- Email: [email protected]
p15 pathway to promote growth of colon cancer cells
164 Friday 21 November 2014 Conclusion: STAT3 ASO effects in immune and stromal cells in preclinical models and patient samples suggest that immune modulatory effects may contribute to the activity associated with early clinical responses. These data provide additional clinical hypotheses for AZD9150. 502 POSTER MI130004, an antibody–drug conjugate including a novel payload of marine origin: Evidences of in vivo activity 1 P.M. Aviles1 , M.J. Guillen1 , J.M. Dominguez1 , M.J. Munoz-Alonso ˜ , L.F. Garcia-Fernandez1 , M. Garranzo1 , V. Martinez1 , A. Francesch1 , 1 1 1 1 S. Munt , C.M. Galmarini , C. Cuevas . PharmaMar S.A., Preclinical, Colmenar Viejo (Madrid), Spain
Background: Antibody–drug conjugates (‘ADCs’) have emerged as powerful tools for the treatment of cancer, combining the potency of cytotoxic molecules with the selectivity of antibodies targeted towards specific antigens exclusively present in cancer cells. The identification of such potent molecules is one of the most critical bottlenecks in the generation of ADCs. In this regard marine compounds represent an interesting opportunity worth exploring as they possess the requirements needed to be considered promising payloads. This poster presents data on the potency and selectivity of an ADC (MI130004) conjugating trastuzumab with a compound of marine origin. Materials and Methods: PM050489, a marine compound belonging to the PharmaMar collection, was modified to include a suitable linker containing a maleimide group to enable conjugation to Cys residues. Conjugation to trastuzumab was performed after reduction of the antibody with a phosphine reagent and the resulting conjugate (MI130004) was purified by size exclusion chromatography. The purified species was tested for its cytotoxic potential against selected cell lines with high (HCC-1954 and SKBR-3) or null (MCF-7 and MBA-MD-231) HER2 expression. To evaluate MI130004 in vivo 5−7 weeks old SCID female mice were subcutaneously implanted with a suspension of 5×106 JIMT-1 cells in 100 ml of cell culture medium. Tumor (ca. 115 mm3 ) bearing animals (N = 10/group) were randomly allocated to receive MI130004 (10 mg/kg) or appropriated control treatments (placebo included). Treatments were administered weekly for 5 consecutive weeks. Antitumor effect was calculated using DT/DC (%), defined as a percentage of the change in tumor size for treated (T) and placebo (C) groups during the placebo-treated survival time (D). Complete tumor regression (CR) was defined when tumor volume <63 mm3 for 2 or more consecutive measurements. Results: MI130004 showed a remarkable selectivity towards cell lines with high HER2 expression (IC50s 0.282 and 0.182 nM against HCC-1954 and SK-BR-3 cell lines respectively) whilst HER2 null cells were unaffected in the range of concentrations tested (up to 300 nM). In vivo, MI130004 also demonstrated efficiency in causing an outstanding tumor reduction, with complete regressions in most of the animals that received the treatment. Conclusions: MI130004 has demonstrated selective and significant in vitro activity in HER2 overexpressing cell lines. In agreement, the treatment with MI130004 demonstrated significant in vivo antitumor activity in mice bearing JIMT-1 xenografts. These results confirm the remarkable potential of marine compounds for the design of new ADCs with therapeutic anticancer properties. 503 POSTER UNC2025: A small molecule inhibitor of merTK with efficacy in mouse melanoma models S. Earp1 , D. Darr2 , A. Holtzhausen2 , A. Zimmermann2 , K. Clark2 , L. Hunter2 , N. Sharpless2 , X. Wang3 , S. Frye3 , D. Graham4 . 1 UNC Lineberger Comprehensive Cancer Center, Chapel Hill, USA; 2 The University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, Chapel Hill, USA; 3 The University of North Carolina at Chapel Hill, Center for Integrative Chemical Biology and Drug Discovery, Chapel Hill, USA; 4 University of Colorado, Cancer Center and Department of Pediatrics, Aurora, USA Background: MerTK, a receptor tyrosine kinase normally expressed in macrophages, epithelium and reproductive tissues, is expressed at higher levels in metastatic melanoma than in primary melanoma or nevi. We have previously shown that treatment of melanoma cell lines with UNC developed MerTK small molecule kinase inhibitors reduces growth and invasiveness (J Clin Invest. 2013; 123: 2257−67). MerTK in macrophages serves to detect and ingest apoptotic material with externalized phosphatidyl serine. In doing so, the MerTK kinase physiologic signal dampens the innate immune inflammatory cytokine response, polarizing macrophages to an M2-wound healing phenotype. Thus, inhibition of MerTK in cancer might have a dual anti-neoplastic action, first inhibiting a tumor cell survival signal produced by aberrantly expressed
