bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 904: The novel C-terminal truncated 90-kDa isoform of topoisomerase IIα, TOP2α/90, is a determinant of etoposide resistance in K562 leukemia cells via heterodimerization with the TOP2α/170 isoform
Authors: Kanagasabai Ragu , Karmahapatra Soumendra , Yu Yang , Hernandez Victor A. , Kientz Corey A. , Kania Evan E. , Elton Terry S. , Yalowich Jack C.
Published: August 2018

DNA topoisomerase IIα (170 kDa, TOP2α/170) is essential in proliferating cells since it resolves DNA topologic entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated TOP2α/90 isoform, detectable in human leukemia K562 cells but more abundantly expressed in a clonal subline, K/VP.5, with acquired resistance to the anticancer agent etoposide (J Pharmacol Exp Ther 2017;360:152-63). TOP2α/90 (786 amino acids) is the translation product of a TOP2α mRNA that retains a processed intron 19. TOP2α/90 lacks the active-site tyrosine-805 (Tyr805) required to generate double-strand DNA breaks as well as the nuclear localization signals present in the TOP2α/170 isoform (1531 amino acids). The function of TOP2α/90 is unknown. Here, we found that TOP2α/90, like TOP2α/170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Importantly, co-immunoprecipitation of endogenous TOP2α/90 and TOP2α/170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2α/90 in K562 cells suppressed, while siRNA-mediated knockdown of TOP2α/90 in K/VP.5 cells enhanced, etoposide-mediated DNA strand breaks compared with similarly treated K562 or K/VP.5 cells transfected with empty vector or control siRNAs, respectively. In addition, forced expression of TOP2α/90 in K562 cells inhibited etoposide cytotoxicity assessed by soft agar colony formation assays. qPCR and immunoassays demonstrated expression of TOP2α/90 mRNA and protein in normal human tissues/cells and in leukemia cells from patients. Together, results strongly suggest that TOP2α/90 expression decreases drug-induced TOP2α-DNA covalent complexes and is a determinant of chemoresistance through a dominant-negative effect related to heterodimerization with TOP2α/170. Alternative processing of TOP2α pre-mRNA, and subsequent synthesis of TOP2α/90, may be an important mechanism regulating the formation and/or stability of TOP2α/170-DNA covalent complexes in response to TOP2α-targeting agents.

Citation Format: Ragu Kanagasabai, Soumendra Karmahapatra, Yang Yu, Victor A. Hernandez, Corey A. Kientz, Evan E. Kania, Terry S. Elton, Jack C. Yalowich. The novel C-terminal truncated 90-kDa isoform of topoisomerase IIα, TOP2α/90, is a determinant of etoposide resistance in K562 leukemia cells via heterodimerization with the TOP2α/170 isoform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 904.
Abstract 905: RICTOR modulation regulates cisplatin sensitivity in lung cancer
Authors: Cheng Haiying , Fan Ni , Borczu Alain , Liu Huijie , Singh Aditi , Wang Feng , Halmos Balazs , Perez-Soler Roman
Published: August 2018

Background: Despite treatment advances, most lung cancer patient will still receive platinum-based chemotherapy and platinum resistance remains a serious clinical problem. RICTOR is a key component of the mTOR complex 2. Activation of RICTOR signaling has been suggested to play critical roles in regulating cancer cell migration, invasion and metastasis. A recent study indicates that RICTOR contributes to cisplatin resistance in human ovarian cancer cells. We hypothesize that activation of RICTOR signaling is associated with platinum resistance in lung cancer. Thus, we tested platinum sensitivities in lung cancer cells and xenograft mouse models with different RICTOR levels. Results: RICTOR expression was found in 79% (99/125) of primary lung tumors and 67% (24/36) lung cancer cells by immunohistochemistry. The expression of RICTOR was significantly higher in squamous cell lung cancer in comparison to lung adenocarcinoma (P<0.05). Overexpression of RICTOR in the Ba/F3 cells resulted in significant cisplatin resistance, whereas down-regulation of RICTOR in H23, HCC827 and H1703 lung cancer cells led to significantly improved cisplatin sensitivity. In addition, the combination of cisplatin and RICTOR knockdown significantly promoted more apoptosis than single treatment. To test the in vivo effects of RICTOR on platinum sensitivity, we performed HCC827 xenograft mouse experiments. Cisplatin alone and RICTOR knockdown alone significantly inhibited tumor growth. Moreover, RICTOR knockdown significantly increased the sensitivities to cisplatin in the xenografts and the combination led to more pronounced tumor reduction than either alone (P<0.05). Conclusion: Our study provides a novel resistance mechanism to platinum treatment in lung cancer and may serve as basis to target RICTOR to overcome platinum resistance.

