bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5083: BSCHL-2046, a lead chalcone derivative from a newly synthesized library, is a potent neddylation inhibitor against the growth of prostate cancer cell lines
Authors: Tippin Matthew A. , Fu Dong Jun , Song Liankun , Pham Victor , Zi Xaolin
Published: August 2019

Members of the chalcone family of molecules and their derivatives have been shown to inhibit various forms of cancer cells. In particular, Flavokawain A has been shown to inhibit prostate cancer cell lines. In order to capitalize on the bioactive nature of chalcone derivatives, we created a library of chalcone derivatives based on the scaffold of Flavokawain A but with an additional anti-cancer moiety. Here we have demonstrated that BSCHL-2046, the most potent member of the library, inhibits cell growth of prostate cancer cell lines including LNCaP, C4-2B, DU145, 22Rv1, and PC3 with IC50’s ranging from 0.44µM-7.5µM. BSCHL-2046 inhibited Ubc12 neddylation in an in vitro assay at an IC50 of 1.5µM and demonstrated complete inhibition at a concentration of 6µM. BSCHL-2046 also reduced Nedd-8 conjugations to both Cullin-1 and Ubc12 in vivo in DU145 cells. The inhibition of neddylation by BSCHL-2046 resulted in an increased expression of p27 and CDT-1. Using molecular modeling, BSCHL-2046 was predicted to bind directly to the Cullin-RING E3 ligase complex. The Cellular Thermal Shift Assay (CETSA, an in vivo assay) demonstrated that BSCHL-2046 significantly decreased heat-induced protein degradation of E3 subunits, supporting BSCHL-2046 directly binding to the complex. Furthermore, BSCHL-2046 at a concentration of 500nM and 1.25µM displayed a 75% to complete inhibition of colony formation of C4-2B cells in soft agar. Taken together, these results demonstrate BSCHL-2046 and its structural analogs are potent neddylation inhibitors that deserve further investigation for the prevention and treatment of prostate cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Matthew A. Tippin, Dong Jun Fu, Liankun Song, Victor Pham, Xaolin Zi. BSCHL-2046, a lead chalcone derivative from a newly synthesized library, is a potent neddylation inhibitor against the growth of prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5083.
Abstract 5087: Carvedilol prevents skin cancer independently of its antioxidant property
Authors: Chen Mengbing , Liang Sherry , Andresen Bradley , Huang Ying
Published: August 2019

Carvedilol is an FDA approved β-blocker traditionally prescribed for cardiovascular diseases. Our previous work demonstrated that carvedilol has preventive activity against ultraviolet radiation (UVR)-induced skin damage, inflammation, and carcinogenesis. However, the photoprotective mechanism for carvedilol remains unknown. Interestingly, not all β-blockers have cancer preventive activity, suggesting that targeting the β-adrenergic receptors may not be responsible for carvedilol’s anticancer activity. Since carvedilol has been shown as a potent antioxidant and such property is not shared by majority of other β-blockers (e.g., metoprolol), we hypothesized that the antioxidant property may be, in part, responsible for carvedilol’s protective activity against photocarcinogenesis. The nontumorous mouse epidermal JB6 P+ cells were subjected to various doses of UVR exposure and H2O2 treatment to identify the optimal doses to examine protective activity. UVR and H2O2dose-dependently reduced cell viability. 25mJ/cm2 UVR and 100 μM H2O2 reduced cell viability detected by Trypan blue exclusion assay to 58.1% and 60.6%, respectively. 25mJ/cm2 UVR and 300 μM H2O2 increased the formation of intracellular fluorescent 2',7'-dichlorofluorescein (DCF) fluorescence, a marker of reactive oxidative species (ROS) detected by flow cytometry, to 5- and 4-fold compared to non-treated control, respectively. The cells were treated before or after UVR and H2O2 exposure with metoprolol, carvedilol or 4-hydroxycarbazole (4-OHC). 4-OHC is an intermediate compound for carvedilol synthesis that possesses an identical UV absorption profile as carvedilol. Additionally, the carbazole moiety is likely responsible for the antioxidant activity of carvedilol. Carvedilol, but not metoprolol and 4-OHC, was able to inhibit EGF-induced JB6 transformation in soft agar. Carvedilol, but not metoprolol and 4-OHC, also attenuated UVR and H2O2 induced cell death. Carvedilol, but not 4-OHC, inhibited UVR-induced NF-κB and AP-1 promoter activity. In addition, unlike carvedilol, 4-OHC failed to prevent chronic UVR induced skin carcinogenesis in SKH-1 mice. However, 4-OHC and carvedilol similarly attenuated UVR- and H2O2-induced ROS formation in JB6 P+ cells, while metoprolol had no effect. These results indicate that the photoprotective effects of carvedilol are primarily due to an unknown mechanism that is independent of β-adrenergic receptors and reactive oxygen species scavenging.

