Fatty Acid Synthase induced S6Kinase facilitates USP11-eIF4B complex formation for sustained oncogenic translation in DLBCL

Altered lipid metabolism and aberrant protein translation are strongly associated with cancerous outgrowth; however, the inter-regulation of these key processes is still underexplored in diffuse large B-cell lymphoma (DLBCL). Although fatty acid synthase (FASN) activity is reported to positively correlate with PI3K-Akt-mTOR pathway that can modulate protein synthesis, the precise impact of FASN inhibition on this process is still unknown. Herein, we demonstrate that attenuating FASN expression or its activity significantly reduces eIF4B (eukaryotic initiation factor 4B) levels and consequently overall protein translation. Through biochemical studies, we identified eIF4B as a bonafide substrate of USP11, which stabilizes and enhances eIF4B activity. Employing both pharmacological and genetic approaches, we establish that FASN-induced PI3K-S6Kinase signaling phosphorylates USP11 enhancing its interaction with eIF4B and thereby promoting oncogenic translation.

indicated antibodies (C). GAPDH was used as loading control. (D) qRT-PCR analysis of FASN and eIF4B expression in indicated cells treated with defined concentration of C75. Results were normalized with DMSO treated corresponding cells and expressed as mean±SD (n=3). Statistical analysis was performed using Student's t-test (Unpaired two tailed) *p<0.05, α p<0.001vs DMSO treated corresponding cells. (E-F) Indicated cells were infected by shRNA against FASN and cultured in puromycin (0.5-1µg/mL) for stable cells generation. Post selection, cells were exposed for 30 min with Puromycin treatment (3µg/mL) and lysed. Cell lysates were subjected to immunoblotting with anti-Puro antibody (SUnSET assay) (E) or indicated antibodies (F). GAPDH was used as loading control. (G) qRT-PCR analysis of FASN and eIF4B expression in indicated stable cells infected with shRNA against FASN or NT (scrambled). Results were normalized with NT infected corresponding cells and expressed as mean±SD (n=3). Statistical analysis was performed using Student's t-test (Unpaired two tailed) *p<0.05, α p<0.001 vs NT infected corresponding cells.

Supplementary Figure 5: eIF4B is required for DLBCL proliferation
(A) Total number of colonies grown in eIF4B depleted SUDHL2 and SUDHL6 in methylcellulose culture. Colony counts were performed on the 15 th day of methylcellulose culture. α p<0.001 vs corresponding NT infected control cells. (B and C) Indicated cells were infected by shRNA against eIF4B and cultured in puromycin (0.5-1µg/mL) for stable cells generation. Post selection, cells were exposed for 30 min with Puromycin treatment (3µg/mL) and lysed. Cell lysates were subjected to immunoblotting with anti-Puro antibody (SUnSET assay) (B) or indicated antibodies (C). (D & E) qRT-PCR analysis of FASN and eIF4B expression in indicated stable cells infected with shRNA against eIF4B or NT (scrambled). Results were normalized with NT infected corresponding controls and expressed as mean±SD (n=3). *p<0.05, **p,0.01, ***p<0.005, α p<0.001 vs NT infected corresponding cells. GAPDH was used as reference gene.

Supplementary Figure 6: eIF4B regulates translation of oncogenes
(A) Indicated eIF4B depleted stable cells were lysed and lysates were probed for the defined antibodies. (B) qRT-PCR analysis for expression of defined genes in indicated stable cells infected with shRNA against eIF4B or NT (scrambled). Results are expressed as mean±SD (n=3). Statistical analysis was performed using Student's t-test (Unpaired two tailed) *p<0.05, **p<0.01, ***p<0.005 α p<0.001 vs NT infected corresponding cells. (C) Exponentially growing eIF4B depleted stable SUDHL4 and SUDHL6 cells were cultured with MG132 (10µM) for 2 h and cell lysates were probed for the defined antibodies. N: scrambled; 1: sheIF4B-1; 2: sheIF4B-2 (D) Total cell lysates from SUDHL2 were subjected to immunoprecipitation with eIF4B antibodies, followed by RNA isolation and qRT-PCR to detect the enrichment of the defined genes. Results were normalized total RNA from cell lysates and expressed relative percentage to mock sample enriched RNA. Values are expressed as mean±SD and statistical analysis was performed using Students t-test, *p<0.005, **p<0.01, *** p<0.005 vs corresponding mock samples.

Supplementary Figure 7:
Densitometric quantification of the immunoblots in Figure Figure 13A. Values were normalized with corresponding NT infected corresponding cells and were represented as mean±SD for n=3. Statistical analysis was performed using Student's t-test (Unpaired two tailed) *p<0.05, **p,0.01, vs NT infected corresponding cells. (C) qRT-PCR analysis for expression of defined genes in indicated stable cells infected with shRNA against FASN or NT (scrambled). Results are expressed as mean±SD (n=3). Statistical analysis was performed using Student's t-test (Unpaired two tailed) *p<0.05, **p,0.01, ***p<0.005, α p<0.001 vs NT infected corresponding cells.

