Poster Session – New Molecular Targets Friday 9 November 2012 173

colon malignancies, among others. In this preclinical study we assessed theantitumor effects of the novel Plk inhibitor, TAK-960, against CRC models,including cell lines and patient-derived xenografts.Methods: The anti-proliferative effects of TAK-960 as a single agentand in combination with irinotecan (SN38) or cetuximab were assessedusing an assay that measures DNA content (CyQUANT). Synergy wascalculated using Calcusyn software while evaluation of downstream effectormolecules and apoptosis was assessed by immunoblotting. Patient-derivedCRC xenografts were implanted into athymic nude mice and tumor growthinhibition (TGI) was evaluated following treatment with TAK-960 alone or incombination with standard agents (irinotecan or cetuximab).Results: CRC cell lines were quite sensitive to TAK-960 with IC50 valuesranging from 0.007 to 1 umol/L. While no synergy was observed in theKRAS WT CRC cell lines in the cetuximab combination groups, additivityto mild synergy was observed in the KRAS MT CRC cell lines exposedto the SN38 combination. Modulation of down stream effector moleculeswas observed following exposure to TAK-960, including pHistone H3 andp73. Interestingly, against patient-derived xenograft models, synergy wasdifficult to assess in the KRAS WT models due to the exquisite sensitivityto cetuximab, while some of the KRAS MT xenografts did demonstrate TGIin the irinotecan combination groups that was supra-additive.Conclusion: The PLK inhibitor TAK-960 demonstrated robust single-agentanti-proliferative effects against CRC cell lines in vitro, whereas synergywas not observed when combined with cetuximab or SN38. However,there were supra-additive effects noted in several patient-derived KRAS MTxenografts treated with TAK-960 and irinotecan, supporting the evaluationof this regimen in this patient population with limited therapeutic options.

563 POSTEREx Vivo Culture of Fresh Tumor Tissues From Patients Assessing

Pharmacodynamic (PD) Markers of OTX008, a Novel Galectin-1

Inhibitor: Relevance to Phase I Trial

L. Astorgues-Xerri1, M.e. Riveiro2, A. Tijeras-raballand3, N. Muller4,K. Rezai5, M. Albuquerque4, V. Paradis4, K. Noel6, E. Raymond1,S. Faivre1. 1INSERM U728, Beaujon University Hospital, Clichy,France; 2Oncology Therapeutic Development, Preclinical department,Clichy, France; 3AAREC Filia Research, Beaujon University Hospital,Clichy, France; 4INSERM U773, Beaujon University Hospital, Clichy,France; 5Curie Institute, Rene Huguenin Hospital, Saint Cloud, France;6Oncoethix SA, Preclinical department, Lausanne, Switzerland

