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Co-inhibition of NF-κB and JNK is synergistic in TNF-expressing human AML.

Volk A, Li J, Xin J, You D, Zhang J, Liu X, Xiao Y, Breslin P, Li Z, Wei W, Schmidt R, Li X, Zhang Z, Kuo PC, Nand S, Zhang J, Chen J, Zhang J - J. Exp. Med. (2014)

Bottom Line: We determined that TNF stimulation drives the JNK-AP1 pathway in a manner parallel to NF-κB, leading to the up-regulation of anti-apoptotic genes in LC.We found that we can significantly sensitize LC to NF-κB inhibitor treatment by blocking the TNF-JNK-AP1 signaling pathway.Our data suggest that co-inhibition of both TNF-JNK-AP1 and NF-κB signals may provide a more comprehensive treatment paradigm for AML patients with TNF-expressing LC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology Program, Department of Biology, Loyola University Chicago, Chicago, IL 60660.

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c-Jun mediates the Tnf-Jnk survival signal in LC. (A) LC and Tnfr−/− LC, grown in medium containing 10% FBS, were pretreated with BAY or SP6 before addition of growth factors and Tnf. Anti-apoptotic and proliferation-related signals were examined by Western blotting. (B) HSPC and LC were pretreated with BAY followed by Tnf stimulation. p-c-Jun and c-Jun expression were examined by Western blotting. (C) HSPC and Tnfr−/− HSPC as well as LC and Tnfr−/− LC were transduced with DN-AP1 and sorted for CFU assay. (D and E) LC and Tnfr−/− LC were transduced with DN-AP1-GFP and were observed in suspension culture for reduction of GFP+ cell percentage (D) or sorted for CFU with or without 20 ng/ml Tnf treatment (E). (F) DN-AP1–transduced LCs were treated with Tnf or vehicle and assayed for cell death. (G) c-Jun knockdown by specific shRNA was verified by Western blotting. (H) c-Jun shRNA-transduced HSPC and LC were treated with TNF or vehicle and plated in methylcellulose for CFU assay. (I) c-Jun knockdown in LC enhances cell death when treated with Tnf. (J) Experiment model, a mechanism by which Jnk signal promotes survival of LC. Results shown are mean values ± 1 SD from three independent trials. * indicates P < 0.05 significant difference when compared with vehicle in E, F, H, and I, vector-only (Migr1) in C as determined by Student’s t test two-tailed analysis. # indicates P < 0.05 significant difference when compared with indicated conditions. A, B, D, and G are representative results from three independent trials.
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fig6: c-Jun mediates the Tnf-Jnk survival signal in LC. (A) LC and Tnfr−/− LC, grown in medium containing 10% FBS, were pretreated with BAY or SP6 before addition of growth factors and Tnf. Anti-apoptotic and proliferation-related signals were examined by Western blotting. (B) HSPC and LC were pretreated with BAY followed by Tnf stimulation. p-c-Jun and c-Jun expression were examined by Western blotting. (C) HSPC and Tnfr−/− HSPC as well as LC and Tnfr−/− LC were transduced with DN-AP1 and sorted for CFU assay. (D and E) LC and Tnfr−/− LC were transduced with DN-AP1-GFP and were observed in suspension culture for reduction of GFP+ cell percentage (D) or sorted for CFU with or without 20 ng/ml Tnf treatment (E). (F) DN-AP1–transduced LCs were treated with Tnf or vehicle and assayed for cell death. (G) c-Jun knockdown by specific shRNA was verified by Western blotting. (H) c-Jun shRNA-transduced HSPC and LC were treated with TNF or vehicle and plated in methylcellulose for CFU assay. (I) c-Jun knockdown in LC enhances cell death when treated with Tnf. (J) Experiment model, a mechanism by which Jnk signal promotes survival of LC. Results shown are mean values ± 1 SD from three independent trials. * indicates P < 0.05 significant difference when compared with vehicle in E, F, H, and I, vector-only (Migr1) in C as determined by Student’s t test two-tailed analysis. # indicates P < 0.05 significant difference when compared with indicated conditions. A, B, D, and G are representative results from three independent trials.

Mentions: To determine whether Tnf–Jnk stimulates survival/proliferation in LC by inducing survival/proliferation-related gene expression, we cytokine-starved LC to reset the internal signaling to an unstimulated basal rate and then pretreated the cells with either SP6 or BAY to block Jnk or NF-κB activity, respectively. After SP6 or BAY treatment, we stimulated the cells with Tnf plus four hematopoietic cytokines (five total growth factors, 5GF) to examine the expression of which survival/proliferation-related genes are regulated through Jnk. We found that c-Jun, JunB, c-Flip, Mcl1, and cyclin D1 are down-regulated in Tnfr−/− LC compared with TnfrWT LC, suggesting that these are Tnf target genes. Among them, cytokine-induced c-Jun, Mcl-1, and c-Flip expression were repressed by Jnk inhibitor treatment but not by NF-κB inhibitor, suggesting they are Tnf-Jnk–specific targets. We found that xIap1 was not reduced in Tnfr−/− LC but was reduced by the addition of SP6, suggesting that xIap1 is a Jnk-specific, but Tnf-independent, survival gene. JunB and Cyclin D1 were unaffected by inhibitors of either Jnk or NF-κB. The elevated levels of Bcl-2 and Bcl-xL in Tnfr−/− LC compared with TnfrWT LC likely indicate the presence of a feedback mechanism due to the lack of Mcl-1, c-Flip, and xIap1. (Fig. 6 A).


