AML1 IN NORMAL AND LEUKEMIC CELLS

AML1/ cbfβ转录因子复合物是人类急性白血病中最常见的遗传改变靶标之一，在多达三分之一的急性髓细胞和淋巴细胞白血病中，通过染色体诱导的重排或点突变被靶向。我的实验室之前的工作已经证明，AMNL1通常作为一个主要的调控转录开关，对最终造血系统的形成至关重要。在我们的初步数据中，我们现在扩展了这一观察结果，表明AML1/CBFbeta以剂量依赖的方式建立了一个转录级联，这是发育中的胚胎主动脉-性腺中肾区（AGM）形成最终造血干细胞（hsc）所必需的。此外，AML1/ cbfβ水平的细微变化会引起造血干细胞在时间和空间上的巨大变化，将它们从AGM中的正常位置转移到卵黄囊中。由染色体重排诱导的ABL1/ cbfβ改变引发的白血病似乎至少部分源于正常AML1/ cbfβ的部分显性负抑制，导致造血干细胞自我更新和成熟的改变。然而，重要的是，我们的初步数据清楚地表明，AML1-ETO本身不足以诱导白血病，而必须配合继发性遗传改变来转化HSC。基于这些观察，我们的工作假设是，HSC的功能需要一定的阈值水平的AML1/CBFbeta。将复合物活性降低到低于这一水平的遗传变化直接改变了HSC的生长，导致一群“白血病前期”细胞必须获得继发性突变才能产生完整的白血病表型。为了直接解决这一假设，在Specific Aim 1中提出了实验，该实验将利用我实验室最近产生的小鼠条件AML1-ETO敲入来定义能够与AML1-ETO合作诱导白血病的继发性突变谱。在Specific Aim 2中，我们将扩展这些研究，以确定家族性和散发性AML病例中鉴定的AML1突变如何通过在其种系中含有这些突变的小鼠的产生而易患白血病。总之，这些研究应该为核心结合因子白血病的分子病理学提供关键的见解。此外，通过这些努力建立的小鼠模型应该被证明是有价值的试剂，通过它来评估针对AML1-ETO或其结合的核共抑制因子的药物的潜在治疗用途。

The AML1/CBFbeta transcription factor complex is one of the most frequent targets of genetic alterations in human acute leukemia, being targeted in up to one-third of acute myeloid and lymphoblastic leukemia by either chromosomal induced rearrangements, or point mutation. Prior work from my laboratory has demonstrated that AMNL1 normally functions as a master regulatory transcriptional switch that is essential for the formation of the definitive hematopoietic systems. In our preliminary data, we now extend this observation to show that AML1/CBFbeta establishes, in a dose-dependent manner, a transcriptional cascade that is required for the formation of definitive hematopoietic stem cells (HSCs) in the aorta-gonad mesonephros region (AGM) of the developing embryo. Moreover, subtle alterations in the level of AML1/CBFbeta induces dramatic changes in the temporal and spatial generation of HSCs, shifting them from their normal position in the AGM to the yolk sac. The initiation of leukemia by chromosomal rearrangement-induced-induced alteration in ABL1/CBFbeta appears to result at least in part, from a partial dominant negative inhibition of normal AML1/CBFbeta, leading to alterations in the self-renewal and maturation of HSCs. Importantly, however, our preliminary data clearly demonstrates that AML1-ETO alone is insufficient to induce leukemia, but rather must cooperate with secondary genetic alterations to transform HSC. Based on these observations, our working hypothesis is that a certain threshold level of AML1/CBFbeta is required for the function of HSC. Genetic changes that decrease the activity of the complex below this level directly alter HSC growth, leading to a pool of "pre-leukemic) cells that must acquire secondary mutations before they can generate a full leukemic phenotype. To directly address this hypothesis, experiments are proposed in Specific Aim 1 that will utilize a conditional AML1-ETO knock in-mouse that was recently generated in my laboratory to define the spectrum of secondary mutations able to cooperate with AML1-ETO to induce leukemia. In Specific Aim 2, we will extend these studies to determine how AML1 mutations identified in familial and sporadic cases of AML predispose to leukemia through the generation of mice containing these mutations in their germline. Together these studies should provide critical insights into the molecular pathology of the core-binding factor leukemia. Moreover, the murine models developed through these efforts should prove to be valuable reagents through which to assess the potential therapeutic use of drugs targeted toward either AML1-ETO or its bound nuclear co-repressors.