Sb590885

WM115, WM266-4, A375, SK-MEL28, SK-MEL2 and RMPI-7951 were purchased at ATCC; WM35, WM983A, WM983B, WM239A, WM3211 and Lu1205 at Coriell Institute and WM1346 at Wistar Institute. WM115, WM266-4, A375 and SK-MEL28 were cultured in DMEM/FBS 10% (v/v), SK-MEL2, 501mel and RPMI-7951 in RPMI-1640/FBS 10% (v/v), WM35, WM983A, WM983B, WM3211, WM1346 and Lu1205 in MCDB153 medium with 20% Leibovitz L-15 medium (v/v), 2% FBS heat inactivated (v/v), 5 μg/mL insulin and 1.68 mM CaCl2. Cell lines were authenticated for mutations in BRAF and NRAS by sequencing within the time frame of the experiments. PLX4032, SB590885, AZD6244, AS703026 and MK2206 were from Selleck Chemicals, Z-VAD-FMK and actinomycin D from Sigma-Aldrich, LY294002 from Calbiochem, G594 competitive inhibitor of AKT was provided by Genentech.

SJCRH30, RD, and CCD-18Co cells were seeded at 2 × 10⁴ cells/well in 96-well plates. The candidate drugs were treated for 24 h. AZ628, BIX02189, fedratinib, nutlin-3a, VX-11e, SB590885, NSC207895, tubastatin A, CX-5461, navitoclax, GSK1070916 and the FDA-approved drug library were purchased from Selleckchem. GW.44175 was purchased from Cayman chemical. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide, Sigma) cell viability detection reagent diluted in serum free medium was added to each well and incubated for 2 h at 37°C. The supernatant was removed, and 50 μL DMSO was added to dissolve the precipitate. Absorbance was measured at 570 nm using a Molecular Devices VersaMax microplate reader and SoftMax® Pro 5 software. IC₅₀ was calculated with GraphPad Prism 7 software.

The TT cell line (MTC, human) was obtained from the European Collection of Cell Cultures (Sigma-Aldrich, Milano, Italy) and cultured in RPMI 1640 (Gibco - Life Technologies, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (FBS; Gibco), L-glutamine (2 mM) and penicillin-streptomycin (100 IU/ml–100 μg/ml respectively). Adherent monolayer cells were maintained in T-75 culture flasks and incubated at 37°C with 5% CO₂ until they achieved 85% confluence. Cells were detached using 0.25% trypsin-EDTA (Sigma-Aldrich) and plated in T-75 flasks at a density of 2 × 10⁶ cells.
RAF265 was kindly provided by Novartis International (Basel, Switzerland), SB590885 and ZSTK474 were purchased from Selleck Chemicals (Houston, TX, USA). The powders were dissolved in a 10 mM stock solution in dimethyl sulfoxide (DMSO), following the manufacturer’s instructions.

Male wild-type C57Bl/6J mice (7–8 weeks of age) were from Charles River (U.K.). Drug delivery used Alzet osmotic pumps (models 1007D or 1004; supplied by Charles River), filled according to the manufacturer's instructions in a laminar flow hood using sterile technique. Mice were treated with vehicle only [DMSO/PEG mix: 50% (v/v) DMSO, 20% (v/v) polyethylene glycol 400, 5% (v/v) propylene glycol, 0.5% (v/v) Tween 80 made up to 100% with H₂O], or 0.5 mg/kg/d SB590885 (Selleck Chemicals) or 3 mg/kg/d encorafenib (MedChemExpress) dissolved in DMSO/PEG mix. In some studies, mice also received a minipump containing angiotensin II (AngII) for delivery at 0.8 mg/kg/d or acidified PBS vehicle. Minipumps were incubated overnight in sterile PBS (37°C) prior to implantation. Implantation was performed under continuous inhalation anaesthesia using isoflurane (induction at 5%, maintenance at 2–2.5%) mixed with 2 l/min O₂. A 1 cm incision was made in the mid-scapular region and mice were given 0.05 mg/kg (s.c.) buprenorphine (Ceva Animal Health Ltd.) to repress post-surgical discomfort. Minipumps were implanted portal first in a pocket created in the left flank region of the mouse. Wound closure used a simple interrupted suture with polypropylene 4-0 thread (Prolene, Ethicon). Mice were allowed to recover singly and returned to their home cage once fully recovered.

Lysis buffer contained 150 mm NaCl, 1.0% Triton X-100, and 50 mm Tris (pH 8). CHAPS immunoprecipitation buffer contained 0.5% CHAPS in HEPES buffer. PLX4720 was obtained from Sai Advantium Pharma Limited (Pune, India). PD0325901 was obtained from Santa Cruz Biotechnology (Dallas, TX). Gefitinib, crizotinib, imatinib, and lapatinib were obtained from LC Laboratories (Woburn, MA). SB590885, vemurafenib were obtained from Selleck Chemicals. S63845 was obtained from Chemgood (Glen Allen, VA). PI-103 was kindly donated by J. Engelman (Massachusetts General Hospital). Actinomycin D was obtained from Sigma-Aldrich (St. Louis, MO). A1210477 was obtained from Active Biochem (Hong Kong). AZD4320 and AZD5991 were kindly provided by Astra-Zeneca (Waltham, MA). Unless indicated, dose of the targeted therapies used was 1 µm for 24 h, except for trametinib which was used at 100 nm.