【作者】 吕晓东；

【导师】 宋永平；

【作者基本信息】 郑州大学 ， 病理与病理生理学， 2018， 博士

【摘要】 慢性粒细胞白血病(CML)是慢性白血病中最常见的一种类型,约占成人白血病的15%。在全球范围内,慢性粒细胞性白血病的发病率为1～2/10万,在中国约为0.36～1/10万,以中老年人为主。BCR-ABL1蛋白的小分子酪氨酸激酶抑制剂(TKI)-伊马替尼(IM)的出现,彻底改变了 CML的治疗方式,使CML患者每年的相关死亡率从10～20%降到了 1～2%,10年生存期也由10～20%提高到80～90%。TKI药物的不断出现,为维持CML患者的长期缓解状态带来了美好的前景。IM使CML从一种致命的恶性疾病变成了 一种慢性疾病。然而,约20～40%新诊断的CML慢性期患者,最终因为药物的不良反应或耐药需要更换治疗方式。耐药的主要原因可分为BCR-ABL1酪氨酸激酶依赖性耐药和非依赖性耐药,依赖性耐药主要与]00多种酪氨酸激酶区突变相关。而在非依赖性耐药中,虽然TKI药物成功抑制了 BCR-ABL1激酶的活性,但是旁路信号通路的激活,弥补了酪氨酸激酶活性的损失,可促使CML细胞持续增殖,凋亡下调,最终导致TKI耐药。然而,引起BCR-ABL1非依赖性耐药的相关机制尚未明确。鉴于上述原因,本研究针对CML患者的BCR-ABL1酪氨酸激酶非依赖性耐药,通过临床病例信息、骨髓/外周血样本检测和体外细胞模型构建等方式,利用高通量测序以及CRISPR/Cas9基因编辑等实验技术,初步探讨了 BCR-ABL1非依赖性耐药相关的异常基因、信号通路及耐药机制。第一部分慢性粒细胞白血病早期分子学反应与伊马替尼耐药的临床研究目的探讨CML-CP患者早期分子学反应与IM耐药之间的关系。方法1.采用Sanger法测序,检测IM耐药患者的BCR-ABL1激酶区突变情况,明确其耐药类型;2.分析早期分子学反应与IM治疗、耐药的关系;3.统计学处理:年龄和时间等变量采用中位数描述,不同组间分类资料的比较采用χ2检验,两样本定量资料的比较采用方差齐性检验和独立样本t检验,P<0.05为差异具有统计学意义,采用SPSS 20.0进行统计分析。结果1.198例未检出激酶区突变的IM耐药患者,早期服用羟基脲、马利兰或干扰素作为初始治疗药物的比例较高(43%),且非IM药物使用时间越长,后期出现无激酶区突变的耐药患者比例越高;2.初始使用非1M药物的时间越长,患者BCR-ABL13mon>10%的风险越高,IM耐药时无激酶区突变的比例越高;3.当BCR—ABL13mon≤10%,BCR-ABL16mon>1%的患者中,耐药发生时检出激酶区突变的比例较高。结论1.CML患者IM治疗前,干扰素和羟基脲、马利兰等细胞毒药物的使用,可能会造成患者的BCR-ABL1酪氨酸激酶非依赖性耐药;2.BCR-ABL1酪氨酸激酶非依赖性耐药,可能会导致CML患者3个月时的分子学反应较差,但不影响IM出现耐药的时间:3.CML患者6个月时BCR-ABLl水平升高,提示可能出现BCR-ABL1激酶区突变导致的继发耐药。第二部分高通量测序技术筛选慢性粒细胞白血病BCR-ABL1非依赖耐药相关基因目的高通量测序技术筛选CML患者中BCR-ABL1酪氨酸激酶非依赖性耐药相关的异常基因。方法1.采用高通量测序技术,以口腔粘膜为对照,筛选BCR-ABL1非依赖性耐药相关基因;2.采用Sanger法测序,验证347例CML耐药患者中相关基因突变频率;3.检测相关基因表达水平,进一步分析可能存在的耐药原因;4.统计学处理:数据用均数±标准差描述,不同组间分类资料的比较采用χ2检验,两样本定量资料的比较采用方差齐性检验和独立样本t检验,P<0.05为差异具有统计学意义,采用SPSS 20.0进行统计分析。结果1.19例无BCR-ABL1激酶区突变的IM耐药患者,采用高通量测序技术筛查578个肿瘤相关基因,筛选结果提示,KMT2CK339N和RUNX1 H214P可能与BCR-ABL1非依赖性耐药相关;2.无激酶区突变的198例CMLIM耐药患者中,检出25例KMT2CK339N突变,其突变频率明显高于携带激酶区突变的耐药患者,而RUNX1 H214P在二组间的突变频率较低,且无明显差异;3.KMT2C mRNA表达水平与IM耐药类型及KMT2C K339N突变无相关性。结论1.KMT2C K339N不伴随KMT2C mRNA表达水平的改变,提示K339N点突变可能造成KMT2C蛋白功能学上的异常;2.KMT2C K339N可能激活相关旁路信号通路,导致CML患者BCR-ABL1酪氨酸激酶非依赖性耐药。第三部分KMT2C调控H3K4单甲基化与慢性粒细胞白血病BCR-ABL1非依赖耐药的机制研究目的进一步明确KMT2CK339N点突变与BCR-ABL1酪氨酸激酶非依赖性耐药的关系及机制。方法1.采用Western Blot法,分析在CML中,KMT2CK339N与不同H3K4甲基化修饰间的关系;2.通过 Real-time RT-PCR 和 Western Blot 法,分析 KMT2C K339N 与PI3K/AKT信号通路间的关系;3.统计学处理:数据用均数±标准差描述,两样本定量资料的比较采用方差齐性检验和独立样本t检验,P<0.05为差异具有统计学意义,采用SPSS 20.0进行统计分析。结果1.H3K4甲基化水平分析,MT2CK339N患者的H3K4mel水平明显低于KMT2CWT患者,而H3K4me2和H3K4me3在二者间均没有明显差异;2.KMT2CK339N患者中PTEN和P21的水平均明显下降,AKT Thr308、AKT Ser473 和 XIAP 的水平明显升高,而 PI3K p110α AKT、BCL-2、BAX 和 P27的表达水平均与KMT2CWT患者没有明显差异。结论1.KMT2C K339N可能通过下调H3K4mel水平来激活旁路信号通路,引起BCR-ABL1酪氨酸激酶非依赖性耐药;2.H3K4mel下调可能通过抑制PTEN表达,进而磷酸化AKT,调节细胞周期抑制蛋白P21和凋亡抑制蛋白XIAP的表达水平,最终影响CML细胞的非依赖性耐药。第四部分CRISPR/Cas9构建KMT2CK339N K562细胞模型及相关机制研究目的构建KMT2CK339N K562细胞模型,进一步明确相关机制。方法1.采用CRISPR/Cas9结合ssODN,构建KMT2CK339N K562细胞模型;2.使用CCK8细胞增殖实验、EdU细胞增殖实验和甲基纤维素克隆形成实验,进一步明确KMT2CK339N K562的细胞增殖情况;3.使用Annexin V-FITC/PI双染细胞凋亡实验,进一步明确KMT2CK339N K562的细胞凋亡情况;4.采用 Real-time RT-PCR 和 Western Blot 法,进一步明确KMT2C K339N突变与H3K4单甲基化及P13K/AKT信号通路的关系;5.统计学处理:数据用均数±标准差描述,两样本定量资料的比较采用方差齐性检验和独立样本t检验,P<0.05为差异具有统计学意义,采用SPSS 20.0进行统计分析。结果1.使用CRISPR/Cas9,结合ssODN,将K562细胞定点敲入KMT2CK339N点突变,构建KMT2CK339N K562细胞模型;2.KMT2CK339NK562细胞增殖能力提高,而凋亡水平下降;3.KMT2CK339N K562细胞的H3K4的单甲基化水平下降,而PTEN、P21、BCL-2、BAX 和 P27 的水平均明显下降,AKTThr308、AKT Ser473 和XIAP 的水平明显升高,而PI3K p110α和AKT的表达水平无明显变化。结论KMT2C K339N突变可影响自身组蛋白甲基转移酶功能,下调H3K4me1水平,进而抑制P13K/AKT信号通路中负性调节因子PTEN的表达,上调AKT的磷酸化水平,影响CML细胞的凋亡和增殖,最终导致CML患者的BCR-ABL1酪氨酸激酶非依赖性耐药。更多还原