Citation Format: Mengbing Chen, Sherry Liang, Bradley Andresen, Ying Huang. Carvedilol prevents skin cancer independently of its antioxidant property [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5087.
Abstract 5090: Epitope mapping of mouse TERT for vaccine development
Authors: Song Yurong , Marshall Jason , Kerr Travis , Pinto Ligia , Sei Shizuko , Shoemaker Robert
Published: August 2019

Telomerase reverse transcriptase (TERT) is one of two essential components of telomerase, an enzyme complex that generates and maintains telomeres. Telomerase is expressed mainly in embryonic and adult stem cells. Telomere biology has recently been implicated in the pathogenesis of a variety of diseases, and mutations in telomerase components result in a predisposition to solid malignancies. More recent findings show that TERT is not only expressed in late stage primary tumor cells and metastatic cells, but also expressed in incipient cancer stem cells and/or tumor-initiating cells, indicating that TERT has essential roles at every stage of tumorigenesis. However, the clinical benefit of a TERT (hTERT)-targeting vaccine, given as a single agent or in combination with chemotherapeutic agents, has been limited in patients with advanced cancer, possibly due to insufficient epitope coverage as well as tumor-induced immune suppression. Thus, targeting TERT in tumor-initiating cells in early stage lesions may be more effective in preventing cancer development and progression. To develop cancer preventive TERT vaccines, novel immunogenic epitopes must be first identified and evaluated in a relevant preclinical model of tumorigenesis. The goal of this project is to identify the epitopes of mouse TERT with high immunogenicity using MHC I-peptide binding assay by flow cytometry analysis. The in vitro peptide-induced MHC Class I stabilization assay was carried out using RMA/S cell line, which was derived from a Rauscher leukemia virus-induced C57BL/6 T cell lymphoma and is deficient in transporter associated with antigen processing (TAP). Results of screening the overlapping peptide library will be presented.

Funded by NCI Contract No. HHSN261200800001E

Citation Format: Yurong Song, Jason Marshall, Travis Kerr, Ligia Pinto, Shizuko Sei, Robert Shoemaker. Epitope mapping of mouse TERT for vaccine development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5090.
Abstract 510: Selective and potent CDK8 inhibitors enhance NK cell activity and promote tumor surveillance
Authors: Hofmann Marco H. , Mani Rajeswaran , Engelhardt Harald , Impagnatiello Maria A. , Carotta Sebastian , Kerenyi Marc A. , Lorenzo-Herrero Seila , Boettcher Jark , Scharn Dirk , Arnhof Heribert , Zoephel Andreas , Schnitzer Renate , Gerstberger Thomas , Rajgolikar Girish , Goswami Swagata , Vasu Sumithira , Ettmayer Peter , Gonzalez Segundo R. , Pearson Mark , McConnell Darryl B. , Kraut Norbert , Muthusamy Natarajan , Moll Juergen
Published: August 2019

Natural killer (NK) cells play a pivotal role in controlling cancer. Activation of NK cells is tightly regulated by multiple extracellular receptors and internal signaling nodes. The production of perforin and granzyme B, key NK cytolytic molecules, is regulated by the suppressive signaling intermediate Cyclin Dependent Kinase 8 (CDK8) through phosphorylation and inhibition of STAT1S727.

We identified highly potent and selective CDK8 inhibitors, including BI-9811 and BI-1347, that we previously reported as valuable probe compounds (Hofmann M.H. et al., AACR Annual Meeting 2017, Abstract 4630). CDK8 inhibition promoted activation of NK cells but had no direct cytotoxic activity on the majority of cancer cell lines tested. Single agent treatment with the CDK8 inhibitor BI-1347 increased the survival of mice bearing melanoma and breast cancer allografts. In addition to this, treatment with BI-1347 resulted in enhanced NK-cell mediated lysis of myeloid leukaemia and lymphoma cells.

We evaluated whether increased efficacy could be observed upon activation of both the innate and the adaptive immune system by combining the CDK8 inhibitor with a SMAC mimetic. SMAC mimetic treatment enhanced T-cell activity and increased NK cell number in the murine EMT6 breast cancer model. Increased survival upon combination treatment was dependent on a pulsatile schedule of the CDK8 inhibitor BI-1347, which permitted activation of NK cells but avoided a hypo-responsive steady state. These results demonstrate that CDK8 inhibitors could be used either as a single agent in hematological cancers or be combined with modulators of the adaptive immune system to inhibit the growth of solid tumors.

Citation Format: Marco H. Hofmann, Rajeswaran Mani, Harald Engelhardt, Maria A. Impagnatiello, Sebastian Carotta, Marc A. Kerenyi, Seila Lorenzo-Herrero, Jark Boettcher, Dirk Scharn, Heribert Arnhof, Andreas Zoephel, Renate Schnitzer, Thomas Gerstberger, Girish Rajgolikar, Swagata Goswami, Sumithira Vasu, Peter Ettmayer, Segundo R. Gonzalez, Mark Pearson, Darryl B. McConnell, Norbert Kraut, Natarajan Muthusamy, Juergen Moll. Selective and potent CDK8 inhibitors enhance NK cell activity and promote tumor surveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 510.
Abstract 5102: Deep learning method for the classification of CNV based on the next generation target sequencing
Authors: Lang Jidong , Tian Geng
Published: August 2019

Background: Copy number variations (CNVs) are of great importance to many cancers. Recently, the next-generation sequencing has made detecting CNV by sequencing possible. Traditional CNV determination methods involve expensive experiments, which is costly. Recently, several algorithms were proposed the detection of CNV, but none of them could utilize only the coverage information at a specific gene to make the determination of CNV at the gene.

