Fig. 1

Genetic or pharmacological ISR inhibition sensitizes CML cells to imatinib in vitro and in vivo. A The levels of PERK, eIF2α and phosphorylated eIF2α (S51P) protein estimated by western blot in wild-type (wt), or stably transfected eIF2α S51A and S51A shUTR K562 mutants. Arrows indicate wt (lower) and mutated (40 kDa higher) eIF2α bands. Tubulin was used as a loading control. The full-length membranes were properly cut based on the protein marker size according to target protein sites prior to hybridization with primary antibodies (see Materials and Methods). Cropped blots are presented; original non-cropped membranes are shown in the Supplementary Fig. S9. B Apoptotic (Annexin V-positive) cells detected by flow cytometry in K562 wt, eIF2αS51A and S51A shUTR mutant cells in untreated conditions or after treatment with 0,5 and 1 μM imatinib. Data are shown as a percentage of dead cells. Statistical analysis: Unpaired Student’s t-test with Welch’s correction (*p ≤ 0.05; **p ≤ 0.005; *** p ≤ 0.0005). C Schematic graph of the integrated stress response (ISR) signaling pathway with the site of ISR inhibitors - ISRIB and GSK157 action (PKR – Protein Kinase R, PERK – PKR-like ER kinase, HRI – Heme-regulated eIF2α kinase, GCN2 – General Control Non-depressible protein 2, eIF2α – eukaryotic translation initiation factor-2α, ATF4 – Activating Transcription Factor 4, CHOP – CCAAT/enhancer-binding protein (C/EBP) homologous protein, GADD34 – Growth Arrest and DNA damage-inducible protein 34, PP1 – protein phosphatase 1). D CHOP and GADD34 mRNA expression levels measured by RT-qPCR. Wild-type K562 cells were preconditioned with either ISRIB or GSK157 inhibitors in indicated concentrations for 2 hours, followed by ISR induction with 100 nM thapsigargin for 2 hours to mimic ISR in vitro. For mRNA analysis the level of not treated cells (control) was used as a reference =1. Statistical analysis: unpaired Student’s t-test with Welch’s correction and p ≤ 0,05 was estimated as significant (*p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.001; ****p ≤ 0.0005). E The workflow of the in vivo Xenograft experiment. Mice were: not treated (n = 12); or treated with: imatinib (n = 14); imatinib and GSK157 (n = 13); imatinib and ISRIB (n = 12). F Left panel - the number of mice which developed tumors upon all tested conditions. Right panel – pictures of tumors isolated form representative experiment (n = 5 mice for each condition). G The tumor mass indicating viability and proliferation potential isolated from mice in indicated variants. Tumors grown in mice injected with K562 cells and treated with imatinib were used as a control = 100%. Statistical analysis: Unpaired Student’s t-test, F-test to compare variances (*p ≤ 0.05; **p ≤ 0.005)