bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1516: Automated circulating DNA purification from large volumes of plasma
Authors: Withers Sydnor T. , Dressler Mary , Cowan Cristopher A.
Published: May 2015

The circulating DNA found in human plasma is enriched for genomic DNA from minority tissues such as tumors, fetuses, and transplanted tissue. This sub fraction of blood has become an area of interest for development and disease as the nucleic acid recovered may represent a source of biomarkers for study. One milliliter of plasma typically yields between 1 ng and 50 ng of DNA. The current commercial DNA purification methods limit researchers' ability to scale their samples in both volume and number. Automated solutions are not available to process large numbers of samples with volumes greater than 1 ml, and manual methods can process up to 5 ml of plasma. Here we present a fully automated solution to extract DNA from 1-64 samples up to 8 ml in volume. The system provides an intuitive user interface to select run options and enter barcodes for reagents and samples. Any combination of sample volumes may be processed together in a single run. Samples are eluted in 50 µl of nuclease-free water in either plates or tubes. We have found that the performance of our system meets or exceeds common commercial alternatives over their common sample input volume.

Citation Format: Sydnor T. Withers, Mary Dressler, Cristopher A. Cowan. Automated circulating DNA purification from large volumes of plasma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1516. doi:10.1158/1538-7445.AM2014-1516
Abstract 1521: Quantitation of HER2 gene amplification using digital PCR
Authors: Li Kelly , Do Devin , Hegerich Patricia , Ping Bruno , Keys David , Majumdar Nivedita , Jackson Stephen , Cifuentes Francisco , Chen Caifu
Published: May 2015

Gene amplification is a common genetic abnormality in many types of cancers and has been implicated in playing important roles in cancer development. The most common types of amplified genes are oncogenes and drug-resistance genes. For example, Human epidermal growth factor receptor 2 (HER2) gene amplification, occurring in about 10-30% of breast cancer cases, is strongly associated with aggressive tumor progression and poor prognosis. HER2 also presents an effective therapeutic target, and several HER2 targeting agents have been approved for treatment of HER2 positive breast cancer by FDA. Analysis of HER2 gene amplification is important not only in selecting right patients for HER2 targeting agents but also monitoring the response to therapy and predicting clinical outcomes. Currently, fluorescence in situ hybridization (FISH) and immunohistochemical (IHC) analysis are standard testing approaches for HER2 gene amplification. However, the challenge for these methodologies is that HER2 gene amplification is less quantitative, especially with some samples whose HER2/CEP17 ratio fall into equivocal range, and the workflow is labor intensive. We have developed digital PCR (dPCR) technology on the QuantStudio™ 3D Digital PCR System and quantify gene copy numbers from zero to eight with high accuracy and precision. We analyzed FFPE samples for HER2 gene amplification and our data show that dPCR provides a robust and quantitative detection, simple workflow and a quick turn around time. This study demonstrate that dPCR quantitation of gene amplification could potentially be used to study any loci that are amplified in cancer.

Citation Format: Kelly Li, Devin Do, Patricia Hegerich, Bruno Ping, David Keys, Nivedita Majumdar, Stephen Jackson, Francisco Cifuentes, Caifu Chen. Quantitation of HER2 gene amplification using digital PCR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1521. doi:10.1158/1538-7445.AM2014-1521
Abstract 1522: Direct quantitative PCR of LINE repeats to quantify CFC-DNA in serum
Authors: Charlot David Jean , Bhalla Megha , Krishnan Raj , Tu Gene
Published: May 2015

Background: Cell free circulating (cfc)-HMW DNA is a proposed diagnostic and prognostic biomarker for malignant tumors. Apoptotic DNA (less than 200 bp) is present in the circulation of normal healthy individuals whereas DNA released from dead cancer cells produces HMW DNA (greater than 400 bp). We developed LINE assay for direct serum/plasma quantitation of cfc-DNA concentration and integrity. LINE (Long Interspersed Elements) is highly conserved repetitive element in human genome with ∼500,000 copies/g.e. These large numbers of copies allow cfc DNA quantitation from small sample volume.

Methods: Sera of 15-30 CRC and Lung cancer patients (early and late stage) from retrospective studies, 5 CRC patients (late stage) from prospective studies along with healthy normals were characterized for cfc-DNA quantity and integrity by LINE Assay. All the samples were prescreened for hemolysis by Potassium assay. Quantitative real-time PCR of LINE repeats was performed with 2 sets of primers (less than 200 bp and greater than 400 bp) amplifying different lengths of DNA. We used 10ul serum directly as a template for qPCR without DNA purification. DNA integrity was determined as a ratio of concentration of longer over shorter LINE fragments.

Results: LINE Assay has a detection limit of 0.08 pg of DNA. Both CRC and lung cancer patients from Retrospective studies have significantly high HMW DNA concentration and DNA integrity as compared to healthy normals (statistically significant at p-value less than 0.05). ROC curves of HMW DNA for detecting early and late stage CRC and Lung have areas under the curves of greater than 0.80. High concentration of HMW DNA along with high DNA integrity also follows the trend of treatment of CRC patients from prospective studies.

