JAK1突变在急性淋巴细胞白血病中的功能研究

发布时间：2018-07-11 19:04

本文选题：急性淋巴细胞白血病 + 基因突变　；参考：《中国人民解放军军事医学科学院》2017年博士论文

【摘要】：急性淋巴细胞白血病(Acute lymphoblastic leukemia,ALL)是造血干细胞或淋巴前体细胞发育停滞引起的恶性克隆性疾病,遗传学异常在ALL的发病过程中扮演重要角色,不仅参与驱动疾病的发生,同时也与复发和疾病进展相关,可用于危险度分级和靶向治疗。ALL是儿童最常见的恶性肿瘤,儿童ALL对常规化疗反应敏感,预后较好,5年无病生存率可达到90%。由于随年龄增加预后不良的遗传学异常更多见,且成人对化疗的敏感性和治疗效果都远不如儿童,成人ALL预后仍较差,总体生存率仅为30%-40%,白血病相关并发症的发生率和复发风险也都相对较高。而可应用于ALL的靶向药十分有限,目前只有针对BCR-ABL1的酪氨酸激酶抑制剂。因此进一步了解ALL的遗传学特征,发现可能导致疾病发生或促进疾病进展和复发的新的遗传学变异,找到有效的新靶点和新的治疗手段,是目前ALL治疗研究的热点。在前期工作中,本课题组利用全外显子组测序的方法在4例成人B-ALL患者中检测到体细胞获得性JAK1突变,并在53例患者中进行验证,获得三个新的JAK1杂合性突变,分别为S646P,A639G和P960S。明确JAK1突变在ALL中的作用,对丰富ALL的发病机制和发现新的治疗靶点有着重要意义。JAK基因编码一种非受体型酪氨酸激酶(JAK,Janus Kinase),包括JAK1、JAK2、JAK3和TYK2四个成员,通过JAK/STAT通路传导细胞因子下游信号,调控靶基因的表达,参与细胞增殖、分化和免疫调控等过程。JAKs激酶在造血生成中也有重要作用,研究发现一半以上的骨髓增殖性肿瘤(Myeloproliferative neoplasms,MPNs)患者存在JAK2V617F突变,经体内外功能实验证实该突变是MPNs发生的一个关键因素,而且JAK1/2激酶抑制剂ruxolitinib已被FDA批准用于中、高危骨髓纤维化的治疗。在JAK2 V617F阴性的部分MPNs患者中也发现存在JAK2 12号外显子上的其他突变,提示JAK2异常是MPNs发生的重要机制和有效的治疗靶点。基于在ALL中的新发现,作为同家族成员的JAK1激酶是否也存在不可忽视的作用,是本研究关注的重点。为明确新发现的JAK1突变是否是ALL发生的一个重要机制,在本研究中重点对JAK1突变在ALL的发生频率和与预后的相关性,以及在体外和体内的恶性转化功能进行深入的探讨研究。首先,本课题组再次收集152例ALL患者骨髓DNA样本,由于前期发现的JAK1突变集中在14和21号外显子,针对这两个外显子设计特异的引物,利用PCR扩增联合sanger测序的方法,在2例患者中分别检测到jak1y652h和n973k突变。结合前期结果,在209例all患者中共检测到5例jak1点突变,2例为t-all,3例为b-all。其中,y652h和p960s在cosmic中已有报道,n973k为dbsnp数据库中已报道的snp(singlenucleotidepolymorphism),s646p和a639g为首次发现的突变位点,通过blast比对不同物种jak1蛋白氨基酸序列,发现5个突变所影响的残基在进化过程中具有高度保守性。总结jak1突变在all中发生率约为2.4%,其中在t-all发生率为10.5%,明显高于b-all中的发生率1.6%(p0.05)。回顾分析突变阳性患者的临床特征,观察到5例患者中4例(80%)出现了早期或晚期复发,且对化疗耐药,提示jak1突变可能与all的复发和耐药存在一定相关性。随后选择3个突变位点(a639g、s646p和p960s)进行体外功能实验验证,以已知的获得功能性突变jak1v658f作为阳性对照,构建插入野生型或突变型jak1的慢病毒表达载体plv-egfp(2a)puro,利用转染试剂将质粒转入hek293v细胞中,通过westernblot检测jak1突变对下游信号通路的影响,结果表明,jak1s646p、p960s和v658f突变能够组成性激活jak1自身和下游stat3、erk1/2信号分子的磷酸化。通过慢病毒包装、浓缩和纯化,感染目的细胞,构建稳定表达野生型或突变型jak1的baf3和nalm-6细胞系,利用cck-8检测细胞增殖和生长曲线,提示s646p和v658f突变能导致baf3细胞不依赖细胞因子生长,台盼蓝拒染结果提示s646p和v658f突变能抵抗撤去il-3引起的细胞凋亡,利用流式细胞术检测细胞周期发现,s646p突变组baf3细胞和nalm-6细胞s/g2期比例增加,促进细胞的增殖。以上细胞学实验结果证新发现的jak1s646p突变是一种获得功能性突变,与jak1v658f作用类似,能够导致细胞发生恶性转化和下游信号通路的过度活化。此外,在细胞学实验中观察了jak1突变对jak1/2激酶抑制剂ruxolitinib的敏感性。westernblot结果显示ruxolitinib能抑制jak1自身和stat3、erk1/2蛋白的磷酸化。用不同浓度的ruxolitinib处理稳定转染的baf3细胞,cck-8检测细胞增殖,结果提示ruxolitinib能够抑制野生型和突变型baf3细胞的增殖,而对携带bcr-abl1突变的k562细胞无影响,表达s646p和v658f突变的baf3细胞对ruxolitinib的敏感性高于野生型和其他突变组,提示jak1基因可能成为有效的治疗靶点。最后,为了研究表达s646p突变的baf3细胞在体内的成瘤作用,将稳定转染空载体,野生型JAK1和S646P突变的BaF3细胞通过尾静脉分别接种给三组Balb/c裸鼠。接种30天后,S646P组裸鼠外周血中白细胞计数和GFP阳性细胞比例明显高于空载体和野生型对照组,在裸鼠处死后,观察到S646P突变导致裸鼠肝、脾明显增大,骨髓中原始细胞和GFP阳性细胞比例增高,提示S646P突变作为一种获得功能性突变导致细胞发生恶性转化,促进其在体内发生类恶性肿瘤样浸润和侵袭。以上结果证实在本实验新发现的体细胞获得性JAK1 S646P突变是一种获得功能性突变,体外和体内实验中均证实存在恶性转化能力,可能影响疾病的进展和复发,而且对JAK1/2激酶抑制剂敏感,可能会成为一个新的治疗靶点。基于临床和基础研究的结果,将JAK1基因扩展至ALL患者分子学检测中将有助于预后评估和靶向治疗的发展,尤其是对于复发难治的患者,JAK抑制剂很可能会成为一种新的有效的治疗手段。
[Abstract]:Acute lymphoblastic leukemia (ALL) is a malignant clonogenic disease caused by stagnation of hematopoietic stem cells or lymphoid cells. Genetic abnormalities play an important role in the pathogenesis of ALL. They are not only involved in the development of the disease, but also related to the recurrence and disease progression, which can be used for risk classification. And targeted therapy.ALL is the most common malignant tumor in children. Children's ALL is sensitive to conventional chemotherapy and has a better prognosis. 