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.

急性髓系白血病（AML）是一种异质性疾病，具有多种基因突变和染色体异常。核心结合因子 (CBF) 白血病，即具有影响转录因子基因 RUNX1 或 CBFB 的易位或倒位的白血病，约占成人急性髓系白血病 (AML) 的 24% 和儿童急性淋巴细胞白血病的 25%。编码的蛋白质 RUNX1 和 CBFbeta 形成异二聚体来调节基因表达，它们都是从斑马鱼到人类的脊椎动物造血所需的。大量的临床研究表明，CBFB-MYH11和RUNX1-ETO这两个CBF白血病常见的融合基因是CBF白血病患者诊断、预后和残留病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，但目前的化疗与显着的发病率和死亡率相关，并且长期生存率（>5年）仅约为50-60%。 多年来，我们利用小鼠模型和多种研究工具来表征CBFB-MYH11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敲入小鼠模型来研究 CBFB-MYH11。使用这些小鼠模型，我们证明 Cbfb-MYH11 在确定性造血过程中显着抑制 Runx1 和 Cbfb 功能，导致杂合子 Cbfb-MYH11 敲入胚胎完全缺乏确定性造血功能。我们还表明，Cbfb-MYH11 对于白血病来说是必需的，但还不够，并且我们能够在小鼠模型中识别出协同遗传事件。我们已经生成了表达截短的 Cbfb-MYH11 的敲入小鼠模型，以确定 CBFbeta-SMMHC 功能域的重要性。总体而言，我们的实验室在该领域被公认为是了解 CBFB-MYH11 白血病的主要贡献者。 在上一财年，我们重点使用小鼠模型来研究 CBFB-MYH11 的白血病发生机制。在第一个具体目标中，我们通过 CBFB-MYH11 确定 RUNX1 对于白血病发生是否重要。先前对正常 RUNX1 和 CBFβ 功能的显性负性抑制被认为是 CBFβ-SMMHC 的潜在机制。然而，最近我们发现 Cbfb-MYH11 敲入胚胎具有原始造血缺陷，这似乎不是由 RUNX1 抑制造成的（Hyde 等人，Blood，2010）。此外，表达 RUNX1 结合能力降低的修饰 CBFβ-SMMHC 蛋白的敲入小鼠比表达全长 CBFβ-SMMHC 的敲入小鼠更快患白血病（Kamikubo 等人，Cancer Cell，2010）。这些发现表明 RUNX1 抑制可能对白血病发生并不重要，并提出了 CBFβ-SMMHC 可能独立于 RUNX1 诱导白血病的可能性。 为了检验这一假设，我们使用了三个 Runx1 缺陷模型来确定 RUNX1 是否是 CBFβ-SMMHC 白血病发生所必需的。在同样为 Runx1-/- 或具有半显性阴性 Runx1 等位基因 Runx1+/lz 的 Cbfb+/MYH11 胚胎中，由 Cbfb-MYH11 诱导的原始造血缺陷得到挽救，即使 Runx1 缺陷胚胎不具有原始造血功能缺陷。 在成人的最终造血过程中，CBFβ-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。这些缺陷也可以通过半显性阴性等位基因 Runx1+/lz 或条件 Runx1 null 来挽救。最后，Cbfb+/MYH11、Runx1+/lz 或 Cbfb+/MYH11、条件性 Runx1 缺失小鼠的白血病发展显着延迟。总的来说，我们的研究结果表明 RUNX1 活性是 Cbfb-MYH11 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 CHD7 和 CBFB-MYH11 在白血病发生方面的潜在合作。染色质结构域解旋酶 DNA 结合蛋白 7 (CHD7) 与 RUNX1 相互作用并在造血过程中抑制 RUNX1 功能。我们假设 CHD7 也在 CBFB-MYH11 的白血病发生中发挥作用，因为 CBFB-MYH11 需要 RUNX1 来治疗白血病。为了检验这一假设，我们将条件 Chd7 敲除小鼠 (Chd7f/f) 与 Cbfb-MYH11 敲除小鼠杂交，产生表达 Cbfbeta-SMMHC 但缺乏 CHD7 的转基因小鼠。我们发现这些转基因小鼠的造血祖细胞群明显低于对照小鼠，这可能是由于细胞增殖减少所致。重要的是，这些转基因小鼠发展为白血病的时间比仅表达 CBFbeta-SMMHC 的小鼠要长得多。我们还表明，CHD7 是 RUNX1-CBFbeta-SMMHC 转录复合物的伙伴，并且 CHD7 可以增强 RUNX1 和 CBFbeta-SMMHC 靶基因的转录。这些数据表明，CHD7 缺陷通过抑制 RUNX1 调节转录和细胞增殖的活性来抑制 Cbfb-MYH11 诱导的白血病发生。