bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2474: Automated somatic variant classifier to reduce false positives identified by tumor normal variant callers
Authors: Harley Alena S. , Lau Corine K. , Shinbrot Eve
Published: August 2019

Accurate identification of somatic mutations is key to targeted cancer therapies. However, due to the complexity of next-generation sequencing, there are many variables that give rise to sequencing and alignment artifacts. Most sequencing workflows using tumor normal paired samples rely on the intersection or union of mutations by more than one somatic variant caller such as Mutect2, Strelka, VarScan2, Muse, and SomaticSniper. Yet, they still contain a number of false positive and false negative calls. Therefore, there is a need for a manual review of variants using Integrative Genomics Viewer (IGV) to incorporate information that is difficult to take into account in automated workflows.

To alleviate the burden of manual inspection, we present an automated classification method to pre classify detected somatic variants into true or false classes for further manual review.

Methods: We created a false positive classifier that utilizes automated IGV visualization of somatic variants. Our somatic workflow involves tumor normal DNA sequencing, BWAMEM alignment, deduplication of reads via Picard, somatic calling using two somatic callers, followed by the union of the detected variants, SnpEff annotation, and extracting nonsilent variants with variant allele fractions greater than 5 percent. We used the data for 1,413 nonsilent variants from a set of 10 tumor normal pairs of various cancer types. The variants were scored by cancer analysts using IGV. Initial false positive rate was 28 percent. We fine tuned Residual Deep Neural Network which utilizes weights from the network with the same architecture trained on ImageNet. Discriminative fine tuning was performed in two stages: first unfreezing the last fully connected layer only, and then unfreezing all the layers and training with differential learning rates.

Results and Conclusion: Our method achieves 93 percent overall accuracy. The classifier greatly reduces the false positive rate for the set of variant calls from 28 percent down to 4 percent at the expense of calling 3 percent of true variants false. Previously, deep neural networks have been shown to yield the state of the art performance in multiple domains including image processing, speech, and text processing. In this work, we describe a way to fine tune Residual Neural Network trained on a large image data set to perform false positive variant detection in tumor-normal variant alignment snapshots from IGV. Our general approach that significantly reduces false positive rate of putative variants can be extended to any subset of somatic callers.

Citation Format: Alena S. Harley, Corine K. Lau, Eve Shinbrot. Automated somatic variant classifier to reduce false positives identified by tumor normal variant callers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2474.
Abstract 2485: Somatic analysis services with best practice workflows in a cloud-based platform
Authors: Tsuji Junko , Hollinger Andrew , MacBeth Alyssa , Grander Brian R. , Rickles-Young Micah , Bowers Tera , Cibulskis Carrie , Lennon Niall
Published: August 2019

With advances in high-throughput sequencing technologies and analytical tools, genomic analysis of tumors has led to the identification of various important somatic mutations that shed light on diagnosis, prevention, and treatment for cancer. However, detecting somatic variants is not a trivial task in terms of the technical aspects (e.g. filtering germline events and removing a variety of noises in tumor samples) and computational resources to handle large-scale cohort analysis. There is also a demand for maintaining stable software versions and the workflows for studies over extended periods of time, that need consistency and traceability, such as clinical trials. We introduce here, the Translational Analysis Group (TAG), a team which deploys, validates, and conducts scalable analytical workflows in a secure, cloud-based environment. We maintain 29 well-tested workflows with best practice methods and ample resources for both somatic and germline analysis. Our cloud platform, FireCloud, enables us to run workflows at any scale. Since May 2017, our team has performed nearly 10,000 analyses for mutation detection (SNV, InDel, CNV, and SV) and cohort analysis on tumor samples and cell-free DNA samples. TAG offers a range of options for somatic and germline variant detection, from legacy pipelines through recently validated contemporary pipelines to allow for continuity across long running projects.

Citation Format: Junko Tsuji, Andrew Hollinger, Alyssa MacBeth, Brian R. Grander, Micah Rickles-Young, Tera Bowers, Carrie Cibulskis, Niall Lennon. Somatic analysis services with best practice workflows in a cloud-based platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2485.
Abstract 2487: Application of RegTools to TCGA samples for the identification of tumor-specific splice variants
Authors: Cotto Kelsy C. , Feng Yang-Yang , Skidmore Zachary L. , Griffith Obi L. , Griffith Malachi
Published: August 2019

The interpretation of variants in cancer is frequently focused on direct protein coding alterations. However, this analysis strategy excludes somatic mutations in non-coding regions of the genome and even exonic mutations may have unidentified non-coding consequences. To address this issue, we created RegTools (Feng et al. 2018), a free, open-source software package that integrates analysis of somatic variant calls from genomic data with splice junctions extracted from transcriptomic data in order to efficiently identify variants that may cause aberrant splicing in tumors. Initially, this software was developed using 188 cancer samples from 4 cancer types and enabled us to identify important non-coding variants which would otherwise be undervalued or perhaps even completely ignored by traditional methods and annotators. Here, we present the application of RegTools to over 10,000 TCGA samples in an effort to identify somatic variants that are associated with alternative splicing patterns within these tumors. In order to perform this analysis, we obtained all tumor samples that had somatic variant VCF files and RNA-seq bam files from the Genomic Data Commons (GDC). Included within these samples are more than 700 solid tissue normal samples, which will be used as a panel of normals in order to filter out variants and splicing events that are not tumor specific. We plan to present on the significance of our findings when analyzing these samples within their respective tumor type and across all tumor types. We also plan to present on whether these aberrant splicing events that result from somatic mutations might interfere with predicted neoantigens pVAC-Seq (Hundal et al. 2016). RegTools is freely available and open source (www.regtools.org).

