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In vitro screening of clinical drugs identifies sensitizers of oncolytic viral therapy in glioblastoma stem-like cells

Gene Therapy volume 22, pages 947–959 (2015)Cite this article

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Abstract

Oncolytic viruses (OV) have broad potential as an adjuvant for the treatment of solid tumors. The present study addresses the feasibility of clinically applicable drugs to enhance the oncolytic potential of the OV Delta24-RGD in glioblastoma. In total, 446 drugs were screened for their viral sensitizing properties in glioblastoma stem-like cells (GSCs) in vitro. Validation was done for 10 drugs to determine synergy based on the Chou Talalay assay. Mechanistic studies were undertaken to assess viability, replication efficacy, viral infection enhancement and cell death pathway induction in a selected panel of drugs. Four viral sensitizers (fluphenazine, indirubin, lofepramine and ranolazine) were demonstrated to reproducibly synergize with Delta24-RGD in multiple assays. After validation, we underscored general applicability by testing candidate drugs in a broader context of a panel of different GSCs, various solid tumor models and multiple OVs. Overall, this study identified four viral sensitizers, which synergize with Delta24-RGD and two other strains of OVs. The viral sensitizers interact with infection, replication and cell death pathways to enhance efficacy of the OV.

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References

  1. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 2009; 10: 459–466.

    Article  CAS  Google Scholar 

  2. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005; 352: 987–996.

    Article  CAS  Google Scholar 

  3. Lang FF, Conrad C, Gomez-Manzano C, Tufaro F, Yung W, Sawaya R et al. First-in-human phase I clinical trial of oncolytic delta-24-rgd (dnx-2401) with biological endpoints: implications for viro-immunotherapy. Neuro Oncol 2014; 16 (Suppl 3): iii39.

    Article  Google Scholar 

  4. Lamfers ML, Grill J, Dirven CM, Van Beusechem VW, Geoerger B, Van Den Berg J et al. Potential of the conditionally replicative adenovirus Ad5-Delta24RGD in the treatment of malignant gliomas and its enhanced effect with radiotherapy. Cancer Res 2002; 62: 5736–5742.

    CAS  Google Scholar 

  5. Kaufmann JK, Chiocca EA . Oncolytic virotherapy for gliomas: steps toward the future. CNS Oncol 2013; 2: 389–392.

    Article  CAS  Google Scholar 

  6. Suzuki K, Fueyo J, Krasnykh V, Reynolds PN, Curiel DT, Alemany R . A conditionally replicative adenovirus with enhanced infectivity shows improved oncolytic potency. Clin Cancer Res 2001; 7: 120–126.

    CAS  Google Scholar 

  7. Fuxe J, Liu L, Malin S, Philipson L, Collins VP, Pettersson RF . Expression of the coxsackie and adenovirus receptor in human astrocytic tumors and xenografts. Int J Cancer 2003; 103: 723–729.

    Article  CAS  Google Scholar 

  8. de Jonge J, Berghauser Pont LM, Idema S, Kloezeman JJ, Noske D, Dirven CM et al. Therapeutic concentrations of anti-epileptic drugs do not inhibit the activity of the oncolytic adenovirus Delta24-RGD in malignant glioma. J Gene Med 2013; 15: 134–141.

    Article  CAS  Google Scholar 

  9. Berghauser Pont LM, Kleijn A, Kloezeman JJ, van den Bossche W, Kaufmann JK, de Vrij J et al. The HDAC inhibitors scriptaid and LBH589 combined with the oncolytic virus Delta24-RGD exert enhanced anti-tumor efficacy in patient-derived glioblastoma cells. PLoS One 2015; 10: e0127058.

    Article  Google Scholar 

  10. US National Institutes of Health. Available at http://dtp.cancer.gov〉 2014, (14.10.15).

  11. Balvers RK, Kleijn A, Kloezeman JJ, French PJ, Kremer A, van den Bent MJ et al. Serum-free culture success of glial tumors is related to specific molecular profiles and expression of extracellular matrix-associated gene modules. Neuro Oncol 2013; 15: 1684–1695.

    Article  Google Scholar 

  12. Lee J, Kotliarova S, Kotliarov Y, Li A, Su Q, Donin NM et al. Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 2006; 9: 391–403.

