Fig 1: MYC overexpression creates a dependency on eIF4A for the expression of prosurvival proteins.(A) Immunoblots showing MCL1, BCL-xL, and BCL-2 protein levels in response to 24 hours of specified treatments in control and MYC-overexpressing resistant lines. (B and C) Immunoblots showing interactions of MCL1 and BCL-xL with immunoprecipitated proapoptotic BIM and BAX proteins, respectively, in response to specified drug treatments in control and MYC-overexpressing resistant lines. The GAPDH immunoblots denoted by asterisks, which serve as a loading control, have been duplicated from the left side of that panel, because immunoblots were all generated from the same gel and membrane. (Right) Immunoblots confirm ectopic expression of MYC by cDNAs.
Fig 2: Suppression of prosurvival proteins underlies the therapeutic response to combined eIF4A and RAS pathway inhibitors.(A) Bar graphs depicting the effects of either KRASi (left, middle) or MEKi (right) in sensitive cell lines in the presence of the indicated siRNA pools. The fold change in cell number was calculated after 72 hours of treatment (versus day 0) in response to 100 nM MRTX849 (KRASi) or 50 nM trametinib (MEKi). Data are represented as means ± SD. n = 3. The siCDK4 studies were performed separately; however, control values were similar (primary data are shown in Supplemental Figure 4). *Complete genetic ablation of MCL1 alone in H23 cells resulted in cell death, preventing further analysis. (B) Bar graphs depicting fold changes in cell numbers in cells treated for 72 hours (versus day 0) with 100 nM S63845 (MCL1i) or 1 μM navitoclax (BCL-xL/BCL-2i) or 1 μM venetoclax (BCL-2i) combined with either 100 nM MRTX849 (KRASi) or 50 nM trametinib (MEKi). Data are represented as means ± SD. n = 3. ***P < 0.001; ****P < 0.0001, 1-way ANOVA and Tukey’s post hoc test. (C) Bar graphs depicting fold change in cell number in cells treated for 72 hours with 500 nM palbociclib (CDK4/6i) combined with either 100 nM MRTX849 (KRASi) or 50 nM trametinib (MEKi). Data are represented as means ± SD. n = 3. **P < 0.01; ***P < 0.001; ****P < 0.0001, 1-way ANOVA and Tukey’s post hoc test.
Fig 3: eIF4A and RAS pathway inhibitors cooperatively suppress the expression of prosurvival BCL-2 family proteins, cyclin D1, and CDK4.(A) Heatmap depicting protein expression of established eIF4A-regulated targets in H23- and H1944-sensitive lines after 24 hours of treatment with vehicle (DMSO) or eFT226 (eIF4Ai). Protein expression was normalized by GAPDH. Raw data from Western blots are shown in Supplemental Figure 3. (B and C) Immunoblots of MCL1, BCL-xL, BCL-2, CDK4, and cyclin D1 protein levels in sensitive cell lines after 24 hours of specified treatments. *For H1944, the loading control is the same as shown in Figure 2B because protein expression was tested using the same membrane. (D) Immunoblots of the same targets in resistant cell lines after 24 hours of specified treatments.
Fig 4: Reconstitution with survival proteins prevents cell death in response to eIF4A and KRAS G12C inhibitors.(A) (Left) Bar graphs depicting fold changes in cell numbers of H23 and H1792 cells ectopically expressing BCL2L1 (BCL-xL), BCL2, or MCL1 cDNAs treated for 72 hours with combined 25 nM eFT226 (eIF4Ai) and 100 nM MRTX849 (KRASi). Data are represented as means ± SD. n = 3. (Right) Immunoblots confirming overexpression of BCL-xL, BCL-2, and MCL1 by cDNAs. (B) Immunoblots showing interactions of MCL1 and BCL-xL with immunoprecipitated proapoptotic BIM and BAX proteins, respectively, in response to specified drug treatments in sensitive H23 cells.
Supplier Page from DNASU for MCL1 (Homo sapiens) in pDONR221 (Gateway donor/master vector)