Background: Galectin-1, a multifunctional lectin, modulates cancer cellproliferation and tumor angiogenesis. OTX008, a synthetic molecule,reduces galectin-1 binding to carbohydrate inducing a conformationalchange. We previously showed that OTX008 inhibits cancer cell pro-liferation, normalizes angiogenesis, and decreases galectin-1 proteinexpression, inducing its translocation to the nucleus in cultured humancancer cells and xenograft models. This study compared PD effectsinduced by ex vivo treatment of tumor samples with OTX008 and PD effectsobserved in a biopsy sample taken following OTX008 treatment in a first-in-man clinical study.Material and Methods: To evaluate whether OTX008 48h- ex vivoexposure could modulate galectin-1 protein expression and localizationin tumor samples, biopsies were obtained from two patients with head &neck carcinoma and from a patient under OTX008 single agent treatmentwith an advanced heavily treated cutaneous angiosarcoma of the scalpthat progressed under chemotherapy. Tumor biopsies were cut in 300 mmthick slices using a tissue slicer. Each slice was randomly selected andexposed to different concentrations of OTX008 (10nM, 1mM and 10mM)and sunitinib (5mM) for 48h. At the end of the treatment, samples wereanalyzed by immunohistochemistry (IHC) or immunofluorescence (IF). IHCanalysis also was performed on tumor biopsy samples taken after 21 daystreatment with OTX008. OTX008 plasma concentrations were measuredby a validated UPLC-MS/MS method.Results: A 2-compartment open model adequately described the totalOTX008 time-concentration curve with linear elimination (following adose of 65mg SC, Cmax=7132 ng/ml; Tmax 0.5 to 1h; AUC=125mg.h/l;T1/2=5.6 h); OTX008 plasma concentrations were >1mM for >48h. BaselineIHC and IF data showed that tumor biopsies expressed high levels ofgalectin-1, primarily located in the cytoplasm of cancer cells. 48h ex vivoexposure to �10nM OTX008 significantly decreased galectin-1 proteinlevels compared to untreated and sunitinib-treated samples (p < 0.05) andwas associated with nuclear translocation of galectin-1 in head & neckcarcinoma and angiosarcoma samples. The biopsy sample taken after 21days of OTX008 treatment showed similar effects, with decreased intensityof galectin-1 staining compared to the pre-treatment sample.Conclusion: Our results show that ex vivo tumor tissue PD analysismay correlate with post-treatment PD data in patients with solid tumorsparticipating in clinical trials. Modulation of galectin-1 tissue staining couldbe a suitable marker to assess on-target effects of OTX008 treatment inclinical studies.

564 POSTERAnti-tumour Activity of the Focal Adhesion Kinase Inhibitor

GSK2256098C in Ovarian Cancer

D. Doughty Shenton1, K. McLeod1, M. Muir1, A. Kinnaird1, C. Gourley1,N.O. Carragher1, M.C. Frame1, V.G. Brunton1. 1Edinburgh CancerResearch Centre, Edinburgh, United Kingdom

Background: Ovarian cancer is the fifth most common cancer in womenin the UK and has a 5 year survival rate of only 30−40%. Current standardof care is surgery and platinum based chemotherapy and although initialresponses occur in some cases, disease recurrence and the emergence ofresistance is a major problem. New approaches to treatment are thereforerequired. Increased understanding of the molecular basis of cancer hasled to the development of new molecularly targeted therapies. One suchtarget is the tyrosine kinase focal adhesion kinase (FAK) which is over-expressed in a number of solid tumours including ovarian cancer where itis associated with shorter overall survival. Here we have assessed the anti-tumour effects of a small molecule kinase inhibitor of FAK, GSK2256098C,in primary human ovarian xenografts and cell lines.Methods: The effect of GSK2256098C on the growth of 11 human ovariancell lines in both 2-dimensional (2D) and 3-dimensional (3D) cultures wasdetermined and effects on cell signalling determined by western blotting.6 primary human ovarian xenografts were established in CD-1 nude mice.Mice received either vehicle (0.5% HPMC + 0.2% Tween) or GSK2256098C(75mg/kg) BID by oral gavage. Tumour growth was recorded by twiceweekly caliper measurements. At the end of the study reverse protein arrayand immunohistochemical analysis was carried out on the tumours.Results: There was no significant effect of GSK2256098C on the 2Dproliferation of any of the 11 cell lines, with IC50 values all >5mM. Thebasal level of phosphorylation of FAK on tyrosine (Y) 397 varied significantlybetween the cell lines and where measureable this was inhibited followingtreatment with GSK2256098C. However, GSK2256098C was able to inhibitthe 3D proliferation of 2 out of 4 ovarian cell lines tested and thiswas associated with reduced FAK Y397 phosphorylation. There was asignificant reduction in tumour growth in vivo following GSK2256098Ctreatment in one of the six primary xenografts. Reverse protein arrayanalysis showed that there were changes in a number of signallingmolecules including those of the TGFb and Wnt pathways.Conclusions: GSK2256098C inhibited FAK kinase activity, using areduction in FAK Y397 as a read-out of activity. GSK2256098C was ableto inhibit the proliferation of a sub-set of human ovarian tumours in vivoand in 3D indicating that FAK kinase inhibitors may have some utilityin the treatment of ovarian cancer. Further mapping of the signallingpathways that are altered in responsive and sensitive tumours followingGSK2256098C treatment will help define potential combination strategiesand identify possible biomarkers.