Citation Format: Haiying Cheng, Ni Fan, Alain Borczu, Huijie Liu, Aditi Singh, Feng Wang, Balazs Halmos, Roman Perez-Soler. RICTOR modulation regulates cisplatin sensitivity in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 905.
Abstract 912: uPA as a topoisomerase-1 protective agent in lung fibroblast cells
Authors: Ge Liang , Gnanamony Manu , Stepanova Victoria , Dinh Dzung H. , Gondi Christopher S.
Published: August 2018

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, with approximately 53,000 people in the U.S. diagnosed with the disease every year. The Gondi lab has previously shown that poor survival rates in pancreatic cancer are correlated with increased expression of urokinase plasminogen activator (uPA), a serine protease. Our previous data has shown for the first time that uPA binds to topoisomerase-1 (TOPO-1), in addition to the interaction of uPA with various transcription factors. Our current research is aimed at elucidating the mechanism of the interaction between uPA and TOPO-1, with the goal of demonstrating a protective role for uPA in the regulation of TOPO-1 function in the presence of TOPO-1 inhibitors. Methods: Two knock-out lung fibroblast cell lines were used. One cell line was a uPA knock-out line, while the other was a uPA knock-out line with a uPA expression vector transfected into the cells. Transfection was performed using Chariot transfection vector. The cell lines were treated with topoisomerase inhibitors TX1111 and SN38. DNA replication activity was measured using flow cytometry as a surrogate for TOPO-1 activity. Controls included untreated cells, cells treated with empty Chariot, and cells treated with Chariot containing TXC peptide. Results and Discussion: In the presence of topoisomerase inhibitors TX1111 and SN38, uPA knock-out lines demonstrated incomplete cell division relative to controls. In uPA-expressing lines, cell division activity was retained in the presence of topoisomerase inhibitors. Our data demonstrates for the first time that uPA serves a protective role in preventing the inhibition of TOPO-1 by topoisomerase inhibitors. This indicates uPA as a potential new drug target in the treatment of chemoresistant pancreatic cancer. Further studies to be performed include visualizing the degree of DNA-replication using DNA combing, and subsequently expanding this study to pancreatic cancer cells.

Citation Format: Liang Ge, Manu Gnanamony, Victoria Stepanova, Dzung H. Dinh, Christopher S. Gondi. uPA as a topoisomerase-1 protective agent in lung fibroblast cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 912.
Abstract 914: COX-2 expression is associated with chemoresistance through cancer stemness property in head and neck squamous cell carcinoma
Authors: Saito Shin , Ozawa Hiroyuki , Imanishi Yorihisa , Sekimizu Mariko , Watanabe Yoshihiro , Ito Fumihiro , Ikari Yuichi , Nakahara Nana , Kameyama Kaori , Ogawa Kaoru
Published: August 2018

Introduction: COX-2 is one of the two isoforms of cyclooxygenase, an enzyme that catalyzes the conversion of arachidonic acid to prostaglandins. COX-2 is considered to be associated with progression in various cancers, and its expression has been reported to have an impact on poor prognosis in head and neck squamous cell carcinomas (HNSCCs). Furthermore, COX-2 expression has been shown to be associated with resistance to anticancer drugs. However, the precise mechanism of COX-2 for chemoresistance in HNSCC is not fully elucidated. The aim of the present study was to investigate the impact of COX-2 on cancer stem cell property, and to reveal its effect on chemoresistance by in vitro and clinicopathological assays. Methods: We examined the chemoresistance to docetaxel with or without COX-2 inhibitor (celecoxib) in HNSCC cell lines (FaDu, Detroit512) by MTS assays. To evaluate the association of COX-2 expression with stemness property, we assessed alteration of expression levels of the genes related to cancer stem cell (CSC) property after exposure to celecoxib by quantitative real-time PCR. We also performed a sphere formation assay using ultra low attachment dishes and microscopic imaging. Moreover, we analyzed immunohistochemical expressions of COX-2 and clinicopathological factors by using matched samples of pre-treatment biopsy and surgical specimens from hypopharyngeal carcinoma patients who had undergone trans-oral tumor resection with preoperative induction chemotherapy including docetaxel. Results: Cell survival rate under exposure to docetaxel was decreased by addition of celecoxib. COX-2 inhibitor downregulated the expression of Oct3/4 and Nanog. Sphere formation was inhibited by co-culture with COX-2 inhibitor, especially in FaDu cells. Immunohistochemical study in biopsy specimens revealed a negative correlation between COX-2 expression in biopsy specimens and pathological effect of induction chemotherapy in surgical specimens. Conclusion: The present study suggests that COX-2 expression is associated with cancer stemness property, and inhibition of COX-2 has a possibility to enhance chemosensitivity in HNSCCs. Furthermore, initial COX-2 expression in biopsy specimens may predict the effect of chemotherapy in HNSCCs. These results indicate that COX-2 can be an attractive target for the treatment of HNSCC.

Citation Format: Shin Saito, Hiroyuki Ozawa, Yorihisa Imanishi, Mariko Sekimizu, Yoshihiro Watanabe, Fumihiro Ito, Yuichi Ikari, Nana Nakahara, Kaori Kameyama, Kaoru Ogawa. COX-2 expression is associated with chemoresistance through cancer stemness property in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 914.
Abstract 921: High-throughput functional evaluation of variants of unknown significance in ERBB2
Authors: Nagano Masaaki , Kohsaka Shinji , Ueno Toshihide , Kojima Shinya , Ikegami Masachika , Takeda Ikuko , Mano Hiroyuki
Published: August 2018

