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Aurora-B mediated snail phosphorylation is essential for mitotic spindle checkpoint and for preventing chromosomal instability in breast cancer
abstracts
Annals of Oncology 7PD
Synergistic anti-cancer activity of auranofin with anti-PD-L1 therapy in triple negative breast cancer
P. Raninga1, A. Lee2, D. Sinha1, Y-Y. Shin2, D. Mittal1, M. Kalimutho1, K.K. Khanna1 1 Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Brisbane, Australia, 2Institute for Radiological Research, Radiation Biology Research Center, Taoyuan, Taiwan
8PD
Automatic interpretation of cancer genomes creates the largest repository of tumour genetic driver events
F. Martınez Jime´nez, F. Mui~ nos, O. Pich, A. Gonzalez-Perez, N. L opez-Bigas Biomedical Genomics, Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Background: Tumor genome sequencing is becoming widely available in the clinical setting. However, the interpretation of tumor somatic variants remains an important challenge to implement precision cancer medicine. Methods: Here, we present IntOGen, a platform aimed for tumor genome interpretation. The first component of the platform is an automatic pipeline for cancer driver gene identification across cohorts. This pipeline implements several bioinformatics methods to detect driver genes, on the basis of signals of positive selection in their mutational pattern across tumor cohorts. It also annotates several oncogenomic features that shed light onto the role of driver genes in the development of each malignancy. The second component of the platform leverages these features to identify the
Disclosure: All authors have declared no conflicts of interest.
Volume 30 | Supplement 5 | October 2019
Aurora-B mediated snail phosphorylation is essential for mitotic spindle checkpoint and for preventing chromosomal instability in breast cancer
B. Xu, M. Xiao, X. Li Breast Cancer Center, Tianjin Medical University Cancer Institute & Hospital, Tianjin, China Background: Snail is a transcriptional repressor that promotes epithelial-to-mesenchymal transition (EMT). Snail is upregulated in breast cancer and it is associated with chromosomal instability (CIN) and metastasis. The mitotic spindle assembly checkpoint (SAC) is a critical mechanism to prevent CIN. Extensive studies have investigated signaling pathways that involve in protein-protein interactions and post translational modifications. However, it not known how mitosis might be impacted by transcriptional regulation. Due to the fact that Snail is a transcriptional regulator and that Snail is upregulated in breast cancer with CIN, it is critical to understand whether and how abnormally regulated Snail contributes to breast cancer CIN via transcriptional regulation. Methods: We used siRNA to knockdown Snail in breast caner cell lines to look for a change in SAC. We developed a phosphor-specific antibody to study Aurora-B mediated phosphorylation. Site specific mutagenesis was used to generate phosphorylation site mutants, and exogenous proteins were expressed in cells to look for an effect in SAC and CIN. Results: We found that Snail is an essential element that is required for the SAC. Genetic depletion of Snail in breast cancer cells by siRNA led to a defect in activation of SAC (measured by accumulation of Histone Serine 10 phosphorylation in response to spindle damaging agents). Interestingly we also found that the Snail protein level was significantly enhanced in the presence of nocodazole. Further, we found that Snail upregulation was through phosphorylation in an Aurora-B dependent manner. We identified a serine site that can be phosphorylated by Aurora-B. Using a phosphor-specific antibody we have developed, we further proved that the phosphorylation event was initiated in mitosis. Functional studies reveal a critical role for Snail phosphorylation in activation of the SAC and prevention of CIN. Conclusions: 1. Snail is required for mitotic spindle checkpoint activation; 2. Snail is phosphorylated by Aurora B in mitosis; 3. Aurora-B medicated Snail phosphorylation is required for optinal activation of the spindle checkpoint and prevention of chromosomal instability in breast cancer. Legal entity responsible for the study: The authors. Funding: Tianjin Medical University Cancer Institute and Hospital. Disclosure: All authors have declared no conflicts of interest.
