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308 Androgen deprivation therapy sensitizes prostate cancer cells to T-cell killing through androgen receptor dependent modulation of the apoptotic pathway
Poster Session – Molecular Targeted Agents I metastases in the lung and liver, respectively, after intravenous injection in Tie2-hPTX3 mice. Also, the orthotopic growth of syngeneic pancreatic and mammary tumor cells was significantly reduced after injection in Tie2hPTX3 mice and led to increased survival compared to control mice. Finally, double transgenic TRAMP/Tie2-hPTX3 mice showed a significant delay of multistage prostate tumor onset and progression in respect to TRAMP mice. Our findings demonstrate for the first time that in vivo delivery of PTX3 exerts a dramatic impact on tumor growth, vascularization and metastasis. These results have set the basis for the identification of a low molecular weight PTX3-derived molecule that recapitulates the FGF-trap activities of PTX3 and exhibits promising therapeutic potential for FGF-dependent tumors. 308 POSTER Androgen deprivation therapy sensitizes prostate cancer cells to T-cell killing through androgen receptor dependent modulation of the apoptotic pathway S. Gameiro1 , A. Ardiani1 , A.R. Kwilas1 , R.N. Donahue1 , J.W. Hodge1 . 1 National Cancer Institute, CCR/LTIB, Bethesda MD, USA Despite recent advances in diagnosis and management, prostrate cancer remains the second most common cause of death from cancer in American men, after lung cancer. Failure of chemotherapies and hormonedeprivation therapies is the major cause of death in patients with castrationresistant prostate cancer (CRPC). Currently, the androgen inhibitors enzalutamide and abiraterone are approved for treatment of metastatic CRPC. Enzalutamide is a second-generation androgen antagonist that has no agonist activity. Abiraterone is a cytochrome P450 inhibitor that blocks adrenal and intratumoral androgen production. Here we show for the first time that both enzalutamide and abiraterone renders prostate tumor cells more sensitive to T cell-mediated lysis through immunogenic modulation, and that these immunomodulatory activities are androgen receptor (AR)-dependent. In studies reported here, the NAIP gene was significantly down-regulated in human prostate tumor cells treated in vitro and in vivo with enzalutamide. Functional analysis revealed that NAIP played a critical role in inducing CTL sensitivity. Amplification of AR is a major mechanism of resistance to androgen-deprivation therapy (ADT). Here, we show that enzalutamide enhances sensitivity to immune-mediated killing of prostate tumor cells that overexpress AR. The immunomodulatory properties of enzalutamide and abiraterone provide a rationale for their use in combination with immunotherapeutic agents in CRPC, especially for patients with minimal response to enzalutamide or abiraterone alone, or for patients who have developed resistance to ADT. 309 POSTER RNA sequencing and in silico analysis identifies an unannotated antisense long non-coding RNA involved in cancer progression S. Inoue1 , K. Horie-Inoue2 , K. Ikeda2 . 1 Univ of Tokyo, Dept of Anti-aging Med, Tokyo, Japan; 2 RCGM Saitama Med Univ, Div of Gene Regulation & Signal Transduction, Saitama, Japan Background: Long non-coding RNAs (lncRNAs) are RNAs that have no obvious coding capacities with their length >200 bp and widely transcribed across the entire genome in spatial and temporal specific manners. The biological roles of lncRNAs as tumor suppressors or activators are emerging in various types of cancer. Identification and functional analyses will be required for lncRNAs involved in cancer progression. Material and Methods: Here we attempt to identify a functional hormonedependent lncRNA in hormone-dependent cancer. We performed RNA sequencing in estrogen-sensitive or hydroxytamoxifen-resistant MCF7 breast cancer cells and conducted in silicoscreening of lncRNAs based on our sequencing data together with integrative annotation open databases of lncRNAs. The contribution of hormonal regulation to the expressions of lncRNAs is also investigated by mapping estrogen receptor binding sites across the genome. lncRNA expression was examined in several cancer cells by qRT-PCR. Effects of lncRNA knockdown on cell proliferation, cell cycle, and cell migration were evaluated by the transduction of specific siRNAs into cancer cells. Whether lncRNA knockdown influences in vivo tumor formation was evaluated by the injection of siRNAs into xenograft tumors in nude mice. Animal studies were approved by the Institutional Animal Care and Use Committee. Results: An unannotated estrogen-dependent lncRNA, denoted as BClnc-X, was identified, which is located in the vicinity of transcriptional start site and in the antisense direction of a coding gene. The genomic position of BClnc-X also includes a functional estrogen binding site. BClnc-X is expressed in prostate cancer cells as well as breast cancer cells. We also found that BClnc-X is expressed in clinical breast cancer samples. Specific siRNAs against BClnc-X substantially repressed the proliferation