Supplementary Figure 14: FASN inhibition leads to ubiquitin mediated proteosomal degradation of eIF4B.
(A) Indicated cells were treated with C75 (indicated concentration) for 14 h followed by treatment with MG132 (10µM) for 4 hours. Lysates were probed for the indicated antibodies. (B) Densitometric quantification of the immunoblots in Figure 4C and Supplementary Figure 14A for the indicated cells. Values were normalized with corresponding NT infected corresponding cells and were represented as mean±SD for n=6. Statistical analysis was performed using one-way ANOVA followed by Bonferroeni's post hoc test. **p<0.01, ****p<0.001 vs DMSO treated corresponding cells, a p<0.05, vs C75 treated corresponding cells (w) infected corresponding cells. (C) Cartoon showing FASN inhibition leads to eIF4B proteosomal degradation. (D) Lysates from SUDHL2 was immunoprecipitated with FASN antibody and probed for eIF4B. (E) String analysis of eIF4B interacting partners. Enlarged box represents eIF4B interaction with reported DUBs (string.db.org).

Supplementary Figure 15:
(A) Densitometric quantification of the immunoblots in Figure 4F. Values were normalized with GFP transfected cells and were represented as mean±SD for n=3. Statistical analysis was performed using Student's t-test (Unpaired two tailed) *p<0.05, vs GFP transfected cells. (B & C) Densitometric quantification of Figure 4G and Figure 4H. Values were normalized with GFP (B) or USP11 (C) transfected untreated cells and were represented as mean±SD for n=3. Statistical analysis was performed using Student's t-test (Unpaired two tailed) ***p<0.005, vs NT infected corresponding cells.

Supplementary Figure 16: USP11 expression promotes translation.
(A) Indicated cells were infected USP11 or its catalytically inactive mutant (USP11 CS ) and selected on puromycin. Post selection cells were cultured in puromycin (3µg/ml) for 30 min and lysates were probed for indicating antibodies. GFP transduced cells were used as corresponding controls. GAPDH was used as loading control. (B) Indicated USP7 depleted stable cells lysates were probed for indicating antibodies. GAPDH was used as loading control. (C) 293T cells were transfected with pYIC and indicated amount of eIF4B and or GFP. Post transfected, lysates were resolved and probed for indicated antibodies. GAPDH was used loading control. (D) Total cell lysates from indicated cells were subjected to immunoprecipitation with eIF4B antibodies, followed by RNA isolation and qRT-PCR to detect the enrichment of the defined genes. Results were normalized total RNA from cell lysates and expressed relative percentage to mock sample enriched RNA. Values are expressed as mean±SD. (E) Indicated eIF4B depleted stable cells were lysed and lysates were probed for the defined antibodies.

Supplementary Figure 17:
Densitometric quantification of the immunoblots in Supplementary Figure 16A & 23. Values were normalized with corresponding GFP infected corresponding cells and were represented as mean±SD for n=3. Statistical analysis was performed using Student's t-test (Unpaired two tailed) *p<0.05, **p,0.01 vs USP11 infected corresponding cells.

Supplementary Figure 18: USP11 directly regulates translation
(A) Indicated USP11 depleted stable cells were cultured in presence of puromycin (3µg/ml) for 30 min and lysates were probed for indicating antibodies. GAPDH was used as loading control. (B) qRT-PCR analysis for expression of defined genes in indicated stable cells infected with shRNA against USP11 or NT (scrambled). Results were normalized with NT infected corresponding controls and expressed as mean±SD (n=3). *p<0.05, **p,0.01, ***p<0.005, α p<0.001 vs NT infected corresponding cells.

Supplementary Figure 19:
(A & B) Densitometric quantification of the immunoblots in Figure 5A (A) and Supplementary Figure 18A (B). Values were normalized with corresponding NT infected corresponding cells and were represented as mean±SD for n=3. Statistical analysis was performed using Student's t-test (Unpaired two tailed) n non-significant, *p<0.05, **p,0.01, ***p<0.005, α p<0.001 vs NT infected corresponding cells.

Supplementary Figure 21: USP11 expression is required for eIF4B-sensitive gene translation
(A) Indicated USP11 knockdown cell lysates were probed for indicated antibodies. Actin was used as loading control. (B) qRT-PCR analysis for checking mRNA expression of indicated genes in USP11 knockdown cells. Results were normalized with NT infected corresponding controls and expressed as mean±SD (n=3). *p<0.05, **p,0.01, ***p<0.005 vs NT infected corresponding cells. (C) Exponentially growing USP11 depleted TMD8 & SUDHL6 cells were treated with MG132 (10µM) for 2 hours and lysates were probed for the indicated antibodies. Actin was used as loading control.