【作者】 古莹；

【导师】 徐荣臻；

【作者基本信息】 浙江大学 ， 内科学， 2011， 博士

【摘要】 研究背景：慢性粒细胞白血病(chronic myeloid leukemia, CML)是一种发生于造血干细胞水平的血液系统恶性克隆增生性疾病,在我国CML占总白血病发病率的20%左右。CML患者的骨髓细胞中可检测到特异性的Ph染色体,它是由于t(9;22)(q34；q11)染色体易位形成的，从而导致BCR-ABL基因融合并表达210KD的BCR-ABL融合蛋白。BCR-ABL蛋白在慢性粒细胞白血病细胞恶性增殖过程中发挥着重要作用,因而成为Ph+慢性粒细胞白血病的重要治疗靶点。第一代以BCR-ABL蛋白为靶标的分子靶向药物--伊马替尼(Imatinib,IM)已成为治疗CML的一线药物。然而,令人遗憾的是,伊马替尼类分子靶向药物不能完全清除患者体内BCR-ABL阳性白血病细胞克隆,并不能使多数CML患者获得长期缓解，停药后很快复发,其中一些患者对伊马替尼类分子靶向药物产生耐药。并且,越来越多的临床和实验研究资料显示,单用分子靶向药物只能控制慢性期CML,而对加速期和急变期CML基本无效,更不能治愈CML。随着近年来不断深入的研究.揭示白血病干细胞(leukemia stem cell, LSC)是白血病复发和耐药的主要根源,而伊马替尼类分子靶向药物对白血病干细胞无效。因为白血病干细胞通常处在休眠状态,很难被伊马替尼类分子靶向药物和经典化疗药物清除,从而留下白血病复发根源。CML患者要获得长期缓解和治愈,不仅需要杀死快速增殖的白血病细胞,而且还要清除处在休眠期的白血病干细胞。已有研究表明很多蛋白,如β-catenin, NF-κB等分子,与白血病干细胞的存活、自我更新和凋亡抵抗有关。但是这些分子在正常造血干细胞中也起着相似的作用,没有明显的特异性。因此,鉴定白血病干细胞特异性的蛋白靶标,并继而研发新型分子靶向药物就显得十分迫切。小檗胺(Berbamine, BBM)是从我国中草药小檗属植物中提取的一种双苄基异喹啉类生物碱药物。多年来一直广泛应用于临床,如抗炎和白细胞减少的治疗。我们前期研究结果显示,在体外小檗胺可以有效抑制对伊马替尼耐药的CML细胞的增殖,但对正常造血细胞增殖没有明显抑制作用。这表明小檗胺很可能对CML干细胞具有特异性杀伤作用,这将对CML的根治具有重要的临床意义。因此本研究将对其具体作用机制进行初步探讨。研究目的：1、研究天然小分子化合物小檗胺(BBM)对裸鼠慢性粒细胞白血病细胞移植瘤的清除作用。2、筛选并鉴定BBM抗慢性粒细胞白血病作用的靶分子。3、阐明该靶分子调控慢性粒细胞白血病干细胞存活、自我更新和凋亡抵抗的分子机制,从而明确BBM对慢性粒细胞白血病细胞清除作用的内在分子机制。研究内容：1天然小分子化合物小檗胺(BBM)对裸鼠慢性粒细胞白血病细胞移植瘤的清除作用研究我们分别用急变期CML细胞敏感株K562-S和耐受伊马替尼(IM)的耐药株K562-R(经流式细胞仪测定含1-2% CD34+白血病干细胞)建立裸鼠荷瘤模型。结果显示,荷瘤裸鼠连续口服小檗胺(150mg/kg,每天1次)10天后,70%(14/20)K562-S白血病细胞异种移植瘤完全消退；荷瘤裸鼠连续口服小檗胺(100mg/kg,每天3次)10天后,其体内伊马替尼耐药的K562-R白血病细胞移植瘤抑制率达到90.91%(10/11)。而且在实验观察期间没有发现明显的药物毒副作用。2小檗胺抗慢性粒细胞白血病作用靶分子CaMKIIγ激酶的鉴定本部分联合应用小檗胺亲和基质,蛋白质谱和Western blot技术方法发现并证实：BBM能与活化的钙离子/钙调素依赖的蛋白激酶Ⅱγ(Calcium/calmodulin-dependent protein kinase IIγ,简称CaMKIIγ)发生特异性作用。进一步应用计算机分子对接模型(molecular docking model)模拟显示小檗胺主要以范德华力(van der Waals forces)与CaMKII y激酶P44, V47,164, V93, P109, V112和L162氨基酸残基结合,并且其位置正好位于CaMKIIγ激酶的ATP结合袋(ATPbinding pocket)。而Western blot结果显示BBM处理白血病细胞后,CaMKIIγ的磷酸化水平(pCaMKIIγ)显著降低,而CaMKIIγ总蛋白水平没有明显变化。这提示小檗胺很可能通过与ATP竞争性结合CaMKIIγ激酶而抑制其磷酸化水平。3 CaMKIIγ激酶调控慢性粒细胞白血病干细胞存活、自我更新和凋亡抵抗的分子机制研究3.1 CaMKIIγ激酶在CD34+白血病细胞中特异性异常激活应用流式细胞分选技术分选出CML干细胞,用Western blot技术对其CaMKIIγ激酶表达水平进行检测和分析。并检测CaMKIIγ激酶在CML干细胞与正常造血干细胞中的表达水平。发现CaMKIIγ激酶在CD34+的CML干细胞中呈特异性高表达,并且其磷酸化水平也较高,而在相应CD34-的CML细胞和正常造血干细胞中表达水平很低。3.2 CaMKIIγ激酶与CML干细胞的存活密切相关,且促进LSC的自我更新应用特异性反义核酸下调K562细胞CaMKIIγ激酶表达水平,观察对细胞增殖的影响,结果显示,下调CaMKIIγ激酶表达水平后,白血病克隆形成能力被明显抑制。利用细胞转染技术上调K562细胞CaMKIIγ激酶表达水平,观察CD34+的干细胞数目,以及对克隆形成能力的影响。发现上调CaMKIIγ激酶表达水平可以增加CD34+的干细胞比例以及增强细胞克隆形成能力。3.3 CaMKIIγ激酶促进裸鼠CML异种移植瘤的成瘤能力将稳定转染pcDNA3.1-EGFP和pcDNA3.1-CaMKIIγ-EGFP的K562细胞分别接种至同一只裸鼠两侧皮下。通过比较两侧异种移植瘤的大小来观察上调CaMKIIγγ激酶水平对白血病细胞增殖能力的影响。结果显示CaMKIIγ激酶高表达可以增强K562细胞裸鼠异种移植瘤成瘤能力。且发现稳定转染CaMKIIγ的K562细胞裸鼠异种移植肿瘤中,干细胞样的原始肿瘤细胞数量显著高于对照组。3.4 CaMKIIγ激酶促进慢性粒细胞白血病干细胞存活,自我更新和凋亡抵抗的机制通过Western blot技术检测,CaMKIIγ激酶高表达对与LSC存活、自我更新和凋亡抵抗密切相关的重要信号分子蛋白β-catenin、GSK3β和Caspase-2等表达水平的影响。结果显示,稳定高表达CaMKIIγ的K562细胞中,p-GSK和β-catenin的蛋白表达水平显著升高,Caspase-2的表达水平明显下降。而且流式分选结果显示CML患者骨髓细胞中CD34+的CML干细胞内β-catenin蛋白高表达。提示CaMKIIγ激酶可能通过磷酸化GSK3导致其活性被抑制,进而解除GSK3对P-catenin的抑制作用,最终实现对Wnt/β-Catenin信号通路正调控,促进LSC存活和自我更新；另一方面可能通过抑制Caspase-2活性,使细胞产生凋亡抵抗。3.5 CaMKIIγ激酶可能是调控GSK3/Wnt/β-catenin和线粒体(?)Caspase-2信号通路的重要分子开关,且能与GSK3分子形成信号转导复合体本部分实验通过免疫共沉淀的方法,进一步鉴定及证实慢性粒细胞白血病干细胞内CaMKIIγ激酶主要信号转导复合体的核心组分。