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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fig4: Jnk functions as a Tnf-induced, NF-κB–independent survival signal in LC, while being a death signal in HSPC. (A) HSPC and LC were treated with individual hematopoietic cytokines for 15 min. Jnk, Erk, p38, and Akt activities were analyzed by Western blot. (B) HSPC and LC were pretreated with BAY before stimulation with TNF. p65 and Jnk activities were analyzed by Western blot. (C) HSPC and LC were treated with indicated doses of p38 inhibitor SB253580, Jnk inhibitor SP600125, and Erk inhibitor PD98059 for 12 h and then seeded for CFU assay. DMSO treatment was used as a negative control. (D) LCs pretreated with JNKi-I (L) or SP600125 were analyzed for Jnk and c-Jun activity after 15 min of Tnf stimulation. (E) HSPC and LC were treated with increasing concentrations of SP6 overnight, and then seeded for CFU assay. Numbers of CFU were normalized to the corresponding vehicle-treated group. (F) HSPC and LC were treated in parallel with indicated doses of JNKi-1 (L) overnight, and then plated in methylcellulose. Numbers of CFU were normalized to the corresponding vehicle-treated group. (G) HSPC and LC were treated in suspension culture with increasing concentrations of SP6 overnight. Cell death was analyzed by Annexin-V/7AAD staining. (H) LC and HSPC were treated with Jnk inhibitor SP6 and Tnf individually or in combination overnight, and then seeded for CFU assay. Numbers of CFU were normalized to the corresponding vehicle-treated group. (I) HSPCs were treated overnight with indicated doses of Tnf and SP6. Cell death is shown as Annexin V+ cells. Values shown are mean ± SD normalized to vehicle-treated control from three independent trials. * (P < 0.05) and ** (P < 0.01) indicate significant difference when compared with vehicle-treated control in C, E, F, and H as determined by Student’s t test two-tailed analysis. # indicates P < 0.05 and ## indicates P < 0.01 significant difference when compared with indicated conditions. A, B, and D are representative results of three independent trials.
To search for the signaling pathway which mediates the contradictory effects of Tnf stimulation in LC and HSPC, we compared the activities of Jnk1/2, Erk1/2, p38, and Akt, all known Tnf-stimulated signaling mediators, between LC and HSPC after stimulation with either Tnf or the individual hematopoietic cytokines used in our cultures. We found that although all of these signaling mediators can be activated by Tnf and some other cytokines in both LC and HSPC, Jnk is only activated by Tnf but not any of the other cytokines (Fig. 4 A).