541 The Histone Methyltransferase EZH2 is Required for Cell Growth in Diffuse Large B-cell Lymphoma

166 Friday 9 November 2012 538 POSTER Finding New Signalling Pathways That Contribute to Cancer Pathogenesis I.J. Majewski1 , Z. Xue1 , L. Mittempergher1 , M. Michaut1 , R. Kluin2 , J. Peeters3 , A. Bosma1 , E. Leproust4 , L.F. Wessels1 , R. Bernards1 . 1 NKI-AVL, Molecular Carcinogenesis, Amsterdam, The Netherlands; 2 NKI-AVL, Central Genomics Facility, Amsterdam, The Netherlands; 3 Agendia NV, R&D, Amsterdam, The Netherlands; 4 Agilent Technologies, R&D Chemistry, Santa Clara California, USA Background: Our research applies next-generation sequencing to identify signalling pathways that drive the development of cancer and those that influence therapy response. Kinases are important mediators of cell signalling and are good targets for small molecule inhibitors. We’ve developed an exon capture system that allows us to profile cancer samples for kinase mutations. A key strength of this platform is that it enables detection of point mutations and copy number alterations simultaneously. Material and Methods: A pooling strategy was developed to enable highthroughput capture and sequencing. DNA and RNA were extracted from sections of frozen tumours, cell lines or matched normal tissue (from blood or lymph nodes). Sequencing was performed on the Illumina HiSeq2000; we obtained >150-fold average coverage for the majority of target regions, providing a high degree of sensitivity for detecting sequence variants. Results: The kinome sequencing technology was validated in cell line models and xenografts and we have since expanded this work to survey 700 patients with breast cancer, colon cancer, or ovarian cancer. These studies are revealing oncogenic signatures that are characteristic of each cancer type. For example, in our breast cancer cohort we confirmed a high frequency of PI3K pathway alterations in lobular breast cancer (ILC), around 50%, but also found a subset of tumours that carried alterations in the MAPK pathway (e.g. MAP3K1, MAP2K4, MAP3K11). These pathways were much less frequently altered in triple negative (TN) tumours. Conclusion: We have documented numerous sequence variants, structural alterations and expression changes that influence kinase genes in human cancers. We are using representative cell line models and functional genomics approaches to investigate the PI3K and MAPK pathways in ILC and to validate new therapeutic targets. 539 POSTER BET Bromodomain Inhibition Targets Both c-Myc and IL7R in Acute Lymphoblastic Leukemia C. Ott1 , N. Kopp1 , L. Bird1 , R. Paranal1 , J. Qi1 , T. Bowman2 , S. Rodig2 , A. Kung3 , D. Weinstock1 , J. Bradner1 . 1 Dana-Farber Cancer Institute, Department of Medical Oncology, Boston MA, USA; 2 Brigham and Women’s Hospital, Department of Pathology, Boston MA, USA; 3 Dana-Farber Cancer Insitute, Department of Pediatric Oncology, Boston MA, USA The development of chemical inhibitors of proteins involved in epigenetic regulation of gene expression has led to the emergence of a limited number of promising cancer therapeutic agents specifically designed to target aberrant gene regulatory factors. These factors generally modify either chromatin or DNA by adding (‘writing’) or removing (‘erasing’) specific chemical moieties involved in oncogenic gene expression programs. Recently, small molecule inhibitors of ‘reader’ chromatin effector proteins have been developed. We have investigated the therapeutic potential of JQ1, an inhibitor of the BET class of human bromodomain proteins [1], in B- and T-cell acute lymphoblastic leukemia (ALL). Bromodomains bind to post-translationally acetylated lysines of histones and mediate assembly of macromolecular protein complexes required for transcriptional activation and polymerase elongation. JQ1 is a novel thieno-triazolo-1,4-diazapine that binds with high affinity into the acetyllysine binding pocket of BET