Mechanistic and translational studies of CBF leukemia

CBF白血病的机制和转化研究

• 批准号: 9152701
• 负责人: Paul Liu
• 金额: $ 96.17万
• 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/9152701
• 关键词: Accounting; Acute Myelocytic Leukemia; Adult; Adult Acute Myeloblastic Leukemia; Affect; Alleles; Animal Model; Binding; Biological Markers; Blood; CBFB gene; Cell Proliferation; Chemicals; Childhood Acute Lymphocytic Leukemia; Chromosome abnormality; Clinical; Clinical Research; Communities; Complex; Core-Binding Factor; DNA-Binding Proteins; Data; Defect; Development; Diagnosis; Disease; Disease remission; Dominant-Negative Mutation; Embryo; Event; Gene Expression; Gene Mutation; Gene Targeting; Genetic; Genetic Transcription; Genomic approach; Genomics; Goals; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Intramural Research Program; Knock-in Mouse; Knockout Mice; Length; MYH11 gene; Modeling; Molecular; Monitor; Morbidity - disease rate; Mus; National Human Genome Research Institute; Pathogenesis; Patients; Play; Population; Process; Proteins; RUNX1 gene; Repression; Research; Residual Tumors; Resources; Role; Scientist; Staging; Stem cells; Technology; Testing; Time; Transcription factor genes; Transgenic Animals; Transgenic Mice; Translating; Translational Research; United States National Institutes of Health; Vertebrates; Zebrafish; cancer cell; chemotherapy; clinical practice; fusion gene; genetic approach; genomic tools; helicase; human MYH11 protein; improved; leukemia; leukemia treatment; leukemogenesis; man; mortality; mouse model; outcome forecast; progenitor; tool; transcription factor; translational study; tumorigenesis

Acute myeloid leukemia (AML) is a heterogeneous disease with diverse gene mutations and chromosomal abnormalities. Core binding factor (CBF) leukemias, those with translocations or inversions that affect transcription factor genes RUNX1 or CBFB, account for approximately 24% of adult acute myeloid leukemia (AML) and 25% of pediatric acute lymphocytic leukemia. The encoded proteins, RUNX1 and CBFbeta, form a heterodimer to regulate gene expression, and they are both required for hematopoiesis in vertebrate animals from zebrafish to man. Extensive clinical studies have demonstrated that CBFB-MYH11 and RUNX1-ETO, the two common fusion genes in CBF leukemia, are the best biomarkers for diagnosis, prognosis, and residual disease monitoring of CBF leukemia patients. Even though CBF leukemias have better initial remission rate and better prognosis than most AML cases, current chemotherapy is associated with significant morbidity and mortality, and the long-term survival (>5 year) is only around 50-60%. Over the years we have used mouse models and a variety of research tools to characterize the CBFB-MYH11 fusion gene, determine the effect of the encoded protein, CBFbeta-SMMHC, on normal hematopoiesis, and understand the leukemogenesis process associated with the fusion gene. We have generated both conventional and conditional knock-in mouse models to study CBFB-MYH11. Using these mouse models we have demonstrated that Cbfb-MYH11 dominantly inhibits Runx1 and Cbfb function during definitive hematopoiesis, resulting in complete lack of definitive hematopoiesis in the heterozygous Cbfb-MYH11 knockin embryos. We also showed that Cbfb-MYH11 is necessary but not sufficient for leukemia, and