title: Interferon-γ-induced upregulation of immunoproteasome subunit assembly overcomes bortezomib resistance in human hematological cell lines.
abstract: BACKGROUND
  Despite encouraging results with the proteasome inhibitor bortezomib in the treatment of hematologic malignancies, emergence of resistance can limit its efficacy, hence calling for novel strategies to overcome bortezomib-resistance. We previously showed that bortezomib-resistant human leukemia cell lines expressed significantly lower levels of immunoproteasome at the expense of constitutive proteasomes, which harbored point mutations in exon 2 of the PSMB5 gene encoding the β5 subunit. Here we investigated whether up-regulation of immunoproteasomes by exposure to interferon-γ restores sensitivity to bortezomib in myeloma and leukemia cell lines with acquired resistance to bortezomib.
  
  
  METHODS
  RPMI-8226 myeloma, THP1 monocytic/macrophage and CCRF-CEM (T) parental cells and sub lines with acquired resistance to bortezomib were exposed to Interferon-γ for 24-48 h where after the effects on proteasome subunit expression and activity were measured, next to sensitivity measurements to proteasome inhibitors bortezomib, carfilzomib, and the immunoproteasome selective inhibitor ONX 0914. At last, siRNA knockdown experiments of β5i and β1i were performed to identify the contribution of these subunits to sensitivity to proteasome inhibition. Statistical significance of the differences were determined using the Mann-Whitney U test.
  
  
  RESULTS
  Interferon-γ exposure markedly increased immunoproteasome subunit mRNA to a significantly higher level in bortezomib-resistant cells (up to 30-fold, 10-fold, and 6-fold, in β1i, β5i, and β2i, respectively) than in parental cells. These increases were paralleled by elevated immunoproteasome protein levels and catalytic activity, as well as HLA class-I. Moreover, interferon-γ exposure reinforced sensitization of bortezomib-resistant tumor cells to bortezomib and carfilzomib, but most prominently to ONX 0914, as confirmed by cell growth inhibition studies, proteasome inhibitor-induced apoptosis, activation of PARP cleavage and accumulation of polyubiquitinated proteins. This sensitization was abrogated by siRNA silencing of β5i but not by β1i silencing, prior to pulse exposure to interferon-γ.
  
  
  CONCLUSIONS
  Downregulation of β5i subunit expression is a major determinant in acquisition of bortezomib-resistance and enhancement of its proteasomal assembly after induction by interferon-γ facilitates restoration of sensitivity in bortezomib-resistant leukemia cells towards bortezomib and next generation (immuno) proteasome inhibitors.
authors.forename: Denise
authors.initials: D
authors.lastname: Niewerth
authors.forename: Gertjan J L
authors.initials: GJ
authors.lastname: Kaspers
authors.forename: Yehuda G
authors.initials: YG
authors.lastname: Assaraf
authors.forename: Johan
authors.initials: J
authors.lastname: van Meerloo
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Kirk
authors.forename: Janet
authors.initials: J
authors.lastname: Anderl
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Blank
authors.forename: Peter M
authors.initials: PM
authors.lastname: van de Ven
authors.forename: Sonja
authors.initials: S
authors.lastname: Zweegman
authors.forename: Gerrit
authors.initials: G
authors.lastname: Jansen
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands. j.cloos@vumc.nl.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Cloos
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006680:HLA Antigens / Q000235:genetics / Q000378:metabolism; D019337:Hematologic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D061988:Proteasome Inhibitors / Q000494:pharmacology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011719:Pyrazines / Q000494:pharmacology*; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: D001897:Boronic Acids; D006680:HLA Antigens; D015395:Histocompatibility Antigens Class I; D009842:Oligopeptides; C542291:PR-957; D061988:Proteasome Inhibitors; D021122:Protein Subunits; D011719:Pyrazines; D000069286:Bortezomib; C524865:carfilzomib; D007371:Interferon-gamma; D046988:Proteasome Endopeptidase Complex