Poster Presentation 31st Lorne Cancer Conference 2019

Jak2V617F and Dnmt3a loss cooperate to induce myelofibrosis through activated enhancer-driven inflammation (#208)

Sebastien Jacquelin 1 , Jasmin M Straube 1 , Leanne Cooper 1 , Therese Vu 1 , Axia Song 1 , Megan Bywater 1 , Eva Baxter 1 , Amy Porter 1 , VCictoria Ling 1 , Joanne Green 1 , Rebecca Austin 1 , Stephen Kazakoff 1 , Nic Waddell 1 , Luke B Hesson 2 , John E Pimanda 3 , Frank Stegelmann 4 , Lars Bullinger 5 , Konstanze Döhner 4 , Raajit K Rampal 6 , Dirk Heckl 7 , Geoffrey GRH Hill 8 , Steven SWL Lane 8
  1. QIMR, Brisbane, QLD, Australia
  2. The Prince of Wales Clinical School, Lowy Cancer Research Centre, Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia
  3. The Prince of Wales Clinical School, Lowy Cancer Research Centre, Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Department of Pathology, School of Medical Sciences, Department of Haematology, Prince of Wales Hospital, , Sydney, NSW, Australia
  4. Department of Internal Medicine III, University Hospital of Ulm, Ulm, Germany
  5. Department of Hematology, Oncology, and Tumorimmunology, Charité University Medicine, Berlin, Germany
  6. Leukemia Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New york, USA
  7. Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany
  8. Immunology, QIMR Berghofer Medical Research Institute, Brisbane, Australia, The Royal Brisbane and Women's Hospital, Brisbane, Australia, University of Queensland, Australia, Brisbane, QLD, Australia

Myeloproliferative neoplasms (MPN) arise following the sequential acquisition of genetic lesions in hematopoietic stem and progenitors cells (HSPCs). The majority of MPN patients, and virtually all patients with polycythemia vera (PV), have a specific mutation in the JAK2 tyrosine kinase known as JAK2V617F. Mutations in genes that regulate epigenetic pathways (e.g. DNMT3A) are more frequently found in advanced stage MPN such as myelofibrosis or acute myeloid leukaemia. DNMT3A catalyzes the addition of methyl groups into active chromatin in CpG-rich regions such as promoter and enhancer sites, leading to gene inactivation. DNMT3A mutations in haematological malignancies generally occurs in the methyltransferase domain and result in loss of function.

To model the combinatorial effects of Dnmt3a loss with Jak2V617F, we used lentivirus delivered CRISPR-Cas9 to disrupt Dnmt3a in Jak2V617F knockin murine HSPCs. Dnmt3a loss led to in vitro immortalization, de novo expression of stemness genes and imprinted genes, together with reduced locus specific DNA methylation. In vivo, Dnmt3a loss cooperated with Jak2V617F to induce lethal myelofibrosis, characterized by extreme splenomegaly and reticulo-fibrotic infiltration of bone marrow with bone marrow failure.

Transcriptional analysis on Dnmt3a-edited Jak2V617F HSPC revealed aberrant self-renewal and inflammatory signaling. This correlated with common gene expression changes observed in human DNMT3A mutant AML and MPN. We confirmed oligoclonal, on target editing of Dnmt3a locus, and did not observe significant off-target effect using whole exome sequencing. Mechanistically, Dnmt3a loss led to increased chromatin accessibility at active enhancer marks in HSPCs. These were enriched at stem cell genes and inflammatory gene loci annotated within the TNFalpha pathway. Similar findings were confirmed in human AML samples with DNMT3A mutations.

Altogether, these findings identify oncogenic cooperativity between Jak2V617F-driven MPN and Dnmt3a loss, leading to activation of HSPC enhancer driven inflammatory signaling and transformation to advanced myelofibrosis. This work has the potential to shape the development of targeted therapeutic approaches in transformed MPN, through agents that modify enhancer accessibility or by blocking aberrant inflammatory signaling.