Activation of the ISR is a vulnerability for multidrug resistant FBXW7-deficient cells (2022)
Figure 5

Fig. 5. FBXW7 deficiency renders cancer cells vulnerable to ISR inducers.

(A) Compounds with a transcriptional signature similar to that triggered by tigecycline identified by CMap using RNAseq data from tigecycline-treated DLD-1 cells. Compound name, compound class and their similarity scores from both genotypes are shown. Mitochondrial poisons (yellow), ISR-activating compounds (green) and additional compounds (blue) are highlighted.

(B) Normalized viability of FBXW7 +/+ and FBXW7 -/- DLD-1 cells upon treatment with increasing doses of tunicamycin for 72h. Cell nuclei were quantified by high-throughput microscopy (HTM) upon staining with DAPI. The experiment was repeated three times, with two biological replicates per experiment, and a representative example is shown. Data from 2 independent FBXW7 -/- clones is shown. Error bars indicate SD.

(C) Nuclear ATF4 levels quantified by HTM in DLD-1 cells upon treatment with the indicated drugs at 10μM (except tunicamycin (1 μM) and paclitaxel (250 nM)) for 3h. This experiment was performed 3 times, and their quantification is shown. Error bars indicate SD (n=3).

(D) WB illustrating the levels of ATF4 in DLD-1 cells treated as in (C). GAPDH levels are shown as a loading control.

(E) Percentage of viable FBXW7+/+ (green) and FBXW7-/- (red) DLD-1 cells treatment with the indicated drugs at 15 μM for 72h. The culture started with a 1:3 ratio of FBXW7+/+ and FBXW7-/- cells. DMSO was used as a control. The experiment was repeated three times, with two biological replicates per experiment, and a representative example is shown. Error bars indicate SD. Cell percentages were quantified by flow cytometry.

(F) Normalized viability of FBXW7+/+ and FBXW7-/- cells upon treatment with 10μM of PLX-4720, vemurafenib, erlotinib and gefitinib, and 5μM of sorafenib and dasatinib for 72h, in the presence or absence of ISRIB (50nM). Cell nuclei were quantified by high-throughput microscopy (HTM) upon staining with DAPI. Error bars indicate SD (n=3, two biological replicates each).

(G) Heatmap representing of the EGFP median fluorescence intensity (MFI) in DLD-1 cells stably transfected with a reporter construct where a destabilized EFGP is under the control of the human CHOP promoter, after treatment with the indicated drugs. Cells were infected with lentiviral vectors expressing mCherry and sgRNAs against the four ISR kinases as well or an empty vector. Cell percentages were quantified by flow cytometry. EGFP MFI ratios between mCherry positive and negative cells were first calculated and then normalized to the levels observed in the controls for each treatment. This experiment was performed 3 times, and a representative example is shown.

(H) Heatmap representing the viability (calculated as the percentage of mCherry positive cells) in DLD-1 cells treated as in (G). Percentages were normalized against DMSO values and then against those found on the empty vector for each treatment. This experiment was performed 3 times, and a representative example is shown.

Data information: n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001; t-test.