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408 A potent and selective small molecule inhibitor of MCL-1 sensitizes DLBCL cell lines to the BCL-2 selective inhibitor ABT-199
130 Thursday 20 November 2014 targets in lung cancer, we used genetic and/or pharmacological approaches in the abovementioned cells to inactivate AURKA and/or AURKB. Inducible shRNA-mediated knockdown of AURKA or AURKB in KRAS-positive H358 and A549 cell lines, as well as treatment with a dual Aurora kinase inhibitor (AI II, Calbiochem), decreased growth, viability, migration, proliferation, and anchorage-independent growth. In addition, Aurora inhibition in both cell lines efficiently increased cell death by apoptosis. Interestingly, these effects were observed only in the presence of KRAS mutations, and Aurora inhibition had no effect on normal or tumorigenic cells without KRAS mutations or with inhibition of KRAS expression by RNA interference. This suggests that Aurora kinase inhibition therapy can specifically target KRAS transformed cells. Conclusions: In conclusion, our results support our hypothesis that Aurora kinases are important KRAS targets in lung cancer and suggest Aurora kinase inhibition as a novel approach for KRAS-induced lung cancer therapy. 406 POSTER Aflibercept (Zaltrap) directly attenuates the migration and invasion of colorectal cancer cells 1 ´ , M. Ayadi1 , V. Poindessous1 , M. Chiron2 , A. Bouygues1 , P. Mesange E. Dochy3 , T. Andre´ 4 , A. de Gramont4 , A.K. Larsen1 . 1 INSERM & ˆ Universite´ Pierre et Marie Curie Hopital Saint-Antoine, Cancer Biology & Therapeutics Saint-Antoine Research Center, Paris 12, France; 2 Sanofi France, Oncology, Vitry-sur-Seine, France; 3 Sanofi Belgium, Medical Development, Diegem, Belgium; 4 Hopital Saint-Antoine, Medical Oncology, Paris 12, France
Background: Colorectal cancer (CRC) cells express VEGF as well as functional VEGF receptors thereby mediating both paracrine and autocrine VEGF-signaling. Autocrine VEGF-signaling in CRC cells has been associated with resistance to 5-FU and hypoxia as well as with increased migration and invasion. Aflibercept (Zaltrap® also known as zivaflibercept in the United States) is a soluble recombinant protein approved for treatment of metastatic CRC that selectively neutralizes VEGF-A, VEGF-B and PlGF. Material and Methods: HCT-116 CRC cells and their 5-fluorouracil (5-FU) resistant variant were subject to ELISA analysis for the secretion of VEGF-A, VEGF-B and PlGF under normoxia and hypoxia. The Boyden chamber was used to establish the influence of aflibercept on CRC cell migration and invasion. The cytotoxic effects of 5-FU in the absence or presence of aflibercept was determined by the MTT viability assay. Results: The secretion of VEGF-A was significantly up-regulated in the 5-FU resistant cells compared to the parental cells under both normoxia and hypoxia (p < 0.01 and p < 0.001, respectively) as well as in the correspondent tumor xenografts (p < 0.01) whereas the expression of VEGF-B and PlGF was comparable. The increased VEGF-A levels were associated with increased migration of the 5-FU resistant cells, compared to the parental cells, under both normoxia and hypoxia and could be attenuated by aflibercept in both cell lines. Invasion was significantly increased for the 5-FU resistant cells under both normoxia and hypoxia and could be attenuated by aflibercept in the resistant cells under hypoxia. Aflibercept by itself had no detectable influence on the viability of parental or 5-FU resistant cells under normoxia or hypoxia, but was able to increase the sensitivity of the 5-FU resistant cells to 5-FU under hypoxia. Conclusions: Aflibercept directly attenuated the migration of CRC cells under all conditions examined without any influence on the viability. Cells with acquired 5-FU resistance showed up-regulation of VEGF-A associated with increased migration and invasion, especially under hypoxic conditions. Aflibercept inhibited these VEGF-A mediated functions. 407 POSTER Structural basis for inhibition of ligand-dependent and -independent ErbB3 activation by KTN3379 D. Alvarado1 , S. Lee2 , E. Greenlee2 , G.F. Ligon1 , J.S. Lillquist1 , E.J. Natoli1 , J. Amick2 , Y. Hadari1 , J. Schlessinger2 . 