824: Re-engineering vesicular stomatitis virus to abrogate neurotoxicity, circumvent humoral immunity and enhance oncolytic potency

EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 Conclusions: Notch signalling is altered in chemo-resistant GC. The analysed chemo-resistant cell lines partially reflect Notch associated expression changes observed in neoadjuvant treated GC. Notch1 and Notch2 may display divergent functions in this context. A wider panel of resistant cell lines might help improve our understanding of chemo-resistance mechanisms in GC. No conflict of interest. 822 The histone acetyltransferases inhibitor CPTH6 preferentially inhibits proliferation of patient-derived lung cancer stem cells in vitro and in vivo M. Di Martile1 , M. Desideri1 , C. Gabellini1 , A. Eramo2 , S. Carradori3 , D. Secci3 , M. Milella4 , D. Del Bufalo1 , D. Trisciuoglio1 . 1 Regina Elena National Cancer Institute, Experimental Chemotherapy Laboratory, Rome, Italy, 2 Istituto Superiore di Sanita` Rome Italy, Department of Hematology Oncology and Molecular Medicine, Rome, Italy, 3 ‘Sapienza’ University Rome Italy, Department of Drug Chemistry and Technologies, Rome, Italy, 4 Regina Elena National Cancer Institute, Division of Medical Oncology A, Rome, Italy Background: Epigenetic changes, including histone modifications, often occur in cancer. It has been postulated that these changes may arise also in the stem cell compartment, that is responsible for tumor heterogeneity and recurrence. Targeting of epigenetic regulators is emerging as a promising strategy for cancer therapy. In this context, we recently identified the thiazole derivative 3-methylcyclopentylidene-[4-(4 -chlorophenyl)thiazol-2-yl]hydrazone (CPTH6) as a novel pCAF and GCN5 histone acetyltransferases inhibitor (HATi). CPTH6 exerts in vitro anti-tumoral effect in a panel of human tumor cell lines derived from different histotypes. In this study, we evaluated the efficacy of CPTH6 alone or in combinatorial regimes with chemotherapeutic agents, using in vitro and in vivo models of Non Small Cell Lung Cancer (NSCLC). Material and Methods: The biological effect of CPTH6 was analysed in a panel of established NSCLC cell lines and in patient-derived Lung Cancer Stem Cell (LCSC) models. Cell viability was tested by MTT and CellTiter Glo assays. Apoptosis induction and cell cycle perturbation were assessed by flow cytometry. DNA damage induction was revealed by Western Blot and immunofluorescence analyses. Pharmacological interactions between CPTH6 and Pemetrexed or Cisplatin were assessed by conservative isobologram analysis. Nude and NOD/SCID mice were used for in vivo experiments. Results: The HATi CPTH6 reduced cell viability of both NSCLC and LCSC lines in vitro. Interestingly, it was more efficient in LCSC (being IC50 values ranging from 10 to 30 mM) than NSCLC lines (IC50 values >60mM). Furthermore, the growth inhibitory effect seemed to be tightly related to baseline expression of acetylated alpha-tubulin, which was particularly prominent in sensitive LCSC. Unlike LCSC models, in which CPTH6 treatment induces cell cycle perturbation and apoptosis even at low doses, in NSCLC lines CPTH6 triggers a cell cycle perturbation associated to DNA damage. When CPTH6 was used in combination of with Pemetrexed or Cisplatin, it determines a strikingly synergistic reduction in cell viability in both NSCLC and LCSC lines. In vivo experiments confirmed the antitumor efficacy of CPTH6, particularly in LCSCderived models. In fact, in vivo experiments showed a pronounced growth inhibitory effect of CPTH6 on LCSC-136 derived tumors, but only a little impact on H1299 derived tumors growth. Conclusions: Overall, CPTH6 could be a valuable agent for the treatment of NSCLC and it should be further studied as a possible antineoplastic agent. No conflict of interest. 823 3-deazaneplanocin A (DZNep), an inhibitor of histone methyltransferase EZH2, induces apoptosis and reduces cell migration in chondrosarcoma cells N. Girard1 , C. Bazille2 , E. Lhuissier1 , H. Benateau3 , A. Llombart-bosch4 , K. Boumediene1 , C. Bauge1 . 1 Universite´ De Caen Basse-normandie, Ea4652 Milpat, Caen, France, 2 CHU, Service d’Anatomie Pathologique, Caen, France, 3 CHU, Service de Chirurgie Maxillo-Faciale, Caen, France, 4 University of Valencia, Pathology Department, Valencia, Spain Objective: Growing evidences indicate that the histone methyltransferase EZH2 (enhancer of zeste homolog 2) may be an appropriate therapeutic target in some tumors. Indeed, a high expression of EZH2 is correlated with poor prognosis and metastasis in many cancers. The major role of EZH2 is to methylate the lysine 27 on histone H3 (H3K27), leading to gene silencing. Interestingly, 3-Deazaneplanocin A (DZNep), an S-adenosyl-L homocysteine hydrolase inhibitor which induces EZH2 protein depletion, leads to cell death in several cancers and tumors. The aim of this study was to determine whether an epigenetic therapy targeting EZH2 with DZNep may be also efficient to treat chondrosarcomas. Methods: EZH2 expression was determined by immunohistochemistry and western-blot. Chondrosarcoma cell line CH2879 was cultured in the presence of DZNep, and its growth and survival were evaluated by counting adherent cells periodically. Apoptosis was assayed by cell cycle analysis, Apo2.7

