锌指蛋白ZFX维持CML干细胞生长的研究

Zinc finger gene ZFX has been identified as a key regulator of both embryonic stem cells (ESCs) and hematopoietic stem cells (HSCs) for their survival and maintenance of stemness. Several reports have demonstrated that Zfx is aberrantly expressed in various human tumors and implicated in their survival and growth, but its role in human hematological malignancies remains unclear. Chronic myeloid leukemia (CML) originates from normal HSCs acquiring BCR-ABL fusion gene. The CML stem cell is the key for the disease maintenance and relapse. However, the expression and function of ZFX in CML stem cells have not been addressed yet. In our study, we first found that ZFX was up-regulated in the CD34+ cells (including the quiescent subset) from CML patients in comparison to the normal control cells. ZFX silence inhibited the proliferation of leukemic CD34+ cells and impaired their colony-forming cell (CFC) capacities as well. Interestingly, ZFX silence sensitized CD34+ CML cells (both IM sensitive and insensitive samples) to the treatment of IM. We propose to assess the effects of ZFX silence or overexpression in CML stem cells (CD34+CD38-IL1RAP+) on their capacities of LTC-IC (long-term culture initiating-cell), IM sensitivity and xenoengraftment in NOD/SCID mice. In order to identify critical functional targets and regulatory network of ZFX in these cells, microarray, chromatin immunoprecipitate (ChIP) and electrophoretic mobility shift assay (EMSA) will be utilized. It would be the first report to delineate the role of ZFX in CML stem cells, which will deepen the understanding of CML pathology and potentially provide valuable clues for novel therapies against this disease.

ZFX（Zinc finger protein, X-linked）是维持多种干细胞生存和干性（stemness）的锌指蛋白。慢性髓细胞白血病（CML）干细胞是疾病维持和复发的重要因素。预研发现患者CD34+细胞（包括静息期亚群）中ZFX异常高表达；沉默ZFX抑制患者CD34+细胞生长并增强其伊马替尼（IM）敏感性。因此亟待研究ZFX在CML干细胞生长、干性维持及IM敏感性中的作用。本项目将检测沉默或过表达ZFX对CML干细胞（CD34+CD38-LI1RAP+）的长期培养起始细胞（LTC-IC）、免疫缺陷小鼠（NOD/SCID）移植能力和IM敏感性的影响；通过Microarray、染色质免疫共沉淀（ChIP）和凝胶迁移（EMSA）等鉴定ZFX的靶基因，并证实ZFX与其靶基因间的功能拮抗关系。将首次揭示ZFX在CML干细胞中的作用，增进CML致病机制的认识，有望为改善CML治疗提供新策略。

ZFX（Zinc finger protein, X-linked）是调控胚胎干细胞和造血干细胞自我更新能力的重要转录调节因子，维持多种干细胞的生存和“干性”，并参与多种肿瘤的病理过程。慢性髓细胞白血病（CML）是造血干细胞克隆性增殖形成的恶性血液肿瘤，目前临床治疗的一线药物伊马替尼（IM）尚无法治愈该疾病，可能因其无法有效清除CML干细胞。因此，在干细胞水平研究CML显得尤为重要。虽然，已有研究显示小鼠缺失zfx减缓急性T-细胞淋巴细胞白血病（T-ALL）和急性髓细胞白血病（AML）的发生，但ZFX在人类CML干/祖细胞中功能和机制的研究仍匮乏。. 本项目研究表明，与正常人骨髓相比，ZFX在CML慢性期患者CD34+细胞中高表达，在急变期患者CD34+细胞中表达进一步增加；ZFX沉默显著抑制CML患者CD34+细胞的集落生成能力（75±5%），显著高于ZFX沉默对正常人骨髓CD34+细胞的抑制率（44±5%）；ZFX沉默能增强IM耐药性CML患者CD34+的IM敏感性，并促进患者CD34+细胞凋亡。Microarray和qRT-PCR结果表明ZFX沉默抑制WNT3的表达水平；染色质免疫沉淀（ChIP）和荧光素酶（luciferase）报告基因等实验证实ZFX能结合于WNT3的启动子上并调控其活性；沉默WNT3或ZFX沉默在CML细胞中具有相同的功能表型；过表达WNT3能部分恢复ZFX沉默对CML细胞造成的集落抑制和IM敏感性；进一步研究表明ZFX沉默会抑制WNT3/β-catenin信号通路活性及其下游靶基因c-Myc和CCND1的表达。此外，我们发现IM处理导致ZFX蛋白表达下降，与Caspase的激活相关。. 综上，高表达的ZFX通过WNT3/β-catenin信号通路维持CML干/祖细胞的生长和耐药性，而活化的BCR-ABL可通抑制Caspase活性促进ZFX蛋白的稳定性。本研究不仅阐述了ZFX维持CML干/祖细胞生长的机制，而且初步描述了ZFX的上下游网络，将有助于深化对CML致病机制的认知，有望为靶向CML干细胞的新型治疗提供理论依据和实验基础。