Mir-199b, a Candidate Effector of Stress Myelopoiesis and Is Differentially Regulated in Acute Myeloid Leukemia

Recently, we reported the functional role of miR-199b in AML and its prognostic implications where low miR-199b in AML patients correlated with worse overall survival for the FAB-M5 subtype. Herein, we have attempted to define the role of miR-199b in stress myelopoiesis using knockout mice and examine miR-199b's gene regulation. To define the role of miR-199b and understand its functional role in myeloid proliferation and AML, we generated miR-199b knockout mice via CRISPR approach. The deleted region was confirmed by DNA sequencing and silencing of miR-199b transcripts in nucleated peripheral blood cells was verified via RT-PCR. At steady state, except for significantly elevated neutrophil levels in miR-199b KO mice (n=5) compared to control mice (n=5) at 6 weeks of age, there were no significant differences in peripheral blood profiles. Bone marrow derived HSPCs (hematopoietic stem and progenitor cells) from miR-199b and wild type mice were examined for LT-HSC, KL, LSK, MPP, GMP and MEP populations. At steady state no significant differences were observed in all the cohorts except for KL (Lin-Kit+Sca-) population, where miR-199b KO mice exhibited significantly decreased levels compared to wild type mice (p<0.0093). Next, we examined the role of miR-199b under stress myelopoiesis by using 5-Fluouracil (5FU) induced myeloablation model. miR-199b KO mice exhibited significantly elevated levels of CFU-GEMM and CFU-GM compared to wild type mice. miR-199b levels in C57Bl/6 wild type mice were significantly decreased in HSCs post 5FU treatment. Gene profiling studies in HSCs post 5FU in wild type C57BL/6 mice identified three putative highly conserved miR-199b targets. They were Itga8 (Intergrin alpha 8), Zmpste 24 (metalloproteinase) and Scamp1 (secretory carrier membrane protein 1), whose roles are being investigated using miR-199b KO mice. Though we recently showed silencing of miR-199b in AML, the regulation of miR-199b gene expression in normal and leukemic cells is yet to be understood. Here, for the first time, using bioinformatics approach in integrated Genomics Viewer, we mined genomic database ENCODE (Encyclopedia of DNA elements) to identify both transcriptional and epigenetic landscape for miR-199b. Compared to CD34 and CD14, AML lines such as NB4 and HL-60 had relatively more regions of DNAase hypersensitivity. For DNA methylation, via reverse bisulphite sequencing data sets, no aberrant methylation was present between normal CD34 and HL-60, NB4 leukemic cells, which corroborates with our earlier finding of decitabine's ineffectiveness in inducing miR-199b expression in AML cells. Previously we had demonstrated that HDAC inhibitors significantly induced miR-199b expression in THP-1 cells. Here, for CD34 cells, we observed a significant presence of H3K4 and H3K9 methylation in miR-199b promoter via CHiP-seq data sets, implying an active transcriptional status. In CD14+ monocyte progenitors, a similar pattern for H3K4 methylation was observed and as well as significant levels of H3K9 acetylation. However, in CD15+ cells (neutrophil) H3K9 methylation was absent. Interestingly, in HL-60 and NB4 AML cells, H3K4me3 histone modifications peaked directly above the miR-199b coding region, whose significance is yet to be understood. For transcription factors, in HL-60 cells, via Chip-seq data sets, we found significant levels of GABP, NRSF and PU.1 binding activity in the upstream region of miR-199b. In NB4 (APL) cells, the Chip-seq data revealed a significant co-presence of cMyc and MAX binding in the upstream region of miR-199b. Further, the binding sites for the respective factors were also tested and confirmed using Genomatix-MatInspector. To experimentally validate these genomics derived data, we treated AML lines (HL-60, NB4 and THP-1) with inhibitors for cMyc (10058-F4), Myc-Max heterodimer (10074-G5), Sp1 (Mithramycin A) and NRSF/REST (X5050) for 24 and 48 hours. Exposure to Myc-Max heterodimer and NRSF/REST inhibitors significantly elevated miR-199b levels for HL-60 cells. For THP-1 cells, treatment with Myc-Max and Sp1 inhibitors significantly elevated miR-199b levels. Further functional assays are required to identify the biological roles of miR-199b in AML. In terms of miR-199b gene expression, we observe a differential regulation pattern in normal and leukemic cells, which in part may reflect the molecular heterogeneity in AML.