The Cebpa Enhancer in Normal Hematopoiesis and Progression to AML

正常造血和 AML 进展中的 Cebpa 增强剂

基本信息

批准号：
9001485
负责人：
ALAN D FRIEDMAN
金额：
$ 40.5万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-01-01 至 2019-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9001485
关键词：
Acute Myelocytic Leukemia Adhesions Affect Alleles Aplastic Anemia Autophagocytosis Binding Biology Blast Cell Blood Cells CCAAT-Enhancer-Binding Protein-alpha CCAAT-Enhancer-Binding Proteins CEBPA gene CRISPR/Cas technology Categories Clustered Regularly Interspaced Short Palindromic Repeats Cytoplasm DNA Data Dependence Development Disease Disease Progression Dysmyelopoietic Syndromes Enhancers Epigenetic Process Exons FLT3 gene Generations Genes Genetic Transcription Health Hematopoiesis Hematopoietic stem cells Homing Homologous Gene Human In Vitro Individual Investigation Malignant Neoplasms Marrow Mediating Mediator of activation protein Metabolism Methylation Modeling Mus Mutation Myelogenous Myelopoiesis Myeloproliferative disease NUP98 gene Open Reading Frames Pancytopenia Pathway interactions Phenotype Pluripotent Stem Cells Proteins RNA RUNX1 gene Receptor Protein-Tyrosine Kinases Regulatory Element Residual state Route Site Transgenic Model Variant clinical application granulocyte in vivo mRNA Expression monocyte mouse model novel novel strategies prevent progenitor promoter stem transcription factor transcriptome sequencing

项目摘要

DESCRIPTION (provided by applicant): The C/EBPα transcription factor is a master regulator of myeloid development. A model of transformation to acute myeloid leukemia (AML) proposes requirement for type I mutations to stimulate proliferation and type II alterations to block differentiation. Type I changes include expression of Bcr-Abl, FLT3-ITD, or Ras activation. In isolation, these changes generate a myeloproliferative phenotype. We propose that reduction in C/EBPα expression is central to myeloid transformation, in de novo AMLs or those arising from MPD or MDS. We have developed a murine model in which a conserved 450 bp +37 kb Cebpa enhancer is flanked by loxP sites. RUNX1-ETO binds the homologous +41 kb CEBPA enhancer in human AMLs. The enhancer directs expression to GMP and to LT-HSC in a transgenic model, and Cre-mediated enhancer deletion leads to 10- fold reduction in Cebpa RNA and protein, with retention of GMP but impaired terminal myelopoiesis in vivo and indefinite myeloid colony replating in vitro. These phenotypes are reminiscent of those seen with mice expressing the truncated C/EBPαp30 variant, which develop AML by one year, though importantly our model reflects purely reduced C/EBPα activity. We hypothesize that the +37 kb Cebpa enhancer is a key mediator of normal C/EBPα expression and a key target of type II mutations in AML. We propose to evaluate the effect of enhancer deletion on normal hematopoiesis and to determine whether consequent reduction in C/EBPα leads to AML, alone or potentially more rapidly when combined with FLT3-ITD. Mice lacking C/EBPα due to biallelic open reading frame deletion do not develop AML. Residual C/EBPα retained in our murine model may be required for generation of GMP as a substrate for myeloid transformation while preventing their further maturation. Our specific aims are: AIM 1. Determine the effect of Cebpa +37 kb enhancer deletion or mutation on normal hematopoiesis. We will assess dependence of Cebpa mRNA expression on the +37 kb enhancer in stem/progenitor subsets, will assess the effect of enhancer deletion on stem/progenitor proliferation, survival, homing, and differentiation, and will assess the effect of enhancer mutations via CRISPR on Cebpa expression and enhancer epigenetics. AIM 2. Determine whether enhancer deletion facilitates de novo transformation or MPD progression. We will determine whether enhancer deletion alone, mediated by Mx1-Cre or Vav-Cre, allows development of AML, will determine whether Cebpa enhancer deletion leads to AML when combined with FLT3ITD, and will use RNA-seq to identify pathways affected by enhancer deletion, alone or with FLT3ITD, in preleukemic stem/progenitors. AIM 3. Determine whether the Cebpa enhancer is targeted epigenetically in murine/human MDS/AML. We will assess pre-leukemic and leukemic enhancer activity and epigenetics in marrow expressing RUNX1-ETO9a, will conduct a similar analysis during myeloid transformation following MDS induced by NUP98-HOXD13, and will determine whether the CEBPA enhancer has reduced activating and increased repressive epigenetics in human AMLs, correlated with CEBPA mRNA expression.

描述（由适用提供）：C/EBPα转录因子是髓样发育的主要调节剂。急性髓样白血病（AML）提案对I型突变的要求的转化模型，以刺激增殖和II型变化以阻断分化。 I型更改包括BCR-ABL，FLT3-ITD或RAS激活的表达。这些变化孤立地产生了髓样增殖表型。我们认为，在从头AML或由MPD或MDS引起的那些中，C/EBPα表达的降低是髓样转化的核心。我们已经开发了一种鼠模型，其中构成450 bp +37 kb Cebpa增强子是LOXP位点的两侧。 Runx1-Eto结合人AML中的同源+41 kb CEBPA增强子。增强剂在转基因模型中将表达引导到GMP和LT-HSC，而CRE介导的增强子缺失导致CEBPA RNA和蛋白质的10倍降低，并且保留GMP，但在体内和不确定的髓类球体中的终末骨髓造成了损害，但降低了末端的骨髓骨膜。这些表型让人联想到那些表达截短的C/EBPαP30变体的小鼠看到的表型，这些变体的发展为AML一年，尽管重要的是我们的模型反映了C/EBPα活性的纯粹降低。我们假设+37 Kb CEBPA增强子是正常C/EBPα表达的关键介质，也是AML中II型突变的关键靶标。我们建议评估增强子缺失对正常造血的影响，并确定与FLT3-ITD结合使用时，C/EBPα的降低是否会导致AML，还是可能会更快。由于双重开放式阅读框架缺失而缺乏C/EBPα的小鼠不会出现AML。在我们的鼠模型中保留的残留C/EBPα可能是GMP作为髓样转化的底物所必需的，同时又可以防止其进一步的成熟。我们的具体目的是：目标1。确定CEBPA +37 Kb增强子缺失或突变对正常造血的影响。我们将评估CEBPA mRNA表达对茎/祖细胞亚集中+37 kb增强子的依赖性，将评估增强子缺失对茎/祖细胞增殖，生存，归因和分化的影响，并将通过CRISPR评估增强子突变对CEBPA表达和增强子表观遗传学的影响。目标2。确定增强子缺失设施是从头转化还是MPD进展。我们将确定仅由MX1-CRE或VAV-CRE介导的增强子删除允许AML的开发是否会确定CEBPA增强子的删除是否会导致AML与FLT3ITD结合使用，并将使用RNA-SEQ使用RNA-SEQ来识别由FLT3ITD或与PREleukeMia in preleukemia in preleukemia in preleukemia in preleukemia synancer deletion所影响的途径。 AIM 3。确定CEBPA增强子是否在鼠/人类MDS/AML中表观遗传。 We will assess pre-leukemia and leukemia enhancer activity and epigenetics in marrow expressing RUNX1-ETO9a, will conduct a similar analysis during myeloid transformation following MDS induced by NUP98-HOXD13, and will determine whether the CEBPA enhancer has reduced activating and increased reflective epigenetics in human AMLs, correlated with CEBPA mRNA expression.