Methods: We collected 1132 samples with 51 mesenchymal-epithelial transition factor (MET) CNV samples and 1081 samples with no CNVs. We split the exons of MET to multiple 50-bp windows with the stride of 40-bp. At each window the supporting reads were counted, and reads numbers at each exons were piled up, generating a matrix. The training set containing 38 CNV-positive matrices and 38 CNV-negative matrices were extracted from all the matrices, leaving the others as the test set. The deep learning network, convolutional neural network (CNN) were applied to distinguish between the CNV-positive matrices and the CNV-negative matrices. For comparison, logistic regression model was also applied for the classification task.

Results: The training set were split into 5 pieces containing 15 samples each and 5-fold cross validation were run for the CNN or the logistic regression model. After running 10 5-fold cross validation, we got an accuracy of 85.73 ± 9.24% for CNN and the average accuracy of 87.07 ± 8.99% for the logistic regression model. We chose 3 best models in both CNN and logistic regression models for the determination of the test set. The CNN models gained the accuracy of 71.84 ± 2.19% while the logistic regression models gained the accuracy of 69.70 ± 2.56%. Furthermore, the Area Under Curve (AUC) of the CNN models were 0.6335 ± 0.0124, higher than that of the logistic regression models which was 0.5666 ± 0.0115.

Conclusions: We proposed a method to determine the samples with CNV by using the convolutional neural network with only the coverage information within the specific region. It provides the possibility of CNV detection for cancer patients using only the next generation sequencing data.

Citation Format: Jidong Lang, Geng Tian. Deep learning method for the classification of CNV based on the next generation target sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5102.
Abstract 5118: TIDE-E: an online evaluator of tumor immune-suppressive function of gene sets
Authors: Fu Jingxin , Zhang Wubing , Jiang Peng , Liu Xiaole S.
Published: August 2019

Recent immunotherapy studies have generated a diverse collection of datasets, covering different aspects of cancer immune evasion. However, to the best of our knowledge, there are no public tools that can systematically rank genes regarding their function on tumor immune evasion and comprehensively evaluate biomarkers across multiple clinical cohorts. Therefore, we presented the TIDE-E (evaluator) website for users to evaluate their gene sets across a large panel of data cohorts (http://tide.dfci.harvard.edu). The website integrated two categories of data resources, human clinical datasets from public immunotherapy studies, TCGA, PRECOG, and METABRIC databases, as well as functional genomics datasets from CRISPR screens in preclinical models. For a user-defined gene set, the web application can test its performance as immunotherapy response biomarkers across multiple clinical studies, and rank the gene members according to their relevance in promoting tumor immune evasion.

Citation Format: Jingxin Fu, Wubing Zhang, Peng Jiang, Xiaole S. Liu. TIDE-E: an online evaluator of tumor immune-suppressive function of gene sets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5118.
Abstract 5131: Breast cancer with insertion or deletion exhibits the immunogenic phenotype
Authors: Yuan Peng , Wang Xue , Zhang Yuzi , Zhao Jing , Cai Shangli , Yin Yongmei
Published: August 2019

Background: Although immunotherapy has been proven to be effective in a wide range of malignant tumors, breast cancer remains to be one of poorly immunogenic tumors and only a small subset of patients with breast cancer achieved benefits from immunotherapy. Therefore, identification of better predictive biomarkers to guide patient selection is highly desirable. It has been reported that insertion or deletion (indels) could create a novel open reading frame and generate more neoantigen which may mediate response to immunotherpay. However, the pattern of indels mutations in breast cancer is still unclear.

Methods: Whole-exome sequencing data, RNA-Seq data and clinical data of 1096 breast tumors from The Cancer Genome Altas (TCGA) database were used to analyze the pattern of indels in breast cancer. Next generation sequencing (NGS) data of 81 breast tumors from clinical dataset were also used to validate the indels mutation pattern in different molecular subtype.

Results: 81.7% (895/1096) of breast tumors in TCGA dataset harbored at least one indels mutation. Hormonal receptor (HR) negative tumors were associated with higher burden of indels mutations than HR positive tumors in both TCGA dataset (P = 0.05) and NGS-clinical dataset (P = 0.003). Indels were significantly correlated with higher TMB and neoantigen level in TCGA cohort (P < 0.0001). In addition, tumors with at least eight indels mutations (cut off as 80% percentile) exhibited even higher TMB (P < 0.0001) and neoantigen (P < 0.0001) level. Among 45 immune related genes, the mRNA expression of 22 genes were significantly higher in tumors with indels mutations, such as LAG3, IL18, IL6, CTLA4 and PDCD1. Indels group also showed a high levels of genome instability in terms of HRD-LOH (P = 0.004), NtAI (P = 0.000), wGII (P = 0.001) and LST (P =0.014).

