AICAr通过打破嘌呤-嘧啶平衡治疗PRPS1基因突变耐药复发ALL的机制研究

The mutation of purine metabolic enzyme PRPS1 leads to resistant to mercaptopurine in leukemia cells, and promotes the relapse of leukemia. The prognosis of patients with PRPS1 mutation is poor, and effective treatment is needed. Our previous study found that purine de novo synthesis pathway is significantly activated in the cells with PRPS1 mutation, and also found that the PRPS1 mutation cells are more sensitive to the small molecule AICAr than the wild type cells, but the molecular mechanism is unclear. We propose a scientific hypothesis: AICAr incorporates into purine de novo synthesis pathway, further increases purine nucleotides in PRPS1 mutation cells, at the same time inhibits pyrimidine synthesis, breaks the purine - pyrimidine nucleotides balance, then causes replication stress, the stalling of transcription machineray and increases transcription errors, finally inhibits cell proliferation. We will study how AICAr regulates the nucleotide metabolism by the method of metabonomics, and how AICAr affects transcription and replication by means of molecular biology research in PRPS1 mutant cell line models, to clarify the molecular mechanism of how AICAr inhibite cell proliferation. At the same time, we intend to verify the therapeutic effect of AICAr on PRPS1 mutant leukemia in the humanized mouse PDX model. These results will shed light on the new treatment of childhood leukemia.

嘌呤代谢酶PRPS1突变导致细胞对巯嘌呤类药物耐药，促进白血病耐药复发。PRPS1基因突变患者预后差，亟需有效的治疗方案。我们前期研究发现PRPS1突变显著激活细胞内嘌呤合成代谢通路，同时还发现PRPS1基因突变细胞比野生型细胞对小分子AICAr更敏感，但其分子机制尚不清楚。根据前期结果，我们提出科学假说：AICAr掺入嘌呤从头合成途径，与PRPS1突变协同作用，增加细胞内嘌呤含量，同时抑制嘧啶合成，打破嘌呤-嘧啶平衡，引起复制压力、转录停滞和转录错误，进而抑制细胞增殖。本项目将在PRPS1突变细胞系模型中，通过代谢组学方法研究AICAr对核苷酸代谢的调控，运用分子生物学手段研究AICAr对转录和复制的影响，以阐明AICAr抑制PRPS1突变细胞增殖的分子机制。同时本项目拟在小鼠人源化PDX模型中验证AICAr对PRPS1突变白血病的治疗效果。本项目研究结果将为儿童白血病的治疗提供新思路。

嘌呤代谢通路重编程在调控巯嘌呤耐药，促进白血病复发中发挥重要功能。PRPS1基因突变患者预后差，亟需有效的治疗方案。PRPS1突变显著激活细胞内嘌呤合成代谢通路，小分子AICAr也能刺激嘌呤合成代谢，同时抑制ALL细胞增殖，但其分子机制尚不清楚。我们通过CRISPR/Cas9敲除细胞，确定了AICAr抑制细胞增殖并不依赖于通过AMPK激活能量代谢这个经典功能，而是通过激活p53。靶向代谢质谱结果表明AICAr提升细胞内嘌呤合成代谢中间产物，导致ATP、dATP、dGTP含量上升，同时通过负反馈抑制PRPS活性，下调嘧啶合成，导致CTP、UTP、dCTP、dTTP含量下降。从而造成嘌呤嘧啶代谢不平衡，进而阻碍RNA转录和DNA复制，其后通过诱导复制压力和DNA双链断裂，引起细胞周期阻滞和细胞凋亡。本项目通过代谢组学方法研究AICAr对核苷酸代谢的调控，运用分子生物学手段研究AICAr对转录和复制的影响，阐明了AICAr抑制ALL细胞增殖的分子机制。本项目研究结果将为AICAr在儿童白血病治疗中的潜在作用提供新认识。