Disturbance of the C/EBP alpha-miR-182 balance impairs granulocytic differentiation and promotes development of acute myeloid leukemia

Abstract

Introduction: Silencing of major myeloid transcription factor C/EBPa by gene mutation, promoter hypermethylation or posttranslational modifications is well described and occurs in ~50% of acute myeloid leukemia (AML) cases. Deregulation of C/EBPa by microRNAs, a class of small non-coding RNAs, as a substantial event during AML development or during myeloid differentiation has not been studied yet. Methods: We screened for C/EBPa dependent miRNAs in inducible K562-C/EBPa-ER cells using Illumina’s Next Generation Sequencing. For in vitro functional studies including gain-of-function and loss-of-function experiments, we utilized common acute myeloid leukemia cell lines, human hematopoietic stem cells from umbilical cord blood, murine hematopoietic stem cells from mouse bone marrow and primary human cells from patients with acute myeloid leukemia. For in vivo investigations, we manipulated Lin-Sca-1+c-Kit+ (LSK) murine hematopoietic progenitor cells by lentiviral infection and transplanted them into lethally irradiated littermates. The resulting phenotype was analyzed by flow cytometry and morphological staining of blood and bone marrow. Results: We identified oncogenic miR-182 as strong regulator of C/EBPa during myeloid differentiation and in AML. Moreover, we uncovered a novel regulatory loop between C/EBPa and miR-182. While C/EBPa blocks miR-182 expression by direct promoter binding during myeloid differentiation, enforced expression of miR-182 leads to reduced C/EBPa protein levels and impairs granulopoiesis in vitro and in a transplantation based mouse model in vivo. In contrast to this, a knockdown of miR-182 expression enhances C/EBPa protein levels in human AML. Furthermore, we observed highly elevated miR-182 expression levels particularly in AML patients with C-terminal CEBPA mutations and thereby uncovered a mechanism how C/EBPa blocks miR-182 expression. Finally by evaluation of TCGA database, we identified miR-182 expression as a strong adverse prognostic factor in cytogenetically high risk AML patients. Conclusions: Taken together, our data demonstrate the importance of a controlled balance between C/EBPa and miR-182 for the maintenance of healthy granulopoiesis and might uncover a novel mechanism for potential treatment strategies in AML. Disclosure: No conflict of interest disclosed