CD77, a promising therapeutical target in gastrointestinal cancer, is epigenetically regulated

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 normal colonic mucosa which obtained from various non-neoplastic conditions were used as control. We studied the following histone modifying enzymes: Enhancer of zeste homolog 2 (EZH2; histone-lysine N-methyltransferase), E1A binding protein p300 (EP300; histone acetyltransferase), DNA topoisomerase 1 (TOP1), Lysine-specific histone demethylase 1A (KDM1A) and 4B (KDM4B). We used quantitative RT-PCR arrays to assess the fold-changes of expression in terms of these enzymes’ genes. Results: Based on the RT-PCR data, we observed increased expressions of TOP1 (~0.9X), EP300 (~0.6X), and EZH2 (~0.5X) genes while KDM1A (~−1.1X) and KDM4B (~−2.3X) showed much lower expression levels, as distinct from normal tissue counterparts. Conclusion: Our data have revealed that the genes responsible for histone modifications are being shown differential expressions in adenocarcinomas with signet ring cells compared to the nonneoplastic cells. We think that up or down-regulation of different sets of histone modification enzymes can have vast effects on the whole genome by making various lengths of chromatin sealed i.e. unable to be expressed. Variations in the patterns of modifications on histone proteins may play pivotal roles in tumor initiation and evolution. As a consequence, this event can be one of the several phenomena underlying different morphological characteristics and biological behavior of malignant tumors. Their roles must be clarified by further studies on different types of tumors. No conflict of interest. 150 Deregulation of IGF2, FZD10, MAPK3, SMAD4 and SRF expression in colorectal cancer M. Ibarrola-Villava1 , N. Tarazona1 , V. Gambardella1 , C. Mongort2 , S. Navarro2 , S. Garcia-Botello3 , S. Rosello1 , A. Cervantes1 , G. Ribas1 . 1 Biomedical Research Institute INCLIVA − University of Valencia, Medical Oncology Unit, Valencia, Spain, 2 Biomedical Research Institute INCLIVA − University of Valencia, Department of Pathology, Valencia, Spain, 3 Clinic University Hospital of Valencia, Department of Surgery, Valencia, Spain Introduction: Colorectal cancer (CRC) is a frequent lethal disease with heterogeneous outcomes and drug responses. Understanding the roles that genetic and epigenetic alterations play in CRC pathogenesis has increased over the last decade and emerging genes have been described related with the disease. The aim of our study was to determine the expression levels of five CRC related genes: IGF2, FZD10, MAPK3, SMAD4 and SRF. Material and Methods: We compared IGF2, FZD10, SMAD4, SRF and MAPK3 mRNA expression levels among colorectal tumour and normal adjacent mucosa samples using quantitative real-time reverse transcription PCR. A total of 56 paired-samples were included in the study. Furthermore, the analysis of 238 mutations across 19 common oncogenes was performed using the Sequenom Oncocarta Panel v1.0 (Sequenom Hamburg, Germany). All study subjects gave written informed consent, and the study was approved by the Biomedical Research Institute INCLIVA Ethics Committee. Results: When compared tumour tissues with adjacent non-tumour tissues a lesser MAPK3 mRNA expression was found in 35 (72.91%) tumours (P = 1.69×10−3 ). Similarly, SMAD4 and SRF expression was slightly lesser in 29 tumour samples although no statistical difference was found (P = 0.19 and P = 0.40, respectively). Although an increased IGF2 expression was detected in tumour tissues, it did not remain statistically significant (P = 0.10). FZD10 expression was null among all the samples analysed. Regarding mutational status, 68.3% of the samples harboured at least one mutation, mainly in the KRAS gene. One expression cluster was identified and most of the samples included are mutated in KRAS G12 position (55%). Conclusions: Our data suggest that MAPK3 may play an important role in CRC and may serve as potential target for therapy. No conflict of interest. 