MOLECULAR PATHOGENESIS OF CHROMOSOME 16 INVERSION IN HUM

人类 16 号染色体倒转的分子发病机制

• 批准号: 6988574
• 负责人: PU PAUL LIU
• 金额: --
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6988574
• 关键词: acute myelogenous leukemia; carcinogenesis; chromosome inversion; developmental immunology; disease /disorder model; hematopoiesis; human genetic material tag; laboratory mouse; microarray technology; molecular oncology; molecular pathology; neoplasm /cancer genetics; transcription factor; transfection; tumor suppressor genes

Chromosome 16 inversion, inv(16), is one of the most common chromosome abnormalities in human acute myeloid leukemia (AML). A fusion gene between the core binding factor B (CBFB) gene and the myosin heavy chain 11 (MYH11) gene is generated by this inversion. CBFB encodes a transcription factor while MYH11 encodes the smooth muscle form of myosin heavy chain (SMMHC). Using a mouse knock-in strategy, we showed that the fusion gene Cbfb-MYH11 is necessary but not sufficient for leukemogenesis. Last year we reported the identification of genes that specifically cooperate with Cbfb-MYH11 in leukemogenesis. Neonatal injection of Cbfb-MYH11 knock-in chimeric mice with retrovirus 4070A led to the development of AML in 2-5 months. Each leukemia sample contained one or a few viral insertions, suggesting that alteration of one gene could be sufficient to synergize with Cbfb-MYH11. The chromosomal position of 67 independent retroviral insertion sites (RIS) was determined, and 90% of the RIS mapped within 10 kb of a flanking gene. In total 54 candidate genes were identified, 6 of them were common insertion sites. This is the first retroviral genetic screen to identify genes that cooperate with a fusion gene linked to the pathogenesis of human leukemia. In order to study the function of Cbfb gene in T cell development, we used a mouse line with floxed exons 5 and 6 of Cbfb inserted 5? to the Cbfb-MYH11 fusion cassette, which produced pseudo-normal mice (loxKI). By crossing the loxKI mice with mice expressing the Cre gene under the control of the T cell-specific Lck promoter (LckCre), we generated LckCre-loxKI double positive mice, in which the floxed exon 5 and 6 were deleted and Cbfb-MYH11 re-expressed only in the thymus when Lck started to express. The LckCre-loxKI mice were viable. However, their thymic development was severely impaired: The size of the thymuses in the mutant mice was about half the normal size, and the total number of thymocytes in the mutant mice was 10-20-fold reduced. FACS analysis of thymocytes from 4 to 12 week old mice showed a developmental blockade at the CD4/CD8-double negative (DN) stage, which was characterized by lower percentage of double positive cells and higher percentage of double negative cells. In addition, the CD4 : CD8 ratio was altered. Furthermore, the mature T cell population size in the spleen of the mutant mice was lower than that of the control mice. Our preliminary data suggested that Cbfb plays an important role in T cell development. The mechanism through which Cbfb affects the T cell development is currently under investigation. It is likely that the phenotype reflects the combined effect of missing all three Runx genes, since the phenotype described here is more severe than either Runx1 or Runx3 null alone. Chromosomal rearrangements affecting RUNX1 and CBFB are common in acute leukemias. These mutations result in the expression of fusion proteins that act in a dominant negative manner to suppress the normal function of the CBFb/RUNX1 complex. In addition, loss-of-function mutations in RUNX1 have been identified in sporadic cases of acute myeloid leukemia (AML) and in association with familial platelet disorder with propensity to develop AML (FPD/AML). In order to examine the hypothesis that decreased gene dosage of RUNX1 may be a critical event in the development of leukemia, we treated chimeric mice generated from Runx1-/- embryonic stem (ES) cells that have homozygous disruption of the RUNX1 gene, as well as Runx1+/- mice with N-ethyl-N-nitrosurea (ENU). The heterozygous Runx1+/- mice did not show increased incidence of any malignancy. On the other hand, we observed an increased incidence of precursor T-lymphoblastic lymphoma in Runx1-/- compared to wild-type chimeras, and confirmed that the tumors were of ES cell origin. It was determined by PCR that Runx1-/- ES cells contributed to the T cell progenitor population in the chimeras prior to leukemia development, which may explain the tissue-specificity of the malignancy we observed. Our results suggest that deficiency of Runx1 can indeed predispose mice to hematopoietic malignancies.

