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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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Tnf signal inactivation sensitizes LC to NF-κB inhibitor treatment but protects healthy HSPC from such treatment. (A and B) Tnf stimulation of NF-κB activity in both LC and HSPC as shown by p65 (NF-κB1) nuclear localization (values in parenthesis are mean similarity dilate peak, sample size = 5,000 cells) as determined by ImageStreamX in A or by p65 phosphorylation in B. Bars, 10 µM. (C) HSPC and LC stimulated with individual cytokines show different patterns of p65 phosphorylation. (D and E) HSPC and LC were treated in parallel with indicated doses of BAY11-7085 (BAY) for CFU assay in D or analyzed for cell death in E. (F) HSPC and Tnfr−/− HSPC were treated with increasing doses of BAY and plated for CFU assay. (G) HSPC and Tnfr−/− HSPC were transduced with IκBαSR-GFP. GFP+ cells were sorted and plated for CFU assay. The number of CFUs in each genotype of HSPC was normalized to vector-only-transduced corresponding genotype control. (H) LC and Tnfr−/− LC were treated with increasing doses of BAY and plated for CFU assay. (I) LC and Tnfr−/− LC were transduced with IκBαSR-GFP. GFP+ cells were sorted and plated for CFU assay. The number of CFUs in each genotype of HSPC or LC was normalized to vector-only-transduced corresponding genotype control. (J) LCs were co-treated with BAY and TNF. CFUs were measured 1 wk after treatment. (K) Experiment model. All values shown are mean ± SD normalized to vehicle-treated control, performed on three independent trials. * (P < 0.05) and ** (P < 0.01) indicate significant reduction compared with corresponding vehicle groups or vector-only groups 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.
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fig3: Tnf signal inactivation sensitizes LC to NF-κB inhibitor treatment but protects healthy HSPC from such treatment. (A and B) Tnf stimulation of NF-κB activity in both LC and HSPC as shown by p65 (NF-κB1) nuclear localization (values in parenthesis are mean similarity dilate peak, sample size = 5,000 cells) as determined by ImageStreamX in A or by p65 phosphorylation in B. Bars, 10 µM. (C) HSPC and LC stimulated with individual cytokines show different patterns of p65 phosphorylation. (D and E) HSPC and LC were treated in parallel with indicated doses of BAY11-7085 (BAY) for CFU assay in D or analyzed for cell death in E. (F) HSPC and Tnfr−/− HSPC were treated with increasing doses of BAY and plated for CFU assay. (G) HSPC and Tnfr−/− HSPC were transduced with IκBαSR-GFP. GFP+ cells were sorted and plated for CFU assay. The number of CFUs in each genotype of HSPC was normalized to vector-only-transduced corresponding genotype control. (H) LC and Tnfr−/− LC were treated with increasing doses of BAY and plated for CFU assay. (I) LC and Tnfr−/− LC were transduced with IκBαSR-GFP. GFP+ cells were sorted and plated for CFU assay. The number of CFUs in each genotype of HSPC or LC was normalized to vector-only-transduced corresponding genotype control. (J) LCs were co-treated with BAY and TNF. CFUs were measured 1 wk after treatment. (K) Experiment model. All values shown are mean ± SD normalized to vehicle-treated control, performed on three independent trials. * (P < 0.05) and ** (P < 0.01) indicate significant reduction compared with corresponding vehicle groups or vector-only groups 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.

Mentions: Previous reports showed that NF-κB activity is significantly increased in LC and LSC (Guzman et al., 2001). We confirmed such elevated NF-κB activity in our murine LC compared with HSPC (unpublished data). In addition, we found that NF-κB activity in LC is cytokine-dependent. p65 (NF-κB) activity was turned off when LCs were incubated in a cytokine-free medium and was restored upon Tnf stimulation as measured by both nuclear localization (Fig. 3 A) and p65 phosphorylation (Fig. 3 B). We also found that all hematopoietic cytokines used in our cultures induced NF-κB activity in LC while only Tnf stimulated NF-κB in HSPC, suggesting how NF-κB activity can be elevated in subtypes of LC which do not express TNF (Fig. 3 C). Consistent with a previous report (Guzman et al., 2001), our murine LCs are more sensitive to NF-κB inhibition by BAY11-7085 (a reversible small molecule inhibitor of IκBα phosphorylation, BAY hereafter) in vitro as shown by a reduction in CFU (Fig. 3 D) and an increase in cell death (Fig. 3 E) when compared with HSPC.


