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Subsequently we treated NBs cell lines with the pharmacological inhibitor of MRE11 exo-nuclease activity, mirin. We monitored its biological effects on proliferation (MTS assay, FACS), DNA damage (Neutral comet assay) and survival (Trypan blue exclusion Test, Tunel assay, PARP and Caspase 3 cleavage) and its effects on the DDR pathway (WB). Moreover we tested mirin in the treatment of human neuroblastoma (LAN5) xenografts, in vivo. Tumors were measured and then analyzed for DDR and apoptotic markers by WB and IHC. Results: Analysis of multiple datasets in the R2 database, indicated that MRE11 and RAD50 are significantly more expressed in MNA compared to MYCN single copy primary neuroblastomas. MRE11 or NBS1 RNAi-mediated knock down impaired proliferation and colony formation in a MYCN-dependent way. Pharmacological inhibition of the MRN complex via mirin, selectively induced cell death in MNA compared to MNSC cells or non-neuroblastoma cancer models. While mirin caused accumulation of 53BP1 foci, a marker of DNA damage associated with replication stress, it also inhibited the ATR/CHK1-dependent checkpoint/s, preventing any arrest in the S and G2 phases of the cell cycle. In contrast, mirin induced early occurrence of DNA double strand breaks and a typical DDR characterized by ATM, H2AX and p53 phosphorylation culminating in the accumulation of pro-apoptotic p53 target genes. Gain and loss of function experiments confirmed that mirin-induced cell death in MNA cells is p53 dependent. Injection of mirin encapsulated in nanoparticles significantly inhibited tumor growth in a neuroblastoma xenograft model. Drug-treated tumors were characterized by an high rate of apoptosis and DDR activation. Conclusion: Overall, we believe that our experiments provide a solid proof of principle that targeting the MRN complex can have significant effects in the treatment of MNA NBs. No conflict of interest. 611 The challenges of an autologous cell therapy product in clinical trials R. Smith1 , M. Bryan1 , K. Campbell2 , L. Cooney1 , F. Gilbert3 , T. Hamill1 , A. Scott4 , K.J. Williams1 . 1 TC BioPharm, Clinical, Motherwell, United Kingdom, 2 TC BioPharm, Quality Control, Motherwell, United Kingdom, 3 TC BioPharm, Production, Motherwell, United Kingdom, 4 TC BioPharm, Operations, Motherwell, United Kingdom Introduction: The cell therapy product is culture-expanded autologous gd T lymphocytes which presents many challenges for clinical trials. These include collecting starting material from each patient, shipping to the production facility, manufacturing the product specifically for each patient, then releasing and administering final product at the clinical trial sites. These challenges limit sites that can be included in a clinical trial and the patients who can be enrolled. Material and Method: Studies have shown that the proliferative capacity of gd T cells varies between individuals. We have developed a pre-screen proliferation assay to identify the patients who proliferate to the capacity required for autologous product manufacture. Shelf-lives of the proliferation assay blood sample and final product are limited, therefore shipment time between the clinic and manufacturing facility geographically restricts sites that can be involved. The Company has been conducting studies to extend shelf-life of the sample and product. Results and Discussion: To manufacture this autologous product, starting material is collected from each patient using an apheresis procedure to extract their own cells. Apheresis can be a demanding procedure, thus to ensure patients do not undergo the process unnecessarily, a pre-screening proliferation assay is performed to confirm proliferative capacity of gd T cells for each patient. This involves obtaining a 10 ml blood sample from a patient, with a limited shelf-life, currently qualified to 6 hours. This therefore geographically limits which clinical sites can be included in a clinical trial. The nature of the product also causes logistical issues. Manufacture of the product is a continuous 14-day process, thus the manufacturing facility must maintain close communication with both final