TRIP13介导骨髓瘤发生、发展和耐药及其靶向干预的研究

Chromosomal instability (CIN) plays an important role in the pathogenesis and drug resistance of multiple myeloma (MM). Thyroid hormone receptor-interacting protein 13 (TRIP13) is one of the most key genes that regulate the CIN of MM. However, the explicit function of TRIP13 and its mechanism of regulating the pathogenesis and drug resistance of MM remain unclear. Previous studies in our laboratory first reported that overexpression of TRIP13 induced MM drug resistance while silencing of the gene inhibited MM cell growth in vitro and in vivo, suggesting that TRIP13 could be used as a novel target for MM-targeted therapy and reversing drug resistance. Our previous work has determined the crystal structure of human wild type TRIP13 protein for the first time and a small molecule compound, DCZ0415, which specifically targets TRIP13 has also been identified. Additionally, the compound which has been conferred the national patent of China is capital of inhibiting the growth of Velcade-resistant MM cells, suggesting the roles of DCZ0415 in killing of MM, reversing drug resistance and sensitizing chemotherapy. Based on subjects including primary human MM cells, MM cell lines, MM mouse models, and drug-resistant MM cells, further investigations including clinical samples and experiments both in vitro and in vivo will be carried out to validate the hypothesis of the anti-myeloma effects of DCZ0415 on drug-resistant MM. Additionally, gene editing, ChIP, Co-IP and other techniques will be applied to explore the related molecular mechanism as well as the downstream genes of TRIP13, which will finally be confirmed by series of functional rescue experiments. Our work will contribute to acquiring independent intellectual property rights of the first anti-MM small molecule compound targeting TRIP13, which provides a new idea for targeted intervention therapy of MM.

染色体不稳定性（CIN）在多发性骨髓瘤（MM）发生和耐药中起着重要作用，TRIP13是调控MM中CIN的关键基因，但其调控MM发生和耐药机制仍不清楚。我们前期研究发现：过表达TRIP13促进MM耐药，沉默TRIP13在体内外均抑制MM生长。我们也首次解析了人源野生型TRIP13蛋白晶体结构，并获得与TRIP13在体外有特异性结合的小分子化合物DCZ0415（获国家专利号），该化合物可有效抑制万珂耐药MM生长。据此我们推测：DCZ0415能有效抗MM、甚至逆转耐药和增敏化疗。为验证该假设，拟选取原代细胞、MM细胞株、动物模型和耐药细胞，通过体外、体内实验及临床样本进一步验证DCZ0415抗耐药MM效应，结合基因编辑、ChIP、Co-IP等技术探索相关分子机制，找到其下游基因，并通过功能挽救实验证实TRIP13的作用机理，以期获得有自主知识产权的抗MM先导药物，为MM靶向干预治疗提供新思路。

染色体不稳定性（CIN）在多发性骨髓瘤（MM）发生和耐药中起着重要作用，TRIP13是介导CIN的关键基因，但其调控MM发生和耐药机制仍不清楚。我们首次解析了人源野生型TRIP13蛋白晶体结构，并获得与TRIP13在体外有特异性结合的小分子化合物DCZ0415（获国家专利号），该化合物可有效抑制万珂耐药MM生长。本研究采用基因表达谱分析、CRISPR-Cas9基因编辑、流式细胞仪检测、双荧光素酶报告实验、染色质免疫共沉淀技术和裸鼠皮下移植瘤动物模型等实验方法，围绕 CIN 调控基因 TRIP13在MM发生、发展及耐药过程中的调控作用、分子机制及其干预策略进行深入的研究。 . 研究发现：（1）小分子化合物DCZ0415在体外对MM具有最为明显的抑制效果；进一步Pull-down以及核磁共振谱实验（NMR）证实小分子化合物DCZ0415能够结合TRIP13蛋白；表面等离子共振（SPR）测定显示DCZ0415和TRIP13亲和常数值为2.42±1.26mM；（2）DCZ0415通过靶向TRIP13抑制非同源末端连接及NF-κB活性而促进细胞凋亡、抑制细胞增值进而起到抗骨髓瘤的作用，并与传统抗MM药物马法兰（melphalan）及帕比司他产生联合致死作用；（3）TRIP13和NF-κB信号通路相关蛋白RPS3、IKKβ有相互作用，且通过PKCδ调控 NF-κB non-Rel亚基RPS3的磷酸化，并激活NF-κB信号通路；（4）TRIP13的异常过表达激活了去泛素化酶USP5的转录，并促进组蛋白甲基转移酶EZH2的去泛素化，从而介导MM干性并促进对BTZ的抵抗；（5）TRIP13抑制剂（DCZ0415）和EZH2抑制剂（GSK126）的联合治疗通过抑制体外和体内的骨髓瘤干性（MMSC）表型介导协同抗骨髓瘤效应。.本人以通讯作者发表标注本基金的SCI论文13篇，影响因子（IF）共101.841分，其中IF大于10分的4篇，IF在5-9分之间的5篇，IF小于3分的4篇。本基金第一标注的SCI论文3篇，IF共22.641分；本基金第二标注的SCI论文5篇；本基金第三标注的SCI论文2篇; 本基金第四标注的SCI论文3篇。申请并授权国家发明专利5项。研究成果荣获“优秀论文三等奖”。参加国内学术交流21次。培养博士研究生2名。