    Article  CAS  Google Scholar 

  13. Cheema TA, Wakimoto H, Fecci PE, Ning J, Kuroda T, Jeyaretna DS et al. Multifaceted oncolytic virus therapy for glioblastoma in an immunocompetent cancer stem cell model. Proc Natl Acad Sci USA 2013; 110: 12006–12011.

    Article  CAS  Google Scholar 

  14. Buijs PR, van Eijck CH, Hofland LJ, Fouchier RA, van den Hoogen BG . Different responses of human pancreatic adenocarcinoma cell lines to oncolytic Newcastle disease virus infection. Cancer Gene Ther 2014; 21: 24–30.

    Article  CAS  Google Scholar 

  15. NIH Small Molecule Repository (SMR). Evotec. http://nihclinicalcollection.com/. (last accessed september 2014).

  16. Chou TC, Talalay P . Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984; 22: 27–55.

    Article  CAS  Google Scholar 

  17. Luo Z, Sheng J, Sun Y, Lu C, Yan J, Liu A et al. Synthesis and evaluation of multi-target-directed ligands against Alzheimer's disease based on the fusion of donepezil and ebselen. J Med Chem 2013; 56: 9089–9099.

    Article  CAS  Google Scholar 

  18. Singh N, Halliday AC, Thomas JM, Kuznetsova OV, Baldwin R, Woon EC et al. A safe lithium mimetic for bipolar disorder. Nat Commun 2013; 4: 1332.

    Article  Google Scholar 

  19. Tsuneizumi T, Babb SM, Cohen BM . Drug distribution between blood and brain as a determinant of antipsychotic drug effects. Biol Psychiatry 1992; 32: 817–824.

    Article  CAS  Google Scholar 

  20. Wang W, Yang Y, Ying C, Li W, Ruan H, Zhu X et al. Inhibition of glycogen synthase kinase-3beta protects dopaminergic neurons from MPTP toxicity. Neuropharmacology 2007; 52: 1678–1684.

    Article  CAS  Google Scholar 

  21. Leonard BE . A comparison of the pharmacological properties of the novel tricyclic antidepressant lofepramine with its major metabolite, desipramine: a review. Int Clin Psychopharmacol 1987; 2: 281–297.

    Article  CAS  Google Scholar 

  22. Jiang H, White EJ, Rios-Vicil CI, Xu J, Gomez-Manzano C, Fueyo J . Human adenovirus type 5 induces cell lysis through autophagy and autophagy-triggered caspase activity. J Virol 2011; 85: 4720–4729.

    Article  CAS  Google Scholar 

  23. Bartlett DL, Liu Z, Sathaiah M, Ravindranathan R, Guo Z, He Y et al. Oncolytic viruses as therapeutic cancer vaccines. Mol Cancer 2013; 12: 103.

    Article  Google Scholar 

  24. Berghauser Pont LM, Spoor JK, Venkatesan S, Swagemakers S, Kloezeman JJ, Dirven CM et al. The Bcl-2 inhibitor Obatoclax overcomes resistance to histone deacetylase inhibitors SAHA and LBH589 as radiosensitizers in patient-derived glioblastoma stem-like cells. Genes Cancer 2014; 5: 445–459.

    PubMed  PubMed Central  Google Scholar 

  25. Berghauser Pont LM, Naipal K, Kloezeman JJ, Venkatesan S, van den Bent M, van Gent DC et al. DNA damage response and anti-apoptotic proteins predict radiosensitization efficacy of HDAC inhibitors SAHA and LBH589 in patient-derived glioblastoma cells. Cancer Lett 2014; 356: 525–535.

    Article  Google Scholar 

  26. Balvers RK, Lamfers ML, Kloezeman JJ, Kleijn A, Berghauser Pont LM, Dirven CM et al. ABT-888 enhances cytotoxic effects of temozolomide independent of MGMT status in serum free cultured glioma cells. J Transl Med 2015; 13: 74.

    Article  Google Scholar 

  27. Pollard SM, Yoshikawa K, Clarke ID, Danovi D, Stricker S, Russell R et al. Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell 2009; 4: 568–580.