bromodomains (including BRD2, BRD3, BRD4, and BRDT). We show that JQ1 potently reduces the viability of ALL cell lines with high-risk cytogenetics. Among the most sensitive B-ALL lines were those with rearrangements of CRLF2 which is overexpressed in approximately 10% of B-ALL. CRLF2 heterodimerizes with the IL7 receptor (IL7R) and signals through JAK2, JAK1, and STAT5 to drive proliferation and suppress apoptosis. Activating mutations in IL7R have also been described in a subset of both B- and T-ALL. As previously observed, JQ1 induced the downregulation of MYC transcription, loss of BRD4 at the MYC promoter, and the reduced expression of c-Myc target genes. Strikingly, JQ1 also downregulated IL7R transcription and depleted BRD4 from the IL7R promoter. This decrease in IL7R transcription led to a dramatic decrease in IL7R surface expression, leading to reduced JAK2 and STAT5 phosphorylation. Genome-wide expression profiling demonstrated a restricted effect of JQ1 on transcription with MYC and IL7R being among the most downregulated genes. In fact, IL7R was the only cytokine receptor in CRLF2-rearranged B-ALL cells significantly downregulated by

Poster Session – New Molecular Targets JQ1 treatment. This specific effect on IL7R transcription was also observed in high-expressing B and T-ALL cell lines. In mice xenografted with primary human CRLF2-rearranged B-ALL, JQ1 suppressed c-Myc expression and STAT5 phosphorylation, and significantly prolonged survival. These results demonstrate that BET bromodomain inhibition is a promising therapeutic strategy for ALL. Moreover, the surprising finding that JQ1 also targets IL7R expression suggests that bromodomain inhibitors may be especially useful in IL7R-mutated ALL and other disorders dependent on IL7R. References [1] Filippakopoulos et al, 2010. Selective inhibition of BET bromodomains. Nature, 468: 1067–1073. 540 POSTER Regulation and Function of the Nek11 Kinase in Colorectal Cancer S.R. Sabir1 , N.K. Sahota1 , G.D. Jones2 , A.M. Fry1 . 1 University of Leicester, Department of Biochemistry, Leicester, United Kingdom; 2 University of Leicester, Department of Cancer Studies and Molecular Medicine, Leicester, United Kingdom Background: Nek11 is one of eleven members of the human serine/ threonine NIMA-related protein kinase family, named Nek1 to Nek11. Of these, Nek1, Nek10 and Nek11 are implicated in the DNA damage response with Nek11 playing a central role in the G2/M checkpoint. In response to ionizing radiation, the ATM/ATR kinases phosphorylate and activate Chk1. Chk1 phosphorlyates Cdc25A at serine 76 and Nek11 at serine 273. This activates Nek11, which then phosphorylates serine 82 on Cdc25A. Together these phosphorylation events promote binding of SCF- - -b-TrCP- to Cdc25A leading to its degradation. As a result Cdk1 remains inactive and cells are arrested at the G2/M transition. Furthermore, it has been reported that Nek11 expression is upregulated in colorectal tumours. We therefore propose that targeting Nek11 may abrogate the DNA damage response and that, in combination with radiotherapy or DNA damaging agents, this may have clinical benefit in colorectal cancer patients. Materials and Methods: Firstly, we have used a qPCR approach to examine expression of four Nek11 splice variants in a range of human cancer cell lines. Secondly, we have generated stable U2OS cell lines expressing each of the four isoforms for the purpose of studying subcellular localization, kinase activity and functional response to different DNA damaging agents. Finally, we have optimized immunohistochemistry of Nek11 to enable us to examine Nek11 expression in primary tumours. Results: qPCR data showed that although all four Nek11 isoforms were expressed in each of the colorectal cancer cell lines, HCT116, HT29, SW480 and SW620, and the normal colorectal cell line, HCEC, the longest isoform was consistently more highly expressed compared to the others. Interestingly, the