Gdc0973 cobimetinib

WM115, RPMI7951, SKMEL24, WM793, SKMEL28, SKMEL5, A375, SKMEL3, and MALM3M human melanoma cell lines were purchased from ATCC. 501MEL and WM9 cell lines were kindly provided by Dr Robert Ballotti and M4BE cell line by Dr Thibault Voeltzel (Centre Léon Bérard). All these BRAF^V600 human melanoma cell lines were cultured in DMEM complemented with 10% FBS (Cambrex) and 100 U/ml penicillin–streptomycin (Invitrogen). In order to authenticate the cell lines, the expected major genetic alterations were verified by NGS sequencing. The absence of mycoplasma contamination was checked every 3 weeks with the MycoAlert detection kit (Lonza). Patient‐derived short‐term cultures (< 10) were established from BRAF^V600 metastatic melanomas, before treatment for GLO and C‐09.10, or after acquisition of resistance to vemurafenib for ESP and GOKA. C‐09.10 were kindly provided by Dr Robert Ballotti (Nice). These short‐term cell cultures were grown in RPMI complemented with 10% FBS and 100 U/ml penicillin–streptomycin. PLX4032/vemurafenib and GDC0973/cobimetinib were purchased from Selleck Chemicals (Houston, TX, USA) and reconstituted in DMSO. Generation of A375‐R and SKMEL5‐R resistant models was performed by treating cells chronically with increasing doses of PLX4032 for 2–3 months. All BRAFi‐resistant cell lines were permanently cultured in the presence of 3 μM PLX4032.

The following drugs and compounds were used: ampicillin (AppliChem, Omaha, NE, USA), blasticidin (InvivoGen, San Diego, CA, USA), cisplatin (1 mg/ml solution, Teva, Petach Tikwa, Israel), cycloheximide (Sigma-Aldrich, St. Louis, MO, USA), DMSO (AppliChem, Omaha, NE, USA), doxycycline (AppliChem, Omaha, NE, USA), Fura-2 AM (Thermo Fisher Scientific GmbH, Schwerte, Germany), hygromycin (AppliChem, Omaha, NE, USA), MG132 (Sigma-Aldrich, St. Louis, MO, USA), the vemurafenib isoform PLX4720 (Selleckchem Houston, TX, USA), GDC-0973/cobimetinib (Selleckchem Houston, TX, USA), puromycin (Merck, Darmstadt, Germany), trametinib (Selleckchem Houston, TX, USA), Neg4 ((oxolan-2-yl)methyl-4-(6-bromo-2H-1,3-benzodioxol-5-yl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate, ChemDiv, San Diego, CA, USA) and Cpd1 (2-phenoxyethyl 4-(2-fluorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate, ChemDiv, San Diego, CA, USA). Neg4 and Cpd1 were dissolved in DMSO at a stock concentration of 10 mM and diluted 1:1000 in media. 3-O-(3,4,5-trimethoxybenzoyl)-(−)-epicatechin (TMECG) was synthesized as described previously^{55 (link)} and was made available by JN Rodríguez-López.