Conclusions: LINE Assay is a robust, highly sensitive, and high-throughput method to measure different sizes of cfc-DNA. Our experiments demonstrate that this sizing capability helps to calculate DNA integrity which is a strong indicator for treatment monitoring in prospective studies and cancer staging in solid tumors. The high sensitivity of the LINE assay helps in direct DNA quantitation in serum.

Citation Format: David Jean Charlot, Megha Bhalla, Raj Krishnan, Gene Tu. Direct quantitative PCR of LINE repeats to quantify CFC-DNA in serum. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1522. doi:10.1158/1538-7445.AM2014-1522
Abstract 1523: Molecular typing of Chinese gastrointestinal stromal tumors using a multigene next generation sequencing panel
Authors: Xu Zhi , Hu Zhibin , Huo Xinying , Tang Chuanning , Chen Si-Yi , Chen Jinfei
Published: May 2015

Identifying mutations in individual tumor is critical to improve the efficacy of cancer therapy by matching targeted drugs to specific mutations. Gastrointestinal stromal tumor (GIST) is a stromal or mesenchymal subepithelial neoplasm affecting the gastrointestinal tract and it frequently contains an activating mutation in either the KIT or platelet-derived growth factor A (PDGFRA) gene. Although GIST is highly responsive to several selective tyrosine kinase inhibitors, combined use of inhibitors targeting other mutations is needed to further prolong survival. In this study, we aim to screen and identify genetic mutations in GIST for targeted therapy using the new Ion Torrent next-generation sequencing platform. By utilizing the Ion Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes from DNA extracted from formalin-fixed and paraffin-embedded (FFPE) samples of 125 human gastrointestinal stromal tumors, set up stringent parameters for reliable variant calling by filtering out potential raw base calling errors, and identified frequent mutations in PDGFRA, KIT, APC, MLH1, MET, RET, and PIK3CA. Moreover, frequent missense mutations were detected in MLH1 (12%), MET (16.80%), KIT (33.60%), and PIK3CA (41.60%) genes. The mutations in MLH1 gene were frequently detected in the exons 2, while MET gene was frequently mutated in the exon 12. In addition, we identified missense mutations in other genes, including FLT3, KRAS, PDGFRA, and STK11, at lower frequencies. This study demonstrates the utility of using Ion Torrent sequencing to efficiently identify human cancer mutations. With the finding of the frequent mutations in MLH1 and MET genes, in addition to KIT, PDGFRA and PIK3CA, in GISTs, this study provides a molecular basis for clinically developing new drugs targeting these gene mutations for GIST therapy.

Citation Format: Zhi Xu, Zhibin Hu, Xinying Huo, Chuanning Tang, Si-Yi Chen, Jinfei Chen. Molecular typing of Chinese gastrointestinal stromal tumors using a multigene next generation sequencing panel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1523. doi:10.1158/1538-7445.AM2014-1523
Abstract 1536: Whole genome sequencing of high-grade treatment-naïve prostate tumors
Authors: Decker Brennan J. , Karyadi Danielle M. , Karlins Eric , Davis Brian W. , Tillmans Lori S. , Thibodeau Stephen N. , Ostrander Elaine A.
Published: May 2015

Prostate cancer (PCa) will impact one in six American men and many low-risk men endure therapy, while other men die of PCa. Optimal treatment selection depends on distinguishing indolent tumors from those that will cause prostate cancer specific mortality (PCSM). To date, sequencing of prostate tumor DNA has highlighted the fact that prostate tumors develop differently than other cancers. Instead of recurrent protein-altering mutations, many prostate tumors harbor “closed chain” structural rearrangements that may mediate tumor evolution. However, we do not yet understand which somatic mutations are involved in aggressive disease and PCSM.

To address this crucial question, we are using whole genome sequencing to pinpoint somatic genetic features in tumor/normal paired samples from four PCSM and six non-PCSM patients with treatment-naïve, Gleason 8+ tumors. Tumor and normal DNA were sequenced to a mean coverage of 95.3X and 48.3X, respectively. Tumor DNA purity ranged from 49%-79% and tumor ploidy ranged from 1.96 to 2.22. To define the genomic landscape of aggressive PCa, we will compare our findings with published studies and genotype high-priority candidates in at least 50 additional low- and high-grade tumors. We will also compare somatic mutations in PCSM versus non-PCSM cases and use network analysis to identify pathways that may be involved in progression and metastasis. Finally, we will characterize the clonal evolution of PCa tumors using deep digital sequencing to distinguish mutations that were present in the original tumor clone from subclonal mutations that arose after oncogenesis. In this way, we aim to identify potential oncogenic drivers and candidate mediators of progression and metastasis.

Ultimately, we aim to 1) Uncover somatic mutations that are specific to aggressive tumors 2) Determine whether PCSM tumors have unique genetic features, and 3) Understand the role of clonal evolution in oncogenesis and progression. As more tumors are sequenced, this approach may yield valuable markers for diagnosis, prognosis and treatment selection.

Note: This abstract was not presented at the meeting.

