Next Generation Sequencing and Functional Analysis of Mirna Expression in Acute Myeloid Leukemia Patients with Different FLT3 Mutations: Block of MiR-155 in FLT3-ITD Driven AML Leads to Downregulation of Myeloid Blasts in Vivo

Up to 30% of all acute myeloid leukemias (AMLs) are associated with an activating mutation in the FMS-like tyrosine kinase 3 receptor (FLT3). Two distinct groups of FLT3 mutations are found: (1) the most common are internal tandem duplications (ITDs) of the FLT3 juxtamembrane region, and (2) point mutations within the tyrosine kinase domains (TKDs). While FLT3-TKD mutations seem to have no prognostic relevance in AML, patients bearing an FLT3-ITD mutation have a significantly worse outcome compared with AML patients with wild-type FLT3 (FLT3-WT). MicroRNAs (miRNAs) are small (~22 bp) noncoding RNAs, which regulate protein expression posttranscriptionally by recruitment of the RNA-induced silencing complex (RISC) to the 3′-untranslated region (3′-UTR) of target mRNAs. We and others have shown that miRNAs are crucial regulators in myeloid differentiation and in leukemogenesis. Furthermore, it was shown that several miRNAs have a prognostic impact. Hence, we hypothesized that the different FLT3 mutations lead to altered miRNA expression.

To find different expression patterns of miRNAs, we performed next generation sequencing of normal karyotype bone marrow patient samples with FLT3-WT (n=5), FLT3-TKD (n=3) and FLT3-ITD (n=3). Sequencing was performed with an Illumina HighScan-SQ sequencer using version 3 chemistry and flowcell according to the instructions of the manufacturer. For normalization the method of trimmed mean of M values (TMM) was used. Data analyses were performed using the Qlucore Omics Explorer 3.1. In a multi group analyses of miRNA expression pattern, we found 17 significant differentially expressed miRNAs (p ≤ 0.05). The expression of 6 miRNAs (miR-10a-5p, miR-10a-3p, miR-18a-5p, let-7b-3p, miR-155-5p and miR-576-5p) was increased only in the FLT3-ITD associated patient samples. In the FLT3-WT samples we found 8 miRNAs (miR-141-3p, 342-3p, 181a-2-3p, 374b-5p, 30b-5p, 29c-3p, 23b-3p and 125a-3p) with an increased expression. The miR-92a-3p showed an enhanced expression in FLT3-WT and FLT3-ITD patient samples. The multi group analyses showed only 2 miRNAs (miR-3615 and miR-193b-3p) induced in FLT3-TKD patient samples. The two FLT3-ITD induced miRNAs, miR-10a-5p and miR-155-5p were the most abundant and most differentially expressed miRNAs in the screen. From our data we hypothesize that miR-155 and miR-10a could play an important role in disease progression and clinical outcome of FLT3-ITD induced AMLs

To analyze a block of miR-155 in FLT3-ITD driven AML in vivo, we transfected 32D cells, stably expressing human FLT3-ITD, with unspecific scramble or miR-155 specific locked nucleic acids (LNAs (Exiqon)). 24h after transfection, we injected 1x106 cells into C3H mice (scr. n=5; LNA-155 n=5). All animals rapidly developed a leukemia like disease with hepatosplenomegaly. The animals died 17 - 21 days after 32DFLT3-ITD cell injection. We could not observe a difference in survival. In flow cytometry analysis of the peripheral blood we found a strong increase of human FLT3 (huCD135) expressing cells (32DFLT3-ITD) 1 to 2 days before the mice died. At death of the animals we analyzed the accumulation of leukemic cells (32DFLT3-ITD) in bone marrow, spleen and liver by flow cytometry for the human FLT3 (huCD135). Here we could observe a significantly (p≤ 0.05) reduced number of leukemic cells in the bone marrow of the mice with the LNA-155 transfected 32DFLT3-ITD cells in comparison to the group with the scramble transfected 32D cells. In spleen we could not observe a difference in accumulation of leukemic cells, but in the liver we could show a tendentially reduced accumulation of 32DFLT3-ITD cells transfected with LNA-155.

The next generation sequencing screen gives insight into the altered miRNA expression pattern of FLT3-WT, FLT3-TKD and FLT3-ITD related AMLs. The miR-10a-5p and miR-155-5p are highly expressed in FLT3-ITD associated AMLs. The block of the FLT3-ITD induced miR-155 in vivo significantly reduces the accumulation of leukemic cells in the bone marrow of transplanted mice. The results give the evidence that miR-155 could be a novel therapeutic target in FLT3-ITD associated AML.