The Nedd8-Activating enzyme inhibitor MLN4924 thwarts microenvironment- driven NF-kB activation and induces apoptosis in chronic lymphocytic leukemia B cells

J. Claire Godbersen, Leigh Ann Humphries, Olga V. Danilova, Peter E. Kebbekus, Jennifer R. Brown, Alan Eastman, Alexey Danilov

Research output: Contribution to journalArticle

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Abstract

Background: Stromal-mediated signaling enhances NF-kB pathway activity in chronic lymphocytic leukemia (CLL) B cells, leading to cell survival and chemoresistance. Ubiquitination of IkBa may partially account for constitutive activation of NF-kB. MLN4924 is an investigational agent that inhibits the Nedd8- activating enzyme, thereby neutralizing Cullin-RING ubiquitin ligases and preventing degradation of their substrates. Experimental Design: We conducted a preclinical assessment of MLN4924 in CLL. Primary CLL cells were cocultured in vitro with CD40L-expressing stroma to mimic the prosurvival conditions present in lymphoid tissue. The effect of MLN4924 on CLL cell apoptosis, NF-kB pathway activity, Bcl-2 family members, and cell cycle was assessed by flow cytometry, Western blotting, PCR, and immunocytochemistry. Results: CD40L-expressing stroma protected CLL cells from spontaneous apoptosis and induced resistance to multiple drugs, accompanied by NF-kB activation and Bim repression. Treatment with MLN4924 induced CLL cell apoptosis and circumvented stroma-mediated resistance. This was accompanied by accumulation of phospho-IkBa, decreased nuclear translocation of p65 and p52 leading to inhibition of both the canonical and noncanonical NF-kB pathways, and reduced transcription of their target genes, notably chemokines. MLN4924 promoted induction of Bim and Noxa in the CLL cells leading to rebalancing of Bcl-2 family members toward the proapoptotic BH3-only proteins. siRNA-mediated knockdown of Bim or Noxa decreased sensitivity to MLN4924. MLN4924 enhanced the antitumor activity of the inhibitors of B-cell receptor (BCR)-Associated kinases. Conclusions: MLN4924 disrupts NF-kB activation and induces Bim expression in CLL cells, thereby preventing stroma-mediated resistance. Our data provide rationale for further evaluation of MLN4924 in CLL.

Original languageEnglish (US)
Pages (from-to)1576-1589
Number of pages14
JournalClinical Cancer Research
Volume20
Issue number6
DOIs
StatePublished - 2014
Externally publishedYes

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NF-kappa B
Enzyme Inhibitors
B-Cell Chronic Lymphocytic Leukemia
Apoptosis
Noxae
CD40 Ligand
Cullin Proteins
((1S,2S,4R)-4-(4-((1S)-2,3-dihydro-1H-inden-1-ylamino)-7H-pyrrolo(2,3-d)pyrimidin-7-yl)-2-hydroxycyclopentyl)methyl sulphamate
Ubiquitination
Multiple Drug Resistance
Lymphoid Tissue
Ligases
Ubiquitin
Chemokines
Small Interfering RNA
Cell Survival
Cell Cycle
Flow Cytometry
B-Lymphocytes
Research Design

ASJC Scopus subject areas

  • Cancer Research
  • Oncology
  • Medicine(all)

Cite this

The Nedd8-Activating enzyme inhibitor MLN4924 thwarts microenvironment- driven NF-kB activation and induces apoptosis in chronic lymphocytic leukemia B cells. / Godbersen, J. Claire; Humphries, Leigh Ann; Danilova, Olga V.; Kebbekus, Peter E.; Brown, Jennifer R.; Eastman, Alan; Danilov, Alexey.

In: Clinical Cancer Research, Vol. 20, No. 6, 2014, p. 1576-1589.

Research output: Contribution to journalArticle

Godbersen, J. Claire ; Humphries, Leigh Ann ; Danilova, Olga V. ; Kebbekus, Peter E. ; Brown, Jennifer R. ; Eastman, Alan ; Danilov, Alexey. / The Nedd8-Activating enzyme inhibitor MLN4924 thwarts microenvironment- driven NF-kB activation and induces apoptosis in chronic lymphocytic leukemia B cells. In: Clinical Cancer Research. 2014 ; Vol. 20, No. 6. pp. 1576-1589.
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abstract = "Background: Stromal-mediated signaling enhances NF-kB pathway activity in chronic lymphocytic leukemia (CLL) B cells, leading to cell survival and chemoresistance. Ubiquitination of IkBa may partially account for constitutive activation of NF-kB. MLN4924 is an investigational agent that inhibits the Nedd8- activating enzyme, thereby neutralizing Cullin-RING ubiquitin ligases and preventing degradation of their substrates. Experimental Design: We conducted a preclinical assessment of MLN4924 in CLL. Primary CLL cells were cocultured in vitro with CD40L-expressing stroma to mimic the prosurvival conditions present in lymphoid tissue. The effect of MLN4924 on CLL cell apoptosis, NF-kB pathway activity, Bcl-2 family members, and cell cycle was assessed by flow cytometry, Western blotting, PCR, and immunocytochemistry. Results: CD40L-expressing stroma protected CLL cells from spontaneous apoptosis and induced resistance to multiple drugs, accompanied by NF-kB activation and Bim repression. Treatment with MLN4924 induced CLL cell apoptosis and circumvented stroma-mediated resistance. This was accompanied by accumulation of phospho-IkBa, decreased nuclear translocation of p65 and p52 leading to inhibition of both the canonical and noncanonical NF-kB pathways, and reduced transcription of their target genes, notably chemokines. MLN4924 promoted induction of Bim and Noxa in the CLL cells leading to rebalancing of Bcl-2 family members toward the proapoptotic BH3-only proteins. siRNA-mediated knockdown of Bim or Noxa decreased sensitivity to MLN4924. MLN4924 enhanced the antitumor activity of the inhibitors of B-cell receptor (BCR)-Associated kinases. Conclusions: MLN4924 disrupts NF-kB activation and induces Bim expression in CLL cells, thereby preventing stroma-mediated resistance. Our data provide rationale for further evaluation of MLN4924 in CLL.",
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T1 - The Nedd8-Activating enzyme inhibitor MLN4924 thwarts microenvironment- driven NF-kB activation and induces apoptosis in chronic lymphocytic leukemia B cells

