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The NAE inhibitor pevonedistat (MLN4924) synergizes with TNF- α to activate apoptosis

View Article: PubMed Central - PubMed

ABSTRACT

Predicting and understanding the mechanism of drug-induced toxicity is one of the primary goals of drug development. It has been hypothesized that inflammation may have a synergistic role in this process. Cell-based models provide an easily manipulated system to investigate this type of drug toxicity. Several groups have attempted to reproduce in vivo toxicity with combination treatment of pharmacological agents and inflammatory cytokines. Through this approach, synergistic cytotoxicity between the investigational agent pevonedistat (MLN4924) and TNF-α was identified. Pevonedistat is an inhibitor of the NEDD8-activating enzyme (NAE). Inhibition of NAE prevents activation of cullin-RING ligases, which are critical for proteasome-mediated protein degradation. TNF-α is a cytokine that is involved in inflammatory responses and cell death, among other biological functions. Treatment of cultured cells with the combination of pevonedistat and TNF-α, but not as single agents, resulted in rapid cell death. This cell death was determined to be mediated by caspase-8. Interestingly, the combination treatment of pevonedistat and TNF-α also caused an accumulation of the p10 protease subunit of caspase-8 that was not observed with cytotoxic doses of TNF-α. Under conditions where apoptosis was blocked, the mechanism of death switched to necroptosis. Trimerized MLKL was verified as a biomarker of necroptotic cell death. The synergistic toxicity of pevonedistat and elevated TNF-α was also demonstrated by in vivo rat studies. Only the combination treatment resulted in elevated serum markers of liver damage and single-cell hepatocyte necrosis. Taken together, the results of this work have characterized a novel synergistic toxicity driven by pevonedistat and TNF-α.

No MeSH data available.


Pevonedistat+TNF-α cytotoxicity is mediated by caspase-8. (a) H-4-II-E cells were treated with 1 or 10 μM pevonedistat±TNF-α for 16 h. Extracts were western blotted for the pro-enzyme form of the indicated caspases. (b) The schematic of the individual subunits of pro-caspase-8 (p24, p18, and p10) are as indicated. (c) Lysates from cells treated with 1 or 10 μM pevonedistat±TNF-for 8 h were western blotted with antibodies specific for epitopes within the caspase-8 p10 (top) or p18 subunits. The predicted caspase-8 subunits are indicated to the left of the image based on the expected size of the product. (d) Lysates from cells transfected with siRNA oligonucleotides against either a non-targeting control or against caspase-8 were western blotted for full-length caspase-8. (e) Cells were transfected with either a non-targeting control or the caspase-8-A siRNA. Four days later, cells received the indicated treatments and viability was assessed after an additional 48 h. All viability experiments were performed in triplicate, and error bars indicate±S.E.M. Approximate molecular sizes of proteins (in kDa) are given to the right of blots.
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fig3: Pevonedistat+TNF-α cytotoxicity is mediated by caspase-8. (a) H-4-II-E cells were treated with 1 or 10 μM pevonedistat±TNF-α for 16 h. Extracts were western blotted for the pro-enzyme form of the indicated caspases. (b) The schematic of the individual subunits of pro-caspase-8 (p24, p18, and p10) are as indicated. (c) Lysates from cells treated with 1 or 10 μM pevonedistat±TNF-for 8 h were western blotted with antibodies specific for epitopes within the caspase-8 p10 (top) or p18 subunits. The predicted caspase-8 subunits are indicated to the left of the image based on the expected size of the product. (d) Lysates from cells transfected with siRNA oligonucleotides against either a non-targeting control or against caspase-8 were western blotted for full-length caspase-8. (e) Cells were transfected with either a non-targeting control or the caspase-8-A siRNA. Four days later, cells received the indicated treatments and viability was assessed after an additional 48 h. All viability experiments were performed in triplicate, and error bars indicate±S.E.M. Approximate molecular sizes of proteins (in kDa) are given to the right of blots.

Mentions: Pro-caspase-8, and to a lesser extent pro-caspase-3, was cleaved/activated in cells treated with pevonedistat+TNF-α (Figure 3a). Pro-caspase-8 is comprised of three domains (Figure 3b), but only p18 and p10 are proteases.21 Two caspase-8 antibodies specific for different areas of the protein detected the numerous cleavage products that resulted from pevonedistat+TNF-α (Figure 3c). Knockdown of caspase-8 expression with siRNA was optimized with single oligonucleotides (Figure 3d). Compared with control cells treated with pevonedistat+TNF-α (~1% viability), the caspase-8-A siRNA knockdown cells tolerated the treatment (84% viability) over 48 h (Figure 3e). These results clearly demonstrate that caspase-8 mediates the synergistic cytotoxicity of pevonedistat+TNF-α.


