Klf4在急性髓系白血病中的调控机制相关研究

Acute myeloid leukemia (AML) is an aggressive malignancy with poor prognosis. AML is a heterogeneously clonal disorder characterized by immature hematopoietic stem progenitor cells (HSPC) with an increase in the proliferation and an arrest in their maturation and apoptosis. The occurrence and development of AML is governed by both intrinsic and extrinsic factors. The transcription factors (TFs) play a significant role in regulating AML. However, because of the heterogeneity and complexity of the molecular network, how the TFs and relevant target controlling AML remains unclear. Kruppel-like factor 4 (Klf4) is a member of zinc finger transcription factors that play an important role in many fundamental biologic processes including proliferation, differentiation, apoptosis and reprogramming. Klf4 is a bi-functional factor that can either activate or suppress tumor by a cellular context-dependent manner. Our previous works provide a clue that Klf4 might participate in the development and outcome of AML. Therefore, we assume that Klf4 potentially serve as an attractive novel therapeutic target. Thus in this project, combining of overexpression and knock-out assay of an non-irradiation AML mouse model, we will study the impact of Klf4 on AML cell function. By means of gene expression profiling and bioinformatics, we will explore the underlying molecular mechanisms and then validate the function of Klf4 by compounds. The target will also be evaluated in clinical specimens. Our study will identify potential key regulators of AML and offer promise for a new therapeutic target for clinical application of AML.

AML是一类高度侵袭且预后不良的血液系统恶性疾病， 主要由于异常造血干祖细胞恶性克隆性增殖伴凋亡分化障碍而导致，其中转录因子发挥了重要的作用。然而恶性细胞如何利用转录因子及其相关靶基因目前善不明确。Klf4属于锌指结构家族转录因子，广泛参与调控细胞增殖，分化，凋亡和重编程等生物学行为。并以细胞依赖的方式，在恶性肿瘤中扮演着抑癌和促癌的双重作用。本项目组前期研究表明Klf4可能参与抑制AML疾病的发生与转归，因此，我们提出靶向Klf4，可能为AML诊治提供新的切入点。本研究以原代AML小鼠模型为基础，通过过表达和敲除双向实验，研究Klf4作为独立分子在AML中的调控作用；运用生物信息学数据，全面阐明Klf4调控AML的分子机制并明确相关下游靶基因及信号通路；结合公用数据库和临床资料，明确Klf4和靶基因与临床资料的关系，并运用相应小分子进行干预，为研发AML新的临床诊治靶点奠定进一步的基础。

诱导重编程的过程实际上是表观遗传重塑的过程，因此重编程模型为我们研究正常造血干细胞和白血病细胞的表观遗传调控提供了有效手段。我们运用二次重编程系统证明了重编程因子OSKM（Oct4, Sox2, c-Myc, Klf4）的瞬时过表达可选择性去除白血病细胞而对正常HSPC功能影响较小，并将此现象称为ERACE(effective reprogramming- apart cancer elimination)现象。OSKM的瞬时表达即可以有效诱导重编程早期的白血病细胞凋亡，并且有效抑制和逆转白血病多种急性白血病小鼠的生存期（MLL-AF9，MLL-NRIP3，NOTCH1）。通过组学分析发现与分化基因和多能基因“in situ”的组蛋白修饰研究不同，重编程诱导了白血病细胞染色质“global”的修饰改变。在重编程诱导的起始阶段组蛋白H3赖氨酸残基9位点2/3甲基化（H3K9me2/3）甲基转移酶 “writer” Suv39h1，Suv39h2，Setdb1均发生显著降低，另外其去甲基化酶Kdm3a发生显著升高，而直接抑制H3K9me2/3同样取得了延缓白血病进程的效果. 同时，为了检测抑制H3K9甲基化水平是否同样会抑制正常造血干祖细胞，我们同时将H3K9甲基转移酶抑制剂作用于小鼠原代正常HSPC。初步结果显示抑制H3K9甲基化水平一定程度影响正常HSPC增殖并诱导其凋亡，但其抑制作用较白血病细胞明显减轻。同时体外实验显示抑制H3K9甲基化水平显著抑制AML细胞并不明显影响正常HSPC细胞克隆形成能力。此外，抑制人白血病细胞系H3K9甲基化水平明显抑制其增殖能力。通过KEGG数据库初步分析，H3K9甲基转移酶SUV39H1、SETDB1下游抑制抑癌基因Trp53及Pten。上述研究表明重编程因子瞬时过表达通过抑制H3K9me2/3选择性去除了白血病细胞。