Conclusions: Breast tumors with indels mutations exhibited the immunogenic phenotype. Further studies are warranted to investigate the potential value of indels as a predictive biomarker for immunotherapy in breast cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Peng Yuan, Xue Wang, Yuzi Zhang, Jing Zhao, Shangli Cai, Yongmei Yin. Breast cancer with insertion or deletion exhibits the immunogenic phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5131.
Abstract 5135: Genome-wide CRISPR-based screening reveals ABCG2 as a novel drug resistance gene in pancreatic cancer
Authors: Ramaker Ryne C. , Hardigan Andrew A. , Gordon Emily R. , Myers Richard M. , Cooper Sara J.
Published: August 2019

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers and incidence is increasing due to the increase in risk factors such as diabetes and obesity. One reason for the high mortality rate associated with this cancer is the high rate of resistance to commonly used chemotherapies. Our previous work showed a link between patient survival and expression of drug resistance genes, thus we undertook genome-wide CRISPR knockout and activation screens to identify genes conferring resistance to cytotoxic chemotherapies in two pancreatic cancer cell lines. Our screen revealed hundreds of genes with potential roles in drug resistance, many of which have expression levels that correlate with patient survival. We have validated our top candidates by stably expressing individual guide RNAs in each of three pancreatic cell lines, Panc-1, BxPC-3 and MiaPaCa-2 and screening for viability following treatment with three chemotherapeutic agents, oxaliplatin, gemcitabine, and irinotecan. ABCG2, an ABC transporter, was revealed as our top hit, conferring resistance to multiple drugs in multiple cell lines. Our results would suggest that repression of ABCG2 might improve sensitivity to cytotoxic chemotherapies. We test this hypothesis using an ABCG2 inhibitor and demonstrate the effect of this inhibitor on cell viability, drug sensitivity and transcriptomic profiles in the context of cell lines with variable ABCG2 expression.

Note: This abstract was not presented at the meeting.

Citation Format: Ryne C. Ramaker, Andrew A. Hardigan, Emily R. Gordon, Richard M. Myers, Sara J. Cooper. Genome-wide CRISPR-based screening reveals ABCG2 as a novel drug resistance gene in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5135.
Abstract 5136: Adapting long read sequencing technologies for targeted and single cell applications
Authors: Iyer Shruti , Goodwin Sara , McCombie W. Richard
Published: August 2019

Structural variations (SV), a hallmark of genomic instability in cancer, include insertions, deletions, duplications, inversions, or translocations that can either activate oncogenes or inactivate tumor suppressor genes. These variants contribute to the complexity of the cancer genome but have not been successfully resolved yet. While short-read sequencing has aided cancer genomics, it has performed poorly in SV detection, with false positive and false negative rates of 50% or more. Long-read sequencing generates reads that are >10 kb long and has helped identify thousands of genomic features pertinent to cancer that were previously missed by short-read sequencing. Long reads can span SV with a single continuous read, giving a clearer idea of the variation, its position, and size. Currently, long-read sequencing methods are greatly limited by the DNA preparation step, with fragment lengths and yield often compromised by standard extraction methods. The potential to generate reads at the megabase level relies greatly on library preps using large amounts (several μg) of DNA input, which prevents its application to samples like patient tumors that are often limited in DNA. Our overarching goal is to develop a targeted approach that generates large contiguous reads from moderate to low input DNA masses. To this end, we use different extraction, amplification, fragmentation, and enrichment strategies to efficiently target and resolve complex regions of the genome that are often associated with disease conditions. Development of a targeted long-read sequencing strategy using low DNA input would give us the potential to explore the landscape of genetic variation across several individuals and heterogeneous cell populations. We are currently employing and adapting CRISPR-based targeting techniques to enrich for specific regions of the genome from intact cells and/or high-molecular-weight DNA.

Citation Format: Shruti Iyer, Sara Goodwin, W. Richard McCombie. Adapting long read sequencing technologies for targeted and single cell applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5136.
Abstract 5138: Inhibition of kPNB1 and NUPR1 binding increase the anti-cancer drug sensitivity
Authors: Park Chan Hee , Ham Seung Wook , Shin HyeKyoung , Oh Kyung Soo
Published: August 2019

Karyopherin beta 1 (KPNB1, known as importin beta 1) is carrier protein, it is known as transporter of protein from cytoplasm to nucleus through nuclear pore complexes (NPCs) using KPNAs as an adapters. The nuclear protein-1 (NUPR1, known as p8/Com-1) is identified as a transcription factor and it is showed multifunctional mechanisms related to cellular stress response such as serum-starvation, oxidative stress and drug stimulation. In this study, we investigated protein-protein interaction between NUPR1 and KPNB1, it might be a new target for cancer therapy.For the evaluation of binding affinity between protein and inhibition effect by compound 1, we processed the single molecule binding assay. Cytotoxicity of combination treatment with doxorubicin and compound 1 were validated several cancer cell lines such as MDA-MB-231, PC-3, SK-OV-3, and SiHa. Gene expression patterns were analyzed RNA-Seq and validated with qRT-PCR and western-blotting. KPNB1 bind to NUPR1 as a high affinity compare to KPNB1 single treatment assay and there was no difference according to concentration of NUPR1. KPNB1 and NUPR1 protein-protein interaction was inhibited by compound 1 according to concentration of compound 1 showed 71%, 44%, and 31% of inhibition rate with 50nM, 10nM, 2.5nM of compound 1, respectively. In doxorubicin single treatment, NUPR1 move to nucleus more than 90% of cells compare to non-treated MDA-MB-231 cells. Otherwise, NUPR1 was exist both nucleus and cytoplasm more than 90% cells, which is indicate that translocation of NUPR1 to the nucleus was blocked by compound 1 treatment. And combination treatment showed synergistic effect of cytotoxicity in various cancer cells such as such as MDA-MB-231, SiHa, PC-3, and SK-OV-3 (1.4 ~ 6 fold). Additionally, NUPR1 related genes were showed change of gene expression in compound 1 combination compared to doxorubicin single treatment. In these results suggest that translocation mechanism of NUPR1 into nucleus bind to KPNB1 and this binding was inhibited by compound 1 which is new candidate drug combination method for cancer therapy.