151 Methylation profile of candidate genes in gastric cancer with microsatellite instability using high-throughput MALDI-TOF mass array technology: The role of RUNX3 in cancer progression 1 1 , , T. Fleitas1 , M. Ibarrola-Villava1 , M.C. Pena-Chilet ˜ M.J. Llorca Cardenosa ˜ C. Mongort2 , L. Navarro2 , S. Navarro2 , G. Ribas1 , A. Cervantes1 . 1 Biomedical Research Institute INCLIVA- University of Valencia, Department of Medical Oncology, Valencia, Spain, 2 Biomedical Research Institute INCLIVAUniversity of Valencia, Department of Pathology, Valencia, Spain

Introduction: Epigenetic alterations through DNA methylation are known to play a key role in inhibiting the expression of tumour-related genes. Gastric cancers (GC) with microsatellite instability (MSI) are a distinct subgroup, presenting differential molecular, clinicopathological and prognostic patterns, and have been associated with female gender, antral tumour location, hypermethylation (CpG Island Methylation phenotype, CIMP), better survival rate, underexpression of MLH1, mutations in ARID1A, KRAS, HER2 and affection of PIK3/PTEN/mTOR pathway. The aim of our study has been to perform a methylation panel of candidate genes to study the correlation of

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MSI with the hypermethylation of CIMP-related genes and the association with other clinicopathological features. Material and Method: The methylation status of 47 promoter-CpG islands of 5 genes was studied through MALDI-TOF mass spectrometry analysis of basespecifically cleaved amplification in 34 microsatellite stable (MSS) GC samples, 24 MSI, 18 cancer-free GC, 6 MSS cell lines and 4 MSI cell lines. HER2, HER3, ARID1A and RUNX3 expression was determined by immunohistochemistry (IHC). MSI status was analyzed by IHC and by a multiplex PCR amplification of a 5 microsatellite-marker panel and subsequently sequenciation of denaturalized PCR products. Validation of RUNX3 expression by IHC in tumour, normal adjacent mucosa and inflammatory peritumoural infiltrate was performed in 40 GC samples. Results and Discussion: Unsupervised hierarchical clustering of methylation levels showed no aggrupation of MSI samples. Methylation of APC, CDH1, MLH1 and CDKN2A was higher in GC compared to normal samples, showing 15 statistical significant different CpGs. Conversely, RUNX3 was highly methylated in normal tissue comparing to tumour, presenting statistical significant differences in 10 CpGs. Correlated to these findings, IHC analysis of RUNX3 expression showed protein silencing in cancer and normal mucosa, but a high expression in inflammatory peritumoural infiltrate in almost all cases. Methylation analysis of peritumoural infiltrate compared to tumour and normal mucosa will be discussed, in order to correlate epigenetic silencing and protein expression, and to find the roles of RUNX3 in cancer progression by its expression in the inflammatory cells. Conclusion: Hypermethylation of APC, CDH1, MLH1 and CDKN2A genes is associated with GC. RUNX3 high expression in inflammatory infiltrate confirms the importance of RUNX3 expression in GC through inflammatory processes. No conflict of interest.