we were able to identify cooperating genetic events in the mouse models. We have generated knock-in mouse models expressing truncated Cbfb-MYH11 to determine the importance of functional domains of CBFbeta-SMMHC. Overall our lab has been recognized in the field as the major contributor to the understanding of CBFB-MYH11 leukemia. In the last fiscal year we focused on using mouse models to study the mechanisms of leukemogenesis by CBFB-MYH11. In the first specific aim we determined if RUNX1 is important for leukemogenesis by CBFB-MYH11. Previously dominant negative inhibition of normal RUNX1 and CBFβ functions has been considered as a potential mechanism for CBFβ-SMMHC. However, recently we showed that Cbfb-MYH11 knockin embryos have primitive hematopoiesis defects that do not seem to result from RUNX1 repression (Hyde et al., Blood, 2010). Moreover, knockin mice expressing a modified CBFβ-SMMHC protein with decreased RUNX1-binding ability developed leukemia faster than those that express the full-length CBFβ-SMMHC (Kamikubo et al., Cancer Cell, 2010). These findings suggested that RUNX1-repression may not be important for leukemogenesis, and raised the possibility that CBFβ-SMMHC may induce leukemia independent of RUNX1. To test this hypothesis, we have used three Runx1 deficient models to determine if RUNX1 is required for leukemogenesis by CBFβ-SMMHC. In Cbfb+/MYH11 embryos that are also Runx1-/-, or with a semi-dominant-negative Runx1 allele, Runx1+/lz, the primitive hematopoietic defect induced by Cbfb-MYH11 was rescued, even though Runx1 deficient embryos did not have primitive hematopoietic defects. During definitive hematopoiesis in adults, CBFβ-SMMHC increased proliferation of progenitor cells and induced an abnormal pre-leukemic progenitor population. These defects were also rescued by the semi-dominant-negative allele, Runx1+/lz, or a conditional Runx1 null. Finally, leukemia development was significantly delayed in Cbfb+/MYH11, Runx1+/lz or Cbfb+/MYH11, conditional Runx1 null mice. Overall, our findings suggest that RUNX1 activity is required for Cbfb-MYH11-induced hematopoietic defects and leukemogenesis. In the second specific aim we studied the potential cooperation between CHD7 and CBFB-MYH11 for leukemogenesis. The chromodomain-helicase-DNA binding protein 7 (CHD7) interacts with RUNX1 and suppresses RUNX1 function during hematopoiesis. We hypothesized that CHD7 also plays a role in leukemogenesis by CBFB-MYH11, since CBFB-MYH11 requires RUNX1 for leukemia. To test this hypothesis, we crossed conditional Chd7 knockout mice (Chd7f/f) with Cbfb-MYH11 knockin mice to generate transgenic mice expressing Cbfbeta-SMMHC but deficient for CHD7. We found that the hematopoietic progenitor cell populations were significantly lower in these transgenic mice than control mice, which was likely due to reduced cellular proliferation. Importantly, it took much longer time for these transgenic mice to develop leukemia than the mice only expressing CBFbeta-SMMHC. We also showed that CHD7 is a partner of the RUNX1-CBFbeta-SMMHC transcription complex and that CHD7 could enhance transcription of RUNX1 and CBFbeta-SMMHCs target genes. These data indicate that CHD7 deficiency inhibits Cbfb-MYH11 induced leukemogenesis through inhibiting RUNX1 activity in regulating transcription and cellular proliferation.

