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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 is cytotoxic. (a) Cultured rat H-4-II-E cells were treated with pevonedistat in combination with either PBS (gray boxes) or 5 ng/ml TNF-α (black triangles) for 24 h. The LC50 was determined using the least-squares method, and solid lines indicate a non-linear fit of the data. (b) Cells were treated with DMSO (−) or pevonedistat (0.1, 1.0, and 10 μM) for 8 h. Lysates were western blotted for the indicated proteins. NEDD8-cullin (arrowhead) and unbound NEDD8 (arrow) are indicated (c). Viabilities were determined at the indicated time points after treatment with DMSO+5 ng/ml TNF-α (black circles), 10 μM pevonedistat+PBS (gray boxes), or 10 μM pevonedistat+5 ng/ml TNF-α (black triangles). (d) Lysates of cells treated with 1 or 10 μM of pevonedistat±TNF-α for 8 h were western blotted for the indicated apoptotic marker proteins. (e) TUNEL (terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labeling; upper) and DAPI (4,6-diamidino-2-phenylindole; lower) staining of apoptotic cells after 6 h of treatment. 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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fig1: Pevonedistat+TNF-α is cytotoxic. (a) Cultured rat H-4-II-E cells were treated with pevonedistat in combination with either PBS (gray boxes) or 5 ng/ml TNF-α (black triangles) for 24 h. The LC50 was determined using the least-squares method, and solid lines indicate a non-linear fit of the data. (b) Cells were treated with DMSO (−) or pevonedistat (0.1, 1.0, and 10 μM) for 8 h. Lysates were western blotted for the indicated proteins. NEDD8-cullin (arrowhead) and unbound NEDD8 (arrow) are indicated (c). Viabilities were determined at the indicated time points after treatment with DMSO+5 ng/ml TNF-α (black circles), 10 μM pevonedistat+PBS (gray boxes), or 10 μM pevonedistat+5 ng/ml TNF-α (black triangles). (d) Lysates of cells treated with 1 or 10 μM of pevonedistat±TNF-α for 8 h were western blotted for the indicated apoptotic marker proteins. (e) TUNEL (terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labeling; upper) and DAPI (4,6-diamidino-2-phenylindole; lower) staining of apoptotic cells after 6 h of treatment. 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: A synergistic cytotoxicity was identified between pevonedistat and TNF-α in the rat hepatoma H-4-II-E cell line. Comparison of the lethality for 50% of cells (LC50) indicated the combination of pevonedistat+TNF-α was approximately 300-fold more toxic than single-agent pevonedistat (Figure 1a). Knockdown of NEDD8 expression with siRNA, which mimicked the inhibitory effect of pevonedistat, also sensitized cells to TNF-α (Supplementary Figure S1). Western blotting of lysates from H-4-II-E cells treated with 10 μM pevonedistat indicated the disappearance of a band corresponding to NEDD8-cullin (Figure 1b, arrowhead) with concurrent buildup of unbound NEDD8 (arrow). This concentration of pevonedistat also resulted in the accumulation of CRL substrate phospho-IκBα, consistent with previous findings,32 and did not affect the expression of total IκBα. Unless otherwise indicated, the concentrations of 10 μM pevonedistat and 5 ng/ml TNF-α were used in subsequent in vitro experiments. Pevonedistat+TNF-α resulted in a rapid cell death, killing ~95% of cells within 16 h (Figure 1c). Three apoptosis markers (cleaved caspase-3, PARP, and BID) were only cleaved after the combination treatment (Figure 1d). Consistent with this result, a TUNEL assay for nicked DNA confirmed that the pevonedistat+TNF-α treatment resulted in more stained cells than any single-agent treatment (Figure 1e). The pevonedistat+TNF-α synergistic cytotoxicity was replicated in a diverse set of other cell types, including: primary rat hepatocytes and liver Kupffer cells; the rat proximal tubule line NRK-52E; the human acute monocytic leukemia line THP-1; and the human hepatocellular carcinoma line HEP-G2 (Supplementary Figure S2). Of note, THP-1 cells were sensitive to pevonedistat in combination with either the human TNF-α cytokine or with an agonist antibody to human TNF-R (Supplementary Figures S2d and e).