Erb-b2 receptor tyrosine kinase 2 (ERBB2) gene amplification is present in a wide variety of human cancers. ERBB2-targeted therapies have been developed and improved the outcome in patients with ERBB2-positive cancers. Recently, the advent of next-generation sequencing technology has enabled to identify numerous ERBB2 somatic mutations across a variety of cancers, but many of the mutations are still variants of unknown significance (VUS) that await further investigation of clinical relevance. In addition, the sensitivities of those ERBB2 mutations to ERBB2-targeted drugs have not been evaluated systematically. Here we assessed comprehensively the transforming activities as well as drug sensitivities of ERBB2 mutations in a high-throughput manner using the mixed-all-nominated-in-one (MANO) method. We evaluated 55 non-synonymous ERBB2 mutations which are reported recurrently in COSMIC database, and discovered several novel activating mutations which probably drive tumorigenesis. Furthermore, ERBB2 mutations showed varying drug sensitivities to ERBB2-targeted inhibitors. Thus, the MANO method may be a novel approach for assessing VUS, and our findings in this study will be beneficial to deliver personalized medicine to cancer patients harboring ERBB2 mutations.

Citation Format: Masaaki Nagano, Shinji Kohsaka, Toshihide Ueno, Shinya Kojima, Masachika Ikegami, Ikuko Takeda, Hiroyuki Mano. High-throughput functional evaluation of variants of unknown significance in ERBB2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 921.
Abstract 927: TCPA: An open access resource for cancer functional proteomics data
Authors: Li Jun , Liang Han
Published: August 2018

In recent years, tremendous efforts have been made to systematically characterize the molecular profiles of tumor tissues from individuals with cancer, laying a critical foundation for elucidating the molecular basis of tumorigenesis and developing biomarker-based diagnostic, prognostic and therapeutic approaches. In particular, cancer genomic data at the DNA or RNA level are being accumulated at an unprecedented speed. However, comprehensive cancer proteomic data have been relatively limited. Using reverse phase protein arrays (RPPA), we have generated a large collection of cancer protein expression data (>8,000 samples of 32 cancer types from The Cancer Genome Atlas and >650 independent cell lines across 19 lineages).  To facilitate access of the broader research community to these RPPA datasets, we have developed a user-friendly data portal, TCPA (The Cancer Proteome Atlas), which can be accessed at http://tcpaportal.org.

 

TCPA currently has two web applications, one is for patient cohort data and the other is for cell line data. Both of them provide three types of modules: (i) “Summary”: the summary module help users check the detailed information about all the datasets curated in our database and provide a user-friendly interface for downloading the data. (ii) “Analysis”: this module provides common used analyses such as correlation, differential, survival and drug sensitivity analysis; and (iii) “Visualization” module provides approaches to examine the global patterns in a specific dataset. share some major analytic and visualization modules.

 

In summary, TCPA represents a highly valuable resource dedicated to cancer functional proteomic data. Compared with other proteomic databases, the primary advantage of TCPA is the availability of quantitative protein expression data over many large cohorts of well-characterized TCGA patient tumors. Moreover, TCPA provides an unprecedented opportunity to validate the findings from TCGA RPPA data through independent sample cohorts and to help select model tumor cell lines for further functional investigation. These unique features of TCPA greatly increase the utility of TCGA proteomic data for both basic research and clinical applications.

Citation Format: Jun Li, Han Liang. TCPA: An open access resource for cancer functional proteomics data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 927.
Abstract 931: Discovery of ABBV-744, a first-in-class highly BDII-selective BET bromodomain inhibitor
Authors: Sheppard George S. , Wang Le , Fidanze Steven D. , Hasvold Lisa A. , Liu Dachun , Pratt John K. , Park Chang H. , Bui Mai-Ha , Faivre Emily J. , Huang Xiaoli , Lin Xiaoyu , Wilcox Denise M. , Shen Yu , Albert Daniel H. , Magoc Terrance J. , Rajaraman Ganesh , Kati Warren M. , McDaniel Keith F.
Published: August 2018

The BET family of proteins consists of BRD2, BRD3, BRD4 and BRDT, with each of these proteins containing two distinct bromodomains (BDI and BDII). ABBV-075, like other first generation BET family bromodomain inhibitors currently under clinical development, binds to each of the 8 bromodomains with similar affinity and inhibits the proliferation of cancer cells that represent a wide range of tumor types. We hypothesized that selectively targeting specific subsets of the BET bromodomain might abolish this broad spectrum profile such that only the tumor types that are highly addicted to the subtype specific BET bromodomain-mediated transcription would remain sensitive to these selective agents. Both BDI and BDII are highly conserved across BET family members (> 70% identity), suggesting that the generation of compounds that are selective for the BDI bromodomains or the BDII bromodomains might be achievable. Structure-based design targeting the Asp144/His 437 and Ile146/Val439 sequence differences (BRD4 BDI/BDII numbering) led to the identification of structural analogs of ABBV-075 demonstrating greater than 100X selectivity for BRD4 BDII over BRD4 BDI. Further elaboration led to compounds with improved BDII-selectivity and oral bioavailability and the identification of the clinical asset ABBV-744. Disclosures: All authors are employees or former employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

