bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 429: Optimization of an RNA sequencing method for low-quantity degraded samples
Authors: Su Dan , Powers Jason G. , Halsey Thomas A. , Hurban Patrick
Published: August 2018

RNA-sequencing (RNA-seq) is an effective tool for gene expression analysis, promising to be a diagnostic tool for patient stratification and individualized therapy. However, it has been challenging to apply RNA-seq to low-quantity and degraded RNA derived from formalin fixed paraffin embedded clinical samples. The Illumina TruSeq® RNA Access approach, utilizing capture probes targeting known exons to enrich for coding RNAs, has shown high performance to profile poor quality RNA samples at input amounts at or above 20 ng. Here, we have further optimized the Illumina TruSeq® RNA Access method for samples with low quality and low quantity, and have compared results to those obtained with libraries prepared with SMARTer® Stranded Total RNA-Seq- Pico Input Kit. Libraries were prepared using 1 ng and 10 ng of degraded RNA from Banked FFPE liver specimens, and were compared to libraries prepared using the standard RNA Access method with an input of 100 ng. With the optimized low-input RNA Access method, the alignment rate was consistently high (95-97%) across both input amounts, with 82-84% of aligned reads mapped to the transcriptome. This is comparable to results obtained with the standard 100 ng input. Gene detection however, changed as a function of input, with ~ 15,900 genes detected at an input of 10 ng, and ~ 13,500 detected at 1 ng of input. In contrast, the SMARTer method detected a relatively constant number of genes (~ 14,500 genes) at both input amounts, albeit with a lower % aligned reads to transcriptome (46-47%), and an increased % reads aligned to Ribosomal RNA when compared to RNA Access. Thus, while both methods are capable of comparable levels of performance, the RNA Access method is more cost-effective from a sequencing standpoint. These characteristics were maintained across a group of additional FFPE-derived samples tested by both methods.

Taken together, these results demonstrate that an optimized RNA Access method has consistent mapping performance and a high gene detection rate, and is thus suitable for the analysis of low-quantity degraded RNA.

Citation Format: Dan Su, Jason G. Powers, Thomas A. Halsey, Patrick Hurban. Optimization of an RNA sequencing method for low-quantity degraded samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 429.
Abstract 4291: Oncogenic ALK regulates cell cycle progression via CDK1: Implications for therapy
Authors: Scorsone Kathleen A. , Gahlawat Sonal , Tyagi Siddhartha , Orellana Mayra C. , Westbrook Thomas F. , Bernardi Ronald J.
Published: August 2018

Pharmacologic inhibition of oncogenic ALK can yield clinical responses in a variety of cancer types, including non-small cell lung cancer, anaplastic large-cell lymphoma, and neuroblastoma; however, de novo and acquired resistance remain major problems. As is seen with inhibition of other oncogenic receptor tyrosine kinases, ALK inhibition primarily induces G1 cell cycle arrest. However, we and others have also observed a role for receptor tyrosine kinases in regulating G2/M progression. For instance, oncogenic HER2 has been shown to directly phosphorylate tyrosine 15 of CDK1, the mitosis promoting factor. This tyrosine is also the well-known substrate of WEE1 and serves to inactivate CDK1 kinase activity as a critical component of the G2-M checkpoint. In the current study, we aimed to determine if oncogenic ALK functions in a similar capacity. We used recombinant proteins to demonstrate that ALK is also capable of directly phosphorylating tyrosine 15 of CDK1. Further, combined inhibition of ALK and WEE1 with crizotinib and AZD-1775 leads to greater reduction in pY15-CDK1 than either agent alone in ALK-amplified neuroblastoma cells. These findings suggest a model where oncogenic ALK and WEE1 provide redundant regulation of the G2-M checkpoint, which may be critical in cancer cells that have lost some of the usual complement of checkpoint controls, such as p53. Consequently, either ALK or WEE1 activity would be sufficient to maintain appropriate cell cycle regulation, while dual blockade would allow mitosis to proceed unchecked. In agreement with these predictions, we performed live cell imaging and observed that combined inhibition of ALK and WEE1 leads to aberrant mitotic progression and mitotic catastrophe. Further, this combination yields greater anti-proliferative activity than either agent alone in ALK-driven models. Thus, this therapeutic strategy holds great promise for ALK-driven cancers.

Citation Format: Kathleen A. Scorsone, Sonal Gahlawat, Siddhartha Tyagi, Mayra C. Orellana, Thomas F. Westbrook, Ronald J. Bernardi. Oncogenic ALK regulates cell cycle progression via CDK1: Implications for therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4291.
Abstract 4295: ASF1B promotes cell proliferation and regulates cell cycle in human benign prostate hyperplasia (BPH)
Authors: Rani Nisha Dahiya , Ankem Dr. Murali , Damodaran Dr. Chendil
Published: August 2018

Background

Benign Prostate Hyperplasia (BPH) is enlargement of the prostate, which occurs in 50% men above 50 years of age. Alpha-adrenergic blockers and 5-alpha-reductase inhibitors are the standard medical therapy for BPH. However, these treatments are not only partially effective but also cause significant toxicity in patients. Understanding the molecular pathogenesis of BPH may reveal novel preventive or therapeutic targets for BPH.

