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和RUNX 1在斑马鱼造血干细胞（HSCs）形成中的作用;具体目标2，使用人诱导多能干细胞（iPSC）研究家族性血小板疾病（FPD）以及RUNX 1在该疾病中的作用。 在第一个目标中，我们研究了CBFbeta和RUNX 1在斑马鱼早期造血过程中的作用。CBFbeta和RUNX 1形成DNA结合异源二聚体，两者都是HSC产生所需的。然而，CBFbeta在HSC产生中的确切作用仍不清楚。我们利用锌指核酸酶技术产生了两个斑马鱼cbfb无效突变体并进行了表征。cbfb-/-胚胎缺乏明确的造血。与不形成HSC的runx 1突变体不同，新生的runx 1 +/c-myb+ HSC在cbfb-/-胚胎中形成。然而，新生的HSC没有从性腺-中肾（AGM）区域释放。此外，用RUNX 1-CBFbeta相互作用的抑制剂Ro 5 -3335处理的野生型胚胎表型模仿cbfb-/-突变体中的造血缺陷，而不是runx 1-/-突变体中的造血缺陷。最后，我们发现cbfb在HSC发育过程中位于Notch通路的下游。我们的数据表明，runx 1和cbfb需要在两个不同的步骤在早期HSC的发展。这项研究最近发表在Blood上。（Bresciani等人，Blood 124：70，2014）。 此外，我们先前表明，对于runx 1中的ENU诱导的截短突变（runx 1 W84 X/W84 X）纯合的斑马鱼能够从幼虫无血期恢复并发育成成体永久性造血，这表明形成了不依赖runx 1的成体HSC（Sood等人，Blood 115：2806，2010）。因此，在上一财年，我们使用TALEN技术产生了两种新的runx 1突变体，以进一步研究是否存在RUNX 1非依赖性途径来形成成体HSC。 在第二个目标中，我们使用人类iPSC模型来研究家族性血小板疾病（FPD），这是一种由RUNX 1中的杂合种系突变引起的血液疾病，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。 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。 我们从一个患有FPD的家族中的两名患者中获得诱导多能干细胞（iPSCs），并发现FPD iPSCs在体外巨核细胞分化中显示缺陷。 我们通过锌指核酸酶技术介导的基因靶向校正了一个FPD iPSC系中的RUNX 1突变，这导致了培养物中iPSC的巨核细胞生成的正常化。 我们的研究结果证明了用患者特异性iPSC成功地进行了FPD的体外建模，并证实了RUNX 1突变是FPD患者中巨核细胞生成缺陷的原因。 这些发现刚刚被接受发表在《血液》（Connelly et al.，血，电子出版前打印）。

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).