Plx4720

Cell line authentication and BRAF/NRAS mutation detection were previously described (8 (link)-10 (link)). Cells were grown in RPMI media in 5% fetal bovine serum. PGC1α promoter reporter was obtained from Addgene. MITF and TRPM1 promoter reporters were obtained from R. Haq (6 (link)). Selumetinib (AZD6244/ARRY142886), AZD8055 and AZD2014 were from AstraZeneca, PLX4720 was from Plexxikon, and other inhibitors were from SelleckChem. For in vitro treatments, the inhibitors were dissolved in DMSO.

8×10⁵ BP cells were implanted subcutaneously (s.c.) in C57BL/6 mice on the left flank. When tumors reached ~100mm³ mice were given ad libitum BRAFi (PLX4720) chow containing 200 or 417 parts per million (ppm) of PLX4720 or control chow acquired from Plexxikon Inc. For CD8 depletion, 200μg of rat anti-mouse CD8a (clone 2.43), or Rat Ig2b Kappa isotype (LTF-2) antibody was administered intraperitoneally (i.p.) 1 day before tumor implantation and every 3–4 days thereafter until time of sacrifice. For PD-1 or PD-L1 blockade experiments, 100μg of rat anti-mouse PD-1 (29F.1A12), 200μg of anti-PD-L1 (10F.9G2) (25 (link)), or isotype antibody (LTF-2) were administered i.p. on days 1, 3, and 5 following BRAFi or control chow initiation. Tumor volume was calculated as L×(W²/2), length (L) being the longer of the two measurements.

Flt3L (30 μg in 100 μl PBS; Celldex) or control PBS was injected intraperitoneally (9 daily injections, unless otherwise noted). High molecular weight poly I:C (50μg in 50μl; Invivogen) was injected intratumorally. For BRAF inhibition, mice were fed by a chow diet containing 417mg/kg PLX4720 (Plexxikon) for 4 days or control diet.

Glioma cell lines (AM-38, DBTRG-05MG, NMC-G1) were obtained from ATCC or the Japanese Brain Tumor Repository; Melanoma cell lines (A375, WM793, WM9) were obtained from Dr. Martin McMahon (UCSF); B9 cSCC cell line were obtained from Dr. Alain Balmain (UCSF); BT40 pilocytic astrocytoma chunks were obtained under MTA from Nationwide Children’s Hospital (Dr. Peter Houghton). PLX4720 was obtained from Plexxikon Inc. (Berkeley, CA, USA). PD0325901 was obtained from Pfizer Inc. (New York City, NY, USA). PLX4032 and GDC0973 were obtained from Genentech Inc. (South San Francisco, CA, USA).

A short-term culture was established from a patient tumor and used to generate a cohort of xenografts. A single cell suspension of ND238 melanoma cells (1×10⁶) was subcutaneously injected into the lower flanks of 6–8 week old, male NSG mice in a suspension of matrigel/complete media at a ratio of 1:1. PLX4720 200ppm chemical additive diet was irradiated and heat-sealed (Research Diets, New Brunswick, NJ) and fed to mice once tumors were established (20 ). PLX4720 was provided by Plexxikon, Berkeley, CA. When tumors reached 200mm³ animals were randomly assigned into 4 different groups: (a) 50μL concentrated control virus; (b) 50μL concentrated shRRM2 virus; (c) 50μL concentrated control virus + PLX4720 (200 mg/kg); and (d) 50 μL concentrated shRRM2 virus + PLX4720 (200 mg/kg). Virus injections were performed at day 0, day 10 and weekly starting at week 6. Tumor size was assessed twice weekly by caliper measurement. Tumor volume was calculated using the formula WxWxL/2. All animal experiments were approved by The Wistar Institute’s IACUC.

PLX4720 200ppm chemical additive diet was irradiated and heat sealed (Research Diets, New Brunswick, NJ) and fed to mice once tumors were established. PLX4720 was provided by Plexxikon, Berkeley, CA. Encorafenib, binimetinib, VX-11e, capmatinib, and BKM120 were provided by Novartis. For in vivo oral gavage, compounds were suspended in 0.5% carboxymethylcellulose sodium (MC), 0.5% Tween 80 (Encorafenib); 1% MC, 0.5% Tween 80 (binimetinib); 5% ethanol, 20% propylene glycol, 7.4% Tween80 (VX-11e); 0.5% MC, 0.1% Tween80 (capmatinib); 1% MC, 1% Tween80 (BKM120) in water and dosed using feeding tubes (Instech Laboratories, Inc. Plymouth Meeting, PA).