结果显示,应用CaMKIIγ激酶特异性抗体进行免疫共沉淀CaMKIIγ激酶相关信号转导复合体,用Westernblot技术验证出在该复合体中存在β-catenin, GSK3/β和Caspase-2蛋白。应用GSK3/β特异性抗体进行免疫共沉淀,并用Western blot技术验证出CaMKIIγ蛋白能和GSK3/γ结合。以上结果表明CaMKIIγ激酶可能是调控GSK3/Wnt/β-catenin和线粒体/Caspase-2信号通路的重要分子开关,且能与GSK3分子形成信号转导复合体。3.6 CaMKIIγ激酶抑制剂BBM对K562-R细胞中干细胞相关重要分子β-catenin表达水平的抑制作用采用Western blot技术检测BBM作为CaMKIIγ激酶抑制剂,是否可以抑制LSC中与存活,自我更新和凋亡抵抗密切相关的重要信号分子β-catenin蛋白表达水平。结果显示,BBM可以显著抑制慢性粒细胞白血病细胞K562-R的β-catenin蛋白表达水平。研究结论：1、小檗胺不仅杀灭裸鼠体内增殖活跃的人慢性粒细胞白血病细胞,还可能清除处在休眠状态的慢性粒细胞白血病干细胞,并且在治疗剂量下小檗胺对裸鼠没有明显的毒副作用。2. CaMKIIγ激酶是小檗胺抗慢性粒细胞白血病作用的靶分子。小檗胺能特异性结合CaMKIIγ激酶的ATP结合袋结构域,并抑制CaMKIIγ的磷酸化。3. CaMKIIγ激酶可能是调控GSK3/Wnt/p-catenin和线粒体/Caspase-2信号通路的重要分子开关。CaMKIIγ通过磷酸化GSK3导致其活性被抑制,进而解除GSK3对β-catenin的抑制作用,最终实现对Wnt/β-Catenin信号通路正调控,促进LSC存活和自我更新；另一方面可能通过抑制Caspase-2活性,使细胞产生凋亡抵抗。4、小檗胺可能通过直接结合于CaMKIIy激酶的ATP结合袋,从而抑制CaMKIIy激酶磷酸化,导致其活性下调,进而抑制GSK3/Wnt/β-Catenin信号通路,激活Caspase-2,使CML干细胞死亡。更多还原

【Abstract】 Background:Chronic myeloid leukemia (CML), which accounts for approximately 20% of all adult leukemias, is characterized by the presence of the Philadelphia (Ph) chromosome derived from the juxtaposition of Abelson TK gene (Abl) on chromosome 9 to the break point cluster region (Bcr) gene on chromosome 22. The resultant oncogenic Bcr-Abl kinase is essential for the growth of CML cells and has become an attractive target for treatment of Ph+ CML cases. Inhibition of Bcr-Abl with Abl tyrosine kinase inhibitors (TKIs). such as imatinib(IM), is highly effective in controlling but not curing the disease. This is largely due to the inability of these kinase inhibitors to kill leukemia stem cell (LSC) (also known as leukemia-initiating cell) responsible for tumor initiation, drug resistance and relapse in myeloid leukemia. Therefore, eradication of LSCs is required for developing curative therapies for CML, which relies on identification of specific targets in leukemia stem cells. A number of targets, such asβ-catenin, Hedgehog, NF-κB, have been shown to be involved in survival, self-renewal and apoptosis resistance of leukemia stem cells, but they also play similar roles in regulating normal hematopoietic stem cells(HSCs). Currently, it is very challenging to identify LSC-specific therapeutic targets involved in the key functional regulation of LSCs but not normal HSCs.Berbamine, a plant natural compound produced by Berberis amurensis, is a traditional Chinese medicine (TCM) that has been widely used to treat leucopenia and inflammation for decades in China. Our previous studies showed that berbamine and its derivatives potently inhibited the growth of imatinib-resistant CML cells but not normal hematopoietic cells. These data suggested that berbamine might have potential to target leukemia stem cells of chronic myeloid leukemia.Objects:1、To determine the in vivo anti-leukemia activity of the berbamine.2、To identify the specific protein target of Berbamine in leukemia, and understand the interaction between Berbamine and the specific target.3、To investigate the role of the identified target in survival, self-renewal and apoptosis resistance of LSCs, and eventually discover the molecular mechanism underlying the anti-leukemia activity of berbamine. Contents:1 The anti-chronic myeloid