PRPS1基因突变介导儿童急性淋巴细胞白血病复发的分子机制研究

Benefiting from risk-directed treatment and improved supportive cares, considerable advances on treatment of childhood acute lymphoblastic leukemia (ALL) have been made in the past decades, and the long-time survival rates approaches 85%. However, disease recurrence is still unsatisfying even with risk-stratified intensive salvage therapies. It was reported that 15% to 20% children with ALL will have a relapse, in which approximately 85% of patients achieve a second remission and the cure rate after relapse were 30% to 40%. Therefore relapse is one of the most important factors on impeding the further improvement of long-term prognosis of childhood ALL. Even though, the relapse mechanism of childhood ALL is unknown. Based on the results of whole-exome sequencing on 16 trios of de novo, complete remission and relapsed childhood ALL samples, we found several non-synonymous mutations of phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1) and these mutations are all relapse-specific when comparing the capillary electrophoresis sequencing results of paried de novo and complete remission samples. PRPS1 is the rate-limiting enzyme in de novo synthesis of purine nucleotide within the cell and which is correlated with the chemotherapy effect of 6-thioguanine or 6-mercaptopurine used in the maintenance stage of childhood ALL.In this study, several experiments will be used to elucidate the relationship between PRPS1 mutation and the relapse of childhood ALL as well as the mechanism of PRPS1 mutation on childhood ALL relapse. In short, we will compare the results of minimal residule disease (MRD) and the high-deepth sequencing of bone marrow samples of patient with PRPS1 mutation and to establish more reliably relationship of PRPS1 point mutation and the relapse of childhood ALL. Furthermore, we will construct expression vector of each mutant and wild type PRPS1 protein and their enzymatic activity will be detected by high performance liquid chromatography (HPLC) method. Moreover, the conditional lentivirus based wild type and all kinds of mutant PRPS1 as well as RNA interference vector will be constructed and corresponding virus will be packaged. After that, the virus will be transfected to human ALL cell lines, such as REH, MOLT-4, Nalm-6 and Jurkat, and the chemotherapeutic agent (6-thioguanine or 6-mercaptopurine) sensitivities of each cell line will be determined by CCK-8 assay. In addition, the metabolism of 6-thioguanine or 6-mercaptopurine will be detected by mass spectrometry.Through these research, it will be help to clarify the clinical value of PRPS1 gene mutations in childhood ALL relapse and related molecular mechanism to a certain extent.Furthermore, this study will also help to establish the theoretical basis and experimental evidences for the recurrence mechanism and propose new relapse-specific treatment strategies of childhood ALL in the future.

复发是儿童急性淋巴细胞白血病(ALL)预后差的首因，相关机制欠明。PRPS1蛋白是细胞内嘌呤核苷酸从头合成途径的限速酶，其产物介导了6-MP和6-TG在细胞内的活性转化。前期研究中我们在16对儿童ALL的初发、缓解和复发样本的全外显子组测序中发现了PRPS1基因多个复发特异性错义变异，并经毛细管电泳测序证实。本项研究中：1）参考具有PRPS1突变的患儿治疗过程中微量残留病(MRD)检测结果，结合相同时间点突变位点的深度测序结果，建立PRPS1突变和儿童ALL复发之间的关系；2）体外表达PRPS1蛋白，直接对突变性蛋白的活性进行检测；3）制备PRPS1基因的RNA干扰慢病毒以及野生和不同突变型慢病毒，探讨PRPS1基因干扰和过表达前后细胞对6-MP和6-TG的敏感性变化以及细胞内药物的代谢变化；通过上述研究将阐明PRPS1突变致疾病复发的分子机制，以进一步阐明儿童白血病的复发机制。

复发是儿童急性淋巴细胞白血病(ALL)预后差的首因，相关机制未明。PRPS1蛋白是细胞内嘌呤核苷酸从头合成途径的限速酶，其产物介导了6-MP和6-TG在细胞内的活性转化。前期研究中我们在16对儿童ALL的初发、缓解和复发样本的全外显子组测序中发现了PRPS1基因多个复发特异性错义变异，并经毛细管电泳测序证实。本项研究中我们参考具有PRPS1突变的患儿治疗过程中微量残留病(MRD)检测结果，结合相同时间点突变位点的深度测序结果，建立了PRPS1突变和儿童ALL复发之间的关系；通过体外表达PRPS1蛋白，直接对突变性蛋白的活性进行了检测，进一步明确了PRPS1突变介导白血病复发的分子机制；通过制备PRPS1基因的RNA干扰慢病毒以及野生和不同突变型慢病毒，研究了PRPS1基因干扰和过表达前后细胞对6-MP和6-TG的敏感性变化以及细胞内药物的代谢变化。除此之外，我们还对105对急淋初发、缓解和复发样本的全基因组测序，获得了大量疾病复发的相关信息，对于深入挖掘PRPS1在导致儿童白血病复发中的作用机制有了深入的认识。我们还设计了针对主要克隆变异位点的高深度测序panel，针对诸如PRPS1、NT5C2、TP53、NR3C1等位点进行了高达10万倍的深度测序，揭示了PRPS1等诱导肿瘤细胞克隆演化的规律。并且设计了针对在上述样本中发现的所有突变位点的靶向捕获panel，针对10859种基因变异位点进行了超高深度测序，用于验证WGS结果。相关研究成果整理后参加了去年的AACR会议，并作为壁报形式展示。本课题对于揭示儿童白血病的复发机制具有重要的科学意义。