565 POSTERTargeting P38d MAPK in Oesophageal Cancer: a Possible Future

Therapeutic Goal?

C. O’Callaghan1, L. Fanning2, A. Houston2, O.P. Barry1. 1UniversityCollege Cork, Pharmacology and Therapeutics, Cork, Ireland; 2UniversityCollege Cork, Medicine, Cork, Ireland

Background: Esophageal cancer is an aggressive tumor which respondspoorly to both chemotherapy and radiation therapy and has a poorprognosis. Approximately half of patients diagnosed with localizedesophageal cancer die of metastatic disease within the first 2 yearsfollowing tumor resection. Thus, a greater understanding of the biology ofoesophageal cancer is needed in order to identify novel therapeutic targets.Among these targets p38 MAPK isoforms are becoming increasinglyimportant for a variety of cellular functions in mammalian cells. Their roles,however, are more complex than previously thought with distinct membersappearing to have different functions. A better understanding of the role(s)of p38 MAPKs may provide useful therapeutic tools for the managementof human cancers.Materials and Methods: We have analysed p38 MAPK isoformexpression in a range of cancer cell lines and corresponding human tissueincluding oesophageal, liver, lung, prostate, colon, renal and pancreaticand observed that over 75% of both cell lines and tissue samples failto express the specific isoform p38d MAPK. To evaluate the role(s)of p38d and active p-p38d MAPK in oesophageal cancer progressionwe developed (i) a series of constructs namely pcDNA3-FLAG-p38dMAPK, pcDNA3-MKK6b(E)-(Gly-Glu)5-FLAG-p38d, pcDNA3-MKK6b-(Gly-Glu)5-FLAG-p38d and pcDNA3-MKK6b(E)-(Gly-Glu)5-FLAG-p38dDN and(ii) stable primary (KE-3, KE-8) and metastatic (OC-1) oesophageal cancercell lines expressing these plasmids.Results: Re-introduction of p38d and active p-p38d proved to be (a) anti-proliferative, (b) anti-migratory and (c) pro-apoptotic. Briefly, we observeda time-dependent decrease in proliferation in oesophageal cancer cell

174 Friday 9 November 2012 Poster Session – New Molecular Targets

lines when stably transfected with p38d MAPK. This anti-proliferative effectwas exacerbated with cells transfected with active p-p38d MAPK (i.e.pcDNA3-MKK6b(E)-(Gly-Glu)5-FLAG-p38d or pcDNA3-MKK6b-(Gly-Glu)5-FLAG-p38d). Using a Boyden chamber and a wound healing assay weobserved a decrease in cellular invasion and migration when p38d orp-p38d MAPK was introduced to both primary and metastatic oesophagealcancer cells. Finally using flow cytometric analysis following staining withFITC-conjugated Annexin V and PI we observed that p38d and p-p38dMAPK confers a greater sensitivity to certain chemotherapeutic drugs butresistance to others.Conclusion: We now provide data for the anti-tumourigenic effect ofone specific p38 MAPK isoform i.e. p38d MAPK in oesophageal cancer.Our research may provide a new potential target for the treatment ofoesophageal cancer and metastases and increase our understanding of themechanisms of oesophageal cancer progression. Interestingly, our findingsmay also be applicable to other cancer types which lack this apparent anti-tumourigenic p38 isoform.

