bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3558: Predicting response to Mcl-1 targeting agents in NSCLC and multiple myeloma
Authors: McEachern Kristen , O’Connor Greg , Cidado Justin , Belmonte Matthew , Barry Evan , Dry Hannah , Secrist Paul , Drew Lisa
Published: July 2016

Mcl-1 is an anti-apoptotic member of the Bcl-2 family of proteins and is frequently amplified or over-expressed in both solid tumors and hematological malignancies, suggesting that its activity may be important for the survival of cancer cells. CDK9 inhibition results in the down regulation of Mcl-1 mRNA and subsequent protein levels by inhibiting transcription and represents an indirect approach to targeting Mcl-1. Mcl-1 can also be targeted directly using an inhibitor that disrupts the Mcl-1 complexes to induce apoptosis.

Using both molecular and pharmacological approaches, we sought to identify predictive biomarkers of Mcl1 dependency in sensitive NSCLC and multiple myeloma cell lines. Here we demonstrate that NSCLC cell lines lacking MCL1 gene copy number gains are not sensitive to siRNA mediated knockdown of Mcl-1 or Mcl-1 inhibition (cell line sensitivity to CDK9 or Mcl-1 inhibition is defined by potency and extent of caspase activation). However, the presence of a copy number alteration does not predict sensitivity to Mcl-1 inhibition. To better understand what the drivers of sensitivity are, we developed quantitative assays on the Peggy platform (a capillary based immunoassay platform by Protein Simple) to measure both Mcl-1 and Bcl-xL protein levels. Using these assays, we show a correlation between sensitivity to a CDK9 or Mcl1 inhibitor and Mcl-1 levels, as well as to the ratio of Mcl-1 to Bcl-xL protein in a NSCLC cell line panel.

These findings were then extended into a panel of multiple myeloma cell lines. While somewhat broad activity for CDK9 or Mcl-1 inhibition is seen across the cell lines tested, a subset of the sensitive lines have MCL1 amplification and express high levels of Mcl-1 protein. Mcl-1 levels alone, however, do not predict for sensitivity across the panel and, similar to NSCLC, the ratio of Mcl1 to Bcl-xL expression has greater positive predictive value.

These results provide the rationale for exploring Mcl-1 copy number alterations and Mcl-1 and Bcl-xL protein levels as predictive biomarkers for tumor response when treating with a CDK9 or Mcl-1 inhibitor in both NSCLC and multiple myeloma.

Citation Format: Kristen McEachern, Greg O’Connor, Justin Cidado, Matthew Belmonte, Evan Barry, Hannah Dry, Paul Secrist, Lisa Drew. Predicting response to Mcl-1 targeting agents in NSCLC and multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3558.
Abstract 3566: Glycosylation of the TNFR1 death receptor controls cell fate
Authors: Holdbrooks Andrew , Schultz Matthew J. , Liu Zhongyu , Bullard Daniel , Bellis Susan L.
Published: July 2016

Activation of the TNFR1 death receptor by TNFα can induce cell survival or cell death signaling, however, the mechanisms regulating this TNFR1 signaling switch are poorly understood. TNFα-induced apoptosis is reported to require higher-order clustering and internalization of activated TNFR1, whereas surface retention of the activated receptor has been shown to promote survival signaling mediated by NFκB and MAPKs. Studies from our group have identified a novel glycosylation-dependent mechanism that controls this signaling switch. Specifically, we have found that TNFR1 localization and activity are regulated by the addition of a distinct sugar, an α2-6 linked sialic acid, by the enzyme ST6Gal-I, a Golgi sialyltransferase whose expression is upregulated in many cancer types. The sialylation effect on TNFR1 was examined in epithelial and monocytic cancer cell lines with forced overexpression or knockdown of ST6Gal-I, as well as in primary monocytes obtained from mice with ST6Gal-I knock-in or knockout. Data from these models indicate that α2-6 sialylation of TNFR1 blocks TNFα-induced apoptosis, and the suggested mechanism underlying this inhibition of apoptosis is the sialylation-driven inhibition of TNFR1 oligomerization and internalization, as observed through immunoblotting, immunocytochemistry and flow cytometry. Considering sialylated TNFR1 is retained on the plasma membrane following activation, we hypothesize that ST6Gal-I functions to not only block TNFR1-mediated cell death but also divert the cell's activity to favor survival. Supporting this hypothesis, cells with elevated levels of ST6Gal-I exhibit heightened expression and activation of many pro-survival factors, such as NFκB, MAPKs and AKT, in addition to decreased activation of caspase-3. Since levels of ST6Gal-I are elevated in multiple tumor cell populations, our group proposes that ST6Gal-I protects tumor cells against TNFα-induced apoptosis within the inflammatory tumor microenvironment. As preliminary support for this concept, peritoneal ascites fluid was collected from ovarian cancer (OC) patients, and the acellular, cytokine-rich fraction was incubated with OC cell lines with or without forced ST6Gal-I expression. Expression of ST6Gal-I conferred strong protection against cell death induced by soluble ascites. Based on these collective findings, we postulate that sialylation of TNFR1 by ST6Gal-I diverts the cellular response to TNFα from apoptosis to survival, providing a mechanism by which tumor cells can evade immune cell killing.