Citation Format: Brennan J. Decker, Danielle M. Karyadi, Eric Karlins, Brian W. Davis, Lori S. Tillmans, Stephen N. Thibodeau, Elaine A. Ostrander. Whole genome sequencing of high-grade treatment-naïve prostate tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1536. doi:10.1158/1538-7445.AM2014-1536
Abstract 1538: Molecular characterization of uterine leiomyomas, histopathological uterine leiomyoma subtypes, and uterine leiomyosarcomas
Authors: Mäkinen Netta , Kämpjärvi Kati , Bützow Ralf , Vahteristo Pia
Published: May 2015

Uterine leiomyomas (ULMs) are the most common tumors of the female genital tract. They originate from the smooth muscle cells of the myometrium. Despite their benign nature, ULMs can cause considerable morbidity, such as excessive bleeding, abdominal pain, and infertility. They are also the most common cause for hysterectomy. Based on histopathology, ULMs can be divided into common leiomyomas and various subtypes, such as cellular, atypical, and mitotically active leiomyomas, that mimic malignancy in one or more aspects. Histopatological ULM subtypes are responsible for approximately 5% of all ULMs. In light of the current literature, ULMs may rarely undergo malignant transformation and develop into a leiomyosarcoma. We have previously reported that mediator complex subunit 12 (MED12) exon 2 is mutated in approximately 70% of ULMs. Our subsequent studies have indicated that both histopathological ULM subtypes (17.5%) and uterine leiomyosarcomas (7%) harbor MED12 exon 2 mutations significantly less frequently than common leiomyomas. The aim of this study is to examine the frequency of other known aberrations in ULMs, such as overexpression of HMGA1 and HMGA2, in histopathological ULM subtypes and uterine leiomyosarcomas. The study material consists of 213 formalin-fixed paraffin-embedded samples including 60 common leiomyomas, 93 histopathological ULM subtypes (cellular, atypical, and mitotically active leiomyomas), and 60 uterine leiomyosarcomas. We have constructed tissue microarrays of these distinct tumor groups for immunohistochemical stainings. In addition to HMGA1 and HMGA2 antibodies, various immunohistochemical markers, commonly used in gynecological pathology, will be utilized to analyze similarities and differences between the tumor groups. It is clinically relevant to identify tumor subtypes with different molecular genetic characteristics which might then lead to improved diagnosis, personalized medical treatments, and prognosis in the future. New knowledge of the mechanisms underlying the potential progression to malignancy is also essential, because, for example in the case of uterine leiomyosarcomas, diagnosis is often accidental and postoperative.

Citation Format: Netta Mäkinen, Kati Kämpjärvi, Ralf Bützow, Pia Vahteristo. Molecular characterization of uterine leiomyomas, histopathological uterine leiomyoma subtypes, and uterine leiomyosarcomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1538. doi:10.1158/1538-7445.AM2014-1538
Abstract 1539: Fusion events in NSCLC cell lines identified by whole transcriptome analysis
Authors: Jung Seung-Hyun , Chung Yeun-Jun
Published: May 2015

Since the discovery of Philadelphia chromosome, human chromosomal rearrangements consequence the great deal within the distinct area of cancer biomarker discovery and targeted therapy. These events are commonly generated by breaking and re-joining the segments on either inter- or intra-chromosomal locations, creating an error within DNA repair mechanism responsible for oncogenic properties and, one of the reasons, causing cancer. Also, alteration in the programing of gene regulation changes the transcriptome structure resulting pathological conditions. Recent advances in the cancer epidemiology predicted the highest death rates within Europe and Korea due to lung cancer representing the leading cause of death within cancer patients. Therefore, we have sequenced the whole transcriptome following the highly sensitive path of RNA-seq with the combination of bioinformatics tools and experimental confirmation. The mRNA was subjected on Illumina HiSeq2000 for paired-end (PE) sequencing and bioinformatics analyses were conducted on Linux based software tools. The PE reads were aligned with Ensemble reference human genome version hg19 using TopHat2 and bowtie1. The fusion events were predicted using two different software packages, deFuse and Tophat-Fusion, and expression analysis was conducted using Cufflinks and R-package cumeRbund. The predicted fusion break-points were confirmed by target specific sanger-sequencing. Through the RNA-seq analysis, we identified a number of fusion events shared by oncogenes in non-small cell lung cancer (NSCLC) cell-lines including EML4:ALK fusion identified in recently established gefitinib resistant SNU2292 cell-line and some novel ones. Moreover, a significant increase in the expression of cell-line specific fusion genes was also observed. Our result will be helpful not only for discovering novel biomarkers but also for understanding the tumorigenesis of NSCLC.

Note: This abstract was not presented at the meeting.

Citation Format: Seung-Hyun Jung, Yeun-Jun Chung. Fusion events in NSCLC cell lines identified by whole transcriptome analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1539. doi:10.1158/1538-7445.AM2014-1539
Abstract 1562: Proteomic analysis of citrullinated targets regulated by the p53-PADI4 pathway
Authors: Tanikawa Chizu , Ueda Koji , Nakagawa Hidewaki , Nakamura Yusuke , Matsuda Koichi
Published: May 2015

We recently reported that p53 regulates protein citrullination through the transcriptional regulation of PADI4 that converts an arginine residue in proteins to a citrulline residue. In response to DNA damage, p53-PADI4 pathway induces citrullination of NPM1, Histone H4, and Lamin C, and subsequently promotes apoptotic pathway. To verify the clinical significance of PADI4 in human carcinogenesis, we screened the mutation of PADI4 in hepatocellular carcinoma and found three non-synonymous mutations among 104 cancer tissues (2.9%). Then we evaluated these three mutations as well as 8 somatic mutations reported by ICGC or COMP projects. We found that 8 among 11 mutations remarkably inhibited the enzymatic activity of PADI4 protein. To identify novel PADI4 substrates, we conducted proteome analysis using HEK293T cells ectopically transfected with wild type or mutant PADI4 expressing plasmid. Among more than 10000 peptides identified by the liquid chromatography MS/MS analysis, 265 peptides (2.2%) were citrullinated in PADI4-introduced cells, while less than 0.4% of total peptides were citrullinated in control or mock transfected cells. Pathway analysis revealed that RNA binding proteins such as hnRNPs are dominantly citrullinated by PADI4, indicating that possible role of protein citrullination in RNA processing.

