Functional and translational studies of RUNX1 and CBFB in hematopoiesis

RUNX1和CBFB在造血中的功能和转化研究

• 批准号: 9572258
• 负责人: PU PAUL LIU
• 金额: $ 70.48万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9572258
• 关键词: Address; Adult; Animal Model; Biological; Bleeding time procedure; Blood; Blood Cells; Blood Platelet Disorders; Blood Platelets; CBFB gene; CD34 gene; CRISPR/Cas technology; Cell Culture System; Cell Line; Cell Therapy; Cells; Chemicals; Clinical; Code; Collaborations; Collection; Communities; Complement; Contusions; Core-Binding Factor; Cultured Cells; Data; Defect; Development; Diagnosis; Disease; Disease model; Embryo; Embryonic Development; Endothelial Cells; Endothelium; Ethylnitrosourea; Exons; Fibroblasts; Fishes; Frequencies; Gene Expression; Generations; Genes; Genetic; Genetic Transcription; Genetic study; Genomic approach; Genomics; Goals; Hematologic Neoplasms; Hematological Disease; Hematologist; Hematopoiesis; Hematopoietic; Hematopoietic System; Hematopoietic stem cells; Human; Image; In Situ; Intramural Research Program; Kidney; Knockout Mice; Lead; Learning; Libraries; Life; Luciferases; MYB gene; Manuscripts; Methodology; Methods; Modeling; Molecular; Monitor; Mosaicism; Mus; Mutate; Mutation; Myelogenous; National Human Genome Research Institute; Nonsense Mutation; Pathogenesis; Pathway interactions; Patients; Pharmacologic Substance; Phase; Phenotype; Platelet Count measurement; Play; Population; Process; Production; Proteins; Proto-Oncogene Proteins c-myb; Publishing; RUNX1 gene; Recurrence; Regulation; Reporter; Reporting; Resources; Risk; Role; Sampling; Scientist; Sister; Stem cells; Technology; Testing; Tissues; Transgenic Animals; Transgenic Organisms; Translating; Translational Research; United States National Institutes of Health; Variant; Zebrafish; base; clinical practice; disease-causing mutation; early embryonic stage; genetic approach; genome editing; genome integrity; genomic tools; hematopoietic stem cell fate; improved; induced pluripotent stem cell; knockout animal; leukemia; leukemia treatment; leukemogenesis; mouse model; mutant; next generation sequencing; programs; reconstitution; screening; transcription factor; transcriptome sequencing; translational study; tumorigenesis; zinc finger nuclease

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 subsequent stages of several 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 studying the role of RUNX1 in the formation of hematopoietic stem cells (HSCs) in zebrafish. In particular, we tried to discover how 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. Surprisingly, we previously discovered 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 have generated three new runx1 mutants using the TALEN and CRISPR-Cas9 technologies. Two of the runx1 mutant lines carry mutations in exon 4 (a deletion of 8 bp, runx1del8/del8, and a deletion of 25 bp, runx1del25/del25), both of which truncate the runt-homologous domain. The third mutation is a large deletion of exons 3 through 8 (runx1del(e3-8)/del(e3-8)), which removes most of the coding region of runx1. All three runx1 mutant lines recapitulated our previous observations as the runx1 mutant embryos lacked the expression of the HSC marker c-myb and failed to initiate definitive hematopoiesis during early embryonic development. However, approximately 40% of the runx1-/- embryos developed into fertile adults with circulating blood cells of multi-lineages, further