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

前言：对大量AML患者进行全基因组测序，通过识别反复发生的突变和描绘共同出现或排他性的模式，拓宽了我们对AML发病机制的洞察力。除了这些观察数据外，(A)许多复发基因突变在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。目的：我们要研究RUNX1、ASXL1和IDH2基因突变的相互作用，以达到以下目的：a)我们将建立体外和体内模型来分析RUNX1、ASXL1和IDH2突变的不同组合对造血干细胞的影响；b)我们将在所建立的模型中测试特定的治疗药物，如IDH2抑制剂，并将研究它们对白血病克隆的影响。c)RUNX1、ASXL1和IDH2突变在老年患者中的突变模式和稳定性将阐明这些突变组合是否在这个队列中更频繁地发生方法：我们将研究RUNX1、ASXL1和IDH2基因在条件CRE-loxP小鼠模型、原代脐血CD34细胞和具有过多体外、体内和体外功能的髓系细胞系中的造血和白血病发生中的作用(例如培养分析、免疫表型鉴定、下一代测序、基因表达谱、GSEA、全局甲基化谱、2-HG水平的测量等)。与条件表达式或条件删除策略相结合。靶向抑制突变IDH2的效果将在条件Idh2R140Q敲入小鼠体内进行分析，并在患者来源的IDH2突变AML细胞和来自Idh2R140Q敲入小鼠的骨髓中进行体外分析。老年AML患者中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中的外显子组测序来确定。复杂的体外和体内AML系统对于更深刻地了解由特定反复发生的AML突变引起的遗传、表观遗传和生化改变是必需的。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.