Citation Format: Chizu Tanikawa, Koji Ueda, Hidewaki Nakagawa, Yusuke Nakamura, Koichi Matsuda. Proteomic analysis of citrullinated targets regulated by the p53-PADI4 pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1562. doi:10.1158/1538-7445.AM2014-1562
Abstract 1605: Pterostilbene, a natural phytoalexin, effectively protects against UVB-induced skin carcinogenesis by increasing antioxidant cellular defenses and preventing mutagenesis
Authors: Sirerol J. Antoni , Pulido Ines , Asensi Miguel , Ortega Angel , Estrela Jose M.
Published: May 2015

Clinical and laboratory studies have demonstrated that skin exposure to ultraviolet radiation (UV) is the main cause of non-melanoma skin cancer (NMSC) (≈ 99%) and melanoma (≈ 95%) development. The procarcinogenic effects of solar exposure are mainly due to UVB radiation (290-320 nm range), the same tight range that produces burning in human skin (erythema), inflammation, oxidative stress, DNA damage, etc. The number of skin cancers around the world keeps increasing and, thus, it is an urgent need to find effective protection remedies. Phytoalexins of polyphenolic structure are naturally occurring compounds involved in the defense against pathogens and environmental stresses in plants. Epidemiological studies confirm that diets enriched in plant polyphenols may offer protection against chronic diseases related to oxidative stress. In this sense polyphenols have shown anticarcinogenic and photoprotective effects (e.g. Clifford JL & DiGiovanni J. Cancer Prev Res 3:132-5, 2010). Seminal studies showed that resveratrol (trans-3,5,4′-trihydroxystilbene; Resv) exerted chemopreventive activity in a model of mouse skin carcinogenesis where topic administration of this polyphenol inhibited multistage carcinogenesis [Jang M et al. Science 275:218-20, 1997]. Pterostilbene (3,5-dimethoxy-4′-hydroxy-trans-stilbene; Pter), a Res analog, shows a higher half-life and more potent anticancer effects in vivo than Res. We compared the potential anti-UVB protection of both polyphenols in a murine model of chronic irradiation (180 mJ UVB/cm2; 3 doses/week; for a total of 30 weeks) that experimentally resembles long-term chronic human exposure to damaging sun radiations. We observed that topical pretreatment of the skin with Pter (1-2 μmol/cm2 per dose, administered right before UB exposure), but not with Resv, fully prevents chronic UVB-induced skin carcinogenesis. The molecular mechanism underlying this protective effect involves a Pter-induced increase in antioxidant skin defenses and a decrease in the rate of mutagenesis.

Citation Format: J. Antoni Sirerol, Ines Pulido, Miguel Asensi, Angel Ortega, Jose M. Estrela. Pterostilbene, a natural phytoalexin, effectively protects against UVB-induced skin carcinogenesis by increasing antioxidant cellular defenses and preventing mutagenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1605. doi:10.1158/1538-7445.AM2014-1605
Abstract 1621: Discovery of pyridyl pyrrolopyridinones as potent and selective CK1γ inhibitors
Authors: Huang Hongbing , Acquaviva Lisa , Bregman Howard , Buchanan John , Chakka Nagasree , DiMauro Erin F. , Dovey Jennifer , Gunaydin Hakan , Hua Zihao , Huang Xin , Huang Liyue , Patel Vinod F. , Martin Matthew W. , Serafino Randy , Wilson Cindy
Published: May 2015

The canonical Wnt pathway regulates the ability of β-catenin to activate specific target genes. Aberrant activation of the Wnt pathway is believed to drive the development and growth of many cancers; for example, APC mutations were observed in >80% of the sporadic colorectal cancers. The casein kinase 1 (CK1) family of serine/threonine protein kinases is highly conserved. Multiple members of the CK1 family are shown to regulate the Wnt pathway through interactions with various proteins. The central role of CK1γ in Wnt signal transduction makes it an attractive target for the treatment of Wnt-pathway dependent cancers. We employed a structure-based approach and identified a series of pyridyl pyrrolopyridinones as potent and selective CK1γ inhibitors. These compounds exhibited good enzyme and cell potency, as well as selectivity against other CK1 isoforms, and favorable rodent PK profiles. Oral dosing of optimized lead compounds resulted in significant inhibition of LRP6 phosphorylation in a mouse tumor PD model.