【Abstract】 Chronic Myeloid Leukemia(CML)is accounting for about 15%of adult leukemia,which is the most common type of chronic leukemia.It can occur at any age and the incidence of the disease increases with age.In the world,the CML incidence is 1～2 cases per 100,000,about 0.36-1 cases per 100,000 in China,and mainly in the elderly.The presence of BCR-ABL1 tyrosine kinase inhibitor(TKI)-imatinib(IM)has completely changed the way of CML treatment,so that the annual mortality rate of CML patients has decreased from 10~20%to 1～2%,and the 10 year survival time has also increased from 10～20%to 80-90%.The continuous emergence of TKI drugs has brought good prospects for the maintenance of long-term remission in CML.IM has transformed CML from a deadly malignant disease to a chronic disease.However,about 20～40%newly diagnosed CML patients in chronic phase,will eventually need to change the treatment because of adverse drug reactions or drug resistance.The main causes of drug resistance can be divided into BCR-ABL1-dependent resistance and BCR-ABL1-independent resistance.Dependent resistance is mainly associated with more than 100 ABL kinase region mutations.In the Independent resistance,although TKI successfully inhibit the activity of BCR-ABL1 kinase,the activation of the accessory signal pathway can make up for the loss of tyrosine kinase activity,to promote the prolifeation of CML cells,leading to TKI resistance.However,less related studies have been made for independent resistance,and its mechanism is still not clear.For the reasons above,this study aimed at to explore the BCR-ABL1-independent resistance in CML.By means of high-througput sequencing and CRISPR/Cas9,we used clinical information,bone marrow/peripheral blood detection and in vitro cell model construction to preliminarily discuss the signaling pathways and related mechanisms.Part I:Clinical study of early molecular response with imatinib resistance in chronic myeloid leukemiaAim:To explore the relationship between the early molecular response and imatinib resistance in CML-CP patients.Methods:1.Sanger sequencing was used to detect the mutation of BCR-ABL1 kinase domain in patients with IM resistance,and to identify the causes of IM resistance.2.Analyzing the relationship between the early molecular response and IM resistance.3.Statistical analysis:Age and time were described as the media.Chi square test was used to analyze the difference between the groups.The difference of parameters between groups was analyzed by homogeneity of variance and t test.P value of 0.05 was Lused to determine statistical significance.SPSS20.0 statistical software was used for statistical analysis.Results:1.In 198 IM-resistant patients without mutation,the proportion of patients with taking