Methods

Normal Human Prostate Epithelial Cells (HPrEpC), RWPE-1 and Benign Prostate Hyperplasia (BPH) were utilized for global sequencing and molecular studies. Western blot analysis was performed to determine the expression of G0/G1 and S-phase and M phase regulators of the cell cycle in BPH and RWPE-1 lysates. Cell cycle and western blot analysis was performed at successive time-points post starvation to understand the dynamics of cell cycle progression in both the cell lines. Knock out experiments were conducted to understand the function ASF1B in normal and BPH cells.

Results

RNA sequencing analysis revealed that the level of ASF1b (Anti-Silencing Function 1B Histone Chaperone) was over 80 times upregulated in BPH cells as compared to healthy prostate epithelial cells. Induction of ASF1B is specific to BPH as confirmed at protein levels. Induction of ASF1B expression correlated in a time-dependent manner with the increased expression of G1 (cdk4, 6 and cyclin D), G1-S (cdk2) and S-phase (cdk2 and cyclin-A) regulators at both transcription and translation levels. The G1-S phase transcriptional enhancers such as E2-F1 were also upregulated with successive time-points. The cell cycle analysis at sequential time-points revealed that the BPH cells entry to S phase was achieved earlier and by higher fraction of cells than the healthy prostate epithelial cells. Knocking down ASF1B expression by siRNA inhibited the proliferation of BPH cells by 30%. The findings so far suggest that ASF1B might be related to hyper-proliferation in BPH cells. The results indicate that ASF1B might be a novel therapeutic target for preventing hyper-proliferation in BPH. Studies are ongoing to validate the expression of ASF1B in clinical samples along with screening of small molecules targeting ASF1B expression specifically. Current study may lead to identification of a novel preventive and/or therapeutic target and related treatment for BPH.

Conclusion

Our studies suggest that ASF1b is a G1 and S phase regulator which are critical phases for hyper-proliferative BPH cells.

Citation Format: Nisha Dahiya Rani, Dr. Murali Ankem, Dr. Chendil Damodaran. ASF1B promotes cell proliferation and regulates cell cycle in human benign prostate hyperplasia (BPH) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4295.
Abstract 4298: Targeting the DNA damage checkpoint kinase Chk1 induces multiple pathways of cytotoxicity
Authors: Warren Nicholas , Ditano Jennifer , Eastman Alan
Published: August 2018

DNA damaging agents are standard therapies for many cancers, but result in limited long-term clinical benefit. DNA damage checkpoints arrest DNA synthesis and provide time for repair and recovery. Using DNA checkpoint inhibitors to sensitize cancer cells to traditional chemotherapeutics is an emerging strategy. However, clinical trials involving Checkpoint Kinase 1 inhibitors (Chk1i) plus DNA-damaging chemotherapies have shown little clinical benefit. This may be due to sub-optimal scheduling of combinations. We have published that delaying Chk1i until 18 h after gemcitabine confers the greatest sensitization of cancer cells. We have now characterized the mechanisms by which the Chk1i, MK-8776, sensitizes human cancer cells to gemcitabine and SN38. SN38, the active metabolite of irinotecan, traps topoisomerase I on DNA resulting in S phase arrest. Chk1i re-activates replication and drives cells into mitosis before the damage from SN38 is repaired. Co-incubation with a CDK1/2 inhibitor surprisingly failed to prevent S phase progression, but did block mitotic entry and prevented further DNA damage. Conversely, the replication-associated kinase, CDC7, is essential for Chk1i-mediated S phase abrogation. CDC7 is presumably required to activate dormant origins of replication. Gemcitabine inhibits ribonucleotide reductase, thus preventing production of deoxyribonucleotides. Addition of Chk1i 18 h following gemcitabine causes aberrant activation of DNA helicases, but without deoxyribonucleotides, this results in excessive single-stranded DNA. Eventually, the level of single-stranded DNA exceeds cellular stores of the protective protein, RPA, which leads to nucleolytic cleavage and double-strand DNA breaks. Chk1i-mediated helicase activation is again dependent on CDC7, but insensitive to a CDK1/2 inhibitor. Concurrent incubation with gemcitabine plus MK8776 does not activate DNA helicases. These data provide a rationale for a delayed administration of Chk1i when patients are receiving gemcitabine. These investigations have detailed how Chk1i elicits distinct mechanisms of cytotoxicity depending on the context of DNA damage. Understanding of these mechanisms may help identify biomarkers to predict patient response. Furthermore, the clinical development of several Chk1i have been terminated due to unfavorable toxicity, but the cause of toxicity remains unknown. The work described here may help the clinical development by determining whether the observed toxicities are due to on- or off-targets.