16号染色体倒位（inv（16））是人类急性髓细胞白血病（AML）中最常见的染色体异常之一。通过该倒位产生核心结合因子B（CBFB）基因和肌球蛋白重链11（MYH 11）基因之间的融合基因。CBFB编码转录因子，而MYH 11编码平滑肌形式的肌球蛋白重链（SMMHC）。 使用小鼠敲入策略，我们表明，融合基因Cbfb-MYH 11是必要的，但不足以白血病的发生。去年，我们报道了在白血病发生中与Cbfb-MYH 11特异性合作的基因的鉴定。新生儿注射逆转录病毒4070 A的Cbfb-MYH 11基因敲入嵌合小鼠导致在2-5个月内发生AML。每个白血病样本都含有一个或几个病毒插入，这表明一个基因的改变可能足以与Cbfb-MYH 11协同作用。确定了67个独立的逆转录病毒插入位点（RIS）的染色体位置，90%的RIS定位在10 kb的侧翼基因内。共鉴定出54个候选基因，其中6个为常见插入位点。这是第一个逆转录病毒基因筛选，以确定与人类白血病发病机制相关的融合基因合作的基因。 为了研究Cbfb基因在T细胞发育中的功能，我们用Cbfb基因第5和第6外显子的floxed突变体插入5？Cbfb-MYH 11融合盒，其产生假正常小鼠（loxKI）。通过将loxKI小鼠与在T细胞特异性Lck启动子（LckCre）控制下表达Cre基因的小鼠杂交，我们产生了LckCre-loxKI双阳性小鼠，其中floxed外显子5和6被删除，并且当Lck开始表达时，Cbfb-MYH 11仅在胸腺中重新表达。LckCre-loxKI小鼠存活。然而，它们的胸腺发育严重受损：突变小鼠的胸腺大小约为正常大小的一半，突变小鼠的胸腺细胞总数减少了10-20倍。对4 - 12周龄小鼠胸腺细胞的FACS分析显示，在CD 4/CD 8-双阴性（DN）阶段发育阻滞，其特征在于双阳性细胞百分比较低，双阴性细胞百分比较高。此外，CD 4：CD 8比率也发生了变化。此外，突变小鼠脾脏中的成熟T细胞群体的大小低于对照小鼠。我们的初步数据表明，Cbfb在T细胞发育中起着重要作用。Cbfb影响T细胞发育的机制目前正在研究中。表型可能反映了缺失所有三个Runx基因的组合效应，因为这里描述的表型比单独的Runx 1或Runx 3 null更严重。 影响RUNX 1和CBFB的染色体重排在急性白血病中很常见。这些突变导致融合蛋白的表达，其以显性负性方式抑制CBFb/RUNX 1复合物的正常功能。此外，在散发性急性髓性白血病（AML）病例中发现了RUNX 1的功能丧失突变，并与家族性血小板疾病伴AML倾向（FPD/AML）相关。为了检验RUNX 1基因剂量减少可能是白血病发展中的关键事件这一假设，我们处理了由RUNX 1基因纯合破坏的Runx 1-/-胚胎干（ES）细胞产生的嵌合小鼠，以及用N-乙基-N-亚硝基脲（ENU）处理Runx 1 +/-小鼠。杂合子Runx 1 +/-小鼠未显示任何恶性肿瘤的发生率增加。另一方面，与野生型嵌合体相比，我们观察到Runx 1-/-中前体T淋巴母细胞淋巴瘤的发生率增加，并证实肿瘤是ES细胞来源的。通过PCR确定Runx 1-/- ES细胞在白血病发展之前贡献于嵌合体中的T细胞祖细胞群体，这可以解释我们观察到的恶性肿瘤的组织特异性。我们的研究结果表明，Runx 1的缺陷确实可以使小鼠容易患造血系统恶性肿瘤。