Citation Format: Hongbing Huang, Lisa Acquaviva, Howard Bregman, John Buchanan, Nagasree Chakka, Erin F. DiMauro, Jennifer Dovey, Hakan Gunaydin, Zihao Hua, Xin Huang, Liyue Huang, Vinod F. Patel, Matthew W. Martin, Randy Serafino, Cindy Wilson. Discovery of pyridyl pyrrolopyridinones as potent and selective CK1γ inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1621. doi:10.1158/1538-7445.AM2014-1621
Abstract 1623: 1,4,5,8-Tetrakis-[(2-N,N-dimethylaminoethyl)amino]anthraquinone as a potential anticancer agent: its synthesis, characterization and anticancer properties
Authors: Ishmael Don R. , Adelsteinsson Orn
Published: May 2015

A number of compounds based on the base compound 1,4,5,8 tetrahydroxyanthraquinone have anticancer properties. One compound of this class mitoxantrone bis-5,8 hydroxyethylaminoethylamino 1,4 dihydroxy anthraquinone has been on the market a number of years and is approved by the FDA for the treatment of: acute myelogenous leukemia, various lymphomas, breast cancer, sarcoma and the non-malignant condition multiple sclerosis. However this drug has a number of drawbacks: (1) for most cancer therapies is considered less effective than the anthracyclines, (2) although it has less cardiotoxicity than anthracyclines it does still has significant cardiotoxicity, (3) it is known to rapidly induce multi-drug resistance factors, and (4) has significant bone marrow toxicity. The class of compounds of 1,4-Bis aminoalkylamino class work against cancer cells by intercalating DNA in the major groove and inhibition of topoisomerase II enzyme. There have been a number of attempts to improve the class of drugs by adding different functional groups. However no compound of this class has exceeded the utility of mitoxantrone. Attempts to make derivatives of leuco 1,4,5,8-tetrahydroxyanthraquinone utilizing the replacement of all four hydroxyl groups for the purpose additional DNA binding or alkylation has been unsuccessful. It was discovered that 1,4,5,8-tetrachloroanthraquinone can be used as a base compound for production of compounds with potential anticancer activity. The compound 1,4,5,8-Tetrakis-[(2-N,N-dimethylaminoethyl)amino]anthraquinone was synthesized and highly purified. It was found to have greater cytotoxicity in the screening assay than mitoxantrone or adriamycin. The cytotoxicity screening was done using the BOT-2 human breast cancer cell line. This compounds may have an advantage over the Bis-derivatives with: 1) greater cytotoxicity in screening assay, 2) cheap intermediates, 3) high yields, 4) increased number of potential active groups sites and 7) removal of the para dihydroxyl groups from the cytotoxic 1,4 bisaminoalkylaminoanthraquinone. This might result in reducing the potential for cardiotoxicity. The 1,4,5,8-Tetrakis-[(2-N,N-dimethylaminoethyl)amino]anthraquinone was purified to 97.8 purity by HPLC. Initial screening in vivo mouse model B16 melanoma was done. The response was the same for mitoxantrone with considerably less toxicity.

Citation Format: Don R. Ishmael, Orn Adelsteinsson. 1,4,5,8-Tetrakis-[(2-N,N-dimethylaminoethyl)amino]anthraquinone as a potential anticancer agent: its synthesis, characterization and anticancer properties. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1623. doi:10.1158/1538-7445.AM2014-1623
Abstract 1633: Novel specific- and dual- tryptophan-2,3-dioxygenase (TDO) and indoleamine-2,3-dioxygenase (IDO) inhibitors for tumor immunotherapy
Authors: Mautino Mario R. , Metz Richard A. , Jaipuri Firoz , Waldo Jesse , Kumar Sanjeev , Marcinowicz-Flick Agnieszka , Potturi Hima , Adams James T. , Allen Clarissa Van , Vahanian Nicholas N. , Link Charles J.
Published: May 2015

Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase is an central immune checkpoint mechanism that controls peripheral immune tolerance and tumor resistance to immune attack. The IDO pathway mediates immunosuppressive effects by expression of the IDO1 gene by tumor cells and/or host antigen presenting cells. Tryptophan depletion and generation of kynurenine generate downstream signals through GCN2, WARS, mTOR and AHR that ultimately lead to anergy of tumor-specific effector CD8+ T cells and activation of Tregs. High expression of IDO correlates negatively with clinical prognosis in patients with a variety of malignancies. Recently, it has been described that tumors can also achieve immunosuppressive effects mediated by tryptophan degradation through expression of TDO, an enzyme functionally and structurally related to IDO1. Therefore, development of small molecules that can prevent tryptophan degradation mediated by IDO1, TDO or both is a prime goal for immunotherapy of cancer. Here we report the discovery of a novel class of imidazoisoindole-based compounds that show potent and selective inhibition of TDO and IDO1, as well as compounds that show potent dual inhibition of both TDO and IDO1. We present structure-activity relationship studies that explore the molecular determinants of potency and specificity for TDO and IDO1. Compounds of these classes were tested for activity in in vitro assays using purified recombinant human and murine TDO and IDO1 as well as in cells expressing IDO1 or TDO. Many of these compounds are 10-200 fold more potent (IC50 < 50 nM) than previously published TDO-specific inhibitors. The pharmacokinetic properties of these compounds are being investigated and lead optimization efforts are in progress.