Citation Format: Nicholas Warren, Jennifer Ditano, Alan Eastman. Targeting the DNA damage checkpoint kinase Chk1 induces multiple pathways of cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4298.
Abstract 4299: Mutual influence of ROS, pH and CLIC1 membrane protein in the regulation of G1/S phase progression in human glioblastoma stem cells
Authors: Verduci Ivan , Peretti Marta , Raciti Federica Maddalena , Carlini Valentina , Patarozzi Alessandra , Barozzi Sara , Garrè Massimiliano , Sertic Sarah , Costa Alex , Daga Antonio , Barbieri Federica , Florio Tullio , Mazzanti Michele
Published: August 2018

Glioblastoma (GB) is the most lethal, aggressive and diffuse brain tumor. The main challenge of successful treatment is targeting the cancer stem cell (CSC) sub-population responsible for tumor origin, progression and recurrence. Chloride Intracellular Channel 1 (CLIC1), highly expressed in CSCs, is constitutively present in the plasma membrane where it is associated with chloride ion permeability. In vitro, CLIC1 inhibition leads to a significant arrest of GB CSCs in G1 phase of the cell cycle. Furthermore, CLIC1 knockdown impairs tumor growth in vivo. Here we demonstrate that CLIC1 membrane localization and function is specific for GB CSCs. Mesenchymal stem cells (MSCs) do not show CLIC1-associated chloride permeability and inhibition of CLIC1 protein function has no influence on MSC cell cycle progression. Investigation of the basic functions of GB CSCs reveals a constitutive state of oxidative stress and cytoplasmic alkalinization compared with MSCs. We now report that these three elements are temporally linked during CSC G1/S transition. Impeding CLIC1-mediated chloride current prevents both intracellular ROS accumulation and pH changes. CLIC1 membrane functional impairment results in GB CSCs resetting from an allostatic tumorigenic condition to a homeostatic steady state. In contrast, inhibiting NADPH oxidase and the NHE1 proton pump results in cell death of both GB CSCs and MSCs. Our results show that CLIC1 membrane protein is crucial and specific for GB CSC proliferation, and is a promising pharmacological target for successful brain tumor therapies.

This work was supported by grant n.16713, IG 2015 to MM Italian Association for Cancer Research (AIRC).

Citation Format: Ivan Verduci, Marta Peretti, Federica Maddalena Raciti, Valentina Carlini, Alessandra Patarozzi, Sara Barozzi, Massimiliano Garrè, Sarah Sertic, Alex Costa, Antonio Daga, Federica Barbieri, Tullio Florio, Michele Mazzanti. Mutual influence of ROS, pH and CLIC1 membrane protein in the regulation of G1/S phase progression in human glioblastoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4299.
Abstract 4309: SPCIP2 enhances breast cancer cell proliferation, cell cycle and tumor formation by expression and interaction with Y1
Authors: Byun Hyunjung , Lee Changhoon
Published: August 2018

SPCIP2 is a member of the serine/threonine protein kinase family. However, the working mechanism of SPCIP2 in cellular functions has not yet been properly understood. In this study, we found that SPCIP2 directly interacts with Y1, a multifunctional protein that can act as a regulator of transcription and translation. Furthermore, we determined the binding domains required for the interaction between SPCIP2 and Y1. The ectopic expression of SPCIP2 activates cell growth by inducing Y1. Binding activates is confirmed by SPCIP2-dependent increase of Y1 expression, as well as by the induced cell proliferation and tumor formation. Also, expression of siSPCIP2 and siY1 is found to induce cell cycle arrest and apoptotic activity in breast cancer cells. These insights provide evidence that SPCIP2 induces cell proliferation and tumor formation via expression and interaction with Y1.

Citation Format: Hyunjung Byun, Changhoon Lee. SPCIP2 enhances breast cancer cell proliferation, cell cycle and tumor formation by expression and interaction with Y1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4309.
Abstract 4312: 3-Hydroxyterphenyllin induces S phase arrest in ovarian cancer cells
Authors: Chen Yi C.
Published: August 2018

Epithelial ovarian cancer is the fifth most common cause of cancer-related death among women in the United States. 3-Hydroxyterphenyllin is a secondary metabolite of Aspergillus fungi. In this study, we evaluated 3-hydroxyterphenyllin as a potential anti-cancer agent using human ovarian cancer cells. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO2. The [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, flow cytometry and western blot analysis were used to confirm that 3-hydroxyterphenyllin exhibited a potent growth inhibitory effect against human ovarian cancer OVCAR-3 and A2780/CP70 cells. 3-hydroxyterphenyllin (2, 4, 8, 12 and 16 μM) reduced the viability of OVCAR-3 and A2780/CP70 cells in a concentration-dependent manner. 3-hydroxyterphenyllin (2, 4 and 8 μM) induced S phase cellular arrest for OVCAR-3 and A2780/CP70 cells by upregulating CyclinB1 and Cdc25A expression and inhibiting Cdk4, CyclinA2 and CyclinE1 expression. These results suggest that 3-hydroxyterphenyllin has the potential to fight ovarian cancer cell growth by preventing progression past the S phase of the cell cycle.