152 The Shiga Toxin receptor Gb3/CD77, a promising therapeutical target in gastrointestinal cancer, is epigenetically regulated M. Perl1 , M. Gehrmann2,3 , U. Nitsche1 , G. Multhoff2,3 , L. Johannes4,5 , ¨ Munchen, K.P. Janssen1 . 1 Klinikum rechts der Isar − Technische Universitat ¨ Surgery, Munich, Germany, 2 Klinikum rechts der Isar − Technische 3 ¨ Munchen, Universitat ¨ Radiation Oncology, Munich, Germany, Helmholtz Zentrum Munchen ¨ − Deutsches Forschungszentrum fur ¨ Gesundheit und Umwelt, Clinical Cooperation Group CCG “Innate Immunity in Tumor Biology”, Munich, Germany, 4 Institut Curie, CNRS UMR3666, Paris, France, 5 Institut Curie, INSERM U1143, Paris, France Introduction: Gastrointestinal cancer comprises some of the tumor entities with worst prognosis. Therefore, new tools for screening and targeted drug delivery are urgently needed. We have previously shown that the non-toxic B subunit of the bacterially derived Shiga Toxin (StxB) can be exploited for tumor targeting, since it binds with high affinity to the glycosphingolipid Gb3/CD77 at the cell surface. Following receptor binding, StxB gets taken up rapidly along a retrograde Golgi-ER route, thereby avoiding lysosomal degradation. Gb3 is overexpressed in colon cancer and its metastases, stomach and pancreatic cancer, and StxB coupled to radioactive or fluorescent probes can be used for tumor detection in murine models. Moreover, we could demonstrate targeted delivery of cytotoxic compounds to tumor cells by StxB. Thus, the Gb3-ligand STxB is a promising tool for cancer therapy. However, the molecular mechanisms leading to aberrant biosynthesis of Gb3, as well as the consequences of elevated Gb3 levels for malignancy are yet unknown. Material and Method: Regulation mechanisms of Gb3 biosynthesis were analyzed in patient samples of gastric (n = 50) and colon cancer (n = 66), and established cell lines. We quantified Gb3 expression by flow cytometry, fluorescence microscopy and thin layer chromatography, and compared the results to clinical and molecular parameters. Additionally, cells were treated in vitro with pharmacological agents, or with StxB coupled to the topoisomerase I inhibitor SN38. Results and Discussion: SN38 coupled covalently to StxB showed greatly enhanced cytotoxicity, when compared to the clinically approved SN38 prodrug irinotecan in cancer cells overexpressing Gb3 on their surface while sparing Gb3 negative cells. However, we found that only a fraction of clinical samples, and cancer cell lines from gastric, pancreatic, esophageal or colon cancer express Gb3. We could show that Gb3 surface exposure is critically dependent on expression of the enzyme Gb3-synthase (A4GALT), and Gb3 levels are tightly correlated with A4GALT expression in cancer cell lines. A4GALT presents several CpG-islands in its presumed regulatory region. In Gb3 deficient gastric (AGS, MKN45) and colon cancer (DLD1) cell lines, A4GALT expression as well as Gb3 surface levels could be restored by inhibition of DNA methylation or histone deacetylation. In contrast, inhibition of glucosylceramide synthase or histone methylation had no effect on A4GALT expression. Preliminary evidence indicates aberrant CpG methylation of the A4GALT promoter in the Gb3-deficient cell line DLD1. Conclusion: These findings not only allow to stratify patients for future STxB-based targeted therapies, they offer a possible explanation for

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deregulated biosynthesis of glycosphingolipids in cancer cells by epigenetic reprogramming. No conflict of interest. 153 Exploiting DNA damage repair defects for effective targeting of acute myeloid leukaemia by PARP inhibitors M.T. Esposito1 , L. Zhao2 , T.K. Fung2 , J. Rane2 , A. Wilson2 , N. Martin3 , J. Gil4 , A.Y. Leung5 , A. Ashworth6 , C.W.E. So7 . 1 University of East London, Health Sport and Bioscience, London, United Kingdom, 2 King’s College London, Haematological Medicine Leukemia and Stem cell biology group, London, United Kingdom, 3 Centre de Recerche en cancerologie de Lyon, Senescence Escape Mechanisms Lab, Lyon, France, 4 Imperial College London, Medical Research Council Clinical Sciences Centre- Cell Proliferation group, London, United Kingdom, 5 The University of Hong Kong, Department of Medicine, Hong Kong, Hong Kong, 6 University of California- San Francisco UCSF, Hellen Diller Family Comprehensive Cancer Center, San Francisco, USA, 7 King’s College London, Haematological Medicine- Leukemia and Stem cell biology group, London, United