Citation Format: Mario R. Mautino, Richard A. Metz, Firoz Jaipuri, Jesse Waldo, Sanjeev Kumar, Agnieszka Marcinowicz-Flick, Hima Potturi, James T. Adams, Clarissa Van Allen, Nicholas N. Vahanian, Charles J. Link. Novel specific- and dual- tryptophan-2,3-dioxygenase (TDO) and indoleamine-2,3-dioxygenase (IDO) inhibitors for tumor immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1633. doi:10.1158/1538-7445.AM2014-1633
Abstract 1752: Search for effective therapies for canine and human osteosarcoma using novel therapeutic agents
Authors: Yang Yating , Bartczak Ewa , Yuzbasiyan-Gurkan Vilma
Published: May 2015

Osteosarcoma (OS) is the most common primary bone tumor of both humans and dogs. Despite advances in treatment of other cancers, the survival rates for OS have stagnated for the past decades. Therefore, there is a great need to identify novel and effective treatments. Several classes of novel small molecules with anti-tumor activities have become available in the last few years. Among these are tyrosine kinase inhibitors (TKIs) which are a class of potential anti-tumor medications that are able to inhibit kinase activity of many receptor tyrosine kinases. Another class of molecules target inhibition of the proteasome which induces cell death through various mechanisms. We hypothesized that one or more of such drugs would be effective against canine and human OS cell lines. Our preliminary data identified three TKIs (sunitinib, sorafenib, gefitinib), and a proteasome inhibitor (bortezomib) as having good growth inhibition effects in low concentrations on three canine OS cell lines (D17, Abrams, and Gracie) and two human OS cell lines (SAOS2 and U2OS) in a series of drug screening assay. We then determined the exact IC50 concentration of each drug, capable of inhibiting proliferation by 50%. The IC50 values of bortezomib range from 0.01 to 0.22 μM and the values for sorafenib were from 6 to nearly 9 μM across different cell lines tested, which were all within or close to achievable plasma concentration and tolerable levels. In addition, sunitinib, sorafenib, and bortezomib were shown to decrease cell migration ability in the wound healing assay. With the promising results from in vitro studies, these small molecule inhibitors are good candidates for further evaluation in treating human and canine OS.

Citation Format: Yating Yang, Ewa Bartczak, Vilma Yuzbasiyan-Gurkan. Search for effective therapies for canine and human osteosarcoma using novel therapeutic agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1752. doi:10.1158/1538-7445.AM2014-1752
Abstract 1756: Novel irreversible EGFR tyrosine kinase inhibitor, DBPR112, as a therapeutic candidate for lung adenocarcinoma
Authors: Shiao Hui-Yi , Hsu Tsu-An , Lin Wen-Hsing , Yang Tsong-Toh , Hsieh Hui-Fang , Chen Chiung-Tong , Yeh Teng-Kuang , Hsieh Hsing-Pang
Published: May 2015

Lung cancer is the leading cause of cancer death in the world and non-small cell lung cancer (NSCLC) accounts for 85% of the lung cancer deaths. In recent years, EGFR tyrosine kinase inhibitors (TKIs) have showed remarkable effects in patients with certain genetic alternations in the EGFR proteins. However, the clinical benefits of two first-generation EGFR TKIs (gefitinib and erlotinib) have been limited due to the acquired resistance from patients within 9-12 months after treatment, therefore, the discovery of next-generation EGFR-TKIs poses an utmost priority.

Our team at IBPR has identified DBPR112 as a potent EGFR-TKI, showing IC50 of 2 nM in HCC827 cells, EGFRWild-Type (IC50: 10 nM) and EGFRL858R/T790M (IC50: 70 nM), which are comparatively better than gefitinib and similar to that of BIBW2992 (afatinib, developed by Boehringer Ingelheim, and approved by FDA in 2013). DBPR112 (10, 50 and 100 mg/kg/day, 5 days/wk for 2 weeks) was orally effective against the growth of human lung HCC827 tumors subcutaneously xenografted in nude mice. A dramatic reduction in tumor size was noted with DBPR112 treatment at 50 and 100 mg/kg/day, while displaying negligible body weight loss in all dosing groups. In addition, the pharmacokinetics properties of DBPR112 are superior to those of BIBW2992; demonstrating the potential of DBPR112 as a therapeutic candidate for the treatment of lung adenocarcinoma with EGFR mutations.

This NRPB-granted 4-year top-down project was commenced in Dec. 2012 and aimed at accomplishing a comprehensive program of pre-clinical development and phase I study. To date, several pre-clinical studies and progress were steadily accomplished, including non-GMP production of 5 kg DBPR112 (tox-lot), preliminary analysis methods development, pre-formulation study, pharmacokinetics/metabolism studies and preliminary toxicology evaluation. GLP-toxicity studies, GMP-production of DBPR112 (clinical-lot) and formulation for clinical use will be scheduled in 2014 (2nd year of this top-down project), and we look forward to filing an IND application in early 2015.

