MiR-143 Targets ERK5 in Granulopoiesis and Predicts Outcome of Patients with Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a very heterogeneous disease of the hematopoietic system which is characterized by a fast progress with poor survival options. Despite intensive research standard treatment strategies rely since decades on the same chemotherapeutics, and the overall survival options still remain poor.

In recent years, there is an emerging role of microRNAs (miRNAs) in the regulation of cancer development and resistance to cancer therapies. Mechanistically, miRNAs regulate gene expression through modulation of multiple target mRNAs by mRNA degradation or block of translation. It is shown that miRNAs can act as tumor suppressors and oncogenes dependent on the type of cancer. The miRNA-143/145 cluster is shown to be down-regulated in most cancers and is able to modulate tumorigenesis by targeting tumor associated genes. Several miR-143/145 targets are well described oncogenes, such as ERK5, DNMT3A or Bcl2. Interestingly, recent studies showed that the miR-143/145 cluster can regulate multidrug resistance proteins (MDR) in various cancer types and that enforced expression of miR-143/145 leads to increased sensitivity to various drugs. Besides its well-studied function mainly in solid tumors the role of the miR-143/145 cluster or miR-143 alone especially in the hematopoietic system remains largely unknown.

In this study, we report a previously unrecognized function of miR-143 in granulopoiesis. Based on our observation that hematopoietic cells undergoing granulocytic differentiation exhibit increased miR-143 expression, we show that the absence of miR-143 in mice results in a reduced number of mature granulocytes but not monocytes in blood and bone marrow. Additionally, we show that overexpression or ablation of miR-143 resulted in accelerated granulocytic differentiation or block of differentiation in several in vitro systems, including leukemic cell lines and G-CSF treated CD34+ HSPCs, respectively.

To identify the mRNA targets affected by miR-143 in hematopoietic cells we performed Argonaut2-RNA-Immunoprecipitation coupled with next generation sequencing. Here we could demonstrate a strong enrichment of members of the MAPK- and mTOR-pathways in cells overexpressing miR-143. Besides several potential targets of miR-143 we found ERK5 as particularly interesting since ERK5 was found to be a central mediator of cell survival, proliferation, motility, differentiation and apoptotic regulation of normal cells. To test whether miR-143 overexpression would also affect ERK5 protein abundance we performed western blot analysis of ERK5 and activated ERK5 in hematopoietic cells infected with miR-143 O/E constructs. Here we could show that miR-143 leads to reduced ERK5 protein levels in these cells. Since deregulation of both factors miR-143 as well as ERK5 have been implicated in the response to chemotherapeutic agents we analyzed the impact of miR-143 expression on treatment response. Here we show that AML patients receiving 5-azacytidine (Vidaza) and respond to the therapy had significant higher miR-143 levels than patients which did not respond. To test whether miR-143 expression would affect the response to chemotherapeutics, we overexpressed miR-143 in leukemic cells followed by Vidaza and AC220 treatment. Our results show a significant increase of apoptosis in cells overexpressing miR-143. Finally, we observed an association of high miR-143 expression levels with a higher probability of survival in two different cohorts of patients with acute myeloid leukemia. Taken together, we identified miR-143 as a relevant factor in granulocytic differentiation, which expression may be useful as prognostic factor and as new target for therapeutic strategies in AML.