Fig. 3: JNK activation in LKB1-deficient cells underlies dependency on MCL-1.

A Phosphoproteomic analysis of isogenic KRAS-mutant NSCLC cell lines treated with 0.1 µM of trametinib for 48 h. B Left: Differential enrichment of phosphopeptide signatures in trametinib-treated isogenic cell line pairs. NES – normalized enrichment scores. Right, individual phospho-sites of JNK1 downstream substrates are annotated. C Change in phospho-JNK in response to MAPK + MCL-1 inhibition in isogenic H2030 and H358 cells (data are mean and S.E.M., each dot represents an independent biological replicate, N = 3-4, H2030: **p = 0.0031, H358: ***p = 0.0008, ***p = 0.0009, paired-parametric t test, 2-sided). D Change in cell number of H2030 EV cells with siJNK1 + 2 or negative control (siNC) after treatment with 0.1 µM trametinib or 1 µM sotorasib in combination with 1 µM AMG 176 quantified by live-cell imaging. Data are mean and S.E.M. of 3 technical replicates. E H2030 EV or LKB1 cells were transfected with siRNAs targeting JNK1 and 2 or siNC and then treated with sotorasib (S) or trametinib (T) ± AMG 176 (A) and viability was determined after 3 days. Each dot represents an independent biological replicate (N = 3, TA: *p = 0.017, SA: *p = 0.028, unpaired-parametric t test, two-sided). F Schematic of siRNA knockdown of LKB1 effectors in H2030 LKB1 cells. G H2030 EV or LKB1 cells transfected with corresponding siRNAs were treated with sotorasib or trametinib in the absence or presence of AMG 176 (1 µM) and viability was determined after 3 days. Each dot represents an independent biological replicate (N = 3, ****p = 0.00001, unpaired-parametric t test, 2-sided). H, I Cells were transfected with the indicated siRNAs and then treated with trametinib (0.1 µM) or sotorasib (1 µM) for 48 h, AMG 176 for 4 h or trametinib (0.1 µM) or sotorasib (1 µM) for 48 h followed by AMG 176 for 4 h. J siPP1B restores sensitivity (∆AUC) to combined sotorasib or trametinib + AMG 176. Each dot represents an independent biological replicate (N = 3, ****p = 0.00001, 2-way ANOVA). K HA-tagged WT NUAK1 (WT) or GKKK NUAK were over-expressed in H2030 isogenic cells and the interaction of NUAK1 and PP1B was assessed by Co-IP. Source data are provided as a Source Data file.