Citation Format: Andrew Holdbrooks, Matthew J. Schultz, Zhongyu Liu, Daniel Bullard, Susan L. Bellis. Glycosylation of the TNFR1 death receptor controls cell fate. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3566.
Abstract 3575: Ferroptosis: A novel nonapoptotic cell death overcomes head and neck cancer resistance to cisplatin
Authors: Jang Hye Jin , Park Jin Young , Kim Eun Hye , Kim Ji Won , Kwon Minsu , Roh Jong-Lyel
Published: July 2016

Ferroptosis was recently identified as a novel form of iron-dependent cell death different from apoptosis or necrosis. Inhibition of key molecules related to ferroptosis, cystine-glutamate antiporter (xCT) and glutathione peroxidase (GPX4), may induce the eradication of cancer cells resistant to conventional chemotherapy or radiotherapy. The present study investigated whether ferroptosis overcome head and neck cancer (HNC) resistant to cisplatin treatment. Three cisplatin-resistant HNC cell lines (AMC-HN3R, -HN4R and -HN9R), their parental lines, and other human HNC lines were used. The effects of cysteine and glutamate alteration and pharmacological and genetic inhibition of xCT were assess by measuring viability, death, total and cytoplasmic lipid reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm), protein expression, and preclinical mouse tumor xenograft models. Conditioned media with no cysteine or high glutamate supplement induced ferroptosis of both cisplatin-sensitive and -resistant HNC cells without apparent change of necrosis and apoptosis markers, RIP1 and RIP3 and casplase-3 expression, and ΔΨm. This was blocked by antioxidants trolox or N-acetyl cysteine pretreatment. Sulfasalazine, artensunate, and erastin inhibited the growth of HNC cells and accumulated cytoplasmic lipid ROS via induction of ferroptosis and inhibition of xCT protein expression. Genetic silencing of xCT with siRNA or shRNA treatment also induced the effective cell death (ferroptosis) of resistant HNC cells. These effects were blocked with trolox or ferrostatin-1 pretreatment. Pharmacological and genetic inhibition of xCT significantly sensitized resistant HNC cells to cisplatin in vitro and in vivo. Cystine deficiency or glutamate excess induces ferroptosis in HNC cells. Further, pharmacological and genetic inhibition of xCT overcomes the cisplatin resistance of HNC cells by inducing ferroptosis.

Citation Format: Hye Jin Jang, Jin Young Park, Eun Hye Kim, Ji Won Kim, Minsu Kwon, Jong-Lyel Roh. Ferroptosis: A novel nonapoptotic cell death overcomes head and neck cancer resistance to cisplatin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3575.
Abstract 3577: Pan-cancer analysis of mutagenesis by APOBEC cytidine deaminases
Authors: Gordenin Dmitry A.
Published: July 2016

The elucidation of mutagenic processes that shape cancer genomes is a fundamental problem whose solution promises insights into new treatment, diagnostic, and prevention strategies. We and others previously identified mutation clusters and genome-wide mutagenesis bearing the single-strand DNA (ssDNA)-specific APOBEC cytidine deaminase signature at 5′-tC-3′ motifs across many cancer types. Because of high abundance of APOBEC mutagenesis in several cancer types, we have developed a statistical approach capable of identifying cancer samples with APOBEC mutagenesis pattern based on WGS as well as on WES mutation data. This analysis, called Pattern of Mutagenesis by APOBEC Cytidine Deaminases (P-MACD), has been recently integrated into the Broad Institute GDAC Firehose and is currently available for Firehose standard and AWG-customized runs. The results of analysis using the P-MACD package will be discussed, including correlations with molecular and clinical features of TCGA cancer samples as well as relative roles of different APOBEC enzymes in cancer mutagenesis.

Citation Format: Dmitry A. Gordenin. Pan-cancer analysis of mutagenesis by APOBEC cytidine deaminases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3577.
Abstract 3590: A Neil1 DNA glycosylase germline variant induces genomic instability and cellular transformation
Authors: Sweasy Joann B. , Galick Heather , Kathe Scott , Wallace Susan
Published: July 2016

NEIL1 DNA glycosylase is a bifunctional enzyme that recognizes oxidized pyrimidines and formamidopyrimidines, and appears to remove damage in advance of the replication fork. The rs5745906 SNP of the NEIL1 gene has a minor allele frequency of 1% and is a G to A base substitution that encodes the G83D NEIL1 protein. Interestingly, this mutation is also present in cholangiocarcinomas. Unlike WT NEIL1, the G83D variant is not able to recognize oxidized bases efficiently due to a shift in the void filling residues that stabilize the DNA duplex once a damaged base has been extruded into the glycosylase substrate binding pocket. The goal of our study was to determine if the G83D NEIL1 variant exhibits a cancer-related phenotype in cells. We found that unlike wild-type (WT) NEIL1, expression of G83D in MCF10A immortal human breast epithelial cells induces genomic instability and cellular transformation. Expression of G83D but not WT NEIL1, in MCF10A cells leads to replication fork stalling and slow replication fork velocity. Our results suggest that G83D is unable to remove oxidative DNA damage at the replication fork, resulting in genomic instability and cellular transformation. Our results are consistent with the idea that individuals who harbor the G83D NEIL1 variant are at increased risk for cancer.

