Fig. 3

Synergistic effect of eFT-508 and adriamycin on the in vitro chemosensitivity of BC cell lines. A BC cell lines are chemoresistant to adriamycin with high values of IC₅₀. Exponentially growing cultures of BC cell lines, MDA-MB-231, MDA-MB-468, MCF7, T47D, and SUM149, were treated with increasing concentrations of adriamycin for 24 h. Cell viability was evaluated using MTT assay, and percent viability after 24 h were plotted. No significant difference was observed between the cell lines for any of the concentrations of adriamycin tested. The selection criteria of drug dilution were scalar dilution factor 3 and then factor 2. B Differential sensitivity of BC cell lines to Tomivosertib (eFT-508). Same as A but was treated with increasing concentrations of eFT-508. All cell lines tested had more chemosensitivity to eFT-508 compared to the same concentration of adriamycin. MDA-MB-468 and MDA-MB-231 were more chemosensitive compared to the other three cell lines. The selection criteria of drug dilution involved scalar dilution factor 3 and then factor 2. ^#P < 0.05; ^##P < 0.01; **P = 0.001; ***P < 0.001. C Adriamycin, in combination with eFT-508, showed a higher in vitro therapeutic index. Same as A but instead treated with both adriamycin and eFT-508. Combination therapy resulted in significantly less IC₅₀ in all cell lines compared to monotherapy. The IC₅₀ in the MDA-MB-468 cells was significantly lower compared to the other four cell lines. The selection criteria of drug dilution included scalar dilution factor 3 and then factor 2. Data in A–C are represented as staggered plots, with each point representing an experimental replicate (n = 3). D Western blot was used to detect the expression of PD-L1 in BC cell lines. E Western blot was used to detect the expression of PD-L2 in BC cell lines. F Treatment with eFT-508 resulted in the inhibition of all five cell lines, and Western blot was used to detect the expression of PD-L1 and PD-L2 in cell lines. G Treatment with eFT-508 resulted in the suppression of MNK1/2-mediated phosphorylation of eIF4E in all five cell lines. Immunoblot analysis of phosphorylated eIF4E (S209) and total eIF4E in the cell lines treated with eFT-508. Shown are representative blots from three biological replicates. GAPDH was used as a loading control. H Treatment with eFT-508 resulted in the inhibition of global translation. Representative polysome profile obtained from the post-nuclear extract of vehicle or eFT-508 treated MDA-MB-231 cells. Treatment with eFT-508 resulted in the lower 80S and polysome peaks. Similar results were obtained in the other four cell lines as well (data not shown). I Results in E were additionally verified using the SUrface SEnsing of Translation (SUnSET) technique to assess changes in global translation. Untreated or eFT-508-treated MCF7 and MDA-MB-231 cells were labeled with puromycin for 15 min. Shown are immunoblot analyses of lysates made from these cells using an anti-puromycin antibody (top). The membrane was subsequently stained with Coomassie Blue to ensure equal protein loading across lanes. Shown are representative blots from three experiments. J Treatment with adriamycin had no noticeable effect on global translation. Untreated or adriamycin-treated MCF7 and MDA-MB-231 cells were labeled with puromycin for 15 min. Shown are immunoblot analyses of lysates made from these cells using an anti-puromycin antibody (top). The membrane was subsequently stained with Coomassie Blue to ensure equal protein loading across lanes. Shown are representative blots from three experiments