Citation Format: Hui-Yi Shiao, Tsu-An Hsu, Wen-Hsing Lin, Tsong-Toh Yang, Hui-Fang Hsieh, Chiung-Tong Chen, Teng-Kuang Yeh, Hsing-Pang Hsieh. Novel irreversible EGFR tyrosine kinase inhibitor, DBPR112, as a therapeutic candidate for lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1756. doi:10.1158/1538-7445.AM2014-1756
Abstract 177: Obesity promotes growth of ovarian cancer through adipose stem cells
Authors: Zhang Yan , Solley Travis , Lu Karen , Wei Caimiao , Nowicka Alesandra , Klopp Ann
Published: May 2015

Introduction: Intraperitoneal spread to visceral adipose is a frequent event and a major cause of morbidity and mortality in women with ovarian cancer. Excess visceral adiposity increases the risk of ovarian cancer. We hypothesized that obesity effects may be mediated by the adipose-derived stem cell population (ASC) to enhance the initiation and progression of ovarian cancer.

Results: Intraperitoneally injected ID8 ovarian cancer cells grew significantly faster in mice with diet-induced obesity (p<0.05). To determine if ASC mediated this effect, ASC were then isolated from the subcutaneous (SC-ASC) and visceral adipose (V-ASC) of mice with diet-induced obesity and lean mice. ASC from all sources expressed same mesenchymal cell surface markers and similarly increased ID8 cell proliferation and survival following paclitaxel treatment. There was no significant difference in ID8 migration in response to ASC conditioned media from lean and obese mice. ASC from obese mice exhibited impaired differentiation into adipocytes and osteocytes in-vitro as compared to ASC from lean mice. 106 renilla luciferase expressing ID8 were co-injected intra-peritoneally with equal number of different ASC isolates into C57Bl6 mice. SC-ASC from obese mice promoted growth of intraperitoneal tumor and the formation of metastasis as compared to SC-ASC from lean mice (p<0.05). There was no significant difference in the effect of V-ASCs from obese and lean mice on ID8 cells. There was trend towards more rapid tumor growth in mice injected with V-ASC as compared to SC-ASC from lean mice. Ascites volume was highest in mice injected with SC-ASC derived from obese mice. SC-ASC from obese mice increased tumor vascularity while ASC from obese mice increased the formation of intra-tumoral perilipin expressing adipocytes.

Conclusions: SC-ASC from obese mice promoted tumor growth more than ASC from lean mice suggesting that obesity alters the ASC population to enhance tumor-promoting effects. Obese derived ASC exhibit impaired differentiation potential, which may be linked to tumor promoting effects. Future studies will focus on understanding the mechanisms by which obesity alters the ASC population to impact tumor progression.

Citation Format: Yan Zhang, Travis Solley, Karen Lu, Caimiao Wei, Alesandra Nowicka, Ann Klopp. Obesity promotes growth of ovarian cancer through adipose stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 177. doi:10.1158/1538-7445.AM2014-177
Abstract 1782: The ubiquitin protease UBP43 is a target for KRAS mutant lung cancers
Authors: Mustachio Lisa Maria , Chinyengetere Fadzai , Lu Yun , Hu Shanhu , Kawakami Masanori , Tafe Laura J. , Danilov Alexey , Sekula David J. , Ma Tian , Freemantle Sarah J. , Dmitrovsky Ethan
Published: May 2015

KRAS is frequently mutated in human lung cancers. Lung cancers with mutant KRAS are often resistant to current therapies. There is therefore a need to develop new ways to target these tumors with KRAS mutations. We previously found that the protein modification pathway containing the ubiquitin-like interferon-stimulated gene 15 (ISG15) destabilizes cyclin D1 and represses growth and tumorigenicity of lung cancer cells. This study substantially extends that prior work by reporting that inhibition of the ubiquitin protease UBP43, which removes ISG15 from protein substrates, also destabilizes KRAS protein levels. In a screen of murine lung cancer cells with KRAS mutations, the lines with highest KRAS protein levels had the highest levels of UBP43 protein, implying a direct link between UBP43 and KRAS. Stable overexpression of UBP43 in murine lung cancer cells increased cancer cell growth and stability of KRAS protein. In contrast, knockdown of UBP43 in murine and human lung cancer cell lines had the opposite effects on growth and KRAS protein stability. Co-immunoprecipitation experiments established an interaction between ISG15 and both wild-type KRAS and mutant KRAS. UBP43 immunohistochemical staining of human lung cancer cases showed a marked increase in UBP43 expression in KRAS mutant versus KRAS wild-type lung cancers. Preliminary findings in newly engineered mouse models revealed an increase in lung cancers in KRAS mutant/UBP43 wild-type as compared to KRAS mutant/UBP43 null mice. This finding provides a genetic basis for targeting UBP43 in lung cancers. Taken together, these studies broaden the role of UBP43 as an antineoplastic target to include an unmet medical need. This is to combat lung cancers that harbor KRAS mutations.

Citation Format: Lisa Maria Mustachio, Fadzai Chinyengetere, Yun Lu, Shanhu Hu, Masanori Kawakami, Laura J. Tafe, Alexey Danilov, David J. Sekula, Tian Ma, Sarah J. Freemantle, Ethan Dmitrovsky. The ubiquitin protease UBP43 is a target for KRAS mutant lung cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1782. doi:10.1158/1538-7445.AM2014-1782
Abstract 1807: Pre-clinical evaluation of Mcl-1 inhibition through maritoclax in acute myeloid leukemia
Authors: Liu Qiang , Doi Kenichiro , Gowda Krishne , Barth Brian M. , Claxton David , Amin Shantu , Loughran Thomas P. , Wang Hong-Gang
Published: May 2015

