞(IC50分别为1.30nmol/L,2.56nmol/L)的生长,对Ba/F3P210 T315I无效。另外,为进一步探讨FB2抗Imatinib耐药的作用机制,对细胞内Bcr-Abl、c-Src和Lyn蛋白的磷酸化水平进行检测,结果与体外活性测定一致,FB2能够明显抑制Ba/F3 P210 WT及Y253F细胞Bcr-Abl、c-Src和Lyn蛋白的自身磷酸化水平,但对Ba/F3 P210 T315I细胞仅能抑制c-Src和Lyn蛋白活性。同时在对细胞周期的影响的研究中发现,FB2在纳摩尔水平可使Ba/F3 P210 (WT、Y253F)细胞发生G0/G1期阻滞,将药物浓度增加至微摩尔水平能增加Ba/F3 P210 T315I细胞G0/G1期细胞比例。最后,通过尾静脉注射不同类型CML细胞株建立白血病动物模型评价FB2治疗CML的作用。无药物处理的对照组小鼠很快发展为白血病小鼠,生存期在27～75天,FB2可以明显延长包括K562敏感株、Lyn激酶过表达的K562/G7.0耐药株、Ba/F3 P210野生株及Imatinib耐药Y253F点突变株,四种不同白血病小鼠模型的生存期,且具有良好的剂量效应关系。在动物实验观察周期均在三个月以上,FB2治疗组动物对药物耐受性均较好,无体重下降现象出现,一定程度上反映了该药毒副作用小的特点。综上,FB2作为一种酪氨酸激酶抑制剂,具有明确的Bcr-Abl及Src激酶抑制作用,可能成为治疗CML并克服除T315I突变的Imatinib耐药的新的治疗药物。伊马替尼(Imatinib)是首个用于肿瘤临床治疗的小分子靶向药物,因其能特异性作用于Bcr-Abl酪氨酸激酶,而在临床上广泛应用于费城染色体阳性的慢性髓系白血病(CML)及急性淋巴细胞性白血病(ALL),并取得了卓越的治疗效果。目前临床应用Imatinib标准剂量为400mg/d,对于CML慢性期患者有效,但对进展期CML患者疗效较差,在治疗后数周或数月内复发或进展至急变期。另外,约有60%的慢性期患者在用药六年后可维持原来的剂量,而近40%患者对Imatinib敏感性降低,需要增加剂量或选择其他治疗方式。因此,Imatinib耐药现象的发生逐渐受到人们的关注。各国学者通过不同的研究方法对于Imatinib耐药机制进行了深入的研究,目前发现的耐药机制包括治疗依从性不好、胃肠道吸收减少、结合的蛋白发生改变、肝药酶加速药物代谢、药物外排增加、Bcr-Abl融合基因扩增或蛋白表达增加以及融合基因发生位点突变等。对于耐药机制的研究方法除通过分析临床发生Imatinib耐药的患者样本所发生的改变以外,体外建立Imatinib细胞耐药株也是研究的重要途径。经过前期的研究本实验室已经建立了K562细胞Imatinib耐药株,并对其耐药机制做了初步的探讨。本研究在此基础上增加耐药株对Imatinib的耐药浓度至7μmol/L建立K562/G7.0细胞,然后观察一系列Imatinib不同耐药程度的K562/G细胞系包括K562/G01、K562/G3.0、K562/G5.0、K562/G7.0的生生物学特性及耐药机制的变化,并通过蛋白质组学技术分析不同耐药株及K562亲本株之间的差异蛋白,以期望发现新的克服Imatnib耐药的分子靶点。首先通过MTT法观察K562及不同耐药细胞株对Imatinib的耐药程度,其IC50值分别为0.36、4.84、14.06、36.65、51.26μmol/L,细胞生长曲线结果显示各细胞株倍增时间无明显变化,另外,通过流式细胞术检测各细胞株之间周期分布的差异,结果表明随耐药浓度的增加处于G2/M期的细胞呈现递增趋势,以上结果表明,体外建立的K562不同耐药程度的Imatinib耐药株生物学特性有一定的改变。此后,本实验对不同K562/G系列细胞的耐药机制进行研究。通过对不同细胞株Abl的ATP结合区基因序列进行扩增后测序未发现耐药细胞中有位点突变现象。而后通过免疫印迹杂交的方法检测各细胞株Imatinib耐药相关蛋白的表达情况,结果显示耐药程度不同各细胞表现一定的耐药机制的改变,其中K562/G01细胞Bcr-Abl、p-gp及Lyn均较K562细胞表达水平增高,随着耐药浓度的增加K562/G3.0细胞p-gp表达降低,Bcr-Abl及Lyn表达持续增加,耐药程度进一步增加,K562/G5.0及K562/G7.0细胞中Bcr-Abl表达水平反而降低,表现为Lyn依赖型耐药机制。这种耐药机制的转变部分解释了国内外很多学者所建立的不同耐药细胞株耐药机制的差异。最后通过蛋白质组学的技术进一步观察了不同耐药株及K562细胞之间蛋白表达的差异。经过二维凝胶电泳分离得到各耐药株及K562亲本株蛋白表达图谱,其中发现具有明显差异的14个蛋白点,通过基质辅助激光解析电离飞行时间质谱技术进一步分析,结合双向电泳图谱上相应点的表观等电点、分子量综合分析成功鉴定9个差异蛋白,其中与肿瘤关系密切的包括波形蛋白、膜突蛋白、埃兹蛋白及膜联蛋白-1,这些蛋白的表达水平与细胞耐药程度也有一定的关系。综上所述,Imatinib耐药是一个复杂的多因素作用的结果,不同的研究方法显示了不同的耐药机制。本研究通过比较不同耐药程度的Imatinib耐药细胞株探索和发现了随耐药浓度的改变其耐药机制发生不同程度的转变,另外蛋白质组学的研究发现了在耐药株中表达升高的蛋白,为进一步寻找Imantinib耐药机制提供了实验基础。
【Abstract】 Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder that is characterized by the Ph chromosome. The reciprocal translocation between the chromosomes 9 and 22 results in the Ph chromosome, and produces a fusion gene known as Bcr-Abl.This fusion gene encodes a chimeric protein which turns on a dysregulated tyrosine kinase activity and drives CML. In CML, a p210 Bcr-Abl isoform is initially expressed in haematopoietic stem cells (HSCs) capable of giving rise to both differentiated myeloid and lymphoid progeny.The traditional therapy of CML includes Hydroxycarbamide, Myleran, Meisoindigo, interferon with high side effects. The biology of CML has enabled preclinical and clinical oncology researches with targeted therapies. Imatinib is a small molecule inhibitor of the Bcr-Abl tyrosine kinase that produces clinical remissions in CML patients with minimal toxicity. Imatinib is now frontline therapy for CML. However, despite the stunning efficacy of this agent, resistance or intolerance to