Thursday 20 November 2014 101 and migration of MCF7 cells, as well as decreased the percentages of cells in S phase of cell cycle. BClnc-X knockdown could also reduce tumor formation in mice models. Conclusions: We consider that BClnc-X will contribute to the progression of cancer by putatively associating with hormone signaling. BClnc-X will be applied to clinical management of breast cancer as a potential molecular target for diagnostic and therapeutic options for the advanced disease. 310 POSTER Inhibition of Trk-driven tumors by the pan-Trk inhibitor RXDX-101 D. Anderson1 , M. Ciomei2 , P. Banfi2 , S. Cribioli2 , E. Ardini2 , A. Galvani2 , G. Li1 . 1 Ignyta Inc, San Diego CA, USA; 2 Nerviano Medical Sciences, Nerviano, Italy The Trk family of kinases, which include TrkA, TrkB and TrkC, are high affinity receptors for the neurotrophin family of nerve growth factors. Deregulated kinase activities of Trk family members due to chromosome rearrangements, gene mutations, splicing variants and overexpression have been shown to be associated with tumorigenesis and poor prognosis in a number of cancer types. Particularly, several chromosomal rearrangements involving TrkA have been reported in lung, colorectal, papillary thyroid, glioblastoma, melanoma and other cancers, and are believed to be the key oncogenic driver in these tumors. Therefore oncogenic Trk may represent a promising therapeutic target in Trk-driven tumors. RXDX-101 is an orally available, potent and selective ATP-competitive panTrk, ROS1 and ALK inhibitor, with comparable activities against TrkA, TrkB and TrkC in biochemical and cell based assays (IC50s <10 nM). In KM12, a human colorectal cancer cell line driven by constitutively active TrkA fusion TPM3-NTRK1, RXDX-101 exhibited in vitro anti-proliferative activity with an IC50 of 17 nM, accompanied by inhibition of TrkA phosphorylation and concomitant inactivation of downstream effectors, PLCgamma1, AKT and ERK, as well as cell cycle arrest and apoptosis. In mice bearing KM12 xenografts, treatment with RXDX-101 resulted in tumor regression and durable stasis under either intermittent or continuous dosing regimens, accompanied by sustained intratumoral inhibition of phospho-TrkA and PLCgamma1. In these studies, RXDX-101 was well tolerated during the course of treatment. In conclusion, our data indicates that RXDX-101, a potent and selective pan-Trk inhibitor currently in clinical development, is an attractive targeted agent for Trk-driven tumors. 311 POSTER IGF-1R inhibition induced activation of Yes/SFK acts as a by-pass resistance pathway in rhabdomyosarcoma X. Wan1 , C. Yeung1 , C. Heske1 , A. Mendoza1 , L.J. Hlman1 . 1 National Cancer Institute, Pediatric Oncology Branch, Bethesda, USA Objective: Development of clinical resistance has been common in IGF-1R block and represents a significant hindrance and limits treatment efficacy in the clinic. Thus, identifying the mechanisms of acquired resistance to IGF-1R blockade is a major goal. The aim of this study was to identify the molecular mechanisms responsible for acquired resistance to IGF-1R targeted therapy in rhabdomyosarcoma. Method: Expression profiles of IGF components and SFKs were analyzed by cDNA microarray. Antiproliferative effects of anti-IGF-1R agents and Src family kinase inhibitors alone or in combination were tested in vitro in multiple rhabdomyosarcoma (RMS) cell lines and in vivo using RD and Rh30 xenografts. Western blot and immunoprecipitation were performed to identify potential resistance mechanisms to IGF-1R inhibition. Results: cDNA microarray revealed that both IGF components and Yes are highly expressed in RMS. IGF-1R blockade either with R1507, an antibody against IGF-1R, or with BMS-754807, a small molecular inhibitor against of IGF-1R/IR, results in an increase in Yes activation, which is associated with resistance to IGF-1R blockade in RMS. Combining anti-IGF-1R agents with SFK inhibitors resulted in blockade of IGF-1R inhibition induced activation of Yes/SFK and displayed enhanced antitumor activity in vitro and in vivo. Conclusions: Our data demonstrate that IGF-1R inhibition induced activation of Yes/SFK may act as a by-pass resistance pathway. Cotargeting both IGF-1R and SFK shows advantageous antitumor activity in vitro and in vivo. Our findings may be of particular relevance clinically since both Yes and IGF components are overexpressed in RMS. Dual inhibition of IGF-1R and SFK may have a broader and enhanced clinical benefit for RMS patients.
