380 Identification & Biological Characterization of a Highly Potent, Irreversible Inhibitor of FGFR, TAS-2985

116 Thursday 8 November 2012 on targeting the interactions between these signaling pathways and key converging downstream effectors. This study aimed to determine how the tumorigenic phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), tumorsuppressive phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and transforming growth factor-beta (TGF-beta) pathways are integrated via the metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1). Moreover, we assessed how the novel anti-tumor agent, Dp44mT, may target these integrated pathways through its ability to increase NDRG1 expression. Materials and Methods: Primary cultures of normal human prostate epithelial cells and the prostate cancer cell lines, PC-3 and DU145, were incubated with Dp44mT for 24 h, and the expression of proteins of interest were analyzed by western blotting. The effects of Dp44mT on cell cycle distribution and proliferation were also examined in these cells. Results: We demonstrated that Dp44mT increased levels of PTEN, which is commonly mutated or deleted in prostate cancer, and decreased phosphorylation of ERK1/2 and SMAD2L, which are regulated by oncogenic Ras signaling. Importantly, we observed that the effects of Dp44mT on NDRG1 and p-SMAD2L expression were more marked in prostate cancer cells than normal prostate epithelial cells. This may partly explain the anti-tumor selectivity of these agents. To examine the role of NDRG1 in mediating these effects, we silenced NDRG1 using shRNA, and observed increased phosphorylation of tumorigenic AKT, ERK1/2 and SMAD2L and decreased PTEN levels, while NDRG1 over-expression induced the opposite effect. Furthermore, NDRG1 silencing significantly reduced the ability of Dp44mT to suppress p-SMAD2L and p-ERK1/2 levels. Conclusions: These results demonstrate an important role for NDRG1 in mediating the tumor-suppressive effects of Dp44mT in prostate cancer via selective targeting of the PI3K/AKT, TGF-beta and ERK pathways. Importantly, these studies may aid in the development of more specific treatments for prostate cancer patients. 380 POSTER Identification & Biological Characterization of a Highly Potent, Irreversible Inhibitor of FGFR, TAS-2985 H. Sootome1 , H. Fujita1 , H. Ochiiwa1 , K. Ito1 , T. Sagara1 , M. Chiba1 , H. Hirai1 , Y. Iwasawa1 , T. Utsugi1 . 1 TTaiho Pharmaceutical Co Ltd., Tsukuba Research Center, Tsukuba, Japan Background: FGFs (fibroblast growth factors) and their receptors (FGFRs) play crucial roles in regulation of cell proliferation, survival, migration and differentiation. Deregulation of FGFR expression has been reported in multiple cancers, including breast, bladder, lung, gastric, endometrial and multiple myeloma. FGFRs are overactivated by several mechanisms such as gene amplification, translocation and mutations in a variety of cancers. FGFRs may represent an important target in the treatment of cancer. In this report, we present a novel small molecule inhibitor of FGFR, TAS-2985, and its biological characterization in vitro. Materials and Methods: In vitro enzyme inhibition of FGFRs was determined by using purified FGFR1−4 and peptide substrates. Cellular phosphorylation of FGFR2 was assayed by ELISA (R&D Systems) or by Western blot. For growth inhibition assay, cells were treated with TAS-2985 for 3 days, and living cells were determined by using CellTiter-GloTM which measures cellular ATP. Results: TAS-2985 was a potent inhibitor of FGFR1−4 enzymes with IC50 at sub nM levels. TAS-2985 is highly selective for FGFR versus other serine/threonine and tyrosine kinases. In vitro, TAS-2985 inhibited cellular phosphorylation of FGFRs as well as intercellular signaling pathways downstream of FGFR. In a cell proliferation assay, TAS-2985 potently suppressed growth of human cancer cell lines in a FGFR-dependent manner. In a panel of gastric cancer cell lines, this compound selectively inhibited growth of cell lines with FGFR gene abnormalities. Conclusion: TAS-2985 is a highly potent, selective, irreversible FGFR inhibitor. It potently inhibited growth of human cancer cell lines selectively, in a FGFR-dependent manner. 381 POSTER Investigation of RasGRP3 Expression and Function in Human Breast Cancers and Breast-derived Ductal Adenocarcinoma Cell Lines Z. Nagy1 , E. Orosz1 , I. Kovacs ´ 2 , M. Tor ¨ ok ¨ 2 , T. B´ıro´ 1 , P.M. Blumberg3 , G. Czifra1 . 1 University of Debrecen, Department of Physiology, Debrecen, ´ Hospital, Department of Pathology, Debrecen, Hungary; 2 Gyula Kenezy Hungary; 3 NIH National Cancer Institute, Center for Cancer Research, Bethesda MD, USA RasGRP3 is a member of the Ras guanine nucleotide releasing protein (RasGRP) family of the Ras-specific guanine nucleotide exchange factors. These proteins play important role in the regulation of the activity of Ras signaling pathway which constitutive activation is demonstrated in many