isoforms show different subcellular localisation patterns with the two longer isoforms being predominantly cytoplasmic and the shorter isoforms being localised to both the nucleus and cytoplasm. Using an antibody generated in-house to examine expression of Nek11 in tumour tissue, preliminary data suggest that expression is increased at the apical surface of lumen in colorectal tumour tissue. Conclusions: Nek11 is expressed as four alternatively spliced isoforms that show different mRNA expression levels and protein localization patterns. We are now examining their activity in response to DNA damaging agents to determine how they contribute to the DNA damage response in colorectal cancer cells. 541 POSTER The Histone Methyltransferase EZH2 is Required for Cell Growth in Diffuse Large B-cell Lymphoma P. Trojer1 , S. Garapaty1 , F. Lan1 , V. Balsubramanian2 , E. Chan1 , C. Hatton3 , R. Campbell4 , R. Cummings4 , E. Normant2 , B. Bryant3 . 1 Constellation Pharmaceuticals, Biology, Cambridge, USA; 2 Constellation Pharmaceuticals, Pharmacology, Cambridge, USA; 3 Constellation Pharmaceuticals, Bioinformatics, Cambridge, USA; 4 Constellation Pharmaceuticals, Lead Discovery, Cambridge, USA Lysine methyltransferases and demethylases were identified as transcriptional co-regulators functioning by either preserving particular chromatin methylation states or by controlling placement and removal of histone lysine methylation marks to promote dynamic changes in gene expression. The development of small molecule methyltransferase and demethylase inhibitors provides a novel approach to affect the regulation of transcription, and thus potentially allowing interference with aberrant transcriptional programs as observed, for instance, in cancer. Enhancer of Zeste Homolog 2 (EZH2), the major histone H3 lysine 27 (K27) methyltransferase, is widely implicated in tumor progression. The presence of a recurrent mutation of a single tyrosine residue in the EZH2 catalytic

Poster Session – New Molecular Targets domain in germinal center B-cell like diffuse large B-cell lymphoma (GCBDLBCL) and follicular lymphoma suggests that these cancers might be dependent on altered EZH2 molecular function, the mutation facilitating the conversion of a H3K27 di-methylated to a tri-methylated state. Inhibition of the EZH2 catalytic activity will provide a new therapeutic approach to treat human cancers, especially lymphomas carrying activating mutations. Constellation has identified, characterized and optimized potent, selective and reversible EZH2 small molecule inhibitors as well as studied the biological impact of such inhibition. We find that pharmacological inhibition of EZH2 causes cell viability defects with cell lines harboring EZH2 mutations being the most sensitive. Genome-wide mapping of EZH2 and H3K27me3 sites in the absence and presence of the compound revealed that the EZH2 inhibitor caused significant changes to the local chromatin modification landscape, however only a subset of these alterations translated into gene expression changes. The EZH2-controlled gene signature and the functional impact of altered expression on GCBDLBCL cell survival will be discussed. 542 POSTER The Experimental Anti-cancer Drug Mitochondrially-targeted Vitamin E Succinate Inhibits Mitochondrial Transcription J. Rohlena1 , J. Truksa1 , L. Dong2 , M. Vondrusova1 , K. Kluckova1 , J. Neuzil1 . 