Citation Format: Yi C. Chen. 3-Hydroxyterphenyllin induces S phase arrest in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4312.
Abstract 4324: Systematic characterization of A-to-I RNA editing in cancer development
Authors: Liang Han
Published: August 2018

Adenosine (A) to inosine (I) RNA editing, mediated by ADAR enzymes, is a well-established RNA modification mechanism. Recent studies show that A-to-I RNA editing introduces a large number of nucleotide changes in both coding and noncoding regions in cancer transcriptomes. First, to elucidate the contribution of RNA editing to proteomic diversity in human cancer, we performed an integrated analysis of The Cancer Genome Atlas (TCGA) genomic data and proteomic data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC). We detected a considerable contribution of RNA editing in terms of amino acids changes induced per tumor sample in breast cancer. We validated some of these editing events at both RNA and protein levels, and further experimentally demonstrated the functional effects of some edited protein (e.g., increased proliferation, migration, and invasion of cancer cells in vitro). Second, we systematically characterized the miRNA editing hotspots across 20 cancer types from TCGA miRNA sequencing data and independently validated the vast majority of them by perturbation experiments in several cancer cell lines. These miRNA editing events show extensive correlations with key clinical variables and other molecular drivers. Focusing on the RNA editing hotspot in miR-200b, a key tumor metastasis suppressor, we found that the miR-200b editing level correlates with patient prognosis opposite to that pattern for the wild-type miR-200b expression. We further experimentally showed that in contrast to wild-type miRNA, the edited miR-200b can promote cell invasion and migration through its impaired ability to inhibit ZEB1/ZEB2 and acquired concomitant ability to repress new targets including LIFR, a well-characterized metastasis suppressor. Our results highlight the contribution of A-to-I RNA editing in cancer development and suggest its translational potential as both biomarkers and therapeutic targets.

Citation Format: Han Liang. Systematic characterization of A-to-I RNA editing in cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4324.
Abstract 4325: Cisplatin treatment of testicular cancer introduces long-term changes to the epigenome
Authors: Rounge Trine B. , Bucher-Johannessen Cecilie , Page Christian M. , Haugen Trine B. , Fosså Sophie D. , Haugnes Hege S. , Grotmol Tom
Published: August 2018

Testicular cancer (TC) survival rates have increased substantially over the last decades, largely due to the introduction of cisplatin (CP) chemotherapy. This treatment is, however, associated with increased risk of developing metabolic syndrome (MetS), defined according to the National Cholesterol Education Program. We aimed to investigate if CP treatment were associated with epigenetic changes, and whether these changes render TC survivors susceptible for developing MetS later in life.

We included 279 Norwegian TC survivors with and without CP treatment and MetS, matched on age at blood sampling (Table 1). The TC survivors were re-examined on average 16 years after the orchiectomy, and for some patients, CP treatment. Whole genome DNA methylation profiles were measured with MethylationEPIC BeadChip and analyzed with the R package minfi. We used a linear regression model adjusting for smoking, age and cell type composition to identify CP differentially methylated CpG sites and logistic regression adjusting for smoking and age to identify differentially methylated CpG sites associated with MetS. Gene enrichment analyses were based on Fisher's exact test using KEGG and Reactome pathways.

32 and 15 differentially methylated CpG sites were associated with CP treatment after adjusting for multiple testing with False Discovery Rate (FDR) and Bonferroni correction, respectively. The PTK6-RAS-MAPk pathway was significantly enriched with the FDR significant CpGs (p-value < 0.1). We could not identify FDR significant differentially methylated CpGs associated with MetS with our sample size.

In conclusion, our results suggest that CP treatment has long-term effects on the epigenome. Genes involved in double-strand break repair, consistent with the cytotoxicity of CP treatment, are among the differentially methylated CpGs. Our top list of differentially methylated CpGs associated with MetS (lowest unadjusted p-values), should be further explored in a larger sample set of TC survivors.

Table 1: Overview of sample characteristicsCP+ MetS+CP- MetS+CP+ MetS-CP- MetS-N613411569Mean age at surgery31312730Mean age at sample collection49484443Time between surgery and sample collection17181714

Citation Format: Trine B. Rounge, Cecilie Bucher-Johannessen, Christian M. Page, Trine B. Haugen, Sophie D. Fosså, Hege S. Haugnes, Tom Grotmol. Cisplatin treatment of testicular cancer introduces long-term changes to the epigenome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4325.
Abstract 4327: The epigenetic effects of benzo[a]pyrene exposure
Authors: Galea Francesca , White Paul A. , Arlt Volker M. , Vineis Paolo , Flanagan James M.
Published: August 2018

