Cooperating mutations in AML: Functional dissection of the cooperating network of recurrent mutations of RUNX1, ASXL1 and IDH2

大型AML患者队列的全基因组测序通过识别复发性突变和描述相互共发生或排他性的模式，扩大了我们对AML发病机制的了解。除了这些观察数据(a)许多复发性基因突变在AML发病机制中的确切作用，(b)它们与其他突变的相互作用，以及(c)它们的治疗相关性仍有待阐明。因此，需要精确的体外和体内模型。目的：我们希望表征RUNX1、ASXL1和IDH2的复发性和相关突变之间的相互作用，实现以下目标：a)我们将建立体外和体内模型，分析RUNX1、ASXL1和IDH2突变的不同组合对造血干细胞的影响。b)特异性治疗剂，如IDH2抑制剂将在我们建立的模型中进行测试，并将研究它们对白血病克隆的影响。c)老年患者中RUNX1、ASXL1和IDH2突变的突变模式和稳定性将阐明这些突变的组合是否在该队列中更频繁地发生，从而允许在这一预后不利的老年AML患者队列中采用更具体的靶向方法。方法:我们将研究RUNX1， ASXL1和IDH2基因在条件Cre-LoxP小鼠模型，原代脐带血CD34+细胞和髓系中造血和白血病发生中的作用，并结合条件表达或条件删除策略，使用大量体外，体内和离体功能技术（例如培养分析，免疫表型分型，下一代测序，基因表达谱，GSEA，全局甲基化谱，2-HG水平测量等）。我们将在条件Idh2R140Q敲入小鼠体内，以及在患者来源的IDH2突变AML细胞和Idh2R140Q敲入小鼠骨髓中，分析靶向抑制突变IDH2的效果。老年AML患者RUNX1、ASXL1和IDH2突变的突变模式和稳定性将通过配对诊断和复发样本的外显子组测序来确定。透视图：这种方法可以很容易地转移到表征其他不同的合作突变集。复杂的体外和体内AML系统对于更深入地了解由特定复发性AML突变引起的遗传、表观遗传和生化改变的功能是必需的。此外，这些模型将为进一步研究新的靶向治疗提供可靠的系统。

Introduction: Genome-wide sequencing of large AML patient cohorts has broadened our insight into AML pathogenesis by identifying recurrent mutations and delineating patterns of mutual co-occurrence or exclusivity. Besides these observational data (a) the exact role of many recurrent gene mutations in the pathogenesis of AML, (b) their interactions with other mutations, and (c) their therapeutic relevance still remain to be elucidated. Therefore, precise in vitro and in vivo models are required.Aim: We want to characterize the interplay between the recurrent and associated mutations of RUNX1, ASXL1 and IDH2 addressing the following aims:a) We will generate in vitro and in vivo models to analyze the effects of different combinations of RUNX1, ASXL1 and IDH2 mutations on hematopoietic stem cells.b) Specific therapeutic agents e.g. IDH2-inhibitors will be tested in our established models and their effect on leukemic clones will be investigated.c) Mutational patterns and stability of RUNX1, ASXL1 and IDH2 mutations in elderly patients will elucidate if combinations of these mutations occur more often in this cohort, allowing a more specific targeted approach in this prognostic unfavorable elderly AML patient cohort.Methods: We will study the role of RUNX1, ASXL1 and IDH2 genes in hematopoiesis and leukemogenesis in conditional Cre-LoxP mouse models, primary cord blood CD34+ cells, and myeloid cell lines with a plethora of functional in vitro, in vivo and ex vivo techniques (e.g. culture assays, immunophenotyping, next generation sequencing, gene expression profiles, GSEA, global methylation profiles, measurement of 2-HG levels, etc.) combined with a conditional expression or a conditional deletion strategy. The effect of a targeted inhibition of mutant IDH2 will be analyzed in vivo in conditional Idh2R140Q knock-in mice and ex vivo in patient-derived IDH2-mutant AML cells and bone marrow from Idh2R140Q knock-in mice.Mutational patterns and stability of RUNX1, ASXL1 and IDH2 mutations in elderly AML patients will be identified by exome sequencing in paired diagnosis and relapse samples.Perspective: This approach can be easily transferred to characterize other different sets of cooperating mutations. Sophisticated in vitro and in vivo AML systems are mandatory for a more profound functional knowledge of genetic, epigenetic and biochemical alterations caused by specific recurrent AML mutations. Moreover, these models will provide reliable systems for further investigation of novel targeted therapies.