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。

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.