Novel C/EBPalpha replacement therapy by microRNA mimics in acute myeloid leukemia

急性髓系白血病（Acute myeloid leukemia， AML）是一种恶性造血系统疾病，急性髓系白血病患者的预后仍然很差。转录因子C/EBP α在骨髓祖细胞的正常分化和随后的细胞死亡中起重要作用。在50%以上的AML病例中发现C/EBP α通过不同的机制失活，例如在10%的病例中发现cebpa基因突变失活。这导致分化受阻和未成熟胚细胞的积累。因此，在治疗上重新激活AML中的C/EBP Alpha功能将是有利的，从而迫使AML原细胞正常分化和死亡。MicroRNAs是一类小的非编码rna，被认为是正常造血和AML发展的重要调节因子。我们之前已经证明C/EBPα诱导microrna miR-223， miR-34a和miR-30c在C/EBPα突变的AML中下调。在这个拟议的项目中，我们将遵循创新的想法，通过替换下游的microrna来恢复野生型C/EBP Alpha功能，从而模仿“C/EBP Alpha”，而不是“固定”C/EBP Alpha本身。我们计划在CEBPA敲除（KO）小鼠以及具有C/EBP α突变的AML患者样本中使用全球下一代测序筛选进一步鉴定C/EBP α诱导的microrna。我们将先前发现的MicroRNA miR-223， miR-34a和miR-30c与CEBPA KO小鼠中从我们的初步数据中下调的其他MicroRNA结合到我们称为“Alpha MicroRNA Group 2016”（AMG-16）的鸡尾酒中。利用PEI纳米颗粒作为高效无毒的递送系统，我们将在各种体外AML细胞系和AML患者样本模型中测试我们的鸡尾酒。此外，体内小鼠模型，如使用AML患者来源的异种移植（PDX）细胞的C/EBP alpha KO小鼠和NSG小鼠，将进一步弥合研究与临床之间的差距。随后，将对microrna进行功能研究并分析其生物学作用。具体目标是：1。体外和体内C/ ebp α-诱导的microrna的鉴定和表征2. 体外AML模型系统中microRNA模拟物C/EBP替代疗法的研究3. 在体内AML模型系统中利用microRNA模拟物开发C/EBP α替代疗法。我们假设，与单一模拟治疗相比，多重模拟将显示出附加的治疗效果。总之，我们将通过microRNA模拟物在AML中建立一种新的C/EBP α替代疗法。因此，我们试图为临床提高AML治疗成功率的新治疗策略奠定基础。

Acute myeloid leukemia (AML) is a malignant disease of the hematopoietic system, and the outcome of patients suffering from AML is still very poor. The transcription factor C/EBP Alpha plays an important role in the normal differentiation of myeloid progenitor cells and subsequent cell death. An inactivation of C/EBP Alpha via different mechanisms is found in over 50% of AML cases, e.g. by inactivating mutations of the CEBPA-gene in 10% of cases. This leads to a block in differentiation and an accumulation of immature blast cells. Thus, it would be advantageous to re-activate C/EBP Alpha function in AML therapeutically, thus forcing the AML blasts to differentiate normally and die. MicroRNAs, a class of small non-coding RNAs, were identified as important regulators of normal hematopoiesis and AML development. We have previously shown that C/EBPα induces microRNAs miR-223, miR-34a, and miR-30c which are downregulated in C/EBP Alpha mutated AML. Within this proposed project we will follow the innovative idea to restore wild-type C/EBP Alpha function by replacing downstream microRNAs and thus mimicking “C/EBP Alpha-ness”, instead of "Fixing" C/EBP Alpha itself. We plan to identify further C/EBP Alpha-induced microRNAs using a global next-generation sequencing screen in CEBPA knock-out (KO) mice, and, additionally, AML patient samples with C/EBP Alpha-mutations. We will combine the previously discovered microRNAs miR-223, miR-34a and miR-30c with further microRNAs downregulated in CEBPA KO mice from our preliminary data into a cocktail we have called "Alpha MicroRNA Group 2016" (AMG-16). Utilizing PEI nanoparticles as a highly efficient and non-toxic delivery system we will test our cocktail in various in vitro AML cell lines and AML patient sample models. In addition, in vivo mouse models, such as C/EBP alpha KO mice and NSG mice with AML patient derived xenografts (PDX) cells will further bridge the gap between research and clinic. Subsequently, the microRNAs will be functionally investigated and their biological role analyzed. The specific aims are: 1. Identification and charaterization of C/EBPα-induced microRNAs in vitro and in vivo; 2. Development of a C/EBP alpha replacement therapy by microRNA mimics in AML model systems in vitro; 3. Development of a C/EBP alpha replacement therapy by microRNA mimics in AML model systems in vivo. We hypothesize that multiple mimics will show an additive therapeutic effect in comparison to single mimic therapy. Taken together, we will establish a novel C/EBP alpha replacement therapy by microRNA mimics in AML. Thereby, we attempt to establish the basis for a new therapeutic strategy to clinically enhance AML treatment success.