    Article  CAS  Google Scholar 

  28. Jiang P, Mukthavaram R, Chao Y, Bharati IS, Fogal V, Pastorino S et al. Novel anti-glioblastoma agents and therapeutic combinations identified from a collection of FDA approved drugs. J Transl Med 2014; 12: 13.

    Article  Google Scholar 

  29. Alonso MM, Jiang H, Yokoyama T, Xu J, Bekele NB, Lang FF et al. Delta-24-RGD in combination with RAD001 induces enhanced anti-glioma effect via autophagic cell death. Mol Ther 2008; 16: 487–493.

    Article  CAS  Google Scholar 

  30. Lamfers ML, Fulci G, Gianni D, Tang Y, Kurozumi K, Kaur B et al. Cyclophosphamide increases transgene expression mediated by an oncolytic adenovirus in glioma-bearing mice monitored by bioluminescence imaging. Mol Ther 2006; 14: 779–788.

    Article  CAS  Google Scholar 

  31. Alonso MM, Gomez-Manzano C, Jiang H, Bekele NB, Piao Y, Yung WK et al. Combination of the oncolytic adenovirus ICOVIR-5 with chemotherapy provides enhanced anti-glioma effect in vivo. Cancer Gene Ther 2007; 14: 756–761.

    Article  CAS  Google Scholar 

  32. Ulasov IV, Sonabend AM, Nandi S, Khramtsov A, Han Y, Lesniak MS . Combination of adenoviral virotherapy and temozolomide chemotherapy eradicates malignant glioma through autophagic and apoptotic cell death in vivo. Br J Cancer 2009; 100: 1154–1164.

    Article  CAS  Google Scholar 

  33. Holzmuller R, Mantwill K, Haczek C, Rognoni E, Anton M, Kasajima A et al. YB-1 dependent virotherapy in combination with temozolomide as a multimodal therapy approach to eradicate malignant glioma. Int J Cancer 2011; 129: 1265–1276.

    Article  Google Scholar 

  34. Idema S, Lamfers ML, van Beusechem VW, Noske DP, Heukelom S, Moeniralm S et al. AdDelta24 and the p53-expressing variant AdDelta24-p53 achieve potent anti-tumor activity in glioma when combined with radiotherapy. J Gene Med 2007; 9: 1046–1056.

    Article  CAS  Google Scholar 

  35. Geoerger B, Grill J, Opolon P, Morizet J, Aubert G, Lecluse Y et al. Potentiation of radiation therapy by the oncolytic adenovirus dl1520 (ONYX-015) in human malignant glioma xenografts. Br J Cancer 2003; 89: 577–584.

    Article  CAS  Google Scholar 

  36. McKenzie BA, Zemp FJ, Pisklakova A, Narendran A, McFadden G, Lun X et al. In vitro screen of a small molecule inhibitor drug library identifies multiple compounds that synergize with oncolytic myxoma virus against human brain tumor-initiating cells. Neuro Oncol 2015; 17: 1086–1094.

    Article  CAS  Google Scholar 

  37. Diallo JS, Le Boeuf F, Lai F, Cox J, Vaha-Koskela M, Abdelbary H et al. A high-throughput pharmacoviral approach identifies novel oncolytic virus sensitizers. Mol Ther 2010; 18: 1123–1129.

    Article  CAS  Google Scholar 

  38. Passer BJ, Cheema T, Zhou B, Wakimoto H, Zaupa C, Razmjoo M et al. Identification of the ENT1 antagonists dipyridamole and dilazep as amplifiers of oncolytic herpes simplex virus-1 replication. Cancer Res 2010; 70: 3890–3895.

    Article  CAS  Google Scholar 

  39. Bieler A, Mantwill K, Dravits T, Bernshausen A, Glockzin G, Kohler-Vargas N et al. Novel three-pronged strategy to enhance cancer cell killing in glioblastoma cell lines: histone deacetylase inhibitor, chemotherapy, and oncolytic adenovirus dl520. Hum Gene Ther 2006; 17: 55–70.

    Article  CAS  Google Scholar 

  40. Liikanen I, Monsurro V, Ahtiainen L, Raki M, Hakkarainen T, Diaconu I et al. Induction of interferon pathways mediates in vivo resistance to oncolytic adenovirus. Mol Ther 2011; 19: 1858–1866.