AU - Godbersen, J. Claire

AU - Humphries, Leigh Ann

AU - Danilova, Olga V.

AU - Kebbekus, Peter E.

AU - Brown, Jennifer R.

AU - Eastman, Alan

AU - Danilov, Alexey

PY - 2014

Y1 - 2014

N2 - Background: Stromal-mediated signaling enhances NF-kB pathway activity in chronic lymphocytic leukemia (CLL) B cells, leading to cell survival and chemoresistance. Ubiquitination of IkBa may partially account for constitutive activation of NF-kB. MLN4924 is an investigational agent that inhibits the Nedd8- activating enzyme, thereby neutralizing Cullin-RING ubiquitin ligases and preventing degradation of their substrates. Experimental Design: We conducted a preclinical assessment of MLN4924 in CLL. Primary CLL cells were cocultured in vitro with CD40L-expressing stroma to mimic the prosurvival conditions present in lymphoid tissue. The effect of MLN4924 on CLL cell apoptosis, NF-kB pathway activity, Bcl-2 family members, and cell cycle was assessed by flow cytometry, Western blotting, PCR, and immunocytochemistry. Results: CD40L-expressing stroma protected CLL cells from spontaneous apoptosis and induced resistance to multiple drugs, accompanied by NF-kB activation and Bim repression. Treatment with MLN4924 induced CLL cell apoptosis and circumvented stroma-mediated resistance. This was accompanied by accumulation of phospho-IkBa, decreased nuclear translocation of p65 and p52 leading to inhibition of both the canonical and noncanonical NF-kB pathways, and reduced transcription of their target genes, notably chemokines. MLN4924 promoted induction of Bim and Noxa in the CLL cells leading to rebalancing of Bcl-2 family members toward the proapoptotic BH3-only proteins. siRNA-mediated knockdown of Bim or Noxa decreased sensitivity to MLN4924. MLN4924 enhanced the antitumor activity of the inhibitors of B-cell receptor (BCR)-Associated kinases. Conclusions: MLN4924 disrupts NF-kB activation and induces Bim expression in CLL cells, thereby preventing stroma-mediated resistance. Our data provide rationale for further evaluation of MLN4924 in CLL.

AB - Background: Stromal-mediated signaling enhances NF-kB pathway activity in chronic lymphocytic leukemia (CLL) B cells, leading to cell survival and chemoresistance. Ubiquitination of IkBa may partially account for constitutive activation of NF-kB. MLN4924 is an investigational agent that inhibits the Nedd8- activating enzyme, thereby neutralizing Cullin-RING ubiquitin ligases and preventing degradation of their substrates. Experimental Design: We conducted a preclinical assessment of MLN4924 in CLL. Primary CLL cells were cocultured in vitro with CD40L-expressing stroma to mimic the prosurvival conditions present in lymphoid tissue. The effect of MLN4924 on CLL cell apoptosis, NF-kB pathway activity, Bcl-2 family members, and cell cycle was assessed by flow cytometry, Western blotting, PCR, and immunocytochemistry. Results: CD40L-expressing stroma protected CLL cells from spontaneous apoptosis and induced resistance to multiple drugs, accompanied by NF-kB activation and Bim repression. Treatment with MLN4924 induced CLL cell apoptosis and circumvented stroma-mediated resistance. This was accompanied by accumulation of phospho-IkBa, decreased nuclear translocation of p65 and p52 leading to inhibition of both the canonical and noncanonical NF-kB pathways, and reduced transcription of their target genes, notably chemokines. MLN4924 promoted induction of Bim and Noxa in the CLL cells leading to rebalancing of Bcl-2 family members toward the proapoptotic BH3-only proteins. siRNA-mediated knockdown of Bim or Noxa decreased sensitivity to MLN4924. MLN4924 enhanced the antitumor activity of the inhibitors of B-cell receptor (BCR)-Associated kinases. Conclusions: MLN4924 disrupts NF-kB activation and induces Bim expression in CLL cells, thereby preventing stroma-mediated resistance. Our data provide rationale for further evaluation of MLN4924 in CLL.

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