Poster Session – Molecular Targeted Agents II MerTK and second reversing the immune suppressive tumor-associated macrophage phenotype in the tumor’s microenvironment. Methods: We treated genetically-engineered melanoma mouse models (GEMMs) using UNC2025, a small molecule MerTK inhibitor with sub nanomolar potency in vitro, high oral bioavailability, and pharmacokinetic properties that allowed the drug to be delivered solubilized in mouse chow. We used this mode of delivery to treat the Tria mouse (Tyrosinase Hras transgene in an INK4a/Arf null background) either as sporadic tumors as they arose or by using a Tria tumor-derived cell line implanted orthotopically in NSG mice. We also treated mouse melanomas when they arose in a mutant BRAF/PTEN null melanoma GEMM. Results: Previously, we reported the Tria GEMM melanomas exhibited growth inhibition of the initial tumor when treated with UNC2025. Here we update those results with expanded numbers (N = 12) and show that the median survival is extended from 21.5 days in vehicle treated animals to 70 days in UNC2025 treated animals (p = 0.005). There were three initial complete responses not seen with in over 10 types of therapy in our experience using this model. In addition, UNC2025 also significantly (n = 5 vehicle vs. treated each group p < 0.01) slowed the growth of the Tria tumors implanted in NSG mice. There were no complete responses and the result was not as substantial as that seen in the immune-competent GEMM potentially suggesting that MerTK inhibition had both anti-tumor cell and immuno-mediated anti-tumor effects. In addition, UNC2025 treatment of spontaneously arising established melanoma in Braf/PTEN deleted mice significantly suppressed the growth of the initial tumors versus vehicle treated mice (n = 4 p < 0.006). Conclusions: UNC2025 has desirable PD/PK properties allowing its efficacious use in melanoma pre-clinical models. Further experiments will be needed to determine anti-tumor mechanism and determine if innate immune stimulatory mechanisms plays a role. 504 POSTER Frequent loss-of-function mutations in MLK4 suppresses signaling in the JNK-cJUN-p21/p15 pathway to promote growth of colon cancer cells A. Marusiak1 , N. Stephenson1 , H. Baik1 , E. Trotter1 , Y. Li2 , E. Testoni1 , K. Blyth3 , S. Mason3 , L. Puto4 , C. Miller2 , T. Hunter4 , O. Sansom3 , J. Brognard5 . 1 Cancer Research UK Manchester Institute, Signaling in Cancer Group, Manchester, United Kingdom; 2 Cancer Research UK Manchester Institute, Computational Biology Group, Manchester, United Kingdom; 3 Beatson Cancer Research UK, Colorectal Cancer and Wnt Signalling Group, Glasgow, United Kingdom; 4 Salk Institute for Biological Studies, San Diego, USA; 5 Cancer Research UK Manchester Institute, SNC, Manchester, United Kingdom MLK4 is a member of the mixed-lineage kinase (MLK) family that regulates the activity of specific MAPK pathways. MLKs are activated by environmental stress, cytokines and growth factors and phosphorylate JNK, p38 and ERK signaling pathways. Mutations have been identified in MLK4 in various human cancers, especially in colon cancer at a frequency of approximately 7%. The aim of this study is to assess the contribution of these mutations in the process of tumor development and assess the signaling pathways influenced downstream of mutant MLK4. Biochemical and structural analysis of MLK4 mutations imply that the majority are lossof-function (LOF). Furthermore, reconstitution of MLK4 WT in colon cancer cells harboring these inactivating mutations reduced viability, proliferation and the ability to form colonies. Reduced tumor growth was observed following reconstitution of MLK4 WT in mouse xenograft models. Finally, restoring the function of MLK4 WT in these colon cancer cell lines induced selective activation of the JNK pathway and its downstream targets, cJUN, ATF3 and p21/p15. In summary, these results indicate that the presence of LOF mutations in MLK4 will suppress signaling in the JNK pathway to promote proliferation. 505 POSTER The anti-ErbB3 antibody, EV20, counteracts vemurafenib resistance in BRAF-mutated colorectal cancer stem cells G. Sala1 , P.R. Prasetyanti2 , D. Barcaroli3 , S. Volpe3 , E. Capone3 , C. Rossi3 , R. Carollo4 , M. Todaro4 , G. Stassi4 , J.P. Medema2 , S. Iacobelli1 , V. De Laurenzi3 . 1 “G. d’Annunzio University”, Mediapharma s.r.l., Chieti, Italy; 2 Academic Medical Center University of Amsterdam, Center for Experimental Molecular Medicine, Amsterdam, Netherlands; 3 “G. d’Annunzio University”, Department of Experimental and Clinical Sciences, Chieti, Italy; 4 University of Palermo, Central Laboratory of Advanced Diagnosis and Biomedical Research (CLADIBIOR), Palermo, Italy Background: Approximately 10% of metastatic colorectal cancers harbor V600E BRAF mutation and this has been correlated with resistance to
