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Enhancement of the antigen-specific T cell immune responses and therapeutic antitumor effects generated by therapeutic HPV DNA vaccine by histone deacetylase inhibitor
Abstracts / Gynecologic Oncology 125 (2012) S3–S167
cisplatin treatment, including c-jun, ERCC1, XPD, and XRCC1. This block in upregulation of c-jun was concurrent with a change in the phosphorylation pattern of the c-jun protein, shifting that pattern from a Ser63/73 dominant pattern to a Thr91/93 dominant pattern. A2780-CP70 cells were treated at their cisplatin IC50, and DNA repair was assessed after pretreatment with anti-Gli1 shRNA or scrambled shRNA control. Control cells repaired 78% of platinum-DNA adducts at 12 h compared to 33% repair in cells pretreated with anti-Gli1 shRNA, a 2.4 fold difference. Pretreatment of A2780-CP70 cells with anti-Gli1 shRNA resulted in supra-additive cell killing with cisplatin, shifting the cisplatin IC50 from 30 uM to 5 uM. Pretreatment of these cells with cyclopamine did not shift the cisplatin IC50. Conclusions: We conclude that the transcriptional protein Gli1 is important in the upregulation of these 3 DNA repair genes in human ovarian cancer cells, thereby strongly influencing platinum-DNA adduct repair and cellular sensitivity to cisplatin. This Gli1 role has cjun as an intermediate in the pathway. doi:10.1016/j.ygyno.2011.12.333
333 Dasatinib (BMS-35482) potentiates the activity of gemcitabine and docetaxel in leiomyosarcoma cell lines M. Lopez Acevedo1, D. Teoh2, D. Adams1, A. Nixon1, J. Jingquan1, L. Grace1, R. Whitaker1, A. Alvarez-Secord1. 1Duke University Medical Center, Durham, NC, 2University of Minnesota, Minneapolis, MN. Objective: (1) To explore the activity of dasatinib alone and in combination with gemcitabine and docetaxel in leiomyosarcoma (LMS) cell lines; and (2) to determine if SRC pathway is inhibited by dasatinib in leiomyosarcoma cell lines. Methods: Three leiomyosarcoma cell lines were treated with single agent gemcitabine, docetaxel, and dasatinib, as well as the following combinations: dasatinib–gemcitabine; dasatinib–docetaxel; and gemcitabine– docetaxel. Cell viability was measured using the ATPlite luminescence detection assay. Dose–response curves were constructed and the coefficient of drug interaction (CDI) was calculated. The level of SRC activation (pSRC) was determined in the uterine LMS cell lines using Meso Scale Discovery (MSD) multi-array immunogenicity assay. Results: RESULTS: The maximal growth inhibitory effect of dasatinib was 42%, 56%, and 49% in SK-UT-1, SK-UT-1B, and SKLMS-1 cell lines, respectively. The addition of a minimally active concentration of dasatinib (IC25) decreased the IC50 of each cytotoxic agent by 3–4 fold. The average CDIs for the combination of gemcitabine–dasatinib were 0.69 (SD±0.11), 0.67 (SD±0.07) and 0.87 (SD±.0.05) for SK-UT-1, SKUT-1B, and SKLMS-1, respectively. The average CDIs for combination docetaxel–dasatinib were 0.85 (SD±0.05), 0.87 (SD±0.01) and 0.93 (SD±0.03) for SK-UT-1, SK-UT-1B, and SKLMS-1, respectively. Growth inhibition for combination gemcitabine–docetaxel was 42%; gemcitabine–dasatinib, 79%; and docetaxel–dasatinib, 46% for SK-UT-1 cells. Growth inhibition for combination gemcitabine–docetaxel was 92%; gemcitabine–dasatinib, 85%; and docetaxel–dasatinib, 96% for SK-UT-1B cells. In SK-UT-1 dasatinib inhibited SRC kinase activity by 83.5% at 30 nM; 92.2% at 100 nM and 98.33% at 500 nM. In SK-UT-1B dasatinib inhibited SRC kinase activity by 89.20% at 30 nM; 88.94% at 100 nM and 95.14% at 500 nM. Conclusions: Dasatinib, through inhibition of the SRC pathway, demonstrated strong potentiation effect in combination with either gemcitabine or docetaxel in leiomyosarcoma. Based on our preclinical data and known activity of gemcitabine and docetaxel, further evaluation of dasatinib in combination with these agents for the treatment of uterine leiomyosarcoma is warranted. doi:10.1016/j.ygyno.2011.12.334
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334 Enhancement of the antigen-specific T cell immune responses and therapeutic antitumor effects generated by therapeutic HPV DNA vaccine by histone deacetylase inhibitor R. Alvarez1, C. Chen2, C. Hung2, T. Kang2, S. Lee3, T. Wu3. 