Brequinar

Drugs were spotted using a Tecan D300e dispenser (Tecan, Mannedorf, Switzerland) at the following concentrations with log2 dilution series. pyrazofurin (SML1502, Sigma-Aldrich) (200 μM–5 μM for U2OS and hTERT-RPE1; 200 μM–2 μM for MCF7), MPA (M5255, Sigma-Aldrich) (100 μM–0.05 μM), 6MP (38171, Merck) (100 μM–0.05 μM), glutamine starvation (2 mM-7.8 μM), brequinar (5.08321 Sigma-Aldrich) (200 μM–10 μM), and leflunomide (L5025, Sigma-Aldrich) (400 μM–10 μM), hydoxyurea (H8627, Sigma-Aldrich) (5 mM–0.05 mM). Five hundred cells per well were seeded on 384-well cell culture plates, and viability was assessed with a resazurin assay and read on a Hidex Sense or Molecular Devices ID5 plate reader after 4 days in technical triplicates. Uridine (U3750, Sigma-Aldrich) rescue was performed at a concentration of 500 μM for 24 h. 2-DG (10 mM) was added to cultured cells for 48 h. Doxorubicine (D1515, Sigma-Aldrich), actinomycin D (A1410, Sigma-Aldrich), olaparib (AZD2281, Selleckchem), camptothecin (C9911, Sigma-Aldrich), nutlin3a (SML0580, Sigma-Aldrich).

ASOs were designed and synthesized by IONIS Pharmaceuticals under a collaborative agreement. The list of mouse and human ILF2 ASOs used in this study are included in Supplementary Table 1. NT and ILF2 ASOs were prepared in culture medium supplemented with 10% fetal bovine serum to achieve the indicated concentrations. ASOs were delivered to the cells by free uptake. For in vitro single-agent assays, KMS11, JJN3, MM1R, H929, and RPMI-8226 cells were initially treated with 0.1, 0.5, 1, 2, or 2.5 μM ASOs for 7 days. ASOs were added every 2 days together with fresh media. For combination therapy studies, the cells were treated with melphalan (Sigma), bortezomib (Tocris), brequinar (Sigma), IACS-010759 (IACS), NSC105808 (Chemspace), or C5 (AOB9082, Aobious, Inc.) at the concentrations and times indicated in the figure legends in the presence or absence of NT or ILF2 ASOs (KMS11, 0.5 μM; JJN3, 1 μM; RPMI-8226, 1 μM; MM1R, 1 μM; H929, 2 μM).
Primary PCs isolated from MM patients and healthy donors were treated with NSC105808 at the concentrations and times indicated in the figure legends prior to being analyzed.

The in vitro inhibition of hDHODH was measured using an N-terminally truncated recombinant hDHODH enzyme as described previously [36 (link)]. Briefly, the hDHODH concentration was adjusted in a way that an average slope of approximately 0.2 AU/min served as the positive control (e.g., without inhibitor). The standard assay mixture contained 60 µM 2,6-dichloroindophenol (Sigma, St. Louis, MO, USA), 50 µM decylubiquinone (Sigma), and 100 µM dihydroorotate (Sigma). The hDHODH enzyme with or without at least six different concentrations of the compounds was added, and measurements were performed in 50 mM TrisHCl, 150 mM potassium chloride (KCl) (Merck), and 0.1% Triton X-100 (Sigma) at pH 8.0 and at 30 °C [37 (link)]. The reaction was started by adding dihydroorotate and measuring the absorption at 600 nm for 2 min. For the determination of the IC50 values, each data point was recorded in triplicate. DHODH inhibitors Cmp 1, Cmp 2, Cmp 3, Cmp 4, and Cmp 5 were provided by Immunic Therapeutics. Brequinar, uridine, 2′-deoxyuridine, cytidine, and 2′-deoxycytidine were purchased from Sigma. UCK2 inhibitor, UCK2-IN-20874830 was obtained from ChemDiv (San Diego, CA, USA).