10P
Development of chimeric antigenic receptor (CAR) against VEGFR2 for solid tumour treatment
L-S. Ai, Y-Y. Wu, J-Y. Huang Biologics Development Department Institute of Biologics, Development Center for Biotechnology, Taipei, Taiwan Background: Cancer is known to be the second cause of death worldwide. Vascular endothelial growth factors (VEGFs) regulate blood vessel development, the overexpressed of VEGF/VEGFR2 are often associated with angiogenesis, invasion, metastasis and prognosis in cancers. Targeting VEGFR2 by monoclonal antibodies has been associated with a survival benefit across multiple malignancies including lung, gastric and colorectal cancers. In addition to anti-VEGFR2 targeted therapies, immunotherapy including checkpoint blockade, adoptive cell therapy, and chimeric antigen receptor T cell (CART) acts as potential strategies to cure cancer. In this study, we propose VEGFR2-CART as a potential therapeutic strategy for different solid tumors treatment, and its therapeutic efficacy will be further investigated and verified. Methods: In this study, the anti-VEGFR2 single chain fragment variable (scFv) antibodies were screened by using an automated high throughput phage display system. The binding affinity various CDR sequences to VEGFR2 determined by binding ELISA. We generated CART by transducing human PBMCs with a VEGFR2-CAR 2nd generation lentiviral vector. Cytotoxic ability of the generated CART was assessed by a LDH assay. To evaluate the biologic effect of VEGFR2-CART cells, a breast xenowas performed by injecting HCC1428 cells subcutaneously into the flank of mice. Seventy-two VEGFR2 clones with various CDR sequences with the range of affinities of 10-8 to 10-10 M were selected. Cell toxicity assays showed that of VEGFR2-overexpressed FS293 cells were killed by VEGFR2-CART cells. To the biologic effect of VEGFR2-CART cells, a breast xenograft model was perby injecting HCC1428 cells subcutaneously into the flank of NOD/SCID mice. ts demonstrate that the VEGFR2-CART cells significantly inhibit the growth due to reduced tumor sizes and weights. ions: The cytotoxicity assay showed that the VEGFR2-overexpressed FS293 apparently killed by VEGFR2-CART cells. The antitumor effects of the RT cells have been proved in a murine tumor model, suggesting that targeting VEGFR2 with CART cells can be a novel strategy in cancer immunotherapy. Legal entity responsible for the study: Development Center for Biotechnology. Funding: The government of the Republic of China (Taiwan). Disclosure: All authors have declared no conflicts of interest.
doi:10.1093/annonc/mdz238 | v3
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz238.008/5576273 by guest on 24 October 2019
Background: Triple-negative breast cancers (TNBCs) are very aggressive and lethal forms of breast cancer with no effective targeted therapy. TNBCs spontaneously metastasize to distant organs including lungs, bone, and brain. Neo-adjuvant chemotherapies and radiotherapy remains a mainstay of treatment with only 25-30% of TNBC patients responding. Thus, there is an unmet clinical need to develop novel therapeutic strategies for TNBCs. Methods: We tested the anti-cancer activity of auranofin monotherapy on primary tumor growth and metastasis in vitro and in vivo. Further, using in vitro analysis we conceptualized the PD-L1 dependent resistance against auranofin which we validated in vivo using syngeneic TNBC model. Results: Expression of the thioredoxin pathway genes is significantly upregulated in TNBC patients compared to non-TNBC patients and correlated with adverse survival outcomes. Treatment with auranofin, an FDA-approved thioredoxin reductase inhibitor caused specific cell death and impaired the growth of TNBC grown as spheroids in 3D culture. Further, auranofin treatment exerted a significant in vivo anti-tumor activity in multiple TNBC models including syngeneic 4T1.2 model, human MDA-MB-231 xenograft, and PDX model by inhibiting thioredoxin redox activity. Auranofin also significantly inhibited the invasion potential of TNBC cells in vitro and significantly inhibited lung metastasis in 4T1.2 syngeneic model in vivo by reducing the expression of various EMT markers. We for the first time showed that auranofin increased CD8þVe T-cells tumor infiltration in vivo and upregulated immune checkpoint PD-L1 expression in ERK1/2-MYC-dependent manner. Moreover, combination of auranofin with anti-PD-L1 monoclonal antibody synergistically impaired the growth of 4T1.2 primary tumor. Conclusions: Our data provides a novel therapeutic strategy using auranofin in combination with anti-PD-L1 antibody for TNBCs and warrants further clinical investigation for TNBC patients. Since the success rate of anti-PD-L1 therapy is very low in TNBC patients, our data provides a novel strategy to use auranofin with anti-PD-L1 therapy to that may enhance the efficacy of immune checkpoint therapy in TNBC patients. Legal entity responsible for the study: The authors. Funding: Cure Cancer Australia & Can Too Foundation Project grant [ID 1147230] to Prahlad Raninga National Health & Medical Research Council (NH&MRC) Program Grant [ID 1017028] to Kum Kum Khanna Perpetual IMPACT Philanthropy project grant [IPAP201602001] to Kum Kum Khanna and Murugan Kalimutho. Disclosure: All authors have declared no conflicts of interest.
