bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 533: XIAP BIR domain suppresses miR-200a expression and subsequently promotes EGFR protein translation and anchorage-independent growth of bladder cancer cell
Authors: Liao Xin , Huang Chao , Li Jingxia , Huang Chuanshu
Published: July 2017

The X-linked inhibitor of apoptosis protein (XIAP) is a well-known potent apoptosis suppressor, and also participates in cancer cell biological behaviors, therefore attracting great attentions as a potential antineoplastic therapeutic target for past years. We found here that lacking of XIAP expression resulted in a remarkable suppression of EGFR expression, consequently leading to the deficient of anchorage-independent cell growth. Further study demonstrated that BIR domain of XIAP was crucial for regulating the EGFR translation by suppressing the transcription and expression of miR-200a. Mechanistic studies indicated that BIR domain activated the PP2A activity by decreasing the phosphorylation of PP2A at Tyr307 in its catalytic subunit, PP2A-C. Such activated PP2A prevented the deviant phosphorylation and activation of MAPK kinases/MAPKs, their downstream effector c-Jun, and in turn inhibiting transcription of c-Jun-regulated the miR-200a. Collectively, our study uncovered a novel function of BIR domain of XIAP in regulating the EGFR translation, providing significant insight into the understanding of the XIAP overexpression in the cancer development and progression, further offering a new theoretical support for using XIAP BIR domain and EGFR as targets for cancer therapy.

Citation Format: Xin Liao, Chao Huang, Jingxia Li, Chuanshu Huang. XIAP BIR domain suppresses miR-200a expression and subsequently promotes EGFR protein translation and anchorage-independent growth of bladder cancer cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 533. doi:10.1158/1538-7445.AM2017-533
Abstract 5332: ISG15 knockdown inhibits growth of pancreatic cancer
Authors: Burks Julian , Smith Jill
Published: July 2017

Background: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease driven by mutated KRAS with an average survival of only 3-6 months from the time of diagnosis. Interferon-Stimulated Gene 15 (ISG15) is a 15 kDa protein induced by Type I interferons (IFN-α and IFN-β) and is a member of the ubiquitin-like superfamily of proteins. The ISG15 pathway is highly elevated in various malignancies where it has been shown to stabilize KRAS protein expression. However, very little is known about the role of the ISG15 pathway in PDAC. The purpose of this project is to investigate the expression and proliferative effects of the ISG15 pathway in PDAC.

Methods: The ISG15 knockdown cells were created using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing system. ISG15 was stably knocked-down in murine Panc02 pancreatic cancer cells by transfection with an ISG15 CRISPR construct from Genscript (pSpCas9 BB-2A-Puro (PX459)) and treated with media containing puromycin (2 μg/ml) for selection. Knockdown was confirmed by RT-PCR. The effect of ISG15 knockdown on PDAC cell growth in vitro was determined by comparing viable cell counts on wild-type and ISG15 knockdown Panc02 cells 48 hours after plating. The effect of ISG15 knockdown on pancreatic tumorigenesis was determined by subcutaneous inoculation of syngeneic immune competent C57BL/6 mice with 1x106 Panc02 wild-type or ISG15 knockdown cells. Tumor volumes were measured weekly and after 3 weeks the mice were euthanized, tumors dissected and weighed.

Results: ISG15 CRISPR treated cells had a 50% reduction in ISG15 mRNA expression as compared to wild-type cells. ISG15 knockdown in Panc02 PDAC cells significantly reduced cell growth as compared to wild-type cells (p=0.030). Tumors formed from ISG15 knockdown Panc02 cells significantly had a 74% reduction in tumor volume (p=0.031) and 78% reduction in tumor weight (p=0.029) as compared to wild-type cells.

Conclusion: The ISG15 pathway contributes to PDAC by increasing cell growth and the tumorigenic potential of pancreatic cancer cells. Ultimately, strategies to target the ISG15 pathway may lead to improved survival for patients diagnosed with PDAC.