Citation Format: George S. Sheppard, Le Wang, Steven D. Fidanze, Lisa A. Hasvold, Dachun Liu, John K. Pratt, Chang H. Park, Mai-Ha Bui, Emily J. Faivre, Xiaoli Huang, Xiaoyu Lin, Denise M. Wilcox, Yu Shen, Daniel H. Albert, Terrance J. Magoc, Ganesh Rajaraman, Warren M. Kati, Keith F. McDaniel. Discovery of ABBV-744, a first-in-class highly BDII-selective BET bromodomain inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 931.
Abstract 938: Detection of actionable mutations in plasma cfDNA samples from patients with non-small cell lung carcinoma using a novel amplicon-based Firefly NGS assay
Authors: Wang Lin , Weng Li , Chen Xiao , Li Min , Guo Qiaomei , Yu Wenjun , Wittkop Tobias , Wang Hongyan , Fahem Malek , Lin Shengrong , Zhao Grace Q. , Lou Jiatao
Published: August 2018

Background: Detection of EGFR, KRAS and BRAF mutations can help guide cancer treatment for non-small cell lung cancer (NSCLC) patients. To identify an easy to use, accurate, multiplex molecular diagnostic assay, we evaluated the performance of a novel next-generation sequencing (NGS)-based cell-free DNA (cfDNA) assay, Firefly assay, which employs a concatemer-based noise suppression mechanism with an amplicon workflow.

Methods: Performance of amplicon based Firefly assay, with a panel covering EGFR, BRAF, and KRAS mutations designed for targeted therapy selection of NSCLC was first evaluated using a cfDNA reference standard and blank control samples. This panel was then used to analyze plasma cfDNA samples from 134 NSCLC cancer patients and 50 non-cancerous controls, and results were compared with tumor tissue ARMS and cfDNA ddPCR results.

Results: Firefly assay demonstrated superior sensitivity and specificity with median detection of 100% at allele frequency of 0.1% for 20ng of cfDNA and zero false positive in all blank control samples. In cfDNA from plasma collected before treatment, EGFR mutation detection by Firefly assay was 94% concordant with tumor tissue ARMS. Firefly assay demonstrated strong per-variant detection-rate concordance (98%) and allele frequency concordance (R2 = 0.95) when compared with cfDNA ddPCR result.

Conclusions: The amplicon based Firefly assay offers multiplex capacity, de novo variant detection, high sensitivity and specificity. Thus, Firefly assay is a kitable NGS solution for cfDNA analysis, which can help guide targeted therapy selection, drug resistance detection, and disease monitoring in NSCLC and other cancer patients.

Citation Format: Lin Wang, Li Weng, Xiao Chen, Min Li, Qiaomei Guo, Wenjun Yu, Tobias Wittkop, Hongyan Wang, Malek Fahem, Shengrong Lin, Grace Q. Zhao, Jiatao Lou. Detection of actionable mutations in plasma cfDNA samples from patients with non-small cell lung carcinoma using a novel amplicon-based Firefly NGS assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 938.
Abstract 950: New strategy to sensitize pancreatic and bladder cancer to gemcitabine
Authors: Bayle Simon , Vena Francesca , Monastyrskyi Andrii , Roush William , Duckett Derek
Published: August 2018

Pancreatic Ductal Adenocarcinoma (PDAC), the major histological subtype of pancreatic cancer displays few symptoms for early detection and the majority of PDAC patients are diagnosed with advanced or metastatic disease. PDAC patients with advanced disease lack durable responses to chemotherapy and have a dismal prognosis. Gemcitabine is the cornerstone of care for advanced and metastatic PDAC. While the underlying mechanisms leading to gemcitabine resistance are likely to be multifactorial, there is a strong association between activating gemcitabine metabolism pathways and clinical outcome. Accordingly, strategies that lead to upregulation of signaling pathways and enzymes involved in generating the active metabolite may lead to improved outcome in PDAC.

We have developed a potent, selective, small molecule inhibitor of casein kinase-1 delta (CK1δ) and demonstrated that inhibition of CK1δ activity specifically compromises the growth, survival and invasion of breast cancer cells that overexpress CK1δ. Notably, our lead compound, SR-3029 provokes pre-clinical tumor regression of triple negative breast cancer (TNBC), including lung metastatic TNBC and basal-like PDX breast cancer models. We observed that CK1δ is upregulated in other cancers including bladder cancer and demonstrated that SR-3029 acts in synergy with DNA damaging cytotoxic agents. Notably, the combination of gemcitabine with SR-3029 resulted in synergistic antiproliferative activity and impaired colony formation in both pancreatic and bladder cancer cells. Mechanistic studies demonstrate that silencing CK1δ or inhibition of CK1δ activity by SR-3029 induces a significant upregulation of deoxycytidine kinase (DCK; rate limiting enzyme for gemcitabine activation) both at the RNA and protein levels, which results in increased cellular DCK and its active gemcitabine metabolite. We evaluated the effect of SR-3029 alone and in combination with gemcitabine in an orthotopic pancreatic tumor mouse model and observed much improved tumor growth inhibition with combination treatment, concomitant with increased expression of DCK.

In conclusion, our study suggests that CK1δ plays signaling roles in DNA metabolism and the combination of CK1δ inhibition with gemcitabine holds promise as a future therapeutic option for metastatic pancreatic cancer as well as other cancers.