Citation Format: Kelsy C. Cotto, Yang-Yang Feng, Zachary L. Skidmore, Obi L. Griffith, Malachi Griffith. Application of RegTools to TCGA samples for the identification of tumor-specific splice variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2487.
Abstract 2490: R2: Genomics analysis and visualization platform
Authors: Koster Jan , Volckmann Richard , Zwijnenburg Danny , Molenaar Piet , Versteeg Rogier
Published: August 2019

Given the complexity, omics data like RNA-seq are often analyzed by bioinformaticians, and not the wetlab researchers that performed the experiments. With the biomedical researcher (with limited or no bioinformatics skills) in mind as the end-user, we developed the user-friendly, publicly accessible R2 platform (http://r2.amc.nl), enabling biomedical scientists to work with their own data from anywhere at any time. R2 (cited in >850 peer-reviewed scientific publications) consists of an omics database, coupled to an extensive set of interactive tools to analyze/visualize the datasets. Interactive analyses within the software are highly connected, allowing quick navigation between various aspects of the datamining process. Besides password protected private data, R2 also contains an extensive body of publicly available omics data, enabling users to relate their findings to other diseases / tissues, or as validation in integrative analyses. Next to gene expression (bulk as well as single cell), R2 is also being employed for the integration, analysis and visualization of copynumber, SNP, methylation, miRNA, drug response, ChIP-seq, and NGS exome/WGS DNA sequencing data. Analyses include correlation, differential expression, gene sets, gene ontology, transcription factor binding sites, tSNE, PCA, k-means, Kaplan Meier survival scans, signature creation etc. Visualizations include various gene oriented plots, heatmaps, Circos plots, embedded genome browser, Venn diagrams, etc. The real power of the platform lies in the chaining of results of analyses; e.g. the results of a differential expression analysis can be further trimmed down using a Kaplan Meier analysis that in turn can be used for a Gene Ontology over-representation analysis. Furthermore, the webserver allows for overviews across different datasets (such as MegaSampler and 2D distribution), where a user can harness the power of thousands of measurements. Recently, integrated analyses for personalized medicine, where copy number, mRNA expression, methylation and mutation data are combined into a comprehensive overview, occasionally supplemented with PDX/organoid drug response data. These serve clinical decision making for a patient, but as growing cohorts also form a treasure trove for scientific discovery. R2 provides a central starting point from where data mining and analysis paths can be followed within one environment. Data/analyses can be shared between R2-users via our community options, making R2 an outstanding environment for discovery, hypothesis testing and scientific collaboration.

Citation Format: Jan Koster, Richard Volckmann, Danny Zwijnenburg, Piet Molenaar, Rogier Versteeg. R2: Genomics analysis and visualization platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2490.
Abstract 252: Tumor treating fields (TTFields) affect blood brain barrier (BBB) integrity in vitro and in vivo
Authors: Kessler Almuth F. , Schaeffer Clara M. , Burek Malgorzata , Ruschig Ursula , Tempel-Brami Catherine , Voloshin Tali , Giladi Moshe , Salvador Ellaine , Ernestus Ralf-Ingo , Löhr Mario , Förster Carola , Hagemann Carsten
Published: August 2019

Background: The blood-brain barrier (BBB) may impede application of drugs to the brain for treatment of malignant brain tumors, in particular glioblastoma multiforme (GBM). Alternating electric fields with intermediate frequency and low intensity, called Tumor Treating Fields (TTFields), are an established novel adjuvant treatment modality for GBM. Here, the effect of TTFields on BBB permeability is analyzed.

Material and Methods: After TTFields treatment with a frequency of 100-300 kHz for up to 72 h, immortalized murine brain capillary endothelial cells (cerebEND) grown on cover slips and transwell inserts were stained for immunofluorescent assessment of the tight junction proteins Claudin-5 and ZO-1. Transendothelial electrical resistance (TEER) was applied to investigate BBB integrity. Moreover, BBB permeability was determined by fluorescein isothiocyanate (FITC) staining followed by flow cytometry. For in vivo analysis, the increase in vessel permeability was quantified by utilizing i.v. injected Evans Blue (EB) in rats during TTFields application to the brain (100 kHz, 72 h).

Results: The BBB was disturbed by treatment with TTFields as tight junction proteins were delocalized from the cell boundaries to the cytoplasm with maximal effects at 100 kHz. TTFields application significantly reduced the BBB integrity by 65% and significantly increased the BBB permeability for 4 kDa large molecules. Initial recovery of the cell morphology was observed 48 h post-treatment and a complete recovery could be detected after 96 h, indicating a reversibility of the TTFields effect on the BBB. Average accumulation of EB in the rat brain was significantly increased by TTFields application to the rats head.

