title: Synergistic apoptosis induction in leukemic cells by the phosphatase inhibitor salubrinal and proteasome inhibitors.
abstract: BACKGROUND
  Cells adapt to endoplasmic reticulum (ER)-stress by arresting global protein synthesis while simultaneously activating specific transcription factors and their downstream targets. These processes are mediated in part by the phosphorylation-dependent inactivation of the translation initiation factor eIF2alpha. Following restoration of homeostasis protein synthesis is resumed when the serine/threonine-protein phosphatase PP1 dephosphorylates and reactivates eIF2alpha. Proteasome inhibitors, used to treat multiple myeloma patients evoke ER-stress and apoptosis by blocking the ER-associated degradation of misfolded proteins (ERAD), however, the role of eIF2alpha phosphorylation in leukemic cells under conditions of proteasome inhibitor-mediated ER stress is currently unclear.
  
  
  RESULTS
  Bcr-Abl-positive and negative leukemic cell lines were used to investigate the functional implications of PP1-related phosphatase activities on eIF2alpha phosphorylation in proteasome inhibitor-mediated ER stress and apoptosis. Rather unexpectedly, salubrinal, a recently identified PP1 inhibitor capable to protect against ER stress in various model systems, strongly synergized with proteasome inhibitors to augment apoptotic death of different leukemic cell lines. Salubrinal treatment did not affect the phosphorlyation status of eIF2alpha. Furthermore, the proapoptotic effect of salubrinal occurred independently from the chemical nature of the proteasome inhibitor, was recapitulated by a second unrelated phosphatase inhibitor and was unaffected by overexpression of a dominant negative eIF2alpha S51A variant that can not be phosphorylated. Salubrinal further aggravated ER-stress and proteotoxicity inflicted by the proteasome inhibitors on the leukemic cells since characteristic ER stress responses, such as ATF4 and CHOP synthesis, XBP1 splicing, activation of MAP kinases and eventually apoptosis were efficiently abrogated by the translational inhibitor cycloheximide.
  
  
  CONCLUSIONS
  Although PP1 activity does not play a major role in regulating the ER stress response in leukemic cells, phosphatase signaling nevertheless significantly limits proteasome inhibitor-mediated ER-stress and apoptosis. Inclusion of specific phosphatase inhibitors might therefore represent an option to improve current proteasome inhibitor-based treatment modalities for hematological cancers.
authors.affiliation: Department of Vascular Cell Biology, Max Planck Institute for Molecular Biomedicine, Muenster, Germany. hannes.drexler@mpi-muenster.mpg.de
authors.forename: Hannes C A
authors.initials: HC
authors.lastname: Drexler
mesh_terms: D051701:Activating Transcription Factor 4 / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002934:Cinnamates / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004357:Drug Synergism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016313:Genes, abl; D006801:Humans; D020014:K562 Cells; D007938:Leukemia / Q000188:drug therapy*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D019284:Thapsigargin / Q000633:toxicity; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D051742:Transcription Factor CHOP / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D000071717:X-Box Binding Protein 1
chemical_list: C497109:ATF4 protein, human; D002934:Cinnamates; C497249:DDIT3 protein, human; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D061988:Proteasome Inhibitors; D012333:RNA, Messenger; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C000605543:XBP1 protein, human; C496827:salubrinal; D051701:Activating Transcription Factor 4; D051742:Transcription Factor CHOP; D019284:Thapsigargin; C517189:PPP2CA protein, human; D054648:Protein Phosphatase 2; D010744:Phosphoric Monoester Hydrolases; D046988:Proteasome Endopeptidase Complex; D013890:Thiourea