Functional and translational studies of RUNX1 and CBFB in hematopoiesis

RUNX1和Cbfb不仅在白血病发生中起重要作用，而且也是正常造血的关键调节因子。这两个基因在造血干细胞形成的最早阶段以及许多血统的许多后续阶段都是必需的。多项研究表明，RUNX1和Cbfb控制的正常转录程序的失调可能是白血病发生的重要机制。因此，更好地了解RUNX1/Cbfb转录程序以及RUNX1和Cbfb在正常造血中的作用将有助于更好地理解白血病的发生机制。 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。在第一个特定目标中，我们一直在确定Cbfb和RUNX1在斑马鱼造血干细胞(HSCs)形成中的作用。特别是，我们试图发现在没有RUNX1的情况下是否可以形成HSCs。以前的研究表明，在胚胎发育过程中，RUNX1是从血液内皮细胞中产生确定的造血干细胞(HSCs)所必需的。例如，RUNX1基因敲除的小鼠胚胎缺乏所有确定的血统，不能存活超过胚胎第13天。然而，我们最近的研究表明，在RUNX1(runx1W84X/W84X)中存在ENU诱导的无义突变的纯合斑马鱼能够从幼虫无血阶段恢复并发育成具有多血统造血功能的可生育成体，这表明形成了独立于RUNX1的成体造血干细胞。然而，我们的发现是基于单一的斑马鱼突变系，这需要在独立的突变体中进行验证。 为了进一步研究成体造血干细胞的形成是否存在RUNX1非依赖的途径，我们利用转录激活器样效应核酸酶(TALEN)技术产生了两个新的RUNX1突变体。这些突变导致移码和过早终止，导致RUNX1(RUNX1-/-)功能丧失。与之前描述的RUNX1基因敲除突变相似，这些新的RUNX1/-突变胚胎未能发育出明确的造血。共聚焦显微镜的延时记录证实在RUNX1-/-胚胎中没有HSC的出现。RUNX1-/-幼虫在受精后8~14天失去循环原始血细胞并失去血液。然而，一周后，他们逐渐恢复了循环中的血细胞。最终，大约40%的RUNX1-/-突变体发育成具有多血统循环血细胞的可育成人。综上所述，我们的数据与之前描述的runx1W84X/W84X表型一致，并支持存在RUNX1不依赖于RUNX1的HSC形成和明确的造血机制。在下一财年，我们将进行研究，以发现独立于RUNX1的HSC的形成途径。 在第二个目的中，我们一直在使用人诱导多能干细胞(IPSCs)来研究RUNX1在人类造血中的功能。我们从家族性血小板紊乱的患者身上培养出了IPSCs，这是一种常染色体显性疾病，具有血小板数量减少和白血病易感性增加的特点。我们还一直在与国家高级翻译科学中心的科学家合作，研究将IPSCs分化为造血细胞的培养条件。最后，我们一直在使用基因组技术来确定IPSCs的基因组完整性。近年来，ipscs的基因组完整性得到了广泛的研究，其中一个核心问题仍然是ipscs基因组变异的起源。以前的研究已经报道了关于ipscs何时发生基因组变异的相互矛盾的结果，无论这些变异是从父母的体细胞遗传来的，还是在重新编程过程中产生的。我们采用了一种独特的方法来确定基因组变异的历史，通过从相同的亲本成纤维细胞中获得单细胞克隆性成纤维细胞系和克隆IPSC系，并应用下一代测序技术来检测DNA序列变异。此外，我们通过SNP阵列询问了更大的DNA改变，以确定每个样本中的结构变异。全外显子组测序(WES)和靶向深度重测序显示，在WES子代细胞中最初被鉴定为从头变异体的大多数单核苷酸变异体和小内含子变异体是罕见的预先存在的变异体，等位基因频率非常低。深度重新测序显示，在WES中发现的全部变异中，只有不到0.1%在亲本成纤维细胞中完全缺失。与亲代成纤维细胞相比，IPSC和成纤维细胞亚克隆中往往会出现拷贝数变异(CNV)，但每个IPSC/成纤维细胞亚克隆只携带1-3个CNV。总体而言，我们的数据显示，在iPSCs中存在的99.9%以上的基因组变异是从双亲体细胞遗传的，而不是作为重新编程的结果。

RUNX1 and CBFB are not only important for leukemogenesis but they are also key regulators of normal hematopoiesis. These two genes are required during the earliest steps of hematopoietic stem cell formation and in many subsequent stages of many blood lineages. Multiple studies suggest that dysregulation of the normal transcriptional program controlled by RUNX1 and CBFB is likely to be an important mechanism for leukemogenesis. Therefore, better understanding of the RUNX1/CBFB transcriptional program and the roles of RUNX1 and CBFB in normal hematopoiesis will lead to better understanding of the mechanisms for leukemogenesis. We have been pursuing two specific aims in this project in the last fiscal year. In the first specific aim, we have been determining the roles of CBFB and RUNX1 in the formation of hematopoietic stem cells (HSCs) in zebrafish. In particular, we tried to discover if HSCs could form in the absence of RUNX1. Previous studies suggest that RUNX1 is required for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium during embryo development. For example, Runx1 knockout mouse embryos lack all definitive blood lineages and cannot survive past embryonic day 13. However, we recently showed that zebrafish homozygous for an ENU-induced nonsense mutation in runx1 (runx1W84X/W84X) were able to recover from a larval bloodless phase and develop to fertile adults with multi-lineage hematopoiesis, suggesting the formation of runx1-independent adult HSCs. However, our finding was based on a single zebrafish mutant line, which requires verification in independent mutants. In order to further investigate