Figure 1.

Type II RAFi+MEKi forestalls and overcomes acquired resistance in BRAF^MUT, NF1^MUT, KRAS ^MUT and NRAS ^MUT cancers.

A, Clonogenic growth (14 days) of indicated inhibitor-naïve BRAF^V600 or NF1 mutant melanoma, BRAF^V600MUT or KRAS^MUT colorectal carcinoma (CRC), KRAS^MUT non-small cell lung carcinoma (NSCLC), and KRAS^MUT (vs. KRAS^WT) pancreatic ductal adenocarcinoma (PDAC) cell lines after treatment with vehicle (DMSO), BGB-283 (0.5 μM), trametinib (0.02 μM), or trametinib (0.02 μM) plus BGB-283 (0.5 μM) or alternatively with RAF709 (0.5 μM), binimetinib (0.02 μM), or binimetinib (0.02 μM) plus RAF709 (0.5 μM). Data representative of two replicates.

B, Clonogenic growth (15 days, left two sub-lines; 10 days, right two sub-lines) of indicated type I RAFi+MEKi double-drug resistant (DDR; hereafter all names of sub-lines with acquired drug resistance shown in red text) BRAF^V600E melanoma sub-lines. DDR sub-lines were maintained on both PLX4032 (1 μM) plus AZD6244 (1 μM), either PLX4032 or AZD6244, or withdrawn from both inhibitors. On top of these four conditions, a type II RAFi (BGB-283 at 1 μM or RAF-709 at 1 μM) was added. Data representative of two replicates.

C, D, Tumor volumes of PDAC (n=2; XWR6, left; XWR7, right, c) and NSCLC (n=2; LBM013-P, left; TM00302, right, d) PDXs in mice that were treated with vehicle, trametinib (3 mg/kg/day PO), BGB-283 (20 mg/kg/day PO), or the combination of trametinib and BGB-283. Vehicle or inhibitor treatments were started at tumor volumes of ~500 mm³. N=5 tumors per group; means ± SEMs.

E, Clonogenic growth (5 or 14 days, upper left panel) of indicated inhibitor-naïve NRAS^MUT melanoma cell lines after treatment with vehicle (DMSO), BGB-283 (0.5 μM), trametinib (0.02 μM), or trametinib (0.02 μM) plus BGB-283 (0.5 μM). Data representative of two replicates. Tumor volumes of NRAS^MUT (n=3; NRAS_PDX3, top; NRAS_PDX4, middle; NRAS_PDX1, bottom) PDXs in mice that were treated with vehicle, trametinib (3 mg/kg/day PO), BGB-283 (20 mg/kg/day PO), or the combination of trametinib and BGB-283. Vehicle or inhibitor treatments were started at tumor volumes of ~500 mm³. N=5 tumors per group; means ± SEMs.

F, (Top) Clonogenic growth (10 days) of indicated inhibitor-naïve NRAS^MUT melanoma cell lines after treatment with vehicle (DMSO) or indicated concentration of MEKi (trametinib) or ERKi (SCH772984). Data representative of two replicates. (Bottom) Western blots of lysates from NRAS^MUT melanoma cell lines treated (2 hours) with vehicle (DMSO) or the same concentrations of MEKi or ERKi (Top) with indicated antibodies.

G, Clonogenic growth (15 or 30 days) of indicated inhibitor-naïve NRAS^MUT melanoma cell lines after treatment with vehicle (DMSO) or trametinib, SCH772984, or the type I RAFi PLX-4032 (15 day only) at the indicated concentrations, ± BGB-283. Over-confluent cultures without BGB-283 co-treatment were terminated early and not shown for day 30. Data representative of two replicates.

H, As in G, except NRAS^MUT melanoma sub-lines (single-drug resistant or SDR, indicated in red) with acquired MEKi (trametinib) resistance were used. Day 15 cultures are shown except those marked with * (which indicates day 30 cultures). Cultures marked with # display the drug addiction phenotype.