Kingdom Introduction: Inhibitors of Poly-ADP-Ribose Polymerase (PARPi) have been successfully developed and approved by FDA for treatment of ovarian cancer carrying mutations in DNA damage response (DDR) genes BRCA1/BRCA2. In Acute Myeloid Leukemia (AML) patients, these mutations are extremely rare. However, chromosomal rearrangements generate chimeric oncofusion proteins that, by acting as transcriptional regulators, impair DDR gene expression. This prompted us to test the efficacy of using PARPi in AML. Materials and Methods: PARPi were tested in vitro and in vivo. In vitro experiments were carried out in mouse and human leukemic cell lines. Mouse leukemic cells expressing the oncofusion gene of interest were generated by Retroviral Transduction Transformation Assay (RTTA). Results: Leukemic cells driven by the oncofusion genes, AML1-ETO and PML-RARa, are sensitive to PARP inhibition whereas cells harbouring MLLAF9 translocation are resistant. Treatment of AML1-ETO and PML-RARa leukemic cells with PARPi induces apoptosis, senescence, cell cycle arrest and differentiation in vitro and significantly prolongs survival in vivo. By using gH2AX and RAD51 as markers of DNA damage and HR (Homologous Recombination) we showed that AML1-ETO and PML-RARa cells accumulate DNA damage and are defective in recruiting RAD51 to DNA damage foci upon PARPi treatment. Further analysis revealed that the expression of a number of genes that are involved in the HR pathway are reduced in AML1-ETO and PML-RARa cells including Rad51, Brca2 and Rpa1. This suggests that AML-ETO and PML-RARa are sensitive to PARPi as result of defective DDR. We showed that HOXA9, a key downstream target of MLL-fusions plays a critical role in promoting expression of HR genes and thus providing evidence by which MLL-AF9 are resistant to PARPi. Depletion of Hoxa9 reduces the expression of Rad51 and Brca2 in MLL-AF9 cells and confers PARPi sensitivity in MLL-AF9 leukemic cells, compromising its ability to form colonies, repair DNA damage and prolongs survival in mouse models. Conversely, HOXA9 overexpression rendered AML1-ETO and PML-RARa cells resistant to PARPi. Likewise, pharmacological suppression of Hoxa9, using GSK3 inhibitor LiCl, can also sensitize MLL-AF9 cells to PARPi and prolongs survival in our mouse model. Discussion: Our data indicate that PARPi might offer a new therapeutic strategy for patients with AML1-ETO or PML-RARa translocations. More importantly, we showed for the first time that HoxA9 can activate a potential DNA repair back-up pathway. PARPi in combination with pharmacological inhibitors of HOXA9 may represent a novel avenue for tailored therapeutic targeting of the aggressive MLL leukaemia. Conflict of interest: Ownership: Alan Ashworth: Patents related to the development of PARP inhibitors with AstraZeneca. Advisory Board: Alan Ashworth: Consulting or Advisory Role: Genentech, Sun Pharma, GlaxoSmithKline, Novartis. 154 Targeted next generation sequencing of Bulgarian prostate cancer patients finds new somatic mutations and reflects disease heterogeneity D. Kachakova1 , A. Vlahova2 , K. Mihova1 , A. Mitkova1 , I. Popov1 , E. Popov3 , S. Christova2 , C. Slavov3 , V. Mitev1 , R. Kaneva1 . 1 Molecular Medicine Centre, Department of Medical Chemistry and Biochemistry- Medical University of Sofia, Sofia, Bulgaria, 2 General and Clinical Pathology Clinic- Alexandrovska University Hospital, Department of General and Clinical Pathology- Medical University of Sofia, Sofia, Bulgaria, 3 Clinic of Urology- Alexandrovska University Hospital, Department of Urology- Medical University of Sofia, Sofia, Bulgaria Background: Prostate cancer (PC) continues to be most frequently occurring cancer in men after lung cancer and it is second leading cause of cancer related deaths among men worldwide. Despite its clinical significance little is known about its etiology. Novel therapeutic approaches are needed. PC shows biological heterogeneity which likely reflects underlying genomic