1University of Alabama at Birmingham, Birmingham, AL, 2Johns Hopkins Medical Institutions, Baltimore, MD, 3Johns Hopkins Kimmel Cancer Center, Baltimore, MD. Objective: Multimodality treatments that combine conventional cancer therapies with antigen-specific immunotherapy have emerged as promising approaches for the control of cancer. Antigen-specific immunotherapy is an attractive approach for the treatment of cancers since it has the potency to specifically eradicate systemic tumors and control metastases without damaging normal cells. A favorable approach for antigen-specific immunotherapy is the use of DNA vaccines due to their safety, stability and ease of preparation. We have previously developed therapeutic human papillomavirus (HPV) DNA vaccines encoding calreticulin (CRT) linked to HPV-16 E7 antigen (CRT/E7) to enhance therapeutic HPV DNA vaccine potency. The CRT/ E7 DNA vaccine has been tested in early phase clinical trials. Methods: In the current study, we explore the employment of a new histone deacetylase (HDAC) inhibitor, AR-42 (formerly known as OSU-HDAC42), in combination with CRT/E7 DNA vaccine in a preclinical model for its potential to enhance E7-specific CD8+ T cell immune responses as well as antitumor effects against E7expressing tumors. AR-42 has also been in use in early phase clinical trials. Results: We found that treatment with AR-42 led to upregulation of MHC class I molecules in E7-expressing TC-1 tumor cells, rendering the tumor cells more susceptible to E7-specific CD8+ T cell-mediated killing. In addition, treatment with AR-42 enhanced the expression of the protein encoded by the DNA construct in transfected dendritic cell line DC-7. More importantly, treatment with combination of AR42 and CRT/E7 DNA vaccine led to improved antigen-specific CD8+ T cell immune responses as well as therapeutic antitumor effects in TC1 tumor-bearing mice. Conclusions: Our data indicate that AR-42 can enhance therapeutic HPV DNA vaccine potency in generating improved antigen-specific immune responses and antitumor effects. These findings have important implications for future clinical translation. doi:10.1016/j.ygyno.2011.12.335
335 Lapatinib induces downregulation of matrix metallopeptidase 1 and deactivation of ERK1/2 and PI3K pathway in endometrial cancer C. Lai1, C. Lin2, A. Chao1, S. Hsueh2, T. Wang1. 1Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan, China, 2Chang Gung Memorial Hospital, Taoyuan, Taiwan, China. Objective: We aim to investigate the molecular mechanisms underlying endometrial cancer cells when treated with lapatinib (Tykerb®), a dual tyrosine kinase inhibitors of EGFR and HER2. Methods: Affymetrix oligonucleotide microarrays were used to screen the differential gene expression of endometrial cancer cells treated with lapatinib. The RNA or protein expression levels of the genes were confirmed in clinical specimens of endometrial cancer and normal endometrial tissues by real-time quantitative PCR and immunohistochemical staining (IHC). The histoscore was the percentage of positive cells multiplied by its staining intensity that ranged from 0 to 300. Endometrial cancer cell lines were cultured
cisplatin treatment, including c-jun, ERCC1, XPD, and XRCC1. This block in upregulation of c-jun was concurrent with a change in the phosphorylation pattern of the c-jun protein, shifting that pattern from a Ser63/73 dominant pattern to a Thr91/93 dominant pattern. A2780-CP70 cells were treated at their cisplatin IC50, and DNA repair was assessed after pretreatment with anti-Gli1 shRNA or scrambled shRNA control. Control cells repaired 78% of platinum-DNA adducts at 12 h compared to 33% repair in cells pretreated with anti-Gli1 shRNA, a 2.4 fold difference. Pretreatment of A2780-CP70 cells with anti-Gli1 shRNA resulted in supra-additive cell killing with cisplatin, shifting the cisplatin IC50 from 30 uM to 5 uM. Pretreatment of these cells with cyclopamine did not shift the cisplatin IC50. Conclusions: We conclude that the transcriptional protein Gli1 is important in the upregulation of these 3 DNA repair genes in human ovarian cancer cells, thereby strongly influencing platinum-DNA adduct repair and cellular sensitivity to cisplatin. This Gli1 role has cjun as an intermediate in the pathway. doi:10.1016/j.ygyno.2011.12.333
333 Dasatinib (BMS-35482) potentiates the activity of gemcitabine and docetaxel in leiomyosarcoma cell lines M. Lopez Acevedo1, D. Teoh2, D. Adams1, A. Nixon1, J. Jingquan1, L. Grace1, R. Whitaker1, A. Alvarez-Secord1. 1Duke University Medical Center, Durham, NC, 2University of Minnesota, Minneapolis, MN. Objective: (1) To explore the activity of dasatinib alone and in combination with gemcitabine and docetaxel in leiomyosarcoma (LMS) cell lines; and (2) to determine if SRC pathway is inhibited by dasatinib in leiomyosarcoma cell lines. Methods: Three leiomyosarcoma cell lines were treated with single agent gemcitabine, docetaxel, and dasatinib, as well as the following combinations: dasatinib–gemcitabine; dasatinib–docetaxel; and gemcitabine– docetaxel. Cell viability was measured using the ATPlite luminescence detection assay. Dose–response curves were constructed and the coefficient of drug interaction (CDI) was calculated. The level of SRC activation (pSRC) was determined in the uterine LMS cell lines using Meso Scale Discovery (MSD) multi-array immunogenicity assay. Results: RESULTS: The maximal growth inhibitory