5 years of disease free survival can reach 90%. due to the increase of genetic abnormalities with poor prognosis with age, and the sensitivity and treatment effect of adult to chemotherapy is far inferior to that of children, and the prognosis of adult ALL is still poor, and the overall survival of adult is still poor. The rate is only 30%-40%, the incidence of leukemia related complications and the risk of relapse are also relatively high. But the target drugs that can be used for ALL are very limited, and only BCR-ABL1 tyrosine kinase inhibitors are currently available. Therefore, further understanding of the genetic characteristics of ALL and the discovery of new remains that may lead to disease or to promote disease progression and recurrence In the previous work, we have detected the somatic cell acquired JAK1 mutations in 4 adult B-ALL patients by the method of exon sequencing in 4 cases of adult B-ALL, and obtained three new JAK1 heterozygosity mutations in the previous work. Do not define the role of JAK1 mutation in ALL for S646P, A639G and P960S.. It is important for the pathogenesis of ALL and the discovery of new therapeutic targets. The.JAK gene encodes a non receptor tyrosine kinase (JAK, Janus Kinase), including JAK1, JAK2, and four members, conducting downstream signals of cytokines through the pathway and regulating the downstream signals. The expression of target genes and the involvement of.JAKs kinase in cell proliferation, differentiation and immunoregulation also plays an important role in hematopoiesis. The study found that more than half of the Myeloproliferative neoplasms (MPNs) patients have JAK2V617F mutations. The mutation is a key cause of the occurrence of MPNs in vivo and in vivo. The JAK1/2 kinase inhibitor ruxolitinib has been approved by FDA for the use of FDA for the treatment of high-risk myelofibrosis. Other mutations in exons of JAK2 12 are found in the JAK2 V617F negative part of MPNs patients, suggesting that JAK2 abnormalities are an important mechanism for the occurrence of MPNs and an effective therapeutic target. Based on a new discovery in ALL, it is the same as the same Whether the JAK1 kinase of family members can not be ignored also is the focus of this study. In order to clarify whether the newly discovered JAK1 mutation is an important mechanism for the occurrence of ALL, this study focuses on the frequency of the JAK1 mutation in ALL and the correlation with the prognosis, as well as the malignant transformation function in vitro and in vivo. First, we once again collected 152 cases of ALL patients' bone marrow DNA samples. Due to the early detection of JAK1 mutations in 14 and 21 exons, specific primers were designed for the two exons and jak1y652h and n973k mutations were detected in 2 patients with PCR amplification and Sanger sequencing. In 209 patients with all, 5 cases of Jak1 point mutation were detected, 2 were T-ALL, 3 were b-all., y652h and p960s were reported in cosmic. N973k was the reported SNP (singlenucleotidepolymorphism) in the dbSNP database. The residues affected by the present 5 mutations have high conservatism in the evolution process. The incidence of Jak1 mutation in all is about 2.4%, and the incidence of T-ALL is 10.5%, obviously higher than that in B-ALL (P0.05). The clinical features of the mutant positive patients were reviewed, and 4 of the 5 patients (80%) had an early or late recurrence. The Jak1 mutation may be related to the recurrence and resistance of all. Then 3 mutation sites (a639g, s646p and p960s) were selected to perform in vitro functional test, and the known functional mutation jak1v658f was used as the positive control to construct the Lentivirus Expression Vector, which was inserted into the wild type or mutant Jak1 (plv-egfp). 