The Cebpa Enhancer in Normal Hematopoiesis and Progression to AML

 描述(申请人提供)：C/eBPα转录因子是髓系发育的主要调节因子。一个转化为急性髓系白血病(AML)的模型提出，I型突变需要刺激增殖，II型突变需要阻断分化。I型改变包括bcr-Abl、Flt3-ITD表达或RAS激活。孤立地，这些变化产生骨髓增殖性表型。我们认为，C/EBPα表达的减少是髓系转化的中心，无论是在新生AML中，还是在MPD或MDS中。我们已经建立了一种小鼠模型，在该模型中，保守的450bp+37kb的CEBPA增强子的两侧是loxP位点。RUNX1-ETO与人AML中同源的+41kb CEBPA增强子结合。在转基因模型中，增强子导向GMP和LT-HSC的表达，而Cre介导的增强子缺失导致CEBPA RNA和蛋白质减少10倍，保留GMP，但在体内损害了终末骨髓生成，并在体外复制了不确定的髓系集落。这些表型让人想起那些表达截短的C/eBPαP30变体的小鼠，这会在一年后发展为急性髓系白血病，尽管重要的是我们的模型反映了纯粹的C/eBPα活性降低。我们推测+37kb的CEBPA增强子是C/EBPα正常表达的关键调节因子，也是急性髓系白血病II型突变的关键靶点。我们建议评估增强子缺失对正常造血的影响，并确定C/EBPα的继发性降低是否导致急性髓系白血病，单独或联合Flt3-ITD时可能更快。双等位基因开放阅读框缺失导致C/EBPα缺失的小鼠不会发生急性髓系白血病。在我们的小鼠模型中残留的C/EBPα可能是产生GMP所必需的，作为髓系转化的底物，同时阻止它们进一步成熟。目的：1.检测CEBPA+37kb增强子缺失或突变对正常造血功能的影响。我们将评估CEBPA mRNA表达对干/祖细胞亚群中+37kb增强子的依赖性，将评估增强子缺失对干/祖细胞增殖、存活、归巢和分化的影响，并将 评估通过CRISPR的增强子突变对CEBPA表达和增强子表观遗传学的影响。目的2.确定增强子缺失是否促进从头转化或MPD进展。我们将确定单独由Mx1-CRE或Vav-CRE介导的增强子缺失是否允许AML的发生，当与FLT3ITD结合时，将确定CEBPA增强子缺失是否导致AML，并将使用RNA-SEQ来识别在白血病前干祖细胞中受增强子缺失单独或与FLT3ITD一起影响的通路。目的3.确定CEBPA增强子在小鼠/人MDS/AML中是否具有表观遗传学靶向性。我们将评估白血病前期和白血病增强子活性和骨髓中表达RUNX1-ETO9a的表观遗传学，将在NUP98-Hoxd13诱导的MDS后的髓系转化过程中进行类似的分析，并将确定CEBPA增强子是否减少了人AML的激活和增加了与CEBPA mRNA表达相关的抑制性表观遗传学。

 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.