title: Identification of markers that functionally define a quiescent multiple myeloma cell sub-population surviving bortezomib treatment.
abstract: BACKGROUND
  The mechanisms allowing residual multiple myeloma (MM) cells to persist after bortezomib (Bz) treatment remain unclear. We hypothesized that studying the biology of bortezomib-surviving cells may reveal markers to identify these cells and survival signals to target and kill residual MM cells.
  
  
  METHODS
  We used H2B-GFP label retention, biochemical tools and in vitro and in vivo experiments to characterize growth arrest and the unfolded protein responses in quiescent Bz-surviving cells. We also tested the effect of a demethylating agent, 5-Azacytidine, on Bz-induced quiescence and whether inhibiting the chaperone GRP78/BiP (henceforth GRP78) with a specific toxin induced apoptosis in Bz-surviving cells. Finally, we used MM patient samples to test whether GRP78 levels might associate with disease progression. Statistical analysis employed t-test and Mann-Whitney tests at a 95% confidence.
  
  
  RESULTS
  We report that Bz-surviving MM cells in vitro and in vivo enter quiescence characterized by p21(CIP1) upregulation. Bz-surviving MM cells also downregulated CDK6, Ki67 and P-Rb. H2B-GFP label retention showed that Bz-surviving MM cells are either slow-cycling or deeply quiescent. The Bz-induced quiescence was stabilized by low dose (500nM) of 5-azacytidine (Aza) pre-treatment, which also potentiated the initial Bz-induced apoptosis. We also found that expression of GRP78, an unfolded protein response (UPR) survival factor, persisted in MM quiescent cells. Importantly, GRP78 downregulation using a specific SubAB bacterial toxin killed Bz-surviving MM cells. Finally, quantification of Grp78(high)/CD138+ MM cells from patients suggested that high levels correlated with progressive disease.
  
  
  CONCLUSIONS
  We conclude that Bz-surviving MM cells display a GRP78(HIGH)/p21(HIGH)/CDK6(LOW)/P-Rb(LOW) profile, and these markers may identify quiescent MM cells capable of fueling recurrences. We further conclude that Aza + Bz treatment of MM may represent a novel strategy to delay recurrences by enhancing Bz-induced apoptosis and quiescence stability.
authors.affiliation: Division of Hematology and Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, NY, 10029, USA. alfred.adomako@mssm.edu.
authors.forename: Alfred
authors.initials: A
authors.lastname: Adomako
authors.affiliation: Division of Hematology and Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, NY, 10029, USA. veronica.calvovidal@mssm.edu.
authors.forename: Veronica
authors.initials: V
authors.lastname: Calvo
authors.affiliation: Division of Hematology and Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, NY, 10029, USA. noa.biran@mssm.edu.|Tisch Cancer Institute, Mount Sinai School of Medicine, New York, NY, 10029, USA. noa.biran@mssm.edu.
authors.forename: Noa
authors.initials: N
authors.lastname: Biran
authors.affiliation: Division of Hematology and Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, NY, 10029, USA. keren.osman@mssm.edu.|Tisch Cancer Institute, Mount Sinai School of Medicine, New York, NY, 10029, USA. keren.osman@mssm.edu.
authors.forename: Keren
authors.initials: K
authors.lastname: Osman
authors.affiliation: Division of Hematology and Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, NY, 10029, USA. ajai.chari@mssm.edu.|Tisch Cancer Institute, Mount Sinai School of Medicine, New York, NY, 10029, USA. ajai.chari@mssm.edu.
authors.forename: Ajai
authors.initials: A
authors.lastname: Chari
authors.affiliation: Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, 5005, Australia. james.paton@adelaide.edu.au.
authors.forename: James C
authors.initials: JC
authors.lastname: Paton
authors.affiliation: Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, 5005, Australia. adrienne.paton@adelaide.edu.au.
authors.forename: Adrienne W
authors.initials: AW
authors.lastname: Paton
authors.affiliation: Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, NY, 10029, USA. kateri.moore@mssm.edu.
authors.forename: Kateri
authors.initials: K
authors.lastname: Moore
authors.affiliation: Division of Hematology and Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, NY, 10029, USA. denis.Schewe@uksh.de.
authors.forename: Denis M
authors.initials: DM
authors.lastname: Schewe
authors.affiliation: Division of Hematology and Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, NY, 10029, USA. julio.aguirre-ghiso@mssm.edu.|Department of Otolaryngology, Mount Sinai School of Medicine, New York, NY, 10029, USA. julio.aguirre-ghiso@mssm.edu.|Tisch Cancer Institute, Mount Sinai School of Medicine, New York, NY, 10029, USA. julio.aguirre-ghiso@mssm.edu.|Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, NY, 10029, USA. julio.aguirre-ghiso@mssm.edu.
authors.forename: Julio A
authors.initials: JA
authors.lastname: Aguirre-Ghiso
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001374:Azacitidine / Q000008:administration & dosage; D000069286:Bortezomib / Q000008:administration & dosage*; D002470:Cell Survival / Q000187:drug effects; D051361:Cyclin-Dependent Kinase 6 / Q000096:biosynthesis* / Q000235:genetics; D000091342:Endoplasmic Reticulum Chaperone BiP; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D008875:Middle Aged; D009101:Multiple Myeloma / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D009364:Neoplasm Recurrence, Local / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D023041:Xenograft Model Antitumor Assays; D054462:p21-Activated Kinases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D000069286:Bortezomib; C515907:PAK2 protein, human; D054462:p21-Activated Kinases; C495911:CDK6 protein, human; D051361:Cyclin-Dependent Kinase 6; D001374:Azacitidine