Fig. 6: ABL1/2 drive ERK reactivation during resistance by promoting MAP3K activation.

a M14-BMR cells were engineered to express vector or HA-tagged constitutively active ERK2 (GOF)³⁷. ERK2-GOF expression is shown on the upper right. Vector or ERK2-GOF-expressing cells were plated, treated with D/T (50 nM/10 nM) in the absence or presence of nilotinib (nilo; 2.5 μM) for 7 days, wells washed, cells incubated in media lacking drugs for an additional 9 days, and stained with crystal violet. Quantitation of mean ± SEM for n = 4 independent experiments is shown on the right. ***p = 0.00084, two-sample t-test (two-sided). b Primary human paired melanoma datasets (RNAseq-77940, 50535, 65185, EGAS00001000992; microarray-50509) were analyzed for ABL1 and MYC mRNA levels pre-/post-BRAFi, MEKi or BRAFi/MEKi treatment. Spearman’s correlation coefficients were used to quantify correlations. Correlation (r), 95% confidence limits (in parentheses), and p values are shown. c M14-BMR or parental M14 cells were treated with the indicated drugs as in Fig. 5 for 24 h, and lysates blotted with antibodies. A representative experiment from n = 3 independent experiments is shown. Veh = vehicle. d M14-BMR cells expressing either vector (vec) or IPTG-inducible shRNA targeting ABL1/2 were treated with IPTG (1 mM) for 5 days prior to plating, treated with DMSO (vehicle) or D/T (100 nM/20 nM) for 24 h, and lysates blotted with the indicated antibodies. A representative experiment from n = 4 independent experiments is shown. e M14-BMR cells were transfected with MAP3K1, MAP4K1, or scrambled siRNA, cells replated and treated with D/T (50/10 nM) −/+ nilotinib (2.5 μM) for 24 h, and lysates blotted with antibody (left). A representative experiment from n = 3 independent experiments is shown. (right) 293T cells were transfected with vector or MAP3K1 (exogenous form runs at 200 kDa), and lysates blotted after 48 h. A representative experiment from n = 2 independent experiments is shown. Vec = empty vector. f, g ABL1 (f) or MAP3K1 (g) immunoprecipitates (IP) from M14-BMR-treated cells, were blotted with the indicated antibodies. Representative experiments from n = 4 (f) and n = 3 (g) independent experiments are shown. Input is whole-cell lysate. Rabbit (f) or mouse (g) IgG served as a negative isotype controls. D/T = 100/20 nM; nilotinib = 2.5 μM; BV02 = 5 μM.