Citation Format: Chan Hee Park, Seung Wook Ham, HyeKyoung Shin, Kyung Soo Oh. Inhibition of kPNB1 and NUPR1 binding increase the anti-cancer drug sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5138.
Abstract 5141: T-cell receptor repertoire analysis by next-generation sequencing peripheral blood mononuclear cells from multiple myeloma or smoldering multiple myeloma patients
Authors: Zhang Yong , Petrovskaya Svetlana , Scott Emma C. , Santana-Quintero Luis , Ghazanchyan Tigran , Rosenburg Amy , Rao V. Ashutosh , Blumenthal Gideon M. , Theoret Marc , Pazdur Richard , Beaver Julia A. , Kazandjian Dickran
Published: August 2019

Multiple myeloma (MM) is a hematological malignancy mostly assessed by bone marrow involvement, so it is attractive to develop a method using peripheral blood. We hypothesized that peripheral blood mononuclear cells (PBMCs) may replace tedious and invasive bone marrow biopsies as a biomarker. In this study, we aimed to investigate if T-cell receptor (TCR) repertoire has clinical significance in MM or Smoldering MM (SMM) diagnosis or prognosis. Using baseline genomic DNA (PBMC) from 24 patients with SMM and 31 patients with MM, we performed next-generation sequencing on the Illumina NextSeq 500 platform by immunoSEQ TCRB Kits from Adaptive Biotechnology to quantitatively assay the complementarity determining region 3 (CDR3) of human T-cell receptor β (TCRβ) gene. To evaluate T-cell diversity and clonality, we performed pipeline analysis by ImmunoAnalyzer from Adaptive Technology, and downloaded sequencing data with manual analysis by JMP13 software. Compared to TCRB data from 280 normal control (Age > 40) PBMCs (Nat Genet. 2017, 49(5):659-665), we found less TCR templates from both MM and SMM baseline based on size of TCRB repertoire, and less TCR rearrangements or unique clonotypes based on structure of TCRB repertoires. Only MM baseline showed higher maximum clonotype frequency. No difference of T-cell clonality was found from MM and SMM. Furthermore, we performed the same sequencing on 18 SMM patients at different treatment time points including chemotherapy after cycle 8, 20, and 32. Our results showed patient samples after cycle 8 had less TCR templates and TCR rearrangements compared to those from the other three time points although no significant difference were found in maximum clonotype frequency or T-cell clonality. TCR overlap analysis will be further performed to track potential clonotype changes. Thus, we identified TCR repertoire analysis may have potential clinical significance as biomarker assay for MM and SMM patients.

Citation Format: Yong Zhang, Svetlana Petrovskaya, Emma C. Scott, Luis Santana-Quintero, Tigran Ghazanchyan, Amy Rosenburg, V. Ashutosh Rao, Gideon M. Blumenthal, Marc Theoret, Richard Pazdur, Julia A. Beaver, Dickran Kazandjian. T-cell receptor repertoire analysis by next-generation sequencing peripheral blood mononuclear cells from multiple myeloma or smoldering multiple myeloma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5141.
Abstract 5146: ATP-binding cassette transporter, ABCG2, activation is responsible for chemo-resistant, SIK3 low expressed ovarian cancer
Authors: Hsu Keng-Fu , Liang Yu-Ling , Wu Chin-Han , Shih Neng-Yao
Published: August 2019

Background: Epithelial ovarian cancer (EOC) has a high tumor-associated mortality rate among the gynecological cancers because of chemo-resistant development.

Materials and Method: The human EOC cell lines, SKOV3, OVRCA4, were obtained from the American Type Culture Collection (ATCC). For determine ATP-binding cassette transporters and SIK3 expression, Western blot and Quantitative real-time polymerase chain reaction (qRT-PCR) were used. We used a lentiviral small-hairpin RNA (shRNA) system to knock down SIK3 in the ovarian cancer cell lines. Microarray data derived from SKOV3, OVRCA4 cells expressing sh-luc, sh-SIK3 (01), and sh-SIK3 (63) were analyzed by MetaCore and Ingenuity Pathways Analysis (IPA). Top 10 up-and down-regulated genes and other metastasis-related genes and 5 most highly enriched networks were identified. For determining cell viability when cells treated with cisplatin and Taxol, WST-1 assay was used. Immunohistochemistry and quantification of ABCG2 and SIK3 were performed in EOC tissues. Statistical analysis of OS and PFS were performed using SPSS statistical software (Version 22.0; IBM Corp, Armonk, NY). Survival curves were generated using the Kaplan-Meier method, and differences in survival were assessed using the log-rank test.

