Fig. 1: SBI-756 prevents eIF4E-eIF4G interaction and cap-dependent translation.

a Cells were tested for eIF4E:eIF4G1 association via Proximity Ligation Assay (PLA). OCI-LY1 DLBCL cells were treated for 4 h with either vehicle (DMSO), MLN0128 100 nM, Rapamycin 10 nM or increasing concentrations of SBI-756 (250–750 nM). Scale bar = 33 µm. Representative images of at least three fields are shown. b Quantification of eIF4E:eIF4G1 interaction for each treatment. The signal indicating eIF4E:eIF4G interaction was measured from the entire field for each treatment (single channels acquired) and was normalised to the number of cells imaged (DAPI staining indicating cells/image). Relative ratios are graphed. *p < 0.05; **p < 0.01. n = 3 or 4, as indicated. Paired one-sample t-test. c OCI-LY1 or Mino1 cells were electroporated to introduce the dual-luciferase reporter in which 5’ cap-dependent untranslated region (5’-UTR) was conjoined to a Renilla (Renilla reniformis) luciferase reporter, while the Internal Ribosomal Entry Site (IRES) was conjoined to a firefly (Photinus pyralis) luciferase reporter. Cells were treated for 16 h with vehicle (DMSO), MLN0128 100 nM, or increasing concentrations of SBI-756 (250–750 nM). Both Renilla luciferase (cap-dependent translation) and firefly luciferase (IRES dependent translation) signals were measured and each was normalized to vehicle contol. *p < 0.05, **p < 0.01, ***p < 0.005. One-sample t-test vs. DMSO control. n = 3. Viability of d OCI-LY1 and e Mino1 cells treated for 48 h with increasing venetoclax concentrations in combination with vehicle (DMSO) control or various inhibitors as indicated. Viability was assessed using Annexin V and PI staining. *p < 0.05; **p < 0.01; ****p < 0.001. We performed independent t-tests (unpaired t-tests) and compared each treatment group to vehicle treated group. We also performed an adjustment for multiple comparisons for each t-test performed. We calculated IC50 values for each cell line tested based of the viability assays performed: f OCI-LY1 IC50s–venetoclax 23.6 nM, SBI-756 209.7 nM; g Mino1 IC50s–venetoclax 749.9 nM, SBI-756 340.3 nM. Isobologram plots were graphed based on Chou-Talalay method for synergy calculation (combination index)¹¹ using median effect method for cell lines treated for 48 h with combinations of SBI-756 and venetoclax at fixed ratios.