Citation Format: Joann B. Sweasy, Heather Galick, Scott Kathe, Susan Wallace. A Neil1 DNA glycosylase germline variant induces genomic instability and cellular transformation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3590.
Abstract 3596: KLLN protects genomic stability by maintaining H3K9 trimethylation (H3K9me3) at the pericentric heterochromatin
Authors: Sankunny Madhav , Nizialek Emily , Niazi Farshad , Eng Charis
Published: July 2016

Dysregulation in the maintenance of chromatin organization resulting in genomic instability is a major driving force for inappropriate development and carcinogenesis. Tumor suppressor genes play a critical role in this regulation through the maintenance of epigenetic marks. Our study focuses on the role of KLLN in the maintenance of pericentric H3K9 trimethylation (H3K9me3) and genomic stability. Germline hypermethylation of KLLN resulting in decreased KLLN expression has been linked to Cowden cancer-predisposition syndrome (CS) in PTEN mutation negative patients. KLLN is a tumor suppressor gene necessary for p53-mediated apoptosis. The protein mediates S-phase arrest and is known to have DNA binding ability. Here, we first used chromatin immunoprecipitation-based sequencing (ChIP-seq) to investigate regions of KLLN binding on the genome and compared it to regions of H3K9me3 enrichment. We used H3K9 specific histone methyltransferase (HMT) activity assay and immunoblotting to measure levels of H3K9me3. Immunostaining was used to study the localization of KLLN, and micronuclei frequency and aberrations in chromosome number was used to assess chromosomal instability. Analysis of ChIP-seq shows enrichment of KLLN binding in regions of H3K9me3. Overexpression of KLLN using plasmid-based transfection correlates with increased H3K9 methyltransferase activity and increased global H3K9me3, while loss of KLLN expression through siRNA-mediated knockdown had an opposite effect. We also established that KLLN localizes to pericentric regions, and loss of KLLN results in dysregulation of pericentric heterochromatin, with consequent chromosomal instability manifested by increased micronuclei frequency and numerical chromosomal aberrations. KLLN regulation of H3K9me3 could be correlated with its interaction with deleted in breast cancer (DBC1). DBC1 is a known inhibitor of SUV39H1, a H3K9 specific histone methyltransferase responsible for maintenance of pericentric heterochromatin. We hypothesize that KLLN sequesters DBC1 through their interaction, thereby abrogating DBC1 inhibition of SUV39H1, resulting in maintenance of pericentric heterochromatin and genomic stability. Our study therefore suggests a critical role for KLLN in the deterrence of tumor initiation and progression.

SIGNIFICANCE: Our study demonstrates a clear role for KLLN in maintenance of chromatin organization thereby maintaining genomic stability, which begins to uncover the basis of KLLN as a tumor suppressor and as a susceptibility gene for inherited cancers.

Citation Format: Madhav Sankunny, Emily Nizialek, Farshad Niazi, Charis Eng. KLLN protects genomic stability by maintaining H3K9 trimethylation (H3K9me3) at the pericentric heterochromatin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3596.
Abstract 3597: RPS3 regulates melanoma growth and sensitivity to DNA damage and predicts a poor prognosis by targeting ADT3
Authors: Tian Yun , Qin Lijun , Guo Wei , Zhang Changlin , Shi Dingbo , Liu Tianze , Li Wenbing , Wang Jingshu , Li Yixin , Qin Ge , Yu Wendan , Xiao Xiangsheng , Kang Tiebang , Huang Wenlin , Deng Wuguo
Published: July 2016

Melanoma, a malignant skin cancer, is resistant to DNA damage-mediated therapy. Discovering and identifying novel therapeutic targets for melanoma is urgently required. In this study, we found that ribosome protein S3 (RPS3) regulated melanoma growth and the sensitivity of melanoma to DNA damage by targeting ADP/ATP translocase 3 (ADT3). Knockdown of RPS3 inhibited cell proliferation and sensitized melanoma cells to DNA damage. RPS3 knockdown also promoted ADT3 translocation to mitochondrial of melanoma cells when exposed to DNA damage. RPS3 interacted with ADT3 in melanoma cells. Knockdown of ADT3 reduced its co-localization with RPS3. RPS3 could not obviously sensitize melanoma cells to DNA damage with ADT3 knockdown. In addition, we found that Lys18 in the death-induce-domain of RPS3 protein played a critical role in the interaction between RPS3 and ADT3. Mutation at Lys18 site could deadlock ADT3 and attenuate cell apoptosis in melanoma cells. Knockdown of RPS3 also inhibited tumor growth in a melanoma mouse model in vivo, but overexpression of the RPS3-18 mutation rescued the growth. Furthermore, we showed that the patients with higher expressed RPS3 had a much shorter median survival, whereas the patients with higher levels of ADT3 had a much longer median survival. Collectively, our results indicate that RPS3 cooperates with ADT3 to regulate the sensitivity of melanoma to DNA damage and suggest that the RPS3/ADT3 pathway is a potential therapeutic target for human melanoma. Grant support: This work was supported by the funds from the National Natural Science Foundation of China (81472178, 81272195) and the State “973 Program” of China (2014CB542005).