Conclusion: In the future, TTFields could be utilized to deliver drugs generally unable to cross the BBB to the central nervous system as TTFields at a frequency of 100 kHz are potentially able to disrupt the BBB. The data presented on in vitro and in vivo application of TTFields to permeabilize the BBB may be a rationale for a phase I clinical trial and clinical application in the future.

Citation Format: Almuth F. Kessler, Clara M. Schaeffer, Malgorzata Burek, Ursula Ruschig, Catherine Tempel-Brami, Tali Voloshin, Moshe Giladi, Ellaine Salvador, Ralf-Ingo Ernestus, Mario Löhr, Carola Förster, Carsten Hagemann. Tumor treating fields (TTFields) affect blood brain barrier (BBB) integrity in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 252.
Abstract 253: Protein-tyrosine phosphatase 1B (PTP1B) as a therapeutic target in glioblastoma
Authors: Bartolomé Ruben , Jaén Marta , Burdiel Miranda , Balyasnikova Irina V. , Casal Ignacio
Published: August 2019

New targeted therapies are urgently needed in glioblastoma (GBM). IL13Rα2 receptor is considered a highly specific receptor for GBM cells and has been proposed for therapeutic purposes. Here, we aimed to identify the protein interaction network of IL13Rα2 and its mediators in GBM searching for novel associated therapeutic targets.

We investigated the IL13Rα2 protein interaction network in U251 GBM cells using immunoprecipitation followed by mass spectrometry. Different cell lines were used for validation. Two glioblastoma cell lines were used for testing proliferation, adhesion, migration and invasion. Claramine, a PTP1B inhibitor, was tested to study inhibition of GBM cells growth in cultured cells and in animal experiments. In vivo experiments with NSG mice were performed after treatment with PTP1B inhibitors.

Using a proteomic approach, we identified the association of the PTP1B phosphatase with IL13Rα2 in U251 GBM cells. Although GBM cell lines were positive for PTP1B, the highest PTP1B expression was found in GBM clinical samples. Moreover, “in silico” dataset analysis showed PTP1B association with poor outcome in GBM patients. PTP1B silencing inhibited the IL-13-promoted activation of Src (Tyr419), AKT and ERK1/2 in all the cell lines tested causing a significant effect on IL-13-mediated migration and invasion, and proliferation at a lower extent, of GBM cells. Therefore, PTP1B-silencing reverted most of the pro-metastatic capacities of the IL-13/ IL13Rα2 signaling axis. Further immunoprecipitation of PTP1B showed its association with a large number of NF-κB and NOTCH-related proteins in GBM cells. Both pathways are highly relevant for cancer proliferation and invasion. Claramine, a selective inhibitor of PTP1B, caused similar results to PTP1B silencing, inhibiting the dephosphorylation of Src Tyr527, migration and invasion in GBM. Moreover, treatment with Claramine caused a complete regression of GBM xenografts in NSG mice and total inhibition of liver metastatic growth in nude mice inoculated with colorectal cancer cells.

In conclusion, our results provide new insights into the mechanisms underlying IL13Rα2-driven GBM aggressiveness after identifying PTP1B as a critical mediator of IL-13/IL13Rα2 signaling. A PTP1B inhibitor such as Claramine showed a great value as a therapeutic candidate in GBM. PTP1B inhibitors might be highly effective to prevent GBM progression and invasion.

Citation Format: Ruben Bartolomé, Marta Jaén, Miranda Burdiel, Irina V. Balyasnikova, Ignacio Casal. Protein-tyrosine phosphatase 1B (PTP1B) as a therapeutic target in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 253.
Abstract 2531: Genomic landscape of breast cancer patients in Korea: Early data from K-MASTER project
Authors: Sung Jae Sook , Chung Hee-Joon , Choi Yoon Ji , Chang Won Jin , Choi Jung Yoon , Kim Ju Won , Park Kyong Hwa , Kim Yeul Hong
Published: August 2019

The K-MASTER cancer precision medicine diagnosis and treatment enterprise (K-MASTER project) is a precision medicine cancer treatment clinical trial platform, which uses NGS assay to screen the actionable mutations in 10,000 patients with refractory solid tumors and assigns the patients to matched treatment. K-MASTER project was initiated in Korea since June 2017. From October 2017, we have analyzed next-generation (NGS)-based cancer panel to detect cancer genomic variations in breast cancer. This study is ongoing with 36 sites. Tumor DNA was analyzed by CancerSCAN (Samsung Genomic Institute) which allows to detect variations of 375 genes. As of October 31 in 2018, total 164 breast cancer patients were submitted and 95 samples were analyzed. The failure rate of sequencing was 4.1% of patients with samples submitted. The mean depth of CancerSCAN was 701x. Average turn-around time (TAT) of CancerSCAN was 3 weeks.In Korean breast cancer patients, variations were mainly identified in TP53 (63.6%), PIK3CA (35.2%), BRCA1 (25.0%), BRCA2 (23.9%), ERBB2 (23.9%), PTCH1 (21.6%), NOTCH1 (19.3%), FGFR1 (12.5%), PTEN (11.4%) and MET (9.1%). Further analysis will confirm amplification and deletion in breast cancer. Until March 2019, we will finish the genomic profiling of 350 breast cancer patients and will be reported. In conclusion, this project is efficient to detect actionable variations in breast cancer. This novel knowledge provides an intriguing background to investigate new target approaches and to progress precision medicine. (This research was supported by a grant of 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 (Grant number: HI17C2206).)