The NAE inhibitor pevonedistat (MLN4924) synergizes with TNF- α to activate apoptosis
Pevonedistat+TNF-α cytotoxicity is mediated by caspase-8. (a) H-4-II-E cells were treated with 1 or 10 μM pevonedistat±TNF-α for 16 h. Extracts were western blotted for the pro-enzyme form of the indicated caspases. (b) The schematic of the individual subunits of pro-caspase-8 (p24, p18, and p10) are as indicated. (c) Lysates from cells treated with 1 or 10 μM pevonedistat±TNF-for 8 h were western blotted with antibodies specific for epitopes within the caspase-8 p10 (top) or p18 subunits. The predicted caspase-8 subunits are indicated to the left of the image based on the expected size of the product. (d) Lysates from cells transfected with siRNA oligonucleotides against either a non-targeting control or against caspase-8 were western blotted for full-length caspase-8. (e) Cells were transfected with either a non-targeting control or the caspase-8-A siRNA. Four days later, cells received the indicated treatments and viability was assessed after an additional 48 h. All viability experiments were performed in triplicate, and error bars indicate±S.E.M. Approximate molecular sizes of proteins (in kDa) are given to the right of blots.
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Related In: Results  -  Collection

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fig3: Pevonedistat+TNF-α cytotoxicity is mediated by caspase-8. (a) H-4-II-E cells were treated with 1 or 10 μM pevonedistat±TNF-α for 16 h. Extracts were western blotted for the pro-enzyme form of the indicated caspases. (b) The schematic of the individual subunits of pro-caspase-8 (p24, p18, and p10) are as indicated. (c) Lysates from cells treated with 1 or 10 μM pevonedistat±TNF-for 8 h were western blotted with antibodies specific for epitopes within the caspase-8 p10 (top) or p18 subunits. The predicted caspase-8 subunits are indicated to the left of the image based on the expected size of the product. (d) Lysates from cells transfected with siRNA oligonucleotides against either a non-targeting control or against caspase-8 were western blotted for full-length caspase-8. (e) Cells were transfected with either a non-targeting control or the caspase-8-A siRNA. Four days later, cells received the indicated treatments and viability was assessed after an additional 48 h. All viability experiments were performed in triplicate, and error bars indicate±S.E.M. Approximate molecular sizes of proteins (in kDa) are given to the right of blots.
Mentions: Pro-caspase-8, and to a lesser extent pro-caspase-3, was cleaved/activated in cells treated with pevonedistat+TNF-α (Figure 3a). Pro-caspase-8 is comprised of three domains (Figure 3b), but only p18 and p10 are proteases.21 Two caspase-8 antibodies specific for different areas of the protein detected the numerous cleavage products that resulted from pevonedistat+TNF-α (Figure 3c). Knockdown of caspase-8 expression with siRNA was optimized with single oligonucleotides (Figure 3d). Compared with control cells treated with pevonedistat+TNF-α (~1% viability), the caspase-8-A siRNA knockdown cells tolerated the treatment (84% viability) over 48 h (Figure 3e). These results clearly demonstrate that caspase-8 mediates the synergistic cytotoxicity of pevonedistat+TNF-α.

View Article: PubMed Central - PubMed

ABSTRACT

Predicting and understanding the mechanism of drug-induced toxicity is one of the primary goals of drug development. It has been hypothesized that inflammation may have a synergistic role in this process. Cell-based models provide an easily manipulated system to investigate this type of drug toxicity. Several groups have attempted to reproduce in vivo toxicity with combination treatment of pharmacological agents and inflammatory cytokines. Through this approach, synergistic cytotoxicity between the investigational agent pevonedistat (MLN4924) and TNF-α was identified. Pevonedistat is an inhibitor of the NEDD8-activating enzyme (NAE). Inhibition of NAE prevents activation of cullin-RING ligases, which are critical for proteasome-mediated protein degradation. TNF-α is a cytokine that is involved in inflammatory responses and cell death, among other biological functions. Treatment of cultured cells with the combination of pevonedistat and TNF-α, but not as single agents, resulted in rapid cell death. This cell death was determined to be mediated by caspase-8. Interestingly, the combination treatment of pevonedistat and TNF-α also caused an accumulation of the p10 protease subunit of caspase-8 that was not observed with cytotoxic doses of TNF-α. Under conditions where apoptosis was blocked, the mechanism of death switched to necroptosis. Trimerized MLKL was verified as a biomarker of necroptotic cell death. The synergistic toxicity of pevonedistat and elevated TNF-α was also demonstrated by in vivo rat studies. Only the combination treatment resulted in elevated serum markers of liver damage and single-cell hepatocyte necrosis. Taken together, the results of this work have characterized a novel synergistic toxicity driven by pevonedistat and TNF-α.

No MeSH data available.