    Article  CAS  Google Scholar 

  41. Jiang H, Gomez-Manzano C, Aoki H, Alonso MM, Kondo S, McCormick F et al. Examination of the therapeutic potential of Delta-24-RGD in brain tumor stem cells: role of autophagic cell death. J Natl Cancer Inst 2007; 99: 1410–1414.

    Article  CAS  Google Scholar 

  42. Abou El Hassan MA, van der Meulen-Muileman I, Abbas S, Kruyt FA . Conditionally replicating adenoviruses kill tumor cells via a basic apoptotic machinery-independent mechanism that resembles necrosis-like programmed cell death. J Virol 2004; 78: 12243–12251.

    Article  Google Scholar 

  43. Baird SK, Aerts JL, Eddaoudi A, Lockley M, Lemoine NR, McNeish IA . Oncolytic adenoviral mutants induce a novel mode of programmed cell death in ovarian cancer. Oncogene 2008; 27: 3081–3090.

    Article  CAS  Google Scholar 

  44. Kleijn A, Kloezeman J, Treffers-Westerlaken E, Fulci G, Leenstra S, Dirven C et al. The In Vivo Therapeutic Efficacy of the Oncolytic Adenovirus Delta24-RGD Is Mediated by Tumor-Specific Immunity. PLoS One 2014; 9: e97495.

    Article  Google Scholar 

  45. Schleuning M, Brumme V, Wilmanns W . Growth inhibition of human leukemic cell lines by the phenothiazine derivative fluphenazine. Anticancer Res 1993; 13: 599–602.

    CAS  PubMed  Google Scholar 

  46. Gil-Ad I, Shtaif B, Levkovitz Y, Dayag M, Zeldich E, Weizman A . Characterization of phenothiazine-induced apoptosis in neuroblastoma and glioma cell lines: clinical relevance and possible application for brain-derived tumors. J Mol Neurosci 2004; 22: 189–198.

    Article  CAS  Google Scholar 

  47. Hwang MK, Min YK, Kim SH . Calmodulin inhibition contributes to sensitize TRAIL-induced apoptosis in human lung cancer H1299 cells. Biochem Cell Biol 2009; 87: 919–926.

    Article  CAS  Google Scholar 

  48. Lin YK, Leu YL, Yang SH, Chen HW, Wang CT, Pang JH . Anti-psoriatic effects of indigo naturalis on the proliferation and differentiation of keratinocytes with indirubin as the active component. J Dermatol Sci 2009; 54: 168–174.

    Article  CAS  Google Scholar 

  49. Hoessel R, Leclerc S, Endicott JA, Nobel ME, Lawrie A, Tunnah P et al. Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases. Nat Cell Biol 1999; 1: 60–67.

    Article  CAS  Google Scholar 

  50. Rahman SH, Bobis-Wozowicz S, Chatterjee D, Gellhaus K, Pars K, Heilbronn R et al. The nontoxic cell cycle modulator indirubin augments transduction of adeno-associated viral vectors and zinc-finger nuclease-mediated gene targeting. Hum Gene Ther 2013; 24: 67–77.

    Article  CAS  Google Scholar 

  51. Mok CK, Kang SS, Chan RW, Yue PY, Mak NK, Poon LL et al. Anti-inflammatory and antiviral effects of indirubin derivatives in influenza A (H5N1) virus infected primary human peripheral blood-derived macrophages and alveolar epithelial cells. Antiviral Res 2014; 106: 95–104.

    Article  CAS  Google Scholar 

  52. Hertel L, Chou S, Mocarski ES . Viral and cell cycle-regulated kinases in cytomegalovirus-induced pseudomitosis and replication. PLoS Pathog 2007; 3: e6.

    Article  Google Scholar 

  53. Hsuan SL, Chang SC, Wang SY, Liao TL, Jong TT, Chien MS et al. The cytotoxicity to leukemia cells and antiviral effects of Isatis indigotica extracts on pseudorabies virus. J Ethnopharmacol 2009; 123: 61–67.