Citation Format: Jae Sook Sung, Hee-Joon Chung, Yoon Ji Choi, Won Jin Chang, Jung Yoon Choi, Ju Won Kim, Kyong Hwa Park, Yeul Hong Kim. Genomic landscape of breast cancer patients in Korea: Early data from K-MASTER project [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2531.
Abstract 2533: The genomic and transcriptomic landscape of left versus right sided breast cancer in 410 cases
Authors: Newton Yulia , Szeto Christopher , Vaske Charles , Reddy Leia , Reddy Sandeep
Published: August 2019

Background: Studies have shown that unilateral breast cancer is more common on the left (L) than right (R) and that the L breast is often larger than the R. No clear explanation for this finding has been described in the literature. Hypotheses range from complex genomic effects to environmental, hormonal, and anatomical causes.

Methods: We performed a retrospective analysis of data from the NantHealth database of 410 commercial cases where WGS (35x germline, 75x somatic) or WES (∼150x). Primary and metastatic breast tumors were included in the analysis. Of these 410 cases, 39.3% were R, 38.5% were L, and 22% were unspecified. Bases adjacent to variant sites were used to determine genomic context (i.e. mutation signatures) as previously reported. Additionally, paired deep whole transcriptomic sequencing (RNA-Seq; ∼200x106 reads per tumor) was performed on 376 of the 410 cases. Nant Breast Cancer Intrinsic Subtypes sorting based on RNAseq assay classified breast tumors into 4 subtypes (LumA/B, Basal-like, and HER2-enriched).

Results: Luminal A subtype was significantly overrepresented in R v L (OR=0.53, p=0.015). Similarly, R-side cases were significantly enriched for estrogen response gene expression by GSEA. However, only slight differential expression of ESR1 and MKI67 was observed specifically in individuals between 40 and 50 years old. Germline variants in the following genes were associated with sidedness: MEN1, KIT, JAK2, TET2, EP300, MET, FLCN, and KMT2D, and somatic variants within the following genes were associated with sidedness: CACNA1E, AGRN, RIMS2, TTYH3, KRTAP10-6, and DDX60L. However, significance of the variant frequency differences was lost after multiple-hypothesis correction. No significant differences between L and R were noted in tumor stage, HER2, ER, PR, somatic CNV, and mutation signatures, including signature 3 (BRCAness).

Conclusions: Despite performing WGS/WES a paucity of differentially mutated genes were seen between left and right sided breast tumors. RNAseq analysis reveals that right sided breast cancers may be more likely to involve ESR1-signaling and be Luminal A subtype. Right-sided ESR1-signaling may be especially relevant in perimenopausal cases, however the current cohort is underpowered to achieve significance in this analysis.

Citation Format: Yulia Newton, Christopher Szeto, Charles Vaske, Leia Reddy, Sandeep Reddy. The genomic and transcriptomic landscape of left versus right sided breast cancer in 410 cases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2533.
Abstract 2538: p53 activation induces cell cycle arrest by promoting DREAM and RB repression of cell cycle genes
Authors: Schade Amy E. , Fischer Martin , DeCaprio James A.
Published: August 2019

p53 activation results cell cycle arrest at the G1/S checkpoint and the repression of early (G1/S) and late (G2/M) cell cycle gene expression in a p21 dependent manner. The mechanism how p53 and p21 repress cell cycle gene expression is unclear. p21 is known to repress CDK2 which is a key inhibitor of the pocket protein family members RB and p130. During a normal cell cycle, p130 as part of the DREAM complex (DP1, RB-like p130, E2F4, and MuvB) and RB cooperate to repress expression of early (G1/S) and late (G2/M) cell cycle gene expression. DREAM binds and represses late (G2/M) cell cycle promoters through MuvB binding and early (G1/S) cell cycle promoters though repressor E2F binding. RB is only able to bind and repress early (G1/S) cell cycle promoters through its interaction with activator E2Fs. The requirement for DREAM and RB for control of cell cycle gene expression after p53 activation was unknown. We hypothesized that DREAM and RB would cooperate to repress cell cycle gene expression after DNA damage. Using primary human foreskin fibroblast cells lacking RB and/or p130, we found repression of early and late cell cycle gene expression is differentially regulated after p53 activation. We found that while RB is required for repression of early (G1/S) cell cycle genes after p53 activation, DREAM is required for repression of expression of late (G2/M) cell cycle genes. Surprisingly, DREAM was unable to repress early cell cycle genes after p53 activation, suggesting that RB is the key regulator of G1/S gene expression during cellular stress like DNA damage. Further, the requirement for DREAM to repress late cell cycle gene expression underlies the importance of DREAM to prevent expression of mitosis inducing genes in a state like DNA damage when mitosis could result in propagation of mutated DNA. We found that p107, a protein reported be able to replace p130 in DREAM, does not potently repress early cell cycle genes in HFFs under contact arrest and serum starvation. However, we found that p107 strongly represses expression of late cell cycle genes during G0 in cells lacking p130 alone or in combination with RB. These data provide evidence for a specific role for p107-DREAM not previously appreciated. Together, our data show that control of cell cycle gene expression after p53 activation is split in two: RB represses early cell cycle genes while DREAM represses late cell cycle genes.

