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，它们都是斑马鱼和老鼠等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。 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。使用这些模型，我们表明Cbfb-MYH11对于白血病的发生是必要的，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。 一般认为CBFbeta-SMMHC是RUNX1的显性负抑制因子。然而，最近的发现挑战了CBFbeta-SMMHC介导的RUNX1抑制模型。为了明确RUNX1在Cbfb-MYH11诱导的白血病中的作用，我们将条件RUNX1基因敲除小鼠与条件Cbfb-MYH11基因敲除小鼠进行杂交。我们发现，所有条件Cbfb-MYH11敲门小鼠在5个月内都发生了白血病，而同样失去RUNX1的条件Cbfb-MYH11敲门小鼠没有发生白血病，这种作用是细胞自主的。重要的是，在Cbfb-MYH11敲除RUNX1的条件性Cbfb-MYH11敲打小鼠中，异常髓系祖细胞(AMPs)减少并消失，AMPs是由Cbfb-MYH11在骨髓中诱导的白血病启动细胞群。对AMP细胞的RNA-SEQ分析表明，与增殖、分化抑制和白血病起始相关的基因在条件性Cbfb-MYH11敲除小鼠和同样失去RUNX1的条件性Cbfb-MYH11敲打小鼠中差异表达。此外，通过染色质免疫切割测序(CHIC-SEQ)，我们观察到RUNX1/CBFbeta-SMMHC靶基因在条件性Cbfb-MYH11敲打小鼠细胞中显著丰富，尤其是在下调的基因中，表明RUNX1和CBFbeta-SMMHC主要通过直接靶基因结合而共同发挥基因表达的激活作用。 小鼠模型的数据表明，RUNX1在Cbfb-MYH11诱导的白血病发生中是不可或缺的，它与CBFbeta-SMMHC一起调节与产生功能性AMP群体相关的关键基因。然而，从分子上讲，我们对RUNX1和CBFbeta-SMMHC如何协同工作知之甚少。因此，在本财年，我们已经开始与NHLBI的蒋建森博士团队合作，利用结构生物学工具研究RUNX1和CBFbeta-SMMHC之间的相互作用。此外，我们正在建立新的小鼠模型，使用CRISPR介导的靶向策略来标记内源RUNX1、CBFbeta和CBFbeta-SMMHC蛋白。我们希望这样的模型将有助于在小鼠的造血细胞和白血病细胞中研究这些蛋白质的功能。

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 such as zebrafish and mice. 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. 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 leukemia development process associated with the fusion gene. We have generated both conventional and conditional knock-in mouse models to study CBFB-MYH11. Using these models we showed that CBFB-MYH11 is necessary but not sufficient for leukemia development, 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 a major contributor to the understanding of CBFB-MYH11 leukemia. It is generally considered that CBFbeta-SMMHC is a dominant negative repressor of RUNX1. However, recent findings challenge the RUNX1-repression model for CBFbeta-SMMHC mediated leukemogenesis. To definitively address the role of Runx1 in CBFB-MYH11 induced leukemia, we crossed conditional Runx1 knockout mice with conditional Cbfb-MYH11 knockin mice. We found that all conditional Cbfb-MYH11 knockin mice developed leukemia in 5 months while no leukemia developed in conditional Cbfb-MYH11 knockin mice that also lost Runx1, and this effect was cell autonomous. Importantly, the abnormal myeloid progenitors (AMPs), a leukemia initiating cell population induced by Cbfb-MYH11 in the bone marrow, decreased and disappeared in conditional Cbfb-MYH11 knockin mice that also lost Runx1. RNA-seq analysis of AMP cells showed that genes associated with proliferation, differentiation blockage and leukemia initiation, were differentially expressed between conditional Cbfb-MYH11 knockin mice and conditional Cbfb-MYH11 knockin mice that also lost Runx1. In addition, with chromatin immunocleavage sequencing (ChIC-seq) assay, we observed a significant enrichment of RUNX1/CBFbeta-SMMHC target genes in cells from conditional Cbfb-MYH11 knockin mice that also lost Runx1, especially among down-regulated genes, suggesting that RUNX1 and CBFbeta-SMMHC mainly function together as activators of gene expression through direct target gene binding. The data from the mouse models indicate that RUNX1 is indispensable for Cbfb-MYH11 induced leukemogenesis by working together with CBFbeta-SMMHC to regulate critical genes associated with the generation of a functional AMP population. However, molecularly we know very little on how RUNX1 and CBFbeta-SMMHC work together. Therefore, in this fiscal year, we have started to collaborate with Dr. Jiansen Jiangs group in NHLBI to study the interaction between RUNX1 and CBFbeta-SMMHC with structural biological tools. In addition, we are generating new mouse models using CRISPR-mediated targeting strategies to tag the endogenous RUNX1, CBFbeta, and CBFbeta-SMMHC proteins. We hope such models will facilitate functional studies of these proteins in hematopoietic and leukemia cells in the mice.