product shipment courier and clinicians at the trial sites, to ensure that a patient is available for treatment on the specified day. If a patient misses their treatment visit, that dose cannot be retained, and a new dose will be manufactured. The final product also has a limited shelflife of 36 hours, and is therefore shipped under quarantine to clinics pending final QP certification, allowing for some geographically challenging sites to be included in the trial. In 6 months we have been able to improve the shelf life of the final product from 24 hours to 36 hours. Conclusion: The main challenge to success of any clinical trial with this product, is the shelf-life of the pre-screening blood sample and the final product. By conducting more shelf-life studies, it is considered that the shelf-life of the materials can be extended. This will expand the potential geographical area for clinical trial sites, and allow more patients to be enrolled and ultimately treated with autologous cell therapy. Conflict of interest: Ownership: Angela Scott, Karen J Williams and Rachel Smith

612 Idelalisib sensitivity and mechanisms of disease progression in relapsed TCF3-PBX1 acute lymphoblastic leukemia S. Eldfors1 , H. Kuusanmaki ¨ 1 , M. Kontro2 , M.M. Majumder1 , A. Parsons1 , 3 ¨ , H. Edgren1 , T. Pemovska1 , O. Kallioniemi1 , K. Wennerberg1 , N. Gokbuget T. Burmeister4 , K. Porkka2 , C. Heckman1 . 1 University of Helsinki − FIMM, Institute for Molecular Medicine Finland, Helsinki, Finland, 2 University of Helsinki and Helsinki University Central Hospital Cancer Center, Department of Hematology, Helsinki, Finland, 3 Goethe Unversity, Department of Medicine ¨ II, Frankfurt, Germany, 4 Charite´ Universtatsmedizin Berlin, Department of Hematology- Oncology and Tumorimmunology, Berlin, Germany Background: TCF3-PBX1 is a recurrent gene fusion in B-cell-precursor lymphoblastic leukemia (BCP-ALL) resulting from translocation t(1;19). The TCF3-PBX1 protein is a transcription factor that directly up-regulates pre-B-cell receptor genes, causing constitutive activation of pre-B-cell receptor signaling. TCF3-PBX1 BCP-ALL patients typically respond to chemotherapy; however, many relapse and develop resistant disease with few effective treatment options. In this study, we aimed to identify novel targeted drugs for treating TCF3-PBX1 BCP-ALL by profiling leukemic cells from a 25-year-old patient with relapsed disease. In addition, we sought to identify molecular mechanisms of disease pathogenesis and progression. Material and Methods: Bone marrow (BM) aspirates and a skin biopsy were collected from the patient at diagnosis and relapse. The sensitivity of BM cells was assessed against a library of 302 anti-neoplastic drugs. To identify molecular mechanisms underlying the pathogenesis and progression of the disease, exome and RNA-seq was performed. Drugs effective at inhibiting the viability of the patient cells were further investigated using TCF3-PBX1+ BCP-ALL and control cell lines. Results and Discussion: Drug sensitivity testing showed that index patient’s leukemic cells were sensitive to several classes of targeted drugs, including PI3K, mTOR, BET and HDAC inhibitors. Among the most effective was PI3K delta (p110d) inhibitor idelalisib approved for CLL and follicular lymphoma. Testing of samples from the patient, positive control CLL and healthy controls, showed that the BCP-ALL and CLL cells were similarly sensitive to idelalisib. To determine whether idelalisib sensitivity is common to all BCP-ALLs, we tested the sensitivity of TCF3-PBX1 positive (n = 3) and negative (n = 3) BCPALL cell lines. Two TCF3-PBX1+ cells lines were sensitive while negative cell lines were resistant. Idelalisib sensitivity of TCF3-PBX1+ BCP-ALL cells was further supported by evidence showing TCF3-PBX1 directly regulates expression of PIK3CD, the gene encoding p110d. RNA sequencing of the relapse samples showed high CXCR4 expression, which was not observed in a cohort of diagnostic phase TCF3-PBX1 BCP-ALLs (N = 15). CXCR4 mediates interactions with CXL12 expressing BM stromal cells and induces contact mediated drug resistance. Idelalisib inhibits CXCR4 signaling, providing a rationale for using this drug to counter drug resistance. The patient’s leukemia acquired mutations to TP53 at relapse. In addition, the patient’s leukemic cells had an MTOR mutation, which was associated with high sensitivity to mTOR inhibitors, which has not been observed before in TCF3-PBX1 BCP-ALL. Conclusions: Our results suggest idelalisib is a promising treatment for patients with TCF3-PBX1 BCP-ALL, while other drugs could be useful depending on the genetic context of individual patients. No conflict of interest. 