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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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Co-inhibition of NF-κB and JNK is synergistic in TNF-expressing human AML. (A) CFU from human LC lines treated with indicated doses of SP, BAY, or combination. (B) Nuclear fractionation analysis showing NF-κB activity in AML cell lines. (C) Several TNF-high–expressing AML cell lines were treated with TNF and BAY individually or in combination. Levels of p-JNK and p-cJUN were examined by Western blotting. (D) Primary AML cells were treated with indicated doses of BAY, SP, or combination, and then assessed for CFU. Right axis indicates relative TNF mRNA expression levels relative to average healthy donor PB stem cells (PBSCs; n = 3). mRNA transcripts were set to GAPDH as control. ∼ indicates increased TNF mRNA level compared with PB stem cell control, P < 0.05. * indicates P < 0.05 when compared with vehicle-treated control, unless otherwise noted, as determined by one-way ANOVA. # indicates P < 0.05 significant difference when compared with indicated conditions. Values shown are mean values ± 1 SD from three independent trials. B and C are representative results from three independent trials. (E) Proposed mechanism for TNF function in AML.
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fig8: Co-inhibition of NF-κB and JNK is synergistic in TNF-expressing human AML. (A) CFU from human LC lines treated with indicated doses of SP, BAY, or combination. (B) Nuclear fractionation analysis showing NF-κB activity in AML cell lines. (C) Several TNF-high–expressing AML cell lines were treated with TNF and BAY individually or in combination. Levels of p-JNK and p-cJUN were examined by Western blotting. (D) Primary AML cells were treated with indicated doses of BAY, SP, or combination, and then assessed for CFU. Right axis indicates relative TNF mRNA expression levels relative to average healthy donor PB stem cells (PBSCs; n = 3). mRNA transcripts were set to GAPDH as control. ∼ indicates increased TNF mRNA level compared with PB stem cell control, P < 0.05. * indicates P < 0.05 when compared with vehicle-treated control, unless otherwise noted, as determined by one-way ANOVA. # indicates P < 0.05 significant difference when compared with indicated conditions. Values shown are mean values ± 1 SD from three independent trials. B and C are representative results from three independent trials. (E) Proposed mechanism for TNF function in AML.

Mentions: To study whether combination treatment using both NF-κB and JNK signaling inhibitors is also additive in eliminating human LC, we compared the responses of human AML cell lines to the treatments. By incubating the LC with NF-κB and JNK inhibitors individually or in combination for 12 h and then seeding for CFU to examine the reduction of clonogenic LC, we observed additive inhibitory effects of the two inhibitors in almost all AML cell lines. We noted that the sensitivity of AML cell lines to such additive inhibitory effects are correlated to levels of TNF expression (Fig. 1 D). Cell lines RS4:11 (B-ALL), K562 erythroid LC, and ML-2 that had a reduced response also express lower levels of TNF (Fig. 8 A), whereas the TNF-expressing cell lines, including HL-60 (M2/3), ML-2 (M5), Molm-13 (M5), U937 (M5), NB4 (M3), and THP-1 (M5), showed exceptional sensitivity to the combined inhibitor treatment. In addition, we found that, although all the sensitive cell lines showed TNF-stimulated NF-κB–independent JNK–c-JUN activation (Fig. 8 C), the most sensitive AML cell lines (U937, NB4, and THP1) had increased basal levels of NF-κB and JNK-c-JUN activities as shown by elevated nuclear p65 and total p-JNK/p-c-JUN (Fig. 8, B and C). We also confirmed the correlation of additive effects of our combination treatment to TNF expression in primary human LC isolated from newly diagnosed AML patients. TNF expression accurately predicted sensitivity to combined BAY and SP6 inhibitor treatment in each case. Interestingly, one AML patient sample was sensitive to combined inhibitor treatment but did not express TNF (Fig. 8 D). We are currently investigating the role of proinflammatory cytokines other than TNF in promoting NF-κB–independent survival signals in this AML patient sample. Collectively, our studies suggest that the combination treatment of NF-κB and JNK inhibitors might be a useful pharmacological strategy for AML patients whose LC produce TNF.