leukemia activity of the berbamine in vivo.To determine the in vivo anti-leukemia activity of the berbamine, we evaluated the effects of this compound on human CML cells K562 xenografts in nude mice by oral administration. We investigated the in vivo anti-leukemia activity of berbamine against IM sensitive-K562 (K562-S) leukemia cells in nude mice. The body weights in control and berbamine-treated groups were 16.69±2.92g and 17.61±1.70g, respectively. K562-R2 Berbamine could specifically interact with the CaMKII-y kinase in chronic myeloid leukemiaWe used berbamine as the bait to identify the putative targets that specifically regulate LSCs. The results shows that berbamine directly interacted with CaMKII y kinase in leukemia cells and inhibit its kinase activity. The phosphorylation level of CaMKII y decreased prominently in the CML cells with the berbamine treatment, while the total CaMKII y protein do not change. To gain an insight into the mechanism by which berbamine inhibited CaMKIIγactivity, we built a model of the CaMKIIγin complex with calmodulin by using Modeller based on the X-ray crystal of CaMKIIδ/calmodulin complex. And the result shows that Berbamine molecule is located in the ATP binding pocket of CaMKIIγkinase. 3 CaMKII y kinase regulates survival, self-renewal and apoptosis resistance of LSCs through GSK3/Wnt/p-catenin and mitochondrial/Caspase-2 pathways3.1 CaMKII y kinase is aberrantly activated in LSCs.To understand whether CaMKII y kinase expression is related with CML, we evaluated both total and phosphorylated CaMKII y protein levels in CD34+leukemia stem cells and normal hematopoietic stem cells. To detect CaMKII y levels in rare LSCs and HSCs, we sorted both CD34+leukemia stem cells and HSCs.We observed that CaMKII y kinase is aberrantly activated in LSCs.3.2 CaMKII y kinase is essential for survival of LSC and promotes self-renewal of LSCsWe evaluated the effect of CaMKII y expression inhibition on the growth of leukemia colonies using a colony forming assay, and observed that down-regulation of CaMKII y expression markedly inhibited the growth of CML cells in vitro, suggesting that down-regulation of CaMKII y expression diminishes the oncogenic potential of leukemia cells.To assess the effects of enhanced expression of CaMKII y on the self-renewal of tumor stem/progenitor cells of CML, we established a stable CML cell line with high CaMKII y expression using human CML K562 cells transfected with the CaMKII y-EGFP expression plasmid and G418 selection. We found that enhanced expression of CaMKII y led to an increase in the number of colonies. More importantly, the size of the colonies was larger in the cells stably transfected with CaMKII y-EGFP than that in the control. These results indicate that CaMKII y enhances the colony-forming ability and proliferative capacity of leukemia cells through promoting the survival and self-renewal of leukemia stem cells.3.3 CaMKIIγkinase promotes survival and self-renewal of LSCs in vivoWe investigated the proliferation and differentiation of enhanced CaMKIIγexpression leukemia cells in vivo. Enhanced CaMKIIγ-EGFP expression K562 leukemia cells were