Results: Knockdown SIK3 expression upregulates ABCG2 expression, activity as well as resistance to chemotherapeutic agents. The ABCG2 and SIK3 expression levels in 204 EOC patient were mutually reversed. In all stages of the disease, high expression of SIK3 (SIK3-H) was associated with a significant better OS, PFS, compared with low expression of SIK3 (SIK3-L) (127.0 vs 46.0 months, 66.0 months vs 26.0 months respectively). Patients with SIK3-L and ABCG2-H carried the worst prognosis.

Conclusion: (1) High expression of SIK3 indicates a better prognosis in primary ovarian cancer and serous type disease, especially in advanced serous disease. (2) Ovarian cancer patients with SIK3-L and ABCG2-H expression carried the worst prognosis due to chemoresistance

Citation Format: Keng-Fu Hsu, Yu-Ling Liang, Chin-Han Wu, Neng-Yao Shih. ATP-binding cassette transporter, ABCG2, activation is responsible for chemo-resistant, SIK3 low expressed ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5146.
Abstract 515: Progesterone receptor inhibition of efficient type I interferon signaling in breast cancer
Authors: Walter Katherine Rose , Goodman Merit , Hagan Christy
Published: August 2019

A vast majority (65-70%) of breast cancers express the progesterone receptor (PR), a nuclear receptor that is activated in response to its ligand progesterone. Compelling clinical trial data have suggested that progestins have a role in breast cancer development, independent of the widely studied estrogen receptor. The mechanism by which this occurs, however, is vastly understudied.

Traditionally, PR exerts its effects following ligand activation by translocating to the nucleus and binding to DNA where it affects the transcription of a variety of genes involved in growth, survival, and proliferation. Our lab has identified a novel subset of genes that have altered expression following PR activation. Interferon Stimulated Genes (ISGs)–primarily activated in response to interferon signaling (viral response pathway) –are repressed when PR is activated by its ligand.

In attempting to elucidate a mechanism by which PR exerts this effect, we have investigated crosstalk of PR with canonical interferon signaling proteins and have found that PR interacts with these proteins and significantly reduces their ability to become activated. STAT1, a crucial protein involved in the interferon signaling cascade, is unable to efficiently be phosphorylated when PR is activated with ligand. ChIP experiments have also shown that PR inhibits the machinery necessary for ISG transcription from efficiently binding ISG promoters, possibly explaining the reduction in ISG expression observed following PR activation. Because of the importance of this particular signaling cascade in normal cell biology, multiple mechanisms most likely exist to fully inhibit interferon signaling and experiments are currently underway to identify other possibilities to better define PR’s functionality in this context.

Evasion of the immune system is considered a Hallmark of Cancer and our preliminary data suggest a potential mechanism by which breast cancer is able to accomplish this. Activation of type I interferon signaling is an early step in marking tumors for immune clearance. By repressing ISG protein expression, it is possible that these tumors are able to avoid detection by host immune cells leading to tumor establishment and subsequent progression. This novel role of PR and progestins offers insight that could aid in improving upon established estrogen only-based therapies for prevention and treatment of hormone dependent breast cancers.

Citation Format: Katherine Rose Walter, Merit Goodman, Christy Hagan. Progesterone receptor inhibition of efficient type I interferon signaling in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 515.
Abstract 5158: Hypoxia alters the release and size distribution of extracellular vesicles in pancreatic cancer cells to support their adaptive survival
Authors: Patton Mary C. , Zubair Haseeb , Khan Mohammad Aslam , Singh Seema , Singh Ajay P.
Published: August 2019

Pancreatic tumors are characterized by poor vasculature and fibrous stromal tissue networksthat create an extensive hypoxic tumor microenvironment. Extracellular vesicles (EVs) play important roles in pancreatic tumor pathobiology by supporting inter-cellular communications. They arebroadly classified into three subtypes: exosomes (Exo; 30-150 nm), microvesicles (MVs; 100 nm-1 µm) and apoptotic bodies (Abs; 1-5 µm), according to their cellular origin. Here, we studied the effect of hypoxic stress on the release kinetics and size distribution of EVs in pancreatic cancer cells. Further, we also investigated the role of different EV subtypes in adaptive survival of pancreatic cancer cells under hypoxia.Our data demonstrated that under hypoxic conditions, pancreatic cancer cells (MiaPaCa and AsPC1) shed greater amount of EVs with most noticeable changes recorded for the small size EVs. Moreover, all EVs (small, moderate, large) showed a shift towards reduced size depending upon the extent of hypoxia. When examined for subtype-specific markers, we observed mixed profiles. Thrombospondin (marker for Abs) and Arf6 (marker for MVs) were exclusively detected in large and moderate size fractions, respectively, under both normoxia and hypoxia. However, CD9 (marker for Exo) was detected in both small and moderate size EVs under hypoxia. Furthermore, in release kinetics studies we observed cyclic increases in accumulation of EV subtypes under hypoxic conditions. In addition, our data demonstrated that EVs from hypoxic cancer cells promoted survival of cancer cells under hypoxia,with small EVs being the most active. Altogether, our findings establish that hypoxia alters shedding of EVs in supporting adaptive survival of pancreatic cancer cells; associated differences could possibly be exploited for effective cancer management.

