Mechanisms of DNMT3A-mutation induced sensitization for 5-Azacytidine in AML

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Genetic mutations determine therapy response for many targeted therapies. DNMT3A mutations are among the most frequent mutations that occur in AML. In a recent clinical trial we discovered that older AML patients with DNMT3A mutations in codon 882 (most common mutation) are extraordinarily sensitive to the combination of the DNMT inhibitor 5-Azacytidine and chemotherapy. In vitro experiments further support the hypothesis that DNMT3A-R882 mutations sensitize for 5-Azacytidine therapy. Thus, DNMT-inhibitors appear to act as targeted epigenetic therapy for DNMT3A mutant AML. The aim of this project is to analyze the mechanisms of this sensitization and to exploit the knowledge for further epigenetic therapy development in AML. For this purpose we are going to analyze a DNMT3A-R882H Knock-in mouse that we developed for this project as well as primary AML specimens from patients with/without DNMT3A mutation. To identify in which circumstances DNMT3A mutations in R882H sensitize for DNMT-inhibitor therapy we are going to analyse pre-leukemic and leukemic stem cells from DNMT3A-mutant knock-in versus wild-type mice. Analyses will be performed in vitro and in vivo for differences in leukemia initiation and therapy response to 5-Azacytidine (and chemotherapy response). Second, we are going to characterize mechanisms of DNMT-inhibitor sensitivity for DNMT3A-R882 mutants. We are going to identify interaction partners for DNMT3A-mutant and wild-type protein in murine and human samples by Mass-Spec. The effects of 5-Azacytidine onto RNA transcription and affected genes will be explored in murine and human cells with/ without DNMT3A mutation. Relevant genes and potential mechanisms will be compared to data from the clinical trial (methylation and RNA-Seq) as well as to results from a previous shRNA library screen. Relevant mechanisms for 5-Azacytidine sensitization will be functionally verified by shRNA based approaches. We are going to analyze the effects of 5-Azacytidine monotherapy and the combination with cytarabine onto human leukemia blasts that carry DNMT3A-mutations/wildtype or specific shRNAs for candidate genes in immunodeficient mice. Main focus is to see, which of the identified mechanisms can be utilized to render 5-Azacytidine more effective in DNMT3A wild-type patients. Overall, this project will identify mechanisms of DNMT3A mutation induced sensitization for 5-Azacytidine and the utilization of these mechanisms for targeted epigenetic therapy in AML with DNMT3A WT.