613 Digoxin is a modifier increasing platinum drug anticancer activity E. Dudko1 , V. Chernov1 , T. Bogush1 , Y. Dyakova1 , V. Kirsanov2 , Z. Shprakh3 , A. Kamensky4 , B. Polotsky5 , S. Tjulandin6 , E. Shestakova1 . 1 N.N. Blokhin Russian Cancer Research Center, Laboratory of medical chemistry, Moscow, Russian Federation, 2 N.N. Blokhin Russian Cancer Research Center, Department of surgery, Moscow, Russian Federation, 3 N.N. Blokhin Russian Cancer Research Center, Deputy director, Moscow, Russian Federation, 4 Lomonosov Moscow State University, Department of human and animal physiology, Moscow, Russian Federation, 5 N.N. Blokhin Russian Cancer Research Center, Department of thoracic surgery, Moscow, Russian Federation, 6 N.N. Blokhin Russian Cancer Research Center, Department of clinical pharmacology and chemotherapy, Moscow, Russian Federation Introduction: Aerobic glycolysis (Warburg effect) is a fundamental hallmark of cancer cells. Glycolysis inhibition by cardiac glycosides increases anticancer drug toxicity towards tumor cells in vitro. The purpose of the study was to reveal such an effect in vivo, studying the influence of cardiac glycoside digoxin (Dig) on anticancer effect of platinum-drug cisplatin (Cis), which activity is associated with energy-dependent reparation of DNA damages. Materials and Methods: Breast cancer Ehrlich ascitis tumor was chosen as a model because of known high level of aerobic glycolysis in the cells. The investigation was conducted on female CBA/Lac mice. Dig 1 mg/kg was injected one hour before Cis administration. Both drugs were injected intraperitoneally (ip). The results were analyzed using Mann–Whitney and Student criteria (significant difference at p < 0.05). Results and Discussion: 1. Linear dosage-effect dependence was shown in 2.5 and 5.0 mg/kg of Cis, and the latter one is a maximal effective dose

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 because of Cis toxicity at dosage escalation. 2. Dig enhanced antitumor activity of Cis: survival time increase of tumor-bearing mice treated with Dig+Cis 2.5 mg/kg was 79%, as compared to 42% under Cis 2.5 mg/kg action only. 3. Cis 2.5 mg/kg+Dig efficacy was higher in comparison with Cis 5.0 mg/kg only: survival time increase of tumor-bearing mice treated with Dig+Cis 2.5 mg/kg was 79% as compared to 51% under Cis 5.0 mg/kg action only. 3. The differences in survival time of tumor-bearing mice between the groups «Cis 2.5 mg/kg» and «Cis 2.5 mg/kg+Dig», as well as between the groups «Cis 2.5 mg/kg+Dig» and «Cis 5.0 mg/kg», were highly significant (p < 0.0001) independently of which control was taken for comparison: tumor-bearing mice without treatment or with Dig injection. Conclusion: On the ascitic Ehrlich tumor-bearing mice, it was shown that ip injection of digoxin one hour before ip injection of cisplatin increases survival time of the tumor-bearing mice compared to cisplatin monotherapy. The amelioration of cisplatin efficacy under digoxin action exceeded the treatment effect of two-fold higher dose, and was more exhibited than the highest anticancer effect revealed in this experimental model. So, digoxin is an effective modifier increasing platinum drug anticancer activity. According to design and results of the study, we believe that clinical trial of cisplatin combination with digoxin in intraperitoneal chemotherapy of ascitic ovarian cancer is important in terms of revealing amelioration of treatment efficacy. Acknowledgment: Supported by grants: RFBR (15-04-06991-a, 16−34-01049mol-a) and President of Russian Federation (MK-7709.2016.7). No conflict of interest. 