9PD
Annals of Oncology 7PD
Synergistic anti-cancer activity of auranofin with anti-PD-L1 therapy in triple negative breast cancer
P. Raninga1, A. Lee2, D. Sinha1, Y-Y. Shin2, D. Mittal1, M. Kalimutho1, K.K. Khanna1 1 Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Brisbane, Australia, 2Institute for Radiological Research, Radiation Biology Research Center, Taoyuan, Taiwan
8PD
Automatic interpretation of cancer genomes creates the largest repository of tumour genetic driver events
F. Martınez Jime´nez, F. Mui~ nos, O. Pich, A. Gonzalez-Perez, N. L opez-Bigas Biomedical Genomics, Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Background: Tumor genome sequencing is becoming widely available in the clinical setting. However, the interpretation of tumor somatic variants remains an important challenge to implement precision cancer medicine. Methods: Here, we present IntOGen, a platform aimed for tumor genome interpretation. The first component of the platform is an automatic pipeline for cancer driver gene identification across cohorts. This pipeline implements several bioinformatics methods to detect driver genes, on the basis of signals of positive selection in their mutational pattern across tumor cohorts. It also annotates several oncogenomic features that shed light onto the role of driver genes in the development of each malignancy. The second component of the platform leverages these features to identify the
Disclosure: All authors have declared no conflicts of interest.
Volume 30 | Supplement 5 | October 2019
Aurora-B mediated snail phosphorylation is essential for mitotic spindle checkpoint and for preventing chromosomal instability in breast cancer
B. Xu, M. Xiao, X. Li Breast Cancer Center, Tianjin Medical University Cancer Institute & Hospital, Tianjin, China Background: Snail is a transcriptional repressor that promotes epithelial-to-mesenchymal transition (EMT). Snail is upregulated in breast cancer and it is associated with chromosomal instability (CIN) and metastasis. The mitotic spindle assembly checkpoint (SAC) is a critical mechanism to prevent CIN. Extensive studies have investigated signaling pathways that involve in protein-protein interactions and post translational modifications. However, it not known how mitosis might be impacted by transcriptional regulation. Due to the fact that Snail is a transcriptional regulator and that Snail is upregulated in breast cancer with CIN, it is critical to understand whether and how abnormally regulated Snail contributes to breast cancer CIN via transcriptional regulation. Methods: We used siRNA to knockdown Snail in breast caner cell lines to look for a change in SAC. We developed a phosphor-specific antibody to study Aurora-B mediated phosphorylation. Site specific mutagenesis was used to generate phosphorylation site mutants, and exogenous proteins were expressed in cells to look for an effect in SAC and CIN. Results: We found that Snail is an essential element that is required for the SAC. Genetic depletion of Snail in breast cancer cells by siRNA led to a defect in activation of SAC (measured by accumulation of Histone Serine 10 phosphorylation in response to spindle damaging agents). Interestingly we also found that the Snail protein level was significantly enhanced in the presence of nocodazole. Further, we found that Snail upregulation was through phosphorylation in an Aurora-B dependent manner. We identified a serine site that can be phosphorylated by Aurora-B. Using a phosphor-specific antibody we have developed, we further proved that the phosphorylation event was initiated in mitosis. Functional studies reveal a critical role for Snail phosphorylation in activation of the SAC and prevention of CIN. Conclusions: 1. Snail is required for mitotic spindle checkpoint activation; 2. Snail is phosphorylated by Aurora B in mitosis; 3. Aurora-B medicated Snail phosphorylation is required for optinal activation of the spindle checkpoint and prevention of chromosomal instability in breast cancer. Legal entity responsible for the study: The authors. Funding: Tianjin Medical University Cancer Institute and Hospital. Disclosure: All authors have declared no conflicts of interest.
