Co-inhibition of NF-κB and JNK is synergistic in TNF-expressing human AML.
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.
Affiliation: Molecular Biology Program, Department of Biology, Loyola University Chicago, Chicago, IL 60660.
- JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors/genetics/metabolism*
- Leukemia, Myeloid, Acute/genetics/metabolism*/pathology
- NF-kappa B/antagonists & inhibitors/genetics/metabolism*
- Tumor Necrosis Factor-alpha/genetics/metabolism*/pharmacology
- Blotting, Western
- Cell Line, Tumor
- Cell Survival/drug effects/genetics
- Cells, Cultured
- Gene Expression Regulation, Leukemic/drug effects
- HL-60 Cells
- K562 Cells
- Leukemia, Monocytic, Acute/genetics/metabolism
- Leukemia, Myelomonocytic, Acute/genetics/metabolism
- Leukemia, Promyelocytic, Acute/genetics/metabolism
- Mice, Knockout
- Receptors, Tumor Necrosis Factor/genetics/metabolism
- Reverse Transcriptase Polymerase Chain Reaction
- Signal Transduction/drug effects/genetics
- Transcription Factor AP-1/genetics/metabolism
- U937 Cells
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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.
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).