hydroxyurea,melphalan or interferon as initial therapy was higher(43%).And along with the non IM drugs use,the proportion of IM-resistant patients with no kinase domain mutation was higher.2.The longer the initial use of non IM drugs.the higher risk for the patients with BCR-ABL13mon>10%.and the higher proportion of patients with no mutation in kinase domain.3.The possibility of detecting kinase region mutations was higher in 34 patients with BCR-ABL13mon≤10%and BCR-ABL16mon>1%.Conclusions:1.CML patients initial treatment with interferon,hydroxyurea and melphalan,may cause the BCR-ABL1-independent resistance.2.BCR-ABL1-independent resistance may cause poor molecular response at 3 months.but not affect the occurrence time of IM resistance.3.Increasing BCR-ABL1 levels at 6 months is suggested the possibility of secondary resistance due to mutation in BCR-ABL1 kinase region.Part II:Screening of genes related with BCR-ABL1-independent resistance in chronic myeloid leukemia by high-throughput sequencingAim:To explore genetic abnormality related with BCR-ABL1-independent resistance by Ihigh-throughput sequencing.Methods:1.Genes related with BCR-ABL1-independent resistance were screened with oral mucosa as the control and using high-throughput sequencing.2.Sanger sequencing was used to verify screened gene mutations in 347 CML patients with IM resistance.3.Analyzing the expression level of screened genes to further analyze the possible causes of BCR-ABL1-independent resistance.4.Statistical anzalysis:Data were described as mean±standard deviation.Chi square test was used to analyze the difference between the groups.The difference of parameters between groups was analyzed by homogneity of variance and t test.P value of 0.05 was used to determine statistical significance.SPSS20.0 statistical software was used for statistical analysis.Results:1.19 IM-resistant CML patients with no mutations in kinase domain were screened for 578 tumor related genes by high-throughput sequencing.The results showed that KMT2C K339N and RUNX1 H214P might lead to dysfunction of related genes after screening for multiple conditions.2.In 198 IM-resistant CML.patients with no mutations.25 cases with KMT2C K339N were detected.The frequency of KMT2C K339N was significantly higher than that with mutation in kinase domain.While the mutation frequency of RUNX1 H214P between the two groups was not significantly different.3.There was no correlation between the expression level of KMT2C mRNA and BCR-ABLl-independent resistance.Conclusions:1.KMT2C K339N do not change with the expression level of KMT2C mRNA.This showes that KMT2C K339N may cause the abnormal function of KMT2C protein.2.KMT2C K339N activates related signaling pathways leading to BCR-ABL1-independent resistance in CML patients.Part III:To analyzing the relationship between H3K4 monomethylation regulated by KMT2C and BCR-ABL1-independent resistance in chronic myeloid leukemia Aim:To further clarify the relationship and mechanism between KMT2C K339N and BCR-ABL1-independent resistance.Methods:1.The