Citation Format: Amy E. Schade, Martin Fischer, James A. DeCaprio. p53 activation induces cell cycle arrest by promoting DREAM and RB repression of cell cycle genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2538.
Abstract 254: Discovery of dual inhibitors of NQO1 and GSTP1 for malignant glioblastoma treatment
Authors: Lei Kecheng , Luo Shilin , Jin Lingjing , Ye Keqiang
Published: August 2019

Glioblastoma (GBM) is a highly aggressive form of neoplasia due to its frequently overexpressed or mutant epidermal growth factor receptor (EGFR). NADPH quinone Oxydoreductase1 (NQO1) and glutathione-S-transferase Pi 1 (GSTP1) are commonly upregulated in GBM. NQO1 and GSTP1 are enzymes with dual roles that decrease the formation of reactive oxygen species (ROS), which mediates the oxidative stress and GBM cell proliferation. To search for a small molecule that blocks the malignant cancer proliferation, we conducted a high-throughput Screening (HTS) and identified a small molecule inhibitor (5-methyl-N-(nitro-1,3-thiazol-2-yl)-3-phenylisoxazole-4-carboxamide) as “HIT” that binds to both NQO1 and GSTP1 with high affinity and selectivity. The “HIT” blocks NQO1 and GSTP1 enzyme activity and mediates apoptosis in GBM via caspase activation. Inactivation of NQO1 and GSTP1 with siRNA or HIT resulted in imbalanced redox homeostasis, leading to mitigated cancer cell proliferation in vitro and in vivo. Thus, optimization of “HIT”, the dual NQO1 and GSTP1 inhibitor, may provide a novel strategy to antagonizing GBM.

Citation Format: Kecheng Lei, Shilin Luo, Lingjing Jin, Keqiang Ye. Discovery of dual inhibitors of NQO1 and GSTP1 for malignant glioblastoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 254.
Abstract 2544: Overexpression of the Polo-like kinase 4 (PLK4) contribute to tumor metastasis
Authors: Fung Yeung Sai , Ching Yick Pang
Published: August 2019

The Polo-like kinase 4 (PLK4) is an important mitotic kinase that lies at the head of centriole duplication pathway. Aberration of PLK4's level in cancer cell often results in supernumery centrosome. PLK4 transcripts was upregulated in a number of cancers including breast, colon gastric and lung cancer, and PLK4 overexpression had been linked to chemoresistance and cancer metastasis. In our preliminary data, knocking down PLK4 in 97L and HLE Hepatocellular Carcinoma (HCC) cell reduced their migration and growth rate. Overexpression of PLK4 in well-differentiated hepatoma HepG2 and Huh7 cell showed enhancement in migration. Centrinone B, a highly potent PLK4 inhibitor, inhibits HCC cells' colony-forming ability and wound healing rate. To gain the insight into the mechanism how PLK4 contribute to cell migration, microtubule regrowth assay was carried out. The results indicating that PLK4 overexpressing cell had a higher rate of microtubule nucleation, which could contribute to directional movement of the cell. Further supporting that PLK4 contribute to HCC progression, we look into the clinical samples. Using qRT-PCR, we found PLK4 transcript is upregulated in a local cohort of HCC samples (n=48), we also confirmed that PLK4 is upregulated in the TCGA and two GEO available datasets. Data from the cancer dependency map showed PLK4 is a common essential gene in a panel of HCC cell line. Suggesting targeted therapy against PLK4 might be useful in reducing tumor growth or metastasis.

Citation Format: Yeung Sai Fung, Yick Pang Ching. Overexpression of the Polo-like kinase 4 (PLK4) contribute to tumor metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2544.
Abstract 2545: Nek2 and other mitotic kinases are downstream of the E2F activators, and may modulate the epithelial-to-mesenchymal transition in breast cancer
Authors: Rivera-Rivera Yainyrette , Marina Mihaela , Saavedra Harold I.
Published: August 2019

