Doxorubicin-induced loss of DNA topoisomerase II and DNMT1- dependent suppression of MiR-125b induces chemoresistance in ALK-positive cells

Systemic anaplastic large-cell lymphoma (ALCL) is a childhood T cell neoplasm defined by the presence or absence of translocations that lead to the ectopic expression of anaplastic lymphoma kinase (ALK), with nucleophosmin-ALK (NPM-ALK) fusions being the most common. Polychemotherapy involving doxorubicin is the standard first-line treatment but for the 25 to 35% of patients who relapse and develop resistance the prognosis remains poor. We studied the potential role of the microRNA miR-125b in the development of resistance to doxorubicin in NPM-ALK(+) ALCL. Our results show that miR-125b expression is repressed in NPM-ALK(+) cell lines and patient samples through hypermethylation of its promoter. NPM-ALK activity, in cooperation with DNA topoisomerase II (Topo II) and DNA methyltransferase 1 (DNMT1), is responsible for miR-125b repression through DNA hypermethylation. MiR-125b repression was reversed by the inhibition of DNMTs with decitabine or the inhibition of DNA topoisomerase II with either doxorubicin or etoposide. In NPM-ALK(+) cell lines, doxorubicin treatment led to an increase in miR-125b levels by inhibiting the binding of DNMT1 to the MIR125B1 promoter and downregulating the pro-apoptotic miR-125b target BAK1. Reversal of miR-125b silencing, increased miR-125b levels and reduced BAK1 expression also led to a lower efficacy of doxorubicin, suggestive of a pharmacoresistance mechanism. In line with this, miR-125b repression and increased BAK1 expression correlated with early relapse in human NPM-ALK(+) ALCL primary biopsies. Collectively our findings suggest that miR-125b could be used to predict therapeutic outcome in NPM-ALK(+) ALCL.

Total RNA from cell lines and CD4-positive cells were extracted using the Trizol reagent (Ambion), according to the manufacturer's instructions. MiRNAs were reverse transcribed using the Universal cDNA Synthesis Kit II (Exiqon). The expression of miR-125b, RNU1A1 and SNORD44 (used as reference genes) was measured by quantitative PCR using a specific Exiqon PCR primer set (Supplementary Table 1) and a SYBR qPCR Premix Ex Taq (Tli RNaseH Plus) Kit from Takara. Messenger RNAs (mRNAs) were reverse transcribed using a SuperScript II Reverse Transcriptase (Invitrogen), according to the manufacturer's recommendations. Data are presented as relative quantities of target RNA. The high-throughput qPCR analysis method used for measuring BAK1 expression was performed using the primers listed in Table  S1 and the BioMark 96 × 96 gene expression platform, according to the manufacturer's instructions (Fluidigm).

MiRNA expression microarray
Microarray experiments were carried out using 1-color hybridizations on a human miRNA microarray (V3, 8 × 15K) (Agilent, Cat No: G4471A-021827): one glass slide formatted with eight high-definition 15 K arrays, based on the Sanger miRbase (release 12.0), with 866 human and 89 human viral miRNA probes represented. The mean normalized signal from the biological replicates was used for comparative expression analysis. A paired t-test with Benjamini-Hochberg correction (P value ≤ 0.05) was used to identify miRNAs differentially-expressed between tumor and benign tissues. The fold change in expression between tumor and benign samples was calculated from the normalized values.

DNA bisulfite treatment and bisulfite pyrosequencing
Genomic DNA (gDNA) was extracted using either the QIAamp DNA Mini Kit (Qiagen) or the Allprep DNA/ RNA/miRNA Kit (Qiagen), according to the manufacturer's instructions. Bisulfite conversion was then conducted with 500 ng gDNA, using the MethylEdge Bisulfite Conversion System (Promega). Two regions of interest in the miR125-b promoters, containing 7 and 6 CpGs respectively, were amplified by PCR (PCR PyroMark Kit, Qiagen), with specific primers designed by the PyroMark Assay Design software (Qiagen), one of which was biotinylated for subsequent binding of the PCR products to streptavidin-coated sepharose beads. Methylation analysis was performed on the PyroMark Q24 Pyrosequencing System (Qiagen) using specific sequencing primers (Supplementary Table 1) and products and protocols supplied by the manufacturer.

Protein extraction and Western blotting analyses
Total cell lysates were prepared in RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 4 mM EDTA, 0.5% Triton X100 and 0.2% SDS). Protein concentrations were determined using a Bradford assay (Biorad). The primary antibodies used are listed in Supplementary  Table 2. HRP-conjugated anti-mouse (Jackson Immuno Research) or anti-rabbit antibodies (Cell Signaling) were used as secondary antibodies. Densitometric analysis was performed using GeneTools software from Syngene.

Cell proliferation assay
The CellTiter 96AQueus One Solution provided by Promega was used to assess cell proliferation/survival, according to the manufacturer's instructions.

Annexin V/propidium iodide (PI) double staining assay
Cells were harvested and incubated with recombinant annexin V-pacific blue at a final concentration of 1 g/ml for 5 min at room temperature. Just before analysis by flow cytometry, PI was added to all samples to a final concentration of 10 g/ml. The percentage of cells annexin V positive is indicated.

Supplementary Figure 1: NPM-ALK represses the expression of miR-125b in ALCL human and mouse models. (A)
Assessment of miR-125b expression by qRT-PCR in wild type mice (WT, n = 6) or NPM-ALK transgenic mice containing a Tet-OFF system treated (+) or not (-) with crizotinib (n = 4). SNORD202 expression served as the internal control, and the relative ratio of mmu-miR-125b expression was expressed as 2 -ΔΔCt relative to WT mice. (B and C) Protein levels of NPM-ALK, p-NPM-ALK and GAPDH were assessed by western blotting in KARPAS-299 and COST cells treated or not (PBS) with crizotinib (Crizo) (B) or transfected with either an irrelevant siRNA as the negative control (si-CTL) or an siRNA targeting ALK mRNA (si-ALK) (C). Data represent means ± SEM (bars) from 3 independent experiments. * P < 0.05 and *** P < 0.0001; unpaired 2-tailed Student's t test.