Fig. 3: AXL/eIF4E-regulated ER stress-relief UPR underpins drug resistance and intratumor heterogeneity in KRAS-mutant lung cancer.

a A549 and A549R cells without or with brefeldin A (5 µg/ml) treatment (6 h) are profiled for secreted proteins. Shown are representative data of three independent experiments (n = 3). b Transcriptional quantification of UPR genes in A549R and A549 cells by qRT-PCR. c Immunoblots of A549 and A549R cells treated onalespib (0.5 µM) for the indicated time. d–g Dose–response curves to tunicamycin (d), MG-132 (d), and inhibitors to PERK and JNK (e–g) assayed on resistant (A549R, H358R) versus parental (A549, H358) cells. Representative results of clonogenic assays are shown to the right. h, i A549R cells transfected with PERK-specific and control siRNAs were subjected to immunoblotting (h) and dose–responsive curves to pemetrexed and trametinib (i). Growth curves of the transfected cells are also shown (h). j Protein lysates (200 μg) prepared from A549R cells treated with onalespib or vehicle were subjected to coimmunoprecipitation with HSP90 and PERK antibodies. Proteins in the precipitates (IP) and the starting material (input) were visualized by western blotting. k Volcano plots of transcriptomic profiling (RNAseq) of the A549_holo and A549_para clones. The X-axis is the fold-change (log2) of individual genes in the holoclone versus paraclone. Genes significantly downregulated (adjusted p value <0.05) in the holo compared to paraclones are shown in green and genes significantly upregulated in the A549_holo versus A549_para are marked in red. Note that HSP90AA1 and HSP90AB1 (encoding HSP90) and the genes involved in EMT, AXL signaling, and the UPR are enriched in the A549_para clone. l–n Dose–response curves to MTA and the MEK inhibitors PD0325901 and trametinib (l), inhibitors of AXL, eIF4E, and JNK (m) and HSP90 (n) as determined on A549_holo_clone (black) and A549_para_clone (red) subpopulations. Data are mean ± s.d. of three biological replicates (n = 3)