Citation Format: Simon Bayle, Francesca Vena, Andrii Monastyrskyi, William Roush, Derek Duckett. New strategy to sensitize pancreatic and bladder cancer to gemcitabine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 950.
Abstract 963: Screening of neoantigen-specific T cells in head and neck cancer and establishment of T-cell receptor-engineered T cells with cytotoxic reactivity
Authors: Ren Lili , Matsuda Tatsuo , Deng Boya , Kiyotani Kazuma , Kato Taigo , Park Jae-Hyun , Seiwert Tanguy , Vokes Everett , Agrawal Nishant , Nakamura Yusuke
Published: August 2018

Neoantigen-reactive T cells are considered to be crucial mediators for anti-tumor activity in adoptive T cell transfer (ACT) immunotherapy although the efficacy of ACT using in vitro expanded tumor-infiltrating T lymphocytes (TILs) has been limited. Since characterization of T cell receptor (TCR) repertoires of TILs before and after in vitro expansion has not well investigated, we in present study performed whole exome sequencing (WES) and transcriptome analysis, and selected candidate neoantigen epitopes to induce cytotoxic T lymphocytes (CTLs) in 20 patients with head and neck cancer (10 frozen tissues and 10 freshly resected tumors). 36 potential neoantigen peptides including missense somatic mutations were examined for induction of neoantigen-reactive cytotoxic T cells in vitro using patients' derived dendritic cells. We have so far confirmed three neoantigen-reactive T cells and obtained the sequence information of a pair of TCR alpha and beta chains, which were identical to those identified in resident dominant TILs in the original tumors. We cloned the TCR genes into T lymphocytes and constructed the neoantigen-reactive T-cell receptor-engineered (TCR-engineered) T cells, which showed HLA-restricted neoantigen-reactive cytotoxic activity. In addition, we compared the TCR composition of TILs before/after in vitro expansion in 10 fresh head and neck cancers, and found that TCR clonotypes in original TILs and in vitro expanded TILs are drastically different except for those between expanded CD8 cells and resident TILs in three tumors, which had unusually high mutational burden due to mutations in the mismatch repair genes (MSH2, MSH6) or a DNA polymerase (POLE) gene. Our data also suggest that some expanded TILs are likely to be cancer-specific T cells and may be good cell sources for ACT immunotherapy and that the mutational load might be a good biomarker to screen effective TIL treatment. We demonstrate establishment of an effective and rapid protocol to generate neoantigen-specific T cells and to identify neoantigen-specific TCRs for individual patients, which makes TCR-engineered T cells applicable for the clinical use.

Citation Format: Lili Ren, Tatsuo Matsuda, Boya Deng, Kazuma Kiyotani, Taigo Kato, Jae-Hyun Park, Tanguy Seiwert, Everett Vokes, Nishant Agrawal, Yusuke Nakamura. Screening of neoantigen-specific T cells in head and neck cancer and establishment of T-cell receptor-engineered T cells with cytotoxic reactivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 963.
Abstract 965: Deleterious effects of MAPK pathway hyperactivation in BRAF-mutant melanoma
Authors: Leung Grace P. , Feng Tianshu , Shirley Matthew D. , Sigoillot Frederic D. , Ruddy David A. , Freeman Alyson K. , Jaskelioff Mariela , Stuart Darrin D.
Published: August 2018

The most frequent genetic alterations in melanoma are gain-of-function mutations in BRAF, which result in addiction to RAF-MEK-ERK signaling. Given the centrality of this pathway in many cancers, inhibitors have been developed against various nodes in this signaling axis. Despite the success of these inhibitors in treating BRAFV600E mutant tumors, a major problem is the inevitable emergence of drug resistance. The mechanisms of acquired resistance often include reactivation of the MAPK pathway, thus efforts to combat resistance have used vertical combination strategies to inhibit multiple nodes of the pathway. Previous work from our group showed that vemurafenib-resistant melanoma tumors displayed increased levels of MAPK signaling as indicated by elevated phospho-ERK. Strikingly, cell lines derived from these resistant tumors were addicted to vemurafenib, and removal of drug caused loss in cell viability. Consistently, in vivo, the tumors regressed upon cessation of drug treatment, which was accompanied by elevated MAPK signaling. Recent studies have reported similar results for in vitro models of melanoma resistant to MAPK inhibitors, which are addicted to drug in an ERK2-dependent manner. Together these data suggest that extreme levels of MAPK signaling are not tolerated by tumor cells. To further characterize this phenomenon, we generated models of inducible MAPK hyperactivation in BRAFV600E melanoma cells. Using these model systems, we confirmed that increasing the levels of MAPK signaling can hinder tumor cell proliferation, and demonstrated that MAPK inhibitors can rescue this phenotype. Intriguingly, the effect of MAPK hyperactivation varied depending on the RAS/RAF mutation status of the tumor cells. Upon induction of MAPK hyperactivation in sensitive cells, there was no cell cycle arrest, but instead cell death occurred, which was accompanied by activation of caspases. On a global level, MAPK hyperactivation resulted in transcriptome changes that were opposite to those produced by treatment with an ERK inhibitor. This phenomenon of MAPK hyperactivation-induced cell death was also observed in vivo, where complete tumor regression was observed in a xenograft model. Ongoing studies seek to further define the mechanism of cell death, which could suggest potential avenues to leverage hyperactivation of MAPK signaling as a therapeutic approach.

