Mechanistic and translational studies of CBF leukemia

急性髓系白血病(AML)是一种具有多种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形成异源二聚体来调节基因表达，它们都是从斑马鱼到人的脊椎动物造血所必需的。广泛的临床研究表明，Cbfb-MYH11和RUNX1-ETO这两个在CBF白血病中常见的融合基因是CBF白血病患者诊断、预后和残留疾病监测的最佳生物标志物。尽管CBF白血病比大多数AML患者有更好的初始缓解率和预后，但目前的化疗与显著的发病率和死亡率有关，长期生存(&gt；5年)只有50-60%左右。 多年来，我们利用小鼠模型和各种研究工具对Cbfb-MYH11融合基因进行了表征，确定了编码蛋白CBFbeta-SMMHC对正常造血的影响，并了解了与融合基因相关的白血病发生过程。我们已经建立了常规和条件敲入小鼠模型来研究Cbfb-MYH11。利用这些小鼠模型，我们证明了Cbfb-MYH11在确定的造血过程中主要抑制RUNX1和Cbfb的功能，导致杂合子Cbfb-MYH11敲击胚胎完全缺乏确定的造血。我们还表明Cbfb-MYH11对白血病是必要的，但不是充分的，我们能够在小鼠模型中识别协同遗传事件。我们已经建立了表达截短的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认为是了解Cbfb-MYH11白血病的主要贡献者。 在上一财年，我们主要使用小鼠模型来研究Cbfb-MYH11的白血病发生机制。在第一个特定目的中，我们通过Cbfb-MYH11确定RUNX1是否在白血病发生中起重要作用。以前，正常的RUNX1和CBF功能的显性负抑制被认为是CBF-SMMHC的潜在机制。然而，最近我们发现Cbfb-MYH11敲门胚胎存在原始造血缺陷，这些缺陷似乎不是RUNX1抑制造成的(Hyde等人，布拉德，2010)。此外，与表达全长CBF-SMMHC蛋白的小鼠相比，表达RUNX1结合能力降低的修饰CBF-SMMHC蛋白的敲门小鼠发生白血病的速度更快(Kamikubo等人，癌细胞，2010)。这些发现表明，RUNX1抑制对白血病的发生可能并不重要，并增加了CBF-SMMHC可能诱导独立于RUNX1的白血病的可能性。为了验证这一假设，我们使用了三种RUNX1缺陷模型来确定RUNX1是否是CBF-SMMHC发生白血病所必需的。在RUNX1-/-的Cbfb+/MYH11胚胎或RUNX1+/LZ为半显性阴性RUNX1+/LZ的胚胎中，即使RUNX1缺陷的胚胎没有原始的造血缺陷，Cbfb-MYH11诱导的原始造血缺陷也被挽救了。在成人明确的造血过程中，CBF&#946；-SMMHC促进了祖细胞的增殖并诱导了异常的白血病前期祖细胞群体。这些缺陷也被半显性负等位基因RUNX1+/LZ或条件性RUNX1空修复。最后，Cbfb+/MYH11、RUNX1+/LZ或Cbfb+/MYH11条件性RUNX1缺失小鼠的白血病发展明显延迟。总体而言，我们的发现表明RUNX1活性是Cbfb-MYH11诱导的造血缺陷和白血病发生所必需的。 在第二个特定目标中，我们研究了CHD7和Cbfb-MYH11在白血病发生中的潜在合作。染色域-解旋酶-DNA结合蛋白7(CHD7)与RUNX1相互作用，在造血过程中抑制RUNX1的功能。我们推测CHD7也在Cbfb-MYH11的白血病发生中起作用，因为Cbfb-MYH11需要RUNX1来治疗白血病。为了验证这一假设，我们将条件CHD7基因敲除小鼠(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数量明显低于对照小鼠，这可能是由于细胞增殖减少所致。重要的是，与只表达CBFbeta-SMMHC的小鼠相比，这些转基因小鼠患白血病的时间要长得多。我们还发现CHD7是RUNX1-CBFbeta-SMMHC转录复合体的合作伙伴，CHD7可以促进RUNX1和CBFbeta-SMMHC靶基因的转录。这些结果表明CHD7缺乏通过抑制RUNX1的转录调节和细胞增殖活性来抑制Cbfb-MYH11诱导的白血病的发生。

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.