Konference: 2015 20th Congress of the European Hematology Association - účast ČR

Kategorie: Maligní lymfomy a leukémie

Téma: Poster

Číslo abstraktu: P178

Autoři: Alexander Arthur Wurm; Meritxell Alberich-Jorda, PhD; B.Sc. Polina Zjablovskaja; Dennis Gerloff; Daniela Braeuer-Hartmann; Christiane Katzerke; Jens-Uwe Hartmann; MD Stephan Fricke; Nadja Hilger; Daniel G. Tenen; MD Dietger W Niederwieser; MD Gerhard Behre


The transcription factor CCAAT enhancer binding protein alpha (C/EBPα) is a master regulator of granulopoiesis and is inactivated in approximately 50% of all acute myeloid leukemia (AML) cases. MicroRNAs, a class of small non-coding RNAs, were identified as important regulators of normal hematopoiesis and leukemia development. We have already shown that microRNAs, such as miR-223, miR-34a and miR-30c, are essential elements in C/EBPα triggered granulocytic differentiation. But to our knowledge nothing is known about inactivation of C/EBPα by microRNAs in acute myeloid leukemia.


Functional characterization of miR-182 in AML.


We used Next Generation Sequencing to detect novel C/EBPα related microRNAs. To investigate the function of miR-182, we used several in vitro and in vivo systems, including cell lines, primary AML cells and a transplantation based mouse model.


In this study, we identified a novel network between C/EBPα, miR-182 and E2F. In a next generation sequencing approach based on inducible K562-C/EBPα-ER cell line, we found miR-182 strongly downregulated by wildtype C/EBPα. We could further demonstrate an inverse correlation between C/EBPα protein amount and miR-182 expression level in several biological systems, including leukemic cell lines, G-CSF treated primary human CD34+ progenitor cells in vitro and sorted murine bone marrow subpopulations in vivo. Additionally, we found elevated miR-182 expression levels in lineage-negative, c-kit-positive myeloid progenitors of CEBPA Knock-Out mice.

To discover the mechanism how miR-182 is blocked by C/EBPα, we analyzed the minimal promoter region of miR-182 and performed chromatin immunoprecipitation (ChIP). Here, we could demonstrate a strong binding of C/EBPα to the miR-182 promoter, particularly to a conserved E2F binding site. Because E2F is a well known inhibitor of C/EBPα function, we tested whether E2F also effects miR-182 expression. An overexpression of E2F1 in U937 cells leads to an elevated miR-182 expression level. In addition, we measured the expression of miR-182 in bone marrow from AML patients regarding to their CEBPA mutation status. We could show that only patients with mutations in the C-terminal region of C/EBPα showed increased miR-182 expression levels, while patients with N-terminal CEBPA mutations revealed no abnormal miR-182 expression compared to healthy donors or AML patients with no CEBPA mutation. The C-terminal domain of C/EBPα is necessary for E2F inhibition. These findings illustrate the importance of C/EBPα-E2F interaction during miR-182 regulation.

Next, we found a highly conserved binding site of miR-182 in the 3’UTR of CEBPA itself, suggesting a possible negative feedback loop. To test this, we performed overexpression of miR-182 in U937 cells, umbilical cord blood mononuclear cells (UCB-MNCs) and primary blasts from AML patients. Here, we observed a strong reduction of C/EBPα protein after miR-182 overexpression in all cell types. Furthermore, we could demonstrate a direct binding of miR-182 to the 3’UTR of CEBPA via luciferase activity assay.

Finally, we were interested in the functional impact of miR-182 in myeloid differentiation and leukemia development. We showed that stable overexpression of miR-182 blocks granulocytic differentiation in G-CSF treated 32D cells and enhances replating capacity of primary mouse bone marrow mononuclear cells. Moreover, we could demonstrate that enforced expression of miR-182 in mouse Lin-Sca-1+c-Kit+ early hematopoietic progenitors by lentiviral infection leads to a strong reduction of mature Mac1+Gr1+ granulocytes in the peripheral blood in vivo.


Taken together, we identified miR-182 as novel oncogenic microRNA that directly blocks C/EBPα during myeloid differentiation and leukemia development. Thus, our data display a potential new strategy for therapeutics in C/EBPα dysregulated AML.

Keyword(s): Acute myeloid leukemia, Gene regulation, Oncogene, Transcription factor


Datum přednesení příspěvku: 12. 6. 2015