566 POSTERMechanisms of Resistance to MEK Inhibitor AZ6244 in KRAS Mutant

Metastatic Colorectal Cancer

V. Gambino1, C. Sun1, A. Prahallad1, A. Tzani1, W. Grernrum1,L. Mittempergher1, R. Bernards1. 1NKI-AVL, Molecular Carcinogenesis,Amsterdam, The Netherlands

Background: KRAS mutation (codons 12 or 13) occurs in 40% ofmetastatic colon rectal cancers (mCRC) and it is an absolute predictorof resistance to the EGFR targeted agents. These mutations in advancedCRC are associated with poor prognosis and there is no effective targetedtherapy available for these tumor types to date.Heterogeneous response to MEK inhibitor in KRAS mutant CRC has beenobserved in both in vitro and in vivo experiments, while no clinical data isavailable for CRC patients in respect of the mutational status of KRAS.To identify biomarkers of differential drug response and eventually findrational drug combinations that overcome the unresponsiveness of CRCto MEK inhibition, we use shRNA-based screen for genetic determinantsof MEK inhibitor sensitivity.Material and Methods: To identify down-regulated genes that conferresistance to the MEK inhibitor AZ6244 we screened a genome wideshRNA bar code library in a sensitive KRASG12V colon rectal cell line(SKCO-1). The genome wide library used consists of over 150,000 hairpinstargeting 15,000 human genes.In parallel, we performed a negative selection shRNA-based screen in aMEK inhibitor resistant KRASG12V CRC cell line (SW480) for kinaseswhose inhibition restores sensitivity to AZ6244. The Kinome library usedrepresents the full complement of 518 human kinases and 17 additionalkinase-related genes.Results from the two genetic screenings will be presented.

567 POSTERA Novel Focal Adhesion Kinase Inhibitor (PF-04554878) Decreases

Growth and Induces Apoptosis in Pancreatic Neuroendocrine Tumor

Cells

R. Francois1, S. Lu2, D. He2, M. Zajac-Kaye1, S. Hochwald2. 1Universityof Florida, Anatomy & Cell Biology, Gainesville Florida, USA; 2Universityof Florida, Surgery, Gainesville Florida, USA

Background: Pancreatic neuroendocrine tumors (PNETs) are poorlyunderstood and there are few effective therapies. Focal adhesion kinase(FAK) is an important tyrosine kinase implicated in cell survival signalingand found to be overexpressed in many malignancies.Methods: By Western blot, the expression of FAK was determined infrozen human PNETs compared to matched normal human pancreas. FAKexpression was also evaluated in pancreatic samples from a conditionalMen1−/− murine model of PNET progression. The compound, PF-04554878(Pfizer), is an ATP competitive kinase inhibitor of FAK. The in-vitro effectsof this compound on cell signaling, viability, clonogenicity, anchorage-independent growth and apoptosis in human pancreatic neuroendocrinecells (BON-1, QGP-1, and CM) were evaluated.Results: FAK was found to be overexpressed in human PNETs comparedto matched normal pancreas. Increased FAK activation was also observedin tumors compared to normal pancreas in a conditional transgenic mousemodel of PNET progression. PF-04554878 not only caused a decrease incell viability with an IC50 of 5 mM in human pancreatic neuroendocrine celllines, but also inhibited clonogenicity and anchorage independent growthin a dose-dependent manner. Of note, PF-04554878 caused a statisticallysignificant dose-dependent increase in apoptosis by TUNEL staining andPARP cleavage in all cell lines tested. These effects were associated witha decrease in tyrosine phosphorylation (Y397) of FAK.Conclusion: A novel compound that inhibits the kinase activity of FAKresults in decreased PNET viability, anchorage dependent and independent

growth, and induction of apoptosis. These effects appear to be mediatedthrough downregulation of p-FAK (Y397). This compound deserves furtherstudy as a novel treatment strategy in human PNETs.

568 POSTERDesign of PTX008 That Allosterically Targets Galectin-1 to Inhibit

Tumor Growth in Mice

R.P.M. Dings1, J.I. Levine2, L. Astorgues-Xerri3, N. Kumar1, M. Serova3,J. MacDonald4, E. Raymond3, T.R. Hoye2, K.H. Mayo1. 1University ofMinnesota, Biochemistry Molecular Biology and Biophysics, Minneapolis,USA; 2University of Minnesota, Chemistry, Minneapolis, USA; 3INSERMU728, Medical Oncology, Clichy, France; 4PepTx, Inc, Excelsior, USA