1 Kolltan Pharmaceuticals Inc., Department of Research, New Haven, USA; 2 Yale School of Medicine, Department of Pharmacology, New Haven, USA Background: The goal of this study is to understand the structural basis for inhibition of ligand-dependent and ligand-independent activation of ErbB3 by KTN3379, an antibody in phase 1 clinical development. Materials and Methods: X-ray crystallography was used to solve the structure of KTN3379 Fab in complex with the complete ErbB3 ectodomain. Results: Here, we report that the monoclonal antibody KTN3379 robustly inhibits both mechanisms of ErbB3 activation in different tumor model settings. The crystal structure of the KTN3379 Fab in complex with the full ErbB3 extracellular domain reveals that the antibody binds with very
Poster Session – Paediatric Oncology high affinity to a novel and unique epitope in the boundary of domains 2 and 3, and locks the receptor in an inactive conformation. Thus, KTN3379 interferes with the first step of ErbB3 activation, which is required for both ligand-dependent and ligand-independent signaling. Using structureguided mutations, we improved the affinity of the antibody-receptor complex even further by engineering additional contacts of the Fab to domain 2 in ErbB3. Conclusions: Our studies uncover the structural basis for the double mechanism of action of KTN3379 and define a unique epitope within ErbB3. The mechanism of inhibition described here should be also applicable to EGFR and ErbB4. 408 POSTER A potent and selective small molecule inhibitor of MCL-1 sensitizes DLBCL cell lines to the BCL-2 selective inhibitor ABT-199 D.C. Phillips1 , Y. Xiao2 , L. Lam1 , E. Litinovic3 , L. Roberts-Rapp1 , A.J. Souers1 , J.D. Leverson4 . 1 AbbVie Inc., Discovery Oncology, North Chicago, USA; 2 AbbVie Inc., Early Discovery Oncology, North Chicago, USA; 3 Abbott Molecular Inc., Des Plaines, USA; 4 AbbVie Inc., Oncology Development, North Chicago, USA Background: Aberrant expression and/or function of BCL-2 family proteins, which are essential regulators of apoptosis, contribute to the development of cancer. Diffuse Large B-cell lymphoma (DLBCL) represents the most common subtype of non-Hodgkin’s lymphoma (NHL). As a population, DLBCL cell lines positive for the t(14;18) translocation and/or possessing elevated BCL-2 copy number (BCL-2High ) are sensitive to navitoclax or the BCL-2 selective inhibitor ABT-199. Despite this, some BCL-2High cell lines remain resistant to either agent. Herein we utilize novel, selective and potent inhibitors of MCL-1 (A-1210477) or BCL-xL (A-1155463) to evaluate the role of these proteins in resistance to navitoclax and ABT-199 in DLBCL. Materials and Methods: We assessed the cytotoxicity of navitoclax, ABT-199, A-1155463 alone or in combination with the MCL-1 inhibitor A-1210477 or the CDK9 inhibitor flavopiridol in a panel of navitoclaxresistant DLBCL cell lines. Synergistic potential was subsequently determined via Bliss analysis. Additionally, expression of BCL-2 family members was determined by luminex and the mechanism of synergy determined using MesoScale ELISA. Results: We show that the MCL-1 specific inhibitor A-1210477 sensitizes navitoclax-resistant DLBCL cell lines to apoptosis. Chemical segregation of this synergy with the BCL-2 selective inhibitor ABT-199 or BCL-xL selective inhibitor A-1155463 indicated that MCL-1 and BCL-2 are the two key anti-apoptotic targets for sensitization. Synergy between navitoclax and A-1210477 in the majority of BCL-2Low DLBCL cell lines was BCL-xL driven. The CDK9 inhibitor flavopiridol down-regulated MCL-1 expression and synergized with ABT-199 in BCL-2High DLBCL cell lines, correlating with the synergy observed between ABT-199 and A-1210477. Conclusions: Collectively these data emphasize that BCL-2 status is predictive of ABT-199 efficacy in DLBCL not only as a single agent, but also in the adjuvant setting with anti-tumorigenic agents that modulate MCL-1 levels. These studies also potentially identify a patient population (BCL-2Low ) that would benefit from BCL-xL (navitoclax) driven combination therapy. Disclosures: DCP, YX, LL, LRR, AJS & JDL are AbbVie employees and are stock holders. The design, study conduct and financial support were provided by AbbVie. AbbVie participated in the data generation, interpretation of data, review and approval of this publication. EL is an employee of Abbott Molecular Inc.
