The Role of MLL-AF9 in Acute Myeloid Leukemia

造血干细胞和白血病干细胞(HSC/LSC)的生存依赖于与骨髓微环境中的细胞外基质、可溶性因子和细胞成分的相互作用。这些相互作用被认为是为了促进干细胞的静止和它们在利基中的保留，并影响关于对称或不对称自我更新分裂的决定。参与这些决定的信号现在才刚刚被识别出来，可能涉及在干细胞中很重要的经典分子通路，包括WNT/Catenin、Notch、KitL/Kit和SDF/CXCR4信号。这些信号中的每一个都直接或间接地影响Rho家族的小GTP酶蛋白，包括RAC家族。这些蛋白质在干细胞在巢内的保留以及生存、增殖和分化决策中起着至关重要的作用，而这些决策对于维持造血和维持干细胞室的完整性至关重要。我们最近证明，在一种新的人类白血病模型中，激活的RAC增加。在这个模型中，MLL-AF9(MA9)白血病癌基因被用来转化原代人类CD34+细胞，并在免疫缺陷小鼠中促进快速急性髓系白血病(AML)。用RAC抑制剂NSC23766抑制RAC-GTP的形成，或shRNA抑制RAC的表达，在对正常人CD34+脐血细胞几乎没有抑制作用的条件下，可以有效地促进细胞凋亡和抑制细胞增殖。Rac的抑制和细胞凋亡的诱导与Bcl2蛋白表达的缺失密切相关，尤其是作为rac2信号转导下游靶点的Bclxl。我们建议使用我们新开发的白血病模型来研究Rac信号在MLL白血病细胞中的作用，方法是通过体外测试与Rac活性相关的表型，包括迁移、存活、黏附和增殖(Aim1)。我们将使用异种移植系统和药理学以及RNAi方法在体内扩展这些结果，以抑制RAC活性和Bcl-XL功能(目标2)。这种结合的方法应该使我们能够确定RAC信号通路在白血病中的重要性，并开始剖析MA9 AML中RAC成瘾的具体机制。相关性(参见说明书)：我们最近证明，人类血液干细胞在引入后可以被诱导成白血病。

Hematopoietic as well as leukemic stem cells (HSC/LSC) are dependent on interactions with extracellular matrix, soluble factors and cellular components of the bone marrow (BM) microenvironment for their survival. These interactions are hypothesized to promote the quiescence of the stem cell and their retention in the niche and influence the decisions regarding symmetric versus asymmetric self-renewal divisions. The signals that contribute to these decisions are just now being identified and likely involve the classical molecular pathways shown to be important in stem cells, including WNT/catenin, Notch, KitL/Kit and SDF/CXCR4 signaling. Each of these signals impacts directly or indirectly on the Rho family of small GTPase proteins, including the Rac family. These proteins play an essential role in the retention of HSC in the niche and in the survival, proliferation and differentiation decisions that are critical for sustaining hematopoiesis and maintaining the integrity of the stem cell compartment. We have recently demonstrated that activated Rac is increased in a novel model of human leukemia. In this model, the MLL-AF9 (MA9) leukemia oncogene is used to transform primary human CD34+ cells and promotes a rapid acute myeloid leukemia (AML) in immunodeficient mice. Suppression of Rac-GTP formation using the Rac inhibitor NSC23766, or shRNA knockdown of Rac expression, could effectively promote apoptosis and suppress proliferation under conditions where little or no inhibitory effect could be detected on normal human CD34+ cord blood cells. The inhibition of Rac and the induction of apoptosis correlate with the loss of Bcl protein expression, especially of Bcl-xL, which is an identified downstream target of Rac2 signaling. We propose to use our newly developed leukemia model to study the role of Rac signaling in MLL leukemia cells using in vitro assays measuring well-characterized phenotypes associated with Rac activity, including migration, survival, adhesion and proliferation (Aim1). We will extend these results in vivo using a xenograft system and a pharmacologic as well as RNAi approach to inhibit Rac activity as well as Bcl-xL function (Aim 2). This combinatorial approach should enable us to determine the importance of the Rac signaling pathway in leukemia and begin to dissect the specific mechanisms involved in the Rac addiction in MA9 AML. RELEVANCE (See instructions): We have recently shown that human blood stem cells can be induced to make leukemia upon introduction