Fig. 3: Blocking ABL1/2 activity reverses resistance to BRAF + MEK inhibitors.

a, b CellTiter Glo viability assays using parental and BRAF/MEK-inhibitor resistant M14 cells treated with nilotinib (nilo; 2.5 μM) in the absence/presence of BRAFi/MEKi (dabrafenib/trametinib; D/T) for 72 h. Results are mean ± SEM for three independent experiments performed in triplicate. Actual p values left→right: a 0.0015, <0.0001 using two-sided, two-sample t-tests; b 50/10: 0.031, 0.008, 0.0018; 100/20: 0.0022, 0.0085, 0.00031; 150/25: 0.00037, 0.00018, <0.0001 using two-sample t-tests (two sided). c–e Colony assays. Cells were treated with vehicle, D/T (100 nM/20 nM) in the absence or presence of nilotinib (2.5 μM), GNF-5 (GNF; 10 μM), or ponatinib (pona; 100 nM) for 7 days, washed, and incubated in the absence of drugs for an additional 6 days (c, e) or treated for 13 days (d). f Cells were treated with nilotinib (M14-BMR-5 μM; Mel1617-BMR-4 μM) in the absence or presence of D/T (150 nM/25 nM) for 96 h, and detached and attached cells lysed and subjected to western blotting. Representative blots from n = 2 independent experiments are shown. V = vehicle. g M14-BMR cells expressing scrambled (shScr) or IPTG-inducible shRNA targeting ABL1 and ABL2 (shABL1/2) were treated with IPTG (1 mM) for 5 days prior to plating, treated with vehicle or D/T (150 nM/25 nM) for 72 h, and cell viability assessed with CellTiter Glo. Results are mean ± SEM for two vector and two shRNA clones and three independent experiments for each, expressed as a percentage of vehicle (DMSO). ***p = 7.5e−5 using a two-sample t-test (two sided). Veh = vehicle. Knockdown efficiency (western blots) is shown on the right. h M14 BRAFi/MEKi in vivo-resistant cells were obtained by treating mice containing M14 xenografts (200 mm³) with vehicle or D/T and establishing a cell line from resistant tumor tissue on day 49. Graph is mean ± SEM, n = 5 mice/group. i, j The established line from h was incubated with nilotinib (2.5 μM if doses are not indicated) in the absence/presence of D/T (100 nM/20 nM, if not indicated), and viability assessed with CellTiter Glo assay (mean ± SEM for n = 3 independent experiments performed in triplicate, i); and colony formation assessed as above (growth/treatment for 17days; j) *p < 0.05, **p ≤ 0.01. ***p < 0.001 using two-sided, one-sample t-tests. Actual p values (left→right): 0.0023, 0.00097, 0.0003, 0.0057, 0.00026, 0.00019, 0.00012, 0.00019, and <0.0001. For all experiments, BRAFi/MEKi-resistant lines were maintained in D/T (100 nM/20 nM) but were cultured for 2 days without D/T prior to plating for experiments.