Citation Format: Grace P. Leung, Tianshu Feng, Matthew D. Shirley, Frederic D. Sigoillot, David A. Ruddy, Alyson K. Freeman, Mariela Jaskelioff, Darrin D. Stuart. Deleterious effects of MAPK pathway hyperactivation in BRAF-mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 965.
Abstract 966: Oncogenic signaling pathways differentially regulate clathrin-mediated endocytosis in cancer cells
Authors: Xiao Guan-Yu , Schmid Sandra
Published: August 2018

Clathrin-mediated endocytosis (CME), the major endocytic pathway, regulates the rates of internalization of receptors as well as their downstream signaling activities. Recently, our lab has discovered that signaling can reciprocally regulate CME in cancer cells. The crosstalk between signaling and CME contributes to abnormal trafficking of signaling receptors and altered downstream signaling, leading to enhanced cancer metastasis. Accordingly, it is important to understand the nature and extent of interactions between CME and intracellular signaling in cancer cell biology. To address this issue, we targeted a set of prioritized kinases that are often implicated in cancer-relevant signaling pathways using validated pharmacologic inhibitors and compared their effects on CME function using different models for human noncancerous and cancer cells. We found that inhibition of several kinases selectively affected CME function in cancer cells. Notably, ERK inhibition had the most significant and consistent effects on CME activity across different types of cancer cells, while not affecting CME in several noncancerous cell lines tested. We established that a second ERK inhibitor, and importantly inhibition of the essential upstream kinase MEK, had the same cancer cell-specific effects on CME activity. Furthermore, we found that inhibition of ERK dramatically reduced the rate of clathrin-coated pit (CCP) initiation selectively in cancer cells. Characterization of one potential ERK substrate, FCHSD2, showed that knockdown of FCHSD2 had the similar cancer cell-specific effects on CME activity and on CCP initiation rate and that the cells depleted for FCHSD2 were insensitive to ERK inhibition. Surprisingly, the expression level of FCHSD2 is positively correlated with higher cancer patient survival rate, suggesting that FCHSD2 might be a tumor suppressor regulated by ERK. Our study provides new insight into the mechanisms and consequences of the reciprocal regulation of signaling and CME in cancer cells.

Citation Format: Guan-Yu Xiao, Sandra Schmid. Oncogenic signaling pathways differentially regulate clathrin-mediated endocytosis in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 966.
Abstract 967: Targeting notch one notch above
Authors: Ucar-Bilyeu Deniz A. , MATOSSIAN Margarite D. , Barnes VAN Hoang , Hossain Fokhrul M. , Gupta Mohit , BURKS HOPE E. , WRIGHT THOMAS D. , Cavanaugh Jane , Flaherty Patrick , Burow Matthew E. , Miele Lucio
Published: August 2018

Triple negative breast cancer (TNBC) is a molecularly heterogeneous, clinically aggressive disease group that is highly prevalent among African-Americans and younger patients. Standard chemo/radio therapy often produces clinical responses, but recurrence and metastasis are unfortunately common. Metastatic disease is generally incurable. Chemo/radiotherapy has been shown to induce EMT and enrich a chemo-resistant cancer stem cell-like (CSC) population in TNBC. CSCs are thought to drive disease recurrence. Notch signaling, particularly Notch1, is critical for maintenance of TNBC CSC. Expression of Notch1 and its ligand Jagged1 are correlated with poor prognosis. Efforts to pharmacologically target Notch directly have been impaired by the systemic toxicity of the Gamma Secretase Inhibitors (GSI) used, and by the fact that Notch1 also plays a key role in anti-tumor adaptive immunity. Therapeutic agents that target Notch signaling in breast cancer cells indirectly and selectively are a potentially attractive strategy. However, no such target has been identified to date. We have found that the MAPK5-ERK5 kinase pathway, which contains at least two druggable targets, functions as a master regulator of Notch signaling in TNBC cells. ERK5 knockout TNBC cells have dramatically decreased expression of Notch receptors, ligands and targets. In vivo, these cells form barely detectable tumors that do not metastasize and express lower levels of Notch1 and its ligand Jagged1. Using in silico screening method, we have identified a small molecule compound that targets MAP2K5 (MEK5) and decreases phosphorylation of MAPK7 (ERK5). Expression of ERK5 is associated with poor prognosis in TNBC. Consistent with ERK5KO cells, suppression of ERK5 phosphorylation decreased the amount of Notch1 and Jagged1 protein and mRNAs. More importantly, a selective MEK5 inhibitor, SC-181, reversed EMT and reduced the CD44hi/CD24lo CSC population in TNBC cells without suppressing T-cell proliferation. Treatment with nanomolar concentration of this compound decreased the number and size of mammospheres in a dose- dependent manner. Our preliminary results suggest that targeting the MEK5-ERK5 pathway is a promising strategy to selectively target Notch signaling in TNBC CSC without systemic Notch inhibition.

