Cooperation of Dnmt3a R878H with Nras G12D promotes leukemogenesis in knock-in mice: a pilot study

Abstract

BackgroundDNMT3A R882H, a frequent mutation in acute myeloid leukemia (AML), plays a critical role in malignant hematopoiesis. Recent findings suggest that DNMT3A mutant acts as a founder mutation and requires additional genetic events to induce full-blown AML. Here, we investigated the cooperation of mutant DNMT3A and NRAS in leukemogenesis by generating a double knock-in (DKI) mouse model harboring both Dnmt3a R878H and Nras G12D mutations.MethodsDKI mice with both Dnmt3a R878H and Nras G12D mutations were generated by crossing Dnmt3a R878H knock-in (KI) mice and Nras G12D KI mice. Routine blood test, flow cytometry analysis and morphological analysis were performed to determine disease phenotype. RNA-sequencing (RNA-seq), RT-PCR and Western blot were carried out to reveal the molecular mechanism.ResultsThe DKI mice developed a more aggressive AML with a significantly shortened lifespan and higher percentage of blast cells compared with KI mice expressing Dnmt3a or Nras mutation alone. RNA-seq analysis showed that Dnmt3a and Nras mutations collaboratively caused abnormal expression of a series of genes related to differentiation arrest and growth advantage. Myc transcription factor and its target genes related to proliferation and apoptosis were up-regulated, thus contributing to promote the process of leukemogenesis.ConclusionThis study showed that cooperation of DNMT3A mutation and NRAS mutation could promote the onset of AML by synergistically disturbing the transcriptional profiling with Myc pathway involvement in DKI mice.

Endothelial lipase promotes acute myeloid leukemia progression through metabolic reprogramming

“…The Dnmt3aR878H/WT KI mice were crossed with NrasG12D/WT KI mice to generate the DKI mouse model (Mx1-Cre; Dnmt3aR878H/WT; NrasG12D/WT). DKI mice developed more aggressive AML than Dnmt3aR878H/WT KI mice after PIpC induction [16]. Since most leukemic cells were Gr1+ cells in the AML mice, we further analyzed the RNA-sequencing data (Supplementary Tables 1-3) of Gr1+ bone marrow cells and revealed that 1,129 genes were differentially expressed in DKI mice versus wild type mice.…”

“…Generation of AML mouse models and transcriptomic analysis. The conditional knock-in AML mouse models harboring Dnmt3aR878H/WT, NrasG12D/WT and both mutations were established respectively as previously described [16]. Mice were used according to animal care standards.…”

“…The mice exhibited leukemic phenotypes and were euthanized 4 months after pIpC injection. Gr1+ cells from the bone marrow of each group were sorted for RNA-sequencing as previously described [16]. Gene Ontology (GO) functional analysis was performed and visualized using R packages including ClusterProfiler version 3.14.3 and GOplot.…”

“…In our previous work, we established a conditional knock-in (KI) mouse model harboring Dnmt3aR878H/WT, which exhibited moderate AML after induction [15]. Then, we revealed that cooperation of Dnmt3aR878H/WT mutation with NrasG12D/WT mutation could induce severe AML by generating a double knock-in (DKI) mouse model [16]. In this work, we compared the Gr1+ cells of the DKI mice harboring both Dnmt3aR878H/WT and NrasG12D/WT mutations and wild type (WT) mice though RNA sequencing.…”

Mouse Models of Frequently Mutated Genes in Acute Myeloid Leukemia

“…In contrast to Nras G12D/+ alone or p53 −/− alone, cooperation of these mutations induces quiescence in megakaryocyte-erythroid progenitors (MEPs) that is sufficient to drive AML development in mice ( Table 2 ; Supplementary Figure S3 ) [ 127 ]. Similarly, cooperation of the NrasG12D mutation with Dnmt3aR878H/DNMT3AR882H activates the Myc pathway and induces AML in mice ( Table 2 ; Supplementary Figure S3 ) [ 128 , 132 ]. NRAS mutations also show synergistic AML development with mutations in other genes such as NPM1 [ 129 ], EZH2 [ 130 ], and IDH2 [ 25 ] ( Table 2 ; Supplementary Figure S3 ).…”