State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China; Tianjin Institutes of Health Science, Tianjin, China.
State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China.
EBioMedicine. 2024 Feb;100:104961. doi: 10.1016/j.ebiom.2023.104961. Epub 2024 Jan 9.
Multiple myeloma (MM) is an incurable hematological malignancy of the plasma cells. The maintenance of protein homeostasis is critical for MM cell survival. Elevated levels of paraproteins in MM cells are cleared by proteasomes or lysosomes, which are independent but inter-connected with each other. Proteasome inhibitors (PIs) work as a backbone agent and successfully improved the outcome of patients; however, the increasing activity of autophagy suppresses the sensitivity to PIs treatment.
The transcription levels of CRIP1 were explored in plasma cells obtained from healthy donors, patients with newly diagnosed multiple myeloma (NDMM), and relapsed/refractory multiple myeloma (RRMM) using Gene expression omnibus datasets. Doxycycline-inducible CRIP1-shRNA and CRIP1 overexpressed MM cell lines were constructed to explore the role of CRIP1 in MM pathogenesis. Proliferation, invasion, migration, proteasome activity and autophagy were examined in MM cells with different CRIP1 levels. Co-immunoprecipitation (Co-IP) with Tandem affinity purification/Mass spectrum (TAP/MS) was performed to identify the binding proteins of CRIP1. The mouse xenograft model was used to determine the role of CRIP1 in the proliferation and drug-resistance of MM cells.
High CRIP1 expression was associated with unfavorable clinical outcomes in patients with MM and served as a biomarker for RRMM with shorter overall survival. In vitro and in vivo studies showed that CRIP1 plays a critical role in protein homeostasis via the dual regulation of the activities of proteasome and autophagy in MM cells. A combined analysis of RNA-seq, Co-IP and TAP/MS demonstrated that CRIP1 promotes proteasome inhibitors resistance in MM cells by simultaneously binding to de-ubiquitinase USP7 and proteasome coactivator PA200. CRIP1 promoted proteasome activity and autophagosome maturation by facilitating the dequbiquitination and stabilization of PA200.
Our findings clarified the pivotal roles of the CRIP1/USP7/PA200 complex in ubiquitin-dependent proteasome degradation and autophagy maturation involved in the pathogenesis of MM.
A full list of funding sources can be found in the acknowledgements section.
多发性骨髓瘤（MM）是一种不可治愈的浆细胞血液恶性肿瘤。蛋白质平衡的维持对 MM 细胞的存活至关重要。MM 细胞中异常升高的副蛋白可被蛋白酶体或溶酶体清除，二者独立但又相互关联。蛋白酶体抑制剂（PI）作为骨干药物发挥作用，成功改善了患者的预后；然而，自噬活性的增加会抑制对 PI 治疗的敏感性。
使用基因表达综合数据库（Gene expression omnibus datasets），在来自健康供体、初诊多发性骨髓瘤（NDMM）和复发/难治性多发性骨髓瘤（RRMM）患者的浆细胞中探索 CRIP1 的转录水平。构建了可诱导 CRIP1-shRNA 的 DOX 诱导型 MM 细胞系和 CRIP1 过表达 MM 细胞系，以探索 CRIP1 在 MM 发病机制中的作用。通过不同 CRIP1 水平的 MM 细胞系检测增殖、侵袭、迁移、蛋白酶体活性和自噬。通过串联亲和纯化/质谱（Tandem affinity purification/Mass spectrum，TAP/MS）进行共免疫沉淀（Co-immunoprecipitation，Co-IP），以鉴定 CRIP1 的结合蛋白。采用小鼠异种移植模型确定 CRIP1 在 MM 细胞增殖和耐药中的作用。
CRIP1 高表达与 MM 患者的不良临床结局相关，并作为总生存期更短的 RRMM 的生物标志物。体外和体内研究表明，CRIP1 通过双重调节 MM 细胞中蛋白酶体和自噬的活性，在蛋白质平衡中发挥关键作用。RNA-seq、Co-IP 和 TAP/MS 的综合分析表明，CRIP1 通过同时与去泛素化酶 USP7 和蛋白酶体共激活因子 PA200 结合，促进 MM 细胞对蛋白酶体抑制剂的耐药性。CRIP1 通过促进 PA200 的去泛素化和稳定，促进蛋白酶体活性和自噬体成熟。
我们的研究结果阐明了 CRIP1/USP7/PA200 复合物在泛素依赖性蛋白酶体降解和自噬成熟中的关键作用，这些过程涉及 MM 的发病机制。
完整的资金来源清单可在致谢部分找到。