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)

Co-inhibition of NF-κB and JNK is synergistic in TNF-expressing human AML. (A) CFU from human LC lines treated with indicated doses of SP, BAY, or combination. (B) Nuclear fractionation analysis showing NF-κB activity in AML cell lines. (C) Several TNF-high–expressing AML cell lines were treated with TNF and BAY individually or in combination. Levels of p-JNK and p-cJUN were examined by Western blotting. (D) Primary AML cells were treated with indicated doses of BAY, SP, or combination, and then assessed for CFU. Right axis indicates relative TNF mRNA expression levels relative to average healthy donor PB stem cells (PBSCs; n = 3). mRNA transcripts were set to GAPDH as control. ∼ indicates increased TNF mRNA level compared with PB stem cell control, P < 0.05. * indicates P < 0.05 when compared with vehicle-treated control, unless otherwise noted, as determined by one-way ANOVA. # indicates P < 0.05 significant difference when compared with indicated conditions. Values shown are mean values ± 1 SD from three independent trials. B and C are representative results from three independent trials. (E) Proposed mechanism for TNF function in AML.
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fig8: Co-inhibition of NF-κB and JNK is synergistic in TNF-expressing human AML. (A) CFU from human LC lines treated with indicated doses of SP, BAY, or combination. (B) Nuclear fractionation analysis showing NF-κB activity in AML cell lines. (C) Several TNF-high–expressing AML cell lines were treated with TNF and BAY individually or in combination. Levels of p-JNK and p-cJUN were examined by Western blotting. (D) Primary AML cells were treated with indicated doses of BAY, SP, or combination, and then assessed for CFU. Right axis indicates relative TNF mRNA expression levels relative to average healthy donor PB stem cells (PBSCs; n = 3). mRNA transcripts were set to GAPDH as control. ∼ indicates increased TNF mRNA level compared with PB stem cell control, P < 0.05. * indicates P < 0.05 when compared with vehicle-treated control, unless otherwise noted, as determined by one-way ANOVA. # indicates P < 0.05 significant difference when compared with indicated conditions. Values shown are mean values ± 1 SD from three independent trials. B and C are representative results from three independent trials. (E) Proposed mechanism for TNF function in AML.
Mentions: To study whether combination treatment using both NF-κB and JNK signaling inhibitors is also additive in eliminating human LC, we compared the responses of human AML cell lines to the treatments. By incubating the LC with NF-κB and JNK inhibitors individually or in combination for 12 h and then seeding for CFU to examine the reduction of clonogenic LC, we observed additive inhibitory effects of the two inhibitors in almost all AML cell lines. We noted that the sensitivity of AML cell lines to such additive inhibitory effects are correlated to levels of TNF expression (Fig. 1 D). Cell lines RS4:11 (B-ALL), K562 erythroid LC, and ML-2 that had a reduced response also express lower levels of TNF (Fig. 8 A), whereas the TNF-expressing cell lines, including HL-60 (M2/3), ML-2 (M5), Molm-13 (M5), U937 (M5), NB4 (M3), and THP-1 (M5), showed exceptional sensitivity to the combined inhibitor treatment. In addition, we found that, although all the sensitive cell lines showed TNF-stimulated NF-κB–independent JNK–c-JUN activation (Fig. 8 C), the most sensitive AML cell lines (U937, NB4, and THP1) had increased basal levels of NF-κB and JNK-c-JUN activities as shown by elevated nuclear p65 and total p-JNK/p-c-JUN (Fig. 8, B and C). We also confirmed the correlation of additive effects of our combination treatment to TNF expression in primary human LC isolated from newly diagnosed AML patients. TNF expression accurately predicted sensitivity to combined BAY and SP6 inhibitor treatment in each case. Interestingly, one AML patient sample was sensitive to combined inhibitor treatment but did not express TNF (Fig. 8 D). We are currently investigating the role of proinflammatory cytokines other than TNF in promoting NF-κB–independent survival signals in this AML patient sample. Collectively, our studies suggest that the combination treatment of NF-κB and JNK inhibitors might be a useful pharmacological strategy for AML patients whose LC produce TNF.

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