慢性髓性白血病微泡多重定向调控造血干/祖细胞促疾病进展

Chronic myeloid leukemia (CML) is a clonal malignant disorder of a pluripotent hematopoietic stem cell characterized by the presence of the reciprocal translocation t(9;22)(q34;q11), which generates the Philadelphia (Ph) chromosome. CML represents an important paradigm for understanding the molecular evolution of cancer because it was the first cancer shown to be initiated at the hematopoietic stem cell level by BCR-ABL1; and the first cancer found to undergo blastic transformation. Notwithstanding the remarkable success in treating patients in CML-CP with ABL1 kinase inhibitors such as imatinib, the dismal outcome of blast crisis (CML-BC) patients underscores the need for a better understanding of the mechanisms responsible for the disease progression. Besides, diagnostic and therapeutic strategies that predict and prevent CML progression represent compelling unmet medical needs. It has been reported that during myelogenous blast crisis, abnormal granulocyte macrophage progenitors (GMP) acquired self-renewal potential and function as leukemic stem cells; and the acute lymphoblastic leukemia (ALL)-initiating cells may result from B-lymphoid progenitors. However, the precise molecular mechanisms driving malignant reprogramming of progenitors into LSC in BC CML have remained elusive. Microvesicles (MV), also termed to as microparticles, have been described as a new and important mechanism of intercellular signal communication via fusing with the plasma membrane of the target cell and discharging their cargo into the cytosol. By facilitating the horizontal transfer of bioactive molecules such as proteins, RNAs and microRNAs, MVs are now thought to have vital roles in tumor invasion and metastases, inflammation, coagulation, stem-cell renewal and transformation of healthy cells. We have demonstrated that BCR-ABL1-positive MVs could initiate malignant transformation of hematopoietic stem/progenitor cells (Leukemia, 2014, doi:10.1038/leu.2014.51). The aim of this subject is to investigate whether MV plays a role in the transformation of blast crisis. In this subject, clinical observation and mice models will be performed to prove that MV participates in the crisis transformation of CML. Besides, we will figure out the key recipient cell of leukemogenesis and the mechanisms that lead to the leukemia transformation induced by MV; as well as compounds in MV that could alter the transformation. Findings of this subject will sheed much-needed light on molecular mechanisms driving malignant reprogramming of progenitors into LSC in BC CML. It also presented a unique opportunity to prospectively study the changes undergone by progenitors, leading to their malignant transformation, serving as a convenient and operable model for investigating leukemogenesis.

慢性髓性白血病(CML)如何由相对惰性向预后极差的进展期演变仍不明了，近来发现白血病状态重编程造血干/祖细胞(HSPC)是CML进展的关键机制。通过前一个项目的研究，我们发现CML细胞分泌的微泡(MV)携带疾病中枢分子BCR-ABL1，并能够体外恶性转化HSPC；同时，从MV切入符合CML进展的疾病特点。由此我们推测：MV介导了CML状态对HSPC的恶性转化，参与疾病进展。本课题拟在前期研究基础上，通过体内外实验及临床研究进一步证明MV参与CML向急性白血病转化；明确MV转化的确切细胞组分，证明其通过投递外源性RNA发挥效应，并初步阐明HSPC白血病化的分子机制；此外，研究不同CML-MV中miRNA的表达差异及组装机制。本项目的完成，将有助于发现CML疾病进展及其异质性的新机制，MV可望发展为CML进展的早期预警信号及关键阻遏靶标；同时本课题还为研究白血病发生提供了一种极佳的模型选择。

慢性髓性白血病（CML）进展期患者预后极差，机制不明，近来研究表明CML细胞释放的微泡（MV）可能是参与恶性转化造血干/祖细胞（HSPC）的重要介质，参与疾病进展。本项目认为BCR-ABL1-MV携带的microRNA分子群是加速恶性转化的必要因素。我们对恶性转化不同阶段进行RNA-seq和miRNA-seq测序，一共检测到18614个基因和1425个miRNAs表达，经生物信息学分析筛选到了3717个差异表达基因（DEGs）和143个差异表达miRNAs（DEMs）。在此基础上，我们筛选癌症相关的差异基因，并据此建立了miRNAs、TFs以及肿瘤通路相关基因三者间的共调控网络，发现在转化的每个阶段受miRNAs和TFs调控而激活或抑制的癌症相关通路并不相同，而第三阶段大多数调节因子和通路显著失调，免疫相关通路如免疫系统、NFkB和Notch信号被抑制，而细胞增殖和能量相关通路包括细胞周期、氧化磷酸化、DNA复制与修复通路则被激活。鉴于以上发现，我们建立了细胞周期、DNA复制与修复以及Notch信号通路共调控网络。除此之外，我们发现MVs中高表达的miR-146b-5p与多个TF协同调控肿瘤相关基因，而NUMB-STAT-miR-146b这一前反馈环可能是恶性转化的重要分子机制。我们通过向K562-MVs中转染miR-146b-5p模拟物或抑制剂发现miR-146b-5p高表达加速细胞恶性转化进程；而伊马替尼联合实验也证实BCR-ABL1是转化过程中必不可少的分子，miR-146b-5p则可能是BCR-ABL1的协同加速因子。我们进一步探索深层机制发现：miR-146b-5p可能通过靶向抑癌基因NUMB使其沉默而加速恶性转化；在此基础上，我们对高水平miR-146b-5p是否增强了靶细胞基因组不稳定性进行了评估，发现K562-MVs中miR-146b-5p的高表达在诱导过程中会诱发靶细胞出现双链DNA断裂等基因组不稳定性成瘤。综上所述，我们发现了MVs恶性转化过程中的网络式共调控机制，深入研究了MVs中miR-146b-5p协同BCR-ABL1诱导靶细胞恶性转化的分子机制，并探讨了这类基因改变对靶细胞生物学行为的影响。