MOLECULAR PATHOGENESIS OF CHROMOSOME 16 INVERSION IN HUMAN LEUKEMIA

人类白血病 16 号染色体倒转的分子发病机制

• 批准号: 6162550
• 负责人: P P 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/6162550
• 关键词: acute myelogenous leukemia; carcinogenesis; chromosome inversion; disease /disorder model; fusion gene; hematopoiesis; human genetic material tag; laboratory mouse; model design /development; molecular oncology; molecular pathology; neoplasm /cancer genetics; oncogenes; protein structure function; transcription factor; transfection; tumor suppressor genes

A pericentric chromosome 16 inversion, inv(16)(p13;q22), is present in almost 100% of patients with the M4Eo subtype of acute myeloid leukemia. A fusion gene between CBFB, the gene for a subunit of transcription factor CBF/PEBP2, and MYH11, which codes for smooth muscle myosin heavy chain, is generated by this chromosome 16 inversion. This fusion gene is believed to play a key role in leukemogenesis. We recapitulated this fusion gene in mice by inserting a human MYH11 cDNA into mouse Cbfb gene by homologous recombination (denoted "knock-in", or KI). Embryos heterozygous for the fusion gene had defects in both primitive and definitive hematopoiesis and died in midgestation due to widespread CNS hemorrhages. The phenotype provides very powerful evidence for a dominant negative function of Cbfb-MYH11 in the CBF pathway, and demonstrates the importance of this fusion gene in the regulation of growth and differentiation of hematopoietic cells, which may contribute to leukemogenesis. To study the the effect of the fusion gene on adult hematopoiesis and its leukemogenic capability, we are creating a conditional KI using the Cre-lox system. Under this design, the fusion gene CBFB-MYH11 is not transcribed until the DNA sequence between lox sites is deleted by Cre-lox recombination. This way, mice carrying the fusion gene (lox-KI) will survive to adulthood and the fusion gene will then be activated by expressing Cre within selected cells. We have already obtained viable adult mice carrying the lox-KI gene and are testing different systems for Cre expression. Using green fluorescent protein and immunofluorescence we observed that the CBFb protein was distributed equally in the cytoplasm and nucleus of 3T3 cells. CBFa was normally localized to the nucleus and co-expression with the heterodimeric partner CBFb resulted in exclusive nuclear localization of CBFb. The fusion protein Cbfb-SMMHC on the other hand appeared to assemble into cytoskeletal and higher order structures, and was able to trap CBFa into these structures. This observed CBFa sequestration may explain the dominant negative function of this inv16 fusion protein. To assess whether the hemorrhages observed in Cbfb-MYH11 KI embryos were a consequence of the fusion gene's expression in CNS, or resulted from impaired coagulation due to lack of megakaryocytopoiesis, Cbfb expression was analyzed by in-situ hybridization. Whole mounts and sections of E10.5-E12.5 embryos showed high levels of expression in dorsal root and cranial nerve ganglia, in addition to diffused expression throughout the embryos. Such patterns suggest a direct role of Cbfb-MYH11 expression in the hemorrhages in the similarly regionalized sites.

16号染色体臂间倒位inv（16）（p13;q22）存在于 几乎100%的急性髓性白血病M4 Eo亚型患者。 CBFB与转录亚基基因之间的融合基因 因子CBF/PEBP 2和MYH 11，其编码平滑肌肌球蛋白重 链，是由16号染色体倒位产生的。这种融合基因是 被认为在白血病发生中起着关键作用。我们概括了一下 通过将人MYH 11 cDNA插入小鼠Cbfb基因中在小鼠中的融合基因 通过同源重组（表示为“敲入”或KI）。胚胎 融合基因的杂合子在原始和 明确的造血，并在妊娠中期死于广泛的中枢神经系统 你说什么？表型为显性遗传提供了非常有力的证据， Cbfb-MYH 11在CBF通路中的负功能，并证明了 这种融合基因在生长调节中的重要性， 造血细胞的分化，这可能有助于 白血病发生研究融合基因对成鲤的影响 造血及其致白血病能力，我们正在创造一个 使用Cre-lox系统的条件KI。在这种设计下， 基因CBFB-MYH 11直到lox之间的DNA序列才被转录 位点通过Cre-lox重组而缺失。这样，携带 融合基因（lox-KI）将存活至成年，并且融合基因将 然后通过在选定的细胞内表达Cre来激活。我们有 已经获得了携带lox-KI基因的存活成年小鼠， 测试Cre表达的不同系统。使用绿色荧光 蛋白和免疫荧光，我们观察到CBFb蛋白是 3 T3细胞胞质和胞核中分布均匀。CBFA是 通常定位于细胞核，并与 异源二聚体伴侣CBFb导致特异性核定位， CBFb。另一方面，融合蛋白Cbfb-SMMHC似乎 组装成细胞骨架和更高级的结构，并能够 将CBFa捕获到这些结构中。这种观察到的CBFa隔离可能 解释了这种inv 16融合蛋白的显性负功能。到 评估在Cbfb-MYH 11 KI胚胎中观察到的胚胎发育是否是 融合基因在CNS中表达的结果，或由 由于缺乏巨核细胞生成和Cbfb表达而导致凝血功能受损 通过原位杂交进行分析。整座和部分 E10.5-E12.5胚胎在背根中显示高水平的表达， 颅神经节，除了在整个脑内弥漫表达外， 胚胎这种模式表明Cbfb-MYH 11表达在细胞凋亡中的直接作用。 在类似的区域化网站的地图。