The NAE inhibitor pevonedistat (MLN4924) synergizes with TNF- α to activate apoptosis
Pevonedistat+TNF-α is cytotoxic. (a) Cultured rat H-4-II-E cells were treated with pevonedistat in combination with either PBS (gray boxes) or 5 ng/ml TNF-α (black triangles) for 24 h. The LC50 was determined using the least-squares method, and solid lines indicate a non-linear fit of the data. (b) Cells were treated with DMSO (−) or pevonedistat (0.1, 1.0, and 10 μM) for 8 h. Lysates were western blotted for the indicated proteins. NEDD8-cullin (arrowhead) and unbound NEDD8 (arrow) are indicated (c). Viabilities were determined at the indicated time points after treatment with DMSO+5 ng/ml TNF-α (black circles), 10 μM pevonedistat+PBS (gray boxes), or 10 μM pevonedistat+5 ng/ml TNF-α (black triangles). (d) Lysates of cells treated with 1 or 10 μM of pevonedistat±TNF-α for 8 h were western blotted for the indicated apoptotic marker proteins. (e) TUNEL (terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labeling; upper) and DAPI (4,6-diamidino-2-phenylindole; lower) staining of apoptotic cells after 6 h of treatment. 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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fig1: Pevonedistat+TNF-α is cytotoxic. (a) Cultured rat H-4-II-E cells were treated with pevonedistat in combination with either PBS (gray boxes) or 5 ng/ml TNF-α (black triangles) for 24 h. The LC50 was determined using the least-squares method, and solid lines indicate a non-linear fit of the data. (b) Cells were treated with DMSO (−) or pevonedistat (0.1, 1.0, and 10 μM) for 8 h. Lysates were western blotted for the indicated proteins. NEDD8-cullin (arrowhead) and unbound NEDD8 (arrow) are indicated (c). Viabilities were determined at the indicated time points after treatment with DMSO+5 ng/ml TNF-α (black circles), 10 μM pevonedistat+PBS (gray boxes), or 10 μM pevonedistat+5 ng/ml TNF-α (black triangles). (d) Lysates of cells treated with 1 or 10 μM of pevonedistat±TNF-α for 8 h were western blotted for the indicated apoptotic marker proteins. (e) TUNEL (terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labeling; upper) and DAPI (4,6-diamidino-2-phenylindole; lower) staining of apoptotic cells after 6 h of treatment. 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: A synergistic cytotoxicity was identified between pevonedistat and TNF-α in the rat hepatoma H-4-II-E cell line. Comparison of the lethality for 50% of cells (LC50) indicated the combination of pevonedistat+TNF-α was approximately 300-fold more toxic than single-agent pevonedistat (Figure 1a). Knockdown of NEDD8 expression with siRNA, which mimicked the inhibitory effect of pevonedistat, also sensitized cells to TNF-α (Supplementary Figure S1). Western blotting of lysates from H-4-II-E cells treated with 10 μM pevonedistat indicated the disappearance of a band corresponding to NEDD8-cullin (Figure 1b, arrowhead) with concurrent buildup of unbound NEDD8 (arrow). This concentration of pevonedistat also resulted in the accumulation of CRL substrate phospho-IκBα, consistent with previous findings,32 and did not affect the expression of total IκBα. Unless otherwise indicated, the concentrations of 10 μM pevonedistat and 5 ng/ml TNF-α were used in subsequent in vitro experiments. Pevonedistat+TNF-α resulted in a rapid cell death, killing ~95% of cells within 16 h (Figure 1c). Three apoptosis markers (cleaved caspase-3, PARP, and BID) were only cleaved after the combination treatment (Figure 1d). Consistent with this result, a TUNEL assay for nicked DNA confirmed that the pevonedistat+TNF-α treatment resulted in more stained cells than any single-agent treatment (Figure 1e). The pevonedistat+TNF-α synergistic cytotoxicity was replicated in a diverse set of other cell types, including: primary rat hepatocytes and liver Kupffer cells; the rat proximal tubule line NRK-52E; the human acute monocytic leukemia line THP-1; and the human hepatocellular carcinoma line HEP-G2 (Supplementary Figure S2). Of note, THP-1 cells were sensitive to pevonedistat in combination with either the human TNF-α cytokine or with an agonist antibody to human TNF-R (Supplementary Figures S2d and e).

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.