泛素连接酶RNF6调控慢性髓细胞白血病细胞增殖和存活的机制研究

Chronic myelogenous leukemia (CML) is a hematological malignancy derived from bone marrow stem cells and it is highly associated with dysregulated expression and overactivation of multiple oncogenes. As an important modification manner of protein stability and function, protein ubiquitination is widely involved in cell proliferation, growth and anti-apoptosis by conjugating the small molecule protein ubiquitin to specific substrate proteins. The process of protein ubiquitination requires several key enzymes including ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, and ubiquitin ligases, of which, ubiquitin ligases are able to interact with specific substrate proteins and mediate the final covalent linkage of the ubiquitin molecule. Our recent study revealed that the ubiquitin ligase RNF6 is overexpressed in CML cell lines and primary patients' samples. Overexpression of RNF6 increases CML cell proliferation and resistance to doxorubicin, in contrast, knockdown of RNF6 leads to decreased proliferation of CML cells and growth of a human CML xenograft in nude mice. However, the detailed mechanism is unclear. We hypothesize that, as an oncogenic ubiquitin ligase, RNF6 could modulate CML cell proliferation and survival via regulating the ubiquitination of its specific substrate proteins in CML cells. To confirm this hypothesis, the present proposal will apply the affinity purification-coupled tandem mass spectrometry (AP/MS/MS) to identify the ubiquitination substrates of RNF6 in combination with biochemical and molecular biological methods. The molecular mechanism in which RNF6 promotes CML cell proliferation and survival via ubiquitinating their substrates will be investigated using various cell biological methods. Their clinical significance will be evaluated in CML patients of chronic phase, accelerated phase and blast crisis. This study will provide novel insights in understanding CML pathophysiology and will also provide novel therapeutic stragetgy against CML.

慢性髓细胞白血病（CML）是一类原发于造血干细胞的恶性疾病，其发生发展与多个癌基因的异常活跃以及蛋白质泛素化修饰失调密切关联。泛素连接酶是介导蛋白质泛素化修饰的关键因子。我们最近的研究发现泛素连接酶RNF6在正常骨髓中低表达，但在CML患者骨髓细胞和CML细胞株中高表达。RNF6过表达促进CML细胞的增殖和对阿霉素耐药；而敲低RNF6则显著性抑制CML细胞的增殖并延缓CML小鼠体内移植瘤的生长，但具体机制未明。我们推测作为一个泛素连接酶RNF6可能结合并介导CML细胞中某些重要蛋白的泛素化而调控CML细胞的增殖和存活。为此，本项目中我们拟在前期研究的基础上采用亲和纯化偶联质谱学策略发现RNF6特异性泛素化底物，并结合分子和细胞生物学研究以及临床案例分析，阐明RNF6通过其泛素化底物促进CML细胞增殖和存活的作用机制，为深入了解CML的病理过程和发现潜在治疗靶标提供理论依据。

慢性髓细胞白血病（CML）是骨髓造血干细胞克隆性增殖形成的恶性肿瘤，其发生和恶性进展与癌基因的高度激活和蛋白质泛素化等翻译后修饰密切相关。我们在前期研究中发现泛素连接酶RNF6在正常骨髓细胞中不表达，但是在CML细胞中高表达，促进它们的增殖和存活，而敲低RNF6则抑制K562等CML细胞的存活和小鼠体内CML移植瘤的生长。此外，RNF6在AML和多发性骨髓瘤（MM）细胞中也是高表达，但是RNF6如何参与这些恶性血液病的病理学过程还不清楚。基于RNF6作为一个泛素连接酶的生物学特性，在本项目中，我们通过免疫亲和偶联质谱学策略分析了RNF6的相互作用蛋白质，并发现了多个潜在的RNF6泛素化底物，包括蛋白质合成延长因子（EF2）、波形蛋白（Vimetin, VIM）、糖皮质激素受体（GR）等。进一步从分子和细胞水平上证实了这些候选底物和RNF6间存在相互作用。不同于以往的发现是，RNF6以非典型泛素化的方式介导这些蛋白质发生多聚泛素化，包括GR的K63位多聚泛素化、VIM的K6和K27位泛素化等。通过这种泛素化方式，RNF6稳定其底物蛋白质水平，从而提升这些蛋白质的功能，促进CML和MM细胞的增殖。有趣的是还发现RNF6可以通过自身泛素化降解，并且从RNF6的相互作用蛋白质中发现了RNF6的去泛素化酶USP7。USP7使RNF6发生K48位连接的多聚泛素化，从而提高其稳定性，促进CML和MM细胞的增殖。并且发现抑制USP7或干扰RNF6后能促进相关底物蛋白的下降，从而诱导MM和CML细胞的凋亡。此外，我们发现已知的抗MM药物LBH589和抗CML药物Nilotinib也能促进RNF6发生自身泛素化降解，并与CML和MM的凋亡密切关联。因此，本项目深入发现了RNF6的多个多聚泛素化底物，并阐明了其作用机制，为开发RNF6为靶标的抗CML和MM药物提供了理论基础。