Citation Format: Deniz A. Ucar-Bilyeu, Margarite D. MATOSSIAN, VAN Hoang Barnes, Fokhrul M. Hossain, Mohit Gupta, HOPE E. BURKS, THOMAS D. WRIGHT, Jane Cavanaugh, Patrick Flaherty, Matthew E. Burow, Lucio Miele. Targeting notch one notch above [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 967.
Abstract 97: UBE3A regulates SIRT6 levels to promote tumorigenesis in an ANXA2 dependent manner
Authors: Kohli Saishruti , Das Sanjeev
Published: August 2018

Background: UBE3A is the founding member of the HECT family of E3 ubiquitin ligases, implicated in cervical, prostate and breast cancer. A recent study has reported that UBE3A promotes proliferation and invasiveness of cancer cells by inducing Annexin A2 (ANXA2) levels. We have delineated a cascade in which UBE3A brings about the proteasomal degradation of SIRT6. Furthermore, we have also established that SIRT6 transcriptionally regulates ANXA2 expression. Here, we have confirmed that UBE3A-mediated SIRT6 downregulation and consequent derepression of ANXA2 promotes tumor metastasis.

Methods: HepG2, a Hepatocellular carcinoma cell line, was used to study the effects of the UBE3A-SIRT6-AXA2 axis. HepG2 UBE3A knockdown, SIRT6 knockdown, UBE3A/SIRT6 double knockdown and SIRT6/ANXA2 double knockdown cells were used to examine in-vitro cell proliferation, invasion, plasmin generation and MMP activation. These cell lines were also used for obtaining subcutaneous and orthotopic xenografts in immunodeficient mice and multiple parameters were analyzed to determine tumor growth and metastases.

Results: We observed that upon UBE3A abrogation, cells exhibited reduced tumor-forming potential and resulted in smaller tumors with low metastasis. On the other hand, increased tumorigenesis, and much larger tumors with increased metastasis were observed upon SIRT6 knockdown, which was significantly reversed upon simultaneous knockdown of UBE3A. Furthermore, smaller tumors were observed in case of SIRT6/ANXA2 double knockdown cells as compared to UBE3A/SIRT6 double knockdown cells. These results suggest that UBE3A-mediated ANXA2 upregulation, via SIRT6 degradation, is critical for its oncogenic functions

Conclusion: UBE3A mediated SIRT6 degradation leads to ANXA2 upregulation and hence enhances tumorigenesis. It would be beneficial to identify drugs targeting UBE3A as a therapeutic approach in hepatocellular carcinoma. Alternatively, mechanisms which inhibit ANXA2 activity must be deciphered to identify druggable targets.

Citation Format: Saishruti Kohli, Sanjeev Das. UBE3A regulates SIRT6 levels to promote tumorigenesis in an ANXA2 dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 97.
Abstract 974: Alternative polyadenylation of androgen receptor variants in castration-resistant prostate cancer
Authors: Etten Jamie L. Van , Nyquist Michael , Li Yingming , LaRue Rebecca , Ho Yeung , Yang Rendong , Voytas Daniel F. , Henzler Christine , Dehm Scott M.
Published: August 2018

The androgen receptor (AR), which is activated by androgens, functions in the normal prostate as a master transcriptional regulator. Accordingly, prostate cancer (PCa) presents as an androgen- and AR-dependent disease. Inhibition of AR transcriptional activity with androgen-deprivation therapy (ADT) is effective for patients with locally advanced and metastatic disease. All ADT suppresses AR via interference with its ligand binding domain (LBD)-dependent transcriptional activity. However, development of therapy resistance and transition to castration-resistant prostate cancer (CRPC) represents a major clinical challenge. CRPC circumvents ADT by expressing constitutively active AR variants (AR-Vs) that lack the LBD. Therapies targeting constitutively active AR variants are not currently available. To address this problem, we investigated the mechanisms underlying AR-V expression in CRPC. One particular AR-V, called AR-V7, consists of contiguously spliced exons 1, 2, 3, and a cryptic exon, CE3, and has been shown to be associated with clinical resistance to second-generation AR inhibitors abiraterone and enzalutamide. We noted that CE3 contains a consensus hexameric AAUAAA polyadenylation signal (PAS). We hypothesized that intronic polyadenylation promotes AR-V7 expression in CRPC. To test this, we developed a morpholino that sterically blocked the CE3 PAS to prevent splicing of AR-V7. We have demonstrated that the PAS in CE3 controls expression of AR-V7, as well as a broad array of AR variants arising by splicing of adjacent cryptic exons that use the shared CE3 PAS. Furthermore, our data demonstrate that the cleavage and polyadenylation complex (CPSF complex) is enriched in primary PCa tumors relative to normal tissues. We have also demonstrated that CPSF1, a member of the CPSF complex, controls expression of AR-Vs relative to full length AR and plays a global role in the determination of mRNA isoform length in prostate cancer cells. Collectively, these findings reveal alterations in the cellular machinery governing mRNA cleavage and polyadenylation in prostate cancer, and identify specific opportunities for therapeutic intervention.