615 Induction of EMT is WNT-independent in cisplatin resistant urothelial carcinoma cell lines M.A. Skowron1 , G. Niegisch1 , G. Fritz2 , J.G.H. Van Roermund3 , A. Romano4 , P. Albers1 , W.A. Schulz1 , M.J. Hoffmann1 . 1 Heinrich-Heine-University, Urology, Duesseldorf, Germany, 2 Heinrich-Heine-University, Toxicology, Duesseldorf, Germany, 3 Maastricht University Medical Centre, Urology, Maastricht, Netherlands, 4 Maastricht University Medical Centre, Obstetrics and Gynaecology, Maastricht, Netherlands Background: Failure of cisplatin therapy in urothelial carcinoma (UC) patients originates from chemoresistance. Since the underlying mechanisms have not been clearly defined yet, we have generated cisplatin resistant UC cell lines (UCCs) by long-term treatment and characterized phenotypical and molecular changes associated with the development of cisplatin resistance. We observed morphological changes resembling epithelial–mesenchymal transition (EMT) indicating that UCCs might circumvent cisplatin induced apoptosis by phenotypic plasticity. As EMT is considered to be associated with stemness, chemoresistance, and metastatic potential, we determined the proliferative and clonogenic potential of cisplatin resistant UCCs and investigated the activity of stemness-associated signalling pathways. As combination chemotherapy is used to overcome resistance, cross-resistance towards doxorubicin and gemcitabine was also studied. Material and Method: Cisplatin-resistant UCCs were selected by long-term treatment (LTT) with escalating doses over several months. Sensitivity towards several cytotoxic drugs and population doubling time were assessed by MTT assay. Clonogenic potential and cell cycle distribution were evaluated by Giemsa staining and FACS analysis, respectively. Phenotypical changes were followed on the molecular level by qRT-PCR, immunofluorescence, and reporter assay for EMT markers and WNT-signalling components. Results: LTT-UCCs RT-112 and J82 grew more slowly compared to parental cells, but survived cisplatin treatment better over shorter or longer periods. Strong cross-resistance to gemcitabine, but not to doxorubicin was observed in RT-112-LTT. Treatment with increased doses of cisplatin disturbed the cell cycle of LTT cells, but they did not undergo apoptosis and recovered fully after 10 days. At the molecular level increased expression of Vimentin, Twist, and Zeb1 and decreased E-Cadherin expression corresponded with their EMTlike morphology. In addition, WNT pathway target genes (CTNNB1, AXIN2, CCND1, MYC, PITX2) were induced. However, TOPflash reporter assays revealed no significant activation of canonical WNT-signalling. Accordingly, the WNT-inhibitor niclosamide did not revert cisplatin resistance. Conclusions: Cisplatin-resistant UCCs appear to evade cell death by a phenotypic plasticity resembling EMT that may extend to other therapeutic compounds. Although overexpression of some WNT target genes was observed, phenotypic plasticity appeared not to be substantially mediated by canonical WNT signalling. No conflict of interest.

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616 Elucidation of molecular action mechanisms of cytotoxic palladium derivatives on various cancer cells O. Kacar1 , I. Hatipoglu1 , V. Yilmaz2 , N. Arda3 , E. Ulukaya4 , C.A. Acilan1 . TUBITAK, Marmara Research Center, Genetic Engineering and Biotechnology Institute, Turkey, 2 Uludag University, Faculty of Arts and Sciences, Departments of Chemistry, Turkey, 3 Istanbul University, Faculty of Arts and Sciences, Department of Molecular Biology and Genetics, Turkey, 4 Uludag University, Medical School, Department of Medical Biochemistry, Turkey 1

