Physical Poster + E-Poster Presentation 34th Lorne Cancer Conference 2022

Determining the role of mutant Dnmt3a in the development of haematopoietic malignancies (#123)

Amali Cooray 1 2 , Erin Lawrence 1 2 , Lahiru Gangoda 1 2 , Maggie Potts 1 2 , Andrew Kueh 1 2 , Marco Herold 1 2
  1. Walter and Eliza Hall Institute, Parkville, VIC, Australia
  2. Department of Medical Biology, University of Melbourne, Melbourne, VIC, Australia

Many haematopoietic malignancies carry a heterozygous mutation at amino acid position R882 in the epigenetic regulator DNMT3a. We have developed a model in which we mimicked this mutation in mice. Serial competitive transplant experiments showed that haematopoietic stem and progenitor cells (HSPCs) derived from the DNMT3amut mice have a competitive advantage over WT cells. However, the DNMT3a mutation alone was not sufficient to cause the development of malignancies suggesting that further oncogenic mutations are required. DNMT3amut mice compared to WT controls showed accelerated development of γ-irradiation induced thymic lymphoma. To understand which pathways are differentially affected by γ-irradiation in the DNMT3a mutant background, we performed transcriptomic profiling of DNMT3amut and WT Lineage-Sca1+cKit+ (LSK) cells from irradiated mice. This revealed the p53 signalling pathway being weaker induced in DNMT3amut cells, suggesting a blunted stress response. To identify the cell type within the HSPC pool that showed the blunted p53 response in the DNMT3amut LSKs, we isolated HSPCs from WT and DNMT3amut mice and irradiated them in vitro. Cell death analysis by FACS showed that DNMT3amut LSK except MPP2 cells died at an accelerated pace. However, DNMT3a is most frequently mutated in AML. To identify other aberrantly expressed oncogenes that cooperate with mutant DNMT3a to induce AML, we have developed a CRISPR activation single guide (sg)RNA library which targets genes that are highly expressed in human mutant DNMT3a/NPM1/FLT3ITD compared to DNMT3amut AMLs. Therefore, we crossed our newly engineered CRISPR activation mouse model to the DNMT3a mutant mice, isolated HSPCs from e14 fetal livers and transduced them with our focused sgRNA library, which primarily targets HOX genes.  These engineered HSPCs were transplanted into lethally irradiated recipient mice, which are observed for the development of AML. Once AML has developed, we will purify the malignant cells and identify the enriched sgRNAs by next generation sequencing to identify the combinations of co-drivers in DNMT3amut AMLs.