GPR183调控Notch1降解影响T-ALL进程的分子机制研究

Acute T lymphoblastic leukemia (T-ALL) is highly malignant and easily relapse among the children ALL. The new pathogenesis of T-ALL is much concerned. Our team has been focused on the critical role of β-Arrestin1 (Arrb1), the key signal scaffold protein, on different subtypes of leukemia in recent years. Supported by the previous NSFC fundings, we found that the inhibited Arrb1 expression regulated by miR223 in T-ALL could promote Notch1 degradation and slow down the progression of leukemia, suggesting that the classical G-protein coupled receptor (GPCR), which is combined with Arrb1, may participate in the process of T-ALL. Interestingly, our recent preliminary data showed that there were a lot of GPCR genes bearing missense mutations or abnormal expression of GPCR genes in the bone marrow cells from 16 T-ALL patients, compared with of 4 matched controls, by combined using full genome re-sequencing and transcriptional sequencing techniques. Among them, the expression of GPR183, which plays key roles in the hematological development and malignant cancers, was significantly decreased, though there were no mutations in the coding region and non-coding region of GPR183 nucleic acid sequence. However, the function of GPR183 is not illustrated. Further we applied 7α, 25 - dihydroxycholesterol (7α, 25-OHC), the active agonist of GPR183, to induce the cell apoptosis and inhibit the expression of MYC gene, the downstream gene of Notch1 signal, in T-ALL cells. Based on our previous studies, here, we continuously explore the molecular mechanisms of active GPR183 recruiting Arrb1, to form the complex with ICN1 and the key E3 ubiquitination ligase, to promote Notch1 ubiquitination and degradation, to suppress the downstream signals of Notch1, to promote T-ALL cell apoptosis, and thus to inhibit the disease process, by using the model of T-ALL in vitro and in vivo. This project is very important to uncover the leukemiagenesis and find potential novel drug targets for refractory leukemia.

急性T淋巴细胞白血病(T-ALL)是儿童白血病中恶性程度高与易复发亚型，其新型发病机制备受关注。课题组一直从事信号分子β-Arrestin1 (Arrb1)与白血病的研究，发现Arrb1参与不同亚型白血病进程，提示与其结合的经典G蛋白偶联受体(GPCR)可能参与T-ALL进程。近期预实验中利用重测序与转录组测序，发现T-ALL患者细胞内大量GPCRs突变或表达量明显改变。其中在造血系统发育及肿瘤中有重要功能的GPR183，在T-ALL中表达显著降低，应用GPR183激动剂抑制Notch1下游MYC基因表达，促进T-ALL细胞凋亡。本项目延续以往研究，利用体内外模型，深入探究活化的GPR183如何招募Arrb1，与ICN1和E3泛素化酶形成复合物，促进Notch1泛素化与降解，阻断其下游信号，促进细胞凋亡与抑制T-ALL进程的分子机制，为阐释难治性白血病发病机制、寻找药物新靶点有重要意义。

项目“GPR183调控Notch1降解影响T-ALL进程的分子机制研究（81870126）”围绕儿童白血病中恶性程度高与易复发亚型T-ALL中的多个关键G蛋白偶联受体（GPCR）进行系列而深入地研究。利用体内外T-ALL模型和生物学技术，我们的研究发现GPR183在T-ALL患者细胞与细胞株中呈低表达，激活GPR183或过表达GPR183可抑制T-ALL细胞增殖，主要通过抑制NOTCH1下游信号通路来完成；发现T-ALL患者磷酸鞘氨醇S1P通路基因异常富集，S1P降解酶SGPL1表达下降，S1P合成酶SPHK1/2表达升高，S1P受体中S1PR3表达显著增加，但其余S1PR受体（S1PR1、2、4、5）变化不大，利用S1PR3抑制剂和敲低S1PR3表达可促进体内外T-ALL细胞凋亡，抑制T-ALL进展；发现调控GPCR结合的BArr1的miR-652-5p在T-ALL表达升高，miR-652-5p与TIGAR结合，促进T-ALL细胞中ROS和耗氧率增加，胞外酸化率、丙酮酸及ATP产量降低，促进细胞周期阻滞，敲低miR-652与过表达TIGAR可明显抑制T-ALL体内进展。以上基于GPR183、S1PR3等GPCR在T-ALL中的系列研究为阐释难治性T-ALL的发病新机制、后续寻找药物新靶点奠定了良好的理论基础。