Human B-cell precursor leukemia cell lines (TOM–1, HB11;19, RS4;11, UOC-B6, REH, SUP-B15, NALM6), T-cell leukemia cell lines (HPB-ALL, CCRF-CEM, JURKAT, MOLT–4), AML cell lines (MV4;11, KASUMI–1, NB–4, THP–1, MOLM–13) and cell lines derived from the blast crisis of chronic myeloid leukemia (CML), which manifest as AML (K–562, LAMA–84) and as ALL (BV–173), were used (Table S1). The HB11;19 cell line was kindly provided by Dr. Anthony Ford from the Institute of Cancer Research (London, UK), and the UOC-B6 cell line was provided by Dr Ondrej Krejci (Massachusetts General Hospital, Boston) (14). The rest of the cell lines were purchased from German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany;). The cell lines were negative for mycoplasma contamination and cultivated in RPMI–1640 medium with GlutaMAX™ supplemented with 10% fetal calf serum, penicillin (100U/mL) and streptomycin (100μg/mL) under controlled conditions (37°C, 5% CO2). The cultured cells were split every two to three days and maintained in exponential growth phase.
Bone marrow or peripheral blood samples from untreated children initially diagnosed with B-cell precursor ALL, T-ALL or AML were collected from the Czech Pediatric Hematology Centers. The inclusion criteria were percentage of blasts higher than 80% and high cellularity. Within 24 hours after aspiration, without freezing, the mononuclear cells were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, UK). All the samples were obtained with the informed consent of the children’s parents or guardians as well as the approval of the Ethical Committee of the University Hospital Motol, Prague, Czech Republic, study no. 201528848A.
The isolated blasts were maintained in RPMI–1640 medium with GlutaMAX™ supplemented with 10% fetal calf serum, penicillin (100U/mL) and streptomycin (100μg/mL). For the MTS assay, insulin-transferrin-sodium selenite supplement was added to the culture media (Sigma-Aldrich, St Louis, MO, USA).
Mitochondrial FAO measurement
The cells were incubated for 4 hours in culture medium containing 100μM palmitic acid, 1mM carnitine and 1.7μCi [9,10(n)–3H]palmitic acid (GE Healthcare, UK) in the presence or absence of etomoxir (100μM, Sigma-Aldrich, MO, USA). Medium was collected to analyze the amount of released 3H2O that was formed during the cellular oxidation of [3H]-palmitate (15,16). The procedure was performed as described previously (7). The measurement was performed in three independent experiments.
The cells were incubated for 30 minutes in culture medium with or without 1μM tetramethylrhodamine ethyl ester (TMRE; ThermoFisher Scientific Inc., MA, USA) and were then washed with PBS and analyzed on a flow cytometer according to the manufacturer’s instructions. The level of TMRE staining was expressed as the mean of the TMRE signal in the live cells.
Cell survival and proliferation
To evaluate the cytotoxicity of ASNase, vincristine (VCR), and daunorubicin (DNR), MTS (dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium) assays were performed using a CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega Corporation, Wisconsin, USA) according to the manufacturer’s instructions. To evaluate the combined cytotoxicity of the two drugs, the number of live cells was determined by flow cytometry using DAPI (ThermoFisher Scientific Inc., MA, USA) and AccuCount Blank Particles (Spherotech Inc., IL, USA).
Seahorse extracellular flux analysis
Glycolytic and mitochondrial respiration parameters of leukemia cell lines were measured on a Seahorse analyzers XFe24 and XFp (Agilent Technologies, Inc., CA, USA) using a Glycolysis stress test and a Cell mito stress test. For the Glycolysis stress test, cells were seeded in XF Base medium, pH 7.4, and for the Cell mito stress test, cells were seeded in XF Assay medium, pH 7.4, supplemented with 10mM glucose, 1mM HEPES, pH 7.4, 1mM pyruvate and 0.1% BSA. The cells were plated at a density of 300,000 cells/well in XFe24 or 40,000 cells/well in XFp tissue culture plates coated with CellTak (Corning GmbH, Wiesbade, GER) according to the Agilent Seahorse protocol for seeding suspension cells.
The glycolytic and mitochondrial respiration parameters of the primary leukemia cells were measured on the Seahorse analyzer XFp using the same tests and media as in the case of the cell lines. The primary cells were plated at a density of 500,000 cells/well in XFp tissue culture plates. The procedure was performed as described previously (17).
Final concentrations of the injected drugs were 10mM glucose, 1μM (for cell lines) or 2μM (for primary cells) Oligomycin A and 100mM 2-deoxy glucose (2-DG) in the Glycolysis stress test and 2μM Oligomycin A, 1–4.5μM FCCP (depending on the cell line) and 1μM Rotenone combined with 1μg/ml Antimycin A in the Cell mito stress test.
Genomic DNA isolation and mtDNA quantification
Genomic DNA was isolated from leukemia cell lines using the QIAamp DNA Mini Kit (Qiagen GmbH, Germany) according to the manufacturer’s instructions. To quantify the mtDNA content, two genes as mitochondrial targets (16S rRNA and D-loop genes) and the GAPDH gene as a nuclear target were used. Quantification was performed using real-time PCR as described elsewhere (18).
Electrophoresis and western blotting
Protein lysates were prepared as previously described (19). The proteins (30μg per well) were resolved by NuPAGE Novex 4–12% Bis-Tris Gels (ThermoFisher Scientific Inc., MA, USA) and transferred to a nitrocellulose membrane (Bio-Rad, CA, USA). The membrane was probed overnight with the primary antibodies listed in Table S2. The bound antibodies were detected with the appropriate secondary antibodies conjugated to horseradish peroxidase (Bio-Rad, CA, USA) and visualized using an enhanced chemiluminescence reagent and documented by Uvitec (Cambridge, UK).
Hierarchical clusters were generated in R using the Pheatmap package (distance measure: “Euclidean”, clustering method: “ward.D2”). Linearization method was used to calculate adjusted (bonferoni) p-values for Oligomycin A effect to ASNase, VCR and DNR sensitivity of leukemia cells (Figure 3). Spearman rank correlations were calculated in R using the method “spearman”. p-values in Figure S1K were calculated using an unpaired, two-tailed, Mann-Whitney test in GraphPad Prism 6. Canonical Correlation analysis was done in R.