Co-inhibition of NF-κB and JNK is synergistic in TNF-expressing human AML.

Volk A, Li J, Xin J, You D, Zhang J, Liu X, Xiao Y, Breslin P, Li Z, Wei W, Schmidt R, Li X, Zhang Z, Kuo PC, Nand S, Zhang J, Chen J, Zhang J - J. Exp. Med. (2014)

c-Jun mediates the Tnf-Jnk survival signal in LC. (A) LC and Tnfr−/− LC, grown in medium containing 10% FBS, were pretreated with BAY or SP6 before addition of growth factors and Tnf. Anti-apoptotic and proliferation-related signals were examined by Western blotting. (B) HSPC and LC were pretreated with BAY followed by Tnf stimulation. p-c-Jun and c-Jun expression were examined by Western blotting. (C) HSPC and Tnfr−/− HSPC as well as LC and Tnfr−/− LC were transduced with DN-AP1 and sorted for CFU assay. (D and E) LC and Tnfr−/− LC were transduced with DN-AP1-GFP and were observed in suspension culture for reduction of GFP+ cell percentage (D) or sorted for CFU with or without 20 ng/ml Tnf treatment (E). (F) DN-AP1–transduced LCs were treated with Tnf or vehicle and assayed for cell death. (G) c-Jun knockdown by specific shRNA was verified by Western blotting. (H) c-Jun shRNA-transduced HSPC and LC were treated with TNF or vehicle and plated in methylcellulose for CFU assay. (I) c-Jun knockdown in LC enhances cell death when treated with Tnf. (J) Experiment model, a mechanism by which Jnk signal promotes survival of LC. Results shown are mean values ± 1 SD from three independent trials. * indicates P < 0.05 significant difference when compared with vehicle in E, F, H, and I, vector-only (Migr1) in C as determined by Student’s t test two-tailed analysis. # indicates P < 0.05 significant difference when compared with indicated conditions. A, B, D, and G are representative results from three independent trials.
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fig6: c-Jun mediates the Tnf-Jnk survival signal in LC. (A) LC and Tnfr−/− LC, grown in medium containing 10% FBS, were pretreated with BAY or SP6 before addition of growth factors and Tnf. Anti-apoptotic and proliferation-related signals were examined by Western blotting. (B) HSPC and LC were pretreated with BAY followed by Tnf stimulation. p-c-Jun and c-Jun expression were examined by Western blotting. (C) HSPC and Tnfr−/− HSPC as well as LC and Tnfr−/− LC were transduced with DN-AP1 and sorted for CFU assay. (D and E) LC and Tnfr−/− LC were transduced with DN-AP1-GFP and were observed in suspension culture for reduction of GFP+ cell percentage (D) or sorted for CFU with or without 20 ng/ml Tnf treatment (E). (F) DN-AP1–transduced LCs were treated with Tnf or vehicle and assayed for cell death. (G) c-Jun knockdown by specific shRNA was verified by Western blotting. (H) c-Jun shRNA-transduced HSPC and LC were treated with TNF or vehicle and plated in methylcellulose for CFU assay. (I) c-Jun knockdown in LC enhances cell death when treated with Tnf. (J) Experiment model, a mechanism by which Jnk signal promotes survival of LC. Results shown are mean values ± 1 SD from three independent trials. * indicates P < 0.05 significant difference when compared with vehicle in E, F, H, and I, vector-only (Migr1) in C as determined by Student’s t test two-tailed analysis. # indicates P < 0.05 significant difference when compared with indicated conditions. A, B, D, and G are representative results from three independent trials.
Mentions: To determine whether Tnf–Jnk stimulates survival/proliferation in LC by inducing survival/proliferation-related gene expression, we cytokine-starved LC to reset the internal signaling to an unstimulated basal rate and then pretreated the cells with either SP6 or BAY to block Jnk or NF-κB activity, respectively. After SP6 or BAY treatment, we stimulated the cells with Tnf plus four hematopoietic cytokines (five total growth factors, 5GF) to examine the expression of which survival/proliferation-related genes are regulated through Jnk. We found that c-Jun, JunB, c-Flip, Mcl1, and cyclin D1 are down-regulated in Tnfr−/− LC compared with TnfrWT LC, suggesting that these are Tnf target genes. Among them, cytokine-induced c-Jun, Mcl-1, and c-Flip expression were repressed by Jnk inhibitor treatment but not by NF-κB inhibitor, suggesting they are Tnf-Jnk–specific targets. We found that xIap1 was not reduced in Tnfr−/− LC but was reduced by the addition of SP6, suggesting that xIap1 is a Jnk-specific, but Tnf-independent, survival gene. JunB and Cyclin D1 were unaffected by inhibitors of either Jnk or NF-κB. The elevated levels of Bcl-2 and Bcl-xL in Tnfr−/− LC compared with TnfrWT LC likely indicate the presence of a feedback mechanism due to the lack of Mcl-1, c-Flip, and xIap1. (Fig. 6 A).

Bottom Line: We determined that TNF stimulation drives the JNK-AP1 pathway in a manner parallel to NF-κB, leading to the up-regulation of anti-apoptotic genes in LC.We found that we can significantly sensitize LC to NF-κB inhibitor treatment by blocking the TNF-JNK-AP1 signaling pathway.Our data suggest that co-inhibition of both TNF-JNK-AP1 and NF-κB signals may provide a more comprehensive treatment paradigm for AML patients with TNF-expressing LC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology Program, Department of Biology, Loyola University Chicago, Chicago, IL 60660.

Show MeSH
Related in: MedlinePlus