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。 小鼠条件性敲入模型使我们能够研究白血病前造血细胞的变化。基因表达谱显示，Gata2是一种造血转录因子，在白血病前期Cbfb-MYH11敲打小鼠中表达上调，并在人类inv(16)AML中表达。另一方面，我们也发现了复发的人类CBF-AML患者中GATA2的复发性单等位基因缺失。为了阐明Gata2在Cbfb-MYH11诱发白血病中的作用，我们建立了Gata2杂合敲除的条件性Cbfb-MYH11敲除小鼠。GATA2杂合基因敲除减少了能够在Cbfb-MYH11小鼠中诱发白血病的异常髓系祖细胞。因此，带有Gata2杂合基因敲除的Cbfb-MYH11小鼠发生白血病的潜伏期比那些Gata2基因完整的小鼠更长。有趣的是，带有Gata2杂合基因敲除的白血病细胞在原代和移植小鼠中获得了更多的突变数量，并显示出更具侵袭性的表型。此外，在竞争性移植实验中，带有Gata2杂合基因敲除的白血病细胞表现出更高的再繁殖能力。总之，在Cbfb-MYH11敲门小鼠中，Gata2活性的降低影响了白血病延迟发病的白血病的突变动力学，但矛盾的是，它导致了更具侵袭性的白血病表型，这可能与人类患者的白血病复发或预后不良有关。 通常认为Cbfb-MYH11编码的融合蛋白CBFbeta-SMMHC是RUNX1的主要负阻遏子，与CBFbeta和CBFbeta-SMMHC发生物理作用。然而，RUNX1在CBFβ-SMMHC诱导的白血病发生中的确切作用尚不清楚。为了解决这个问题，我们产生了基于Cre的条件性RUNX1基因敲除和Cbfb-MYH11敲除的小鼠，它们在pIpC(PolyI：C)处理后表达Cbfb-MYH11，但没有RUNX1，以诱导Cre表达。这些表达RUNX1基因的Cbfb-MYH11小鼠在pIpC治疗后一年内没有发生白血病，而所有表达RUNX1正常的Cbfb-MYH11小鼠都发生了白血病，中位生存期为4个月。结果表明，RUNX1在Cbfb-MYH11诱导的白血病发生中起着不可或缺的作用。为了了解RUNX1在Cbfb-MYH11诱导的白血病发生中的作用机制，我们研究了导致白血病细胞产生的异常髓系祖细胞(AMPs)。在表达RUNX1基因的Cbfb-MYH11小鼠中，在pIpC表达后4周，AMP开始减少并消失，提示这是这些小鼠白血病发生失败的关键阶段。我们对AMP群体进行了RNA-seq，我们发现超过1600个基因在表达Cbfb-MYH11的RUNX1缺陷和RUNX1熟练的小鼠之间存在差异表达。这些差异表达的基因中有许多是RUNX1靶基因。在显著丰富的基因组中，有那些与白血病干细胞相关的基因，这表明这些基因对白血病的启动能力很重要。我们正在对AMP群体进行芯片序列分析，以确定RUNX1的缺失如何破坏CBFbeta-SMMHC与靶基因的结合。上述结果表明，RUNX1是CBFbeta-SMMHC调控白血病发生关键基因所必需的。

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. The murine conditional knock-in model allows us to study the pre-leukemic changes in the hematopoietic cells. Gene expression profiling suggests that Gata2, a hematopoietic transcription factor, is a top upregulated genes in preleukemic Cbfb-MYH11 knockin mice and is expressed in human inv(16) AML. On the other hand, we have also identified recurrent monoallelic deletions of GATA2 in relapsed human CBF-AML patients. To clarify the role of Gata2 in leukemogenesis by Cbfb-MYH11, we generated conditional Cbfb-MYH11 knockin mice with Gata2 heterozygous knockout. Gata2 heterozygous knockout reduced abnormal myeloid progenitors, which are capable of inducing leukemia in the Cbfb-MYH11 mice. Consequently, Cbfb-MYH11 mice with Gata2 heterozygous knockout developed leukemia with longer latencies than those with intact Gata2. Interestingly, leukemic cells with Gata2 heterozygous knockout gained higher number of mutations and showed more aggressive phenotype in both primary and transplanted mice. Moreover, leukemic cells with Gata2 heterozygous knockout showed higher repopulating capacity in competitive transplantation experiments. In summary, reduction of Gata2 activity affects mutational dynamics of leukemia with delayed leukemia onset in Cbfb-MYH11 knockin mice, but paradoxically results in a more aggressive leukemia phenotype, which may be correlated with leukemia relapse or poor prognosis in human patients. It is generally considered that CBFbeta-SMMHC, the fusion protein encoded by CBFB-MYH11, is a dominant negative repressor of RUNX1, which physically interacts with CBFbeta and CBFbeta-SMMHC. However, the exact role of RUNX1 in leukemogenesis induced by CBFbeta-SMMHC is not clear. To address this question, we generated mice with both Cre-based conditional Runx1 knockout and Cbfb-MYH11 knockin, which express Cbfb-MYH11 but no Runx1 after pIpC (poly I:C) treatment to induce Cre expression. None of such Runx1-deficient, Cbfb-MYH11 expressing mice developed leukemia up to one year after pIpC treatment, while all Cbfb-MYH11 expressing mice with normal Runx1 developed leukemia with an median survival time of 4 months. The finding indicated that Runx1 is indispensable for Cbfb-MYH11 induced leukemogenesis. To understand the mechanism of Runx1 contribution to Cbfb-MYH11 induced leukemogenesis, we studied the abnormal myeloid progenitors (AMPs) from which the leukemia cells arise. The AMPs started to decrease and disappear 4 weeks after pIpC in Runx1-deficient, Cbfb-MYH11 expressing mice, suggesting that this is a critical stage for the failure of leukemogenesis in these mice. We performed RNA-seq on the AMP population and we found that more than 1600 genes were differential expressed between Cbfb-MYH11 expressing mice that were Runx1-deficient and those that were Runx1-proficient. Many of these differentially expressed genes were RUNX1 target genes. Among the significantly enriched gene sets were those related to leukemic stem cells, suggesting these genes are important for the leukemia initiating ability. We are preforming ChIP-seq on the AMP population to determine how loss of RUNX1 disrupts binding of CBFbeta-SMMHC to target genes. The above results suggest that RUNX1 is required for the regulation of critical genes for leukemogenesis by CBFbeta-SMMHC.