title: The HDAC Inhibitors Scriptaid and LBH589 Combined with the Oncolytic Virus Delta24-RGD Exert Enhanced Anti-Tumor Efficacy in Patient-Derived Glioblastoma Cells.
abstract: BACKGROUND
  A phase I/II trial for glioblastoma with the oncolytic adenovirus Delta24-RGD was recently completed. Delta24-RGD conditionally replicates in cells with a disrupted retinoblastoma-pathway and enters cells via αvβ3/5 integrins. Glioblastomas are differentially sensitive to Delta24-RGD. HDAC inhibitors (HDACi) affect integrins and share common cell death pathways with Delta24-RGD. We studied the combination treatment effects of HDACi and Delta24-RGD in patient-derived glioblastoma stem-like cells (GSC), and we determined the most effective HDACi.
  
  
  METHODS
  SAHA, Valproic Acid, Scriptaid, MS275 and LBH589 were combined with Delta24-RGD in fourteen distinct GSCs. Synergy was determined by Chou Talalay method. Viral infection and replication were assessed using luciferase and GFP encoding vectors and hexon-titration assays. Coxsackie adenovirus receptor and αvβ3 integrin levels were determined by flow cytometry. Oncolysis and mechanisms of cell death were studied by viability, caspase-3/7, LDH and LC3B/p62, phospho-p70S6K. Toxicity was studied on normal human astrocytes. MGMT promotor methylation status, TCGA classification, Rb-pathway and integrin gene expression levels were assessed as markers of responsiveness.
  
  
  RESULTS
  Scriptaid and LBH589 acted synergistically with Delta24-RGD in approximately 50% of the GSCs. Both drugs moderately increased αvβ3 integrin levels and viral infection in responding but not in non-responding GSCs. LBH589 moderately increased late viral gene expression, however, virus titration revealed diminished viral progeny production by both HDACi, Scriptaid augmented caspase-3/7 activity, LC3B conversion, p62 and phospho-p70S6K consumption, as well as LDH levels. LBH589 increased LDH and phospho-p70S6K consumption. Responsiveness correlated with expression of various Rb-pathway genes and integrins. Combination treatments induced limited toxicity to human astrocytes.
  
  
  CONCLUSIONS
  LBH589 and Scriptaid combined with Delta24-RGD revealed synergistic anti-tumor activity in a subset of GSCs. Both HDACi moderately augmented viral infection and late gene expression, but slightly reduced progeny production. The drugs differentially activated multiple cell death pathways. The limited toxicity on astrocytes supports further evaluation of the proposed combination therapies.
authors.affiliation: Department of Neurosurgery, Brain Tumor Center, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Lotte M E
authors.initials: LM
authors.lastname: Berghauser Pont
authors.affiliation: Department of Neurosurgery, Brain Tumor Center, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Anne
authors.initials: A
authors.lastname: Kleijn
authors.affiliation: Department of Neurosurgery, Brain Tumor Center, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Jenneke J
authors.initials: JJ
authors.lastname: Kloezeman
authors.affiliation: Department of Neurosurgery, Brain Tumor Center, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Wouter
authors.initials: W
authors.lastname: van den Bossche
authors.affiliation: Department of Neurosurgery, Harvey Cushing Neuro-Oncology Laboratories, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Johanna K
authors.initials: JK
authors.lastname: Kaufmann
authors.affiliation: Department of Neurosurgery, Utrecht University Medical Center, Utrecht, The Netherlands.
authors.forename: Jeroen
authors.initials: J
authors.lastname: de Vrij
authors.affiliation: Department of Neurosurgery, Brain Tumor Center, Erasmus MC, Rotterdam, The Netherlands; Department of Neurosurgery, Elisabeth Hospital, Tilburg, The Netherlands.
authors.forename: Sieger
authors.initials: S
authors.lastname: Leenstra
authors.affiliation: Department of Neurosurgery, Brain Tumor Center, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Clemens M F
authors.initials: CM
authors.lastname: Dirven
authors.affiliation: Department of Neurosurgery, Brain Tumor Center, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Martine L M
authors.initials: ML
authors.lastname: Lamfers
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects; D001932:Brain Neoplasms / Q000235:genetics / Q000473:pathology* / Q000628:therapy; D002470:Cell Survival; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics; D045643:DNA Repair Enzymes / Q000235:genetics; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D005909:Glioblastoma / Q000235:genetics / Q000473:pathology* / Q000628:therapy; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D006898:Hydroxylamines / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology*; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D051379:Mice; D009154:Mutation; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology; D050130:Oncolytic Virotherapy; D050504:Oncolytic Viruses; D000077767:Panobinostat; D011401:Promoter Regions, Genetic; D011804:Quinolines / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
chemical_list: D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D006898:Hydroxylamines; D007211:Indoles; D039302:Integrin alphaVbeta3; D011804:Quinolines; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C410733:scriptaid; D000077767:Panobinostat; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes