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495 Divergent androgen regulation of UPR pathways drives prostate cancer
Poster Session – Molecular Targeted Agents II 493 POSTER Preclinical pharmacokinetic (PK)/pharmacodynamic (PD)/Efficacy modeling for MLN2480, an investigational pan-RAF kinase inhibitor, in A375 and SKMEL-2 human melanoma xenografts M. Patel1 , J. Chouitar2 , J. Mettetal1 , E. Gangolli3 , S. Balani1 , P. Shimoga1 , K. Galvin2 , W.C. Shyu1 , A. Chakravarty1 , C.J. Zopf1 . 1 Takeda Pharmaceuticals International Co., DMPK, Cambridge, USA; 2 Takeda Pharmaceuticals International Co., Cancer Pharmacology, Cambridge, USA; 3 Takeda Pharmaceuticals International Co., Translational Medicine Oncology, Cambridge, USA Background: MLN2480 is an orally administered, investigational small molecule pan-RAF kinase inhibitor currently in Phase 1 clinical development. Here, we built integrated PK/PD/efficacy models to understand the relationship between MAPK pathway inhibition and efficacy as well as the effect of dose schedule in two melanoma xenograft models, A375 and SKMEL-2. Materials and Methods: We fit a one-compartment PK model to MLN2480 plasma concentration time profiles following a single dose in mice. Tumor pERK levels in xenograft-bearing mice, measured by Western blotting following single (A375) or multiple (SKMEL-2) doses of MLN2480 were fit to a direct or indirect inhibitory Emax model, respectively, to describe the PK/PD relationship between the plasma concentration and %pERK inhibition. Activity in each xenograft was evaluated as the percent growth rate inhibition (%GRI), the percent change between treated and control exponential tumor volume growth rates over the treatment cycle. To connect PK, PD, and efficacy for each xenograft, we simulated the PK and PD profiles over the treatment cycle for each dose group, and estimated the averages of plasma concentration (Cavg ) and %pERK inhibition, respectively, using non-compartmental analysis. Results: From a multiple linear regression, schedule-related parameters are not predictive for drug effect demonstrating schedule-independent activity. For a variety of dose schedules, the relationship between total dose and SKMEL-2 xenograft growth rate is approximately linear (R2 = 0.90, p < 0.0001). A sigmoidal PK/efficacy model captures the relationship between Cavg and %GRI for both xenografts. A375 exhibits greater sensitivity than SKMEL-2 to the same Cavg of MLN2480, with the models predicting tumor regression (Emax = 172% GRI) and stasis (Emax = 102% GRI) as the maximal effects, respectively. Both xenografts have steep, sigmoidal PD/efficacy relationships, which show the same pERK inhibition is associated with greater %GRI in A375, and %GRI saturates with residual pERK (EC90 = 38% in A375, 22% in SKMEL-2). Conclusions: Integrated PK/PD and PK/efficacy modeling for MLN2480 response in A375 and SKMEL-2 xenografts shows a strong relationship between pERK inhibition and preclinical activity, and that maximal effect is achieved without complete inhibition of the MAPK pathway. The findings of pERK inhibition-dependent but not schedule-dependent activity provide translational guidance to clinical dose and schedule selection. 494 POSTER LPA6 promotes growth and tumorigenicity of hepatocellular carcinoma via activation of PIM-3 proto-oncogene kinase C. Lopane1 , V. Goffredo1 , F. Dituri1 , F. De Santis2 , A. Filannino3 , R.C. Betz4 , Y.Y. Li5 , N. Mukaida6 , P. Winter7 , C. Tortorella8 , G. Giannelli9 , C. Sabba` 8 , A. Mazzocca8 . 1 University of Bari School of Medicine, Interdisciplinary Department of Medicine, Bari, Italy; 2 Institute Curie, Centre de Recherche, Paris, France; 3 Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy; 4 Department of Emergency and Organ Transplantation, University of Bonn, Bonn, Germany; 5 Cancer Research Institute, Fudan University Shanghai Cancer Center, Shanghai, China; 6 Division of Molecular Bioregulation, Kanazawa University, Kanazawa, Japan; 7 GenXPro GmbH, Kanazawa University, Frankfurt Main, Germany; 8 Interdisciplinary Department of Medicine, University of Bari School of Medicine, Bari, Italy; 9 Department of Emergency and Organ Transplantation, University of Bari School of Medicine, Bari, Italy Background: Aberrations occurring in hepatocytes during hepatocarcinogenesis confer the tumorigenic properties needed for hepatocellular carcinoma (HCC) development. Overexpression of certain receptors is common among these alterations and recent evidence suggests that the expression of lysophosphatidic acid (LPA) receptors is dysregulated in HCC. However, the contribution of these receptors to the pathogenesis of HCC has not previously been evaluated. We investigated the role of LPA receptor-6 (LPA6) in HCC tumorigenesis and growth and its mechanisms. Material and Methods: We used a gain- and loss-of-function approach to elucidate how LPA6 sustains the HCC tumorigenic process. Additionally, deletion mutants were employed to corroborate the involvement of LPA6 in this process. We also performed an extensive gene profiling analysis
