TRIP13通过NF-κB信号通路介导骨髓瘤耐药的分子机制研究

Resistance to conventional chemotherapeutics plays an important role in the death of multiple myeloma (MM) patients due to failure of treatment. TRIP13 is one of the key genes in regulating the drug resistance of MM. However, the exact mechanism of TRIP13 in regulating the drug resistance of this hematological malignancy remains unclear. We previously found that overexpression of TRIP13 induced drug resistance of MM. Additionally, DCZ0415, a small molecule compound specifically targeting TRIP13 which was first developed by our team, has been found to be capital of reversing drug resistance of MM. Mechanistically, our work has demonstrated that TRIP13 interacts with proteins involved in NF-κB signaling pathway. Additionally, NF-kappa B signaling pathway is involved in TRIP13-mediated MM drug resistance. Hereby, we hypothesize that TRIP13 may mediate drug resistance in MM through NF-κB signaling pathway and targeting the key regulatory targets of TRIP13 would be a novel strategy to reverse MM resistance. Based on this hypothesis, MM cell lines as well as MM animal models will be used to explore the role of TRIP13 in mediating MM resistance through NF-κB signaling pathway both in vitro and in vivo. Besides, methods and techniques, including ChIP-seq, Co-IP in combination with LC-MS/MS, gene editing et al, will be used to explore the molecular mechanism and the key targets of TRIP13 in the regulation of NF-κB signaling pathway, which will finally be confirmed by series of functional rescue experiments. Our work will provide new ideas for the targeted intervention therapy of relapsed and drug-resistant MM patients, which owns potential clinical application and transformation prospects.

细胞耐药在多发性骨髓瘤（MM）治疗失败导致死亡中起重要作用，TRIP13是调控MM耐药的关键基因之一，但其调控MM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。首个我们自主研发的DCZ0415靶向TRIP13能逆转MM细胞耐药。机制研究发现，TRIP13与NF-κB通路存在相互作用，NF-κB信号通路参与了TRIP13介导的MM耐药。据此假设：TRIP13通过NF-κB信号通路介导MM耐药，靶向TRIP13调控的关键靶点，能有效逆转耐药。为验证该假设，拟选取MM细胞和动物模型，通过体外和体内实验进一步验证TRIP13通过NF-κB通路介导MM耐药作用，结合ChIP-seq、Co-IP联合LC-MS/MS、基因编辑等技术，阐明其分子机制，并通过功能挽救实验验证TRIP13调控NF-κB通路的作用机理，为复发、耐药MM的靶向干预治疗提供新思路，具有潜在临床应用和转化前景。

耐药是多发性骨髓瘤（MM）治疗失败导致患者死亡的重要原因之一，TRIP13是调控MM耐药的关键基因之一，但具体机制仍不明确。我们前期研究发现：过表达TRIP13促进MM耐药，靶向TRIP13小分子化合物DCZ0415能有效逆转MM细胞耐药，且前期机制研究提示，TRIP13与NF-κB通路的关键分子存在相互作用。在此研究背景下，我们围绕TRIP13-NF-κB信号通路研究MM 耐药的具体机制。研究重要结果为：（1）通过免液相色谱-质谱/质谱（LC-MS/MS）检测，发现NF-κB信号通路相关蛋白（包括RPS3、IKKβ、NKRF等）可能与TRIP13有相互作用。进一步收集了MM细胞（ARP-1和OCI-MY5）的细胞沉淀，裂解获得蛋白液，通过内源性Co-IP和免疫共聚焦实验，证实TRIP13和RPS3、IKKβ有相互作用；（2）通过GEO网络公共数据库，下载和分析Affymetrix基因芯片检测的正常浆细胞、骨髓瘤患者MM细胞中RPS3 mRNA的数据（ GSE5900 和 GSE6477，RPS3对应的探针是208692_at），结果发现RPS3在原代骨髓瘤细胞中高表达；并在临床标本中验证出相比于健康人PBMC，MM患者中RPS3表达水平增高，结果表明NF-κB non-Rel亚基RPS3在骨髓瘤中高表达，并介导骨髓瘤的进展和耐药；（3）过表达TRIP13能够明显增强RPS3磷酸化水平，通过磷酸化蛋白质谱检测，发现TRIP13可能促进了RPS3 T221位点的磷酸化；（4）进一步研究证实：靶向TRIP13小分子化合物DCZ0415能够抑制万珂耐药MM细胞中NF-κB信号通路相关蛋白激活，并阻滞了NF-κB p65的核转移。这些研究结果为复发、耐药MM 患者的靶向治疗提供新思路，具有潜在临床应用和转化前景。. 本项目在基金委的资助下，已经发表标注本基金号的SCI论著3篇。本课题主要研究内容已经完成，论文目前投稿Haematologica，处于审稿状态。总之，项目进展顺利，待取得更多研究成果后，继续汇报。