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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fig2: Tnf promotes LC growth but represses HSPC growth in vitro. (A) CFU from MA9-LC and HSPC treated with indicated doses of Tnf. The numbers of CFU of each type of cell in Tnf-treated groups were normalized to the CFU number in vehicle-treated group. Genotype results are shown for LC and Tnfr−/− LC. (B and C) LC and Tnfr−/− LC were treated with 20 ng/ml Tnf (B) or 20 µg/ml anti-Tnf mAb (C) in suspension culture. Growth curves of LC were examined by counting the number of live cells daily by trypan blue exclusion. (D) Serial colony-forming ability was compared between LC and Tnfr−/− LC. (E) CFU/20,000 cells shown for Tnfr−/− HSPC and HSPC. (F) Leukemogenic capacity of LC and Tnfr−/− LC was compared by in vivo transplantation. (G) CFUs from AML-ETO and CBFβ-MYH11–transduced murine LCs were treated with Tnf. Expression of Tnf and its receptors are shown by Western blot analysis. (H) Cell cycle was compared between LC and Tnfr−/− LC. (I) Tnfr−/− LC and LC were cultured overnight in the presence of anti-Tnf mAb. Cell death was measured by Annexin V/PI assay. Significance is measured relative to vehicle control in LC, unless otherwise noted. (J) LCs were treated with Tnf with or without Nec-1 overnight, and cell death was measured by Annexin V/PI. Rip3−/− LCs were used as necroptosis-negative controls. (K) HSPC and Tnfr−/− HSPC were cultured in medium with or without exogenous Tnf. Nec1 was used to block necroptosis. (L) HSPCs from WT (Rip3 WT) and Rip3−/− mice were cultured with or without Tnf. Z-IETD-FMK was used to inhibit Caspase 8 activity. Cell death was examined 24 h after Tnf treatment. (M) Tnf secreted by MA9-LC is sufficient to induce cell death in HSPC. WT and Tnfr−/− HSPCs were incubated in fresh medium or 1:5 conditioned/fresh medium from LC culture and treated with or without anti-Tnf mAb. * indicates P < 0.05 when compared with vehicle treated control (A–C and G–M) or WT control (D–F) as determined by Student’s t test two-tailed analysis. # indicates P < 0.05 significant difference when compared with indicated conditions. Values shown are mean ± SD analysis from three independent trials, unless otherwise notated. Western blot in G and readout for H are representative results from three independent trials.
To investigate the role of Tnf in LC and normal HSPC, we generated MA9-transduced Tnfr−/− (Tnf receptors 1 and 2 knockout) and TnfrWT murine LC by infecting HSPC isolated from Tnfr−/− and TnfrWT mice, respectively, with MA9-expressing virus (Fig. 1 E). We found that exogenous Tnf promotes LC growth in methylcellulose colony-forming assays and liquid culture (Fig. 2, A and B) but significantly represses the growth of normal HSPC colonies (Fig. 2 A). Inactivation of Tnf signaling by either Tnfr knockout or a neutralizing monoclonal antibody restricts LC growth but has no obvious effect on normal HSPC (Fig. 2 C-E). Furthermore, transplantation studies demonstrated that Tnfr−/− LC-transplanted mice required a longer latency for leukemia development in vivo than TnfrWT LC (Fig. 2 F). A similar response to exogenous Tnf was also observed in CBFβ-MYH11–transduced murine LC (Tnf-expressing, M4) versus AML-ETO–transduced murine LC (Tnf-nonexpressing, M2), suggesting that such effects are not MA9-LC specific (Fig. 2 G). All these suggest that in these Tnf-producing leukemias, Tnf might function as an enhancer for leukemia development and a repressor for normal hematopoiesis. For simplicity, WT HSPC will be described as HSPC, and murine WT LC will be described as LC. Other genetic deletions, such as Tnfr−/− LC, will be specified.