Additional file 1 of Novel eIF4A1 inhibitors with anti‐tumor activity in lymphoma
Creators: name: Kayastha, Forum
name: Herrington, Noah B.
name: Kapadia, Bandish
name: Roychowdhury, Anirban
name: Nanaji, Nahid
name: Kellogg, Glen E.
name: Gartenhaus, Ronald B.
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. eIF4A1 expression in molecular subtypes of DLBCL in microarray datasets. A) eIF4A1 expression was found to be significantly (***p &lt; 0.001) higher in ABC-DLBCL subtypes compared to GCB and UN-DLBCL in the microarray dataset GSE10846. B) A similar trend was observed in the GSE87371 dataset p = 0.203. Figure S2. Assessment of eIF4A1 specific luciferase assay in Hek293T/17 stables. A) Design of luciferase construct with 5’UTR of eIF4A1 G-quadruplex sequence with the β-actin promoter, negative controls, blank with scrambled sequence and empty test construct. B) eIF4A-3X-Luciferase expressing 293 T cells were transfected with shRNA against eIF4A1 and eIF4B followed by luciferase readout. Statistics were performed using Dunnett's Test. A significant decrease was observed in relative luciferase units in eIF4A1 shRNA groups. ****p &lt; 0.0001 vs non-transfected cells C) Various dose treatments of silvestrol in eIF4A1-3X, blank and empty luciferase Hek293T/17 stables. Percentage inhibition in the reduction of luciferase was calculated relative to DMSO control. The IC50 value was observed to be 10 nM. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s correction analysis. ap &lt; 0.05; cp &lt; 0.001, dp &lt; 0.0001 vs DMSO control groups, αp &lt; 0.05, βp &lt; 0.001, ¥p &lt; 0.0001 vs 1 μM or 10 μM treatment groups. Figure S3. RBF98 activity in luciferase and biochemical assays A) Percentage inhibition of luciferase activity on treatment with RBF98 at 0.1, 1, and 10 µM concentrations in empty/blank luciferase HEK293T/17 stable cell lines. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s correction analysis. ap &lt; 0.05; cp &lt; 0.001, dp &lt; 0.0001 vs DMSO control groups, αp &lt; 0.05, βp &lt; 0.001, ¥p &lt; 0.0001 vs 10 μM treatment groups. B) eIF4A1 titration and measurement of phosphate release with 50 μM ATP and 100 ng/ml of yeast RNA. C) ATP titration to select linear range concentration in presence of 20 ng of eIF4A1 and 100 ng/ml yeast RNA. D) Dose-dependent percentage inhibition of human eIF4A1 in-vitro activity on the treatment of RBF98, compared to DMSO control using inorganic phosphate release assay (SensoLyte kit). IC50 values observed were observed to be 3 µM. Figure S4. RBF98 reduces proliferation and overall translation in DLBCL A) For proliferation assay, Farage (GCB) origin was seeded at a density of 10,000 and treated with 0.5 and 1 µM of RBF98 for up to 72 h. The cell viability was measured at different time points using the trypan blue method. Silvestrol treatment was done at 50 nM as a positive control group. Viability was observed to be decreasing with increasing time in comparison to DMSO control (ap &lt; 0.05; cp &lt; 0.001, dp &lt; 0.0001). B) Effect of RBF98 on DLBCL colony formation. The total number of colonies grown in Toledo (malignant), GMO1528, and GMO13604 (non-malignant) cells on treatment with 0.5 and 1 mM of RBF98. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s correction analysis. ap &lt; 0.05; cp &lt; 0.001, dp &lt; 0.0001 vs DMSO control groups, αp &lt; 0.05, βp &lt; 0.001, ¥p &lt; 0.0001 vs 1 mM treatment groups. C) Puromycin (1 μg/mL) was exposed, post compound treatments for 30 min (SUnSET assay), and cells (Toledo and OCI-LY3) were lysed. Representative immunoblots probed with anti-puromycin, anti-eIF4A1, anti-eIF4E, anti-cMYC, anti-CyclinD1. GAPDH was used as the loading control. Figure S5. Percentage inhibition of luciferase activity. On treatment of RBF197, 203, and 208 respectively at 0.1, 1, and 10 µM concentrations A) empty and B) blank luciferase HEK293T/17 stable cell lines. Average inhibition in relative