imatinib may become increasingly important. Moreover, imatinib does not completely eradicate residual leukemic stem cells and progenitors, which present a persistent risk of desease relapse. Therefore, there is a clear need for CML research to focus on novel targets and targeted drugs.Various mechanisms may contribute to imatinib resistance, and it can be categorized into two broad groups:Bcr-Abl-dependent and Bcr-Abl-independent. The main cause in Bcr-Abl-dependent imatinib resistance involves point mutations in the Abl kinase domain of the fusion protein and over-expression of Bcr-Abl kinase through gene amplification. In addition, the Src family of kinase (SFKs) members Hck and Lyn are over-expressed in some imatinib-resistant patient isolated and cell lines, suggesting that SFKs may be involved in Bcr-Abl-independent imatinib resistance. Abl shares significant sequence homology and remarkable structural resemblance in its active state with Src family members. Several Src inhibitors from various chemical classes, including bosutinib, dasatinib and INNO-406 have been developed. These agents are more effective than imatinib in blocking Bcr-Abl tyrosine kinase autophosphorylation, and these effects extend to point mutantions of Bcr-Abl.FB2 is a novel N-(thiazol-2-yl)pyrimidin-4-amine derivative, and it had shown that FB2 inhibited imatinib-sensitive and resistance CML cell lines with the wild-type Bcr-Abl fusion gene. In this report, it was sought to identify this novel compound for treating Ph+ chronic myeloid leukemia that is potent in blocking Bcr-Abl kinase activity, including point mutations in the kinase domain, and inhibits src kinase activity. To assess its potential as a therapeutic agent, it was investigated the effect of FB2 on survival span of mice inoculated with K562 cells, K562/G7.0 cells and Ba/F3 cells expressing different isoforms of Bcr-Abl (wild-type, Y253F).On the basis of prior structural insights from dual Abl and Src inhibitor Dasatinib, we reasoned that FB2 may impose less stringent conformational requirements on Abl for kinase inhibition, and we therefore purified enzymes to assesse its activity against imatinib-resistant Bcr/Abl mutants. As expected, FB2 was potent at inhibiting nonmutanted Bcr/Abl (Bcr/Abl WT) and mutanted Bcr-Abl (Y253F) kinase activity, while not on mutanted Bcr-Abl with T315I. Furthermore, Src and Lyn protein kinase assays were done and the results exhibited FB2 was a nanomolar inhibitor of two Src kinases (Src and Lyn with IC50 of 1.37 nmol/L,2.83nmol/L, respectively).Ba/F3 p210 cells were obtained by transfecting the IL-3-dependent murine hematopoietic Ba/F3 cell lines with pSRa-p210Bcr-Abl plasmid. The mutations (Y253F and T315I) were introduced into full-length p210Bcr-Abl. MTT assay were performed to investigate the inhibition of FB2 on Ba/F3 p210 (WT, Y253F and T315I) cells. And the mean IC50 values for FB2 were 1.30 and 2.56 nmol/L in Ba/F3 p210 WT and Ba/F3 p210 Y253F cells respectively. However, it had no effects on proliferation of Ba/F3 p210 T315I cells. FB2 inhibited the activities of Bcr-Abl and Src kinases as assayed by reduction of the phosphorylated forms of Bcr-Abl、c-Src and Lyn, respectively. Ba/F3 p210 WT and Y253F cells presented the marked dose-dependent reduction in