Background: The discovery of cisplatin, which is prescribed to almost 50% of cancer patients, has accelerated the design of many novel metal complexes. In this study, two novel palladium (Pd) complexes, [PdCl(terpy)](sac).2H2O (complex 1) and [Pd(sac)(terpy)](sac).4H2O (complex 2) were investigated for their anti-cancer properties. Material and Method: Cell viability was determined using the WST-1 proliferation assay. Apoptosis was assessed based on condensed/fragmented nuclei, gross morphological changes via microscopic analysis, and DNA laddering visualized on agarose gel electrophoresis. DNA double strand breaks were measured. HeLa, MDA-MB-231, and HCT-116 cells were arrested at G1, at the G1/S boundary or in mitosis. Synchronized cells were treated with the Pd compounds for 24, 48, and 72 h. Synchrony was evaluated based on morphology and flow cytometry, and cell viability was measured. Results: The tested compounds induced cytotoxicity in cancer cell lines that originated from various organs, suggesting a broad spectrum of activity. Cells treated with the Pd(II) complex exhibited typical characteristics of apoptosis based on increase in caspase 3/7 activity, presence of fragmented nuclei, DNA laddering, cellular shrinkage and blebbing. DSBs were formed in response to Pd(II) treatment both in vitro and in vivo. Furthermore, our initial data indicated that whilst arresting cells through serum starvation at G1/S had no effect on Pd(II) induced cytotoxicity, cells arrested in M phase appeared to be more resistant to drug treatment. Our results showed that cells were efficiently arrested at the expected cell cycle phase. Treatment of cells with the Pd(II) complexes at specific stage of the cell cycle resulted in similar death rates. The main form of cell death was found to be apoptosis in all treatments tested. Conclusions: The new Pd(II) complex appears to represent a new potent anticancer agent that acts through DNA damage and can induce apoptosis. Arresting cells at the cell cycle phases was accomplished successfully; consequently, these cells were treated with Pd(II) complexes which yielded no significant variation in cell death for HCT-116 and MDA-MB-231; however, HeLa was more sensitive in S/G1. Apoptosis was found to be the leading cause of cell death in all treatments. Indeed, further studies in animal models are required for proof-of-concept before undertaking clinical studies. No conflict of interest. 618 Synthesis, biological characterization and evaluation of molecular mechanisms of novel copper complexes as anticancer agents C. Acilan1 , B. Cevatemre2 , Z. Adıguzel1 , D. Karakas2 , E. Ulukaya3 , N. Arda4 , N. Ribeiro5 , I. Correiae5 , J.C. Pessoa5 . 1 TUBITAK, Marmara Research Center, Genetic Engineering and Biotechnology Institute, Turkey, 2 Uludag University, Faculty of Arts and Sciences, Department of Biology, Turkey, 3 Uludag University, Medical School, Department of Medical Biochemistry, Turkey, 4 Istanbul University, Moelcular Biology and Genetics Department, ´ Istanbul, Turkey, 5 Centro de Qu´ımica Estrutural, Instituto Superior Tecnico, Universidade de Lisboa Background: Three copper(II) complexes, Cu(Sal-Gly)(phen), Cu(SalGly)(pheamine), Cu(Sal-Gly)(phepoxy) are synthesized as potential anticancer agents, and characterized for their interaction with DNA, cytotoxicity, and mechanism of action. Material and Method: The binding ability of the complexes to Calf Thymus DNA was evaluated by competition fluorescence studies with thiazole orange, a known intercatator, UV-Vis spectrophotometric titrations and circular dichroism spectroscopy. DNA damage was determined by cleavage of supercoiled DNA in vitro and induction of 8-oxo-guanidine and gH2AX staining in cells. Apoptosis was assessed by DNA condensation/fragmentation, assessment of mitochondrial membrane potential, Annexin V staining and caspase 3/7 activity. Total ROS measurements were made by DCFDA analysis. Gene expression was evaluated via RT-qPCR analysis. Results and Discussion: Binding constants were evaluated as 1.7×106 , 2.5×106 and 3.2×105 M−1 , for Cu(Sal-Gly)(phen), Cu(Sal-Gly)(pheamine) and Cu(Sal-Gly)(phepoxy), respectively. All compounds induce DNA damage both in vitro and in cell culture. Apoptosis is the main form of cell death based on morphological analyses. Caspase 3/7 activity is elevated in response all Cucomplexes, supporting apoptotic cell death. There is an increase in reactive oxygen species (ROS), suggesting that ROS may mediate DNA damage and antitumor activity. Although the compounds were cytotoxic to all tested cancer cell lines, only Cu(Sal-Gly)(pheamine) displayed significantly lower toxicity towards non-cancer cells, demonstrating selectivity for cancer cells. Therefore, Cu(Sal-Gly)(pheamine) was further tested for molecular changes in response