Citation Format: Yun Tian, Lijun Qin, Wei Guo, Changlin Zhang, Dingbo Shi, Tianze Liu, Wenbing Li, Jingshu Wang, Yixin Li, Ge Qin, Wendan Yu, Xiangsheng Xiao, Tiebang Kang, Wenlin Huang, Wuguo Deng. RPS3 regulates melanoma growth and sensitivity to DNA damage and predicts a poor prognosis by targeting ADT3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3597.
Abstract 360: Detecting intracellular bRAF/cRAF dimerization using a novel luminescent complementation system
Authors: Somberg Richard L. , Schwinn Marie K. , Slater Michael R. , Machleidt Thomas , Cong Mei , Wood Keith V.
Published: July 2016

BRAF mutations can promote constitutive activation of MEK/ERK signaling, leading to unregulated cell proliferation and tumorigenesis. Although inhibitors targeting oncogenic BRAF have shown promise in preventing cell growth, these same inhibitors can paradoxically activate signaling by inducing BRAF interaction with CRAF. Methods for monitoring this undesired outcome are typically low throughput or use kinase binding domains modified with a membrane targeting sequence. We sought to develop a cell-based assay that could reliably detect dimerization of full-length BRAF/CRAF and could potentially be used to rapidly screen inhibitors for modulation of this interaction. To achieve this objective, we utilized a NanoLuc luciferase-based complementation reporter consisting of 1.3 kD (SmBiT) and 18 kD (LgBiT) fusion tags that reconstitute an active luciferase when brought into proximity. In this optimally configured BRAF/CRAF assay, BRAF-LgBiT and CRAF-SmBiT fusions are stably expressed in mammalian cells, and luminescence, indicative of protein interaction, is monitored using a live cell detection reagent. With this assay, we were able to observe rapid and dose-dependent induction of BRAF/CRAF dimerization in response to a panel of kinase inhibitors. The sensitivity and robustness of the assay permitted reliable screening in both 96- and 384-well formats (Z’ factors > 0.9 and 0.6, respectively). Furthermore, the brightness of the reporter allowed the assay to be monitored in individual cells using bioluminescence imaging. These results suggest this BRAF/CRAF complementation assay can enable rapid, high-throughput profiling of kinase inhibitors targeting the RAS/RAF/MEK/ERK signaling pathway.

Citation Format: Richard L. Somberg, Marie K. Schwinn, Michael R. Slater, Thomas Machleidt, Mei Cong, Keith V. Wood. Detecting intracellular bRAF/cRAF dimerization using a novel luminescent complementation system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 360.
Abstract 3614: Comparison and evaluation of somatic mutation using PGM and proton platform in cell free DNA of non-small cell lung cancer patients
Authors: Sung Jae Sook , Lee Jong Won , Kim Boyeon , Lee Saet Byeol , Kwon Nak-Jung , Lee Won-Chul , Kim Hae Mi , Jang Won Jin , Choi Yun Ji , Park Kyung Hwa , Kim Yeul Hong
Published: July 2016

Non-small cell lung cancers (NSCLC) are characterized by a unique pattern of genetic driver mutations, and some of mutations may be used to predict prognosis and targeted treatment such as EGFR TKIs. Cell free (cfDNA) present in the blood stream shows much potential as a useful cancer maker for early diagnosis and cancer progression monitoring. Especially, analyzing the cfDNA with Next Generation Sequencing (NGS) technology allows high through put examination of various genes concurrently at a low cost. However, there are still no standardized methods to identify mutations in cfDNA. In this study, we examined the viability of PGM and Proton platforms. Ion AmpliSeq Cancer Hotspot Panel v2 (Ion Torrent), covering 2800 COSMIC mutations from 50 cancer genes was used to analyze cfDNA of 125 serum samples from NSCLC patients. The on target was 88% and mean depth was 643x using PGM platform. And, the on target was 92% and mean depth was 22,868x in Proton platform. To validate the results of two NGS platforms, we analyzed EGFR status by sanger sequencing in available 100 tumor tissues. EGFR mutations were identified in 34 (34%) by sanger sequencing. EGFR mutations were identified in 32 (25.6%) and 28 (22.4%) by PGM and Proton platform. Interestingly, out of 34 mutations of tumor tissue, EGFR mutations were matched to 11 and 26 in PGM and Proton platform, respectively. These results showed concordance of 76.5% between the tDNA (sanger sequencing) and cfDNA (Proton). In addition, KRAS (codon 12 and 13) mutations were 4 (3.2%) and 18 (14.4%), respectively. In our study, we demonstrated that Proton platform of high depth is a useful assay to identify somatic mutations of cfDNA in NSCLCs. [This research was supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C0066)]

Citation Format: Jae Sook Sung, Jong Won Lee, Boyeon Kim, Saet Byeol Lee, Nak-Jung Kwon, Won-Chul Lee, Hae Mi Kim, Won Jin Jang, Yun Ji Choi, Kyung Hwa Park, Yeul Hong Kim. Comparison and evaluation of somatic mutation using PGM and proton platform in cell free DNA of non-small cell lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3614.
Abstract 362: A homogeneous bioluminescent succinate assay for detection of jumonjiC domain-containing histone demethylase activities
Authors: Alves Juliano , Goueli Said , Zegzouti Hicham
Published: July 2016