Paediatric Oncology 409 POSTER Transcription factor activating protein 2 beta (TFAP2B) mediates neuronal differentiation in neuroblastoma F. Ikram1 , S. Ackermann1 , F. Roels1 , R. Volland1 , B. Hero1 , F. Hertwig1 , 3 H. Kocak1 , D. Dreidax2 , K.O. Henrich2 , F. Berthold1 , P. Nurnberg ¨ , F. Westermann2 , M. Fischer1 . 1 University Hospital of Cologne, Department of Pediatric Oncology, Cologne, Germany; 2 German Cancer Research Center, Department of Tumor Genetics, Heidelberg, Germany; 3 University of Cologne, Cologne Center for Genomics (CCG), Cologne, Germany Background: Induction of tumor cell differentiation by retinoic acid is an important part of current neuroblastoma treatment protocols. The molecular mechanisms underlying differentiation processes in neuroblastoma, however, are still poorly understood. Genes of transcription factors of
Background: Colorectal cancer (CRC) cells express VEGF as well as functional VEGF receptors thereby mediating both paracrine and autocrine VEGF-signaling. Autocrine VEGF-signaling in CRC cells has been associated with resistance to 5-FU and hypoxia as well as with increased migration and invasion. Aflibercept (Zaltrap® also known as zivaflibercept in the United States) is a soluble recombinant protein approved for treatment of metastatic CRC that selectively neutralizes VEGF-A, VEGF-B and PlGF. Material and Methods: HCT-116 CRC cells and their 5-fluorouracil (5-FU) resistant variant were subject to ELISA analysis for the secretion of VEGF-A, VEGF-B and PlGF under normoxia and hypoxia. The Boyden chamber was used to establish the influence of aflibercept on CRC cell migration and invasion. The cytotoxic effects of 5-FU in the absence or presence of aflibercept was determined by the MTT viability assay. Results: The secretion of VEGF-A was significantly up-regulated in the 5-FU resistant cells compared to the parental cells under both normoxia and hypoxia (p < 0.01 and p < 0.001, respectively) as well as in the correspondent tumor xenografts (p < 0.01) whereas the expression of VEGF-B and PlGF was comparable. The increased VEGF-A levels were associated with increased migration of the 5-FU resistant cells, compared to the parental cells, under both normoxia and hypoxia and could be attenuated by aflibercept in both cell lines. Invasion was significantly increased for the 5-FU resistant cells under both normoxia and hypoxia and could be attenuated by aflibercept in the resistant cells under hypoxia. Aflibercept by itself had no detectable influence on the viability of parental or 5-FU resistant cells under normoxia or hypoxia, but was able to increase the sensitivity of the 5-FU resistant cells to 5-FU under hypoxia. Conclusions: Aflibercept directly attenuated the migration of CRC cells under all conditions examined without any influence on the viability. Cells with acquired 5-FU resistance showed up-regulation of VEGF-A associated with increased migration and invasion, especially under hypoxic conditions. Aflibercept inhibited these VEGF-A mediated functions. 407 POSTER Structural basis for inhibition of ligand-dependent and -independent ErbB3 activation by KTN3379 D. Alvarado1 , S. Lee2 , E. Greenlee2 , G.F. Ligon1 , J.S. Lillquist1 , E.J. Natoli1 , J. Amick2 , Y. Hadari1 , J. Schlessinger2 . 1 Kolltan Pharmaceuticals Inc., Department of Research, New Haven, USA; 2 Yale School of Medicine, Department of Pharmacology, New Haven, USA Background: The goal of this study is to understand the structural basis for inhibition of ligand-dependent and ligand-independent activation of ErbB3 by KTN3379, an antibody in phase 1 clinical development. Materials and Methods: X-ray crystallography was used to solve the structure of KTN3379 Fab in complex with the complete ErbB3 ectodomain. Results: Here, we report that the monoclonal antibody KTN3379 robustly inhibits both mechanisms of ErbB3 activation in different tumor model settings. The crystal structure of the KTN3379 Fab in complex with the full ErbB3 extracellular domain reveals that the antibody binds with very
