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OC-0244: Secretome analysis of NSCLC cells: Identification of novel targets for radiosensitization
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ESTRO 33, 2014 potential modulation at the posttranslational level. Interestingly, irradiation induced an acute, dose-dependent increase in ADAM17 activity, which correlated with substrate shedding. Ex vivo analysis of murine blood serum derived from irradiated tumor xenograft-carrying mice support our in vitro results. Targeting ADAM17 with the small molecular inhibitor TMI-005 and siRNA-mediated silencing confirmed that ADAM17 is driving a prosurvival response with subsequent treatment resistance. Pharmacologic inhibition or siRNA-mediated downregulation of ADAM17 in combination with IR reduced proliferation and clonogenicity of the NSCLC cells. Corresponding in vivo experiments are ongoing. Conclusions: Our findings demonstrate that IR significantly activates ADAM17, which results in shedding of multiple survival factors, growth factor pathway activation and treatment resistance in NSCLC cells. We provide a sound rationale for positioning ADAM17 inhibitors as radiosensitizers to improve the treatment of NSCLC.
“when the top level guys look down, they see only shit when the bottom level guys look up, they see only assholes” A quote from one of Toyota’s Quality Managers after implementation of Lean: “We get brilliant results from average people managing brilliant processes”. Another quote however: “…when undertaken on their own, they create islands of excellence which, if they are not linked together, make little difference to the bottom line.” Realizing that everyone’s circle of influence while applying improvements is limited, it should be no surprise that Quality Improvement within a radiotherapy process can only be achieved optimally through multi-professional collaboration. Take home Respect, understanding and communication through all levels of a radiotherapy workflow highly increases the success of Quality Improvement. This should be considered not only in quality teams within a radiotherapy department, but in radiotherapy treatment teams in general: Multi-professional collaboration results in better patient treatment in Radiotherapy.
PROFFERED PAPERS: RADIOBIOLOGY 2: NEW TARGET DISCOVERY AND IMMUNOTHERAPY OC-0244 Secretome analysis of NSCLC cells: Identification of novel targets for radiosensitization A. Sharma1, S. Bender1, O. Riesterer1, A. Broggini-Tenzer1, M. Pruschy1 1 University Hospital Zurich, Radiation Oncology, Zurich, Switzerland Purpose/Objective: Inherent and acquired resistance is a significant impediment to the efficacy of radiotherapy. Primary therapeutic response to ionizing radiation (IR) is imparted by genomic instability and DNA damage, however IR also triggers multiple intracellular signaling processes that lead to the secretion of various para- and autocrine factors. These secreted factors then loop back (autocrine fashion) to modulate multiple pathways eventually driving acquired resistance. Here we investigated treatment-dependent secretion of various auto- or paracrine factors, which might drive acquired rescue mechanisms and which determine the overall radiation sensitivity of the tumor. Materials and Methods: Exhaustive secretome analysis was performed for a wide range of secretory factors by antibody array screening. Secretion kinetics of selected factors were determined across different established tumor cell lines in response to increasing doses of ionizing radiation and in murine blood serum, derived from irradiated tumor xenograft-carrying mice. To determine the relevance of the specific factors, clonogenic survival assays in response to IR were performed with siRNA-targeted tumor cell lines and in presence of respective inhibitors. Results: We performed an exhaustive IR-dependent secretome analysis (>300 factors) and investigated in detail the effect of increasing doses of IR on the expression and tumor cell secretion of amphiregulin, transforming growth factor-a and ALCAM, which represent top hits in the array screening. All these factors were secreted in a dose dependent way from several non–small cell lung cancer (NSCLC) cell lines in response to IR. No changes were observed at the transcriptional level implying