Supported by: NIH T32CA00968 grant

Citation Format: Julian Burks, Jill Smith. ISG15 knockdown inhibits growth of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5332. doi:10.1158/1538-7445.AM2017-5332
Abstract 5333: Monoubiquitination inhibits the actin bundling activity of fascin
Authors: Lin Shengchen , Lu Shuang , Mulaj Mentor , Fang Bin , Keeley Tyler , Wan Lixin , Hao Jihui , Muschol Martin , Sun Jianwei , Yang Shengyu
Published: July 2017

Fascin is the most frequently upregulated actin regulatory protein in metastatic tumors. The elevated expression levels of fascin universally correlate with poor clinical course and shorter survival across different cancer types. It is believed that fascin promotes cancer cell migration and invasion by crosslinking actin filaments into bundles. However, the molecular mehcanisms underlying the regulation of fascin bundling activity are not completely understood. In this study we examine the regulation of Fascin activity by monoubiquitination. IP and LC-MS/MS was used to identify the posttranslational modification of Fascin A novel chemical monoubiquitination method was employed to synthesize monoubiquitinated Fascin. The monoubiquitinated fascin was purified, and the effect of monoubiquitination on fascin bundling activity was determined using low speed sedimentation assay, fluorescence microscopy and transmission electron microscopy. Here we identified monoubiquitination as a novel mechanism that regulates fascin bundling activity and dynamics. The monoubiquitination sites were identified to be K247 and K250, two residues located in a positive charge patch at the actin binding site 2 (ABS2) of fascin. Using a chemical ubiquitination method, we synthesized chemically monoubiquitinated fascin (mUb-fascin) and determined the effects of monoubiquitination on fascin bundling activity and dynamics. Our data demonstrated that monoubiquitination decreased the fascin bundling EC50, delayed the initiation of bundle assembly and accelerated the disassembly of existing bundles. By analyzing the electrostatic properties on the solvent accessible surface of fascin, we proposed that monoubiquitination introduced steric hindrance to interfere with the interaction between actin filaments and the positively charged patch at ABS2. Mutation of the monoubiquitination sites (K247 and K 250) to arginine inhibited fascin monoubiquitination and enhanced pro-migration ability of fascin, supporting the notion that monoubiquitination inhibit fascin activity. We also identified Smurf1 as an E3 ligase regulating the monoubiquitination of fascin. Our findings revealed a previously unidentified regulatory mechanism for fascin, which will have important implications for the understanding of actin bundle regulation under physiological and pathological conditions.

Citation Format: Shengchen Lin, Shuang Lu, Mentor Mulaj, Bin Fang, Tyler Keeley, Lixin Wan, Jihui Hao, Martin Muschol, Jianwei Sun, Shengyu Yang. Monoubiquitination inhibits the actin bundling activity of fascin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5333. doi:10.1158/1538-7445.AM2017-5333
Abstract 5335: Dissecting the ubiquitin pathway using homogenous and sensitive bioluminescent assay
Authors: Goueli Said A. , Hsiao Kevin , Mondal Subhanjan
Published: July 2017

Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins is a fundamental mechanism for regulating protein functions and protein stability thus affecting diverse cellular processes. Several ubiquitin-like (Ubl) molecules have been identified including SUMO, NEDD8, ATG8/15 and ISG15 which share the ubiquitin fold Ub/Ubl proteins are conjugated to lysine residues in substrate proteins through an ATP-dependent enzymatic cascade involving E1-activating enzyme, E2-conjugating enzyme and E3-ligase. To mark proteins for degradation, multiple ubiquitins are covalently attached to produce a Lys48 linked poly-ubiquitin chain. The poly-ubiquitinated proteins are recognized by the 26S proteasome system and deubiquitinated and destroyed. Substrates with ubiquitin chains linked through Lys 6, 11, 27, 29, 33 also regulate protein degradation. Substrates marked with mono-ubiquitination or Lys63 ubiquitin chains are involved in cell signaling independent of degradative functions. Many diseases are associated with dysfunction of ubiquitin signaling, with the E3 ligase class of enzymes are considered to be the most important target. However, in recent years, evidence have accumulated demonstrating the significance of E2 class of enzymes where mutations and impairment of this class lead to severe diseases states including chromosomal instability, cancer predisposition, neurological syndromes, and immunological disorders. Thus, in addition to E3, E2 group of enzymes represent important class of therapeutic targets. To address the need for developing drugs targeting these enzymes, we have developed a novel assay that can be used to screen for modulators of these enzymes by taking advantage of the first ubiquitin activating enzyme E1 which activates free Ub forming a high-energy thioester bond between E1’s active site Cys and the C-terminus of Ub, generating AMP and pyrophosphate (PPi) as products of the reaction. We monitor the activity of these enzymes by determining the concentration of AMP generated in the reactions in a luminescence-based assay. Our assay interrogate not only E1 but also E2 and E3 in a very robust and sensitive manner. The assay is homogenous, bioluminescent, and show a very high signal to background in a very selective manner for each class of the ubiquitin pathway. The assay was successfully tested for ubiquitination, SUMOlyation and Neddylation reactions and thus can be used for screening of enzymes targeting these pathways.