Poster Session – Signal Transduction Modulators cancer types. The RasGRP3 proteins have potential oncogenic effect, the amplification of the gen of the protein is observed in many malignant cancer types, for example in Burkitt’s lymphoma and pre-B-cell leukemia. Our recent investigations have highlighted that RasGRP3 plays a role in tumorigenesis, hence it exerts effect on the proliferation, migration, survival and tumorigenecity of prostate adenocarcinoma-derived and melanoma cells. In light of this potential oncogenic effect we have examined the change of expression and potencial function of RasGRP3 in one of the most malignant cancer type, the breast-derived ductal adenocarcinoma. The RasGRP3 and phosphoRasGRP3 expressions were examined in human ductal adenocarcinoma-derived samples from different grades and in 1 primary ductal adenocarcinoma-derived cell line called BT-474 and 5 different metastatic: JIMT-1, MCF7, SK-BR-3, MDA-MB-453 es ´ T-47D cell lines both in mRNA (Q-PCR) and protein levels (Western blot; immunhistoand cytochemistry). To explore the biological function of the protein RasGRP3 knockdown cultures were created on MCF7 and T-47D cell lines using retroviral transfection. To examine the role of RasGRP3 in the viability of cells annexin-V/PI staining analyzed by flow citometry was performed. To clarify the protein’s function in cell proliferation and in the developement of resistance to the most common clinical chemotherapeutic drugs Tamoxifen and Herceptin CyQuant assay were performed. In addition to observe the RasGRP3 function in tumor formation and maintance, the SCID mouse model was used. According to our results the expression of RasGRP3 and the active phosphoRasGRP3 were elevated both at the mRNA and protein levels on the human ductal adenocarcinoma samples. This expression was increased in the tumor samples compared to the normal. RasGRP3 was found typically in the cytoplasm of the cells, while the phosphoRasGRP3 showed strong nuclear reaction. The RasGRP3 expression of the BT-474 cell line was significantly lower then in the metastatics. The downregulation of RasGRP3 did not induce significant apoptosis or necrosis, but inhibited cell proliferation and sensitizied the T-47D cells to killing by Tamoxifen and Herceptin. In vivo tumor growth in mouse xenografts of both cell lines was decreased. Our results suggest the RasGRP3 may have an importante role in the regulation of cell growth, chemotherapeutic resistance, and tumor formation in breast cancer. 382 POSTER Preclinical Pharmacokinetics of GE-huMab-HER3, a Novel Humanized, Glycoengineered Anti-HER3 Antibody Using PHER3/HER3 Ratio as Pharmacodynamics (PD) Marker G. Meneses-Lorente1 , I. Kolm2 , M. Thomas2 , T. Friess2 , S. Bader3 , C. Meille4 , B. Bossenmaier2 . 1 Roche Products Limited, Clinical Pharmacology, Welwyn, United Kingdom; 2 Roche Diagnostics GmbH, Discovery Oncology, Penzberg, Germany; 3 Roche Diagnostics GmbH, Bioinformatics and Exploratory Data Analysis, Penzberg, Germany; 4 F. Hoffman-La Roche GmbH, Bioinformatics and Exploratory Data Analysis, Basel, Switzerland GE-huMab-HER3 is a novel humanized (hu) and glycoengineered (GE) IgG1 antibody that binds to HER3 with high specificity and affinity. It is currently tested in phase I clinical trials and will be evaluated as a potential therapy for various human solid tumors. To determine the in vivo efficacy, SCID beige mice bearing ~200 mm3 FaDu xenografts were treated once a week for 3 weeks with GEhuMab-HER3 over the dose range from 0.3 mg/kg to 10 mg/kg. In this chronic study, measurements of tumor volume, exposure of GE-huMabHER3 and signalling pathway biomarkers were assessed. In order to evaluate the effect of total HER3 and pHER3 decrease as well as the full pharmacokinetic profile of GE-huMab-HER3 also following a single dose administration, a group of mice bearing FaDu xenografts were treated with single dose of 0.3 mg/kg and 1 mg/kg of GE-huMab-HER3. Full pharmacokinetics, HER3 and pHER3 profiles were assessed in this study. GE-huMab-HER3 showed a dose-dependent effect to inhibit tumor growth in FaDu xenograft models of human cancer, reaching a plateau between 3 mg/kg and 10 mg/kg, together with a dose response decrease of pHER3 and HER3 or even better in the pHER3/HER3 ratio. Trough GEhuMabHER3 concentrations (Ct) from mice administered with the lower doses (0.3 and 1 mg/kg) showed no or only minimal accumulation during the course of treatment from 1st to 3rd administration. Mice dosed with 3 and 10 mg/kg showed increased Ct from the first administration to the 3rd administration. This distinct exposure behaviour was consistent with better efficacy and a decrease of HER3 and pHER3. After single dose administration the pharmacokinetic profile in mice dosed with 0.3 mg/kg and 1 mg/kg correlated with the pHER3/HER3 ratio profile with a maximum PD effect of more than 90% pHER3/HER3 decrease at 1 mg/kg. The faster elimination shown in the 0.3 mg/kg group is consistent with an earlier return of pHER3/HER3 to basal levels. In conclusion the presented studies describes the pharmacokinetic profile of GE-huMab-HER3 in mice, its correlation with antitumor activity in a xenograft model, and confirms the