Depletion of ZBTB38 potentiates the effects of DNA demethylating agents in cancer cells via CDKN1C mRNA up-regulation

DNA methyltransferase inhibitor (DNMTi) treatments have been used for patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and have shown promising beneficial effects in some other types of cancers. Here, we demonstrate that the transcriptional repressor ZBTB38 is a critical regulator of the cellular response to DNMTi. Treatments with 5-azacytidine, or its derivatives decitabine and zebularine, lead to down-regulation of ZBTB38 protein expression in cancer cells, in parallel with cellular damage. The depletion of ZBTB38 by RNA interference enhances the toxicity of DNMTi in cell lines from leukemia and from various solid tumor types. Further we observed that inactivation of ZBTB38 causes the up-regulation of CDKN1C mRNA, a previously described indirect target of DNMTi. We show that CDKN1C is a key actor of DNMTi toxicity in cells lacking ZBTB38. Finally, in patients with MDS a high level of CDKN1C mRNA expression before treatment correlates with a better clinical response to a drug regimen combining 5-azacytidine and histone deacetylase inhibitors. Collectively, our results suggest that the ZBTB38 protein is a target of DNMTi and that its depletion potentiates the toxicity of DNMT inhibitors in cancer cells, providing new opportunities to enhance the response to DNMT inhibitor therapies in patients with MDS and other cancers.

. ZBTB38 and control siRNAs and further exposed to decitabine or not. (B) Quantification of autophagic cells (gate C in panel S5A) in THP-1 and HeLa cells transfected with ZBTB38 and control siRNAs and further exposed to DNMT inhibitors or not. (C) Western blot analysis of autophagic markers P62/SQSTM1 and LC3I-II expression in HeLa cells transfected with ZBTB38 and control siRNAs and further exposed to decitabine or not. (D) Representative FACS analysis of cell cycle phases in THP-1 cells after treatment with ZBTB38 and control siRNAs and further exposed to decitabine or not. (E) Quantification of G1, S and G2/M cells in THP1 and HeLa cells transfected with ZBTB38 and control siRNAs and further exposed to DNMT inhibitors or not.
(F) Representative FACS analysis of apoptosis and necrosis in THP-1 cells after treatment with ZBTB38 and control siRNAs and further exposed to decitabine or not. (G) Quantification of late apoptotic (black bar), early apoptotic (grey bar) and necrotic (light grey bar) cells in THP-1 and HeLa cells transfected with ZBTB38 and control siRNAs and further exposed to DNMT inhibitors or not (DAC: decitabine 1µM, 24 hours; AZA: azacytidine 4µM, 24 hours). (H) Quantification of cell debris in THP-1 and HeLa cells transfected with ZBTB38 and control siRNAs and further exposed to DNMT inhibitors or not (similar condition as panel S6B).   The lack of TP53 expression in THP-1 cells was confirmed by western blot ( Figure 5A).

Western blot analysis.
Cell extracts were prepared as previously described in RIPA buffer supplemented with protease and phosphatase inhibitors (Miotto et al., 2014), resolved on Bolt pre-cast gels (Invitrogen) and then transfered to Immobilon-P membranes (Millipore). The membranes were blocked with 5% fat-free milk in PBS, then incubated overnight at 4 °C with the appropriate primary antibodies.
The membranes were incubated with the cognate secondary antibody coupled to horseradish peroxidase, and revealed using the West Dura kit (ThermoFisher Scientific), in the ChemiSmart 5000 imager (Vilber Lourmat). References of the primary antibodies are provided in Supplementary Table S2. Secondary antibodies coupled with horseradish peroxidase were purchased from Jackson ImmunoResearch.
Digital images were used for semi-quantification of protein expression on the Image J software using GAPDH as the reference.

