Deciphering the Function of KLF4 as a Differentiation Inducer in Hematologic Malignancies

Kruppel-like factor4 (KLF4) is a member of the KLF family transcription factors, well known for its reprogramming capacities to promote iPS cell transformation. In the context of hematopoietic cells, the major role of KLF4 has attributed to its myeloid to monocyte differentiation capacity and considered to work as a tumor suppressor in acute myeloid leukemia (AML) or myeloid dysplastic syndrome (MDS)-derived cells. Ras-Raf-MEK-ERK pathway is consistently up-regulated in these tumor cells and we have previously reported the role of KLF4 as a major differentiation inducer in this setting, however, how does KLF4 induce monocytic differentiation under MEK-ERK pathway activation has remained unknown. We thus addressed this issue and found a novel key mediator of monocytic differentiation in myeloid leukemia cells.

To identify essential downstream factors of KLF4 in myeloid leukemia cells, we first analyzed 3 independent gene expression microarray data sets of AML patients (GSE45194, GSE38810 and GSE22845). AML patients were divided into two groups according to their KLF4 expressions and top 1000 up-regulated genes in KLF4 high-expressing AML patients were extracted. Venn diagram was used to identify the overlapping genes in these data sets and we identified 26 candidate genes possibly involved in KLF4 mediated differentiation in hematologic malignancies. We then performed comprehensive quantitative real-time PCR (qRT-PCR) analysis to examine the expression of all of these candidate genes upon additive KLF4 expression in leukemia cell lines of MOLM-13 and THP-1 cells. Among these genes, KLF4 exceptionally up-regulated the expression of Dihydropyrimidinase like 2 (DPYSL2) over 200-folds. DPYSL2 consists DPYSL gene family. Since previous reports suggest their multiple roles in neuronal differentiation and polarity, as well as in axon growth and guidance, we hereafter focused on this DPYSL2 gene to reveal its veiled function in leukemia cells. Intriguingly, qRT-PCR assay demonstrated that KLF4 uniquely up-regulated the gene expression of DPYSL2 isoform1 among DPYSL family members. We confirmed the specific expression of DPYSL2 isoform1 upon additive KLF4 expression by immunoblotting in AML cells . Chromatin immunoprecipitation (ChIP) assay proved that KLF4 bound directly to the gene promoter region of DPYSL2 isoform1. We next induced the endogenous expression of KLF4 in myeloid leukemia cells using phorbol 12-myristate 13-acetate (PMA) which leads to a rapid and sustained activation of MEK and ERK, ultimately inducing a substantial monocytic differentiation in these cells. PMA treatment induced concomitant expressions of KLF4 and DPYSL2 isoform1 both at mRNA and protein levels in these cells. To assess the function of DPYSL2 isoform1 in myeloid leukemia cells, we generated tetracycline-inducible DPYSL2 isoform1-overexpressing human leukemia cell lines. Upon doxycycline treatment, these leukemia cells differentiated into monocytic lineage with marked CD11b and CD14 cell surface expressions. We next knocked out DPYSL2 isoform1 in KLF4 overexpressing leukemia cells using CRISPR/Cas9 gene modification system and found that the genetically modified cells maintained the undifferentiated state upon KLF4 overexpression. We also demonstrated that shRNA-mediated partial down-regulation of DPYSL2 isoform1 in leukemia cells with enforced KLF4 expressions resulted in mild inhibition of KLF4-induced monocyte differentiation. Taken together, these results underpin the importance of DPYSL2 isoform1 in monocytic differentiation of myeloid leukemia cells.

Our findings offer insight into a novel role of DPYSL2 as a differentiation inducer in hematologic malignancies and may provide a new therapeutic approach for hematologic malignancies.