Krüppel-like Factors KLF4 and KLF2 Regulate microRNA-150 Expression in Myeloid Leukemias

Background: 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) are small single stranded non-coding RNAs 19 to 24 nucleotides in length that regulate expression of tens to hundreds of genes via mRNA degradation or translational repression. MiRNA contributions to normal hematopoiesis have been described and deletion of key miRNA processing enzymes in murine and human cells suggests that miRNA loss contributes to the cancer phenotype and aberrant differentiation in leukemia.

By combining observations of miRNA expression in normal hematopoietic progenitor cells and patient AML cells and high-throughput lentiviral expression library screening approaches in AML cell lines we have identified candidate miRNAs that contribute to altered proliferation and differentiation in AML cells. We have previously established 1) that miR-150 expression is decreased in a large subset of primary patient AML samples, in particular poor risk cytogenetic groups, 2) and that miR-150 re-expression induces myeloid differentiation and decreases cell proliferation of normal hematopoietic progenitor cells and AML cell lines and primary patient cells in part through downregulation of MYB expression. MiR-150 loss is relevant in other hematopoietic and solid tumor malignancies where re-expression inhibits cell proliferation, promotes apoptosis and induces reversal of endothelial to mesenchymal transition.

Transcription factors are important regulators of myeloid differentiation and cell proliferation. Moreover, as highlighted by recent sequencing of the AML genome, alterations in myeloid transcription factors through mutation, gene rearrangement, and altered expression play a significant role in leukemogenesis. Consequently, we have focused on how myeloid transcription factors regulate miRNA expression, specifically for miR-150.

Results: Using 5’RACE from healthy bone marrow RNA, we identified a major transcription start site at 214 basepairs upstream of the pre-miR-150 hairpin. We identified the minimal miR-150 promoter region as -266 to +259 basepairs from the major transcription start site using miR-150 promoter truncation luciferase constructs assayed in myeloid leukemia cell lines (THP-1, K562, and KG1a) and a lymphoid leukemia cell line (Jurkat). We identified DNA binding sites for the Krüppel-like factor (KLF) family of transcription factors that are necessary for miR-150 promoter activity using site-directed DNA mutagenesis of the luciferase reporters. KLFs regulate proliferation, differentiation, pluripotency, migration and inflammation. Depending on cell type and context, KLFs can function as tumor suppressors or oncogenes. To identify which KLF isoforms regulate miR-150 expression, we assayed the ability of KLFs 2, 3, 4, 5, 6, 7, 9, and 10 to induce miR-150 promoter activity using the luciferase reporters and endogenous miR-150 expression by quantitative PCR. KLF2 and KLF4 overexpression increased miR-150 promoter activity in luciferase assays 50-fold and 450-fold respectively in K562 cells. Furthermore, KLF2 and KLF4 induced endogenous miR-150 expression 20-fold and 100-fold respectively as detected by quantitative PCR in both THP-1 and K562 cells. Prior work has established that KLF2 and KLF4 regulate the differentiation of monocytes. We then confirmed that KLF2 and KLF4 overexpression promotes myeloid differentiation of THP-1 cells by flow cytometry and gene expression that was partially reversed by inhibition of miR-150 expression.

Conclusions: Previous studies have determined that KLF2 and KLF4 expression are decreased or absent in a significant subset of AML cases. Our observations suggest that loss of KLF2 and KLF4 expression contributes to decreased miR-150 expression which in turn alters cell proliferation and differentiation. Other studies have implicated the cell cycle inhibitor p21^WAF1/CIP1 and altered PPAR gamma signaling downstream of KLF4. Nonetheless, our mechanistic understanding is limited. Our work suggests that the loss of miR-150 and other miRNAs downstream of these transcription factors also contributes. Understanding the interactions between KLFs, miR-150 and other miRNAs has broader significance as KLF2 and KLF4 expression is altered in other hematopoietic and solid tumors.