In vivo CRISPR editing of DNMT3A in JAK2V617F hematopoietic stem cells induces myelofibrosis

JAK2V617F is present in the majority of patients with myeloproliferative neoplasm (MPN), however, it is unclear how co-occurring mutations in epigenetic regulators (e.g. DNMT3A) in MPN impact disease biology. DNMT3A methylates DNA at cytosine residues in enriched CpG region, and drives promoter hypermethylation. DNMT3A is mutated in advanced MPN (15% of MF and 17% of AML). These mutations sit in the methyltransferase domain and result in reduced activity. To determine the functional effects of Dnmt3a loss in MPN, we used CRISPR-Cas9 technology to edit Dnmt3a function in Jak2V617F murine hematopoietic stem and progenitor cells (HSPC). Jak2V617F LKS+ (Lin-Sca-1highKithigh) HSPCs were infected with a lentivirus encoding Cas9 and sgRNA targeting Dnmt3a (Jak2V617F/ΔDnmt3a) or non-targeting controls (Jak2V617F). In vitro CFU from Jak2V617F/ΔDnmt3a had enhanced serial replating and increased expression of markers Kit and Cd34. CFU RNAseq confirmed Dnmt3a deletion in Jak2V617F/ΔDnmt3a, as well as transcriptional upregulation of key stemness genes, and de novo expression of imprinted genes Igf2 and H19, together with decreased H19 promoter methylation in Jak2V617F/ΔDnmt3a compared to controls. To assess the functional effect of Dnmt3a loss on Jak2V617F driven MPN, we transplanted Jak2V617F/ΔDnmt3a or Jak2V617F LKS+ into irradiated recipients. Jak2V617F/ΔDnmt3a became severely pancytopenic with bone marrow failure, fibrosis and the accumulation of myeloid cells. Jak2V617F/ΔDnmt3a bone marrow showed osteosclerosis, disorganized architecture with dense fibrocellular infiltrate. RNAseq on Jak2V617F/ΔDnmt3a LKS showed strong enrichment in gene sets previously annotated by Dnmt3a-/- in normal HSPC (Challen, Nat Gen 2011) and oncogenic Dnmt3a R878H (Guryanova,Nat Med 2016), together with LT-HSC signatures and PRC2 deregulation. This novel model of mutant Dnmt3a, achieved through in vivo CRISPR-Cas9 editing, links loss of Dnmt3a with acquisition of self-renewal and disease progression in Jak2V617F HSPC. Such knowledge has the potential to inform the development of targeted therapeutic approaches in transformed MPN, a highly chemorefractory disease associated with poor prognosis.