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A kinome-scale synthetic lethality screen reveals an essentiality of CDK13 for 19q12 amplified cancer cells
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EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
Both pan-HER inhibitor and AKT inhibitor abrogate the increased AR activity seen on stimulation with Heregulin. We now have 4 target genes that characteristic of Enzalutamide resistance that could represent either future biomarkers of drug-resistance or potential therapeutic targets in advanced disease. No conflict of interest. 463 Spheroids versus monolayers: are the cells metabolically the same? M. Gkiouli1 , A. Otto1 , J. Hintermair1 . 1 Munich Technical University, Institute of Medical Engineering IMETUM, Garching, Germany Background: Monolayer cultures as tumor models do not mimic well an in vivo tumor. Therefore, 3D cultures are being increasingly used as in vitro models that mimic tumor conditions in a more physiological manner with respect to diffusional limitations of oxygen, nutrients and cellular waste products as well as pH gradients. To be able to evaluate what kind of differences could occur in the metabolic activities when tumor cells are cultured as 3D- as opposed to 2D-cultures, two different breast carcinoma cell lines were analysed for their metabolic responses to limiting nutrient levels and variable pH. Material and Methods: MCF-7 and the more malignant MDA-MB231 cells were cultivated as monolayer and as spheroid cultures with variable extracellular pH and tumor-relevant glucose and glutamine levels. Cell number was determined by nuclei counting. Metabolic activity was measured with a water soluble metabolic tetrazolium assay (WST-1, “proliferation assay”), which reflects the activity of a cell surface membrane NADH-oxidase that is dependent on intracellular NADH. Moreover, lactate production and glucose consumption were determined in cell culture supernatants. Results: The results show that in general tumor cell growth and metabolic activity do not correlate; they are dependent on the extracellular pH and on the concentration and the ratio of glucose / glutamine. In 3D cultures, NADHoxidase activity per cell number is increased up to 10-fold compared to 2D cultures. Moreover, MDA-MB231 cells have a higher NADH oxidase activity than MCF-7 cells. The amount of glucose converted to lactate is higher in 3D than in monolayer cultures, and it is higher in MDA-MB231 cells than in MCF-7 cells. Finally, with increasing acidity, in both cell lines growth as well as NADH-oxidase and glucose consumption become less dependent on the variable levels of nutrients, a common feature for both 2D and 3D cultures. Conclusions: There are differences in the metabolism of spheroids compared to their monolayer cultures, revealing the necessity for recapitulating in the 3D cultures metabolic parameters that have been established in 2D cultures, especially in the context of testing for anticancer activity, as well as for choosing cellular systems for metabolic profiling and imaging. Acknowledgment: This project is supported by the “Wilhelm Sander-Stiftung”, Munich, Germany. No conflict of interest. 465 Development and characterisation of a novel syngeneic, spontaneous breast cancer metastasis model U. Jungwirth1 , G. Qiong1 , S. Wantuch1 , C. Isacke1 . 1 The Institute of Cancer Research, Breast Cancer Now Research Centre, London, United Kingdom Background: The development of novel therapeutic strategies for targeting metastatic disease is restricted by our lack of knowledge of molecular mechanisms underlying the metastatic process. However, it is clear that successful metastatic colonisation involves a close crosstalk between tumour cells and the metastatic microenvironment. Unfortunately, there are no in vitro models that recapitulate all the different steps of the metastatic cascade and in vivo human xenograft and PDX models are transplanted into immunocompromised mice that lack an intact immune system. Material and Methods: In this study we developed two syngeneic, spontaneous Balb/c breast cancer metastasis models, namely D2A1-LuM1 and D2A1-LuM2, from the poorly spontaneous metastasising D2A1 cell line by serial in vivo passaging. We characterised the models in vivo, by immunohistochemistry and in vitro, using 2D and 3D cultures. Furthermore, we performed gene expression profiling to identify differentially modulated pathways. Results: Both D2A1-LuM1 and D2A1-LuM2 spontaneously metastasis from the fat pad of Balb/c mice giving rise to macrometastatic diease in the lungs. The metastatic potential of the two models is consistent with 2D colony growth and 3D tumour spheroid growth assays, showing that the D2A1-Lum1 clone is more aggressive than the D2A1-LuM2 clone. Gene expression profiling identified 218 (111 upregulated, 107 downregulated) differentially expressed genes (with a p-value of 0.001) in the metastatic clones compared to the parental D2A1 cells, including invasion and cell death pathways. Conclusions: Currently researchers rely on the 4T1 spontaneous breast cancer metastasis model. Consequently, there is a need for additional syngeneic models. The metastatic D2A1 clones described here, together with the recently described EO771.LMB metastatic clone, provide important