Citation Format: Mary C. Patton, Haseeb Zubair, Mohammad Aslam Khan, Seema Singh, Ajay P. Singh. Hypoxia alters the release and size distribution of extracellular vesicles in pancreatic cancer cells to support their adaptive survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5158.
Abstract 5160: Extracellular vesicles from scirrhous gastric cancer cells induce premetastatic niche formation for peritoneal metastasis
Authors: Okuno Tomohisa , Yashiro Masakazu , Kushiyama Shuhei , Nishimura Sadaaki , Togano Shingo , Kuroda Kenji , Tamura Tatsuro , Toyokawa Takahiro , Tanaka Hiroaki , Muguruma Kazuya , Hirakawa Kosei , Ohira Masaichi
Published: August 2019

Background: Human scirrhous gastric carcinoma (SGC) is also known as diffusely infiltrating carcinoma, type 4 on Borrmann’s criteria, and linitis plastica-type carcinoma. SGC is characterized by frequent peritoneal metastasis, high proliferation and infiltration activity with extensive fibrosis in the tumor stroma. We previously reported that a soluble factor(s) from SGC cells changed the peritoneum to pre-metastatic niche such as round shape of mesothelial cells and peritoneal fibrosis, which is a favorable environment for cancer cells to metastasize. Extracellular vesicles (EVs; Exosomes) might be one of soluble factor(s) which might change the peritoneal component to pre-metastatic niche. In this study, we investigated the effect of EVs from SGC cells on the pre-metastatic niche formation of the peritoneum.

Methods: Four SGC cell lines, OCUM-2M, OCUM-2MD3, OCUM-12, KATO-III, 2 non-SGC cell lines, MKN74, MKN45, and peritoneal mesothelial cells (PM cells) were used. The effect of EVs from gastric cancer cells on the peritoneum was examined in vitro and in vivo, as follows. PKH26-labeled EVs from OCUM-2MD3 cells were intravenously injected into nude mice for 3 weeks, then we examined the distribution of PKH26-labeled EVs by a fluorescence microscope. Next, we injected EVs intraperitoneally into nude mice, and investigated the morphology of peritoneum. Effect of EVs on the gene expression of PM cells was examined by RT-PCR. Clinical significance of EVs markers, CD9 and CD63, was evaluated by immunohistochemistry using 63 human gastric cancer specimens.

Results: PKH26-labeled EVs frequently distributed to peritoneum and the liver. The morphology of PM cells changed from round shape to spindle shape by EVs addiction in vitro and in vivo. mRNA expression level of ZEB and N-cadherin of PM cells was significantly increased following the addiction of EVs. The high expression of EVs

markers, CD9 and CD63, was significantly correlated with frequent peritoneal metastasis.

Conclusion: EVs from SGC cells might frequently distribute to mesothelial cells of peritoneum. EVs from SGC cells might play an important role for the formation of a pre-metastatic niche on peritoneum by the morphologic change of peritoneal mesothelial cells.

Citation Format: Tomohisa Okuno, Masakazu Yashiro, Shuhei Kushiyama, Sadaaki Nishimura, Shingo Togano, Kenji Kuroda, Tatsuro Tamura, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. Extracellular vesicles from scirrhous gastric cancer cells induce premetastatic niche formation for peritoneal metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5160.
Abstract 5163: Angiocrine induction of colorectal cancer cell HER3 activation and cell survival is independent of CRC mutational status
Authors: Wang Rui , Bhattacharya Rajat , Fan Fan , Ye Xiangcang , Ellis Lee M.
Published: August 2019

INTRODUCTION: Colorectal cancer (CRC) is the second-leading cause of cancer-related death in the United States. A better understanding of the regulation of CRC cell survival pathways is necessary for developing new therapeutic strategies for patients with metastatic CRC (mCRC). We have previously found that endothelial cells (ECs) from the liver, the most common site of CRC metastases, secrete soluble factors in their conditioned medium (CM) that, in turn, increase cell growth and chemoresistance of CRC cells. We also determined that EC-induced CRC cell survival is mediated, in part, by HER3/ERRB3 signaling.

OBJECTIVES: In the current study we sought to determine if alterations in driver mutations mediate the role of liver ECs in activating HER3 signaling and promoting cell survival in CRC cells.

METHODS: Primary ECs from non-malignant liver parenchyma were isolated as previously described (Wang et al., Mol Cancer Res., 2018). CRC cell lines with distinct molecular profiles (MSI status, and wildtype or mutant KRAS, BRAF, and PIK3CA) were incubated with either their own CM (control) or CM from ECs. The effects of CM on CRC cell growth and response to 5-fluorouracil (5-FU) was determined by MTT and colony formation assays. The effect of angiocrine signaling on chemoresistance was also determined by fluorescence-activated cell sorting (FACS) for apoptosis after treating CRC cells with 5-FU, either in control CM or EC CM. HER3 blockade was accomplished by 1) siRNA knockdown or 2) a HER3 antibody.

