Mir-199b, a Novel Tumor Suppressor miRNA In Acute Myeloid Leukemia With Prognostic Implications In FAB-M5 Subtype

Acute myeloid leukemia (AML) manifests a marked heterogeneity in both response to therapy and patient survival, observations that likely reflect its varied pathogenesis. Dysregulation of miRNAs that can act as tumor suppressors or oncogenes can result in tumorigenesis. One miRNA of interest is human miR-199b-5p, which is an intragenic miRNA encoded in the Dynamin 1 gene. Previously we demonstrated that miR-199b was significantly downregulated in AML and targets Podocalyxin and Discoidin Domain Receptor 1. Herein we investigated the functional role of miR-199b in AML and its prognostic implications. We first examined the relative miR-199b expression in steady state hematopoiesis and showed CD33⁺ myeloid progenitors had the highest miR-199b expression (p<0.03). In order to test whether suppression of miR-199b results in myeloproliferation, human bone marrow derived CD34⁺ cells were transduced with anti-miR-199b or control lentivirus particles. To examine the effect of miR-199b silencing on colony forming abilities, a CFU assay was performed. At day 16, silencing of miR-199b in CD34⁺ cells resulted in significant increases in CFU-GM colonies. We then investigated the effect of decreased miR-199b on hematopoietic stem cells (HSC) proliferation via aldehyde dehydrogenase assay. Anti-miR-199b CD34⁺ cells exhibited a significant increase in HSC numbers compared to control CD34⁺ cells demonstrating a proliferative advantage for HSC with decreased miR-199b. Via The Cancer Genome Atlas (TCGA), we analyzed the molecular and clinical characteristics of 166 AML cases to investigate a prognostic role for miR-199b. The Kaplan-Meier curves for high and low expression values of miR-199b and the observed distribution of miRNA expression revealed the highly expressed group had significantly better survival outcomes (p<0.016, log rank test). In addition, miR-199b expression varied by cytogenetic risk category in the least squared mean plot, with significantly higher miR-199b expression for the favorable category compared to the intermediate and poor categories combined (p<0.0001). Therefore, low expression of miR-199b predicts worse survival outcome and higher cytogenetic risk in AML. Additionally, there was significant difference between miR-199b expression across the AML subtypes with particularly low expression found in the M5 subtype. Furthermore, M5 subtype showed a poor prognosis with a one-year survival rate of only 25%, compared with 51% survival in the overall sample (p<0.024). With AML patients grouped into M5 versus other subtypes to look at the dichotomized miR-199b variable, we found that all of the M5 patients have low miR-199b expression (p<0.0001). Due to miR-199b significantly correlating with M5 subtype, the M5 cell line THP-1 was utilized to determine the epigenetic regulation of miR-199b in AML. Recently, epigenetic alterations such as histone modifications and DNA methylation have been shown to deregulate miRNA expression. Therefore, treatment of THP-1 cells for 24 hours with HDAC inhibitors AR-42 (2uM), Panobinostat (0.7uM), or Decitabine (demethylating agent, 5uM) showed miR-199b expression was significantly elevated upon AR-42 and Panobinostat treatment (p<0.0002 and p<0.0001 respectively). Furthermore, AR-42 and Panobinostat treated cells showed drastic apoptosis in both treatments. Thus, epigenetic regulation of miR-199b appears to be via histone modification. To further understand the hematopathological consequences of decreased miR-199b in the onset and development of myeloid leukemia, we employed a transduce / transplant mouse model. The Lin^-Sca-1⁺Kit⁺ (LSK) population was isolated from Ly5.2 C57Bl6 mice, transduced with anti-miR-199b or control lentivirus particles, and transplanted into Ly5.1 C57Bl6 recipient mice. Short term (3.5 weeks) peripheral blood analysis revealed that loss of miR-199b results in significant increase of % neutrophils (p<0.001), % monocytes (p<0.04), and % eosinophils (p<0.02). Further analysis to determine the role of miR-199b in leukemogenesis is ongoing via this mouse model. Taken together, for the first time our results demonstrate that in vivo loss of miR-199b can lead to myeloproliferation and in vitro HDAC inhibitors can restore miR-199b-5p expression and promote apoptosis. Lastly, low miR-199b-5p in AML patients leads to worst overall survival and has prognostic significance for FAB-M5 subtype.