Cb 839

Miltirone (CFN98531, AOBIOUS, CAS 27210-57-7), ST1926 (SML2061, Sigma-Aldrich, CAS 496868-77-0), 2-DG (31060, Sigma-Aldrich, CAS 154-17-6), CB-839 (533717, Sigma-Aldrich, CAS 1439399-58-2), Etomoxir (236020, Sigma-Aldrich, CAS 828934-41-4), and N-acetyl-l-cysteine NAC (A9165, Sigma-Aldrich, CAS 616-91-1) were used at indicated concentrations and duration.

Stock solutions of topotecan (Sigma-Aldrich, #E1383), CB-839 (Sigma-Aldrich, #1439399) and Prexasertib (Biomol, Cayman Chemical, #21490) were prepared in dimethyl sulfoxide (DMSO). Stock solutions were kept at -20°C and were freshly diluted in the culture medium for experiments.

Drugs dissolved in DMSO (stock solution) were added to culture media for last 24 hours, while for control treatments, identical amounts of DMSO in culture media was used. Concentration of drugs as used in this study was based on previously published data (13 (link), 23 (link), 24 (link)). The following chemicals were used: 1 mM aminooxyacetic acid hemihydrochloride (AOA, 4-aminobutyrate aminotransferase inhibitor, Cat. No. C13408, Sigma Aldrich, USA) and 2 μM bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES, glutaminase inhibitor, Cat. No. SML0601, Sigma Aldrich, USA); 500 nM N-[5-[4-[6-[[2-[3-(trifluoromethoxy)phenyl]acetyl]amino]-3-pyridazinyl]butyl]-1,3,4-thiadiazol-2-yl]-2-pyridineacetamide (CB-839, glutaminase 1 inhibitor, Cat. No. 22038, Cayman Chemicals, USA) and 40 μM (3R,5S)-rel-5-[6-(2,4-dichlorophenyl)hexyl]tetrahydro-3-hydroxy-2-oxo-3-furanacetic acid (SB-204990, citrate lyase inhibitor, Cat. No. 15245, Cayman Chemicals, USA). The concentration of 40 μM for SB204990 was chosen based on previous reports showing sufficient inhibition of ACLY in a cell line and to closely mimic plasma concentrations achieved in animal experiments after oral administration (25 (link)).

For glutamine deprivation assay, SKBR3FLAG-KISS1R cells and controls were seeded in 6 cm dishes (400,000 cells each) in glutamine-free RPMI media with dialyzed FBS. Cells were treated with 0.02 mM glutamine, 0.2 mM glutamine, or 2 mM glutamine (Gibco) over 72 h; media was changed every 24 h and cells trypsinized and counted using a hemocytometer at 24 h intervals. For BPTES or CB-839 (Sigma Aldrich) treatment, SKBR3FLAG-KISS1R cells (400,000 cells) were plated in 6 cm dishes. On the following day, these cells were treated with different concentrations of BPTES or CB-839 and cell number counted at 24 h intervals. To determine the effect of c-Myc knockdown on cell growth, SKBR3FLAG-KISS1R cells expressing c-Myc siRNA were cultured in media without glutamine. Media was changed daily and each day cells were counted for each experimental condition.

Oxygen consumption rates (OCR) were measured using a Seahorse Bioscience XF^e96 Extracellular Flux Analyzer. MG63.3 cells were plated at 10,000 cells/well in XF96 cell culture microplates. Following attachment, cells were treated with (1) vehicle, (2) 1 μM CB-839 (Selleck Chemicals), (3) 5 mM metformin (Sigma-Aldrich), or (4) 1 μM CB-839 + 5 mM metformin and incubated for 24 h at 37 °C. Just prior to the Seahorse assay, growth media was replaced with 180 μL of Seahorse XF Base Media (Agilent) supplemented with glucose, glutamine, and sodium pyruvate, and the plate incubated in a 37 °C incubator lacking CO₂ for 45 to 60 minutes. OCR was determined by performing the Cell Mito Stress Test (Agilent) according to the manufacturer’s specifications, as previously described [26 (link)].