The E2F transcription factors have been extensively studied in cancer since they are known for their role in the regulation of cell cycle progression, centrosome duplication, cell survival, and other functions. We recently demonstrated that the E2F activators (E2F1, E2F2, and E2F3) regulate mitosis through the elevated expression of multiple mitotic proteins including Sgo1, Nek2, Hec1, BubR1, and Mps1. We have shown that Mps1 triggers EMT through multiple mechanisms. NIMA-related kinase 2 (Nek2) is a centrosome-associated protein kinase particularly abundant in cells during the G2-M phase, which primary function is to phosphorylate centrosome linker proteins to cause centrosome disjunction and the formation of the bipolar mitotic spindle. Previous results from our lab showed that overexpression of Nek2 in HCC1954 (Her2+ breast cancer cell line) with a knock down for E2F3 rescued a high centrosome amplification pattern and enhances the formation of invasive protrusions. cBioportal analysis using the TCGA database showed Nek2 mRNA alterations in 9.1% of breast cancer patients and is predominantly present in tumors that are ER-, PR-, and Her2- (25%). Similarly, analysis using the METABRIC database showed Nek2 mRNA alterations in 6.1% of breast cancer patients and is predominantly present in tumors from basal (24%), Her2+ (10%), and luminal B (13.9%) subtypes. Based on these results, we hypothesize that Nek2 overexpression has a role in the epithelial-to-mesenchymal (EMT) pathway leading to cell migration and invasion as prelude to metastasis in breast cancer. To address this, we used MCF10A non-tumorigenic mammary cells with or without the overexpression of Nek2 (MCF10A/GFP and MCF10A/GFP-Nek2). Our preliminary data indicates that overexpression of GFP-Nek2 in mammary epithelial cells enhances levels of vimentin, whereas decreased the levels of E-cadherin, suggesting a role of Nek2 in a mesenchymal state. Likewise, siRNA-mediated silencing of Nek2 in mesenchymal breast cancer cells modulates markers of EMT. Because inhibitors are available for Nek2, it would be feasible to target this kinase to combat metastatic HR- breast cancers. Our work will allow the identification of mechanistic molecular pathways influenced by Nek2 role in driving invasion and metastasis to highlight this mitotic kinase as a potential prognostic marker for high-risk breast tumors.

Citation Format: Yainyrette Rivera-Rivera, Mihaela Marina, Harold I. Saavedra. Nek2 and other mitotic kinases are downstream of the E2F activators, and may modulate the epithelial-to-mesenchymal transition in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2545.
Abstract 2547: TERT promoter mutations in B viral humanhepatocarcinogenesis
Authors: Kim Young-Joo , Yoo Jeong Eun , Jeon Youngsic , Chong Jae Uk , Choi Gi Hong , Song Dae-Geun , Jung Sang Hoon , Oh Bong-Kyeong , Park Young Nyun
Published: August 2019

Telomerase reverse transcriptase (TERT) promoter somatic mutations, related to telomerase activation, have been known to frequently occur at two hot spots located at -124 and -146 bp relative to the start codon in various cancers. In the present study, we investigated the occurrence and implications of the genetic alterations of the TERT promoter in B viral hepatocarcinogenesis. TERT promoter mutations, especially -124C>T mutation, obviously enhanced TERT promoter activity in hepatocellular carcinoma (HCC) cell lines. We identified prospero homeobox protein 1 (PROX1) as a novel transcriptional activator for TERT gene through binding to the promoter regions containing two hot spots. PROX1 binding affinity was strong to the mutant TERT promoter harboring a consensus E-twenty six/ ternary complex factor (ETS/TCF) binding sequence (CCGGAA). The incidence of TERT promoter mutations gradually increased according to the progression of human B viral multistep hepaticarcinogenesis, which was found in 9.0% of low grade dysplastic nodules (LGDNs), 13.5% of high grade dysplastic nodules (HGDNs), 27.3% of early HCCs (eHCCs) and 28.4% of progressed HCCs (pHCCs). The occurrence of TERT promoter mutations correlated with lower levels of alpha-fetoprotein (AFP) (p=0.046) and a poor overall survival (p=0.012) in B viral HCC patients. On the contrary to in vitro data, TERT mRNA expression was lower in B viral HCCs with the mutant TERT promoter compared to those without. In addition, mRNA level of PROX1 was not correlated with that of TERT in B viral HCCs, in contrast that such correlation was evident in non-B viral HCCs. Interestingly, induction of stable HBx expression inhibited PROX1-mediated TERT expression in vitro study. In conclusion, our findings suggest that TERT promoter somatic mutations are early events in B viral human multistep hepatocarcinogeneis and HBx can induce a loss of PROX1 function as transcriptional activator for TERT expression.

Citation Format: Young-Joo Kim, Jeong Eun Yoo, Youngsic Jeon, Jae Uk Chong, Gi Hong Choi, Dae-Geun Song, Sang Hoon Jung, Bong-Kyeong Oh, Young Nyun Park. TERT promoter mutations in B viral humanhepatocarcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2547.
Abstract 2548: DNA damage response gene expression in CHK1 inhibitor responsive and resistant mouse models of MYC driven B-cell lymphoma
Authors: Hannaway Nicola L. , Hunter Jill E. , Greystoke Alastair , Perkins Neil D.
Published: August 2019