JumonjiC domain-containing histone lysine demethylases (JMJCs) play a pivotal role in determining the epigenetic status of the genome, leading to either transcriptional repression or activation of target genes by counteracting the activities of histone lysine methyltransferases. Because of their implication in cancer, they have become validated drug targets. Therefore, assays for this enzyme subfamily are desirable in order to facilitate the identification of selective and potent inhibitors for drug discovery and as basic research tools. Since succinate is one of the products of the demethylation reaction catalyzed by these enzymes, an assay that detects succinate would be suitable for monitoring all JMJC activities as well as other succinate-forming enzymes (i.e.: dioxygenases). Thus, a bioluminescent and homogenous succinate detection assay for measuring JMJC activity was developed, and it is performed in a two-step format that relies on converting the succinate product to ATP, and then to light in a robust luciferase reaction. The light output is proportional to succinate concentration from low nM to 15μM, and the assay is highly sensitive and robust, two features that are highly desirable and essential for measuring the activity of the majority of JMJC demethylase subfamilies. Therefore, the succinate detection assay is a simple-to-use method that does not require antibodies or modified substrates. Examples of various applications of this succinate detection assay will be presented, including studies on specificity of different substrates by diverse JMJCs, as well as mode of action studies using specific inhibitors. The development of this succinate detection assay will make it possible to investigate a large number of JMJC demethylases and could have significant impact on diverse areas of Epigenetics research.

Citation Format: Juliano Alves, Said Goueli, Hicham Zegzouti. A homogeneous bioluminescent succinate assay for detection of jumonjiC domain-containing histone demethylase activities. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 362.
Abstract 3625: Beyond SNPs: Utilizing NGS methods to detect copy number alterations at high sensitivity in clinical cancer samples
Authors: Raghunath Sharanya , Wayne Jackie , Shamo Aimee , Bradley Patrick , Abanto Moises Hernandez , Loughmiller David , Gillman Jason , Haslem Derrick S. , Stone Gary , Mishra Pravin , Nadauld Lincoln D.
Published: July 2016

The emergence of novel technologies, including Next-Generation Sequencing, has enabled the identification of actionable variants in clinical specimens, which can then be targeted with molecular therapeutics in a precision medicine approach. In human cancers, Single Nucleotide Polymorphisms (SNPs) have become powerful predictors of response to certain targeted therapies. However, Copy Number Alterations (CNAs) also represent a major class of clinically actionable variants that are frequently identified in clinical cancer samples. CNAs account for more than one third of all actionable mutations. A major question in the clinical application of technologies such as NGS is whether CNAs are faithfully detected; a particularly important question given the prevalence of actionable CNAs in cancer samples. To address this question we prepared tumor samples from eleven distinct patients and compared CNA detection on two different NGS platforms. As an orthogonal method for validation, we compared the CNA detection results to Fluorescent In Situ Hybridization (FISH) performed on the same samples. We observed 20 copy number amplification events across the eleven patient samples according to FISH analysis. By comparison, our internal NGS assay, termed ICG100, detected 19/20 (94%) amplification events, whereas an external, commercially available NGS-based test detected only 12/20 (59%) amplification events. Of the seven amplification events that were not identified by the external NGS test, six were considered clinically actionable variants. These findings highlight the discrepancies that exist amongst NGS library preparation chemistries and their abilities to accurately detect CNAs. The relevance of these findings is particularly underscored by the clinically actionable nature of the variants that were discordantly detected, including a high-level ERBB2 amplification.

Citation Format: Sharanya Raghunath, Jackie Wayne, Aimee Shamo, Patrick Bradley, Moises Hernandez Abanto, David Loughmiller, Jason Gillman, Derrick S. Haslem, Gary Stone, Pravin Mishra, Lincoln D. Nadauld. Beyond SNPs: Utilizing NGS methods to detect copy number alterations at high sensitivity in clinical cancer samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3625.
Abstract 3627: Automated, purification-free, Ion AmpliSeq™ library preparation from FFPE samples using the Ion Torrent™ Ion Chef™ System
Authors: Biorac Tanya , Allen Michael , Peters Francis B. , Van Loy Cristina , Rhodes Kate , Andersen Mark
Published: July 2016

The ability to produce high quality libraries from FFPE samples is ever more important for next-generation sequencing. Quick turnaround time from sample to answer and simple protocols are critical components for this process. The Ion Chef and Ion AmpliSeq Kit for Chef DL8 is an automated system that has proven to reliably generate quality libraries from purified high molecular weight genomic DNA, in addition to formalin-fixed paraffin embedded (FFPE) DNA samples. We have further developed a simple and robust protocol for preparing DNA libraries from FFPE samples with no nucleic acid purification required. Our protocol is compatible with all Ion AmpliSeq panels and generates eight balanced libraries on the Ion Chef Instrument in under 8 hours, with less than 1 hour hands-on time. We observed equivalent or better sequencing performance using this method compared to a 16-hour manual FFPE DNA extraction protocol. This method has shown to work with low, medium, and high input FFPE samples from different tissue types on a single run. We evaluated a variety of FFPE samples using 1 or 2 pool Ion AmpliSeq DNA research panels containing assays designed to known oncogenes. We were able to generate and successfully sequence DNA libraries from a single FFPE slice as small as 2 mm X 2 mm. Minimal sample handling is an advantage for the NGS platform with this robust protocol.

