The significance of DNA Methyltransferase inhibition and DNA Methylation for therapy response in Acute Myeloid Leukemia

• Applicant: Professor Dr. Carsten Müller-Tidow
• Subject Area: Hematology, Oncology
• Term: from 2010 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 171434580

Acute myeloid leukemia (AML) is associated with epigenomic changes including profound alterations in DNA methylation. Origin and relevance of these changes are not entirely clear. Despite intensive chemotherapy, the majority of patients cannot be cured by current therapies. DNA methyltransferase (DNMT) inhibitors such as Decitabine and Azacytidine are not curative and the mechanisms of action are incompletely understood. In the first funding period, we used Reduced Representation Bisulfite Sequencing (RRBS) to identify altered DNA methylation patterns in murine leukemias with increased DNMT3B activity and in Acute Promyelocytic Leukemia (APL) patients. We found out that high risk and age were especially associated with dysregulated DNA methylation in APL patients. In DNMT3B overexpressing cells, leukemia initiation and maintenance were impaired. Murine leukemic blasts with high DNA methylation also showed increased sensitivity towards Azacytidine. We have started to generate murine leukemias with DNMT3B+/- to analyze leukemia induction and therapy response with a slight decrease of DNMT3B activity. The main aim of the new funding period is to identify and modify DNA methylation events that influence therapy response towards cytotoxic drugs and Azacytidine. For this purpose, we are going to analyze the relevant epigenomic changes that determine changes in leukemogenesis and treatment response in the mouse models. Relevant genes will be silenced by genomic editing (TALEN/CRISPR) and/or by shRNA transduction in cell lines and primary blasts. In addition, relevant genomic regions are modulated by targeted epigenomic editing using TALE-SssI constructs. Efficacy and effects onto gene expression, cellular functions and therapy response are tested in vitro and in vivo. As a complementary approach, we are going to perform a biomarker analysis for the AML-AZA clinical trial with 450K methylation bead arrays at the time of diagnosis, after day 5 of Azacytidine exposure and on day 15 after chemotherapy start. One major aim here is to identify DNA methylation patterns that are associated with increased response to standard chemotherapy +/- Azacytidine. These results are compared to the murine leukemia data with defined levels of DNMT activity. In addition, the AML-AZA biobank will provide well characterized specimens to several collaboration partners within the SPP1463.Taken together, these analyses will identify DNA methylation associated therapy response towards Azacytidine and cytotoxic drugs. The functional relevance of the DNA methylation changes will be analyzed to identify the underlying mechanisms.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1463: Epigenetic Regulation of Normal Hematopoiesis and its Dysregulation in Myeloid Neoplasia
• Participating Persons: Dr. Stefanie Göllner; Dr. Utz Olaf Krug; Dr. Eva Schmidt