Program: Oral and Poster Abstracts
Session: 602. Disordered Gene Expression in Hematologic Malignancy, including Disordered Epigenetic Regulation: Poster II
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.
Disclosures: No relevant conflicts of interest to declare.
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