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)

Tnf signal inactivation sensitizes LC to NF-κB inhibitor treatment but protects healthy HSPC from such treatment. (A and B) Tnf stimulation of NF-κB activity in both LC and HSPC as shown by p65 (NF-κB1) nuclear localization (values in parenthesis are mean similarity dilate peak, sample size = 5,000 cells) as determined by ImageStreamX in A or by p65 phosphorylation in B. Bars, 10 µM. (C) HSPC and LC stimulated with individual cytokines show different patterns of p65 phosphorylation. (D and E) HSPC and LC were treated in parallel with indicated doses of BAY11-7085 (BAY) for CFU assay in D or analyzed for cell death in E. (F) HSPC and Tnfr−/− HSPC were treated with increasing doses of BAY and plated for CFU assay. (G) HSPC and Tnfr−/− HSPC were transduced with IκBαSR-GFP. GFP+ cells were sorted and plated for CFU assay. The number of CFUs in each genotype of HSPC was normalized to vector-only-transduced corresponding genotype control. (H) LC and Tnfr−/− LC were treated with increasing doses of BAY and plated for CFU assay. (I) LC and Tnfr−/− LC were transduced with IκBαSR-GFP. GFP+ cells were sorted and plated for CFU assay. The number of CFUs in each genotype of HSPC or LC was normalized to vector-only-transduced corresponding genotype control. (J) LCs were co-treated with BAY and TNF. CFUs were measured 1 wk after treatment. (K) Experiment model. All values shown are mean ± SD normalized to vehicle-treated control, performed on three independent trials. * (P < 0.05) and ** (P < 0.01) indicate significant reduction compared with corresponding vehicle groups or vector-only groups 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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig3: Tnf signal inactivation sensitizes LC to NF-κB inhibitor treatment but protects healthy HSPC from such treatment. (A and B) Tnf stimulation of NF-κB activity in both LC and HSPC as shown by p65 (NF-κB1) nuclear localization (values in parenthesis are mean similarity dilate peak, sample size = 5,000 cells) as determined by ImageStreamX in A or by p65 phosphorylation in B. Bars, 10 µM. (C) HSPC and LC stimulated with individual cytokines show different patterns of p65 phosphorylation. (D and E) HSPC and LC were treated in parallel with indicated doses of BAY11-7085 (BAY) for CFU assay in D or analyzed for cell death in E. (F) HSPC and Tnfr−/− HSPC were treated with increasing doses of BAY and plated for CFU assay. (G) HSPC and Tnfr−/− HSPC were transduced with IκBαSR-GFP. GFP+ cells were sorted and plated for CFU assay. The number of CFUs in each genotype of HSPC was normalized to vector-only-transduced corresponding genotype control. (H) LC and Tnfr−/− LC were treated with increasing doses of BAY and plated for CFU assay. (I) LC and Tnfr−/− LC were transduced with IκBαSR-GFP. GFP+ cells were sorted and plated for CFU assay. The number of CFUs in each genotype of HSPC or LC was normalized to vector-only-transduced corresponding genotype control. (J) LCs were co-treated with BAY and TNF. CFUs were measured 1 wk after treatment. (K) Experiment model. All values shown are mean ± SD normalized to vehicle-treated control, performed on three independent trials. * (P < 0.05) and ** (P < 0.01) indicate significant reduction compared with corresponding vehicle groups or vector-only groups 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.
Mentions: Previous reports showed that NF-κB activity is significantly increased in LC and LSC (Guzman et al., 2001). We confirmed such elevated NF-κB activity in our murine LC compared with HSPC (unpublished data). In addition, we found that NF-κB activity in LC is cytokine-dependent. p65 (NF-κB) activity was turned off when LCs were incubated in a cytokine-free medium and was restored upon Tnf stimulation as measured by both nuclear localization (Fig. 3 A) and p65 phosphorylation (Fig. 3 B). We also found that all hematopoietic cytokines used in our cultures induced NF-κB activity in LC while only Tnf stimulated NF-κB in HSPC, suggesting how NF-κB activity can be elevated in subtypes of LC which do not express TNF (Fig. 3 C). Consistent with a previous report (Guzman et al., 2001), our murine LCs are more sensitive to NF-κB inhibition by BAY11-7085 (a reversible small molecule inhibitor of IκBα phosphorylation, BAY hereafter) in vitro as shown by a reduction in CFU (Fig. 3 D) and an increase in cell death (Fig. 3 E) when compared with HSPC.

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