Target-based and activity-based approaches are two, relatively generaltherapeutic drug discovery strategies. Currently, activity-based drugdiscovery is undergoing a renaissance due to limited predictive valueof in vivo toxicity and efficacy using the target-based approach. Here,we employed a structure-activity-based approach to discover new potentanti-tumor agents. Our design approach took us from de novo designedpeptides to partial peptide mimetics and ultimately to fully non-peptidicorganic-scaffolded topomimetics of the folded peptides. After severalrounds of design optimization, we had two compounds PTX008 andPTX009 that inhibited tumor growth in mouse models by up to 90% at dailydoses of 10mg/Kg. PTX008 targets galectin-1 as an allosteric inhibitor ofcarbohydrate binding. Galectin-1, which plays a key role in cell adhesionand migration, is upregulated and highly expressed in cells within thetumor microenvironment, and tumor growth is attenuated by inhibiting thelectin. PTX008 demonstrates no apparent in vivo toxicity and exhibits anexcellent pharmacodynamics profile in animals. This compound is currentlyin a Phase I clinical trial with cancer patients. As a next generation effort,we improved the anti-tumor activities of PTX008 by chemically modifyingthe topomimetic surface. We found that one of the new agents, PTX013,is particularly effective at inhibiting the growth of several drug resistanthuman cancers via induction of cell cycle arrest in sub-G1 and G0/G1phases. In the syngeneic B16F10 melanoma tumor mouse model, PTX013(0.5mg/Kg) inhibits tumor growth by about 50-fold better than PTX008.A preliminary pharmacodynamics study strongly suggests that PTX013exhibits good in vivo exposure and a relatively long half-life. Overall, thisresearch contributes to the discovery of novel therapeutics as potentiallyuseful agents against cancer in the clinic.

569 POSTERPrognostic Impact of Splicing Variants in Pediatric Brain Tumors: in

Silico Analysis From High Density Microarrays

O. Perez-Gonzalez1, M. Macias-Vega1, R. Cardenas-Cardos1,A. Marhx-Bracho1, R. Rivera-Luna1. 1Instituto Nacional de Pediatria,Cancer Genomics Research Laboratory, Distrito Federal, Mexico

Purpose: Primary Central Nervous System Tumors are the most frequentsolid malignant tumors in childhood, in particular those derived fromastrocytes, aracnoids and ependimum. The prognostic criterion is basedon the anatomic localization, size and surgical accessibility for resection.Never the less, this criteria is not enough to predict a therapeutic successin all patients, neither we know precisely the underlying biological processinvolved in pediatric cerebral tumor’s behavior. The goal of this research isto identify splicing variants from treatment response associated genes inpediatric brain tumors using high density microarrays.Methods: We consider neoplastic tissue samples from pediatric patientswith Ependymomas, Astrocythomas and Meduloblastomas for RNApurification (RNAeasy minikit, Qiagen). Those RNAs With RNA IntegrityNumber greater than 8 Were used for microarray hybridization onthe GeneChip® Human Gene 1.0 ST Array (Affymetrix) according tothe recommended protocol. The statistic analysis was performed withAffymetrix Expression Console v1.1 and Partek’s Genomics Suite, forquality assessment (with Robust Multi-Chip Analysis) and gene expressionprofiles discrimination analysis, respectively. Splicing variants were theresult of ASANOVA algorithm in Partek’s Genomics Suite.Results: We included to the study 46 samples of brain tumors from threehistological types, Ependymomas, Astrocythomas and Meduloblastomas.We found a set of genes differentially expressed according to eachhistological subtype and their treatment response rate. Based on this set,we obtain a subset of genes with significant splicing variants, some of themunreported. Details of these results will be presented at the meeting.Conclusion: These results show that significative gene expression profileswith splicing variants may clearly differentiate ependymomas, astrocytomasand medulloblastomas according to their histological subtype and treatmentresponse rate. Significative splicing variants are being analyzed andvalidated as a prognostic tool in order to provide a more accurate allocationcriteria for antineoplastic treatments in pediatric CNST.