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens formed from the incomplete combustion of organic materials. Exposure to PAHs can be linked to at least 9 different cancer types, including lung and breast cancer. PAH are genotoxic and can form DNA adducts which can either be repaired or lead to mutations. DNA repair can lead to aberrant DNA methylation, however, few studies have investigated the link between PAH exposure and DNA methylation modifications. Here, we have used a mouse model to investigate the epigenetic consequences of PAH exposure. Mice (n=3/group) were treated by oral gavage with 0, 25, 50 and 75 mg/kg bodyweight/day of benzo[a]pyrene (BaP) for 28 days. Lung DNA from these mice was used to prepare Reduced Representation Bisulphite Sequencing (RRBS) libraries which were then sequenced using Illumina HiSeq2500to an average depth of 25x. For each mouse, DNA methylation was averaged over 500 bp tiled windows and differentially methylated regions (DMRs) were identified by comparing the controls (untreated) with all BaP-exposed mice (treated) irrespective of dose. In the treated vs untreated we identified 1815 windows (>25% methylation difference, p<0.05). Of these, we found that DMRs were under-represented in 5' untranslated regions (UTR), promoter, long interspersed nuclear elements (LINEs) regions and CpG islands compared with the genomic distribution of windows. DMRs were found to occur significantly more frequently in transcription termination regions. In addition, we found significantly more hypomethylation events in exon, intergenic, intronic and promoter regions, long terminal repeats (LTR) and short interspersed nuclear elements (SINE) and more hypermethylation in LINEs. Pyrosequencing validation on a selection of these DMRs is ongoing, using DNA from the same mice used in RRBS, plus mice exposed to BaP and other PAHs in lung and other tissues. In summary, these results show that PAHs have an effect on DNA methylation, and that these changes happen mostly within the gene body or intergenically, with significantly less differences observed at traditional gene expression regulators such as promoters. This

implies that the DNA methylation changes effected by BaP exposure may serve another purpose other than gene expression regulation.

Citation Format: Francesca Galea, Paul A. White, Volker M. Arlt, Paolo Vineis, James M. Flanagan. The epigenetic effects of benzo[a]pyrene exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4327.
Abstract 433: Diagnostic targeted sequencing panel for hepatocellular carcinoma genomic screening
Authors: Paradiso Viola , Garofoli Andrea , Tosti Nadia , Perrina Valeria , Lanzafame Manuela , Quagliata Luca , Matter Matthias S. , Wieland Stefan , Heim Markus H. , Piscuoglio Salvatore , Ng Charlotte K. , Terracciano Luigi M.
Published: August 2018

Commercially available targeted or exome panels miss some genes and regions frequently mutated in hepatocellular carcinoma (HCC); we aim to design and benchmark an HCC-specific panel as a robust high-throughput and cost-effective panel for genomic screening in HCC.

We designed an Ion AmpliSeq panel targeting all exons of 33 liver cancer protein-coding genes, recurrently mutated long non-coding RNA genes MALAT1 and NEAT1, the recurrently mutated promoter regions of TERT, WDR74, MED16 and TFPI2, as well as mutation hotspots in 7 additional cancer genes. We profiled DNA from fresh frozen tumor (FFT, n=10, median depth 1495x) and/or formalin-fixed paraffin-embedded (FFPE) tumor with low input DNA (n=36, median depth 530x), and their non-tumoral counterparts from 39 HCCs using the custom panel. We benchmarked the somatic mutations and copy number alterations (CNAs) identified against those from Illumina whole-exome sequencing (WES) of the equivalent FFT samples (median depth 112x).

At least one somatic mutation was identified in 35/39 cases using the custom panel. Median of 2.5 (0-74) and 3 (0-76) mutations were identified in FFT and FFPE samples, respectively. 98% (61/62) of the mutations identified from WES of the 10 FFT samples were successfully recovered using the custom panel, with the identification of an additional 6 and 32 mutations in coding and non-coding regions, respectively. Similarly, all 104 mutations identified from WES were also found based on the analysis of our custom panel of the 36 FFPE biopsies. We identified an additional 18 and 70 somatic mutations in coding and non-coding genes, respectively, using our custom panel, including 2 CTNNB1 activating mutations. Furthermore, copy number analysis revealed high correlation (r = 0.8) in the identification of gains/ amplifications and losses/ homozygous deletions in the genes included in the custom-panel between our custom panel and the WES.

Here, we develop a robust high-throughput and cost-effective tool for HCC genomic screening that allows the identification with high sensitivity and specificity of somatic mutations as well as copy number alterations in routine diagnostic tissue specimens.

Citation Format: Viola Paradiso, Andrea Garofoli, Nadia Tosti, Valeria Perrina, Manuela Lanzafame, Luca Quagliata, Matthias S. Matter, Stefan Wieland, Markus H. Heim, Salvatore Piscuoglio, Charlotte K. Ng, Luigi M. Terracciano. Diagnostic targeted sequencing panel for hepatocellular carcinoma genomic screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 433.
Abstract 4332: Meta-analysis of molecular features associated with aggressive prostate cancer
Authors: Costello James C. , Powers Rani , Goodspeed Andrew , Cramer Scott D.
Published: August 2018