Citation Format: Jamie L. Van Etten, Michael Nyquist, Yingming Li, Rebecca LaRue, Yeung Ho, Rendong Yang, Daniel F. Voytas, Christine Henzler, Scott M. Dehm. Alternative polyadenylation of androgen receptor variants in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 974.
Abstract 996: Host-derived MCP-1 dictates prostate cancer skeletal metastasis in vivo
Authors: Meng Jie , Liang Weiping , Tong Haibo , Keller Evan T. , Zhang Jian , Lu Yi
Published: August 2018

Overexpression of the tumor-derived monocyte chemotactic protein-1 (MCP-1) has been suggested to partially promote prostate cancer bone metastasis, but the function for host-derived MCP-1 in these processes is not well established. To understand the function of host-derived MCP-1 on bone metastasis, we developed a mouse model using intracardiac injection and in vivo selection to obtain tumor cell subpopulations with a higher propensity for bone metastasis. Here, we found that a loss of host MCP-1 (MCP-1 knockout) retarded tumor growth in bone and prolonged survival of mice by intracardiac injection of tumor cells in vivo. Moreover, a decrease in osteolytic bone lesion was observed in MCP-1 knockout mice by bone density analysis, as compared to wild-type control. Systemically, MCP-1 deficiency inhibited the proportion of PMN-and M-MDSC populations with immunosuppressive function. Together, this study indicates that the dysregulated immunity resulting from a loss of host MCP-1 signaling is sufficient to drive PCa bone metastasis, and provides a new perspective for targeting the endogenous MCP-1 pathway as an antimetastatic strategy.

Supported by NSFC Key Project 81130046; NSFC projects 81773146, 81272415; JCYJ20170412152943794, JCYJ20170412154619484, JCYJ20170307105128101, JCYJ2017030711041760; Guangxi Key Projects 2013GXNSFEA053004.

Citation Format: Jie Meng, Weiping Liang, Haibo Tong, Evan T. Keller, Jian Zhang, Yi Lu. Host-derived MCP-1 dictates prostate cancer skeletal metastasis in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 996.
Abstract 999: Unravelling myeloid and T cell compartment interactions through a novel approach to tumor multimodal analysis combining whole slide multiplexed immunofluorescence and gene expression profiling
Authors: Zwing Natalie
Published: August 2018

Understanding the role of myeloid cells in tumor biology requires a systems approach combining phenotypic and functional biomarkers. As a model system, colorectal cancer (CRC) tumors represent well-described clinically relevant immune subtypes. Distinct immune patterns have been reported involving the myeloid cells acting as immune suppressors for CD8 cytotoxic T cells. We hypothesized that myeloid derived suppressor cells (MDSCs) and CD8 T cells co-localize due to their direct interactions.

A cohort of 85 CRC primary tumors were analyzed by multiplexed immunohistochemistry/ immunofluorescence and subsequent quantitative and qualitative whole slide image analysis. To define the functional status of the identified immune cell populations, their corresponding signatures were analyzed by gene expression profiling.

Combining density and distribution of both myeloid cells and T cells as well as their distance to epithelial cancer cells, specific patterns were observed. In both microsatellite stable (MSS) and instable (MSI) tumors, MDSCs showed a comparable stromal distribution. By contrast, in MSI cases only, effector T cells tended to accumulate in the tumor epithelium suggesting they overcome the immunosuppressive environment. Additionally, the assessment of CD8 T cell morphology revealed changes potentially reflecting their cancer cell eliminating properties.

This multimodal analysis represents a novel approach to characterize and understand the role of the myeloid compartment through a combination of morphological and spatial characteristics of the T cell compartment.

Citation Format: Natalie Zwing. Unravelling myeloid and T cell compartment interactions through a novel approach to tumor multimodal analysis combining whole slide multiplexed immunofluorescence and gene expression profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 999.
Abstract CT097: Quantitative MR measurements in glioblastoma patients: Mean diffusivity with tumor treating fields plus standard therapy versus standard treatment alone
Authors: Vymazal Josef , Zacek Radovan , Sroubek Jan , Klener Jan , Rulseh Aaron M.
Published: August 2018

Introduction: Quantitative MR parameters, including those derived from diffusion imaging, may advance knowledge on the biological characteristics of gliomas in both diagnostic and treatment settings. We evaluated quantitative diffusion differences that could be detected in glioblastoma patients treated with standard therapy plus TTFields (Optune) versus standard therapy alone. 

Methods: Twelve patients with glioblastoma were subjected to surgical resection, radiotherapy and temozolomide. Six subjects were additionally treated with TTFields. All patients were followed by MRI every two months (range 210-1036 days) with standard imaging and diffusion tensor imaging until tumor progression. 

Results: Progression-free survival (PFS) was significantly longer in the subgroup treated by TTFields (mean 784 days ± 163 SD) as compared to standard therapy alone (443 days ± 249). No difference was detected between groups in the initial mean diffusivity values measured within the affected white matter near the resection cavity. However the linear regression slope describing mean diffusivity in this region over serial measurements was higher in the subgroup treated by TTFields than in the other group (5.43 ± 2.4 versus -3.77 ± 11.8). Furthermore, we detected significant positive correlation between mean diffusivity slope and PFS.

Discussion: Our data demonstrate increasing mean diffusivity in the affected white matter of glioblastoma patients, which is favorable with positive correlation to PFS seen in steeper regression slopes. The patients treated with TTFields showed greater PFS in comparison to those who did not receive TTFields. There was a corresponding increase in mean diffusivity in the affected white matter of these patients. In the group that received standard therapy, the increase was not as apparent and, if present, the slope was not as steep. Changes in mean diffusivity likely reflect changes in tumor density in the white matter. 

Citation Format: Josef Vymazal, Radovan Zacek, Jan Sroubek, Jan Klener, Aaron M. Rulseh. Quantitative MR measurements in glioblastoma patients: Mean diffusivity with tumor treating fields plus standard therapy versus standard treatment alone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT097.