For Research Use Only. Not for use in diagnostic procedures.

Citation Format: Tanya Biorac, Michael Allen, Francis B. Peters, Cristina Van Loy, Kate Rhodes, Mark Andersen. Automated, purification-free, Ion AmpliSeq™ library preparation from FFPE samples using the Ion Torrent™ Ion Chef™ System. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3627.
Abstract 3632: Adaptive operations and technology platform for nation-scale precision oncology
Authors: Abaan Ogan , Basu Amrita , Deal David , Hultner Michael
Published: July 2016

Precision oncology requires predictive models for therapy selection using variety of biomarkers and clinical features as input. Building and validating these models requires analysis of large numbers of diverse cases in order to relate markers and treatments to positive outcomes. The -omics technologies provide a rich source of genetic and epigenetic markers but demand large compute and storage systems to process the data. Thus, there is an urgent need for scalable and reliable information systems to support nation-scale research and delivery of precision oncology.

At Lockheed Martin, we deliver operational solutions to complex problems. Here, we present our vision for a precision oncology platform. This solution integrates best-in-class capabilities from multiple sources/vendors to support innovation, research and clinical care for a whole nation. We not only thought about the basic -omics based data collection, but also an infrastructure to collect and store data within a compliant privacy and security framework that also facilitates collaborative analytics and data sharing for deeper insight.

Taking a systems engineering approach, we have examined some of the challenges to implement such a platform. For instance, running the basic genomic data processing pipelines to yield variant calls, which in turn will feed the variant store, should be a single scalable workflow. Accounting for multiple data sources, various use cases and selections of tools are at the core of an adaptable workflow. A variant store design that can scale and support a national cohort with an overlaying cohort selection tool are all part of this intricate design.

It is our vision that a systems engineering and integration approach can deliver a unified solution for the national precision oncology roadmap. It is paramount that all the individual pieces should be well tuned to deliver scalability and reliability and simultaneously work in complete harmony. Only then we can process data at-scale needed for finding actionable mutations, designing effective treatments and implementing prevention strategies, affordably and reliably.

Citation Format: Ogan Abaan, Amrita Basu, David Deal, Michael Hultner. Adaptive operations and technology platform for nation-scale precision oncology. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3632.
Abstract 3648: Resolving copy number variations from existing exome SNP data without the need for sophisticated software
Authors: Hansen Marcus C. , Herborg Laura L. , Roug Anne S. , Chotirat Sadudee , Auewarakul Chirayu U. , Kjeldsen Eigil , Hokland Peter
Published: July 2016

Several tools exists to evaluate copy-number variation (CNV) from sequencing read data. Here, the aim is to investigate whether larger CNVs in leukemia can be extracted directly from already called variants - without specialized algorithms.

Methods: 12 cases of leukemia, with 6 paired and 6 unpaired, were included (Table 1). Following exome sequencing and reference alignment variants were called with GATK to produce recalibrated sets in variant call format (VCF). Passed single nucleotide polymorphisms (SNPs) were limited to depth of coverage > 29x. Each chromosome were visualized as frequency scatter plot and smoothened histograms of frequency distribution in order to inspect frequency bands and number of peaks in histograms. Kolmogorov-Smirnov test was employed to compare distributions between paired samples.

Results: None of the AML cases with normal karyotype displayed CNVs and no significant changes in frequency distributions were detected. In the relapse of paired T-ALL a Chr 4 trisomy and a 33×10−6 bp copy-number neutral loss of heterozygosity (CN-LOH) on Chr 9 were detected. Collectively, 7 aberrations confirmed standard cytogenetics, 8 new aberrations were added to the cytogenetical profile and 5 could not be detected as shown in Table 1. The aberrations not detected were, as expected, balanced translocations (3) and microdeletions (2).

Discussion: No previously found large aberrations escaped detection, when disregarding translocations. In addition, other CNVs not detected by conventional cytogenetics were found by SNP analysis. Moreover, the added information is in agreement with the literature: 5% of T-ALL patients display del(11)(p12p13) and +12 is one of the most common cytogenetic changes of CLL, e.g.