Background: Antibody–drug conjugates (‘ADCs’) have emerged as powerful tools for the treatment of cancer, combining the potency of cytotoxic molecules with the selectivity of antibodies targeted towards specific antigens exclusively present in cancer cells. The identification of such potent molecules is one of the most critical bottlenecks in the generation of ADCs. In this regard marine compounds represent an interesting opportunity worth exploring as they possess the requirements needed to be considered promising payloads. This poster presents data on the potency and selectivity of an ADC (MI130004) conjugating trastuzumab with a compound of marine origin. Materials and Methods: PM050489, a marine compound belonging to the PharmaMar collection, was modified to include a suitable linker containing a maleimide group to enable conjugation to Cys residues. Conjugation to trastuzumab was performed after reduction of the antibody with a phosphine reagent and the resulting conjugate (MI130004) was purified by size exclusion chromatography. The purified species was tested for its cytotoxic potential against selected cell lines with high (HCC-1954 and SKBR-3) or null (MCF-7 and MBA-MD-231) HER2 expression. To evaluate MI130004 in vivo 5−7 weeks old SCID female mice were subcutaneously implanted with a suspension of 5×106 JIMT-1 cells in 100 ml of cell culture medium. Tumor (ca. 115 mm3 ) bearing animals (N = 10/group) were randomly allocated to receive MI130004 (10 mg/kg) or appropriated control treatments (placebo included). Treatments were administered weekly for 5 consecutive weeks. Antitumor effect was calculated using DT/DC (%), defined as a percentage of the change in tumor size for treated (T) and placebo (C) groups during the placebo-treated survival time (D). Complete tumor regression (CR) was defined when tumor volume <63 mm3 for 2 or more consecutive measurements. Results: MI130004 showed a remarkable selectivity towards cell lines with high HER2 expression (IC50s 0.282 and 0.182 nM against HCC-1954 and SK-BR-3 cell lines respectively) whilst HER2 null cells were unaffected in the range of concentrations tested (up to 300 nM). In vivo, MI130004 also demonstrated efficiency in causing an outstanding tumor reduction, with complete regressions in most of the animals that received the treatment. Conclusions: MI130004 has demonstrated selective and significant in vitro activity in HER2 overexpressing cell lines. In agreement, the treatment with MI130004 demonstrated significant in vivo antitumor activity in mice bearing JIMT-1 xenografts. These results confirm the remarkable potential of marine compounds for the design of new ADCs with therapeutic anticancer properties. 503 POSTER UNC2025: A small molecule inhibitor of merTK with efficacy in mouse melanoma models S. Earp1 , D. Darr2 , A. Holtzhausen2 , A. Zimmermann2 , K. Clark2 , L. Hunter2 , N. Sharpless2 , X. Wang3 , S. Frye3 , D. Graham4 . 1 UNC Lineberger Comprehensive Cancer Center, Chapel Hill, USA; 2 The University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, Chapel Hill, USA; 3 The University of North Carolina at Chapel Hill, Center for Integrative Chemical Biology and Drug Discovery, Chapel Hill, USA; 4 University of Colorado, Cancer Center and Department of Pediatrics, Aurora, USA Background: MerTK, a receptor tyrosine kinase normally expressed in macrophages, epithelium and reproductive tissues, is expressed at higher levels in metastatic melanoma than in primary melanoma or nevi. We have previously shown that treatment of melanoma cell lines with UNC developed MerTK small molecule kinase inhibitors reduces growth and invasiveness (J Clin Invest. 2013; 123: 2257−67). MerTK in macrophages serves to detect and ingest apoptotic material with externalized phosphatidyl serine. In doing so, the MerTK kinase physiologic signal dampens the innate immune inflammatory cytokine response, polarizing macrophages to an M2-wound healing phenotype. Thus, inhibition of MerTK in cancer might have a dual anti-neoplastic action, first inhibiting a tumor cell survival signal produced by aberrantly expressed