Poster Session – Paediatric Oncology high affinity to a novel and unique epitope in the boundary of domains 2 and 3, and locks the receptor in an inactive conformation. Thus, KTN3379 interferes with the first step of ErbB3 activation, which is required for both ligand-dependent and ligand-independent signaling. Using structureguided mutations, we improved the affinity of the antibody-receptor complex even further by engineering additional contacts of the Fab to domain 2 in ErbB3. Conclusions: Our studies uncover the structural basis for the double mechanism of action of KTN3379 and define a unique epitope within ErbB3. The mechanism of inhibition described here should be also applicable to EGFR and ErbB4. 408 POSTER A potent and selective small molecule inhibitor of MCL-1 sensitizes DLBCL cell lines to the BCL-2 selective inhibitor ABT-199 D.C. Phillips1 , Y. Xiao2 , L. Lam1 , E. Litinovic3 , L. Roberts-Rapp1 , A.J. Souers1 , J.D. Leverson4 . 1 AbbVie Inc., Discovery Oncology, North Chicago, USA; 2 AbbVie Inc., Early Discovery Oncology, North Chicago, USA; 3 Abbott Molecular Inc., Des Plaines, USA; 4 AbbVie Inc., Oncology Development, North Chicago, USA Background: Aberrant expression and/or function of BCL-2 family proteins, which are essential regulators of apoptosis, contribute to the development of cancer. Diffuse Large B-cell lymphoma (DLBCL) represents the most common subtype of non-Hodgkin’s lymphoma (NHL). As a population, DLBCL cell lines positive for the t(14;18) translocation and/or possessing elevated BCL-2 copy number (BCL-2High ) are sensitive to navitoclax or the BCL-2 selective inhibitor ABT-199. Despite this, some BCL-2High cell lines remain resistant to either agent. Herein we utilize novel, selective and potent inhibitors of MCL-1 (A-1210477) or BCL-xL (A-1155463) to evaluate the role of these proteins in resistance to navitoclax and ABT-199 in DLBCL. Materials and Methods: We assessed the cytotoxicity of navitoclax, ABT-199, A-1155463 alone or in combination with the MCL-1 inhibitor A-1210477 or the CDK9 inhibitor flavopiridol in a panel of navitoclaxresistant DLBCL cell lines. Synergistic potential was subsequently determined via Bliss analysis. Additionally, expression of BCL-2 family members was determined by luminex and the mechanism of synergy determined using MesoScale ELISA. Results: We show that the MCL-1 specific inhibitor A-1210477 sensitizes navitoclax-resistant DLBCL cell lines to apoptosis. Chemical segregation of this synergy with the BCL-2 selective inhibitor ABT-199 or BCL-xL selective inhibitor A-1155463 indicated that MCL-1 and BCL-2 are the two key anti-apoptotic targets for sensitization. Synergy between navitoclax and A-1210477 in the majority of BCL-2Low DLBCL cell lines was BCL-xL driven. The CDK9 inhibitor flavopiridol down-regulated MCL-1 expression and synergized with ABT-199 in BCL-2High DLBCL cell lines, correlating with the synergy observed between ABT-199 and A-1210477. Conclusions: Collectively these data emphasize that BCL-2 status is predictive of ABT-199 efficacy in DLBCL not only as a single agent, but also in the adjuvant setting with anti-tumorigenic agents that modulate MCL-1 levels. These studies also potentially identify a patient population (BCL-2Low ) that would benefit from BCL-xL (navitoclax) driven combination therapy. Disclosures: DCP, YX, LL, LRR, AJS & JDL are AbbVie employees and are stock holders. The design, study conduct and financial support were provided by AbbVie. AbbVie participated in the data generation, interpretation of data, review and approval of this publication. EL is an employee of Abbott Molecular Inc.
Paediatric Oncology 409 POSTER Transcription factor activating protein 2 beta (TFAP2B) mediates neuronal differentiation in neuroblastoma F. Ikram1 , S. Ackermann1 , F. Roels1 , R. Volland1 , B. Hero1 , F. Hertwig1 , 3 H. Kocak1 , D. Dreidax2 , K.O. Henrich2 , F. Berthold1 , P. Nurnberg ¨ , F. Westermann2 , M. Fischer1 . 1 University Hospital of Cologne, Department of Pediatric Oncology, Cologne, Germany; 2 German Cancer Research Center, Department of Tumor Genetics, Heidelberg, Germany; 3 University of Cologne, Cologne Center for Genomics (CCG), Cologne, Germany Background: Induction of tumor cell differentiation by retinoic acid is an important part of current neuroblastoma treatment protocols. The molecular mechanisms underlying differentiation processes in neuroblastoma, however, are still poorly understood. Genes of transcription factors of