if a RUNX1-independent pathway exists for the formation of adult HSCs, we generated two new runx1 mutants using the transcription activator-like effector nuclease (TALEN) technology. These mutations cause frameshifts and premature terminations, resulting in loss of function of runx1 (runx1-/-). Similar to previously described RUNX1 knockout mutants, these new runx1-/- mutant embryos failed to develop definitive hematopoiesis. Time-lapse recordings with confocal microscopy confirmed the absence of HSC emergence in the runx1-/- embryos. The runx1-/- larvae lost circulating primitive blood cells and became bloodless between 8 and 14 days post fertilization (dpf). However they gradually regained circulating blood cells one week later. Eventually, about 40% of runx1-/- mutants developed to fertile adults with circulating blood cells of multi-lineages. Taken together, our data is consistent with the previously described runx1W84X/W84X phenotype and supports the existence of a runx1-independent mechanism for HSC formation and definitive hematopoiesis. In the next fiscal year we will conduct research to discover the pathway for the RUNX1-independent HSC formation. In the second aim we have been using human induced pluripotent stem cells (iPSCs) to study the function of RUNX1 in human hematopoiesis. We have generated iPSCs from patients with familial platelet disorder, an autosomal dominant disease with reduced platelet numbers and increased susceptibility to leukemia. We have also been working on the culturing conditions for the differentiation of iPSCs to hematopoietic cells, in collaboration with scientists at the National Center for Advancing Translational Sciences. Finally, we have been using genomic technology to determine the genomic integrity of iPSCs. Genomic integrity of iPSCs has been extensively studied in recent years and one central question that still remains is the origin of genomic variations in iPSCs. Previous studies have reported conflicting results regarding when genomic variations occur in iPSCs, whether they are inherited from the parental somatic cells, or generated during the reprogramming process. We have undertaken a unique approach to determine the history of genomic variations by deriving single cell clonal fibroblast lines and clonal iPSC lines from the same parental fibroblasts and applied next generation sequencing technologies to detect the DNA sequence variations. Moreover, we interrogated larger DNA alterations by SNP arrays to identify structural variations in each samples. Whole exome sequencing (WES) followed by targeted deep re-sequencing revealed that most single nucleotide variants and small indels that were initially identified as de novo variants in the daughter cells from WES were rare pre-existing variants with very low allele frequency. Deep re-sequencing showed that only less than 0.1% of total variants discovered from WES were completely absent in the parental fibroblasts. Copy number variations (CNVs) tend to have emerged de novo in the iPSCs and fibroblast subclones than inherited from the parental fibroblasts, but each iPSC/fibroblast subclone carried only 1-3 CNVs. Overall our data showed that more than 99.9% of genomic variations present in iPSCs are inherited from parental somatic cells rather than as consequences of reprogramming.