title: The Nedd8-activating enzyme inhibitor MLN4924 thwarts microenvironment-driven NF-κB activation and induces apoptosis in chronic lymphocytic leukemia B cells.
abstract: BACKGROUND
  Stromal-mediated signaling enhances NF-κB pathway activity in chronic lymphocytic leukemia (CLL) B cells, leading to cell survival and chemoresistance. Ubiquitination of IκBα may partially account for constitutive activation of NF-κB. MLN4924 is an investigational agent that inhibits the Nedd8-activating enzyme, thereby neutralizing Cullin-RING ubiquitin ligases and preventing degradation of their substrates.
  
  
  METHODS
  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-κB 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-κB activation and Bim repression. Treatment with MLN4924 induced CLL cell apoptosis and circumvented stroma-mediated resistance. This was accompanied by accumulation of phospho-IκBα, decreased nuclear translocation of p65 and p52 leading to inhibition of both the canonical and noncanonical NF-κB 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-κB activation and induces Bim expression in CLL cells, thereby preventing stroma-mediated resistance. Our data provide rationale for further evaluation of MLN4924 in CLL.
authors.affiliation: Authors' Affiliations: Departments of Medicine and Pathology, Dartmouth-Hitchcock Medical Center, Lebanon; Department of Pharmacology and Toxicology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire; and Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.
authors.forename: J Claire
authors.initials: JC
authors.lastname: Godbersen
authors.forename: Leigh Ann
authors.initials: LA
authors.lastname: Humphries
authors.forename: Olga V
authors.initials: OV
authors.lastname: Danilova
authors.forename: Peter E
authors.initials: PE
authors.lastname: Kebbekus
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Brown
authors.forename: Alan
authors.initials: A
authors.lastname: Eastman
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Danilov
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001402:B-Lymphocytes / Q000187:drug effects*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003517:Cyclopentanes / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D011743:Pyrimidines / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D059016:Tumor Microenvironment / Q000187:drug effects; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors*
chemical_list: D000970:Antineoplastic Agents; D003517:Cyclopentanes; D004791:Enzyme Inhibitors; D016328:NF-kappa B; D011743:Pyrimidines; D044764:Ubiquitin-Activating Enzymes; C491802:NAE protein, human; C539933:pevonedistat