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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.


Related in: MedlinePlus

Combination of pevonedistat and elevated TNF-α is toxic to rats. Sprague-Dawley rats (n=8) were administered single doses of vehicle, TNF-α, pevonedistat, or pevonedistat+TNF-α. Scheduled necropsy occurred 24 h later. (a) Representative microscopic images of H&E-stained livers from animals that received the indicated treatments: hepatocyte karyomegaly (white arrowhead), single-cell necrosis (black arrowhead), and neutrophilic infiltration (white arrow). (b) Rats were administered single doses of the indicated compounds; markers of liver injury were analyzed approximately 24 h later. Serum was available for only six animals that received pevonedistat+TNF-α. The group-mean concentrations ALT, AST, and SDH are indicated. Asterisks indicate statistically significant (P<0.01) differences. (c) Livers from 20 rats that received the indicated compounds were western blotted for caspase-8 and cFLIP-L expression. Full-length pro-caspase-8 (arrow) and the cleaved p32 subunit (arrowhead) are indicated. Approximate molecular sizes of proteins (in kDa) are given to the right of blots. (d) Tissue liver extracts from the same 20 rats were used in a caspase-8 activity assay. Each sample was analyzed in triplicate, values averaged, and normalized against the group mean value for vehicle control. Error bars indicate±S.E.M.
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fig6: Combination of pevonedistat and elevated TNF-α is toxic to rats. Sprague-Dawley rats (n=8) were administered single doses of vehicle, TNF-α, pevonedistat, or pevonedistat+TNF-α. Scheduled necropsy occurred 24 h later. (a) Representative microscopic images of H&E-stained livers from animals that received the indicated treatments: hepatocyte karyomegaly (white arrowhead), single-cell necrosis (black arrowhead), and neutrophilic infiltration (white arrow). (b) Rats were administered single doses of the indicated compounds; markers of liver injury were analyzed approximately 24 h later. Serum was available for only six animals that received pevonedistat+TNF-α. The group-mean concentrations ALT, AST, and SDH are indicated. Asterisks indicate statistically significant (P<0.01) differences. (c) Livers from 20 rats that received the indicated compounds were western blotted for caspase-8 and cFLIP-L expression. Full-length pro-caspase-8 (arrow) and the cleaved p32 subunit (arrowhead) are indicated. Approximate molecular sizes of proteins (in kDa) are given to the right of blots. (d) Tissue liver extracts from the same 20 rats were used in a caspase-8 activity assay. Each sample was analyzed in triplicate, values averaged, and normalized against the group mean value for vehicle control. Error bars indicate±S.E.M.

Mentions: The in vivo effects of pevonedistat and TNF-α were assessed in Sprague-Dawley rats. The dose of pevonedistat administered to rats was known from previous investigations to be well tolerated, and the dose of recombinant rat TNF-α activated TNF signaling without toxic side effects.4 Animals within each group (n=8) first received either vehicle or 10 μg/kg TNF-α, followed by either a second vehicle or 120 mg/kg pevonedistat 1 h later. Two animals dosed with the combination treatment exhibited moribund conditions and were euthanized within 10 h. There was a clear difference in liver damage of single-agent versus combination treatments in rats. The incidence and severity of microscopic liver findings for five representative animals from each dose group are presented in Table 1. The livers of animals dosed with pevonedistat+TNF-α had minimal-to-mild single-cell necrosis and neutrophilic infiltration. Representative histological images in Figure 6a illustrate karyomegaly (white arrowhead) in the livers from animals that received pevonedistat alone and necrosis (black arrowhead) and neutrophilic infiltrate (white arrow) in the combination-treated livers. Animals that received the combination treatment had significant ~5-fold elevation of the serum markers alanine transaminase (ALT), aspartate transaminase (AST) and sorbitol dehydrogenase (SDH) compared with those that received single-agent treatments (Figure 6b). Western blotting of liver extracts identified uncleaved caspase-8 (Figure 6c, arrow) in all animals and the p32 fragment of caspase-8 was observed in 9/10 animals that received pevonedistat±TNF-α (arrowhead). Neither p10 nor p18 (data not shown) were detected. Staining of the cleaved cFLIP-L 43-kDa fragment was strongest in samples that also had caspase-8 cleavage. There was a 4-fold elevation of caspase-8 activity in the pevonedistat±TNF-α groups compared with vehicle (Figure 6d). Whether caspase-8 activation was the principle driver of toxicity in rats could not be established.


