DNMT3A mutation leads to leukemic extramedullary infiltration mediated by TWIST1

Genetic alterations are now regarded as important biomarkers for disease evaluation and prognosis assessment. In addition to chromosomal disorders, growing mutation information is recognized in EMI procedure. Some case reports found FLT3-ITD and NPM1 variations in myeloid sarcomas with high frequencies of 15 and 14.4 %, respectively. Extramedullary tumors that carry these two abnormalities are mostly accompanied with cytogenetically normal AML and represent short lifespan, although NPM1 mutation is considered a good prognostic indicator [22, 23]. This observation demonstrates that genetic mutation may independently affect disease progression in systemic leukemia with EMI.

In the present study, genetic lesion located in exon 18 of DNMT3A can promote leukemic cell migration. Meningeal leukemia, where EMI is displayed, could be determined in our NOD/SCID mice transplanted with human leukemic cells carrying D3Amut. Hence, we provide a strong evidence to support the clinical discovery of D3Amut in CSF from CNS relapse patient [10]. Importantly, about 20 % of our AML cases with D3Amuts and whose genetic profiles have been reported before [24] showed CNS leukemia when CSF was detected during disease courses.

D3Amuts are frequently detected in cases diagnosed with M4 or M5 subtypes of AML [25]. D3Amut alone could induce aggressive proliferation of differentiated monocytes [20], thereby suggesting that this mutation underlies the development of monocytic blasts [20]. MLL abnormalities, which are mutually exclusive to D3Amuts in M4/M5 variants [25], are related to extramedullary disease [6]. Our results demonstrate that D3Amut, which represents another group of AML patients with monocytic involvement, might also be associated with EMI.

The role of DNMT3A in cell invasion has been observed in lung cancer. Deletion of DNMT3A promotes tumor progression and enables cells to invade into bronchiole. Remarkably, a pool of genes in charge of cell adhesion and motion is highly expressed in DNMT3A-knockout mice [26]. Therefore, DNMT3A variation may enhance tumor cell invasiveness through altering migrating mechanisms [26]. In our leukemic EMI model, an EMT inducer TWIST1 is highly expressed in OCI-AML3 strains and AML patients’ bone marrow samples because of D3Amuts. EMT occurs in the initiation of metastasis for cancer progression. It enables carcinoma cells to escape cell-cell adhesion and gain migratory phenotype. EMT involvement has been experimentally proven in solid tumors [21]. Recently, a group from Italy reported that EMT-like processes are relevant to acute promyelocytic leukemia development or progression [27]. This result implicates that EMT regulator TWIST1 causes leukemia invasive behavior. Our data further suggest that the aggressive migratory behavior reminiscent of TWIST1 also exists in extramedullary leukemia and could be induced by DNMT3A R882 mutation.

DNMT3A is an epigenetic modifier, and mutation on its catalytic domain can decrease enzymatic activities and affect epigenetic modifications. We analyzed the methylation level of TWIST1 genes in a set of primary AML samples with normal karyotype from the TCGA AML cohort [28]. This set includes 27 and 49 samples with DNMT3A R882 mutations and WT DNMT3A, respectively. Notably, in a region within 500-bp up- and downstream of gene transcriptional start site, R882 mutation group showed hypomethylation (Additional file 1: Figure S7). We suppose that D3Amut may lead to the demethylation of TWIST1 gene, thereby increasing its expression in leukemic cells. Interestingly, SHI-1, a cell line harboring MLL-AF6 translocation derived from an AML-M5 patient, can also invade murine brain [29]. MLL is a histone modifying gene, and MLL rearrangement interferes the normal function of MLL. Therefore, EMI, as one of the features of AML-M4/M5 subtypes, may be partly attributed to epigenetic deregulation.

EMI is one of the reasons for the relapsed and refractory AML. Clinical studies have demonstrated that cells bearing DNMT3A mutant are resistant to conventional chemotherapy but sensitive to high-dose of daunorubicin-based regimen [30, 31]. We suppose that dose-escalated therapy might be useful for clearance of DNMT3A mutated cells, thus disrupting cell mobility. Importantly, in our assays, abrogation of DNMT3A mutant or TWIST1 in leukemic cells reveals an anti-infiltration effect, thereby providing a possible theoretical basis for clinical transformation.