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

急性髓系白血病(AML)是一种恶性的造血系统疾病，患者的预后仍然很差。转录因子C/EBPα在髓系祖细胞的正常分化和随后的细胞死亡中起重要作用。超过50%的AML病例通过不同机制发现C/EBPα失活，例如10%的AML病例通过CEBPA基因突变失活。这会导致分化受阻，幼稚母细胞堆积。因此，在治疗上重新激活AML的C/EBP Alpha功能将是有利的，从而迫使AML原始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发生的重要调节因子。我们先前已经证明，C/EBPα诱导了miR-223、miR-34a和miR-30c，这些microRNAs在C/EBPAlpha突变的急性髓细胞白血病中下调。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microRNAs来恢复野生型C/EBP Alpha的功能，从而模仿“C/EBP Alpha”，而不是“修复”C/EBP Alpha本身。我们计划在CEBPA基因敲除(KO)小鼠以及具有C/EBP Alpha突变的AML患者样本中使用全球下一代测序屏幕来识别进一步的C/EBP Alpha诱导的microRNAs。我们将把先前发现的miR-223、miR-34a和miR-30c与CEBPA KO小鼠中进一步下调的microRNAs结合起来，形成一种鸡尾酒，我们称之为“Alpha microRNA Group 2016”(AMG-16)。利用PEI纳米粒子作为一种高效无毒的递送系统，我们将在各种体外AML细胞株和AML患者样本模型中测试我们的鸡尾酒。此外，具有AML患者来源的异种移植(PDX)细胞的C/EBPαKO小鼠和NSG小鼠等体内小鼠模型将进一步弥合研究和临床之间的差距。随后，将对microRNAs进行功能研究并分析其生物学作用。本研究的具体目标是：1.体外和体内对C/EBPRNA诱导的microRNA的鉴定和鉴定；2.体外模拟急性髓系白血病模型系统中的C/EBPα替代疗法；3.体内模型系统中模拟α的C/EBPAlpha替代疗法的研究。我们假设，与单一的模拟疗法相比，多个模拟疗法将显示出相加的治疗效果。综上所述，我们将通过在AML中模拟microRNA来建立一种新的C/EBPα替代疗法。因此，我们试图为临床上提高AML治疗成功率的新的治疗策略奠定基础。

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.