supporting the presence of RUNX1-independent mechanisms for the generation of HSCs. Live confocal imaging revealed the presence of hematopoietic progenitor cells in the runx1- /- mutants at early stages of embryonic development. Transcriptional profiling of these hematopoietic precursors at both bulk and single cell levels by RNAseq showed that the runx1-null hematopoietic progenitors are different from wildtype cells in global gene expression. On the other hand, the transcriptional profile of the hematopoietic cells in adult kidney is similar between runx1-/- and wildtype adult fish, except for some key myeloid and thrombocyte genes, which were downregulated in the runx1- /- mutants. Taken together, we can now provide four independent evidences that RUNX1-independent pathways for HSC formation and definitive hematopoiesis exist. 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 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. Hematopoietic diseases are an attractive target for iPSC-based cell therapy, because of the ability of hematopoietic stem cells (HSCs) to reconstitute the entire hematopoietic system. However, directing differentiation of iPSCs towards transplantable HSCs has proven to be difficult. In order to make it possible to treat patients suffering from blood disorders using patient-specific iPSCs, there is a great need to establish efficient methods to differentiate human iPSCs to HSCs. iPSC reporter lines that will allow testing and monitoring of directed hematopoietic differentiation of iPSCs to an HSC fate can be very useful for developing such methods. We hypothesize that RUNX1 is a good marker gene for HSC formation since RUNX1 is among the first expressed genes when hemogenic endothelial cells become committed to HSCs. Therefore, we have developed human RUNX1 reporter iPSC lines in which either the luciferase or the tdTomato cassette is inserted to the RUNX1 locus through genome editing using Zinc Finger Nucleases. These RUNX1-reporter lines have been shown to express RUNX1-luciferase or RUNX1-tdTomato protein upon hematopoietic differentiation. We have performed small compound library screening using LOPAC1280 and NCATS Pharmaceutical Collection (NPC) with the established RUNX1-reporter iPSC lines to identify compounds that enhance hematopoietic differentiation. We identified and validated 120 small compounds that were able to enhance RUNX1 expression from the RUNX1-reporter iPSC lines. The top compounds were then further tested for their potential to stimulate hematopoietic differentiation in iPSCs and in zebrafish. Several compounds were able to enhance hematopoietic differentiation potential, as evidenced by increased production of CD34+ cells from iPSCs in culture and increased production of cmyb+ HSCs in the AGM region in zebrafish embryos. Our results demonstrate successful establishment of RUNX1-reporter iPSC lines that can be used to optimize conditions for hematopoietic differentiation and to perform high-throughput chemical screening to identify compounds for more efficient generation of HSCs from iPSCs. Finally, we have been using genomic technology to determine the genomic integrity of iPSCs. Specifically we have been trying to address two important questions. First, if the iPSCs harbor more mutations than other cultured cells due to reprogramming process; and second, where the mutations coming from. From the same fibroblast populations we generated iPSCs and fibroblast