1 Institute of Biotechnology AS CR, Molecular Therapy Group, Prague 4, Czech Republic; 2 Griffith University, School of Medical Science, Southport QLD, Australia Background: The redox-silent vitamin E analogue a-tocopherol succinate (a-TOS) selectively eliminates cancer cells by reactive oxygen species (ROS)-induced apoptosis resulting from the inhibition of mitochondrial complex II (CII). Tagging a-TOS with the triphenyphosphonium group produced mitochondria-targeted vitamin E succinate (MitoVES), which accumulates in mitochondria and displays 10−50 times higher anti-cancer potential. While CII-dependent ROS-mediated apoptosis is an important effector mechanism of MitoVES, the possibility exists that additional factors, perhaps related to mitochondrial targeting, contribute to the elevated anticancer efficacy. Materials and Methods: MCF7 breast cancer and Jurkat T lymphoma cells were treated with MitoVES in vitro, and proliferation and apoptosis rates were assessed. Mitochondrial parameters such as respiration, ROS production, mitochondria membrane potential (DY mi ) and expression of mitochondrial genes were determined. The role of CII was investigated using a CII-deficient cell line. The findings were confirmed in vivo in a murine model of HER2-positive breast cancer (transgenic FVB/N c-neu mice). Results: MitoVES suppressed cell proliferation and mitochondrial biogenesis at sub-apoptotic concentrations. In contrast to untargeted a-TOS, MitoVES inhibited the transcription of mitochondrial genes, in particular the D-LOOP transcript, an effect that was diminished by antioxidants and absent in CII-deficient cells, which fail to produce ROS in response to MitoVES. ROS induction is therefore necessary but not sufficient for DLOOP reduction, as both MitoVES and a-TOS induce ROS, but only MitoVES reduces D-LOOP. MitoVES also dissipates DY mi and affects mitochondrial respiration, providing additional stress to mitochondria. Using other compounds acting on mitochondria, we confirmed that suppression of mtDNA transcript requires ROS production accompanied by DY mi dissipation and/or inhibition of respiration. The results were reproduced in vivo, where administration of MitoVES but not a-TOS to FVB/N c-neu mice reduced D-LOOP in the tumour tissue only, demonstrating selectivity of the agent. Conclusions: Inhibition of mitochondrial transcription and biogenesis represent an important novel aspect of anti-cancer activity of MitoVES, which occurs independently of the apoptogenic activity of the compound and may also be important for other mitochondria-affecting drugs. 543 POSTER Overexpression of Discoidin Domain Receptor 1 (DDR1) in Oral Squamous Cell Carcinoma W.H. Tsai1 , Y.L. Chen1 , H.C. Chen2 , A.J. Cheng3 , K.Y. Chang1 , P.Y. Chu4 , J.R. Hsiao5 , J.Y. Chang1 , S.F. Lin1 . 1 National Health Research Institutes, National Institute of Cancer Research, Miaoli, Taiwan; 2 Chang Gung University, Departments of Biomedical Sciences, Taoyuan, Taiwan; 3 Chang Gung University, Departments of Medical Biotechnology, Taoyuan, Taiwan; 4 St. Martin De Porres Hospital, Department of Pathology, Chiayi, Taiwan; 5 College of Medicine National Cheng Kung University, Department of Otolaryngology, Tainan, Taiwan Overexpression and mutation of protein tyrosine kinases (PTKs) are observed in a variety of human cancers, which are associated with poor

Friday 9 November 2012 167 prognosis. To identify PTKs that are overexpressed in oral cancer, the expressions of eighty-three PTKs in four Taiwanese oral squamous cell carcinoma (OSCC) lines and a pooled cDNA reference were compared by quantitative RT-PCR analysis. Among several hits, we found DDR1 was consistently over-represented in a panel of OSCC cell lines. In addition, by immunohistochemistry analysis of clinical OSCC specimens, we observed that DDR1 concentrations were positively correlated with tumor grades. Similarly, results from microarray analysis of forty tumor/normal-paired oral cancer biopsies revealed an average ~1.5-fold elevation of DDR1 expressions in the tumor parts relative to the normal parts. DDR1 is a non-integrin collagen receptor that has been implicated in a variety of malignancies including breast, prostate, brain, and hepatocellular carcinomas. By using shRNA-mediated depletion of DDR1 in OSCC cells, we found that DDR1 signaling pathway, including phosphorylation of AKT, phosphorylation of ERK1/2 and Bcl-2 expression, plays a critical role in OSCC cell growth. Furthermore, results