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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fig7: Jnk inhibition sensitizes LC to NF-κB inhibitor-induced cell death and partially protects HSPC. (A) CFU from HSPC treated with indicated doses of SP6 and BAY. (B) CFU from LC and Tnfr−/− LC treated with indicated doses of SP6 and BAY. (C) CFU from AML-ETO and CBFβ-MYH11-transduced LC treated with Bay, SP6, or combination. (D) LC and Tnfr−/− LC were transduced with IκBαSR-GFP. GFP+ cells were sorted and plated in methylcellulose with or without SP6 treatment. Values are normalized to vector-only-transduced control cells. (E) LCs were transduced with DN-AP1-GFP. GFP+ cells were sorted and plated in methylcellulose with or without BAY treatment. (F) LCs were transduced with c-Jun-shRNA1; GFP+ cells were sorted and plated in methylcellulose with or without BAY treatment. shSCR transduction was used as control. (G) LCs were treated with indicated inhibitors for 12 h in vitro. Surviving cells were transplanted into lethally irradiated recipient mice. Survival of the recipient mice was analyzed by Kaplan-Meier survival graphing. Leukemia was confirmed at the time of death for each transplant mouse. Five mice were used in each treatment group. (H) Sublethally irradiated mice were engrafted with LC and treated with BAY, TNF mAb, or in combination. Survival of the mice was measured over time and analyzed by Kaplan-Meier survival graphing. Leukemia was confirmed at the time of death for each transplanted mouse. Numbers of mice used are indicated in the panel. All values shown are mean values ± 1 SD from three independent trials. In A–F, * (P < 0.05) and ** (P < 0.01) indicate notated significance when compared with vehicle-treated control as determined by Student’s t test two-tailed analysis. In G and H, * indicates P < 0.05 when compared with vehicle treatment. # indicates P < 0.05 significant difference when compared with indicated conditions. ∼ indicates P < 0.05 in G and H when compared with BAY-treated LC/mice as determined by Log-Rank test.
We observed that LCs lacking Tnf receptors are more sensitive to NF-κB inhibition than their WT counterparts (Fig. 3). We then found that Tnf stimulates Jnk as an NF-κB–independent survival signal in LC, but a death signal in HSPC (Fig. 4), through short Jnk signal duration (Fig. 5) and activation of c-Jun/AP1 in LC (Fig. 6). Therefore, we hypothesize that Jnk inhibition should sensitize LC to NF-κB inhibition while protecting HSPC from the effects of NF-κB inhibition. To investigate this, we treated LC and HSPC with BAY and SP6 individually or in combination. We found that SP6 treatment can partially prevent death and rescue the decrease in CFU of HSPC resulting from high concentrations of BAY (Fig. 7 A). However, in LC, addition of SP6 can sensitize LC to BAY-induced cell death at the same rate as in Tnfr−/− LC (Fig. 7 B). We found that inhibition of both signals eliminated more clonogenic LC than inhibiting either individual signal. Such additive inhibitory effects were also observed in the Tnf-expressing CBFβ-MYH11 LC but not in Tnf-nonexpressing AML-ETO LC (Fig. 7 C). To confirm our inhibitor specificity, we genetically inactivated NF-κB or Jnk–AP1 signaling by transducing LC with IκBαSR, DN-AP1, or c-Jun-shRNA. We found that genetic inactivation of NF-κB or Jnk–AP1/c-Jun was able to sensitize LC to SP6 or BAY, respectively, reproducing the conclusion from our inhibitor combination studies (Fig. 7, D–F).