Bcr-Abl, c-Src and Lyn phosphorylation when treated with FB2 from 0.2 to 5 nmol/L, and its potency of inhibition in c-src phosphorylation was stronger than dasatinib on it. FB2 reduced the level of p-c-Src and p-Lyn in Ba/F3-T315I cells while not the level of p-Bcr-Abl. To determine the antiproliferative effects of FB2 involved growth arrest at specific phases of the cell cycle, flow cytometric studies were performed.Our data showed that, in the low nanomolar range, FB2 induced the inhibition of cell growth and cell cycle progression of Ba/F3 p210 (WT, Y253F) cell lines mainly by inducing the G0/G1 phase arrest, and exhibited the dose-dependent relationship. Increasing the concentration of agent to micromolar range, FB2 could induce Ba/F3 p210 T315I cells arrested in G0/G1 phase.To assess its potential as a therapeutic agent, we studied FB2 in a mouse model of imatinib-resistant, Bcr/Abl dependent disease. Nod/Scid mice were injected intravenously with K562 and K562/G7.0 cells, and Balb/c mice were harbored Ba/F3 cells expressing different Bcr/Abl isoforms. Untreated mice harboring cells expressing nonmutant or imatinib-resistant mutant Bcr/Abl developed aggressive disease, typically resulting in death in 27-75 days. All the three doses tested groups showed significantly prolonged survival span and the increases in survival times were in dose dependent manner. Mice bearing K562 and Ba/F3 p210 cells tolerated administrations of FB2 well, and obvious evidence of toxicity did not occurred in three months.In summary, our current studies demonstrated an effective inhibition of FB2 on Bcr-Abl and Src kinases. These data provide the framework for clinical trials with FB2 in Ph+CML and imtinib-resisitant CML。 Imatinib is the first small molecular drug for targeted therapies, and it is a selective inhibitor of the tyrosine kinase activity of Bcr-Abl fusion oncoprotein. Imatinib is indicated for the treatment of patients with Ph+CML in chronic phase、blast crisis and accelerated phase, and Ph+ALL. Most patients with chronic phase (CP) CML treated with imatinib have well-controlled disease. Imatinib at an oral dose of 400mg daily has now become standard initial treatment for all CML patients who present in CP. It is highly effective, too, in the short term for patients who present in advanced phases, but the duration of response is usually much shorter and the probability of relapse to blastic phase is high. Even for CP, however, the drug is not perfect. Only approximately 60% of patients are still taking imatinib at standard dosage after 6 years, which means that approximately 40% have needed higher doses of imatinib or alternative therapy. Therefore the mechanisms of resistance emerged as a major question.Multiple studies have addressed the problem of imatinib resistance and helped to define the major elements contributing to this occurrence. In simple terms, the mechanisms of imatinib resistance include treatment compliance excessive binding of imatinib to the plasma protein al-acid glycoprotein-1(AGP-1), increase of metabolization by the cytochrome p450 isoenzymes, changes on intracellular uptake of imatinib, Bcr-Abl overexpression and point mutations in the kinase domain of Bcr-Abl. There are many methods to research imatinib resistance. Some imatinib