Enhanced tumor replication stress and genomic instability can lead to disruption of the DNA damage response, leading to tumor survival and dependence on essential checkpoint controls. Drugs to target intact pathways, such as CHK1 inhibitors, are therefore desirable and currently in clinical trials as potential future anticancer therapies. The underlying mechanisms underpinning the response and resistance to CHK1 inhibitor treatment are still unclear. We have demonstrated that NF-κB can regulate the response to oncogene induced DNA replication stress in vivo through regulation of CLSPN (Claspin) gene expression. Claspin is an essential adaptor protein required for the phosphorylation and activation of CHK1 by ATR. Using the Eµ-MYC mouse model of B-cell lymphoma we have found that mutation of NF-κB subunits (knockout of c-Rel or a T505A transactivation domain mutation of RelA) leads to earlier lymphoma onset that correlates with loss of Clspnexpression and inhibition of CHK1 activity. Moreover, while wild type Eµ-MYC lymphomas are highly responsive to CHK1 inhibitor treatment, by contrast Eµ-MYC NF-κB mutant cRel-/- or RelAT505A lymphomas are resistant. To understand more about how MYC driven lymphoma cells respond to CHK1 inhibitor treatment, we have investigated changes in the expression of genes encoding members of DNA damage response pathways. Wild type mice reimplanted with Eµ-MYC lymphoma cells were treated with the CHK1 inhibitor CCT244747 and harvested at multiple fixed timepoints. Quantitative PCR analysis of mRNAs encoding proteins in the ATR/CHK1 and ATM/CHK2 pathways was performed, with RPL13A used as reference gene. Interestingly, while dramatic changes were seen in wild type Eµ-MYC cells, these were not seen in CHK1 inhibitor resistant cRel-/- lymphomas. We observed an upregulation of the DNA damage and cell cycle responsive genes in the CHK1 inhibitor sensitive wild-type Eµ-MYC mice. This is consistent with with previously published in vitro studies and suggests an S phase cell cycle arrest in response to CHK1 inhibitor induced DNA damage. This response, however, is absent in the CHK1 inhibitor resistant Eµ-MYC NF-κB mutant lymphomas. It is hoped that by understanding these differences in gene expression that biomarkers of CHK1 inhibitor response can be identified to help stratify future clinical candidates.

Citation Format: Nicola L. Hannaway, Jill E. Hunter, Alastair Greystoke, Neil D. Perkins. DNA damage response gene expression in CHK1 inhibitor responsive and resistant mouse models of MYC driven B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2548.
Abstract 2553: Integration of genomic studies and the patient-derived xenograft model in nasopharyngeal carcinoma precision medicine research
Authors: Hsu Cheng-Lung , Li Hsin-Pai
Published: August 2019

Patient-derived xenograft (PDX) tumor model has become a new approach to identify druggable tumor mutations, and to screen and evaluate personalized anti-cancer drugs. Here, we established five nasopharyngeal carcinoma (NPC) PDXs in mouse model and performed whole-exome sequencing (WES) and copy number variations (CNVs) analyses. Relatively high incident rate of CNV aberration in cell cycle pathway was detected. Among these five NPC-PDXs, three had cyclin D1 (CCND1) amplification while four had cyclin-dependent kinase inhibitor CDKN2A deletion. Selected inhibitors were tested for their anti-tumor effects in two NPC-PDX mouse models. Palbociclib, a cyclin-dependent kinase inhibitor, revealed efficient anti-tumor effects by inducing G1 arrest in the PDX tumor according to RNA sequencing. In this study, cyclin D1 overexpression was also observed in >90% clinical metastatic NPC tumors (87/91) and was associated with poor outcomes. One NPC patient with liver metastatic who was prescribed with Palbociclib had stable disease and a drop in Epstein Barr virus (EBV) DNA titer. CNV analysis disclosed that plasma CCND1/CDKN2A ratio is correlated with EBV DNA load in NPC patients’ plasma and could serve as a screening test to select potential CDK4/6 inhibitors treatment candidates. Taken together, our findings indicate that integrated information from sequencing-based genomic studies and tumor transcriptomes before and after drug treatment in NPC-PDX models provide novel guidelines for personalized precision treatments.

Citation Format: Cheng-Lung Hsu, Hsin-Pai Li. Integration of genomic studies and the patient-derived xenograft model in nasopharyngeal carcinoma precision medicine research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2553.
Abstract 2558: Regulation of the expression of the DNA repair endonuclease 8-like 3 gene in hepatocellular carcinoma
Authors: Huang Wenya
Published: August 2019

Hepatocellular carcinoma (HCC) is the fifth commonest malignancy in the world, but the molecular mechanism of HCC development is still unclear. Previous studies had showed that the endonuclease 8-like 3 (Neil3) gene, a DNA glycosylase for oxidative DNA repair, overexpressed in most of cancers including HCC. In our previous cDNA microarray analysis in HCC tissues, it was found that the Neil3 gene was highly expressed in mouse and human tumors. The results of real-time RT-PCR analysis in 113 HCC patients showed that the Neil3 gene mRNA was increased in most human tumors. The Neil3 gene expression levels were also highly correlated with HCC recurrence in the patients receiving the hepatectomy surgeries, suggesting that it can serve as a prognostic marker for HCC. In this study we characterized the molecular mechanisms of the Neil3 gene expression regulation in HCC. We analyzed the methylation status in the Neil3 gene promoter regions by DNA methylation array (Agilent) and found that the Neil3 gene promoter was hypo-methylated in HCC tumors. Also, by the promoter reporter assays it was found that the main regulatory region for its transcription activity was from -123 to +94 upstream to the transcription start site. Furthermore, the DNA affinity precipitation assay (DAPA) was performed to screen for the transcription factors that bind to the Neil3 promoter and identified that cell cycle regulator E2F1, Yin Yang 1 (YY1) and histone deacetylase 1 (HDAC1) bind to the Neil3 gene promoter. It suggested that E2F1, YY1 and HDAC1 play important roles in regulating Neil3 gene expression. These transcription factors have been known to enhance cell proliferation and potentially the NEIL3-mediated oncogenic properties. And treatments of the HDAC inhibitor SAHA increased the Neil3 mRNA and protein levels. The functional effects of these pro-oncogenic transcriptional factors to the NEIL3-relevant HCC progression are currently in investigation. The results of these studies will clarify the regulatory mechanisms for the Neil3 gene induction in HCC progression.