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expression using flow cytometry, and by PARP cleavage using western-blot. Cell migration was assessed by wound healing assay. Results: Chondrosarcomas (at least with high grade) express high amount of EZH2, when compared to enchondromas or chondrocytes. In vitro, DZNep inhibits EZH2 protein expression, and subsequently reduces the trimethylation of H3K27 (H3K27me3). Interestingly, DZNep induces cell death of chondrosarcoma cells by apoptosis, while it slightly reduces growth of normal chondrocytes. In addition, DZNep reduces migration of chondrosarcomas. Conclusion: These results indicate that an epigenetic therapy that pharmacologically targets EZH2 may constitute a novel approach to treat chondrosarcomas. Preclinical assays using xenograft of chondrosarcoma in nude mice are in process. No conflict of interest. 824 Re-engineering vesicular stomatitis virus to abrogate neurotoxicity, circumvent humoral immunity and enhance oncolytic potency D. Von Laer1 , A. Muik2 , L.J. Stubbert3 , R.Z. Jahedi4 , Y. Geiß2 , C. Dold1 , R. Tober1 , U. Dietrich2 , H. Miletic4 , J.C. Bell3 . 1 Innsbruck Medical University, Division of Virology, Innsbruck, Austria, 2 Georg-Speyer-Haus, Frankfurt am Main, Germany, 3 Ottawa Hospital Research Institute, Centre for Innovative Cancer Research, Ottawa, Canada, 4 University of Bergen, Department of Biomedicine, Bergen, Norway Background: Oncolytic viruses destroy cancer cells specifically without harming normal tissues and are a highly promising new class of anti-cancer therapeutics. The Vesicular Stomatitis Virus (VSV) is an extremely potent oncolytic agent; however, clinical application has been limited by its devastating neurotoxicity and the rapid induction of a neutralizing antibody response. Here, we sought to exploit the many virtues of replication competent VSV as an anticancer therapeutic, while at the same time mitigating its propensity to infect and destroy normal brain cells. Material and Methods: We removed the VSV glycoprotein G as key neurovirulence deteminant and replaced it with the arenavirus glycoprotein LCMV-GP thereby generating a replicating therapeutic, rVSV(GP). Here, we analyzed safety and efficacy of this novel virus in vitro and in vivo. Results: While there are no doses at which wildtype VSV (wtVSV) can be safely introduced into rodent brains, we found that our chimeric strain rVSV(GP) caused no significant neurotoxicity even at doses of 108 plaque forming units injected directly into the brains of rodents. In addition, rVSV(GP) was a much safer virus when delivered systemically compared to its parental rVSV strain. rVSV(GP), however, was significantly more potent against a spectrum of human cancer cell lines than current oncolytic virus candidates. Furthermore, it retained rVSVs potent oncolytic activity in both syngeneic and xenogeneic orthotopic human glioblastoma models as well as in a syngeneic CT26 colon carcinoma brain metastasis model. Most importantly, in contrast to wtVSV, rVSV(GP) was not inactivated by human serum complement and did not induce a neutralizing antibody response in mice. The lack of neutralizing antibody induction allowed rVSV(GP) to access and replicate within tumour tissue of pre-immunized animals. Thus, rVSV(GP) is the first oncolytic virus that has the potential to fully retain therapeutic efficacy upon repeated therapeutic application. Conclusions: Taken together, rVSV(GP) is an extremely promising new oncolytic virus platform, which does not show any of the major drawbacks that have limited clinical efficacy of oncolytic viruses so far. No conflict of interest. 825 Improved cytotoxic therapy by dual targeting of a5b1 integrin and p53 pathway in glioblastoma M. Dontenwill1 , H. Janouskova1 , G. Renner1 , A.M. Ray1 , F. Noulet1 , L. Choulier1 , N. Etienne-Selloum1 , M. Lehmann1 , I. Lelong-Rebel1 , S. Martin1 . 1 University of Strasbourg, Tumoral Signaling and Therapeutic Targets, Illkirch, France Background: Glioblastoma is the most aggressive and malignant form of brain tumour. Despite advances in standard therapy, including surgical resection followed by radiation and chemotherapy with temozolomide (TMZ), the prognosis for patients with glioblastoma remains unfavourable, with a median survival of 12−18 months. We recently demonstrated that a5b1 integrin is a promising anti-cancer target in high grade glioma. We characterized an inverse crosstalk between this integrin and the tumor suppressor p53. The aim of this study was to investigate the potential benefit of combining a5b1 integrin antagonists with a non genotoxic p53 activator, Nutlin-3a, in glioma cells. Material and Methods: U87MG (p53wt) cells overexpressing or depleted in a5 integrin subunit were treated with K34c (a RGD-like integrin antagonist) and/or Nutlin-3a. U373 (p53 mutant) and LNZ308 (p53 KO) cells were similarly investigated. Cell survival was assessed by clonogenic assays. p53 activation was determined by western blot analysis using a specific phospho-p53 (ser15) antibody and by RT-qPCR analysis of p53 target genes. Apoptosis level was recorded by flow cytometry analysis of Annexin V/PI staining and cleaved