    Article  Google Scholar 

  54. Leclerc S, Garnier M, Hoessel R, Marko D, Bibb JA, Snyder GL et al. Indirubins inhibit glycogen synthase kinase-3 beta and CDK5/p25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer's disease. A property common to most cyclin-dependent kinase inhibitors? J Biol Chem 2001; 276: 251–260.

    Article  CAS  Google Scholar 

  55. Benson JM, Shepherd DM . Dietary ligands of the aryl hydrocarbon receptor induce anti-inflammatory and immunoregulatory effects on murine dendritic cells. Toxicol Sci 2011; 124: 327–338.

    Article  CAS  Google Scholar 

  56. Yuskaitis CJ, Jope RS . Glycogen synthase kinase-3 regulates microglial migration, inflammation, and inflammation-induced neurotoxicity. Cell Signal 2009; 21: 264–273.

    Article  CAS  Google Scholar 

  57. Samudio I, Harmancey R, Fiegl M, Kantarjian H, Konopleva M, Korchin B et al. Pharmacologic inhibition of fatty acid oxidation sensitizes human leukemia cells to apoptosis induction. J Clin Invest 2010; 120: 142–156.

    Article  CAS  Google Scholar 

  58. Driffort V, Gillet L, Bon E, Marionneau-Lambot S, Oullier T, Joulin V et al. Ranolazine inhibits NaV1.5-mediated breast cancer cell invasiveness and lung colonization. Mol Cancer 2014; 13: 264.

    Article  Google Scholar 

  59. Pasqualini R, Koivunen E, Ruoslahti E . Alpha v integrins as receptors for tumor targeting by circulating ligands. Nat Biotechnol 1997; 15: 542–546.

    Article  CAS  Google Scholar 

  60. Balvers RK, Belcaid Z, van den Hengel SK, Kloezeman J, de Vrij J, Wakimoto H et al. Locally-delivered T-cell-derived cellular vehicles efficiently track and deliver adenovirus delta24-RGD to infiltrating glioma. Viruses 2014; 6: 3080–3096.

    Article  Google Scholar 

  61. Galli R, Binda E, Orfanelli U, Cipelletti B, Gritti A, De Vitis S et al. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 2004; 64: 7011–7021.

    Article  CAS  Google Scholar 

  62. Gursel DB, Shin BJ, Burkhardt JK, Kesavabhotla K, Schlaff CD, Boockvar JA . Glioblastoma stem-like cells-biology and therapeutic implications. Cancers 2011; 3: 2655–2666.

    Article  Google Scholar 

  63. Chou TC . Preclinical versus clinical drug combination studies. Leuk Lymphoma 2008; 49: 2059–2080.

    Article  Google Scholar 

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Acknowledgements

This research was partially supported by a Travel Fund of the Royal Dutch Wilhelmina Fund (KWF) and a travel fund of the EUR Trustfonds, Rotterdam, The Netherlands.

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Authors and Affiliations

  1. Department of Neurosurgery, Brain Tumor Center Erasmus MC, Rotterdam, The Netherlands

    L M E Berghauser Pont, R K Balvers, J J Kloezeman, W van den Bossche, S Leenstra, C M F Dirven & M L M Lamfers

  2. Harvey Cushing Neuro-oncology Laboratories, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    M O Nowicki, E A Chiocca & S E Lawler

  3. Department of Immunology, Erasmus MC, Rotterdam, The Netherlands

    W van den Bossche

  4. Department of Bioinformatics, Erasmus MC, Rotterdam, The Netherlands

    A Kremer

  5. Department of Neurosurgery, Massachussets General Hospital, Harvard Medical School, Boston, MA, USA

    H Wakimoto

  6. Department of Viroscience Erasmus MC, Rotterdam, The Netherlands

    B G van den Hoogen

  7. Department of Neurosurgery, Elisabeth Hospital, Tilburg, The Netherlands

    S Leenstra

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  1. L M E Berghauser Pont
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Correspondence to M L M Lamfers.

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Berghauser Pont, L., Balvers, R., Kloezeman, J. et al. In vitro screening of clinical drugs identifies sensitizers of oncolytic viral therapy in glioblastoma stem-like cells. Gene Ther 22, 947–959 (2015). https://doi.org/10.1038/gt.2015.72

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