AML1 IN NORMAL AND LEUKEMIC CELLS

AML1/CBFβ转录因子复合体是人类急性白血病最常见的基因改变靶点之一，通过染色体诱导的重排或点突变，在多达三分之一的急性髓系白血病和淋巴细胞性白血病中成为靶点。我的实验室以前的工作已经证明，AMNL1通常作为一个主调控转录开关发挥作用，这对于最终的造血系统的形成是必不可少的。在我们的初步数据中，我们现在扩展这一观察结果，以表明AML1/CBFbeta以一种剂量依赖的方式建立转录级联，这是在发育中的胚胎的主动脉-性腺中肾区域(AGM)形成确定的造血干细胞(HSCs)所必需的。此外，AML1/CBFbeta水平的细微变化会导致HSC在时间和空间上的生成发生戏剧性的变化，将它们从AGM中的正常位置转移到卵黄囊。染色体重排诱导的ABL1/CBFβ基因突变导致白血病的发生至少部分是由于正常的AML1/CBFβ基因的部分显性负抑制，导致HSCs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单独不足以诱发白血病，而必须与继发性基因改变协同作用才能转化HSC。基于这些观察，我们的工作假设是，HSC的功能需要一定的AML1/CBFβ阈值水平。将复合体的活性降低到这一水平以下的基因变化直接改变了HSC的生长，导致了一批必须获得次级突变才能产生完整白血病表型的“白血病前”细胞池。为了直接解决这一假设，在特定目标1中提出了实验，该实验将利用我实验室最近产生的条件AML1-ETO敲入小鼠来定义能够与AML1-ETO合作诱发白血病的次级突变的谱。在具体目标2中，我们将扩大这些研究，以确定在家族性和散发性AML病例中发现的AML1突变如何通过在其种系中包含这些突变的小鼠的生成而易患白血病。总而言之，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，通过这些努力开发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物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.