resistant cells were derived from CML patients, while it is useful to establish the resistant cells by adding imatinib to the culture. On the basis of the resistant cell line K562/G5.0 which was established by our laboratory before, K562/7.0 cells were induced and could be maintained in liquid culture with 7μmol/L imatinib. Then the biological characteristics of K562/G cell lines including K562/G01, K562/G3.0, K562/G5.0, K562/G7.0, and the changes of resistant mechanisms, were investigated in order to find some new targets to overcome the imatinib resistance.First, cell proliferation assay was performed using MTT, and IC50 for K562, K562/G01, K562/G3.0, K562/G5.0, K562/G7.0 was,0.36,4.84,14.06,36.65 and 51.26μmol/L, respectively. And there was no obviously change of doubling generation time between these cell lines. Furthermore, flow cytometric studies were performed to investigate the distribution of cell cycle, our data showed that cells in G2/M phase were increased gradually accompanied by the increase of resistant concentrations. All these results showed that the biological characteristics were changed between K562/G cell lines and K562 cells.To address whether there were any mutations in the ATP binding site of the Abl kinase domain, thought to be the target of imatinib, the ATP binding domains of K562 sensivive and resistant cells were sequenced and compared these to the the pubished sequences. No mutation was found in the fragment in any of the cell lines. The expression levels of Bcr-Abl, Lyn and p-gp were studied by immunoblotting. Compared with the sensitive parental line, the level of Lyn overexpressed increasing with the degree of resistance. However, the expression of p-gp was increased at lower level in K562/G01 cells and decreased at higher resistance fold in K562/G3.0, K562/G5.0 and K562/G7.0. The Bcr-Abl expression in K562/G3.0 cells was higher than that in K562, while it was reduced in K562/G5.0 and K562/G7.0 which was lower than in K562. The different changes of resistant proteins probably explained that the different results were gotten by different researchers about the resistant mechanisms.Proteomics can provide a global, systemic, and comprehensive approach to the identification and description of the biochemical processes, pathways, and networks involved in both normal and abnormal physiological states at the protein level. As a typical proteomic analysis, two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS) has been used widely in research. Differences in protein expression levels were detected in cell states between K562 and K562/G cell lines by 2-DE, and 14 protein spots showing significant changes were gotten. There were 9 proteins identified via mass spectrometry correctly. Among those protein, Vimentin, Moesin, Ezrin and ANXA1 were correlated with cancer closely.Currently, resistance to imatinib is believed to be a consequence of the interaction of multiple factors. Different research methods may exhibit different resistant mechanisms. Varies of imatinib resistant K562/G cell lines were used to explore the changes of mechanisms, and it was found the higher expression proteins in K562/G cells by 2-DE and MS. All these results were provided experimental basis for further exploration of imatinib resistance.