The NAE inhibitor pevonedistat (MLN4924) synergizes with TNF- α to activate apoptosis
Combination of pevonedistat and elevated TNF-α is toxic to rats. Sprague-Dawley rats (n=8) were administered single doses of vehicle, TNF-α, pevonedistat, or pevonedistat+TNF-α. Scheduled necropsy occurred 24 h later. (a) Representative microscopic images of H&E-stained livers from animals that received the indicated treatments: hepatocyte karyomegaly (white arrowhead), single-cell necrosis (black arrowhead), and neutrophilic infiltration (white arrow). (b) Rats were administered single doses of the indicated compounds; markers of liver injury were analyzed approximately 24 h later. Serum was available for only six animals that received pevonedistat+TNF-α. The group-mean concentrations ALT, AST, and SDH are indicated. Asterisks indicate statistically significant (P<0.01) differences. (c) Livers from 20 rats that received the indicated compounds were western blotted for caspase-8 and cFLIP-L expression. Full-length pro-caspase-8 (arrow) and the cleaved p32 subunit (arrowhead) are indicated. Approximate molecular sizes of proteins (in kDa) are given to the right of blots. (d) Tissue liver extracts from the same 20 rats were used in a caspase-8 activity assay. Each sample was analyzed in triplicate, values averaged, and normalized against the group mean value for vehicle control. Error bars indicate±S.E.M.
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fig6: Combination of pevonedistat and elevated TNF-α is toxic to rats. Sprague-Dawley rats (n=8) were administered single doses of vehicle, TNF-α, pevonedistat, or pevonedistat+TNF-α. Scheduled necropsy occurred 24 h later. (a) Representative microscopic images of H&E-stained livers from animals that received the indicated treatments: hepatocyte karyomegaly (white arrowhead), single-cell necrosis (black arrowhead), and neutrophilic infiltration (white arrow). (b) Rats were administered single doses of the indicated compounds; markers of liver injury were analyzed approximately 24 h later. Serum was available for only six animals that received pevonedistat+TNF-α. The group-mean concentrations ALT, AST, and SDH are indicated. Asterisks indicate statistically significant (P<0.01) differences. (c) Livers from 20 rats that received the indicated compounds were western blotted for caspase-8 and cFLIP-L expression. Full-length pro-caspase-8 (arrow) and the cleaved p32 subunit (arrowhead) are indicated. Approximate molecular sizes of proteins (in kDa) are given to the right of blots. (d) Tissue liver extracts from the same 20 rats were used in a caspase-8 activity assay. Each sample was analyzed in triplicate, values averaged, and normalized against the group mean value for vehicle control. Error bars indicate±S.E.M.
Mentions: The in vivo effects of pevonedistat and TNF-α were assessed in Sprague-Dawley rats. The dose of pevonedistat administered to rats was known from previous investigations to be well tolerated, and the dose of recombinant rat TNF-α activated TNF signaling without toxic side effects.4 Animals within each group (n=8) first received either vehicle or 10 μg/kg TNF-α, followed by either a second vehicle or 120 mg/kg pevonedistat 1 h later. Two animals dosed with the combination treatment exhibited moribund conditions and were euthanized within 10 h. There was a clear difference in liver damage of single-agent versus combination treatments in rats. The incidence and severity of microscopic liver findings for five representative animals from each dose group are presented in Table 1. The livers of animals dosed with pevonedistat+TNF-α had minimal-to-mild single-cell necrosis and neutrophilic infiltration. Representative histological images in Figure 6a illustrate karyomegaly (white arrowhead) in the livers from animals that received pevonedistat alone and necrosis (black arrowhead) and neutrophilic infiltrate (white arrow) in the combination-treated livers. Animals that received the combination treatment had significant ~5-fold elevation of the serum markers alanine transaminase (ALT), aspartate transaminase (AST) and sorbitol dehydrogenase (SDH) compared with those that received single-agent treatments (Figure 6b). Western blotting of liver extracts identified uncleaved caspase-8 (Figure 6c, arrow) in all animals and the p32 fragment of caspase-8 was observed in 9/10 animals that received pevonedistat±TNF-α (arrowhead). Neither p10 nor p18 (data not shown) were detected. Staining of the cleaved cFLIP-L 43-kDa fragment was strongest in samples that also had caspase-8 cleavage. There was a 4-fold elevation of caspase-8 activity in the pevonedistat±TNF-α groups compared with vehicle (Figure 6d). Whether caspase-8 activation was the principle driver of toxicity in rats could not be established.

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-&alpha; 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-&alpha; 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-&alpha;, 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-&alpha; also caused an accumulation of the p10 protease subunit of caspase-8 that was not observed with cytotoxic doses of TNF-&alpha;. 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-&alpha; 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-&alpha;.

No MeSH data available.


Related in: MedlinePlus