Gene expression analysis.
mRNAs were prepared using TriReagent protocol (Sigma-Aldrich; T9424) and followed by a standard phenol-chloroform purification procedure. mRNAs were reverse-transcribed using the SuperScript II (or IV) reverse transcriptase following manufacturer procedures (Thermo Fischer Scientific). cDNAs were analyzed by real-time PCR on a LightCycler ® 480 system (Roche) available at the GENOM'IC platform at Institut Cochin (INSERM U1016, Paris). The list of specific primer pairs is provided in Supplementary Table S3. Relative gene expression level were determined using the 2 -ΔΔ Ct method and the data normalized to the expression of a set of 3 housekeeping genes (GAPDH, MAPK14 and TFRC) as previously described (Miotto et al., 2014).

Flow-cytometry analyses
Flow-cytometry based analysis were performed on a BD Accuri TM C6 (BD Biosciences) available at the CYBIO platform at Institut Cochin (INSERM U1016, Paris). All data were visualized, analyzed and processed on the BD Accuri TM C6 analysis software. Ten thousand to twenty thousand cells were analysis per condition.
Cell cycle analysis were performed using the Click-IT Edu assay kit for flow cytometry using protocols provided by the manufacturer (Thermo Fisher Scientific). Briefly, cells were grown in the presence of 5-ethynyl-2´-deoxyuridine for 40 minutes. Following fixation and Click-IT reaction cells were further stained with 7-aminoactinomycin D to detect total DNA content.
Cell death analysis were performed by co-staining cells with propidium iodide and Annexin V on fresh cells using the Annexin V apoptosis detection kit APC (eBiosciences) following the recommendations of the manufacturer.
Autophagy was monitored by visualizing the intensity of the acidic cellular compartment using acridine orange staining. Cells were incubated with medium containing 1 µg/mL acridine orange (Invitrogen A3568) for 20 minutes, washed once with PBS and the red and green fluorescence quantified by FACS.

GEO datasets and bioinformatics analysis
Expression data from MDS patients treated with a combination of 5-azacytidine and entinostat were retrieved from GSE16625 (Fandy et al., 2009). Patients enrolled in the study were treated with sequential administration of 5-azacytidine and entinostat. 5-azacytidine was administered subcutaneously for 10 consecutive days in doses of 30, 40 or 50 mg/m2 per day. Entinostat (2, 4, 6 or 8 mg/m2) was administered orally on days 3 and 10 of the 5-azacytidine treatment.
CD34-positive samples were obtained before treatment (Day 0) and on day 15. The clinical response of each patient was assessed using International Working Group 2000 criteria and previously reported (Fandy et al., 2009).
Gene expression in the bone marrow of AML and MDS patients was re-analyzed from microarray data deposited as GSE13159 (Haferlach et al., 2010). The cohort comprised 72 control individuals, 542 patients with AML and 206 patients with MDS.

Bone marrow samples and survival outcomes in MDS patients
Bone marrow samples were collected from patients with MDS (n=55)

DNA methylation analyses
LUminometric-based Methylation Assay (LUMA) was performed as previously described (Karimi et al., 2011 Dot blots were conducted using a specific antibody directed against 5-methyl-CpG (Active Motif, 61480) and 5-hydroxymethyl-CpG (Active Motif, 39769) and normalized to total DNA using an antibody directed against single-strand DNA (Millipore, MAB3034) in each samples. Genomic DNAs were heat-denaturated and an equal amount of DNA for each condition spotted on a nylon membrane (Sigma-Aldrich). Following incubation with antibodies and signal detection, intensity of 5(h)mC and total DNA signals were evaluated using Image J software and the ratio 5mC/DNA and 5hmC/DNA calculated.
MeDIP (Methylated DNA immunoprecipitation) analysis was conducted using the Auto-MeDIP kit (Diagenode) using the manufacturer conditions. Briefly, genomic DNA was purified from samples, sonicated to the range of 200 base pairs, denaturated by heat and immunoprecipitated using a methyl-CpG specific antibody (Monoclonal antibody 33D3;