The TMPRSS2:ERG fusion is the most common genomic alteration in prostate cancer, occurring in 40-50% of primary tumors. The frequency of the ERG fusion remains between 40-50% in metastatic tumors, supporting previous findings that the fusion can drive tumor development, but is not sufficient to drive aggressive, lethal disease. Large -omic datasets collected over thousands of patient tumors, such as The Cancer Genome Atlas (TCGA), paired with outcomes present the raw data needed to associate genetic aberrations with markers of aggressive disease. Accordingly, we performed a meta-analysis of over 2,000 patient samples to identify combinations of -omic features associated with aggressive ERG fusion-positive and ERG fusion-negative prostate cancer. The data were collected from 8 independent, publicly available prostate cancer cohorts and cover range of disease phenotypes (e.g., neuroendocrine, castration resistance). Our analysis consisted of training univariate Cox regression models separately for single genomic or pairs of genomic features within each independent dataset. Based on the availability, we explored gene expression, mutation, and copy number alterations, all used to build separate models. We then combined models for a genomic feature across all datasets by weighting individual coefficients by the inverse of their squared error in a fixed effects model; final results are reported as corrected p-values. We compared and contrasted results from combined models within data type (i.e., only gene expression) to models integrating multiple data types. Interestingly, we find that the genomic features associated with aggressive disease differed based on ERG status. We report genomic loci that are altered in all patients, but associate with aggressive disease only in patients with an ERG fusion. We also report loci that co-occur or are mutually exclusive of the ERG fusion and associated with aggressiveness. For example, SPOP mutations are known to be mutually exclusive of ERG fusions. We report a finer stratification of patients with SPOP mutations based on biochemical relapse. Our results present a compressive meta-analysis of molecular features in prostate cancer and identify novel molecular subtypes associated with aggressive disease.

Citation Format: James C. Costello, Rani Powers, Andrew Goodspeed, Scott D. Cramer. Meta-analysis of molecular features associated with aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4332.
Abstract 4338: Introduction of CALR mutations to a human erythroleukemia cell line, HEL, by CRISPR/Cas9 systems
Authors: Yasuda Shunichiro , Aoyama Satoru , Watanabe Daisuke , Kawamata Norihiko
Published: August 2018

[Introduction]Mutations of the calreticulin gene (CALR) are frequently found in patients with myeloproliferative neoplasms (MPN). Five-nucleotide insertion (Ins5) and 52-nucleotide deletion (Del52) in the last exon of the CALR gene are common mutations, generating an altered CALR protein with a mutated peptide sequence at the carboxy end due to the frame-shifts. Cell-lines with such CALR mutations have not been reported yet. CRISPR/Cas9 system is the novel technique commonly used in genetic engineering. CRISPR/Cas9 technology allows us to “edit” genome in mammalian cells. We introduced 46-nucleotide deletion (46Del) in the last exon of the CALR gene in a human erythroleukemia cell line, HEL. This 46Del abnormality lead to generation of an altered CALR protein with the abnormal peptide sequence at the carboxy end as seen in MPN cells with 52Del or Ins5. We compared the CALR-engineered HEL cells with the parental cells in the presence/absence of a Jak2 kinase inhibitor, ruxolitinib (RUX).

[Methods and Results]To introduce 46Del abnormality, we have designed two guide RNAs. We co-transfected vectors encoding the Cas9 with a fluorescence protein as a marker as well as the two guide RNAs into HEL cells using nucleoporation. Fluorescence protein positive cells were isolated and the single cells were cultured in 96-well plates for 3 weeks. Genomic DNA of each the clone was extracted, and genomic status of CALR was examined by PCR/sequencing. We have isolated one clone with 46Del (HEL-46DEL), of which expression of abnormal transcripts was confirmed by RT-PCR/sequencing. We also confirmed expressions of abnormal CALR protein by western blot analysis. Since the JAK2-STAT5 pathway is known to be activated in MPN cells, phosphorylation status of STAT5 in HEL and HEL-DEL46 was examined, showing comparable levels in each the line. Proliferation of HEL and HEL-DEL46 was examined with XTT assay, showing that the two lines proliferated similarly. RUX inhibited proliferation of HEL cells as well as HEL-DEL46. HEL cells have a JAK2 mutation which causes constitutive activation of the JAK2-STAT5 pathway. Introduction of the Del46 mutation into HEL cells might not enhance activation of the pathway because the JAK2 mutation already activates the pathway at the maximum level.

Citation Format: Shunichiro Yasuda, Satoru Aoyama, Daisuke Watanabe, Norihiko Kawamata. Introduction of CALR mutations to a human erythroleukemia cell line, HEL, by CRISPR/Cas9 systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4338.
Abstract 4354: Molecular signature of multiple myeloma progression through single-cell RNA-seq
Authors: Jang Jin Sung
Published: August 2018

Multiple myeloma (MM) is a malignant plasma cell disorder with well-defined primary genetic/cytogenetic abnormalities. Investigating genetic heterogeneity in the MM cells, may have an impact on therapeutic approaches and assessment of response to treatment. Single cell genomic profiling technology provides an opportunity for us to identify genetic heterogeneity during the pathogenesis of MM progression and select the most appropriate clinical interventions. We performed single cell whole transcriptome analysis in 597 CD138 positive cells derived from 15 patients at different stages of MM progression. Using Coefficient of Variation (CV) approach, we identified 790 genes and defined four main groups reflective of increasing levels of aggressiveness at the single cell level. Differential gene expression and gene set enrichment analyses showed that protein homeostasis pathways, unfolded protein response and mTORC1 related gene sets were significantly enriched at the most aggressive level consistent with the MM cell progression and predicts the overall survival. Our results provide novel insights into the cellular heterogeneity within each MM patient and a foundation for therapeutic opportunities including proteasome inhibitors.