RESULTS: CM from liver ECs significantly increased cell growth and chemoresistance in CRC cells regardless of the gene alteration profiles in in vitro assays. We confirmed that EC CM activated the HER3-AKT signaling pathway in CRC cells. Furthermore, we found that HER3 blockade attenuated EC CM-induced AKT activation and cell survival in CRC cells independent of the cell mutational profile.

CONCLUSIONS: Our results demonstrate that HER3 mediates liver EC-induced cell growth and chemoresistance in CRC cells independent of the driver mutational profiles of the cells. The genetic background of CRC cells is not a mediator of EC CM-induced HER3 activation and cell survival. These findings suggest that effects of HER3 targeting of tumor cells in patients with mCRC should be independent of MSI status, or RAS, BRAF, or PIK3CA mutational status.

Citation Format: Rui Wang, Rajat Bhattacharya, Fan Fan, Xiangcang Ye, Lee M. Ellis. Angiocrine induction of colorectal cancer cell HER3 activation and cell survival is independent of CRC mutational status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5163.
Abstract 5164: Signaling crosstalk within prostate tumor microenvironment mediates castrate resistant disease progression
Authors: Tripathi Manisha , Nandana Srinivas , Huang Jen-Ming , Kato Manabu , Mishra Rajeev , Chung Leland , Xin Li , Bhowmick Neil
Published: August 2019

Juxtacrine signaling with the stromal compartment is important to the development of castrate resistance prostate cancer. We found that Notch signaling is upregulated in prostate cancer associated fibroblasts (CAFs) compared with normal tissue associated fibroblasts (NAFs). Further, inhibition of Notch signaling in CAFs resulted in decreased Wnt signaling and decrease in expression of Notch pathway genes. Interestingly, androgen receptor antagonism resulted in increased Notch signaling and the upregulation of downstream Wnts. Further, generation of chimeric tissue recombinant xenografts with human RV1 prostate cancer epithelia with mouse prostate stromal cells with activated Notch signaling that are derived from NICD transgenic mice resulted in increased tumor size compared with wild type control stromal recombinants. Conversely, recombinants derived from prostatic fibroblasts of RBPJ-knockout mice that have a loss of Notch signaling demonstrated diminished tumorigenesis, compared with xenografts with the wild type fibroblasts. In trying to determine the mediator of Wnt-associated paracrine prostate epithelial expansion, we found downstream YAP activation in the RV1 cells. In a reciprocal epithelial-to-fibroblast communication we found that the RV1 cells treated with conditioned media from NICD fibroblasts showed increased IL-6 expression. Conversely, conditioned media from prostate cancer cells caused increased Notch target gene expression in CAFs, which was blocked upon addition of IL-6 neutralizing antibody. Taken together our results suggest that there is a stromal-derived feed-back - feed-forward signaling loop involving Notch and Wnt ligands that activates YAP in the epithelia for paracrine IL-6-dependent Notch activity back in the stromal fibroblasts. Critically, disruption of this cross-talk in the fibroblasts can potentially limit the gain of stem features and castrate resistance of the adjacent epithelia in halting lethal progression of prostate cancer.

Citation Format: Manisha Tripathi, Srinivas Nandana, Jen-Ming Huang, Manabu Kato, Rajeev Mishra, Leland Chung, Li Xin, Neil Bhowmick. Signaling crosstalk within prostate tumor microenvironment mediates castrate resistant disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5164.
Abstract 517: Supraphysiological androgens activate innate immune signaling in prostate cancer
Authors: Mendonca Janet , Owoyemi Olutosin , Rosen Marc , Carducci Michael , Markowski Mark , Antonarakis Emmanuel , Pardoll Drew , Denmeade Samuel , Kachhap Sushant K.
Published: August 2019

Castration resistance prostate cancer (CRPC) first manifests as a sustained rise in the androgen-responsive gene, PSA, consistent with reactivation of a functioning androgen receptor (AR) axis. This observation led to the development of “second-line” therapies aimed at further blocking androgen/AR signaling. Unfortunately, resistance to these agents can also develop quickly. Paradoxically, several studies have suggested that the growth of AR-positive human CRPC cell lines may be inhibited by supraphysiologic levels of testosterone (SupT). These studies suggested that the adaptive reliance on AR signaling by CRPC cells becomes a therapeutic liability that can be exploited through the administration of SupT, which we termed as bipolar androgen therapy (BAT). Understanding how BAT works at the molecular and cellular levels might help in rationally combining BAT with other agents to achieve increased efficacy and tumor responses. Our data indicates that SupT induces DNA double strand breaks (DSBs) in prostate cancer (PCa) cells. Unrepaired DSBs induced by SupT are routed for specialized autophagic degradation, termed nucleophagy. We further show that SupT-induced autophagosomal DNA can activate cytoplasmic DNA sensing pathways and downstream innate immune signaling. Based on our findings, we propose that BAT engages the immune system to inhibit tumor growth. Future combination of BAT with existing immunotherapeutics including immune checkpoint blockade may prove beneficial for treatment of CRPC.

Citation Format: Janet Mendonca, Olutosin Owoyemi, Marc Rosen, Michael Carducci, Mark Markowski, Emmanuel Antonarakis, Drew Pardoll, Samuel Denmeade, Sushant K. Kachhap. Supraphysiological androgens activate innate immune signaling in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 517.