Mir-590-5p, Mir-219-5p, Mir-15b and Mir-628-5p Are Commonly Regulated by IL-3, GM-CSF and G-CSF in Acute Myeloid Leukemia,

Abstract 3520

IL-3, GM-CSF and G-CSF are predominant regulators for growth and differentiation of myeloid progenitors. Interestingly, they all signal via a common JAK2-STAT5 pathway in myeloid progenitor compartments. However, the specific mechanism through which JAK2-STAT5 responds differentially to early-acting and lineage restricted cytokines, particularly in leukemic and stem/progenitor cells, is largely unresolved. Aberrations in IL-3, GM-CSF and G-CSF induced signaling are frequently reported in acute myeloid leukemia (AML). microRNA (miRNA) play several crucial roles during hematopoiesis that include lineage decisions, stem cell progenitor transitions, niche control and other cell functions. Recent investigations have linked aberrant miRNA expression with AML. We hypothesized that a unique response of leukemic myeloid progenitors to IL-3, GM-CSF, and G-CSF are possibly mediated in part by distinct regulation at the miRNA level. Therefore, herein, by utilizing a unique leukemic myeloid cell line, AML-193, that responds to both early and late acting cytokines, we profiled IL-3, GM-CSF and G-CSF regulated miRNA signatures in leukemic myeloid progenitors. For miRNA profiling, AML-193 cells were initially exposed to IL-3 for 3 days followed by GM-CSF for 3 more days and subsequently to G-CSF for 3 days. We then profiled miRNA expression induced by IL-3, GM-CSF and G-CSF in AML-193 cells by treating the respective cohorts post growth factor deprivation with corresponding cytokines. Using SA Bioscience's complete Human V2.0 miRNA Genome Array platform for real-time qPCR-based miRNA expression profiling, we investigated miRNA signatures regulated by IL-3, GM-CSF and G-CSF for n=704 miRNAs in AML-193 cells. Frequencies of false positives were avoided using stringent filters. IL-3 specifically regulated 54 miRNAs and those miRNAs that were highly regulated included miR-362-3p, miR-590-3p, miR-340, miR-24-2, miR-1183 and miR-99a. GM-CSF specifically regulated miRNAs included let-7f, let-7a*, miR-195, miR-122, miR-376-c and miR-33a. G-CSF specifically regulated set included miR-21*, miR-192*, miR-32*, miR-7-1*, miR-545* and miR-37-4a*. Interestingly, 301 miRNAs were commonly regulated by IL-3, GM-CSF and G-CSF. Among the commonly regulated miRNAs, the ones that were subjected to high levels of regulation included miR-590-5p, miR-219-5p, miR-92-a1*, miR-378*, miR-548-3p, miR-29a*, miR-590-3p, miR-203, miR-363, miR-454, miR-340, miR-196a, miR-152, miR-10b, miR-24-2*, miR-10a, miR-182, miR-27a*, and miR-199a-3p. Interestingly, the commonly regulated miRNAs demonstrated a directional regulation in the order of IL-3>GM-CF>G-CSF. Analysis of the potential targets of significantly regulated miRNAs revealed important functional roles in myeloid cell development and differentiation. Conserved targets of miR-590-5p included BMPR2, PCBP2 and KLF3. Targets for miR-219-5p included Smad4 and GADD45b. Targets for miR-362-3p included Sox17, an inhibitor of the Wnt signaling pathway. Furthermore, to identify the JAK2-STAT5 responsiveness of miR-219-5p, miR-362-3p, and miR-590-5p, a STAT5 specific inhibitor (N′-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide) was used prior to cytokine treatment. Interestingly, IL-3 and GM-CSF mediated induction of miR-590-5p, miR-219-5p and miR-362-3p were not significantly affected by STAT5 inhibitor. However, the G-CSF induction of miR-219-5p and miR-590-5p were significantly inhibited by the STAT5 specific inhibitor. In summary, we have discovered for the first time novel miRNA profiles regulated by IL-3, GM-CSF and G-CSF in an acute myeloid leukemia progenitor cell model. Importantly, we have established a cytokine regulated miRNome for leukemic myeloid progenitors and set the stage for future investigations in leukemic stem cells to delineate the pathological roles of dysregulated miRNAs in AML. In addition, our study displayed that G-CSF induced a subset of miRNAs that are principally regulated via the STAT5 circuit in AML. Further, these novel miRNA signatures may have therapeutic implications for targeting dysregulated miRNAs by antagomir strategy or miRNA replacement therapy, paving the way for the development of novel miRNA-based therapeutic interventions in AML.