independent syngeneic models for investigating the molecular and cellular mechanisms underpinning metastatic breast cancer progression. No conflict of interest. 466 A kinome-scale synthetic lethality screen reveals an essentiality of CDK13 for 19q12 amplified cancer cells R. Stark1 , W. Krek1 . 1 Swiss Federal Institute of Technology, Institute of Molecular Health Sciences, Zurich, ¨ Switzerland Introduction: Amplification of the chromosomal locus 19q12 is a common feature among multiple tumor types including stomach, liver, lung, breast and ovarian cancer. The most frequent malignant tumors with 19q12 amplifications are high-grade serous ovarian carcinomas for which therapy and cure options are very limited due to chemoresistance. 19q12 encompasses multiple genes among which two genes, CCNE1 and URI1, have been associated with oncogenic function.CCNE1 encodes Cyclin E which controls G1/S phase transition in complex with CDK2 during cell cycle; URI1 is an unconventional member of the prefoldin family of molecular chaperones (Gstaiger et al, 2003; Theurillat et al., 2011). To identify genes critical for the survival of 19q12 amplified tumors, we performed a kinome-focused shRNA screen in 19q12 amplifed and non-amplifed ovarian cancer cells. Identified kinases could serve as potential therapeutic targets in the treatment of 19q12 amplified cancers. Methods: Using a lentivirally-delivered shRNA library, we performed parallel pooled shRNA screens in 6 ovarian cancer cell lines (3 with 19q12 amplification and 3 lacking the amplicon). Cell line dependencies on 537 kinase-encoding genes were interrogated by 5 shRNAs per gene. The proliferation effect of each shRNA in each cell line was assessed by transducing a population of 2.7 mio cells with one shRNA virus per cell and determining the relative enrichment or depletion of each of the 2684 shRNAs after 12 population doublings using Next Generation Sequencing of the individual hairpin sequences. Results: After quality control, count normalization and differential representation analyses (Robinson et al, 2010, Dai et al, 2014), Analytic Technique for Assessment of RNAi by Similarity (ATARiS) (Shao et al, 2013) was applied, which produces quantitative, gene-level phenotype values that provide an intuitive measure of the effect of gene suppression in each sample. The top 5 hits were validated in a biological context by performing clonogenic assays after applying RNAi with 2 distinct shRNAs. Hit validation was performed in all 6 cell lines and an extended panel of 19q12 non-amplified ovarian cancer and breast cancer cell lines. The number of remaining colonies was determined 14 days after application of the respective knockdowns. From the top 5 hits, CDK13 and CSNK1E (CK1e) were confirmed by subsequent validation studies. Conclusion: By screening the human kinome for potential therapeutic targets specifically required for 19q12 amplified ovarian cancer cell lines, we identified CDK13 and CK1e to be preferentially required for the survival of 19q12 amplified but not non-amplified cancer cells. Thus, CDK13 and CK1e may represent potential targets for therapy in 19q12 amplified cancer cells of diverse origins. No conflict of interest. 467 Mcl-1 dynamics influence mitotic slippage and death in mitosis O. Sloss1 , C. Topham1 , S. Taylor1 . 1 University of Manchester, Faculty of Life Sciences, Manchester, United Kingdom Background: Microtubule-binding drugs are used to treat a variety of cancers, however the link between treatment and tumour cell death is still unclear. In cell culture these drugs induce a mitotic arrest, caused by spindle assembly checkpoint activation. Following a sustained arrest, cells can commit to mitotic death or mitotic slippage (mitotic exit without division). Whereas mitotic slippage is caused by declining Cyclin B1 levels by APC/C ubiquitination, the mitotic death signal is not well defined. Protein levels of anti-apoptotic factor Mcl-1 decline during a mitotic arrest, suggesting that Mcl-1 acts as a mitotic death timer. We investigated the effect of proteasome-mediated degradation of Mcl-1 on mitotic death as well as inhibition of several E3 ligase complexes previously shown to target Mcl-1 for degradation. Additionally, it has been proposed that apoptotic network factors could influence the rate of mitotic slippage. Therefore, we also analysed the effect of Mcl-1 levels on mitotic slippage. Materials and Methods: Two colorectal cell lines (RKO and DLD-1) were treated with a variety of small molecular inhibitors and siRNAs during a mitotic arrest in order to analyse the effect these methods had on Mcl-1 stability in a mitotic arrest. In parallel, time-lapse microscopy was utilised in order to analyse cell fate of populations of cells treated with microtubule-binding drugs in order to see if the changes in Mcl-1 levels could influence the rate of mitotic death and mitotic slippage. In addition, mutant forms of Mcl-1 believed to influence its degradation were over-expressed and similarly analysed. Results: Treatment of cells with a proteasome inhibitor significantly delayed mitotic death and this delay was dependent on increased Mcl-1 levels. Additionally we show that Mcl-1 is synthesised during mitosis and blocking protein synthesis with cycloheximide accelerates mitotic death. Inhibition of