Friday 21 November 2014 161 by MACE to gain insight into genes under the control of LPA6. Finally, we analyzed LPA6 expression in HCC tissues and its correlation with patient survival. Results: We found that RNA-interference-mediated knockdown of LPA6 impaired tumorigenicity in human HCC xenografts and the expression profile of several genes, including proto-oncogene Pim-3 regulated by LPA6 at gene-promoter level. Conversely, overexpression of LPA6 in nontumorigenic HCC cells led to Pim-3 upregulation and the acquisition of a tumorigenic phenotype. Moreover, our experiments with deleted mutants indicate that the STAT3 binding site is important for the transcriptional activation of Pim-3 by LPA6. Lastly, we found that LPA6 overexpression in HCC patients is correlated with high proliferation rates, elevated Pim-3 levels and a worse clinical outcome. Conclusions: Taken together, our data demonstrate a novel tumorigenic mechanism and provide a rationale for therapeutic targeting of the LPA6−Pim-3 axis in HCC. 495 POSTER Divergent androgen regulation of UPR pathways drives prostate cancer Y.J. Arnoldussen1 , M. Storm1 , X. Sheng1 , M. Tesikova1 , Y. Jin1 , H.Z. Nenseth1 , S. Zhao1 , I.G. Mills2 , L. Fazli3 , P. Rennie3 , B. Risberg4 , H. Wæhre4 , H.E. Danielsen4 , G.S. Hotamisligil5 , F. Saatcioglu1 . 1 University of Oslo, Department of Biosciences, Oslo, Norway; 2 University of Oslo, Centre for Molecular Medicine Norway, Oslo, Norway; 3 The Vancouver Prostate Centre, Department of Urologic Sciences, Vancouver, Canada; 4 Oslo University Hospital, Institute for Cancer Genetics and Informatics, Oslo, Norway; 5 Harvard University, Department of Genetics and Complex Diseases, Boston, USA Background: Endoplasmic reticulum (ER) stress resulting from protein misfolding is a common feature of malignant cells. The unfolded protein response (UPR) counteracts ER stress which can profoundly affect the cancer cell phenotype. Methods: Androgen receptor (AR) and UPR gene signatures were compared in a prostate cancer (PCa) cohort. Western analysis and quantitative PCR was used to evaluate the effect of androgens on UPR pathway activation in PCa cells in vitro and in vivo. ChIP assays were used to investigate AR binding to UPR related gene promoters in vivo. IRE1a and XBP-1 knockdown PCa cell lines were generated and used in xenograft experiments. A small molecule inhibitor of IRE1a was used to evaluate its effect on tumor growth in two independent preclinical models in vivo. XBP1 expression in human prostate cancer specimens was determined by immunohistochemistry. Results: The canonical UPR pathways in PCa cells were directly and divergently regulated by androgens which are critical for PCa survival. AR bound to gene regulatory sites and activated the IRE1a branch of UPR, but simultaneously inhibited PERK signaling. IRE1a inhibition, or its target XBP1, profoundly inhibited PCa cell growth in vitro as well as in preclinical models in vivo. Consistently, IRE1a expression decreased in xenografts upon androgen withdrawal and regression. Furthermore, AR and UPR gene expression were positively correlated in human PCa cohorts. Conclusion: Androgens regulate a genetic switch that impacts UPR in PCa cells which suggests that targeting IRE1a signaling may be a novel therapeutic approach in PCa. 496 POSTER Prevalence of MET amplification, MET expression, and MET-related genomic alterations in non-small cell lung cancer (NSCLC) A.L. Ang1 , H. Yang2 , A.A. Anderson1 , R. Tang2 , M.A. Damore1 , R.D. Loberg1 . 1 Amgen Inc., Molecular Sciences & Computational Biology, Thousand Oaks CA, USA; 2 Amgen Inc., Biostatistics − Medical Sciences, Thousand Oaks CA, USA Background: Signaling through the MET receptor tyrosine kinase regulates cellular functions including proliferation, survival, migration, and morphogenesis. The frequency of genomic alterations in MET varies widely among solid tumors and has been reported to be in the range of 2−8% in NSCLC patients who have not developed resistance to tyrosine kinase inhibitors (Cappuzzo et al. 2009). The relationship between MET amplification, MET expression, and additional genomic alterations known to be important in NSCLC biology (e.g., EGFR and PI3K mutations, ALK fusion genes) has not been well established. Materials and Methods: Formalin-fixed, paraffin-embedded (FFPE) NSCLC whole tissue sections (N = 181) were collected at 7 sites in the Eastern United States from patients of various ethnic backgrounds (White or Caucasian, n = 151; Black or African American, n = 28; Asian, n = 1; missing, n = 1). Samples were mostly early-stage primary resections
