title: ABT-888 enhances cytotoxic effects of temozolomide independent of MGMT status in serum free cultured glioma cells.
abstract: BACKGROUND
  The current standard of care for Glioblastoma Multiforme (GBM) consists of fractionated focal irradiation with concomitant temozolomide (TMZ) chemotherapy. A promising strategy to increase the efficacy of TMZ is through interference with the DNA damage repair machinery, by poly(ADP-ribose) polymerase protein inhibition(PARPi). The objective of the present study was to investigate the therapeutic benefit of combination therapy in patient-derived glioma stem-like cells (GSC).
  
  
  METHODS
  Combination therapy feasibility was tested on established GBM cell lines U373 and T98. We developed an in vitro drug-screening assay based on GSC cultures derived from a panel of primary patient tissue samples (n = 20) to evaluate the effect of PARPi (ABT-888) monotherapy and combination therapy with TMZ. Therapeutic effect was assessed by viability, double stranded breaks, apoptosis and autophagy assays and longitudinal microscopic cell monitoring was performed. O-6-methylguanine-DNA methyltransferase (MGMT) status was determined by methylation assay and protein expression by western blots.
  
  
  RESULTS
  PARPi monotherapy was found to decrease viability by more than 25% in 4 of the 20 GSCs (20%) at 10 μM. TMZ monotherapy at 50 μM and 100 μM was effective in 12 and 14 of the 20 GSCs, respectively. TMZ resistance to 100 μM was found in 7 of 8 MGMT protein positive cultures. Potentiation of TMZ therapy through PARPi was found in 90% (n = 20) of GSCs, of which 6 were initially resistant and 7 were sensitive to TMZ monotherapy. Increased induction of double stranded breaks and apoptosis were noted in responsive GSCs. There was a trend noted, albeit statistically insignificant, of increased autophagy both in western blots and accumulation of autophagosomes.
  
  
  CONCLUSIONS
  PARPi mediated potentiation of TMZ is independent of TMZ sensitivity and can override MGMT(-) mediated resistance when administered simultaneously. Response to combination therapy was associated with increased double strand breaks induction, and coincided by increased apoptosis and autophagy. PARPi addition potentiates TMZ treatment in primary GSCs. PARPi could potentially enhance the therapeutic efficacy of the standard of care in GBM.
authors.affiliation: Brain Tumor Center; Department of Neurosurgery, Erasmus MC, Molewaterplein 50, Ee2236, 3015GE, Rotterdam, The Netherlands. r.balvers@erasmusmc.nl.
authors.forename: Rutger K
authors.initials: RK
authors.lastname: Balvers
authors.affiliation: Brain Tumor Center; Department of Neurosurgery, Erasmus MC, Molewaterplein 50, Ee2236, 3015GE, Rotterdam, The Netherlands. m.lamfers@erasmusmc.nl.
authors.forename: Martine L M
authors.initials: ML
authors.lastname: Lamfers
authors.affiliation: Brain Tumor Center; Department of Neurosurgery, Erasmus MC, Molewaterplein 50, Ee2236, 3015GE, Rotterdam, The Netherlands. j.kloezeman@erasmusmc.nl.
authors.forename: Jenneke J
authors.initials: JJ
authors.lastname: Kloezeman
authors.affiliation: Brain Tumor Center; Department of Neurosurgery, Erasmus MC, Molewaterplein 50, Ee2236, 3015GE, Rotterdam, The Netherlands. a.kleijn@erasmusmc.nl.
authors.forename: Anne
authors.initials: A
authors.lastname: Kleijn
authors.affiliation: Brain Tumor Center; Department of Neurosurgery, Erasmus MC, Molewaterplein 50, Ee2236, 3015GE, Rotterdam, The Netherlands. l.m.e.berghauserpont@gmail.com.
authors.forename: Lotte M E
authors.initials: LM
authors.lastname: Berghauser Pont
authors.affiliation: Brain Tumor Center; Department of Neurosurgery, Erasmus MC, Molewaterplein 50, Ee2236, 3015GE, Rotterdam, The Netherlands. c.dirven@erasmusmc.nl.
authors.forename: Clemens M F
authors.initials: CM
authors.lastname: Dirven
authors.affiliation: Brain Tumor Center; Department of Neurosurgery, Erasmus MC, Molewaterplein 50, Ee2236, 3015GE, Rotterdam, The Netherlands. s.leenstra@erasmusmc.nl.|Department of Neurosurgery, St Elisabeth Hospital, Tilburg, The Netherlands. s.leenstra@erasmusmc.nl.
authors.forename: Sieger
authors.initials: S
authors.lastname: Leenstra
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects; D001562:Benzimidazoles / Q000494:pharmacology / Q000627:therapeutic use*; D001932:Brain Neoplasms / Q000188:drug therapy* / Q000473:pathology*; D016895:Culture Media, Serum-Free; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D015254:DNA Modification Methylases / Q000378:metabolism*; D045643:DNA Repair Enzymes / Q000378:metabolism*; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D005910:Glioma / Q000188:drug therapy* / Q000473:pathology*; D006801:Humans; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D000067856:Poly(ADP-ribose) Polymerase Inhibitors / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D000077204:Temozolomide; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: D001562:Benzimidazoles; D016895:Culture Media, Serum-Free; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D025521:Tumor Suppressor Proteins; C521013:veliparib; D003606:Dacarbazine; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D011065:Poly(ADP-ribose) Polymerases; D045643:DNA Repair Enzymes; D000077204:Temozolomide