Fig. 4: JNK phosphorylates BCL-XL to drive an MCL-1 dependent state.

A Co-IP of BIM bound to MCL-1 in H2030 EV (empty vector) and H2030 LKB1 cells after treatment with vehicle, trametinib (0.1 µM) for 24 h or trametinib for 24 h followed by AMG 176 (1 µM) for 4 h. B Time course of BCL-XL S62 phosphorylation in isogenic H2030 and H358 cells by western blot after treatment with 0.1 µM trametinib + 1 µM AMG 176. C Experimental approach for expressing MCL-1 & BCL-XL phospho-site mutants while suppressing endogenous MCL-1 and BCL-XL. Interrogated phosphorylation sites are designated in yellow, phosphomimetic sites in red. D MCL-1 phospho-site mutants do not reduce sensitivity to MCL-1 inhibition (∆AUC). After induction of mutant MCL-1 (or WT control) and knockdown of endogenous MCL-1, H2030 EV cells were treated with trametinib in the absence or presence of AMG 176 (1 µM) and viability was determined after 3 days. Each dot is an independent biological replicate (N = 3). E BCL-XL S62A mutant decreases MCL-1 dependence. After induction of BCL-XL S62A (or WT control) and knockdown of endogenous BCL-XL, H2030 EV cells were treated with sotorasib or trametinib alone or in the presence of AMG 176 (1 µM) and viability was determined after 3 days. Each dot is an independent biological replicate (N = 6, ****p = 0.000001, unpaired-parametric t test, two-sided). H2030 EV cells expressing inducible WT or S62A mutant BCL-XL S62A (F) or H2030 LKB1 cells expressing inducible WT or BCL-XL S62E phosphomimetic (G) were treated with 0.1 µM trametinib or 0.1 µM trametinib in combination with 1 µM AMG 176 and cell number was quantified by live-cell imaging. Data are mean and S.E.M. of 3 technical replicates. V vehicle, T trametinib, A AMG 176, TA trametinib + AMG 176. Source data are provided as a Source Data file. Western blots and immunoprecipitation images are representative of at least 2 independent biological replicates.