Additional file 1 of EIF4A inhibition targets bioenergetic homeostasis in AML MOLM-14 cells in vitro and in vivo and synergizes with cytarabine and venetoclax

Additional file 1: Supplementary Fig. 1. a. Gating strategy to assess the effect of chemotherapy on live AML cells defined using viability 700 stain/FSC-A and CD45.1/FSC-A in mononuclear cells isolated from the bone marrow of mice treated with vehicle (Control, upper row) or chemotherapy (Treated, middle row). The lower row shows unstained controls b. The effect of chemotherapy on p4E-BP, pS6, TMRE or Mitosox staining was assessed in live AML cells. Supplementary Fig. 2. a. Quantification of band intensities for western blots in Fig. 2j was done using ImageJ software ( https://imagej.nih.gov/ij/index.html ). Quantification was performed from 3 independent experiments and data for BCL2, BCL-XL and MCL1 was normalized by actin levels for each experiment. Data is expressed as average +/− SD, relative to DMSO control. * p < 0.05. b-f. MOLM-14 cells were treated with DMSO, 250 nM araC for 48 h, and/or 2.5 nM CR-1-31-B for 24 h. Oxygen consumption (OCR) and extracellular acidification rate (ECAR) measurements are shown in b and d, respectively, and were conducted in an XFe96 Seahorse extracellular flux analyzer and normalized to cell counts in the presence of different stressors. Oligomycin is an inhibitor of mitochondrial complex V (ATP synthase), FCCP is an uncoupler that dissipates mitochondrial proton gradient, Rotenone and Antimycin a (Aa) are inhibitors of Complex I and II, respectively; 2DG is a glucose analogue that inhibits the first step of glycolysis. OCR and ECAR rates were utilized to calculate c oxidative phosphorylation, e glycolytic parameters, and f to estimate the contribution of OxPhos and glycolysis to ATP production. The results represent 3 independent experiments and SEM; ns indicates p > 0.05 and * p < 0.05. Supplementary Fig. 3. a. BM cells from vehicle (control, black bars) or CR-1-31-B treated mice (0.20 mg/kg i.p. daily for 7 days, red bars) were stained with acridine orange/propidium iodide. The bars represent mean absolute cell numbers and SEM, ns indicates p > 0.05. b. Gating strategy for the evaluation of hematopoietic lineages for B cells (B220+), helper T cells (CD4+), cytotoxic T cells (CD8+) and myeloid cells (CD11b + GR-1+). c. Gating strategy for hematopoietic stem and progenitor cells (HSPC) in viable, lineage-negative (Lin−) cells: Sca-1+ and c-kit+ cells (LSK cells), granulocyte-macrophage progenitors (GMPs, CD16/32+CD34+Lin−Sca-1−c-kit+), common lymphoid progenitor (CLPs, CD127+/LSK cells). Lymphoid myeloid progenitors (LMPPs) and HSPC were discriminated by differential expression of CD135 in LSK cells, HSCs enriched within the HSPC gate as CD48−CD150+ (LSK SLAM). d Effect of vehicle (control, black bars) or CR-1-31-B 0.20 mg/kg i.p. daily for 7 days (red bars) in defined HSPC populations. The results are expressed as absolute cell number means and SEM. Differences were assessed by t-test, *p < 0.05. Supplementary Fig. 4. Lymphocyte suspensions from bone marrow were stained with the following mouse specific antibodies: PerCP-Cy5.5-anti-B220 (RA3-6B2; BD), PE-Cy7-anti-CD43 (S7; BD), PE-anti-Ly-51(BP-1; BD), FITC-anti-CD24(M1/69; BD), BV421-anti-IgM (R6-60.2; BD) and BV605-anti-IgD (11-26c.2a; BD). Samples were acquired on LSRFortessa (BD) and data were analyzed with FlowJo software (Treestar). Supplementary Fig. 5. a. Representative gating strategy for BH3 profiling analysis. Cells were gated first on size, followed by elimination of cell doublets (FSC-H vs FSC-A). Dead cells were removed via staining with Aqua Live/Dead (Invitrogen, #L34966). Lastly, cytochrome C retention was measured using AF647 (BD Biosciences, #558709). Alamethicin acts as the negative staining control, as no cells retain cytochrome C, while DMSO acts as the positive staining control. Gates for cytochrome C were set to the negative control (Ala) for each experiment. b. Interaction partners of BH3 peptides and pharmacological inhibitors. The table shows the binding schematic between peptides/inhibitors used in the dynamic BH3 profiling experiments and the BCL2 family of proteins (BCL2, MCL1, BCL-XL). BIM/BID are targeted by all BCL2 family proteins, while PUMA binds them in order to free BIM/BID for apoptotic activation. BAD, ABT-737, and venetoclax primarily target BCL2. Peptides MS1 and NOXA, as well as inhibitor S63845, target MCL1. HRK and inhibitor A-1331852 selectively target BCL-XL.

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