Functional and translational studies of RUNX1 and CBFB in hematopoiesis

在上一个财政年度，我们一直在追求这个项目的两个具体目标。它们是：具体目标1，确定CBFB在斑马鱼HSC形成中的作用;具体目标2，使用人iPSC研究家族性血小板疾病并研究RUNX 1在该疾病中的作用。 CBF和RUNX 1形成DNA结合异二聚体，并且它们都是造血干细胞（HSC）阶段的确定性造血所需的。然而，CBF在HSC发育中的确切作用仍不清楚。为了剖析CBF在HSC的出现和维持中的作用，我们使用锌指核酸酶（ZFN）技术产生了两种斑马鱼cbfb无效突变体。与我们发表的runx 1突变胚胎相似，cbfb-/-胚胎经历了原始造血，但缺乏确定的造血。然而，与runx 1突变体不同的是，在cbfb-/-胚胎中，AGM中HSC的出现不受影响。相反，随后从AGM的HSC动员被阻断，如通过runx 1 + HSC在AGM中的积累以及伴随的尾部造血组织（CHT）中此类细胞的缺乏所证明的。我们发现，cbfb是下游的Notch途径在HSC的发展，因为cbfb的表达扩大Notch转基因胚胎，但废除Notch缺陷的思维炸弹突变体。此外，用RUNX 1-CBF相互作用的抑制剂Ro 5 -3335处理的胚胎表型模仿cbfb-/-突变体中的造血缺陷。总之，我们的数据表明，CBF和功能性CBF-RUNX 1异二聚体不是HSC出现所必需的，但对于早期确定性造血过程中HSC的动员是必不可少的。 （Bresciani等人，提交人姓名pt） RUNX 1的杂合种系突变导致家族性血小板疾病（FPD），这是最早已知的单倍不足疾病之一。患有这种疾病的患者具有缺陷的巨核细胞发育，血小板计数低，出血时间延长，频繁瘀伤，以及在其一生中的某个时间点发生AML的高频率（>35%）。该疾病的临床表现强调了RUNX 1在巨核细胞分化和血小板功能中的关键作用，以及其在早期造血中的作用。由于它是唯一已知的由RUNX 1突变引起的遗传性疾病，FPD是研究RUNX 1在人类造血中功能的良好模型。此外，我们希望我们的研究最终能够更好地管理FPD患者，特别是以细胞疗法的形式，这可能治愈这种疾病。此外，为此目的开发的方法和试剂可适用于许多其他血液病的基于细胞的疗法。 重要的是，没有动物模型可用于FPD：Runx 1杂合子敲除动物（小鼠和斑马鱼）在巨核细胞发育中没有缺陷，并且它们不会发生白血病。 诱导性多能干细胞（iPSC）技术是世纪前十年生物学和医学领域最重要的进展之一。 iPSC具有分化成人体任何细胞类型的潜力，因此它们可用于模拟许多人类疾病。由于没有合适的动物模型可用于研究FPD，FPD中的造血缺陷可以在细胞培养中复制或建模。 我们已经从2名FPD患者的皮肤成纤维细胞中建立了iPSC系，这些FPD患者在RUNX 1基因中具有Y260 X突变。我们证明，在体外，FPD iPSC显示造血分化的缺陷，特别是对巨核细胞。 然后，我们进行锌指核酸酶（ZFN）介导的基因靶向以校正FPD iPSC系之一中的突变。我们可以证明ZFN介导的突变校正挽救了FPD表型，增加了CD 41 + CD 42+巨核细胞的数量。我们使用iPSC系建立FPD模型的创新方法具有通过基因靶向校正突变的能力，为进行FPD的翻译研究提供了独特的工具。 我们将进行分子表征，以了解RUNX 1调节巨核细胞生成的机制以及FPD iPSCs中的潜在缺陷。 (Kwon例如，正在编写手册）

We have been pursuing two specific aims in this project in the last fiscal year. They are: specific aim 1, Determining the roles of CBFB in HSC formation in zebrafish; and specific aim 2, Studying familial platelet disorder and studying the role of RUNX1 in this disease using human iPSCs. CBFβ and RUNX1 form a DNA-binding heterodimer and they are both required for definitive hematopoiesis at the stage of hematopoietic stem cells (HSCs). However, the exact role of CBFβ in the development of HSCs remains unclear. To dissect the role of CBFβ in the emergence and maintenance of HSCs we generated two zebrafish cbfb null mutants using zinc finger nuclease (ZFN) technology. Similar to our published runx1 mutant embryos, cbfb-/- embryos underwent primitive hematopoiesis, but lacked definitive hematopoiesis. Unlike the runx1 mutants, however, the emergence of HSCs in the AGM was unaffected in cbfb-/- embryos. Rather, the subsequent mobilization of the HSCs from AGM was blocked, as evidenced by the accumulation of runx1+ HSCs in the AGM and the concomitant absence of such cells in the caudal hematopoietic tissue (CHT). We found that cbfb was downstream of the Notch pathway during HSC development, since cbfb expression was expanded in Notch transgenic embryos but abrogated in the Notch-deficient mind bomb mutants. Moreover, embryos treated with Ro5-3335, the inhibitor of RUNX1-CBFβ interaction, phenocopied the hematopoietic defects in the cbfb-/- mutants. Overall our data suggest that CBFβ and functional CBFβ-RUNX1 heterodimers are not required for the emergence of HSCs but are essential for the mobilization of HSCs during early definitive hematopoiesis. (Bresciani et al., Manuscript submitted) Heterozygous germline mutations in RUNX1 lead to familial platelet disorder (FPD), which is one of the first known haploinsufficiency diseases. Patients with this disorder have defective megakaryocytic development, low platelet counts, prolonged bleeding times, frequent bruises, and a high frequency (>35%) of developing AML at some point in their lifetime. The clinical manifestations of the disease underscore the critical role of RUNX1 in megakaryocyte differentiation and platelet function, in addition to its role in early hematopoiesis. Since it is the only known inherited disease caused by RUNX1 mutations, FPD is a good model to study RUNX1 function in human hematopoiesis. In addition, we hope our studies will eventually lead to better management of the FPD patients, especially in the form of cell therapy, which is potentially curative of the disease. Moreover, the approaches and reagents developed in this aim can be applicable to cell-based therapies of many other hematological diseases. Importantly, no animal models are available for FPD: Runx1 heterozygous knockout animals (both mouse and zebrafish) have no defects in megakaryocytic development and they do not develop leukemia. The induced pluripotent stem cell (iPSC) technology is one of the most important advances in biology and medicine in the first decade of the 21st century. The iPSCs have the potential to differentiate into any cell type of the human body, so they can be used to model many human diseases. Since no suitable animal models are available to study FPD, the hematopoietic defects in FPD can potentially be replicated or modeled in cell culture. We have established iPSC lines from skin fibroblasts of 2 FPD patients harboring a Y260X mutation in the RUNX1 gene. We demonstrate, in vitro, that the FPD iPSCs display defects in hematopoietic differentiation, particularly towards megakaryopoiesis. We then performed zinc finger nuclease (ZFN) mediated gene targeting to correct the mutation in one of the FPD iPSC lines. We could demonstrate that ZFN-mediated mutation correction rescued the FPD phenotype with increased number of CD41+CD42+ megakaryocytes. Our innovative approach to model FPD using iPSC lines with an ability to correct the mutation by gene targeting provide a unique tool to perform translational studies of FPD. We will perform molecular characterizations to understand the mechanisms through which RUNX1 regulates megakaryopoiesis and the underlying defects in the FPD iPSCs. (Kwon et al., Manuscript in preparation)