STAT5 and NF-κB Induced Oncogenic Mir-155 Directly Targets PU.1 in FLT3-ITD Associated AML
Authors: Gerloff Dennis , Grundler Rebekka , Braeuer-Hartmann Daniela , Katzerke Christiane , Hartmann Jens-Uwe , Mueller-Tidow Carsten , Duyster Justus , Tenen Daniel G. , Niederwieser Dietger , Behre Gerhard 
Published: October 2019

Abstract
Abstract 3515
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 constitutive activated FLT3 signalling, 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. MicroRNA (miRNA) expression is also targeted by FLT3-ITD signaling. MiRNAs are small (19 – 22 nt) non coding RNAs, which regulate protein expression by binding to the 3'UTR of their target mRNAs. MicroRNA-155 (miR-155) was found to be highly expressed in FLT3-ITD associated AML patient samples. Thus, we hypothesized that miR-155 is an oncomiR which is regulated by FLT3-ITD signalling. 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 or FLT3-TKD associated AMLs. In addition we compared the miR-155 expression between 32D cells stably expressing either FLT3-WT or FLT3-ITD. The data show, that miR-155 expression is approximately 10 fold higher in the FLT3-ITD expressing 32D cells. 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 STAT5 and NF-κB (p65) in miR-155 regulation. We show that siRNA mediated knockdown of STAT5 or NF-κB (p65) in MV4;11 cells correlates with reduced miR-155 expression. A knockdown of NF-κB (p65) and STAT5 is able to overcome the FLT3-ITD overexpression induced miR-155 expression in U937 cells. To investigate the STAT5 impact on miR-155 expression in FLT3-ITD associated AML in vivo, we analyzed the miR-155 expression of irradiated C57BL/6 mice, transplanted with bone marrow from MX-Cre STAT5flox/flox mice stably expressing FLT3-ITD, after induced STAT5 knockout. The expression of miR-155 was significantly decreased to 0,4 fold in the STAT5−/− mice compared to STAT5flox/flox. These data reveal that STAT5 plays a pivotal role in FLT3-ITD induced 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 dissociation 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 as well as overexpression of miR-155 reduces the PU.1 protein level in U937 cells to 0.5 fold. However, a block of miR-155 by locked nucleic acids (LNAs) in MV4;11 cells, leads to PU.1 protein increase. Further, by luciferase assay analyses we prove that miR-155 directly targets PU.1 3'UTR.
Altogether we conclude that FLT3-ITD induces the oncogenic miR-155 via STAT5 and NF-κB (p65). The increased miR-155 expression targets PU.1 and enhances the myeloproliferative disorder in acute myeloid leukemia blasts. These data reveal the impact of deregulated miRNAs in cancer development and may provide novel biomarkers and therapeutic targets in leukemia.

Disclosures:
No relevant conflicts of interest to declare.