Poster Presentation 29th Lorne Cancer Conference 2017

Investigating the role of mutant DNMT3A in the Development of Acute Myeloid Leukaemia (#194)

Erin M Lawrence 1 2 , Martin Pal 1 2 , Andrew J Kueh 1 2 , Lin Tai 1 , Andreas Strasser 1 2 , Andrew Wei 3 4 , Marco J Herold 1 2
  1. Molecular Genetics of Cancer, Walter and Eliza Hall Institute, Parkville, Victoria, Australia
  2. Medical Biology, The University of Melbourne, Melbourne, Victoria, Australia
  3. Monash University, Melbourne, Victoria, Australia
  4. The Alfred Hospital, Melbourne, Victoria, Australia

Acute Myeloid Leukaemia (AML) is characterized by aberrant proliferation of clonal myeloid stem and progenitor cells. Mutations in DNA Methyltransferase 3a (DNMT3A) and other epigenetic regulators have been identified as initiating events in AML. DNMT3A is mutated in 22% of AMLs making it one of the most frequently mutated genes. Hetero- and homozygous deletion of DNMT3A can lead to haematological malignancies, however, deletion of DNMT3A does not parallel the missense mutation at the ‘hotspot’ position R882 that produces a dominant-negative protein in human AML. Therefore, we have used CRISPR/ Cas9 technology to recapitulate this DNMT3a mutation in mice by mutating R878 (murine homologue to R882) to histidine. Our CRISPR generated DNMT3Amut mice do not spontaneously develop AML, which is consistent with previous studies that suggest that other oncogenic drivers, such as NRASmut, IDH2mut or FLT3_ITD, are required for malignant transformation. Therefore, I have transduced haematopoietic stem and progenitor cells (HSPCs) derived from mutant DNMT3A and wildtype foetal livers with retroviral constructs encoding for NRASG12D and IDH2R140Q and transplanted them into lethally irradiated mice. We are currently waiting for these mice to develop tumours. These experiments will clarify the role of DNMT3Amut in tumour development in the context of two different oncogenes in a physiologically relevant murine model.   Additionally, we will perform transcriptome and epigenetic analysis in premalignant and malignant cells expressing either mutant or WT forms of DNMT3A combined with different oncogenic drivers using Next Generation Sequencing approaches. This will allow us to identify differentially regulated genes or methylated regions in cells expressing either mutant or wildtype DNMT3A. Importantly, we will not only address a role for DNMT3A in the development, but also the sustained growth of AML. To this end we have introduced an additional sgRNA binding site into the mutant DNMT3A allele. By targeting this allele, we can effectively delete the mutant DNMT3A once AML has developed and track the survival of leukaemic cells in vitro and in vivo. Transcriptome and epigenetic analysis will be performed before and after deletion of DNMT3A to determine genes that are regulated by mutant DNMT3A.