of immunoprecipitationwestern blot analysis showed that the overexpressed DDR1 was tyrosinephosphorylated and known ligands of DDR1 (collagens I and IV) did not further enhance its phosphorylation state, indicating that DDR1 in these OSCC cells is constitutively-active and is ligand-independent. Finally, imatinib mesylate (Glivec/ Gleevec), a potent DDR1 inhibitor, was found to inhibit the growth of OSCC cells in a dose- and time-dependent manner. This result reinforces the role of DDR1 in OSCC cell survival. Collectively, our data suggest that DDR1 could be a novel therapeutic target for oral cancer treatment. 544 POSTER Characterization of Potent and Well Tolerated Second Generation FLT3 Inhibitors M. Ciomei1 , N. Amboldi1 , S. Cribioli1 , D. Donati1 , I. Fraietta1 , A. Galvani1 , C. Nuccio1 , E. Pesenti1 , G. Texido Romero1 , A. Lombardi Borgia1 . 1 Nerviano Medical Sciences Srl, Oncology, Nerviano (Milano), Italy FMS-like tyrosine kinase 3 (FLT3), a member of the class III receptor tyrosine kinase family, is characterized by an autoinhibitory juxtamembrane (JM) domain that docks with the kinase domain to stabilize a catalytically inactive conformation. Mutations or deletions in the JM or in adjacent regions can lead to constitutive kinase activation. FLT3 is mutated in approximately 30% of AML patients, with the so-called internal tandem repeat (ITD) insertion as the predominant mutation. Here, we describe the in vitro and in vivo activity of NMS-P948 and NMSP088, two members of a novel indazole sub-series, which selectively target FLT3, and which retain potent activity against FLT3 secondary mutations known to give rise to acquired resistance to inhibitors in current clinical development which target this kinase. Both compounds were able to arrest in vitro growth of the human AML cell lines MOLM-13 and MV-4−11, which bear the FLT3-ITD rearrangement, with IC50s in the low nanomolar range, and with high selectivity towards cell lines which are not dependent on FLT3. When tested against a panel of BA-F3 cells engineered to be driven by different FLT3-ITD mutants, both NMS-P948 and NMS-P088 compared favorably with quizartinib, a FLT3-selective inhibitor in advanced clinical development. For example, comparative IC50s in proliferation studies were 0.12 nM vs. 0.32 nM for NMS-088 vs. quizartinib respectively in BA-F3 FLT3-ITD (no secondary mutation), 1.9 nM vs. 21.4 nM in BA-F3 FLT3ITD(F691L) and 19.5 nM vs. 31.2 nM in BA-F3 FLT3-ITD(D835V). In vivo studies conducted on mice bearing disseminated MOLM-13 model revealed that both compounds were able to significantly increase survival time following repeated oral administration. Given the properties of NMS-P948 and NMS-P088 in terms of potent and selective activity against FLT3-ITD and clinically relevant secondary mutants, together with favourable exploratory ADME / toxicity profiles, which include good oral bioavailability and low/negligible activity against the hERG potassium ion channel (whose inhibition is associated with potential for QTc interval prolongation), this indazole subclass is currently undergoing intensive investigation towards selection of a candidate for development. 545 POSTER DHT Regulates Serum Response Factor Transcription Activity in Castrate-resistant Prostate Cancer Cell Lines M. Prencipe1 , A. O’Neill1 , G. O’Hurley2 , H. Klocker3 , E.W. Kay4 , W.R. Watson5 . 1 University College Dublin, Conway Instituteof Biomolecular and Biomedical Research, Dublin, Ireland; 2 OncoMark Limited, NovaUCD, Dublin, Ireland; 3 Innsbruck Medical University, Dept of Urology, Innsbruck, Austria; 4 RCSI Education and Research Centre Beaumont Hospital, Dept of Pathology, Dublin, Ireland; 5 University College Dublin, Conway Institute of Biomolecular and Biomedical Research, Dublin, Ireland Advanced prostate cancer is treated by hormone ablation therapy. However, despite an initial response, the majority of men relapse to develop a