Functional and translational studies of RUNX1 and CBFB in hematopoiesis

在上个财政年度，我们一直在追求这个项目的两个具体目标。它们是：特定目标1，确定Cbfb在斑马鱼HSC形成中的作用；以及特定目标2，研究家族性血小板紊乱并利用人IPSCs研究RUNX1在该疾病中的作用。 CBF&946；和RUNX1形成DNA结合的异源二聚体，它们都是在造血干细胞(HSCs)阶段确定的造血所必需的。然而，CBF在造血干细胞发展中的确切作用仍不清楚。为了分析CBF在HSCs出现和维持中的作用，我们利用锌指核酸酶(ZFN)技术产生了两个斑马鱼Cbfb缺失突变体。与我们发表的RUNX1突变胚胎类似，Cbfb-/-胚胎经历了原始造血，但缺乏明确的造血。然而，与RUNX1突变体不同的是，在Cbfb-/-胚胎中，AGM中HSCs的出现不受影响。相反，AGM中随后的HSCs动员被阻断，证据是AGM中RUNX1+HSCs的聚集以及伴随而来的尾部造血组织(CHT)中此类细胞的缺失。我们发现，在HSC发育过程中，Cbfb位于Notch途径的下游，因为Cbfb在Notch转基因胚胎中表达增加，而在Notch缺陷的心灵炸弹突变体中表达被取消。此外，用RUNX1-CBF-1相互作用的抑制剂Ro5-3335处理的胚胎，在Cbfb-/-突变体中出现了表型缺陷。总体而言，我们的数据表明CBF&946；和功能性CBF&RUNX1异源二聚体对于造血干细胞的出现并不是必需的，但对于早期终末期造血过程中的造血干细胞动员是必不可少的。(Bresciani等人，提交手稿) RUNX1基因杂合性胚系突变导致家族性血小板紊乱(FPD)，是最早发现的单倍体功能不全疾病之一。患有这种疾病的患者有有缺陷的巨核细胞发育，血小板计数低，出血时间延长，经常擦伤，在他们一生中的某个时候发展为AML的频率很高(&gt；35%)。这种疾病的临床表现强调了RUNX1在巨核细胞分化和血小板功能中的关键作用，以及它在早期造血中的作用。由于它是唯一已知的由RUNX1突变引起的遗传性疾病，FPD是研究RUNX1在人类造血中功能的一个很好的模型。此外，我们希望我们的研究最终将导致对FPD患者的更好管理，特别是以细胞疗法的形式，这对这种疾病有潜在的治愈作用。此外，为此目的开发的方法和试剂可以适用于许多其他血液病的基于细胞的治疗。 重要的是，FPD尚无动物模型可用：RUNX1杂合基因敲除动物(小鼠和斑马鱼)在巨核细胞发育方面没有缺陷，也不会患白血病。诱导多能干细胞(IPSC)技术是21世纪头10年生物学和医学领域最重要的进展之一。IPSCs具有分化为人体任何细胞类型的潜力，因此它们可以用来模拟许多人类疾病。由于没有合适的动物模型来研究FPD，FPD中的造血缺陷有可能在细胞培养中复制或建模。 我们已经从2例RUNX1基因Y260X突变的FPD患者的皮肤成纤维细胞中建立了IPSC系。我们证明，在体外，FPD iPSCs在造血分化方面表现出缺陷，特别是在巨核细胞分化方面。然后，我们进行了锌指核酸酶(ZFN)介导的基因打靶，以纠正其中一个FPD iPSC株的突变。我们可以证明，ZFN介导的突变纠正挽救了FPD表型，CD41+CD42+巨核细胞数量增加。我们使用IPSC系建立FPD模型的创新方法，能够通过基因靶向纠正突变，为FPD的翻译研究提供了一个独特的工具。我们将进行分子表征，以了解RUNX1调控巨核细胞生成的机制和FPD iPSCs中的潜在缺陷。(权等人，手稿正在准备中)

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)