Conditional knockin of Dnmt3a R878H initiates acute myeloid leukemia with mTOR pathway involvement

Abstract

DNMT3A is frequently mutated in acute myeloid leukemia (AML). To explore the features of human AML with the hotspot DNMT3A R882H mutation, we generated Dnmt3a R878H conditional knockin mice, which developed AML with enlarged Lin − Sca1 + cKit + cell compartments. The transcriptome and DNA methylation profiling of bulk leukemic cells and the single-cell RNA sequencing of leukemic stem/progenitor cells revealed significant changes in gene expression and epigenetic regulatory patterns that cause differentiation arrest and growth advantage. Consistent with leukemic cell accumulation in G 2 /M phase, CDK1 was up-regulated due to mTOR activation associated with DNA hypomethylation. Overexpressed CDK1-mediated EZH2 phosphorylation resulted in an abnormal trimethylation of H3K27 profile. The mTOR inhibitor rapamycin elicited a significant therapeutic response in Dnmt3a R878H/WT mice.have been identified in a subset of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and acute lymphoblastic leukemia (ALL), with DNMT3A R882 being the hotspot (1-4). Clinical features of most AML cases with DNMT3A mutations include preferential involvement of a monocytic lineage (AML-M4 and -M5 subtypes), thrombocytosis, onset at a relatively old age, and poor prognosis (2, 5, 6). Careful genotypephenotype correlations suggest that patients manifest DNMT3A mutations in preleukemic hematopoietic stem cells (HSCs)/multipotent progenitors (MPPs), which exhibit a competitive advantage over normal HSCs. Because these mutations occur at a very early stage among genetic abnormalities, they are likely involved in the development of leukemia (7,8).Functionally, the DNMT3A R882 mutation might disrupt epigenetic regulation. This kind of DNMT3A mutation confers reduced methyltransferase activity and promotes the possibility of dominant-negative consequences compared with the wild-type (WT) allele (2, 9, 10). Moreover, the DNMT3A mutation causes aberrant DNA hypomethylation and up-regulates a series of target genes involved in AML pathogenesis (11-13).In vivo animal tests have shown that the Dnmt3a gene plays an essential role in hematopoiesis regulation. Dnmt3a −/− HSCs expand remarkably, but their differentiation is inhibited when Dnmt3a is conditionally inactivated in the murine hematopoietic system; this phenomenon is consistent with a preleukemic state (7). Moreover, all lethally irradiated mice transplanted with Dnmt3a-deleted HSCs died within 1 y and were diagnosed with a spectrum of malignancies similar to those observed in patients carrying DNMT3A mutations, including MDS, AML, primary myelofibrosis, and ALL, suggesting that Dnmt3a functions as a tumor suppressor (7). With a second hit of mutations in various genes such as N-RAS, C-KIT, or FLT3 in Dnmt3a −/− mice, Dnmt3a deletion induces leukemic transformation (14, 15). Although these results indicate a major role of Dnmt3a deletion in facilitating the development of leukemia, the in vivo roles of DNMT3A mutants in leukemogenesis still need to be addressed. In our previous work, b...

Prognosis and regulation of an adenylyl cyclase network in acute myeloid leukemia

“…By contrast, these drugs elicited no reduction in OCI-AML3 and OCI AML2 cell viability. However, both OCI-AML2 and OCI-AML3 cells were sensitive to 500 nM sapanisertib, with only 50-60% of cells remaining viable after 48 h ( Figure 7C , 7D ), which was consistent with previous reports [ 19 ]. Kasumi-1 and MOLM13 cells were less sensitive to 500 nM sapanisertib, with about 80% of cells remaining viable after 48 h. The IC50 of inhibitors in these AML cell lines were displayed in Table 2 .…”

Sequentially inducible mouse models reveal that Npm1 mutation causes malignant transformation of Dnmt3a-mutant clonal hematopoiesis

“…The most common somatic mutations present in CH are in genes associated with epigenetic modifications, including the DNA methyltransferase DNMT3A , and splicing factors. Several mouse models of DNMT3A -driven CH have been generated by conditional knockout [9], mutant allele knockin [10, 11], or mutant allele overexpression [12]. Further, several studies have identified cooperating mutations that can act synergistically with Dnmt3a knockout or mutation to cause leukemia, such as Flt3 ITD [13, 14], Flt3 ITD and Npm1 c [11], cKit [15], and Kras G12D [16].…”

Expression and prognosis analysis of DNMT family in acute myeloid leukemia

“…More importantly, high incidence of DNMT3A mutation was identified in AML and DNMT3A mutation correlated with poor prognosis in AML [ 12 ]. Functional studies showed that DNMT3A was essential for hematopoietic stem cell (HSC) differentiation and mutated DNMT3A initiated AML [ 13 – 14 ], suggesting DNMT3A acted as a tumor suppressor gene. Although DNMT3B was rarely mutated in AML [ 15 ], studies have proved that loss of DNMT3B accelerated MLL-AF9 leukemia progression and increased expression of DNMT3B in LSC delayed leukemogenesis [ 16 – 17 ].…”