Abstract
The recent discovery of new molecular lesions with prognostic significance in acute myeloid leukemia (AML) is enhancing our understanding of leukemia biology and our ability to identify new therapeutic targets. Previously, using the unique leukemic myeloid progenitor line AML-193, we profiled IL-3-, GM-CSF-, and G-CSF-regulated miRNA signatures. 301 miRNAs were commonly regulated by these three cytokines, and the most highly induced miRNA was miR-590-5p. Herein, we have attempted to define the functional role and clinical relevance of miR-590 in AML. We first examined the relative miR-590 expression in steady state hematopoiesis and showed it was highest at CD34+ and declined its expression through myeloid lineage differentiation (ANOVA, p<0.0001). To functionally determine the role of increased miR-590 expression, we generated a gain-of-function model in human CD34+ hematopoietic stem cells (HSC) via lentivirus transduction. Increased expression of miR-590 in CD34+ cells resulted in significant increases in CFU-GM colonies, strongly suggesting that dysregulation of miR-590 expression may be myeloproliferative. In AML (n=33) and control (n=9) bone marrow samples, miR-590 expression was determined via RT-qPCR. miR-590-5p expression was highly upregulated in 22 of the samples (67%) compared to control subjects. In silico analysis of the miR-590-5p promoter revealed three potential binding sites for STAT5 (-249, -749, -1499). To functionally determine whether STAT5 directly regulates miR-590-5p expression, we performed a ChIP assay, which showed that STAT5 binds to the -749 region of miR-590-5p promoter. To conclusively determine the STAT5 binding sites, we cloned the miR-590 promoter in a luciferase vector and performed site directed mutagenesis for each potential binding site. This assay confirmed that the -749 binding site was the major STAT5 regulatory site for miR-590 (p<0.002). Importantly, constitutive activation of STAT5 is a hallmark of AML associated with FLT3 mutations, therefore, we set out to determine if specific STAT5 and FLT3 inhibitors could decrease miR-590 expression. We pretreated MV4-11 cells, which harbors the FLT-ITD mutation and has increased STAT5 activation, with 100uM STAT5 inhibitor (N′-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide) for 90 minutes or 100nM FLT3 inhibitor (EMD Millipore, 343020) for 12 hours, both of which resulted in significant inhibition of miR-590-5p expression (p<0.05). To evaluate whether the AML samples with high miR-590 expression also possess elevated phospho-STAT5 or phospho-FLT3 levels, we performed immunohistochemistry analysis on a custom-made tissue microarray. In AML samples with high miR-590 levels, increased activation of FLT3 and STAT5 was observed compared to controls. Since FLT3 mutations result in decreased survival and poorer prognosis in AML, it may be that miR-590-5p plays an important role in the pathology of AML associated with dysregulated FLT3 and STAT5. To understand the complete functional role of miR-590 in AML, the predicted targets need to be identified and validated for their roles in leukemogenesis. Upon molecular screening of several predicted targets, FasL was experimentally found to be a conserved target of miR-590. More specifically, 3’UTR analysis of FasL revealed three potential seed sequences for miR-590 which have been verified experimentally via luciferase assay. Furthermore, significantly increased levels of FasL protein and transcript expression was detected in the MV4-11 cells stably expressing anti-miR-590 compared to control cells. Additionally, we identified the levels of Fas/CD95 (FasL receptor) on AML-193 and MV4-11 cell lines and found these cells had high Fas/CD95 expression on the cell surface as analyzed via flow cytometry. In order to determine the physiological significance of Fas/FasL, these cells were treated with soluble FasL (100ng) for 24 hours and apoptosis was analyzed via Annexin V staining. FasL treatment induced increased apoptosis compared to the untreated cells. Taken together, we have identified miR-590 as a candidate oncomiR that is regulated via the STAT5 pathway and targets FasL to promote cell survival. Thus, our data suggests that further understanding of miR-590’s role in AML may lead to development of novel anti-miR-590 therapeutic strategies in AML associated with dysregulated STAT5.
Disclosures:
No relevant conflicts of interest to declare.
DNA Binding Site Selection of Dimeric and Tetrameric Stat5 Proteins Reveals a Large Repertoire of Divergent Tetrameric Stat5a Binding Sites