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
Both pan-HER inhibitor and AKT inhibitor abrogate the increased AR activity seen on stimulation with Heregulin. We now have 4 target genes that characteristic of Enzalutamide resistance that could represent either future biomarkers of drug-resistance or potential therapeutic targets in advanced disease. No conflict of interest. 463 Spheroids versus monolayers: are the cells metabolically the same? M. Gkiouli1 , A. Otto1 , J. Hintermair1 . 1 Munich Technical University, Institute of Medical Engineering IMETUM, Garching, Germany Background: Monolayer cultures as tumor models do not mimic well an in vivo tumor. Therefore, 3D cultures are being increasingly used as in vitro models that mimic tumor conditions in a more physiological manner with respect to diffusional limitations of oxygen, nutrients and cellular waste products as well as pH gradients. To be able to evaluate what kind of differences could occur in the metabolic activities when tumor cells are cultured as 3D- as opposed to 2D-cultures, two different breast carcinoma cell lines were analysed for their metabolic responses to limiting nutrient levels and variable pH. Material and Methods: MCF-7 and the more malignant MDA-MB231 cells were cultivated as monolayer and as spheroid cultures with variable extracellular pH and tumor-relevant glucose and glutamine levels. Cell number was determined by nuclei counting. Metabolic activity was measured with a water soluble metabolic tetrazolium assay (WST-1, “proliferation assay”), which reflects the activity of a cell surface membrane NADH-oxidase that is dependent on intracellular NADH. Moreover, lactate production and glucose consumption were determined in cell culture supernatants. Results: The results show that in general tumor cell growth and metabolic activity do not correlate; they are dependent on the extracellular pH and on the concentration and the ratio of glucose / glutamine. In 3D cultures, NADHoxidase activity per cell number is increased up to 10-fold compared to 2D cultures. Moreover, MDA-MB231 cells have a higher NADH oxidase activity than MCF-7 cells. The amount of glucose converted to lactate is higher in 3D than in monolayer cultures, and it is higher in MDA-MB231 cells than in MCF-7 cells. Finally, with increasing acidity, in both cell lines growth as well as NADH-oxidase and glucose consumption become less dependent on the variable levels of nutrients, a common feature for both 2D and 3D cultures. Conclusions: There are differences in the metabolism of spheroids compared to their monolayer cultures, revealing the necessity for recapitulating in the 3D cultures metabolic parameters that have been established in 2D cultures, especially in the context of testing for anticancer activity, as well as for choosing cellular systems for metabolic profiling and imaging. Acknowledgment: This project is supported by the “Wilhelm Sander-Stiftung”, Munich, Germany. No conflict of interest. 465 Development and characterisation of a novel syngeneic, spontaneous breast cancer metastasis model U. Jungwirth1 , G. Qiong1 , S. Wantuch1 , C. Isacke1 . 1 The Institute of Cancer Research, Breast Cancer Now Research Centre, London, United Kingdom Background: The development of novel therapeutic strategies for targeting metastatic disease is restricted by our lack of knowledge of molecular mechanisms underlying the metastatic process. However, it is clear that successful metastatic colonisation involves a close crosstalk between tumour cells and the metastatic microenvironment. Unfortunately, there are no in vitro models that recapitulate all the different steps of the metastatic cascade and in vivo human xenograft and PDX models are transplanted into immunocompromised mice that lack an intact immune system. Material and Methods: In this study we developed two syngeneic, spontaneous Balb/c breast cancer metastasis models, namely D2A1-LuM1 and D2A1-LuM2, from the poorly spontaneous metastasising D2A1 cell line by serial in vivo passaging. We characterised the models in vivo, by immunohistochemistry and in vitro, using 2D and 3D cultures. Furthermore, we performed gene expression profiling to identify differentially modulated pathways. Results: Both D2A1-LuM1 and D2A1-LuM2 spontaneously metastasis from the fat pad of Balb/c mice giving rise to macrometastatic diease in the lungs. The metastatic potential of the two models is consistent with 2D colony growth and 3D tumour spheroid growth assays, showing that the D2A1-Lum1 clone is more aggressive than the D2A1-LuM2 clone. Gene expression profiling identified 218 (111 upregulated, 107 downregulated) differentially expressed genes (with a p-value of 0.001) in the metastatic clones compared to the parental D2A1 cells, including invasion and cell death pathways. Conclusions: Currently researchers rely on the 4T1 spontaneous breast cancer metastasis model. Consequently, there is a need for additional syngeneic models. The metastatic D2A1 clones described here, together with the recently described EO771.LMB metastatic clone, provide important
