Twist-1在MLL-AF9急性髓系白血病发生和维持中的作用及作用机制

Mixed lineage leukemia (MLL)-AF9 acute myeloid leukemia (AML) is a kind of refractory leukemia with highly malignancy and poor prognosis, which is closely related with the high frequency of leukemia stem cells (LSC). However, the underlying molecular mechanisms of LSCs' effects have not been clarified. It’s now well accepted that Twist-1 may function as a multifunctional proto-oncogene. Overexpression of Twist-1 in solid tumors enhanced tumor-initiating and self-renewal capability. We have first reported when conducting the last research supported by the National Natural Science Foundation of China, that Twist-1 was not only highly expressed in leukemia stem cell candidates of AML, but also was critical for maintenance of leukemia stem/progenitor cell colony forming ability. We further find at present that Twist-1 is selectively highly expressed in MLL-rearranged AML in a data set with information on four distinct subtypes of aberrant karyotype AML. We use the conditional knockout murine model to examine the role of Twist-1 in MLL-AF9-induced AML and further demonstrate that Twist-1 is associated with the maintenance of MLL-AF9 AML. Based on the these findings, here, we mainly investigate whether Twist-1 is critical for the initiation and maintenance of MLL-AF9 AML, whether Twist-1 is an important mediator of MLL-AF9 AML LSC, and its potential pathogenic mechanism. This study will be helpful for revealing the molecular mechanism of MLL-AF9 AML LSC and provide new targets for the research of anti-leukemia drugs.

混合谱系白血病（MLL）-AF9是急性髓系白血病（AML）中一类恶性程度高、预后差的难治性白血病，这与其具有高比例白血病干细胞（LSC）的特性密切相关，但是目前介导LSC作用的分子机制尚未阐明。Twist-1是癌基因，多种实体瘤中促进肿瘤干细胞起始和自我更新能力。我们在进行上一个国家自然科学基金时首次发现， Twist-1高表达于AML患者骨髓LSC中并调控白血病干祖细胞集落形成能力。目前进一步研究发现，Twist-1高表达于有MLL重排的AML患者骨髓细胞，并通过MLL-AF9 AML小鼠模型进一步证实，Twist-1与MLL-AF9 AML的进展相关。本研究在此发现基础上，深入探讨Twist-1是否是MLL-AF9 AML发生和维持的关键因素，是否对维持LSC功能至关重要，并深入探讨其下游分子机制。本课题将有助于揭示AML LSC的分子调控机制，为抗白血病药物研发提供新靶点。

混合谱系白血病（MLL）-AF9是急性髓系白血病（AML）中一类恶性程度高、预后差的难治性白血病，这与其具有高比例白血病干细胞（LSC）的特性密切相关，但是目前介导LSC作用的分子机制尚未阐明。Twist-1是癌基因，高表达于AML患者骨髓LSC中并调控LSC集落形成能力，且特意性高表达于有MLL重排的AML患者骨髓细胞，但其在MLL-AF9白血病的发生和维持作用还不清楚。本项目利用ER-Cre诱导的Twist-1条件敲除鼠模型、 Twist-1过表达模型和MLL-AF9诱导的小鼠AML模型，探讨Twist-1在MLL-AF9 AML发生和维持中的作用及作用机制。发现，在 MLL-AF9 AML小鼠的白血病发生和发展阶段， Twist-1缺失均延长发病小鼠生存期，减少白血病细胞的浸润，促进白血病细胞髓系分化；此外，Twist-1缺失导致LSC的比例和数目减少，自我更新能力下降，细胞凋亡和ROS水平增加，并抑制LSC 粘附、迁移以及归巢能力。进一步，对有MLL重排的AML细胞系 THP-1和Molm-13 以及 AML 原代患者标本中，敲降Twist-1也发现相似的表型。这些结果表明Twist-1是促进MLL-AF9 AML发生和维持的关键因子。机制探索上，通过对Twist-1缺失和对照组LSC进行RNA-seq、ATAC-seq以及蛋白验证，我们发现TWIST-1能够和BRD4蛋白相互作用，激活Wnt5a/Ca2+通路，进而激活RAC1蛋白。在Twist-1过表达的MLL AML白血病模型中，分别利用Brd4抑制剂，Wnt5a抑制剂和Rac1挽救实验，能部分逆转Twist-1导致的促进MLL-AF9白血病发生和进展，以及LSC功能的表型。上述结果提示，转录因子Twist-1在LSC中通过结合BRD4蛋白，调节Wnt5a/Ca2+-Rac1通路，促进MLL-AF9 AML的发生和进展。本研究阐释了调控LSC自我更新的内在新机制，发现了新的关键调控分子Twist-1，为MLL-AF9 白血病的治疗提供新的靶点。