Fig. 2

EIF4A inhibition impairs bioenergetic fitness and survival in a ROS-dependent manner. CR-1-31-B halts mRNA translation in MOLM-14 cells: a (left) MOLM-14 cells were treated for 1 h with vehicle or 10 nM CR-1-31-B. Sub-polysome, light-, and heavy-polysome fractions were obtained by ultracentrifugation using 5–50% sucrose gradients. Positions of 40S and 60S ribosomal subunits, monosomes (80S), and polysomes in the absorbance profiles (y-axis, 254 nm) are shown. The ratio between polysomal/monosomal fractions is shown in the right panel. CR-1-31-B is cytotoxic to AML cells: b KG1A, U937 and MOLM-14 cells were treated with CR-1-31-B (0-10 nM) and viability was determined after 48 h using the CellTiter-Glo assay. c The percentage of apoptotic cells (Annexin V⁺) after treatment with vehicle or 2.5 nM CR-1-31-B for 24 and 48 h was determined using flow cytometry. CR-1-31-B reduces bioenergetic capacity: MOLM-14 cells were treated with vehicle or 2.5 nM CR-1-31-B for 24 h. d Oxygen consumption rate (OCR) and e Extracellular acidification rate (ECAR) profiles were obtained using the Seahorse XFe technology. f OCR and ECAR data were used to estimate the rate of ATP synthesis by mitochondrial respiration or glycolysis and the bioenergetic profiles. Data were normalized by cell counting (10,000 cells/condition). CR-1-31-1B-dependent mitochondrial ROS is cytotoxic in MOLM-14 cells: MOLM-14 cells were treated with vehicle or 2.5 nM CR-1-31B for 48 h. g Mitochondrial membrane potential (mitochondrial MP) and h Mitochondrial superoxide content (mtROS) were quantified by flow cytometry using TMRE and mitoSOX respectively in viable cells. a.u. arbitrary units. i MOLM-14 cells were treated for 24 and 48 h with 2.5 nM CR-1-31B alone or after pre-incubation with 10 μM mitoTEMPO. The percentage of apoptotic (Annexin V⁺) was assessed by flow cytometry normalized to vehicle-treated cells. EIF4A inhibition reduces BCL2 and MCL1 expression levels and synergizes with venetoclax: j MOLM-14 cells were treated as indicated for 24 h. BCL2, BCL-XL and MCL1 levels were assessed by western blot analysis. β-actin was used as loading control. k MOLM-14 cells were treated with CR-1-31-B (0-10 nM) in combination with venetoclax (0-6.25 μM) for 24 h. Viability data was obtained using CellTiter-Glo and synergy between the drugs was assessed using the SynergyFinder application. Data related to Fig. 2j are shown in Supp. Fig. 2a. l Members of the BCL2 family of proteins consist of pro-apoptotic activators and sensitizers, and anti-apoptotic inhibitors, which modulate the activity of effectors through BH3-domain interactions. Disruption of the balance of pro-apoptotic and antiapoptotic BCL2 family proteins results in BAX/BAK activation, cytochrome C release and apoptosis [57, 58]. Highlighted in red are apoptotic inhibitors affected by eIF4A in MOLM-14 cells. m The effect of CR-1-31-B on cytochrome C release was measured as described using flow cytometry [58]. The upper heatmap represents the percentage of cell undergoing cytochrome C release after treatment with DMSO or 2.5 nM CR-1-31-B alone or in combination with the apoptotic activators, sensitizers or inhibitors as indicated. The concentrations of the compounds are indicated in μM. Alamethecin (ALA) is a positive control that induces cytochrome C release in all cells independent of BAX or BAK. The lower heatmap represents apoptotic priming by 2.5 nM CR-1-31-B as calculated by the difference in the percentages of cells undergoing cytochrome C release between CR-1-31B and DMSO treated cells (0-50% scale). The asterisks indicate that CR-1-31-B enhances the percentage of cytochrome C releasing cells (p < 0.05) with respect to DMSO control, as assessed by t-test. Footnote. The bars or circles represent the mean values of at least 3 independent experiments plus/minus the standard error of the mean (SEM) for b, c, g-i or standard deviation for d-f. Comparisons between groups were assessed by One-way ANOVA followed by paired t-test with *p < 0.05. All functional analyses were performed by flow cytometry in viable DAPI-negative cells. Representative gating strategies for cytochrome C release and BH3 peptide/inhibitor targets are shown in Supp. Fig. 5