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和RUNX1在斑马鱼造血干细胞(HSCs)形成中的作用；以及特定目标2，利用人类诱导的多能干细胞(IPSCs)研究家族性血小板紊乱(FPD)和RUNX1在这种疾病中的作用。 在第一个目的中，我们研究了CBFbeta和RUNX1在斑马鱼早期造血中的作用。CBFbeta和RUNX1形成DNA结合的异源二聚体，都是生产HSC所必需的。然而，CBFbeta在造血干细胞产生中的确切作用仍不清楚。我们利用锌指核酸酶技术获得并鉴定了两个斑马鱼Cbfb缺失突变体。Cbfb-/-胚胎缺乏明确的造血功能。与未形成HSCs的RUNX1突变体不同，新生的RUNX1+/c-MYB+HSCs是在Cbfb-/-胚胎中形成的。然而，新生的HSCs没有从主动脉-性腺-中肾(AGM)区释放出来。此外，用RUNX1-CBFβ相互作用抑制剂Ro5-3335处理的野生型胚胎，在Cbfb-/-突变体中表现为造血缺陷，而不是在RUNX1-/-突变体中。最后，我们发现在HSC发育过程中，Cbfb位于Notch途径的下游。我们的数据表明，RUNX1和Cbfb在早期HSC发育的两个不同步骤中都是必需的。这项研究最近发表在《血液》杂志上。(Bresciani等人，《血液124：70,2014》)。 此外，我们之前已经证明，在RUNX1(runx1W84X/W84X)中具有ENU诱导截断突变的纯合斑马鱼能够从幼体无血阶段恢复并发展成鱼最终的造血，这表明形成了独立于RUNX1的成体HSCs(Sood等人，血液115：2806,2010)。因此，在上个财政年度，我们利用TALEN技术产生了两个新的RUNX1突变体，以进一步研究是否存在RUNX1非依赖途径(S)用于成年HSC的形成。 在第二个目的中，我们正在使用人类IPSC模型来研究家族性血小板紊乱(FPD)，这是一种由RUNX1基因杂合胚系突变引起的血液疾病，这是第一批已知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。IPSC技术是21世纪头十年生物学和医学领域最重要的进步之一。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。 我们从一个FPD家族中的两名患者中分离出了诱导多能干细胞(IPSCs)，并发现其在体外的巨核细胞分化方面存在缺陷。我们通过锌指核酸酶技术介导的基因打靶技术，纠正了一株FPD iPSC的RUNX1突变，使培养的iPSC的巨核细胞功能正常化。我们的结果证明了用患者特定的IPSCs成功地建立了FPD的体外模型，并证实了RUNX1突变是FPD患者巨核细胞缺陷的原因。这些发现刚刚被接受发表在《血液》杂志上(Connelly等人，《血液》，印刷前的e-pub)。

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 and RUNX1 in the formation of hematopoietic stem cells (HSCs) in zebrafish; and specific aim 2, Studying the familial platelet disorder (FPD) and the role of RUNX1 in this disease using human induced pluripotent stem cells (iPSCs). In the first aim we study the role of CBFbeta and RUNX1 during early hematopoiesis in zebrafish. CBFbeta and RUNX1 form a DNA-binding heterodimer and are both required for HSC production. However, the exact role of CBFbeta in the production of HSCs remains unclear. We generated and characterized two zebrafish cbfb null mutants using zinc finger nuclease technology. The cbfb-/- embryos lacked definitive hematopoiesis. Unlike runx1 mutants in which HSCs are not formed, nascent, runx1+/c-myb+ HSCs were formed in cbfb-/- embryos. However, the nascent HSCs were not released from the aorta-gonad-mesonephros (AGM) region. Moreover, wild type embryos treated with an inhibitor of RUNX1-CBFbeta interaction, Ro5-3335, phenocopied the hematopoietic defects in cbfb-/- mutants, rather than those in runx1-/- mutants. Finally, we found that cbfb was downstream of the Notch pathway during HSC development. Our data suggest that runx1 and cbfb are required at two different steps during early HSC development. This research has been recently published in Blood. (Bresciani et al., Blood 124: 70, 2014). Moreover, we previously showed that zebrafish homozygous for an ENU-induced truncation mutation in runx1 (runx1W84X/W84X) were able to recover from a larval bloodless phase and develop adult definitive hematopoiesis, suggesting the formation of runx1-independent adult HSCs (Sood et al., Blood 115:2806, 2010). Therefore, in the last fiscal year we generated two new runx1 mutants using the TALEN technology in order to further investigate if RUNX1-independent pathway(s) exists for the formation of adult HSCs. In the second aim we are using the human iPSC model to study familial platelet disorder (FPD), a blood disease caused by heterozygous germline mutations in RUNX1, 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 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 derived induced pluripotent stem cells (iPSCs) from two patients in a family with FPD, and found that the FPD iPSCs display defects in megakaryocytic differentiation in vitro. We corrected the RUNX1 mutation in one FPD iPSC line through gene targeting, mediated by zinc finger nuclease technology, which led to normalization of megakaryopoiesis of the iPSCs in culture. Our results demonstrate successful in vitro modeling of FPD with patient-specific iPSCs and confirm that RUNX1 mutations are responsible for megakaryopoietic defects in FPD patients. These findings have just been accepted for publication in Blood (Connelly et al., Blood, e-pub ahead of print).