Crispr-Cas9 Mediated Disruption Of Dnmt3a In Jakv617f Hematopoietic Stem Cells Accelerates Disease Phenotype And Induces Lethal Myelofibrosis

Myeloid malignancies arise following the sequential acquisition of somatic mutations within hematopoietic stem and progenitor cells (HSPC). JAK2V617F is commonly found in myeloproliferative neoplasms (MPN) such as polycythemia vera, essential thrombocythemia and myelofibrosis. While other mutations (e.g. TET2, DNMT3A) have been found to co-occur in MPN HSPC, it remains unclear how they impact disease biology or progression from early stage disease (i.e. polycythemia or essential thrombocythemia) to advanced stage disease such as myelofibrosis or acute myeloid leukemia. DNMT3A methylates cytosine rich DNA residues (known as CpG islands, and often found in promoters of genes) leading to transcriptional repression. DNMT3A is also recurrently mutated at relatively low frequency in polycythemia vera (5-7%) but mutations are more common in advanced MPN (approximately 15% of MF and 17% of AML, Stegelmann et al. Leukemia 2011; Abdel-Wahab et al. Leukaemia 2011). These mutations are found in the methyltransferase domain and cluster around arginine 882 (e.g. R882H), resulting in loss of DNA binding and reduced catalytic activity. We used CRISPR-Cas9 gene editing technology to disrupt Dnmt3a function in mouse HSPC and assessed for cooperativity together with a conditional, knockin Jak2V617F allele.