The Role of MLL-AF9 in Acute Myeloid Leukemia

造血干细胞和白血病干细胞（HSC/LSC）的存活依赖于与骨髓（BM）微环境的细胞外基质、可溶性因子和细胞组分的相互作用。这些相互作用被假设为促进干细胞的静止和它们在生态位中的保留，并影响关于对称与不对称自我更新分裂的决定。有助于这些决定的信号现在正在被鉴定，并且可能涉及在干细胞中显示重要的经典分子途径，包括WNT/连环蛋白、Notch、KitL/Kit和SDF/CXCR 4信号传导。这些信号中的每一个都直接或间接地影响小GT3蛋白的Rho家族，包括Rac家族。这些蛋白质在HSC在小生境中的保留以及在存活、增殖和分化决定中发挥重要作用，这些决定对于维持造血和维持干细胞区室的完整性至关重要。我们最近已经证明，激活的Rac在一种新的人类白血病模型中增加。在该模型中，MLL-AF 9（MA 9）白血病癌基因用于转化原代人CD 34+细胞，并促进免疫缺陷小鼠中的快速急性髓性白血病（AML）。使用Rac抑制剂NSC 23766抑制Rac-GTP形成，或Rac表达的shRNA敲低，可以有效地促进细胞凋亡和抑制增殖的条件下，很少或没有抑制作用，可以检测到正常的人CD 34+脐带血细胞。Rac的抑制和细胞凋亡的诱导与Bcl蛋白表达的丧失相关，特别是Bcl-xL，其是Rac 2信号传导的鉴定的下游靶标。我们建议使用我们新开发的白血病模型来研究Rac信号传导在MLL白血病细胞中的作用，使用体外测定测量与Rac活性相关的良好表征的表型，包括迁移，存活，粘附和增殖（Aim 1）。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活性以及Bcl-xL功能（目的2）。这种组合方法应使我们能够确定Rac信号通路在白血病中的重要性，并开始剖析MA 9 AML中Rac成瘾的具体机制。相关性（见说明书）：我们最近发现，人类造血干细胞可以诱导后，使白血病的引进

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