Both FLT3-ITd and NPM1 mutations are associated with decreased MLLT3 mRNA expression in acute myeloid leukemia
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BACKGROUND: Acute myeloid leukemia (AML) is a molecularly heterogeneous disease characterized by the accumulation of acquired somatic genetic alterations. The most clinically relevant gene mutations include internal tandem duplications (ITDs) targeting the Fms-Related Tyrosine Kinase 3 ( FLT3 ) and small inserts in the Nucleophosmin ( NPM1 ). Mutations in these genes have been shown to impact survival; however, the functional significance and the mechanisms by which these mutations contribute to the pathogenesis of the disease are still not well defined. PURPOSE: To identify downstream biomarkers indicating dysregulated FLT3 -ITD signaling. These markers may be useful to further explore the functional significance of FLT3 -ITD mutations. METHODS: We established a lentiviral vector-based FLT3 cell line model in order to characterize the dysregulation of the FLT3 signaling. Lentiviral vectors over expressing wild-type FLT3 and two different ITD mutants were transduced into the ALL-1 cell line, and expression profiling was performed using the Affymetrix GeneChip U133 Plus 2.0 microarray. Cell line results were confirmed in two patient cohorts using RT-qPCR, microarray expression profiling, or RNAseq read counts. RESULTS: The mRNA signature associated with the FLT3 -ITD pathway dysregulation was determined in microarray experiments and identified Myeloid/Lymphoid or Mixed-Lineage Leukemia Translocated To 3 ( MLLT3 ) gene as being reduced at the mRNA level in FLT3 -ITD transduced cells. MLLT3 dysregulation was verified using RT-qPCR. This finding was confirmed by analysis of data from The Cancer Genome Atlas (TCGA) obtained on ≥99 intermediate karyotype AML samples showing MLLT3 mRNA suppression in FLT3 -ITD-positive cases (P<0.001). Additionally, low MLLT3 mRNA expression levels showed a strong association with positive NPM1 mutation status in both the TCGA and our own in-house AML patient cohorts (both P<0.001). In contrast to MLLT3 mRNA suppression, the protein levels did not show any association with neither FLT3 -ITD nor NPM1 , indicating the involvement of a post-transcriptional regulatory mechanism. CONCLUSIONS: Our study identified a reduced MLLT3 mRNA expression in cell lines upon induction of FLT3 -ITD. This reduction was confirmed in primary patient material. An additional association between reduced MLLT3 mRNA expression and NPM1 mutations was identified during the course of patient studies. MLLT3 may serve as a biomarker of both the FLT3 and NPM1 pathways with potential utility in the molecular diagnostics laboratory.