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CDK4/CDK6 inhibitors in ALK-positive lymphomas
Methods
Human cell lines, tumoral and normal samples
The two human NPM-ALK+ ALCL cell lines, KARPAS-299 and SU-DHL1, were obtained from DSMZ (German Collection of Microorganisms and Cell Culture, Braunschweig, Germany). The COST cell line was established in our laboratory.28 CD4+ cells were stimulated for two days using CD3/CD28 antibodies coupled to magnetic beads (Dynabeads, Invitrogen, Waltham, USA) in RPMI-1640 with 20% fetal calf serum (FCS). All cells were cultured in RPMI-1640 supplemented with 10% FCS (KARPAS-299 and COST) or 15% FCS (SU-DHL1) supplement- ed with 2 mM L-glutamine, 1 mM sodium pyruvate, and 100 U/mL penicillin/streptomycin (all from Invitrogen, Waltham, USA). Cells were cultured at 37°C with 5% CO2 and maintained in exponential growth phase. Tumor samples from NPM-ALK+ and NPM-ALK– ALCL diagnosed based on morphological and immunophenotypical criteria (according to the last WHO classi- fication) were obtained from our tumor tissue bank. Only cases with at least 50% lymph node involvement (assessed by CD30 staining of frozen biopsies) and good RNA integrity were select- ed from our tumor bank. Studies were conducted in accordance with the Declaration of Helsinki, and with the approval of the relevant ethics committees. Lymph nodes collected from patients with reactive non-malignant disease, who were consid- ered to be healthy donors, were retrieved from the CRB-Cancer du CHU de Bordeaux n. BRIF BB-0033-00036, a member of the “Cancéropôle Grand Sud-Ouest” network.
Cell treatment
Anaplastic large cell lymphoma cell lines were treated with 2 μM decitabine (Sigma-Aldrich, Saint Quentin Fallavier, France) for four days [fresh drug was added at 48 hours (h)] and NPM- ALK activity was inhibited using 500 nM crizotinib (@rtMolecule, Poitiers, France) for three days. ALCL cell lines were treated with 1 mM of PD-0332991 (Selleck Chemicals, Houston, TX, USA) during 24 h.
MiRNA and siRNA transfections
siRNAs (si-CDK6, si-CCNE1, si-CDC25A and si-STAT3) at 0.5 nmol, si-E2F3 (Online Supplementary Table S1) at 1 nmol or miRNA mimics (miR-497-5p: mirVana MC10490 and miR-CTL: mirVana AM17110, Invitrogen, Waltham, USA) were transfect- ed by electroporation according to a previously described proce- dure.17 In the case of co-transfection of several siRNAs, 0.2 nmol of each si-CDK6 and si-CCNE1 and 1 nmol of si-E2F3 were used. The same amount of duplex siRNA (Eurogentec, Angers, France) was transfected and used as negative control.
Purification of biotinylated-miRNA containing complexes
Five million KARPAS-299, COST and SU-DHL1 cells were transfected with 0.25 nmol of biotinylated hsa-miR-497-5p (hsa- miR-497 mercury LNA miRNA mimics premium) or biotinylat- ed-irrelevant miRNA (negative control) (Exiqon, Vedbaek, Denmark). Total cell lysates, 24 h post transfection, were pre- pared in IP-buffer: 25 mM Tris-HCl pH 7.5, 200 mM NaCl, 0.2% Triton-X100, 5 mM MgAc, 1 mM DTT, protease inhibitors (Roche France, Meylan, France) and 0.2 U/mL RNase OUT (Invitrogen, Waltham, USA). The lysates were incubated for 2 h with streptavidin coupled to magnetic beads (Thermofisher, Waltham, USA) previously saturated with 0.5 mg/mL BSA and 0.2 mg/mL yeast tRNA. Purified RNAs were recovered after 5 washes with IP buffer by Trizol reagent-mediated extraction (Invitrogen, Waltham, USA), according to the manufacturer’s
instructions. RNA was reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen, Waltham, USA) and subjected to quantitative PCR. Biotin pull-down efficiency was expressed as a percentage of input. GAPDH amplification was used to remove pull-down background. The specificity of purification was evaluated for each putative target based on the enrichment in the Biotin-miR-497 condition relative to Biotin-CTL.
RNA extraction, reverse transcription and quantitative PCR
Total RNA from cell lines and CD4+ cells were extracted using the Trizol reagent (Invitrogen, Waltham, USA), according to the manufacturer’s instructions. MiRNAs were reverse transcribed using the Universal cDNA Synthesis Kit II (Exiqon, Vedbaek, Denmark). The expression of miR-195, miR-497, RNU1A1 and SNORD44 (used as reference genes) was measured by quantita- tive PCR (qPCR) using a specific Exiqon PCR primer set and SYBR qPCR Premix Ex Taq (Tli RNaseH Plus) Kit (Takara Bio Europe, Saint-Germain-en-Laye, France). Messenger RNAs (mRNAs) were reverse transcribed using Primescript RT reagent kit (Takara Bio Europe, Saint-Germain-en-Laye, France), accord- ing to the manufacturer’s recommendations. qPCR was per- formed using the primers listed in Online Supplementary Table S1.
Protein extraction and Western blotting analyses
Total cell lysates were prepared by incubating ALCL cells in Laemmli 1X buffer (Bio-Rad, Marnes-la-Coquette, France) for 10 minutes (min) on ice. Cells were then subjected to sonication using a Bioblock Vibra-cell 72446 apparatus at 35% of its power (Fisher Scientific, Illkirch, France). Proteins were separated by electrophoresis on SDS-polyacrylamide gels and electroblotted onto nitrocellulose membrane (GE Healthcare Europe, Velizy- Villacoublay, France). Proteins of interest were detected using primary antibodies [(CDC25A 1/500: sc-7389, CDK6 1/2000: CST#3136, CCNE1 1/2000: sc-247, E2F3 1/2000: GTX102302, CDK4 1/2000: sc-260, Rb 1/2000: OP66, phosphor-Rb (ser780) 1/2000: CST#9307)] and HRP -conjugated anti-mouse (Promega, Madison, USA) or anti-rabbit secondary antibodies (Cell Signaling Technology, Danvers, USA). Densitometric analysis was performed using ImageJ software.29
Cell proliferation assays and cell cycle analysis
Cell proliferation was evaluated using CellTiter 96AQueus One Solution (Promega, Madison, USA) and cell counting. For cell cycle analysis, NPM-ALK+ ALCL cells were washed with PBS and fixed overnight at 4°C in 70% ethanol. Cells were then washed with PBS supplemented with 0.1% BSA and incubated for 30 min with 10 mg/mL propidium iodide (Invitrogen, Waltham, USA) and 500 mg/mL of RNAse (Sigma-Aldrich, Saint Quentin Fallavier, France). Thereafter, cells were analyzed with a flow cytometer MACSQUANT VYB (Miltenyi, Paris, France).
Apoptosis luminescent assay
The Caspase-Glo® 3/7 Assay Kit (Promega, Madison, WI, USA) was used to assess cell apoptosis, according to the manu- facturer’s instructions.
Bisulfite conversion and DNA methylation analysis
Genomic DNA was bisulfite converted using the MethylEdge Bisulfite Conversion System (Promega, Madison, USA) follow- ing the manufacturer’s protocol. The region of interest was amplified by PCR and methylation level was measured by pyrosequencing using PyromarkQ24 (Qiagen France, Courtaboeuf, France). (Primers are listed in Online Supplementary Table S1).
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