PRPS2突变对儿童急性淋巴细胞白血病耐药复发的作用机制研究

Drug resistance in childhood acute lymphoblastic leukemia (ALL) is the major cause of treatment failure. However, molecular mechanisms of drug resistance are still elusive. Using whole-exome sequencing, we identify PRPS2 (phosphoribosyl pyrophosphate synthetase 2) mutations only in relapsed childhood ALL, and patients with PRPS2 mutations have a shorter ALL relapse time. We further have found that PRPS2 (phosphoribosyl pyrophosphate synthetase 2) mutants also show drug resistance with a different way from PRPS1 (Nature Medicine 2015), suggesting that PRPS2 is also critical for drug resistance in ALL. We also found that the three amino acid (V103-G104-E105) loop of PRPS2 which is the critical different from PRPS1. Based on our primary results, we will study the mechanism by PRPS2 mutations and its binding/association proteins resist drugs in vitro cell experiments. We will purify PRPS2 and PRPS1 and analyze their enzyme activities using a Kinase-Glo luminescent kinase assay kit, as well as the role of polymerization state and drug resistance. Our work will provide theoretical and experimental basis for system analysis of purine pathways involved in resistance mechanisms in ALL, and will lead to initial accurate treatment for ALL children patients with PRPS mutation-induced relapse.

耐药复发是儿童急性淋巴细胞白血病（ALL）治疗失败而死亡的主要原因，目前对其机制仍知之甚少，因此缺乏有效的治疗手段。前期我们通过全外显子组测序发现嘌呤代谢通路中磷酸核糖焦磷酸合成酶2（PRPS2）发生复发特异性突变，调控了ALL的耐药复发。初步实验表明PRPS2特别的耐药机制与同源同工酶PRPS1明显不同，进一步研究发现PRPS2和PRPS1的一级蛋白序列上最大区别的103-105位的3个氨基酸（3AA）对这两个酶的功能起重要作用。本项目拟在白血病细胞系上研究PRPS2突变的耐药机制，并与PRPS1进行异同分析；在蛋白水平检测突变对酶的活性以及聚合状态的影响，并建立动物模型研究PRPS2突变的耐药作用。从而系统阐明PRPS2突变的耐药机制以及与PRPS1的互作调控关系。本项目将进一步阐明嘌呤代谢通路突变引起儿童ALL耐药复发的作用机制，为药物开发和临床治疗提供科学依据。

肿瘤复发是儿童急性淋巴细胞白血病(ALL)治疗失败的主要原因，但其机制尚不明确。在本项目中，我们证明了磷酸核糖焦磷酸合成酶2 (PRPS2)的突变通过影响PRPS1/2六聚体的稳定性驱动ALL复发。前期通过对急性淋巴细胞白血病样本的超深度测序确定了PRPS2的突变，我们进而评估了PRPS2突变对细胞增殖生长、细胞凋亡和耐药性的影响。进行了体外PRPS2酶活性和ADP/GDP反馈抑制PRPS酶活性的检测。利用超高效液相色谱串联质谱(UPLC-MS /MS)分析了嘌呤代谢物。结合测序数据和临床信息，我们发现仅在接受硫嘌呤治疗的复发性儿童ALL患者中出现PRPS2突变。最终我们证明了功能性PRPS2突变通过影响PRPS1/2六聚体的稳定性，特异性介导嘌呤代谢，从而降低核苷酸反馈抑制PRPS活性，从而增强对6-巯基嘌呤的耐药性。PRPS1和PRPS2的关键区别V103-G104-E105插入PRPS1后，PRPS六聚体的界面发生了严重的空间碰撞，导致其酶活性降低。此外，我们还证实了PRPS2 P173R在异种移植物小鼠模型中增加了巯基嘌呤耐药性。本项目的研究结果阐明了PRPS2突变驱动儿童ALL耐药复发的作用新机制，并发现PRPS2突变可作为儿童ALL复发的生物标志物。