Citation Format: Wenya Huang. Regulation of the expression of the DNA repair endonuclease 8-like 3 gene in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2558.
Abstract 2559: Functional characterization of a RECQL4 mutation in Rothmund Thomson Syndrome
Authors: Wu Tianyi , Walker Robert L. , Pineda Marbin , Lee MiYoung , Zhu Yuelin , Venkitaraman Ashok R. , Meltzer Paul S.
Published: August 2019

Osteosarcoma (OS) is the most common malignancy of the bone, typically diagnosed during the adolescent years. Despite early advances in treatment, OS survival rates have not improved in recent times. Targeted treatment remains elusive because OS is a genetically complex and heterogeneous disease and the low incidence of sporadic OS presents a challenge in dissecting the causes and drivers of tumor development. Type II Rothmund-Thomson Syndrome (RTS), a cancer predisposition disorder, presents a potential model for studying OS pathogenesis as roughly 30% of Type II RTS patients are eventually diagnosed with OS. Type II RTS is associated with germline mutations of the DNA helicase RECQL4 that exclusively target the C-terminal helicase domain thought to be involved in various cellular mechanisms of DNA repair. The N-terminal SLD2-homology domain, required for the assembly of the CMG complex at origins of replication, is spared. We used CRISPR/Cas9 gene editing to generate cell lines containing a common RECQL4 mutation identified in Type II RTS patients to further study its effects. Unexpectedly, a mutation previously reported as a nonsense mutation instead upregulates the activation of a known cryptic splice site in exon 14. This splice variant results in the in-frame deletion of the final 66 amino acids of exon 14. Our cell lines exhibit a mildly increased sensitivity to the DNA double-strand break inducing agent neocarzinostatin consistent with RECQL4’s reported functions in DNA damage repair. We will use next generation sequencing techniques to further characterize DNA damage repair signatures in RTS RECQL4 mutant cell lines.

Citation Format: Tianyi Wu, Robert L. Walker, Marbin Pineda, MiYoung Lee, Yuelin Zhu, Ashok R. Venkitaraman, Paul S. Meltzer. Functional characterization of a RECQL4 mutation in Rothmund Thomson Syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2559.
Abstract 2573: DNA damaging drugs stimulate tRNA cleavage through SLFN11 to inhibit protein synthesis and induce cell death
Authors: Li Manqing , Kao Elaine , Malone Dane , Gao Xia , Wang Jean Y. , David Michael
Published: August 2019

DNA damaging agents (DDAs) based chemotherapies are the first and still the most widely used anticancer treatments. However, quickly developed drug resistance of cancer cells often impedes their efficacies. Two independent transcriptome analyses in vast collections of cancer cell lines discovered that human SLFN11 is the only gene whose expression significantly sensitizes cancer cells to DNA damaging agents (DDAs) including Top1 and Top2 inhibitors, DNA alkylating agents, and DNA synthesis inhibitors. But the mechanism was unclear. We showed previously that SLFN11 inhibits HIV viral proteins syntheses by modulating tRNAs abundances and exploiting the biased codon usage of the virus. We hereby report that upon DDAs treatment, SLFN11 preferentially inhibits translation of genes involved in DNA damage response and repair, including ATR and ATM, based on their distinct codon usages, while the early DNA damage response signaling remains unaffected. Type II tRNAs, which include all serine and leucine tRNAs, are cleaved in a SLFN11-dependent manner in response to DDAs. The syntheses of proteins encoded by genes with high TTA (Leu) codon usage such as ATR display utmost susceptibility to SLFN11 expression due to the extreme low abundance of cognate tRNA-Leu-TAA. Specific inhibition of tRNA-Leu-TAA expression with special antisense oligonucleotides (Gapmers) suppressed ATR protein expression and restored DDA sensitivity of SLFN11-deficient cancer cells. Our study uncovered a novel mechanism of DNA damage response regulation mediated by SLFN11-dependent tRNA cleavage and codon-specific translational inhibition. Further investigation will elucidate the roles of SLFN11 in DNA damage-related tumorigenesis. More importantly, bioinformatics analysis revealed high frequency of codon TTA (Leu) usage in most genes involved in DNA damage response signaling and repair mechanisms including Homology Directed Repair (HDR), Nonhomologous End-Joining (NHEJ), and Mismatch Mediated Repair (MMR). Instead of targeting single gene product, novel therapies based on the modulation of tRNA-Leu-TAA expression would effect by inhibiting the protein synthesis of a wide spectrum of genes involved in DNA damage response and repair, thus preventing the development of drug resistance by cancer cells.

Citation Format: Manqing Li, Elaine Kao, Dane Malone, Xia Gao, Jean Y. Wang, Michael David. DNA damaging drugs stimulate tRNA cleavage through SLFN11 to inhibit protein synthesis and induce cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2573.