Crispr-Cas9 Mediated Disruption of Dnmt3a in JakV617F Hematopoietic Stem Cells Accelerates Disease Phenotype and Induces Lethal Myelofibrosis

Abstract

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.Jak2V617F/ΔDnmt3a-Cas9 but not Jak2V617F/Cas9 controls demonstrated increased HSPC self-renewal and proliferation properties in vitro as evidenced by serial replating in methylcellulose (>5 weeks) and increased colony forming unit capacity. Flow-cytometry analysis of Jak2V617F/ΔDnmt3a-Cas9 revealed enrichment in LKS+ (Lin-Sca-1highKithigh) cells 5 weeks after CRISPR-Cas9disruption of Dnmt3a, and this was associated with increased expression of stemness markers Kit and Cd34 in Jak2V617F/ΔDnmt3a-Cas9 cells. RNAseq was performed on early (week 1, P1) and late culture HSPC (week 5, P5) from Jak2V617F-Cas9 (P1 only) and Jak2V617F/ΔDnmt3a-Cas9 (P1, P5). This confirmed deletion of Dnmt3a in Jak2V617F/ΔDnmt3a-Cas9 but not in Jak2V617F/Cas9 controls. Transcriptional upregulation of Kit and Cd34 were confirmed, as well as other key stem cell genes such as Erg and Angpt1 in Jak2V617F/ΔDnmt3a-Cas9 P5. We observed denovo expression of imprinted genes Igf2 and H19 in Jak2V617F/ΔDnmt3a P5, suggesting impaired DNA methylation in this group. Jak2V617F/ΔDnmt3a-Cas9 P5 were significantly enriched for transcriptional pathways controlling cell cycle progression, oncogenic signatures, and DNA damage. Conversely, Jak2V617F/Cas9 controls were enriched for myeloid differentiation and normal progenitor cell signatures.To assess the effect of Dnmt3a loss on Jak2V617F driven MPN, we transplanted Jak2V617F/ΔDnmt3a-Cas9 or Jak2V617F/Cas9 LKS+ into irradiated B6 recipients. Recipients of Jak2V617F/Cas9 LKS+ developed early stage MPN reminiscent of polycythemia vera with high hemoglobin, white cell count and platelets and was sustained >32 weeks. In contrast, Jak2V617F/ΔDnmt3-Cas9 recipients exhibited a biphasic disease, reminiscent of human myelofibrosis. At 8 weeks, Jak2V617F/ΔDnmt3-Cas9 showed panmyelosis with thrombocytosis (1.38x106/µl vs. 1.14x106/µl controls, p=0.057). However, by 32 weeks, this mice became severely pancytopenic with progressive bone marrow failure (Hemoglobin 121g/L vs. 210g/L controls, p =0.0011; platelets 0.338x106/µl vs. 1.343x106/µl controls, p <0.0001). Jak2V617F/ΔDnmt3-Cas9 mice exhibited extreme splenomegaly associated with reticulin fibrosis and the accumulation of myeloid cells. Bone marrow histology of Jak2V617F/ΔDnmt3-Cas9 revealed osteosclerosis and disorganized architecture and a dense fibrocellular infiltrate and reticulin fibrosis. Flow cytometry revealed impaired erythropoiesis and blocked differentiation. AML was not seen.These data demonstrate new evidence linking loss of Dnmt3a with acquisition of self-renewal in combination with constitutively active Jak2V617F. Importantly, in vivo loss of Dnmt3a accelerates or induces myelofibrotic transformation of the underlying MPN. This work provides new understanding to the factors that promote advanced disease in MPN. Ultimately, such knowledge has the potential to inform the development of novel targeted therapeutic approaches for the treatment of transformed MPN, a highly chemorefractory disease associated with extremely poor prognosis in patients. DisclosuresLane:Janssen: Other: i have done consulting (once) for janssen..