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The clinical activity of decitabine (5‐aza‐2‐deoxycytidine, DAC), a hypomethylating agent, has been demonstrated in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients. However, secondary resistance to this agent often occurs during treatment and leads to treatment failure. It is important to clarify the mechanisms underlying the resistance for improving the efficacy. In this study, by gradually increasing concentration after a continuous induction of DAC, we established the DAC‐resistant K562 cell line (K562/DAC) from its parental cell line K562. The proliferation and survival rate of K562/DAC was significantly increased, whereas the apoptosis rate was remarkably decreased than that of K562 after DAC treatment. In K562/DAC, a total of 108 genes were upregulated and 118 genes were downregulated by RNA‐Seq. In addition, we also observed aberrant expression of DDX43/H19/miR‐186 axis (increased DDX43/H19 and decreased miR‐186) in K562/DAC cells. Ectopic expression of DDX43 in parental K562 cells rendered cells resistant to the DAC. Taken together, we successfully established DAC‐resistant K562 cell line which can serve as a good model for investigating DAC resistance mechanisms, and DDX43/H19/miR‐186 may be involved in DAC resistance in K562.

The K-homology (KH) domain is a nucleic acid–binding domain present in many proteins. Recently, we found that the DEAD-box helicase DDX43 contains a KH domain in its N-terminus; however, its function remains unknown. Here, we purified recombinant DDX43 KH domain protein and found that it prefers binding ssDNA and ssRNA. Electrophoretic mobility shift assay and NMR revealed that the KH domain favors pyrimidines over purines. Mutational analysis showed that the GXXG loop in the KH domain is involved in pyrimidine binding. Moreover, we found that an alanine residue adjacent to the GXXG loop is critical for binding. Systematic evolution of ligands by exponential enrichment, chromatin immunoprecipitation–seq, and cross-linking immunoprecipitation–seq showed that the KH domain binds C-/T-rich DNA and U-rich RNA. Bioinformatics analysis suggested that the KH domain prefers to bind promoters. Using 15N-heteronuclear single quantum coherence NMR, the optimal binding sequence was identified as TTGT. Finally, we found that the full-length DDX43 helicase prefers DNA or RNA substrates with TTGT or UUGU single-stranded tails and that the KH domain is critically important for sequence specificity and unwinding processivity. Collectively, our results demonstrated that the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase.

The tumour suppressor PML (promyelocytic leukaemia protein) regulates several cellular pathways involving cell growth, apoptosis, differentiation and senescence. PML also has an important role in the regulation of stem cell proliferation and differentiation. Here, we show the involvement of the helicase HAGE in the transcriptional repression of PML expression in ABCB5+ malignant melanoma-initiating cells (ABCB5+ MMICs), a population of cancer stem cells which are responsible for melanoma growth, progression and resistance to drug-based therapy. HAGE prevents PML gene expression by inhibiting the activation of the JAK–STAT (janus kinase–signal transducers and activators of transcription) pathway in a mechanism which implicates the suppressor of cytokine signalling 1 (SOCS1). Knockdown of HAGE led to a significant decrease in SOCS1 protein expression, activation of the JAK–STAT signalling cascade and a consequent increase of PML expression. To confirm that the reduction in SOCS1 expression was dependent on the HAGE helicase activity, we showed that SOCS1, effectively silenced by small interfering RNA, could be rescued by re-introduction of HAGE into cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes SOCS1 mRNA unwinding and protein expression in vitro. Finally, using a stem cell proliferation assay and tumour xenotransplantation assay in non-obese diabetic/severe combined immunodeficiency mice, we show that HAGE promotes MMICs-dependent tumour initiation and tumour growth by preventing the anti-proliferative effects of interferon-α (IFNα). Our results suggest that the helicase HAGE has a key role in the resistance of ABCB5+ MMICs to IFNα treatment and that cancer therapies targeting HAGE may have broad implications for the treatment of malignant melanoma.