Leukemogenic Mechanism by genomic mutations of hematopoietic-speci is transcription factor, AML1/RUNX1

造血转录因子AML1(RUNX1)是白血病相关染色体易位的常见靶点。AML1通过染色体易位重排形成嵌合体蛋白，该嵌合体蛋白对野生型AML1具有强大的反式显性抑制作用，有助于白血病的发生，而该分子与DNA接触残基的错义突变也被认为是与白血病相关的突变。在这项研究中，我们在整个动物的背景下评估了这些点突变的后果。我们成功地将每一个突变，R139Q，R174Q，R177Q，或I150ins，通过敲入方法引入小鼠生殖系。当纯合子遗传时，所有的小鼠系都是胚胎致死的，这表明这些等位基因都是功能丧失类型的突变。相比之下，每个突变的杂合子通常出生和发育都很健康。他们有生育能力，一生中没有患上明显的白血病。因此，这些突变似乎不足以导致临床白血病的单等位基因，这与人类疾病的情况形成了鲜明对比。因此，有人建议，需要额外的协作性突变(S)才能发展为该病。为了解决这个问题，我们用R174Q小鼠进行了逆转录病毒感染实验，发现杂合子比它们的野生型小鼠更早发生白血病。这一观察表明，AML1的一个等位基因突变进一步促进了白血病的发病。此外，对白血病基因组中前病毒整合位点的克隆分析揭示了一些候选的合作基因。我们目前正在分析这些基因产物，看看它们是否在人类疾病中起作用。

Hematopoietic transcription factor AML1 (RUNX1) is a frequent target of leukemia-associated chromosome translocations. AML1 is rearranged by chromosome translocations to form chimera protein which acts as a strong trans-dominant inhibitor against wild-type AML1 and contributes to leukemia development, while mis-sense mutations at DNA-contacting residues of the molecule have also been recognized as leukemia-associated mutations recently. In this study we assessed the consequences of these point mutations in context of entire animal. We successfully introduced each of the mutations, R139Q, R174Q, R177Q, or I150ins, into mouse germline through the knock-in approach. All mouse lines were embryonic lethal when inherited homozygously, indicating that these alleles are all loss-of-function type mutations. In contrast, heterozygotes for each mutation were normally born and developed healthy. They were fertile and developed no overt leukemia through their lives. Thus, these mutations seem to be insufficient to cause clinical leukemia as single allele, showing sharp contrast to the case for human diseases. Thus, it was suggested that additional co-operative mutation (s) are required to develop the disease. In order to address this issue, we performed retroviral-infection experiment using R174Q mice, and found that heterozygotes had tendency to develop leukemia earlier than their wild-type littermates did. This observation indicates that one allele mutation of AML1 serves to promote leukemia-onset one step further. In addition, cloning-analysis for proviral integration sites within the leukemia genome revealed a number of candidate co-operative genes. We are currently analyzing these gene products to see if they collaborate in human diseases.