luciferase units was not found to be more than 20% at the given concentrations. Statistical analysis was performed using one-way ANOVA followed by Bonferroni correction analysis. ap &lt; 0.05; cp &lt; 0.001, dp &lt; 0.0001 vs DMSO control groups, αp &lt; 0.05, βp &lt; 0.001, ¥p &lt; 0.0001 vs 10 μM treatment groups. Figure S6. RBF197 attenuates DLBCL growth. A) Concentration–response curves of RBF197 in SUDHL4, OCI-Ly3, DS, and RC cell lines. Linear regression fit of the inhibition values was plotted using Graph pad prism and EC50 values were calculated. The EC50 values were observed to be less than 10 µM. Figure S7. RBF197 and 208 decrease overall translation and eIF4A1 dependent pathway proteins in ABC-DLBCL OCI-Ly3 cell line. A) Representative immunoblots probed with anti-puromycin, anti-eIF4A1, and anti-eIF4E. B, D) Relative fold change in protein levels with on treatment of RBF 197 (B) or RBF208 (D) at 1 and 3 µM concentrations with respect to the DMSO control. GAPDH was used as a loading control C, E) qRT-PCR analysis for the expression of defined genes in RC with the treatment of RBF 197 (C) or RBF208 (E) at 1 and 3 µM. 36B4 was used as a housekeeping gene. Results were normalized with DMSO controls and expressed as mean ± SD (n = 3). F) Representative immunoblots of indicated antibodies on treatment with RBF197 and 208 at 1 and 3 µM concentrations respectively. Vinculin was used as a loading control. G,I) Relative fold change in protein levels of the above-mentioned proteins with on treatment of RBF197 (G) and RBF208 (I) respectively. H, J) Relative mRNA expression data for defined genes with the treatment of RBF197 (H) and RBF208 (J) respectively. 36B4 was used as a housekeeping gene. Results were normalized with DMSO controls and expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s correction analysis. ap &lt; 0.05; cp &lt; 0.001, dp &lt; 0.0001 vs DMSO control groups, αp &lt; 0.05, βp &lt; 0.001, ¥p &lt; 0.0001 vs 3 μM treatment groups. K) Heat map of translation efficiency values for RBF197 and RBF208 in OCI-Ly3 cell line. Figure S8. RBF197 and RBF208 decreases overall translation and eIF4A1 dependent pathway proteins in double-hit lymphoma. A) qRT-PCR analysis for expression of defined genes in RC following treatment of RBF197 or RBF208 at 1 and 3 µM. B) Relative mRNA expression data for defined genes with the treatment of RBF197 and RBF208 respectively. Figure S9. Effect of RBF197 and RBF208 on DLBCL colony formation. A, B) The total number of colonies grown in Farage, SUDHL4, DS, OCI-Ly3 (malignant), GMO17220B (non-malignant) cell lines upon treatment with 1 and 3 µM of RBF197 (A) and RBF208 (B) respectively. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s correction analysis. ap &lt; 0.05; cp &lt; 0.001, dp &lt; 0.0001 vs DMSO control groups, αp &lt; 0.05, βp &lt; 0.001, ¥p &lt; 0.0001 vs 3 μM treatment groups. Figure S10. RBF 197 and RBF208 inhibit human eIF4A1 by RNA clamp mechanism. A) Schematic depiction of the fluorescent duplex unwinding assay. A 32-mer RNA strand is modified on its 5′ end with cyanine 5 (Cy5) and annealed to a complimentary loading strand that is modified on its 3′ end 9-mer with cyanine 3 (Cy3). The 9-mer RNA strand released upon unwinding was trapped by an unlabeled 9-mer DNA oligonucleotide to give single turnover reactions. B) Four-step protocol, 1. Duplex formation (5 µM) by heating at 960C for 2 min, cooling on ice for 5 min. 2. Read stable fluorescence at 0 min with or without compound in presence of human eIF4A1, unlabeled 9mer, and additional duplex. 3. Addition of ATP and record kinetic reading for 30 min. 4. Post 30 min, again add duplex and read the fluorescence intensity after 3 min C) The difference in the increase in the fluorescence was calculated in the presence and absence of RBF197 and RBF208. Concentration–response curves were plotted using a graph pad prism and IC50 was observed at 0.7 and 0.9 µM respectively.