independent syngeneic models for investigating the molecular and cellular mechanisms underpinning metastatic breast cancer progression. No conflict of interest. 466 A kinome-scale synthetic lethality screen reveals an essentiality of CDK13 for 19q12 amplified cancer cells R. Stark1 , W. Krek1 . 1 Swiss Federal Institute of Technology, Institute of Molecular Health Sciences, Zurich, ¨ Switzerland Introduction: Amplification of the chromosomal locus 19q12 is a common feature among multiple tumor types including stomach, liver, lung, breast and ovarian cancer. The most frequent malignant tumors with 19q12 amplifications are high-grade serous ovarian carcinomas for which therapy and cure options are very limited due to chemoresistance. 19q12 encompasses multiple genes among which two genes, CCNE1 and URI1, have been associated with oncogenic function.CCNE1 encodes Cyclin E which controls G1/S phase transition in complex with CDK2 during cell cycle; URI1 is an unconventional member of the prefoldin family of molecular chaperones (Gstaiger et al, 2003; Theurillat et al., 2011). To identify genes critical for the survival of 19q12 amplified tumors, we performed a kinome-focused shRNA screen in 19q12 amplifed and non-amplifed ovarian cancer cells. Identified kinases could serve as potential therapeutic targets in the treatment of 19q12 amplified cancers. Methods: Using a lentivirally-delivered shRNA library, we performed parallel pooled shRNA screens in 6 ovarian cancer cell lines (3 with 19q12 amplification and 3 lacking the amplicon). Cell line dependencies on 537 kinase-encoding genes were interrogated by 5 shRNAs per gene. The proliferation effect of each shRNA in each cell line was assessed by transducing a population of 2.7 mio cells with one shRNA virus per cell and determining the relative enrichment or depletion of each of the 2684 shRNAs after 12 population doublings using Next Generation Sequencing of the individual hairpin sequences. Results: After quality control, count normalization and differential representation analyses (Robinson et al, 2010, Dai et al, 2014), Analytic Technique for Assessment of RNAi by Similarity (ATARiS) (Shao et al, 2013) was applied, which produces quantitative, gene-level phenotype values that provide an intuitive measure of the effect of gene suppression in each sample. The top 5 hits were validated in a biological context by performing clonogenic assays after applying RNAi with 2 distinct shRNAs. Hit validation was performed in all 6 cell lines and an extended panel of 19q12 non-amplified ovarian cancer and breast cancer cell lines. The number of remaining colonies was determined 14 days after application of the respective knockdowns. From the top 5 hits, CDK13 and CSNK1E (CK1e) were confirmed by subsequent validation studies. Conclusion: By screening the human kinome for potential therapeutic targets specifically required for 19q12 amplified ovarian cancer cell lines, we identified CDK13 and CK1e to be preferentially required for the survival of 19q12 amplified but not non-amplified cancer cells. Thus, CDK13 and CK1e may represent potential targets for therapy in 19q12 amplified cancer cells of diverse origins. No conflict of interest. 467 Mcl-1 dynamics influence mitotic slippage and death in mitosis O. Sloss1 , C. Topham1 , S. Taylor1 . 1 University of Manchester, Faculty of Life Sciences, Manchester, United Kingdom Background: Microtubule-binding drugs are used to treat a variety of cancers, however the link between treatment and tumour cell death is still unclear. In cell culture these drugs induce a mitotic arrest, caused by spindle assembly checkpoint activation. Following a sustained arrest, cells can commit to mitotic death or mitotic slippage (mitotic exit without division). Whereas mitotic slippage is caused by declining Cyclin B1 levels by APC/C ubiquitination, the mitotic death signal is not well defined. Protein levels of anti-apoptotic factor Mcl-1 decline during a mitotic arrest, suggesting that Mcl-1 acts as a mitotic death timer. We investigated the effect of proteasome-mediated degradation of Mcl-1 on mitotic death as well as inhibition of several E3 ligase complexes previously shown to target Mcl-1 for degradation. Additionally, it has been proposed that apoptotic network factors could influence the rate of mitotic slippage. Therefore, we also analysed the effect of Mcl-1 levels on mitotic slippage. Materials and Methods: Two colorectal cell lines (RKO and DLD-1) were treated with a variety of small molecular inhibitors and siRNAs during a mitotic arrest in order to analyse the effect these methods had on Mcl-1 stability in a mitotic arrest. In parallel, time-lapse microscopy was utilised in order to analyse cell fate of populations of cells treated with microtubule-binding drugs in order to see if the changes in Mcl-1 levels could influence the rate of mitotic death and mitotic slippage. In addition, mutant forms of Mcl-1 believed to influence its degradation were over-expressed and similarly analysed. Results: Treatment of cells with a proteasome inhibitor significantly delayed mitotic death and this delay was dependent on increased Mcl-1 levels. Additionally we show that Mcl-1 is synthesised during mitosis and blocking protein synthesis with cycloheximide accelerates mitotic death. Inhibition of