10P
Development of chimeric antigenic receptor (CAR) against VEGFR2 for solid tumour treatment
L-S. Ai, Y-Y. Wu, J-Y. Huang Biologics Development Department Institute of Biologics, Development Center for Biotechnology, Taipei, Taiwan Background: Cancer is known to be the second cause of death worldwide. Vascular endothelial growth factors (VEGFs) regulate blood vessel development, the overexpressed of VEGF/VEGFR2 are often associated with angiogenesis, invasion, metastasis and prognosis in cancers. Targeting VEGFR2 by monoclonal antibodies has been associated with a survival benefit across multiple malignancies including lung, gastric and colorectal cancers. In addition to anti-VEGFR2 targeted therapies, immunotherapy including checkpoint blockade, adoptive cell therapy, and chimeric antigen receptor T cell (CART) acts as potential strategies to cure cancer. In this study, we propose VEGFR2-CART as a potential therapeutic strategy for different solid tumors treatment, and its therapeutic efficacy will be further investigated and verified. Methods: In this study, the anti-VEGFR2 single chain fragment variable (scFv) antibodies were screened by using an automated high throughput phage display system. The binding affinity various CDR sequences to VEGFR2 determined by binding ELISA. We generated CART by transducing human PBMCs with a VEGFR2-CAR 2nd generation lentiviral vector. Cytotoxic ability of the generated CART was assessed by a LDH assay. To evaluate the biologic effect of VEGFR2-CART cells, a breast xenowas performed by injecting HCC1428 cells subcutaneously into the flank of mice. Seventy-two VEGFR2 clones with various CDR sequences with the range of affinities of 10-8 to 10-10 M were selected. Cell toxicity assays showed that of VEGFR2-overexpressed FS293 cells were killed by VEGFR2-CART cells. To the biologic effect of VEGFR2-CART cells, a breast xenograft model was perby injecting HCC1428 cells subcutaneously into the flank of NOD/SCID mice. ts demonstrate that the VEGFR2-CART cells significantly inhibit the growth due to reduced tumor sizes and weights. ions: The cytotoxicity assay showed that the VEGFR2-overexpressed FS293 apparently killed by VEGFR2-CART cells. The antitumor effects of the RT cells have been proved in a murine tumor model, suggesting that targeting VEGFR2 with CART cells can be a novel strategy in cancer immunotherapy. Legal entity responsible for the study: Development Center for Biotechnology. Funding: The government of the Republic of China (Taiwan). Disclosure: All authors have declared no conflicts of interest.
doi:10.1093/annonc/mdz238 | v3
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz238.008/5576273 by guest on 24 October 2019
Background: Triple-negative breast cancers (TNBCs) are very aggressive and lethal forms of breast cancer with no effective targeted therapy. TNBCs spontaneously metastasize to distant organs including lungs, bone, and brain. Neo-adjuvant chemotherapies and radiotherapy remains a mainstay of treatment with only 25-30% of TNBC patients responding. Thus, there is an unmet clinical need to develop novel therapeutic strategies for TNBCs. Methods: We tested the anti-cancer activity of auranofin monotherapy on primary tumor growth and metastasis in vitro and in vivo. Further, using in vitro analysis we conceptualized the PD-L1 dependent resistance against auranofin which we validated in vivo using syngeneic TNBC model. Results: Expression of the thioredoxin pathway genes is significantly upregulated in TNBC patients compared to non-TNBC patients and correlated with adverse survival outcomes. Treatment with auranofin, an FDA-approved thioredoxin reductase inhibitor caused specific cell death and impaired the growth of TNBC grown as spheroids in 3D culture. Further, auranofin treatment exerted a significant in vivo anti-tumor activity in multiple TNBC models including syngeneic 4T1.2 model, human MDA-MB-231 xenograft, and PDX model by inhibiting thioredoxin redox activity. Auranofin also significantly inhibited the invasion potential of TNBC cells in vitro and significantly inhibited lung metastasis in 4T1.2 syngeneic model in vivo by reducing the expression of various EMT markers. We for the first time showed that auranofin increased CD8þVe T-cells tumor infiltration in vivo and upregulated immune checkpoint PD-L1 expression in ERK1/2-MYC-dependent manner. Moreover, combination of auranofin with anti-PD-L1 monoclonal antibody synergistically impaired the growth of 4T1.2 primary tumor. Conclusions: Our data provides a novel therapeutic strategy using auranofin in combination with anti-PD-L1 antibody for TNBCs and warrants further clinical investigation for TNBC patients. Since the success rate of anti-PD-L1 therapy is very low in TNBC patients, our data provides a novel strategy to use auranofin with anti-PD-L1 therapy to that may enhance the efficacy of immune checkpoint therapy in TNBC patients. Legal entity responsible for the study: The authors. Funding: Cure Cancer Australia & Can Too Foundation Project grant [ID 1147230] to Prahlad Raninga National Health & Medical Research Council (NH&MRC) Program Grant [ID 1017028] to Kum Kum Khanna Perpetual IMPACT Philanthropy project grant [IPAP201602001] to Kum Kum Khanna and Murugan Kalimutho. Disclosure: All authors have declared no conflicts of interest.
9PD