Poster Session – Molecular Targeted Agents II MerTK and second reversing the immune suppressive tumor-associated macrophage phenotype in the tumor’s microenvironment. Methods: We treated genetically-engineered melanoma mouse models (GEMMs) using UNC2025, a small molecule MerTK inhibitor with sub nanomolar potency in vitro, high oral bioavailability, and pharmacokinetic properties that allowed the drug to be delivered solubilized in mouse chow. We used this mode of delivery to treat the Tria mouse (Tyrosinase Hras transgene in an INK4a/Arf null background) either as sporadic tumors as they arose or by using a Tria tumor-derived cell line implanted orthotopically in NSG mice. We also treated mouse melanomas when they arose in a mutant BRAF/PTEN null melanoma GEMM. Results: Previously, we reported the Tria GEMM melanomas exhibited growth inhibition of the initial tumor when treated with UNC2025. Here we update those results with expanded numbers (N = 12) and show that the median survival is extended from 21.5 days in vehicle treated animals to 70 days in UNC2025 treated animals (p = 0.005). There were three initial complete responses not seen with in over 10 types of therapy in our experience using this model. In addition, UNC2025 also significantly (n = 5 vehicle vs. treated each group p < 0.01) slowed the growth of the Tria tumors implanted in NSG mice. There were no complete responses and the result was not as substantial as that seen in the immune-competent GEMM potentially suggesting that MerTK inhibition had both anti-tumor cell and immuno-mediated anti-tumor effects. In addition, UNC2025 treatment of spontaneously arising established melanoma in Braf/PTEN deleted mice significantly suppressed the growth of the initial tumors versus vehicle treated mice (n = 4 p < 0.006). Conclusions: UNC2025 has desirable PD/PK properties allowing its efficacious use in melanoma pre-clinical models. Further experiments will be needed to determine anti-tumor mechanism and determine if innate immune stimulatory mechanisms plays a role. 504 POSTER Frequent loss-of-function mutations in MLK4 suppresses signaling in the JNK-cJUN-p21/p15 pathway to promote growth of colon cancer cells A. Marusiak1 , N. Stephenson1 , H. Baik1 , E. Trotter1 , Y. Li2 , E. Testoni1 , K. Blyth3 , S. Mason3 , L. Puto4 , C. Miller2 , T. Hunter4 , O. Sansom3 , J. Brognard5 . 1 Cancer Research UK Manchester Institute, Signaling in Cancer Group, Manchester, United Kingdom; 2 Cancer Research UK Manchester Institute, Computational Biology Group, Manchester, United Kingdom; 3 Beatson Cancer Research UK, Colorectal Cancer and Wnt Signalling Group, Glasgow, United Kingdom; 4 Salk Institute for Biological Studies, San Diego, USA; 5 Cancer Research UK Manchester Institute, SNC, Manchester, United Kingdom MLK4 is a member of the mixed-lineage kinase (MLK) family that regulates the activity of specific MAPK pathways. MLKs are activated by environmental stress, cytokines and growth factors and phosphorylate JNK, p38 and ERK signaling pathways. Mutations have been identified in MLK4 in various human cancers, especially in colon cancer at a frequency of approximately 7%. The aim of this study is to assess the contribution of these mutations in the process of tumor development and assess the signaling pathways influenced downstream of mutant MLK4. Biochemical and structural analysis of MLK4 mutations imply that the majority are lossof-function (LOF). Furthermore, reconstitution of MLK4 WT in colon cancer cells harboring these inactivating mutations reduced viability, proliferation and the ability to form colonies. Reduced tumor growth was observed following reconstitution of MLK4 WT in mouse xenograft models. Finally, restoring the function of MLK4 WT in these colon cancer cell lines induced selective activation of the JNK pathway and its downstream targets, cJUN, ATF3 and p21/p15. In summary, these results indicate that the presence of LOF mutations in MLK4 will suppress signaling in the JNK pathway to promote proliferation. 505 POSTER The anti-ErbB3 antibody, EV20, counteracts vemurafenib resistance in BRAF-mutated colorectal cancer stem cells G. Sala1 , P.R. Prasetyanti2 , D. Barcaroli3 , S. Volpe3 , E. Capone3 , C. Rossi3 , R. Carollo4 , M. Todaro4 , G. Stassi4 , J.P. Medema2 , S. Iacobelli1 , V. De Laurenzi3 . 1 “G. d’Annunzio University”, Mediapharma s.r.l., Chieti, Italy; 2 Academic Medical Center University of Amsterdam, Center for Experimental Molecular Medicine, Amsterdam, Netherlands; 3 “G. d’Annunzio University”, Department of Experimental and Clinical Sciences, Chieti, Italy; 4 University of Palermo, Central Laboratory of Advanced Diagnosis and Biomedical Research (CLADIBIOR), Palermo, Italy Background: Approximately 10% of metastatic colorectal cancers harbor V600E BRAF mutation and this has been correlated with resistance to