FLT3-ITD Signaling Induces Oncogenic Mir-155 by NF-κB and STAT5 Pathways In Acute Myeloid Leukemia Thereby Targeting Transcription Factor PU.1,

Abstract 3469

Almost 30% of all acute myeloid leukemias (AML) are associated with an internal tandem duplication (ITD) in the juxtamembrane of FLT3. This mutation leads to constitutively activated FLT3 signaling, which also includes altered FLT3 targets like STAT5. The dysregulation of pathways causes a differentiation block and plays a role in inhibition of hematopoietic transcription factors like PU.1 and C/EBPalpha. Here we report that FLT3-ITD signaling induces the oncogenic miR-155 by NF-κB and STAT5 pathways. Furthermore we demonstrate that miR-155 targets the myeloid transcription factor PU.1. Analysis of FLT3-ITD positive patient samples show an approximately 2 fold higher miR-155 expression compared to FLT3-WT associated AMLs. Besides, the overexpression of FLT3-ITD in myeloid U937 cells increases miR-155 expression 2,2 fold. In contrast, a block of FLT3-ITD signalling in FLT3-ITD associated cell line MV4;11 by protein kinase inhibitors (PKIs), PKC412, SU5614 and CEP701, results in an 80% decreased miR-155 expression. In further experiments we analyzed the role of FLT3-ITD downstream targets NF-κB (p65) and STAT5 in miR-155 regulation. We show that siRNA mediated knockdown of NF-κB (p65) or STAT5 in MV4;11 cells correlates with reduced miR-155 expression. In addition we demonstrate that p65 binds to the miR-155 promoter in MV4;11 cells while the treatment with the PKI CEP701 results in a p65 release from the miR-155 promoter. Furthermore, we prove that miR-155 overexpression in PMA (phorbol 12-myristate 13-acetate) treated U937 cells reduces macrophage differentiation about 50%. In in silico analysis we found PU.1 as a putative miR-155 target. Interestingly, we could show that overexpression of FLT3-ITD and miR-155 reduces the PU.1 protein level in U937 cells, respectively. These data elucidate the regulation and function of miR-155 in FLT3-ITD associated AML and may lead to a better understanding of the role of miRNAs in leukemogenesis.