细胞周期调节蛋白D3（CCND3）在多发性骨髓瘤（MM）中高度表达，并促进MM细胞增殖。在一定的细胞周期阶段，CCND3会迅速降解，这对于MM细胞周期进程和增殖的严格控制是必不可少的。在本研究中，我们研究了调节MM细胞中CCND3降解的分子机制。通过利用亲和纯化耦合串联质谱技术，在人类MM OPM2和KMS11细胞系中鉴定了去泛素化酶USP10与CCND3相互作用。此外，USP10特异性抑制CCND3的K48-链聚泛化和蛋白酶体降解，从而增强其活性。我们证明USP10的N-末端区域（aa. 1-205）对于结合和去泛素化CCND3是不必要的。尽管Thr283对CCND3活性很重要，但在USP10调控的CCND3泛素化和稳定性方面是不需要的。通过稳定CCND3，USP10激活了CCND3 / CDK4 / 6信号通路，磷酸化Rb，并在OPM2和KMS11细胞中上调了CDK4，CDK6和E2F-1。与这些发现一致的是，Spautin-1抑制USP10导致CCND3的K48-链聚泛化和降解的积累，与CDK4 / 6抑制剂Palbociclib协同诱导MM细胞凋亡。在携带有OPM2和KMS11细胞的骨髓瘤异种移植裸鼠中，Spautin-l和Palbociclib的联合给药几乎在30天内抑制了肿瘤生长。因此，本研究确定了USP10作为CCND3的第一个去泛素化酶，发现靶向USP10 / CCND3 / CDK4 / 6轴可能是治疗骨髓瘤的一种新模式。© 2023.作者（s）独家许可给中国科学院上海药物研究所和中国药理学会。

The cell cycle regulator cyclin D3 (CCND3) is highly expressed in multiple myeloma (MM) and it promotes MM cell proliferation. After a certain phase of cell cycle, CCND3 is rapidly degraded, which is essential for the strict control of MM cell cycle progress and proliferation. In the present study, we investigated the molecular mechanisms regulating CCND3 degradation in MM cells. By utilizing affinity purification-coupled tandem mass spectrometry, we identified the deubiquitinase USP10 interacting with CCND3 in human MM OPM2 and KMS11 cell lines. Furthermore, USP10 specifically prevented CCND3 from K48-linked polyubiquitination and proteasomal degradation, therefore enhancing its activity. We demonstrated that the N-terminal domain (aa. 1-205) of USP10 was dispensable for binding to and deubiquitinating CCND3. Although Thr283 was important for CCND3 activity, it was dispensable for CCND3 ubiquitination and stability modulated by USP10. By stabilizing CCND3, USP10 activated the CCND3/CDK4/6 signaling pathway, phosphorylated Rb, and upregulated CDK4, CDK6 and E2F-1 in OPM2 and KMS11 cells. Consistent with these findings, inhibition of USP10 by Spautin-1 resulted in accumulation of CCND3 with K48-linked polyubiquitination and degradation that synergized with Palbociclib, a CDK4/6 inhibitor, to induce MM cell apoptosis. In nude mice bearing myeloma xenografts with OPM2 and KMS11 cells, combined administration of Spautin-l and Palbociclib almost suppressed tumor growth within 30 days. This study thus identifies USP10 as the first deubiquitinase of CCND3 and also finds that targeting the USP10/CCND3/CDK4/6 axis may be a novel modality for the treatment of myeloma.© 2023. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.