OC-0245 A high-throughput siRNA screen identifies several novel determinants of tumour radiosensitivity G. Tiwana1, R. Prevo1, F. Buffa1, D. Ebner2, A. Howarth2, E. Seraia2, K. Chu1, L. Durrant1, G. McKenna1, G. Higgins1 1 University of Oxford, Oncology, Oxford, United Kingdom 2 Target Discovery Institute, Nuffield Department of Medicine, Oxford, United Kingdom Purpose/Objective: The therapeutic window for radiotherapy could be improved if tumour cells could be selectively rendered more sensitive to radiation. Such a strategy depends upon exploiting tumour specific targets, many of which remain to be identified. Materials and Methods: We have therefore conducted a high-throughput siRNA screen using a 96-well plate colony formation assay to identify novel genes involved in tumour cell radiosensitisation. Colony formation was chosen as an endpoint as it is the gold standard for assessing the true replicative potential of a cell following radiation treatment. Two siRNA libraries have been assayed: Dharmacon ON-TARGETplus Kinome and Druggable libraries. The assay consisted of reverse transfecting HeLa cells with 20nM of target gene siRNA on day 1. 72hrs post transfection, cells were lifted and plated into two sets of quadruplicate repeats within 96-well plates. The cells were left to adhere for 4hrs and treatment plates were irradiated at 7 Gy using 6MV photons delivered by Varian Clinac iX Linear accelerator. Plates were incubated for 7-9 days and stained with crystal violet. Colonies were counted and Z-scores calculated.The kinome library was assayed first and hits were validated. Results: Several key determinants of tumour radiosensitivity were identified based on Z values lower than 0. Several genes known to play key roles in intrinsic radiosensitivity such as ATR, ATM, CHEK1 and DNAPKcs were identified, thereby validating the screening technique. We identified five novel genes from the kinome library whose depletion induced HeLa radiosensitisation in 6-well plate CFAs. These experiments were repeated with separate siRNA (Ambion) to ensure these findings were not the result of off-target effects. Depletion of these genes appeared to cause radiosensitisation in several other tumour cell lines as well. Mechanisms behind the radiopotentiation effects for four of the candidate genes have been elucidated. In addition, two candidate genes have prognostic significance associated with recurrence free survival in breast cohorts. Conclusions: We are continuing validation studies on the kinome radiosensitising genes and in the process of validating novel radiosensitising genes from the Druggable ON-TARGETplus library. OC-0246 Screening on aided drug-membrane traversal identifies PI3K inhibitors as targets for improved radiosensitization A. Van Hell1, T. Dijkema1, M. Verheij2 1 The Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital (NKI-AVL), Biological Stress Responses, Amsterdam, The Netherlands 2 The Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital (NKI-AVL), Radiotherapy, Amsterdam, The Netherlands Purpose/Objective: Insufficient accumulation of drugs at their molecular target greatly limits anti-cancer therapy effectiveness. The plasma membrane is the main barrier for a drug to enter the (tumor) cell. We have identified a series of short-chain lipids (SCL), which facilitate the membrane traversal of amphiphilic drugs, e.g. doxorubicin. By concomitant administration of the SCL N-octanoyl-glucosylceramide (GC) with doxorubicin in a liposomal co-formulation, therapeutic efficacy markedly improved in various treatment resistant mouse tumor models.
ESTRO 33, 2014 potential modulation at the posttranslational level. Interestingly, irradiation induced an acute, dose-dependent increase in ADAM17 activity, which correlated with substrate shedding. Ex vivo analysis of murine blood serum derived from irradiated tumor xenograft-carrying mice support our in vitro results. Targeting ADAM17 with the small molecular inhibitor TMI-005 and siRNA-mediated silencing confirmed that ADAM17 is driving a prosurvival response with subsequent treatment resistance. Pharmacologic inhibition or siRNA-mediated downregulation of ADAM17 in combination with IR reduced proliferation and clonogenicity of the NSCLC cells. Corresponding in vivo experiments are ongoing. Conclusions: Our findings demonstrate that IR significantly activates ADAM17, which results in shedding of multiple survival factors, growth factor pathway activation and treatment resistance in NSCLC cells. We provide a sound rationale for positioning ADAM17 inhibitors as radiosensitizers to improve the treatment of NSCLC.
“when the top level guys look down, they see only shit when the bottom level guys look up, they see only assholes” A quote from one of Toyota’s Quality Managers after implementation of Lean: “We get brilliant results from average people managing brilliant processes”. Another quote however: “…when undertaken on their own, they create islands of excellence which, if they are not linked together, make little difference to the bottom line.” Realizing that everyone’s circle of influence while applying improvements is limited, it should be no surprise that Quality Improvement within a radiotherapy process can only be achieved optimally through multi-professional collaboration. Take home Respect, understanding and communication through all levels of a radiotherapy workflow highly increases the success of Quality Improvement. This should be considered not only in quality teams within a radiotherapy department, but in radiotherapy treatment teams in general: Multi-professional collaboration results in better patient treatment in Radiotherapy.