Thursday 20 November 2014 101 and migration of MCF7 cells, as well as decreased the percentages of cells in S phase of cell cycle. BClnc-X knockdown could also reduce tumor formation in mice models. Conclusions: We consider that BClnc-X will contribute to the progression of cancer by putatively associating with hormone signaling. BClnc-X will be applied to clinical management of breast cancer as a potential molecular target for diagnostic and therapeutic options for the advanced disease. 310 POSTER Inhibition of Trk-driven tumors by the pan-Trk inhibitor RXDX-101 D. Anderson1 , M. Ciomei2 , P. Banfi2 , S. Cribioli2 , E. Ardini2 , A. Galvani2 , G. Li1 . 1 Ignyta Inc, San Diego CA, USA; 2 Nerviano Medical Sciences, Nerviano, Italy The Trk family of kinases, which include TrkA, TrkB and TrkC, are high affinity receptors for the neurotrophin family of nerve growth factors. Deregulated kinase activities of Trk family members due to chromosome rearrangements, gene mutations, splicing variants and overexpression have been shown to be associated with tumorigenesis and poor prognosis in a number of cancer types. Particularly, several chromosomal rearrangements involving TrkA have been reported in lung, colorectal, papillary thyroid, glioblastoma, melanoma and other cancers, and are believed to be the key oncogenic driver in these tumors. Therefore oncogenic Trk may represent a promising therapeutic target in Trk-driven tumors. RXDX-101 is an orally available, potent and selective ATP-competitive panTrk, ROS1 and ALK inhibitor, with comparable activities against TrkA, TrkB and TrkC in biochemical and cell based assays (IC50s <10 nM). In KM12, a human colorectal cancer cell line driven by constitutively active TrkA fusion TPM3-NTRK1, RXDX-101 exhibited in vitro anti-proliferative activity with an IC50 of 17 nM, accompanied by inhibition of TrkA phosphorylation and concomitant inactivation of downstream effectors, PLCgamma1, AKT and ERK, as well as cell cycle arrest and apoptosis. In mice bearing KM12 xenografts, treatment with RXDX-101 resulted in tumor regression and durable stasis under either intermittent or continuous dosing regimens, accompanied by sustained intratumoral inhibition of phospho-TrkA and PLCgamma1. In these studies, RXDX-101 was well tolerated during the course of treatment. In conclusion, our data indicates that RXDX-101, a potent and selective pan-Trk inhibitor currently in clinical development, is an attractive targeted agent for Trk-driven tumors. 311 POSTER IGF-1R inhibition induced activation of Yes/SFK acts as a by-pass resistance pathway in rhabdomyosarcoma X. Wan1 , C. Yeung1 , C. Heske1 , A. Mendoza1 , L.J. Hlman1 . 1 National Cancer Institute, Pediatric Oncology Branch, Bethesda, USA Objective: Development of clinical resistance has been common in IGF-1R block and represents a significant hindrance and limits treatment efficacy in the clinic. Thus, identifying the mechanisms of acquired resistance to IGF-1R blockade is a major goal. The aim of this study was to identify the molecular mechanisms responsible for acquired resistance to IGF-1R targeted therapy in rhabdomyosarcoma. Method: Expression profiles of IGF components and SFKs were analyzed by cDNA microarray. Antiproliferative effects of anti-IGF-1R agents and Src family kinase inhibitors alone or in combination were tested in vitro in multiple rhabdomyosarcoma (RMS) cell lines and in vivo using RD and Rh30 xenografts. Western blot and immunoprecipitation were performed to identify potential resistance mechanisms to IGF-1R inhibition. Results: cDNA microarray revealed that both IGF components and Yes are highly expressed in RMS. IGF-1R blockade either with R1507, an antibody against IGF-1R, or with BMS-754807, a small molecular inhibitor against of IGF-1R/IR, results in an increase in Yes activation, which is associated with resistance to IGF-1R blockade in RMS. Combining anti-IGF-1R agents with SFK inhibitors resulted in blockade of IGF-1R inhibition induced activation of Yes/SFK and displayed enhanced antitumor activity in vitro and in vivo. Conclusions: Our data demonstrate that IGF-1R inhibition induced activation of Yes/SFK may act as a by-pass resistance pathway. Cotargeting both IGF-1R and SFK shows advantageous antitumor activity in vitro and in vivo. Our findings may be of particular relevance clinically since both Yes and IGF components are overexpressed in RMS. Dual inhibition of IGF-1R and SFK may have a broader and enhanced clinical benefit for RMS patients.