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

• Applicant: Dr. Friederike Pastore
• Subject Area: Hematology, Oncology
• Term: from 2014 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 257981898

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.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Dr. Ross Levine