There is an urgent and unmet medical need for development of new therapeutics for acute myeloid leukemia (AML). The anti-apoptotic Bcl-2 family protein Mcl-1 plays a critical role in AML cell survival and chemoresistance. Mcl-1 is upregulated in relapsed AML and leukemic stem cells. Inhibition of Mcl-1 expression in AML cells by kinase inhibitors or gene targeting induces apoptosis and suppresses leukemogenesis. However, the impact of targeting Mcl-1 in AML therapy with selective pharmacological inhibitors remains to be assessed. Recently, we discovered a novel Mcl-1 inhibitor, maritoclax (marinopyrrole A), which antagonizes Mcl-1 by targeting Mcl-1 for proteasomal degradation. This unique mechanism of action is in contrast to BH3 mimetics that exert their inhibitory function through disruption of the interactions between pro- and anti-apoptotic Bcl-2 family proteins. In a panel of AML cell lines, the potency of maritoclax positively correlated with Mcl-1 expression and inversely correlated with Bcl-2 and Bcl-xL expression. Maritoclax was efficacious as a single agent and markedly synergized with ABT-737 to induce apoptosis in Mcl-1 overexpressing ABT-737 and multidrug resistant AML cell lines. Moreover, maritoclax was able to overcome stroma-mediated resistance to apoptosis in AML cells. Importantly, maritoclax induced Mcl-1 degradation and apoptotic cell death selectively in clinical AML specimens with high levels of Mcl-1. To evaluate the anti-tumor activity of maritoclax in vivo, we established U937 tumor xenografts in nude mice. As a single agent, maritoclax significantly inhibited the growth of established xenografts at a dose of 20 mg/kg/d intraperitoneally. At this dosing level, no significant difference in body weight change and complete blood count was observed between maritoclax and vehicle treatment groups. Collectively, our studies demonstrated that maritoclax represents a novel class of Mcl-1 inhibitors and that the selective pharmacological inhibition of Mcl-1 may be a viable approach for the treatment of AML.

Citation Format: Qiang Liu, Kenichiro Doi, Krishne Gowda, Brian M. Barth, David Claxton, Shantu Amin, Thomas P. Loughran, Hong-Gang Wang. Pre-clinical evaluation of Mcl-1 inhibition through maritoclax in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1807. doi:10.1158/1538-7445.AM2014-1807
Abstract 1826: Altered expression of miRNAs in acquired trastuzumab resistance
Authors: Howe Karen , Eustace Alex J. , Gullo Giuseppe , Browne Brigid , Aherne Sinead , Crown John , O'Donovan Norma
Published: May 2015

HER2 positive breast cancer accounts for approximately 20-25 % of breast cancers. Trastuzumab, a humanised monoclonal antibody, is approved for the treatment of HER2 positive breast cancer. However, patients with metastatic HER2 positive breast cancer frequently develop resistance to trastuzumab. The aim of this study is to investigate microRNAs (miRNAs) in cell line models of acquired trastuzumab resistance to identifty altered miRNAs which may be biomarkers of resistance.

The trastuzumab resistant cell line, SKBR3-T was established by continuous exposure to trastuzumab (10 µg/ml) for 6 months. MiRNA extracted from SKBR3, and SKBR3-T was profiled using Taqman low density arrays (TLDA). Six differentially regulated miRNAs were identified in the SKBR3-T cells. qRT-PCR assays confirmed that four were significantly altered in the SKBR3-T compared to SKBR3 cells: miR-224 1.6 fold down-regulated (p=0.01), miR-221 5.8 fold up-regulated (p=0.04), miR-222 was 3.8 fold up-regulated (p=0.01) and miR-9 was 2.2 fold up-regulated (p=0.04). In another model of acquired trastuzumab resistance, BT474-Tr, miR-221 and -222 were also up-regulated, miR224 expression was switched off. No significant change in miR-9 was observed in BT474-Tr.

We analysed expression of the four miRNAs in a panel of HER2 positive cell lines which included 6 innate trastuzumab resistant cell lines and 3 innately sensitive cell lines. miR-224 was significantly down-regulated in the 6 resistant cell lines and miR-221 was significantly up-regulated in 3 of the 6 resistant cell lines, compared to the sensitive cell lines. miR-222 and miR-9 were not consistently altered in the resistant cell lines compared to the sensitive cell lines.

We analysed expression of these miRNAs in a small cohort of patients who showed durable complete response (pCR) (9 %) following treatment with trastuzumab and chemotherapy [1], and a control group of patients whose disease progressed following treatment. miRNA was extracted from FFPE tumor samples (n=12) and analysed by qRT-PCR. The mean levels of expression of miR-9, -221, and -222 were higher in non-responders (n=6) than responders (n=6) and the mean levels of miR-224 were lower in the non-responders compared to responders.

These results suggest that miR-9, -221, -222 and -224 may be potential biomarkers of trastuzumab resistance.

1. Gullo, G., et al., Durable complete response following chemotherapy and trastuzumab for metastatic HER2-positive breast cancer. Annals of Oncology, 2012.

Citation Format: Karen Howe, Alex J. Eustace, Giuseppe Gullo, Brigid Browne, Sinead Aherne, John Crown, Norma O'Donovan. Altered expression of miRNAs in acquired trastuzumab resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1826. doi:10.1158/1538-7445.AM2014-1826