Friday 21 November 2014 161 by MACE to gain insight into genes under the control of LPA6. Finally, we analyzed LPA6 expression in HCC tissues and its correlation with patient survival. Results: We found that RNA-interference-mediated knockdown of LPA6 impaired tumorigenicity in human HCC xenografts and the expression profile of several genes, including proto-oncogene Pim-3 regulated by LPA6 at gene-promoter level. Conversely, overexpression of LPA6 in nontumorigenic HCC cells led to Pim-3 upregulation and the acquisition of a tumorigenic phenotype. Moreover, our experiments with deleted mutants indicate that the STAT3 binding site is important for the transcriptional activation of Pim-3 by LPA6. Lastly, we found that LPA6 overexpression in HCC patients is correlated with high proliferation rates, elevated Pim-3 levels and a worse clinical outcome. Conclusions: Taken together, our data demonstrate a novel tumorigenic mechanism and provide a rationale for therapeutic targeting of the LPA6−Pim-3 axis in HCC. 495 POSTER Divergent androgen regulation of UPR pathways drives prostate cancer Y.J. Arnoldussen1 , M. Storm1 , X. Sheng1 , M. Tesikova1 , Y. Jin1 , H.Z. Nenseth1 , S. Zhao1 , I.G. Mills2 , L. Fazli3 , P. Rennie3 , B. Risberg4 , H. Wæhre4 , H.E. Danielsen4 , G.S. Hotamisligil5 , F. Saatcioglu1 . 1 University of Oslo, Department of Biosciences, Oslo, Norway; 2 University of Oslo, Centre for Molecular Medicine Norway, Oslo, Norway; 3 The Vancouver Prostate Centre, Department of Urologic Sciences, Vancouver, Canada; 4 Oslo University Hospital, Institute for Cancer Genetics and Informatics, Oslo, Norway; 5 Harvard University, Department of Genetics and Complex Diseases, Boston, USA Background: Endoplasmic reticulum (ER) stress resulting from protein misfolding is a common feature of malignant cells. The unfolded protein response (UPR) counteracts ER stress which can profoundly affect the cancer cell phenotype. Methods: Androgen receptor (AR) and UPR gene signatures were compared in a prostate cancer (PCa) cohort. Western analysis and quantitative PCR was used to evaluate the effect of androgens on UPR pathway activation in PCa cells in vitro and in vivo. ChIP assays were used to investigate AR binding to UPR related gene promoters in vivo. IRE1a and XBP-1 knockdown PCa cell lines were generated and used in xenograft experiments. A small molecule inhibitor of IRE1a was used to evaluate its effect on tumor growth in two independent preclinical models in vivo. XBP1 expression in human prostate cancer specimens was determined by immunohistochemistry. Results: The canonical UPR pathways in PCa cells were directly and divergently regulated by androgens which are critical for PCa survival. AR bound to gene regulatory sites and activated the IRE1a branch of UPR, but simultaneously inhibited PERK signaling. IRE1a inhibition, or its target XBP1, profoundly inhibited PCa cell growth in vitro as well as in preclinical models in vivo. Consistently, IRE1a expression decreased in xenografts upon androgen withdrawal and regression. Furthermore, AR and UPR gene expression were positively correlated in human PCa cohorts. Conclusion: Androgens regulate a genetic switch that impacts UPR in PCa cells which suggests that targeting IRE1a signaling may be a novel therapeutic approach in PCa. 496 POSTER Prevalence of MET amplification, MET expression, and MET-related genomic alterations in non-small cell lung cancer (NSCLC) A.L. Ang1 , H. Yang2 , A.A. Anderson1 , R. Tang2 , M.A. Damore1 , R.D. Loberg1 . 1 Amgen Inc., Molecular Sciences & Computational Biology, Thousand Oaks CA, USA; 2 Amgen Inc., Biostatistics − Medical Sciences, Thousand Oaks CA, USA Background: Signaling through the MET receptor tyrosine kinase regulates cellular functions including proliferation, survival, migration, and morphogenesis. The frequency of genomic alterations in MET varies widely among solid tumors and has been reported to be in the range of 2−8% in NSCLC patients who have not developed resistance to tyrosine kinase inhibitors (Cappuzzo et al. 2009). The relationship between MET amplification, MET expression, and additional genomic alterations known to be important in NSCLC biology (e.g., EGFR and PI3K mutations, ALK fusion genes) has not been well established. Materials and Methods: Formalin-fixed, paraffin-embedded (FFPE) NSCLC whole tissue sections (N = 181) were collected at 7 sites in the Eastern United States from patients of various ethnic backgrounds (White or Caucasian, n = 151; Black or African American, n = 28; Asian, n = 1; missing, n = 1). Samples were mostly early-stage primary resections