2A) Puro, the plasmid was transferred into hek293v cells by using the transfection reagent, and the effect of Jak1 mutation on the downstream signal pathway was detected by Westernblot. The results showed that the jak1s646p, p960s and v658f mutations were capable of activating the Jak1 itself and the downstream STAT3, the phosphorylation of the erk1/2 signal molecules. The baf3 and Nalm-6 cell lines, which are stable expressing wild or mutant Jak1, are used to detect cell proliferation and growth curves by CCK-8, suggesting that s646p and v658f mutations can lead to baf3 cells not dependent on cytokine growth. Trypan blue staining results suggest that s646p and v658f mutations can resist apoptosis induced by withdrawal IL-3, using flow cytometry. The cell cycle test showed that the proportion of baf3 cells in s646p mutant group and s/g2 phase of Nalm-6 cells increased and promoted cell proliferation. The results of the above cytological test showed that the newly discovered jak1s646p mutation was a functional mutation, similar to jak1v658f, which could lead to the malignant transformation of cells and the over activation of downstream signal pathways. The sensitivity of the Jak1 mutation to the jak1/2 kinase inhibitor ruxolitinib was observed in the cytological experiment. The results showed that ruxolitinib could inhibit the Jak1 itself and the phosphorylation of the STAT3, erk1/2 protein. The stable transfected baf3 cells were treated with different concentrations of ruxolitinib, and the proliferation of the cells was tested CCK-8. The results suggest that ruxolitinib can inhibit the wild. The proliferation of type and mutant baf3 cells has no effect on the K562 cells carrying BCR-ABL1 mutation. The sensitivity of baf3 cells expressing s646p and v658f mutations to ruxolitinib is higher than that of the wild type and other mutation groups, suggesting that the Jak1 gene may be an effective target for treatment. Finally, to study the tumorigenesis of baf3 cells expressing s646p mutation in the body. After 30 days after inoculation, the proportion of white blood cells and GFP positive cells in the peripheral blood of S646P group nude mice was significantly higher than that of the empty and the wild type control group. After the death of the nude mice, the S646P mutation was observed to lead to the liver of nude mice after the death of the nude mice in nude mice. The BaF3 cells of the wild type JAK1 and the S646P mutant were inoculated to the nude mice respectively through the tail vein. The proportion of the spleen increased obviously, the proportion of the original cells in the bone marrow and GFP positive cells increased, suggesting that the S646P mutation, as a kind of functional mutation, resulted in malignant transformation of the cells and promoted the occurrence of malignant tumor like infiltration and invasion in the body. The results confirmed that the newly discovered somatic acquired JAK1 S646P mutation is a kind of acquisition in this experiment. Functional mutations, in vitro and in vivo, have confirmed the existence of malignant transformation ability, which may affect the progression and recurrence of the disease, and may be sensitive to JAK1/2 kinase inhibitors. It may become a new therapeutic target. Based on the results of clinical and basic studies, the extension of the JAK1 gene to ALL patients will contribute to the evaluation of the prognosis. And the development of targeted therapy, especially for relapsed and refractory patients, is likely to be a new and effective treatment for JAK inhibitors.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R733.71

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