Citation Format: Jin Sung Jang. Molecular signature of multiple myeloma progression through single-cell RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4354.
Abstract 4356: SIRT2-deacetylase activity is a potential predictive and prognostic marker in lung cancer patients
Authors: Guo Yang , Song Ha Yong , Raparia Kirtee , Vassilopoulos Athanassios
Published: August 2018

Background: SIRT2 has been described as a tumor suppressor, whereas our previous study using an LSL-KRASG12D mouse model showed that Sirt2 loss induced lung tumorigenesis in vivo through increased KRAS acetylation (K147) and activity. However, there is limited information about the clinical significance of SIRT2 expression and/or activity in lung cancer patients.

Purpose: The aim of this study was to investigate the associations between SIRT2 levels and activity with clinical-pathological characteristics and patient outcomes in lung cancer.

Materials and Methods: SIRT2 expression and activity was analyzed using a tissue microarray of 162 resected lung cancer samples from Northwestern Memorial Hospital. To assess SIRT2 expression, we used an antibody against SIRT2 for immunohistochemistry. To assess SIRT2 activity, we evaluated acetylated levels of two specific deacetylation targets, α-tubulin (K40) and KRAS (K147). For all tested markers, staining intensity of each tissue sample was scored as 0 (no signal), + 1 (weak), + 2 (distinct), + 3 (strong). The percentage of each positive staining was categorized as A (<25%), B (25~50%), C (>50%). Following statistical analysis, it was determined whether SIRT2 expression and activity were associated with several clinical parameters or outcomes. p < 0.05 was assumed statistically significant.

Results: Acetylated α-tubulin and acetylated KRAS in lung tumor samples were significantly correlated with each other (p<0.05), but not with SIRT2 expression. Of note, acetylated α-tubulin was significantly associated with TTF1 expression (p<0.05), staging (p<0.005), pleural metastasis (p<0.005), surgery (p<0.001), and chemotherapy (p<0.005). Acetylated KRAS had significant associations with surgery (p<0.005), lymph nodal status (p<0.05) and pleural metastasis (p<0.05). Kaplan-Meier analysis further indicated that both TTF1 and acetylated α-tubulin were independent predictors of patient survival (p=0.005 and p=0.028, respectively).

Conclusions: Our data implicated that SIRT2 deacetylation activity, but not SIRT2 expression levels, could represent an optimal predictive and prognostic factor in lung cancer. Based on this finding, strategies to increase SIRT2 activity might be favorable in clinical treatment protocols for lung cancer.

Citation Format: Yang Guo, Ha Yong Song, Kirtee Raparia, Athanassios Vassilopoulos. SIRT2-deacetylase activity is a potential predictive and prognostic marker in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4356.
Abstract 4361: Role of Rab GTPases and endosomal adaptor proteins in oncogenic RAS induced formation of perinuclear signaling complexes (PSCs)
Authors: Basu Srikanta , Basu Sandip K. , Salotti Jacqueline , Johnson Peter F.
Published: August 2018

Oncogenic RAS signaling leads to neoplastic transformation in immortalized cells and oncogene induced senescence in primary cells. Our laboratory has found that in both primary and immortalized cells oncogenic RAS induces constitutive perinuclear re-localization of p-ERK, CK2, and the MAPK scaffolding protein, KSR1. KSR1 binds to p-ERK and CK2, forming assemblies that we call “perinuclear signaling complexes” (PSCs). We observe PSCs in all cancer cell lines tested and in KRasG12V-driven mouse lung tumors, indicating that PSC formation is an important and integral characteristic of tumor cells. Our results show that KSR1 is essential for PSCs, as RNAi-mediated depletion of KSR1 or knockout of KSR1 disrupted their formation. We also demonstrated a key role for endosomal trafficking in this localized RAS signaling, as pharmacological inhibition of endocytosis prevented PSC formation. Furthermore, Rab11A, a Rab GTPase involved in trafficking of recycling endosomes, is required for establishment of PSCs and co-localizes with a pool of cellular CK2 and KSR1, but not p-ERK. Moreover, Rab11A knockdown caused decreased proliferation and survival of A549 cells (human KRAS mutant non-small cell lung adenocarcinoma). Our results demonstrate that there may be at least two classes of PSC-containing endosomes, p-ERK bound endosomes and CK2 bound endosomes. We are currently attempting to identify Rab GTPases that may differentiate these two kinds of PSC containing endosomes. Jongsma et al. (Cell, 2016) have shown that endosomes associated with the ubiquitin adaptor proteins EPS15, Tollip or TAXBP1 localize to the perinuclear region via the signaling adaptor, SQSTM1 (p62), which is tethered to the ER. We found that Tollip knockdown in A549 cells disrupted the CK2 bound endosomes but not p-ERK bound endosomes. Tollip knockdown also decreased cell proliferation and cell survival, while increasing apoptosis and autophagy. Whether other adaptor proteins are involved in RAS-mediated PSC formation remains to be determined. Growth factors (GF) also transiently induced PSCs in normal cells with delayed kinetics (4-6 hr). Our findings indicate that transformed cells sustain this novel late phase of GF signaling, localizing RAS pathway kinases to a nuclear-proximal compartment to drive tumorigenesis. We propose that PSCs are hallmarks of cancer cells and may represent useful biomarkers for cancer diagnosis and therapeutic responses.