Thus, we suggest that CNV profiling can be initiated by directly evaluating frequencies from existing variant calls. This may ease implementation of “closed toolboxes”, enable analysis of single unpaired samples and function as a direct complement to array-CGH or FISH to detect CN-LOH. Table 1SamplesPairedCytogeneticsCNV detectedNK-AML5YesNormal karyotypeNoneT-ALL relapse1Yes4+, 3p26.2 and Xp22.2 micro-deletions+4, partial del(9p)AML M01YesNormal karyotypePartial del(11p)AML M31Not(15;17)NoneCML1Not(9;22)NoneT-ALLNodel(5) t(7;14)Partial del(5q), +4q, +9p, del(11p), del(12q)CLL1NoNot analyzed+12AML M01NoNormal karyotypePartial +11

Citation Format: Marcus C. Hansen, Laura L. Herborg, Anne S. Roug, Sadudee Chotirat, Chirayu U. Auewarakul, Eigil Kjeldsen, Peter Hokland. Resolving copy number variations from existing exome SNP data without the need for sophisticated software. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3648.
Abstract 3649: Targeted sequencing of 409 cancer-related genes for somatic mutations and copy number variations in human cancer using the semiconductor sequencers
Authors: Sasaki Yasushi , Koyama Ryota , Nakagaki Takafumi , Tamura Miyuki , Idogawa Masashi , Tokino Takashi
Published: July 2016

Next-generation sequencing technologies have revolutionized cancer genomics research by providing a comprehensive method of detecting somatic cancer genome alterations. In this study, we describe a next-generation semiconductor sequencing protocol for rapid (2 days), standardized, and cost-effective gene analysis for human cancer specimens including FFPE samples. DNA was extracted from 19 human cancer cell lines and 61 human cancer specimens and their corresponding non-cancerous tissues, including oral squamous cell carcinomas and multiple myelomas. Using the Ion Ampliseq Comprehensive Cancer Panel, we sequenced 15992 loci from 409 tumor suppressor genes and oncogenes frequently cited and frequently mutated in human cancers (covered regions = 95.4% of total). Each sample underwent on average 8.3 million sequencing reads after quality filtering. The mean read depths were 461x, and >95% of targeted bases were represented by at least 20 reads. We also detected copy number variations in which segments of the genome can be duplicated or deleted from sequencing data. We found several genetic alterations that may have been associated with the poor prognosis and poor response to chemotherapy of cancer patients. Pathway assessment has shown that somatic aberrations within myeloma genomes are mainly involved in several important pathways, including cell cycle regulation, RTK-MAPK-PI3K and NF-kB. This study demonstrates the utility of using a semiconductor-based sequencing to efficiently identify human cancer mutations. The targeted next generation sequencing using low amounts of FFPE DNA is a valuable tool for high-throughput genetic testing in research and clinical settings.

Citation Format: Yasushi Sasaki, Ryota Koyama, Takafumi Nakagaki, Miyuki Tamura, Masashi Idogawa, Takashi Tokino. Targeted sequencing of 409 cancer-related genes for somatic mutations and copy number variations in human cancer using the semiconductor sequencers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3649.
Abstract 3652: Identification of cancer-specific signaling networks: what is “normal” control
Authors: Anjanappa Manjushree , Hao Yangyang , Edenberg Howard J. , Liu Yunlong , Nakshatri Harikrishna
Published: July 2016

Success of precision medicine depends on definitive identification of cancer-specific alterations in signaling pathways. However, identifying cancer-specific signaling networks is challenging because of lack of proper control tissue for differential gene expression analyses. Most studies in breast cancer utilize tumor-adjacent normal tissue or reduction mammoplasty samples as “normal” controls. We recently reported that breast epithelial cells from healthy donors as well as tumor-adjacent normal are in different differentiation states compared with tumor cells and the differences in differentiation status alone could account for major transcriptome variations between normal and tumor. To overcome these limitations, we propagated breast epithelial cells from three healthy donors (healthy-normal), two high-risk patients, two tumor-adjacent normal (HR/AD-normal) and five tumor samples of different molecular subtypes. Phenotypically defined (CD49f+/EpCAM+) luminal progenitor cells were sorted from these cultures and subjected to RNA-seq analyses. Pathway analysis revealed activation of cell-intrinsic pro-inflammatory signaling in HR/AD-normal cells compared with healthy-normal cells. This signaling network was further amplified in tumor cells. The pro-inflammatory chemokine CCL2, which is overexpressed in highly aggressive breast cancer, and the cytokine TNFRSF11B were elevated in HR/AD-normal luminal progenitor cells. Despite using phenotypically defined cells in the transcriptome analyses, cancer-specific signaling network identification was directly influenced by the type of controls used; healthy-normal or HR/AD-normal. While cancer-enriched PI3K and NF-κB activation was observed when compared to any kind of control, SRC kinase activation was noted only when cells from healthy-normal were used as a control. In general, the number of tumor signaling networks identified using healthy-normal as a control was higher than when compared with HR/AD-normal as a control. These results suggest that considerable attention should be placed on the type of tissues used as control for definitive identification of cancer-specific signaling networks and therapies to target such pathways. Additionally, these data show that non-cancer tissues of breast cancer patients acquire a cell intrinsic pro-inflammatory phenotype, which may be prerequisite for cancer development and potentially an early-detection tool.

