The zinc finger ubiquitin ligase RNF6 has been proposed as a potential therapeutic target in several cancers, but understanding its molecular mechanism of degradation has been elusive. In the present study, we find that RNF6 is degraded via auto-ubiquitination in a manner dependent on its Really Interesting New Gene (RING) domain. We determine that when the RING domain is deleted (ΔRING) or the core cysteine residues in the zinc finger are mutated (C632S/C635S), the WT protein, but not the ΔRING or mutant RNF6 protein, undergoes polyubiquitination. We also identify USP7 as a deubiquitinase of RNF6 by tandem mass spectrometry. We show that USP7 interacts with RNF6 and abolishes its K48-linked polyubiquitination, thereby preventing its degradation. In contrast, we found a USP7-specific inhibitor promotes RNF6 polyubiquitination, degradation, and cell death. Furthermore, we demonstrate the anti-leukemic drug Nilotinib and anti-myeloma drug Panobinostat (LBH589) induce RNF6 K48-linked polyubiquitination and degradation in both multiple myeloma (MM) and leukemia cells. In agreement with our hypothesis on the mode of RNF6 degradation, we show these drugs promote RNF6 auto-ubiquitination in an in vitro ubiquitination system without other E3 ligases. Consistently, reexpression of RNF6 ablates drug-induced MM and leukemia cell apoptosis. Therefore, our results reveal that RNF6 is a RING E3 ligase that undergoes auto-ubiquitination, which could be abolished by USP7 and induced by anti-cancer drugs. We propose that chemical induction of RNF6 auto-ubiquitination and degradation could be a novel strategy for the treatment of hematological malignancies including MM and leukemia.

锌指泛素连接酶 RNF6 已被提议作为几种癌症的潜在治疗靶点，但了解其降解的分子机制一直难以捉摸。在本研究中，我们发现 RNF6通过以依赖于其真正有趣的新基因（RING）域的方式进行自动泛素化。我们确定当 RING 结构域被删除 (ΔRING) 或锌指中的核心半胱氨酸残基发生突变 (C632S/C635S) 时，WT 蛋白而不是 ΔRING 或突变 RNF6 蛋白会发生多泛素化。我们还通过串联质谱法将 USP7 鉴定为 RNF6 的去泛素酶。我们表明 USP7 与 RNF6 相互作用并消除其 K48 连接的多泛素化，从而防止其降解。相比之下，我们发现一种 USP7 特异性抑制剂可促进 RNF6 多泛素化、降解和细胞死亡。此外，我们证明了抗白血病药物尼罗替尼和抗骨髓瘤药物 Panobinostat (LBH589) 在多发性骨髓瘤 (MM) 和白血病细胞中诱导 RNF6 K48 连接的多泛素化和降解。没有其他 E3 连接酶的体外泛素化系统。一致地，RNF6 的重新表达消除了药物诱导的 MM 和白血病细胞凋亡。因此，我们的研究结果表明，RNF6 是一种 RING E3 连接酶，它经历自泛素化，可被 USP7 消除并由抗癌药物诱导。我们提出 RNF6 自身泛素化和降解的化学诱导可能是治疗包括 MM 和白血病在内的血液恶性肿瘤的新策略。