Fig. 2: ABL1/2 inhibition reverses resistance to BRAF inhibitors.

a, c, e Viability (CellTiter Glo) assays for cells treated with vehicle (veh) or nilotinib (nilo) alone (e (right)) in the absence/presence of BRAFi, PLX4720 (doses shown) for 72 h (a, c, e (left)). Nilotinib doses: a 5 or 6 μM; c, e 5 μM. Results are mean ± SEM for three independent experiments performed in triplicate. Additional drug doses are shown in Supplementary Fig. 2a, b. *p < 0.05, **p ≤ 0.01, ***p < 0.001 using two-sample t-tests (two sided). Actual p values (left→right): a 0.0029, 0.00016; c 0.039, 0.0068; e (left) 0.016, 0.043, 0.015; e (right) 0.019, 0.035, 0.035. b, d, f Colony assays. Cells were treated with nilotinib (b 4 μM, d 3 μM; f doses shown) and/or PLX4720 (2.5 μM) for 7 days, wells washed, plates incubated additional days without drugs (451-Lu/M14-5d; Mel1617-6d) until colonies were well visualized, at which time they were stained with crystal violet. g Cells were treated with the indicated drugs for 96 h and detached and attached cells lysed and subjected to western blotting. Nilotinib: Mel1617-BR, 4 μM; M14-BR, 451-Lu-BR, 5 μM. PLX: Mel1617-BR, 4 μM, M14-BR, 3 μM. Representative blots from n = 2 independent experiments are shown. V = vehicle. h Low-invasive WM164 cells, which lack endogenous activated ABL1/2 and harbor BRAF-V600E, were engineered to stably express constitutively active forms of ABL1 and ABL2 (PP) or vectors. Cells were plated, treated with vehicle or the BRAFi, PLX4720, for 7 days, wells washed, incubated with media without drug for 9 days, stained with crystal violet, and colonies manually counted. Levels of ABL1/2 proteins are shown on the right. Cells were maintained in the presence of PLX but removed from drug two days prior to plating for experiments. Colony assay shown is representative of n = 4 (0.5 μM) or n = 2 (1 μM) independent experiments (see Supplementary Fig. 2h). V = vehicle = DMSO.