Citation Format: Manjushree Anjanappa, Yangyang Hao, Howard J. Edenberg, Yunlong Liu, Harikrishna Nakshatri. Identification of cancer-specific signaling networks: what is “normal” control. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3652.
Abstract 3655: Suppression of Plk3 expression by nickel may contribute to lung carcinogenesis
Authors: Li Cen , Park Soyoung , Dai Wei , Xu Dazhong
Published: July 2016

Exposure to nickel (Ni) compounds is known to induce human lung malignancies. Research shows that Ni induces lung cancer mainly through epigenetic and metabolic pathways. Polo-like kinase 3 (Plk3) is a multifunctional protein kinase that involves in tumor initiation and progression. Plk3−/− mice tend to develop highly vascularized lung tumors at an advanced age. Reduced expression of Plk3 was reported in human lung cancers. In this study, we examined the effect of Ni on Plk3 expression in A549 lung carcinoma cells. Western blot showed that NiCl2 reduced the Plk3 protein level in a time- and dose-dependent manner. We then treated A549 cells with cycloheximide for various times to examine the effect of NiCl2 on the half-life of Plk3 protein. We observed a shortened half-life of Plk3 in the presence of NiCl2. A reduced level of Plk3 protein was also found in hypoxia-treated A549 cells. In addition, MG132, a specific proteasome inhibitor, could partially reverse the effect of NiCl2 or hypoxia on the Plk3 protein level. Our cell fractionation experiments showed that majority of the endogenous Plk3 protein is located in the cytoplasm where degradation of Plk3 likely occurs. Transfection of A549 cells with FLAG-tagged ubiquitin (FLAG-Ub) followed by treatment with NiCl2 and/or MG132, immunoprecipitation (IP) with an anti-FLAG antibody, and Western blot with an anti-Plk3 antibody showed that Plk3 was poly-ubiquitinated and that the ubiquitination was significantly enhanced by NiCl2 and/or MG132 treatments. The elevated ubiquitination of Plk3 in response to NiCl2 was further confirmed in 293T cells with co-transfection of Plk3 and FLAG-Ub. Furthermore, overexpression of USP28, a deubiquitinase known to be inhibited by Ni or hypoxia, partially restored the protein level of Plk3 suppressed by NiCl2. Moreover, real-time PCR analysis showed that NiCl2 also reduced the Plk3 mRNA level, in a time- and dose-dependent pattern. Taken together, our data show that NiCl2 suppresses Plk3 expressions by lowering the Plk3 mRNA level and promoting proteasome-mediated degradation of the Plk3 protein. Given the potential role of Plk3 as a tumor suppressor in the lung, suppression of Plk3 expression likely contributes to Ni-induced lung carcinogenesis and tumor progression.

Citation Format: Cen Li, Soyoung Park, Wei Dai, Dazhong Xu. Suppression of Plk3 expression by nickel may contribute to lung carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3655.
Abstract 3658: A novel microtubule associated RNA binding protein matrin 3 act as a tumor suppressor by regulating mitotic spindle organizing proteins in triple negative breast cancers
Authors: Subbarayalu Panneerdoss , Rajamanickam Subapriya , Viswanadhapalli Suryavathi , Onyeagucha Benjamin C. , Eedunuri Vijay K. , Dybdal-Hargreaves Nicholas , Timilsina Santosh , Bansal Hima , Bansal Sanjay , Mohammad Tabrez , Chen Yidong , Herr John C. , Mooberry Susan L. , Rao Manjeet K.
Published: July 2016

Microtubule-targeting drugs are widely used as cancer chemotherapeutic agents and they have been shown to inhibit mitotic progression and interphase signaling. Despite their use as a first line treatment for cancer, many patients develop resistance to microtubule-targeting drugs leading to early relapse and shorter survival. Moreover, the quality of life for patients who do survive is often substantially reduced due to the toxicity associated with these drugs. Therefore, identification of new factors that determine the effectiveness of microtubule-targeting agents will not only facilitate a better understanding of the mechanisms of acquired drug resistance but will also be amenable to therapeutic interventions. We discovered a novel microtubule associated protein “Matrin 3 (MATR3)” that is known to bind to RNA and play a critical role in RNA transport and RNA stabilization. Our results revealed that MATR3 acts as a potent tumor suppressor as it inhibits long-term growth, migration, invasion as well as tumor growth of triple negative breast cancer (TNBC) cells in vivo. We demonstrated that MATR3 overexpression induced cell cycle arrest and apoptosis in TNBC cells. Furthermore, analysis of breast cancer samples showed a significantly lower expression of MATR3 when compared to normal adjacent tissues. Importantly, our RNA immunoprecipitation (RIP)-seq analysis showed that MATR3 controls expression of several mitotic spindle organizing proteins by binding to their RNA. Interestingly, we found that these MATR3 regulated proteins were highly altered in breast cancer patients. In conclusion, we identified a novel RNA-binding protein that inhibits breast cancer growth and progression suppressor by regulating microtubule dynamics.

Citation Format: Panneerdoss Subbarayalu, Subapriya Rajamanickam, Suryavathi Viswanadhapalli, Benjamin C. Onyeagucha, Vijay K. Eedunuri, Nicholas Dybdal-Hargreaves, Santosh Timilsina, Hima Bansal, Sanjay Bansal, Tabrez Mohammad, Yidong Chen, John C. Herr, Susan L. Mooberry, Manjeet K. Rao. A novel microtubule associated RNA binding protein matrin 3 act as a tumor suppressor by regulating mitotic spindle organizing proteins in triple negative breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3658.