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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 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.
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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.

Mentions: 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.


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 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.
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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.
Mentions: 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.

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