Fig. 5: Nilotinib prevents reactivation of ERK-dependent (or ERK-independent) signaling during BRAFi/MEKi resistance.

a, c Parental or BRAFi/MEKi-resistant cells (-BMR) were treated with vehicle, nilotinib (nilo; 2.5 μM), dabrafenib/trametinib (D/T; BRAFi/MEKi; 50/10 nM) or the combination for 24 h, and the resulting lysates probed with the indicated antibodies. Quantitation of key signaling molecules for n = 3 independent experiments (except pERK, n = 4) is shown below; green arrows indicate lanes quantitated. Mean ± SEM *p < 0.05, **p ≤ 0.01, ***p < 0.001 using two-sided, one-sample t-tests. Actual p values (left→right): a 0.045, 0.0099, 0.0033, and 0.004. c **p = 0.0045. b M14-BMR cells expressing either vector (shScr) or IPTG-inducible shRNA targeting ABL1 and ABL2 (shABL1/2; two independent clones are shown) were treated with IPTG (1 mM) for 10 days prior to plating to induce expression, treated with DMSO (vehicle) or D/T (100 nM/20 nM) for 24 h, and lysates blotted with the indicated antibodies. n = 4 biological replicates (using two vector and two shRNA clones). **p ≤ 0.01, ***p < 0.001 using two-sided, one-sample t-tests. Actual p values (left→right): <0.0001, 0.011, 0.0056, and 0.000191. pCRKL (substrate of ABL1/2) is an indirect read-out of ABL1/2 activity and indicates the efficiency of nilotinib-mediated inhibition. For all experiments, BRAFi/MEKi-resistant lines were maintained in dabrafenib (100 nM) and trametinib (20 nM), but were cultured for 2 days without BRAFi/MEKi prior to plating for experiments.