急性骨髓性白血病（AML）是一种具有不同基因突变和染色体异常的异质性疾病。核心结合因子（CBF）白血病，即那些影响转录因子基因RUNX 1或CBFB的易位或倒位的白血病，约占成人急性髓性白血病（AML）的24%和儿科急性淋巴细胞白血病的25%。RUNX 1和CBF β是两种常见的CBF白血病融合基因，是CBF白血病诊断、预后和残留病灶监测的最佳生物标志物。尽管CBF白血病比大多数AML病例具有更好的初始缓解率和更好的预后，但目前的化疗与显著的发病率和死亡率相关，并且长期存活率（>5年）仅为约50- 60%。 多年来，我们使用小鼠模型和各种研究工具来表征CBFB-MYH 11融合基因，确定编码蛋白CBFbeta-SMMHC对正常造血的影响，并了解与融合基因相关的白血病发生过程。我们已经生成了常规和条件性敲入小鼠模型来研究CBFB-MYH 11。使用这些小鼠模型，我们已经证明，Cbfb-MYH 11显性抑制Runx 1和Cbfb的功能，在确定的造血过程中，导致完全缺乏明确的造血在杂合子Cbfb-MYH 11敲入胚胎。我们还表明，Cbfb-MYH 11对于白血病是必要的，但不是充分的，我们能够在小鼠模型中鉴定出协同遗传事件。我们已经产生了表达截短的Cbfb-MYH 11的敲入小鼠模型，以确定CBFbeta-SMMHC功能结构域的重要性。总的来说，我们的实验室在该领域被公认为是理解CBFB-MYH 11白血病的主要贡献者。 在上一个财政年度，我们专注于使用小鼠模型研究CBFB-MYH 11的白血病发生机制。在第一个具体目标中，我们通过CBFB-MYH 11确定RUNX 1是否对白血病发生重要。以前的显性负抑制正常RUNX 1和CBF功能已被认为是CBF-SMMHC的潜在机制。然而，最近我们发现Cbfb-MYH 11敲入胚胎具有原始造血缺陷，其似乎不是由RUNX 1抑制引起的（Hyde et al.，Blood，2010）。此外，表达具有降低的RUNX 1结合能力的修饰的CBF-SMMHC蛋白的敲入小鼠比表达全长CBF-SMMHC的那些更快地发展白血病（Kamikubo等人，Cancer Cell，2010）。这些发现表明RUNX 1抑制可能对白血病发生不重要，并提出了CBF-SMMHC可能独立于RUNX 1诱导白血病的可能性。 为了检验这一假设，我们使用了三种Runx 1缺陷模型来确定RUNX 1是否是CBF-SMMHC引起白血病所必需的。在同样为Runx 1-/-或具有半显性阴性Runx 1等位基因Runx 1 +/lz的Cbfb+/MYH 11胚胎中，由Cbfb-MYH 11诱导的原始造血缺陷被挽救，即使Runx 1缺陷胚胎没有原始造血缺陷。 在成人的确定性造血过程中，CBF-SMMHC增加了祖细胞的增殖，并诱导了异常的白血病前祖细胞群。这些缺陷也被半显性阴性等位基因Runx 1 +/lz或条件性Runx 1 null挽救。最后，在Cbfb+/MYH 11、Runx 1 +/lz或Cbfb+/MYH 11、条件性Runx 1缺失小鼠中，白血病发展显著延迟。总体而言，我们的研究结果表明，RUNX 1活性是Cbfb-MYH 11诱导的造血缺陷和白血病发生所必需的。 在第二个具体目标中，我们研究了CHD 7和CBFB-MYH 11之间在白血病发生方面的潜在合作。染色体结构域-解旋酶-DNA结合蛋白7（CHD 7）与RUNX 1相互作用并抑制造血过程中RUNX 1的功能。我们假设CHD 7也在CBFB-MYH 11的白血病发生中发挥作用，因为CBFB-MYH 11需要RUNX 1治疗白血病。为了验证这一假设，我们将条件性Chd 7敲除小鼠（Chd 7 f/f）与Cbfb-MYH 11敲入小鼠杂交，以产生表达Cbfbeta-SMMHC但缺乏CHD 7的转基因小鼠。我们发现这些转基因小鼠的造血祖细胞数量明显低于对照小鼠，这可能是由于细胞增殖减少。重要的是，这些转基因小鼠比仅表达CBFbeta-SMMHC的小鼠发生白血病所需的时间要长得多。我们还发现CHD 7是RUNX 1-CBFbeta-SMMHC转录复合物的伴侣，并且CHD 7可以增强RUNX 1和CBFbeta-SMMHC靶基因的转录。这些数据表明CHD 7缺陷通过抑制RUNX 1调节转录和细胞增殖的活性来抑制Cbfb-MYH 11诱导的白血病发生。