transplanted into nude mice and EGFP expression leukemia cells were used as the control. As expected, the size of K562 leukemia xenograft tumors with enhanced CaMKIIγ-EGFP expression was larger than that of control K562 xenograft tumors at day 15. Unexpectedly, morphologically undifferentiated stem-like blast cells that highly express CaMKIIγmarkedly increased in the tumor of enhanced CaMKIIγ-EGFP expression K562 xenografts as compared to control K562 xenografts.3.4 CaMKII y kinase regulates the expression level of phosphorylated GSK3-β,β-catenin and Caspase-2 protein.We examinedβ-catenin expression level, and found that enhanced expression of CaMKIIγkinase greatly upregulatedβ-catenin protein level as compared to control with a concomitant increase of phosphorylated GSK3-βprotein. These results suggest that CaMKIIγupregulatesβ-catenin protein via inhibiting phosphorylation of GSK-3/βresulting in survival and self-renewal of LSCs. We also observed that enhanced expression of CaMKIIγmarkedly reduced Caspase-2 protein level in leukemia cells. CaMKIIγmay also maintain apoptosis-resistance through inhibition of Caspase-2.3.5 CaMKIIγkinase is an important molecular to GSK3/Wnt/β-catenin and mitochondrial/Caspase-2 signaling way.To obtain biochemical evidence for this signaling axis, co-immunoprecipitation experiments were carried out. Leukemia cell lysates were incubated with CaMKIIγantibody or GSK3βantibody, and the immune complexes were then purified, separated by SDS-PAGE, and immunoblotted with GSK3βor CaMKIIγorβ-catenin antibody. We observed that GSK3p.β-catenin and Caspase-2 were present in the complex immunoprecipitated by the CaMKII y antibody. As expected, CaMKII y was also present in the complex in a reciprocal immunoprecipitate using GSK3βantibody. Neither CaMKII y nor GSK3P was detected in the immune complex associated with control IgG, validating the specificity of the observed co-association and the presence of CaMKII y/GSK3β/β-catenin signaling axis.3.6 CaMKII y inhibitor berbamine caused a decrease ofβ-catenin proteinP-catenin has been shown to be essential for survival and self-renewal of leukemia stem cells. We demonstrated that treatment of K562 leukemia cells with CaMKII y inhibitor berbamine caused a decrease of P-catenin protein.Conclusions:1、Berbamine eliminates both imatinib-sensitive and-resistant CML xenografts in nude mice. Berbamine showes significant retardation in tumor growth without obvious toxicity in all animal groups.2. Berbamine selectively interacts with CaMKII-y kinase in leukemia cells and directly targets the ATP binding site of CaMKII y kinase.3、CaMKII y kinase is an important molecular to GSK3/Wnt/p-catenin and mitochondrial/Caspase-2 signaling axis. CaMKIIγupregulatesβ-catenin via inhibitory phosphorylation of GSK-3/βof leukemia stem cells. CaMKIIγmay also maintain apoptosis-resistance through inhibitin of Caspase-2.4、Berbamine directly targets the ATP binding site of CaMKII y kinase and inhibits the expression of phosphorylated CaMKIIy kinase. Thereby. Berbamine inhibits Wnt/β-Catenin signaling pathway, activates Caspase-2, and eventually causes the death of leukemia stem cell.更多还原