Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EIF4A inhibition targets bioenergetic homeostasis in AML MOLM-14 cells in vitro and in vivo and synergizes with cytarabine and venetoclax

doi: 10.1186/s13046-022-02542-8

Figure Lengend Snippet: Chemoresistant AML cells show increased mTORC1 activity and mitochondrial function. a In AML, hyperactive mTORC1 signaling is driven by oncogenic signaling (i.e. mutated RAS, FLT3), chemotherapy and microenviromental cues such as cytokines [ – ]. mTORC1 is a multimeric complex containing the catalytic subunit mTOR serine/threonine kinase, and scaffolding unit Raptor. Two key mTORC1 substrates that regulate cap-dependent translation initiation are the eukaryotic initiation factor (eIF) 4E-binding proteins (4E-BPs) and the ribosomal protein S6 kinase 1 (S6K1). mTORC1 phosphorylation inhibits 4E-BPs, which are negative regulators of eIF4E. Active S6K1 phosphorylates rpS6 and the programmed cell death protein 4 (PCD4), a negative regulator of eIF4A. eIF4E cap-binding protein, eIF4G scaffolding protein and eIF4A RNA helicase are subunits of eIF4F, the rate-limiting complex in cap-dependent translation initiation. Activation of mTORC1 releases eIF4E and eIF4A from their negative regulators, 4EBPs and PDCD4, respectively, allowing the assembly of eIF4F. Proposed mechanisms for CR-1-31-B action include (i) direct inhibition of translation of the target mRNA by increasing eIF4A1:RNA binding (clamping) and (ii) depletion of the limited eIF4F pool available for ribosome recruitment leading to a trans-inhibitory effect toward mRNAs that are not directly affected by clamping [ , ]. Among eIF4A targets are the BCL2 family members BCL-2, MCL-1 and BCL-XL . b NSG or C57BL/6 J mice were transplanted with MOLM-14-Luc cells or FM4 cells, respectively. NSG mice were treated with 50 mg/kg araC injected intraperitoneally (i.p.) daily for 5 days. C57BL/6 J mice were treated with 100 mg/kg araC i.p. daily for 5 days with 3 mg/kg doxorubicin i.p. daily concomitantly from days 1-3. Analyses shown in c - k were performed at day 8 post treatment initiation. c Mean and SEM of total FM4 cells (y-axis) in bone marrow of C57BL/6 J mice treated with vehicle (Control, n = 3) or chemotherapy ( n = 4). Significant differences were assessed by two-tailed unpaired t-test, * p < 0.05. d - g Chemoresistant FM4 AML cells were analyzed by flow cytometry to assess for differences on d p4E-BP, e pS6, f mitochondrial membrane potential (Mitochondrial MP) using TMRE and g OP-Puro incorporation. The y-axis represents the median fluorescence intensity and the bars, the mean values and SEM. Significant differences were assessed by two tailed unpaired t-test, * p < 0.05. h AML growth in NSG mice was assessed by in vivo bioluminescence imaging at days 1 and 8. Tumor burden on day 8 was normalized to tumor burden on day 1 (y-axis). The bar represents the mean value, ns indicates p > 0.05. i - k Chemoresistant MOLM-14 were analyzed by flow cytometry to assess for differences on i p4E-BP, j pS6, k Mitochondrial MP using TMRE. The y-axis represents the median fluorescence intensity and the bars, the mean values and SEM. Significant differences were assessed by two-tailed unpaired t-test; ns p > 0.05; MFI: mean fluorescence intensity. Data related to Fig. 1 are shown in Supp. Fig.

Article Snippet: Moreover, cells were incubated with inhibitors targeting BCL2 (Venetoclax, AbbVie), MCL1 (S63845, SelleckChem) and BCL-XL (A-1331852, SelleckChem) or multiple BCL2 proteins (ABT-737, SelleckChem).

Techniques: Activity Assay, Scaffolding, Binding Assay, Phospho-proteomics, Activation Assay, Inhibition, RNA Binding Assay, Injection, Control, Two Tailed Test, Flow Cytometry, Membrane, Fluorescence, In Vivo, Imaging

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EIF4A inhibition targets bioenergetic homeostasis in AML MOLM-14 cells in vitro and in vivo and synergizes with cytarabine and venetoclax

doi: 10.1186/s13046-022-02542-8

Figure Lengend Snippet: 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. a. 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 [ , ]. 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 . 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.

Article Snippet: Moreover, cells were incubated with inhibitors targeting BCL2 (Venetoclax, AbbVie), MCL1 (S63845, SelleckChem) and BCL-XL (A-1331852, SelleckChem) or multiple BCL2 proteins (ABT-737, SelleckChem).

Techniques: Inhibition, Glo Assay, Flow Cytometry, Cell Counting, Membrane, Incubation, Expressing, Western Blot, Control, Activity Assay, Disruption, Activation Assay, Positive Control, Standard Deviation, Functional Assay