Citation Format: Srikanta Basu, Sandip K. Basu, Jacqueline Salotti, Peter F. Johnson. Role of Rab GTPases and endosomal adaptor proteins in oncogenic RAS induced formation of perinuclear signaling complexes (PSCs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4361.
Abstract 4367: The small GTPase ARF6 is necessary for melanomagenesis
Authors: Acosta Lehi , Rogers Aaron , Peng Jingfu , Mueller Alan , Tong Zongzhong , Shin Donghan , Yoo Jae Hyuk , Li Dean Y. , Odelberg Shannon J. , Holmen Sheri L. , Grossmann Allie H.
Published: August 2018

ARF6 is a member of the adenosine diphosphate (ADP)-ribosylation factor (ARF) family of small GTPases and is part of the RAS superfamily. We have shown that pharmacologic inhibition of ARF6 can inhibit proliferation of uveal melanoma and invasion/metastasis of cutaneous melanoma. Most cutaneous melanomas are defined by mutually-exclusive driver mutations in BRAF, NRAS, or NF1. These subtypes share similar mechanisms of oncogenesis because they align along a continuum of RAS signaling. Mutations in the PI3K pathway occur in a fraction of all melanoma subtypes, indicating a selective advantage for PI3K/AKT activation beyond BRAF or RAS activation. We and others have reported ARF6-dependent ERK activation occurring upon either extracellular ligand stimulation or mutant oncoprotein signaling. Recently, ARF6 has also been shown to be upstream of PI3K signaling. Based on these findings, we hypothesized that ARF6 is critical for both the RAF and PI3K arms of the RAS pathway and that loss of ARF6 will impede tumor growth driven by these oncogenes. We tested this hypothesis using human melanoma cell lines and with an inducible, genetically engineered mouse model (GEMM) of melanoma. Our data reveal that pharmacologic inhibition of ARF6 can reduce proliferation of BRAF, NRAS and NF1-mutated melanoma cells. Furthermore, loss of Arf6 causes a reduction in mean tumor size and improves survival in BRAFV600E/CDKN2ANULL/PTENNULL/AKTE17K and BRAFV600E/CDKN2ANULL/PTENNULL mice. These results indicate that ARF6 is necessary for melanomagenesis and for oncogenic BRAF and/or PI3K signaling. Our data suggest that ARF6 may be an actionable node in the RAS pathway and that therapeutic targeting of ARF6 has the potential to improve current treatment methods for progressive disease.

Citation Format: Lehi Acosta, Aaron Rogers, Jingfu Peng, Alan Mueller, Zongzhong Tong, Donghan Shin, Jae Hyuk Yoo, Dean Y. Li, Shannon J. Odelberg, Sheri L. Holmen, Allie H. Grossmann. The small GTPase ARF6 is necessary for melanomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4367.
Abstract 4369: Functional role of RhoGEF FGD4 in promotion of aggressive prostate cancer
Authors: Bossan Alexia , Ottman Richard , Coppola Domenico , Chakrabarti Ratna
Published: August 2018

Hyperactivation of Rho family of GTPases including Rac, Cdc42 and Rho, as a result of deregulation of Rho guanine nucleotide exchange factors (GEF) is commonly noted in various cancers including prostate cancer. A number of GEFs are overexpressed in prostate and breast cancer cells and tissues, and show an association with poor prognosis. Earlier, we showed that a prolonged treatment with androgen receptor antagonist Casodex resulted in up regulation of a GEF FGD4, traditionally associated with hereditary motor and sensory neuropathies including Charcot-Marie-Tooth disease. FGD4 is a Cdc42 specific GEF and contains an actin binding domain FAB, in addition to the conserved FYVE domain, a DH domain for catalytic activity and two PH domains for targeting proteins to the membranes through phosphoinositide binding. Our study revealed that Casodex treatment of prostate cancer cells lead to down regulation of a microRNA cluster miR-17-92a that targets FGD4. Here we show that FGD4 is upregulated in cancerous prostate tissues compared to benign prostatic hyperplasia and PIN. FGD4 protein expression and altered localization gradually increase with increasing tumor grade and aggressiveness. Inhibition of FGD4 expression using siRNAs, reduced cell proliferation, resulted in a G2/M arrest, inhibited cell migration and reduced Cdc42 activity. Inhibition of FGD4 also improved sensitivity of androgen sensitive LNCaP cells to Casodex and PC-3 cells to the microtubule stabilizing drug docetaxel. Our data demonstrate a tumor promoting function of FGD4 in prostate cancer cells and that inhibition of FGD4 expression has a therapeutic benefit for both androgen-dependent and -independent prostate cancer cells.

Citation Format: Alexia Bossan, Richard Ottman, Domenico Coppola, Ratna Chakrabarti. Functional role of RhoGEF FGD4 in promotion of aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4369.