PROFFERED PAPERS: RADIOBIOLOGY 2: NEW TARGET DISCOVERY AND IMMUNOTHERAPY OC-0244 Secretome analysis of NSCLC cells: Identification of novel targets for radiosensitization A. Sharma1, S. Bender1, O. Riesterer1, A. Broggini-Tenzer1, M. Pruschy1 1 University Hospital Zurich, Radiation Oncology, Zurich, Switzerland Purpose/Objective: Inherent and acquired resistance is a significant impediment to the efficacy of radiotherapy. Primary therapeutic response to ionizing radiation (IR) is imparted by genomic instability and DNA damage, however IR also triggers multiple intracellular signaling processes that lead to the secretion of various para- and autocrine factors. These secreted factors then loop back (autocrine fashion) to modulate multiple pathways eventually driving acquired resistance. Here we investigated treatment-dependent secretion of various auto- or paracrine factors, which might drive acquired rescue mechanisms and which determine the overall radiation sensitivity of the tumor. Materials and Methods: Exhaustive secretome analysis was performed for a wide range of secretory factors by antibody array screening. Secretion kinetics of selected factors were determined across different established tumor cell lines in response to increasing doses of ionizing radiation and in murine blood serum, derived from irradiated tumor xenograft-carrying mice. To determine the relevance of the specific factors, clonogenic survival assays in response to IR were performed with siRNA-targeted tumor cell lines and in presence of respective inhibitors. Results: We performed an exhaustive IR-dependent secretome analysis (>300 factors) and investigated in detail the effect of increasing doses of IR on the expression and tumor cell secretion of amphiregulin, transforming growth factor-a and ALCAM, which represent top hits in the array screening. All these factors were secreted in a dose dependent way from several non–small cell lung cancer (NSCLC) cell lines in response to IR. No changes were observed at the transcriptional level implying
OC-0245 A high-throughput siRNA screen identifies several novel determinants of tumour radiosensitivity G. Tiwana1, R. Prevo1, F. Buffa1, D. Ebner2, A. Howarth2, E. Seraia2, K. Chu1, L. Durrant1, G. McKenna1, G. Higgins1 1 University of Oxford, Oncology, Oxford, United Kingdom 2 Target Discovery Institute, Nuffield Department of Medicine, Oxford, United Kingdom Purpose/Objective: The therapeutic window for radiotherapy could be improved if tumour cells could be selectively rendered more sensitive to radiation. Such a strategy depends upon exploiting tumour specific targets, many of which remain to be identified. Materials and Methods: We have therefore conducted a high-throughput siRNA screen using a 96-well plate colony formation assay to identify novel genes involved in tumour cell radiosensitisation. Colony formation was chosen as an endpoint as it is the gold standard for assessing the true replicative potential of a cell following radiation treatment. Two siRNA libraries have been assayed: Dharmacon ON-TARGETplus Kinome and Druggable libraries. The assay consisted of reverse transfecting HeLa cells with 20nM of target gene siRNA on day 1. 72hrs post transfection, cells were lifted and plated into two sets of quadruplicate repeats within 96-well plates. The cells were left to adhere for 4hrs and treatment plates were irradiated at 7 Gy using 6MV photons delivered by Varian Clinac iX Linear accelerator. Plates were incubated for 7-9 days and stained with crystal violet. Colonies were counted and Z-scores calculated.The kinome library was assayed first and hits were validated. Results: Several key determinants of tumour radiosensitivity were identified based on Z values lower than 0. Several genes known to play key roles in intrinsic radiosensitivity such as ATR, ATM, CHEK1 and DNAPKcs were identified, thereby validating the screening technique. We identified five novel genes from the kinome library whose depletion induced HeLa radiosensitisation in 6-well plate CFAs. These experiments were repeated with separate siRNA (Ambion) to ensure these findings were not the result of off-target effects. Depletion of these genes appeared to cause radiosensitisation in several other tumour cell lines as well. Mechanisms behind the radiopotentiation effects for four of the candidate genes have been elucidated. In addition, two candidate genes have prognostic significance associated with recurrence free survival in breast cohorts. Conclusions: We are continuing validation studies on the kinome radiosensitising genes and in the process of validating novel radiosensitising genes from the Druggable ON-TARGETplus library. OC-0246 Screening on aided drug-membrane traversal identifies PI3K inhibitors as targets for improved radiosensitization A. Van Hell1, T. Dijkema1, M. Verheij2 1 The Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital (NKI-AVL), Biological Stress Responses, Amsterdam, The Netherlands 2 The Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital (NKI-AVL), Radiotherapy, Amsterdam, The Netherlands Purpose/Objective: Insufficient accumulation of drugs at their molecular target greatly limits anti-cancer therapy effectiveness. The plasma membrane is the main barrier for a drug to enter the (tumor) cell. We have identified a series of short-chain lipids (SCL), which facilitate the membrane traversal of amphiphilic drugs, e.g. doxorubicin. By concomitant administration of the SCL N-octanoyl-glucosylceramide (GC) with doxorubicin in a liposomal co-formulation, therapeutic efficacy markedly improved in various treatment resistant mouse tumor models.