relationship between KMT2C K339N and the level of H3K4 methylation was analyzed by Western Blot.2.The relationship between KMT2C K339N and PI3K/AKT signaling pathways was analyzed by Real-time RT-PCR and Western Blot.3.Statistical analysis:Data were described as mean1standard deviation.The difference of parameters between groups was analyzed by homogeneity of variance and t test.P value of 0.05 was used to determine statistical significance.SPSS20.0 statistical software was used for statistical analysis.Results:1.The level of H3K4 methylation analysis showed that the level of H3K4mel in KAMT2CK39N patients was significantly lower than that in KMT2CWT patients,and there was no significant difference between the H3K4me2 and H3K4me3 in the two groups.2.The levels of PTEN and P21 in KMT2CK339N patients were significantly decreased,and the levels of AKT Thr308.AKT Selr473 and XIAP increased significantly,while the expression levels of PI3K p110α,AKT,BCL-2,BAX and P27 showed not significant difference.Conclusions:1.KMT2C K339N activates the signal pathway by regulating the level of H3K4mel,causing BCR-ABL1-independent resistance.2.The downregulation of H3K4mel decreases PTEN expression,and then phosphorylates AKT,regulates the expression level of’ P21 and XIAP,and ultimately leads to BCR-ABL1-independent resistance.Part IV:Construction of KMT2CK339N K562 cell model by CRISPR/Cas9 and related mechanism study Aim:To construct the KMT2CK339N K562 cell model and further clarify the related mechanism.Methods:1.KMT2CK339N K562 cell model was constructed by CRISPR/Cas9 and ssODN.2.Ceel proliferation ability of KMT2CK339N K562 was further clarified by using CCK8 cell proliferation test,EdU cell proliferation test and methyl cellulose clone formation test.3.The apoptosis situation of KMT2CK339N K562 was further clarified by using the apoptosis detection of Annexin V-FITC/PI double staining cells.4.Real-time RT=PCR and Western Blot were used to further clarify the relationship among KMT2C K339N.H3K4 methylation and PI3K/AKT pathway.5.Statistical analysis:Data were described as mean±standard deviation.The difference of parameters between groups was analyzed by homogeneity of variance and t test.P value of 0.05 was used to determine statistical significance.SPSS20.0 statistical software was used for statistical analysis.Results:1.K562 cell was constructed as KMT2CK339N K562 cell model by Lusiing CRISPR/Cas9 and ssODN.2.The proliferation ability of KMT2Ck339N K562 cells increased and the level of apoptosis decreased.3.The level of H3K4 monomethylation in KMT2CAK339N K562 cells decreased,while the levels of PTEN,P21,BCL-2,BAX and P27 decreased significantly,and the levels of AKT Thr308,AKT Ser473.and X:IAP increased significantly,while the expression levels of PI3K p110α and AKT were not changed significantly.Conclusions:KMT2C K339N inhibits PTEN expression through down regulating H3K4mel level,and then activates PI3K/AKT signaling pathway,which affects CML cell apoptosis and proliferation,and finally leads to BCR-ABL1-independent resistance.更多还原