Trametinib

Mouse-derived MPNST cell lines described previously [34 (link)] were cultured at 37 °C in a humidified atmosphere in 5% CO₂ in low pH DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (Corning, lot #35070165) and 1% Penicillin Streptomycin (Thermo Fisher Scientific), unless otherwise indicated. Cell lines were verified to be free of Mycoplasma contamination every 6 months by PCR (ATCC). Human-derived MPNST cell lines were cultured as described previously [85 (link)]. Capmatinib (Novartis), trametinib (Novartis), everolimus (Selleckchem), and afuresertib (Selleckchem) solutions were prepared in DMSO. For human cell line [86 (link)] IC₅₀ experiments, drugs were purchased as 10 mM stock solutions (Selleckchem) and handled as previously described [85 (link)]. All cell culture experiments were performed 3 independent times, unless otherwise noted.

Human melanoma cell lines, WM266, M14 and A375 were obtained from the laboratory of Dr. Paolo A. Ascierto at National Cancer Institute of Naples “Fondazione G. Pascale”, Naples, Italy. All these cell lines harbor BRAF-V600 mutations that is V600D for WM266 and V600E for M14 and A375. They all derive from metastatic lesions. In particular, M14 was reported to be established at the University of California Los Angeles (UCLA) from a 33-year-old patient with metastatic melanoma, A375 derived from a 54-year-old female with malignant melanoma, whereas WM266 came from individual lymph-node metastases from a 55-year-old female. BRAFi-resistant counterparts were obtained through sequential increasing treatments of a BRAFi for about two months; for more detail please see our previous work. All melanoma cell lines used were cultured in RPMI supplemented with 10% FBS. Nanoparticles treatments were performed by exposing cells to 30 μg of each LNPs for 72 h in the presence of FBS with the exception of setting experiments shown in Figures S1B and S2. Dabrafenib and trametinib as BRAFi and MEKi, respectively, were obtained by Novartis Farma S.p.A. (Rome, Italy). For biological assays they were used at different concentrations, starting from the highest dose of 3 μM and then diluted 1:2 for ten times.

Human recombinant PD-L1 (Peprotech), nivolumab (anti-PD-1 monoclonal antibody, Bristol Myers Squibb), pembrolizumab (anti-PD-1 monoclonal antibody, Merck), atezolizumab (anti-PD-L1 monoclonal antibody, Genentech), trastuzumab (anti-HER2 monoclonal antibody, Genentech), daratumumab (anti-CD38 monoclonal antibody, Janssen), and trametinib (anti-MEK1/2 small molecule, Novartis) were obtained. Treatment assays with PD-L1 were performed at a concentration of 1 µg/ml as described.^{11 (link)} The following antibodies were used for western blot assay and immunofluorescence (IF): anti-PD-1 (Proteintech), anti-PD-L1 (Abcam), anti-phospho and total ERK (Cell Signaling), anti-GAPDH (Santa Cruz), and anti-beta-actin (Sigma Aldrich). PD1 (PDCD1) (NM_005018) Human Overexpression Lysate (Origene) and empty vector cell lysate (Origene) were used as PD-1 positive and negative controls, respectively.

Alpelisib, everolimus, and trametinib were obtained from Novartis International AG (Basel, Switzerland). Drugs were dissolved in 100% dimethylsulfoxide (DMSO) to a 10 mM concentration and stored at −80 °C before dilution to intermediate concentrations in cell culture medium. In all the pharmacological experiments, control cells were treated with an equivalent 0.1% vehicle DMSO concentration, equivalent to the 0.1% final DMSO concentration in the drug dilution.