subclones, which are identical to each other in terms of their tissue origin and the way they were derived, except for the treatment of reprogramming factors in the case of the iPSC generation. We then performed NextGen sequencing analysis of the iPSCs and fibroblast subclones to detect mutations. Using this approach we were able to compare the mutation profile of the iPSCs with that of the fibroblast subclones, and provide a definitive answer tothe question of if iPSCs have increased mutation burden. Our data reveal that iPSCs have comparable numbers of mutations as their sister fibroblast subclones. Moreover, we demonstrated that >90% of the mutations detected in the iPSCs and the fibroblast subclones were rare, pre-existing, mosaic variants in the parental fibroblast population. Our data therefore strongly demonstrate that iPSC reprogramming is not mutagenic and iPSCs do not contain increased mutation burden. A manuscript reporting these findings has been published earlier this year (Kwon et al., PNAS 114:1964, 2017).

RUNX1和CBFB不仅对白血病生成很重要，而且还是正常造血的关键调节剂。这两个基因是在造血干细胞形成的最早步骤以及几个血统阶段的最早步骤中。多项研究表明，由RUNX1和CBFB控制的正常转录程序失调可能是白血病发生的重要机制。因此，更好地理解RUNX1/CBFB转录程序以及RUNX1和CBFB在正常造血中的作用将使人们更好地了解白血病的机制。 在上一个财政年度，我们一直在该项目中追求两个具体目标。在第一个特定目的中，我们一直在研究runx1在斑马鱼中造血干细胞（HSC）形成中的作用。特别是，我们试图发现在没有RUNX1的情况下如何形成HSC。先前的研究表明，在胚胎发育过程中，来自血肿内皮细胞的定性造血干细胞（HSC）是必需的。例如，runx1敲除小鼠胚胎缺乏所有明确的血统，在胚胎第13天无法生存。令人惊讶的是，我们以前发现，斑马鱼纯合子对于ENU引起的runx1中的胡说突变（Runx1w84x/w84x），可以从较大的血液中恢复过多，并提出了型成年人的次数，并提出了多种肥料的成年人。独立于Runx1的成人HSC。 However, our finding was based on a single zebrafish mutant line, which requires verification in independent mutants. 为了进一步研究成人HSC的形成是否存在runx1独立的途径，我们使用TALEN和CRISPR-CAS9技术生成了三个新的Runx1突变体。 Runx1突变线中的两个携带突变（删除8 bp，runx1del8/del8和25 bp，runx1del25/del25），这两者都截断了Runt-holdomologusous域。第三个突变是外显子3至8（runx1del（e3-8）/del（e3-8））的大删除，该突变删除了Runx1的大多数编码区域。所有三个Runx1突变线都概括了我们先前的观察结果，因为Runx1突变体胚胎缺乏HSC标记C-MYB的表达，并且在早期胚胎发育过程中未能引发明确的造血作用。然而，大约40％的Runx1 - / - 胚胎发展成具有多线循环的血细胞的肥大成年人，进一步支持了与Runx1无关的HSC生成的机制。 实时共聚焦成像揭示了在胚胎发育的早期阶段的Runx1- / - 突变体中存在造血祖细胞。这些造血前体在整体和单细胞水平上通过RNASEQ对这些造血前体进行了转录分析表明，Runx1-Null造血祖细胞与全球基因表达中的野生型细胞不同。另一方面，除了在Runx1- / - 突变体中下调的一些关键的髓样和血小板基因外，成年肾脏中造血细胞的转录分布相似。 综上所述，我们现在可以提供四个独立的证据，即HSC形成和确定的造血症的独立途径存在。 在第二个目标中，我们一直在使用人类诱导的多能干细胞（IPSC）来研究Runx1在人造血中的功能。我们一直在研究与国家前进的转化科学中心的科学家合作的培养条件，以将IPSC分化为造血细胞。造血疾病是基于IPSC的细胞疗法的有吸引力的靶标，因为造血干细胞（HSC）能够重建整个造血系统。 但是，事实证明，将IPSC的分化指向可移植的HSC是很困难的。为了使使用患者特异性IPSC治疗患有血液疾病的患者成为可能，需要建立有效的方法将人IPSC与HSC区分开来。 IPSC记者线将允许对IPSC的有向造血分化对HSC命运进行测试和监测对于开发此类方法非常有用。 我们假设Runx1是HSC形成的良好标记基因，因为Runx1是血液生成内皮细胞致力于HSC时的第一个表达基因之一。因此，我们已经开发了人类Runx1报告基因IPSC系，其中使用锌指核酸酶通过基因组编辑将荧光素酶或TDTOMATO盒插入Runx1基因座。这些Runx1-Reporter系已显示出造血分化后表达Runx1-荧光素酶或Runx1-TDTOMATO蛋白。 我们使用LOPAC1280和NCATS Pharmaceutical Collection（NPC）进行了小型复合库筛选，并使用已建立的Runx1-Reporter IPSC系列进行了识别增强造血分化的化合物。 我们确定并验证了120个小型化合物，这些化合物能够从Runx1-Reporter IPSC系列增强Runx1的表达。然后进一步测试了顶部化合物的刺激IPSC和斑马鱼中造血分化的潜力。 几种化合物能够增强造血分化潜力，这证明了斑马鱼胚胎中AGM区域中IPSC的CD34+细胞的产生增加，并增加了CMYB+ HSC的产生。 我们的结果表明，成功建立了RUNX1-REPORTER IPSC系列，可用于优化造血分化条件并进行高通量化学筛选，以识别化合物，以从IPSC中更有效地生成HSC。 最后，我们一直在使用基因组技术来确定IPSC的基因组完整性。具体来说，我们一直在尝试解决两个重要问题。首先，如果由于重编程过程而与其他培养的细胞相比，IPSC具有更多的突变；其次，突变来源。从相同的成纤维细胞群体中，我们产生的IPSC和成纤维细胞亚克隆，除了在IPSC生成的情况下，除了治疗重编程因子外，它们的组织起源及其衍生方式相同。然后，我们对IPSC和成纤维细胞亚克隆进行了NextGen测序分析以检测突变。 使用这种方法，我们能够将IPSC的突变曲线与成纤维细胞亚克隆的突变曲线进行比较，并提供了一个确定的答案，即IPSC是否增加了突变负担。 我们的数据表明，IPSC具有相当数量的突变，它们是其姐妹成纤维细胞子克隆。 此外，我们证明了在IPSC中检测到的突变的> 90％，成纤维细胞亚克隆很少见，在父母成纤维细胞种群中已有预先存在的马赛克变体。因此，我们的数据强烈表明IPSC重编程不是诱变，而IPSC不包含增加的突变负担。报道这些发现的手稿已于今年早些时候发表（Kwon等，PNAS 114：1964，2017）。