Functional miRNA Expression Library Screen Identifies miRNAs That Alter Proliferation and Differentiation in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by increased self-renewal of leukemia stem/progenitor cells and failure of differentiation to mature myeloid cells. MicroRNAs (miRNAs, miRs) are short non-coding RNAs whose power stems from the ability to regulate hundreds of targets by interfering with gene expression by degrading messenger RNA or blocking protein translation. It has become evident that miRNAs are important regulators of normal cell processes that are altered in cancer, and restoring or inhibiting miRNAs may be a therapeutic strategy in cancer. MiRNAs play a significant role in normal hematopoiesis and differentiation; however, our understanding of how miRNAs contribute to impaired differentiation and proliferation in acute leukemia remains limited.

We have used a combined approach to prioritize miRNAs for investigation for functional roles in leukemogenesis. To identify miRNAs that may play a role in altered cell proliferation or differentiation of AML cells, we used a functional miRNA library screen. We transduced a high-throughput lentiviral library containing 613 precursor miRNAs (Systems Biology) into the AML cell line THP-1 harboring a MLL-AF9 translocation. These cells were then treated with 12-O-tetra-decanoylphorbol-13-acetate (TPA), an agent that induces monocytic differentiation and cell adhesion. After recovery of proliferating cells we compared miRNA expression before and after TPA induced differentiation using quantitative PCR to identify enriched miRNAs (may block differentiation) and miRNAs that drop out (may promote differentiation). To prioritize miRNAs with phenotypic effects that may be relevant to AML, we focused on miRNAs that are differentially expressed in various AML subtypes, using both miRNA-sequencing data from primary pediatric patient AML samples (N=117) and mature miRNA expression of primary adult patient AML samples (N=20).

Selected miRNAs were cloned into a lentiviral expression vector and retested in THP-1 cells to assess whether proliferation or differentiation was altered by expression. In line with previously published data we found enrichment of miR-9-1 and members of the miR-17-92 cluster, and its paralogs miR-106a-363 and miR-106b-25, as inhibitors of myeloid differentiation. In addition, we have validated three miRNAs (miR-590, miR-550A1 and miR-550A2) with previously uncharacterized roles that block differentiation and increase proliferation of THP-1 cells. These phenotypes were confirmed in other AML cell lines, including NB4 cells (PML-RARA translocation) and U937 cells (MLL-AF9 translocation). Increased expression of either miR-590 or miR-550A2 enhances MAPK signaling, as detected by increased phospho-ERK1/2, which influences cell proliferation. We have identified that PPP2R2A, the regulatory B55 alpha subunit of the tumor suppressor protein phosphatase 2A (PP2A), is a miR-590 target. Regulation of this complex by miR-590 could contribute to alterations in MAPK/ERK signaling. To identify additional miR-590 and miR-550A putative targets, we profiled gene expression changes by microarray (Illumina Human HT-12v.4) in THP-1 and NB4 cells expressing miR-590 or miR-550A2 versus control cells. Validation of identified putative targets, as well as functional investigations assessing the impact of the identified miRNAs and targets on AML cell proliferation or differentiation, are underway.

In conclusion, functional miRNA screening is an important tool to prioritize differentially expressed miRNAs identified in primary AML patient samples that alter differentiation and proliferation. Using this approach we have identified new roles for miR-590 and miR-550A2 in AML. Our goal is to determine mechanistically how these miRNAs block differentiation in AML cells and to establish how to target these pathways therapeutically to promote differentiation and AML cell death.