diversity. The oncogenic landscape of PC has become clearer with nextgeneration sequencing (NGS) studies. NGS could help in investigations of the mechanisms of prostate cancerogenesis and finding new therapeutic targets. Material and Methods: Twelve paraffin embedded radical prostatectomy (RP) tissue samples from patients with PC were used in this study. DNA was extracted from part of the RP samples containing more than 75% tumour tissues using QIAamp DNA FFPE Tissue kit. NGS was performed on MiSeq sequencer. Sequencing amplicon libraries were prepared using TruSeq Amplicon Cancer Panel (TSACP), according to the manufacturer’s instructions. For variant calling human genome build 19 was used as reference. Variants were filtered according the following criteria: sequencing quality (GQX above 30), consequence of variants and alternative variant frequency above 5%. Then pathogenic or likely pathogenic variants were considered only. Results and Discussion: Variants passing sequence quality were between 34 and 259 per sample. Among them between 2 and 28 variants per sample have some consequence and are with alternative allele frequency above 5%. In three of the samples there were no pathogenic or likely pathogenic variants meeting the criteria. In the rest of the samples these variants vary between 1 and 10. Six pathogenic or likely pathogenic new mutations (2 missense, 3 stop gain and 1 frameshift) were found in RB1 gene in 4 of the patients. Such variants were also found in PTEN and ABL1 in 3 of the patients. Pathogenic and likely pathogenic variants were detected in APC, ATM, TP53, CDKN2A, FBXW7 and others. Most of these variants were new. Conclusion: The results from this pilot study show that most of the variants with protein consequence are with frequency below or around 5%. This observation could be explained with the heterogenic nature of the prostate tumours. New pathogenic or likely pathogenic variants were discovered which could direct to possible new therapeutic targets. Larger sample size is needed to fully understand the mutation landscape and evaluate the role of the newly identified somatic mutations as possible therapeutic targets and/or prediction biomarkers. No conflict of interest. 155 CRISPR/Cas-9-mediated targeting of TP53 and MYC to investigate antimitotic mode of action S. Littler1 , H. Whalley1 , S. Sousa1 , S. Taylor1 . 1 University of Manchester, MCP, Manchester, United Kingdom Background: Antimitotic drugs, e.g. the taxanes, are used widely to treat breast, ovarian and lung cancer. By inhibiting microtubule dynamics, antimitotic drugs disrupt cell division, causing cells to then die, either directly in mitosis or following “slippage” back into the next interphase. However, the molecular mechanisms governing cell fate following mitotic disruption are poorly understood. It is clear that p53 plays a major role in terms of postmitotic behavior and recently, we showed that MYC is also a major determinant of mitotic cell fate (Topham et al (2015, Cancer Cell)). To determine how p53 and MYC modulate antimitotic chemotherapy responses, we are using CRISPR/Cas-9 approaches in order to correlate p53 and MYC function with mitotic behavior and subsequent cell fate. Material and Method: Utilizing CRISPR/Cas-9 technology, we screened a panel of sgRNAs to identify guides that efficiently target TP53 and MYC in established cell lines. We used immunoblotting to validate gene targeting and characterized the resulting null lines using a variety of assays. We then introduced cDNAs to generate transgenic lines where p53 and MYC are under tetracycline control. Finally, we used donor constructs to introduce exogenous sequences into both TP53 and MYC via homologous-recombination-mediated repair. Results: We identified sgRNAs that efficiently inactivate TP53 and MYC by introducing mutations near the ATG. We have introduced tet-responsive p53 and MYC cDNAs allowing us to fine tune p53 and MYC levels. And finally, we have successfully introduced various fluorescent protein tags into the N-terminus of both p53 and MYC. We have started to analyze these lines using a variety of microscopy-based approaches and we will present our latest results. Conclusions: CRISPR/Cas-9 technology can efficiently target both TP53 and MYC, providing us with an opportunity to measure gene expression dynamics at a single-cell level, both in unperturbed mitosis and in response to antimitotic agents. Future experiments will hopefully enhance our understanding of the mechanisms regulating mitotic cell fate. No conflict of interest. 156 Search for predisposing alleles in Hungarian non-BRCA breast and ovarian cancer families 1 ´ 1 . 1 National Institute of , A. Bozsik1 , J. Papp1 , T. Vaszko´ 1 , E. Olah T. Pocza ´ Oncology, Molecular Genetics, Budapest, Hungary Introduction: Germline mutations in the BRCA1 and BRCA2 genes account for about 7% of all breast cancer cases in Hungary. Near 60% of patients with strong family history of hereditary breast and ovarian cancer (HBOC) syndrome were diagnosed negative for germline mutations in our routine