effect of dasatinib was 42%, 56%, and 49% in SK-UT-1, SK-UT-1B, and SKLMS-1 cell lines, respectively. The addition of a minimally active concentration of dasatinib (IC25) decreased the IC50 of each cytotoxic agent by 3–4 fold. The average CDIs for the combination of gemcitabine–dasatinib were 0.69 (SD±0.11), 0.67 (SD±0.07) and 0.87 (SD±.0.05) for SK-UT-1, SKUT-1B, and SKLMS-1, respectively. The average CDIs for combination docetaxel–dasatinib were 0.85 (SD±0.05), 0.87 (SD±0.01) and 0.93 (SD±0.03) for SK-UT-1, SK-UT-1B, and SKLMS-1, respectively. Growth inhibition for combination gemcitabine–docetaxel was 42%; gemcitabine–dasatinib, 79%; and docetaxel–dasatinib, 46% for SK-UT-1 cells. Growth inhibition for combination gemcitabine–docetaxel was 92%; gemcitabine–dasatinib, 85%; and docetaxel–dasatinib, 96% for SK-UT-1B cells. In SK-UT-1 dasatinib inhibited SRC kinase activity by 83.5% at 30 nM; 92.2% at 100 nM and 98.33% at 500 nM. In SK-UT-1B dasatinib inhibited SRC kinase activity by 89.20% at 30 nM; 88.94% at 100 nM and 95.14% at 500 nM. Conclusions: Dasatinib, through inhibition of the SRC pathway, demonstrated strong potentiation effect in combination with either gemcitabine or docetaxel in leiomyosarcoma. Based on our preclinical data and known activity of gemcitabine and docetaxel, further evaluation of dasatinib in combination with these agents for the treatment of uterine leiomyosarcoma is warranted. doi:10.1016/j.ygyno.2011.12.334
S137
334 Enhancement of the antigen-specific T cell immune responses and therapeutic antitumor effects generated by therapeutic HPV DNA vaccine by histone deacetylase inhibitor R. Alvarez1, C. Chen2, C. Hung2, T. Kang2, S. Lee3, T. Wu3. 1University of Alabama at Birmingham, Birmingham, AL, 2Johns Hopkins Medical Institutions, Baltimore, MD, 3Johns Hopkins Kimmel Cancer Center, Baltimore, MD. Objective: Multimodality treatments that combine conventional cancer therapies with antigen-specific immunotherapy have emerged as promising approaches for the control of cancer. Antigen-specific immunotherapy is an attractive approach for the treatment of cancers since it has the potency to specifically eradicate systemic tumors and control metastases without damaging normal cells. A favorable approach for antigen-specific immunotherapy is the use of DNA vaccines due to their safety, stability and ease of preparation. We have previously developed therapeutic human papillomavirus (HPV) DNA vaccines encoding calreticulin (CRT) linked to HPV-16 E7 antigen (CRT/E7) to enhance therapeutic HPV DNA vaccine potency. The CRT/ E7 DNA vaccine has been tested in early phase clinical trials. Methods: In the current study, we explore the employment of a new histone deacetylase (HDAC) inhibitor, AR-42 (formerly known as OSU-HDAC42), in combination with CRT/E7 DNA vaccine in a preclinical model for its potential to enhance E7-specific CD8+ T cell immune responses as well as antitumor effects against E7expressing tumors. AR-42 has also been in use in early phase clinical trials. Results: We found that treatment with AR-42 led to upregulation of MHC class I molecules in E7-expressing TC-1 tumor cells, rendering the tumor cells more susceptible to E7-specific CD8+ T cell-mediated killing. In addition, treatment with AR-42 enhanced the expression of the protein encoded by the DNA construct in transfected dendritic cell line DC-7. More importantly, treatment with combination of AR42 and CRT/E7 DNA vaccine led to improved antigen-specific CD8+ T cell immune responses as well as therapeutic antitumor effects in TC1 tumor-bearing mice. Conclusions: Our data indicate that AR-42 can enhance therapeutic HPV DNA vaccine potency in generating improved antigen-specific immune responses and antitumor effects. These findings have important implications for future clinical translation. doi:10.1016/j.ygyno.2011.12.335
335 Lapatinib induces downregulation of matrix metallopeptidase 1 and deactivation of ERK1/2 and PI3K pathway in endometrial cancer C. Lai1, C. Lin2, A. Chao1, S. Hsueh2, T. Wang1. 1Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan, China, 2Chang Gung Memorial Hospital, Taoyuan, Taiwan, China. Objective: We aim to investigate the molecular mechanisms underlying endometrial cancer cells when treated with lapatinib (Tykerb®), a dual tyrosine kinase inhibitors of EGFR and HER2. Methods: Affymetrix oligonucleotide microarrays were used to screen the differential gene expression of endometrial cancer cells treated with lapatinib. The RNA or protein expression levels of the genes were confirmed in clinical specimens of endometrial cancer and normal endometrial tissues by real-time quantitative PCR and immunohistochemical staining (IHC). The histoscore was the percentage of positive cells multiplied by its staining intensity that ranged from 0 to 300. Endometrial cancer cell lines were cultured