Citation Format: Said A. Goueli, Kevin Hsiao, Subhanjan Mondal. Dissecting the ubiquitin pathway using homogenous and sensitive bioluminescent assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5335. doi:10.1158/1538-7445.AM2017-5335
Abstract 5336: Characterization of selective covalent inhibitors of USP7
Authors: Wang Feng , Wu Jian , Wang Liqing , Sokirniy Ivan , Wang Hui , Grove Charles , Nguyen Phuong , Bregnard Thomas , Weinstock Joseph , Mattern Michael , Hancock Wayne , Bezsonova Irina , Kumar Suresh
Published: July 2017

The ubiquitin-specific protease 7 (USP7) has emerged as an attractive therapeutic target owing to its critical roles in several cancer signaling pathways as well as its essential role in maintaining Foxp3+ T-regulatory cell (Treg) functions. Pharmacological inhibition of USP7 using Progenra’s small molecule inhibitors resulted in direct anti-tumor activity as well as the unleashing of anti-tumor immunity by suppressing Treg functions. However, the precise mechanism of action these compounds was not well characterized. Using a combination of NMR spectroscopy, mass spectrometry, and single amino acid substitution approaches, we have now demonstrated that our USP7 inhibitors specifically target the catalytic cleft of USP7 and covalently modify its active site cysteine (Cys223). Pharmacokinetic studies revealed sustained irreversible USP7 inhibition after short term inhibitor treatment as well as subsequent changes in the level and ubiquitylation of various pharmacodynamic markers, including the Treg lineage specific transcription factor Foxp3. Detailed knowledge of the mechanism of USP7 inhibition has allowed us to incorporate rational design strategies into the lead optimization to obtain improved

molecules that will serve as the basis of a new class of anti-cancer immunotherapy agents.

Citation Format: Feng Wang, Jian Wu, Liqing Wang, Ivan Sokirniy, Hui Wang, Charles Grove, Phuong Nguyen, Thomas Bregnard, Joseph Weinstock, Michael Mattern, Wayne Hancock, Irina Bezsonova, Suresh Kumar. Characterization of selective covalent inhibitors of USP7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5336. doi:10.1158/1538-7445.AM2017-5336
Abstract 5337: USP26 regulates TGF-β signaling by deubiquitinating and stabilizing SMAD7
Authors: Eichhorn Pieter J.A. , Lui Sarah , Iyengar Prasanna , Jaynes Patrick
Published: July 2017

The amplitude of transforming growth factor-β (TGF-β) signal is tightly regulated to ensure appropriate physiological responses. As part of negative feedback loop SMAD7, a direct transcriptional target of downstream TGF-β signaling, acts as a scaffold to recruit the E3 ligase SMURF2 to target the TGF-β receptor complex for ubiquitin mediated degradation. Here we identify the deubiquitinating enzyme USP26 as a novel integral component of this negative feedback loop. We demonstrate that TGF-β rapidly enhances the expression of USP26 and reinforces SMAD7 stability by limiting the ubiquitin mediated turnover of SMAD7. Conversely, knockdown of USP26 rapidly degrades SMAD7 resulting in TGF-β receptor stabilization and enhanced levels of p-SMAD2. Clinically, loss of USP26 correlates with high TGF-β activity and confers poor prognosis in glioblastoma. Our data identify USP26 as a novel negative regulator of the TGF-β pathway and suggests that loss of USP26 expression may be an important factor in glioblastoma pathogenesis.

Citation Format: Pieter J.A. Eichhorn, Sarah Lui, Prasanna Iyengar, Patrick Jaynes. USP26 regulates TGF-β signaling by deubiquitinating and stabilizing SMAD7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5337. doi:10.1158/1538-7445.AM2017-5337
Abstract 5339: Molecular phylogenetic classification of human papillomaviruses and cancer associated genes: In correlation with cervical cancer manifestation
Authors: Idoko John , Faruk Mohammed , Yaro Jigo , Enemari James , Akpulu Peter
Published: July 2017

Human papillomaviruses (HPVs) are a diverse group of small double stranded Deoxyribonucleic acid viruses implicated in many malignancies including cervical cancer. The viruses are also attributed to premalignant epithelial lesions in this disease. Cervical cancer is a leading course of morbidity and mortality in African women and beyond. We aimed to study the molecular phylogenetic classification of HPVs and cervical cancer associated genes in relation with tumour manifestation. Phylogenetic tree was constructed based on nucleotide sequence alignments using ClustalX 2.1 algorithms to classify 31 different HPV DNA virus types and 10 cervical cancer associated genes. Study of immunohistochemistry markers associated with cervical cancer pathology was performed. The nucleotide sequences used include HPV- 1 (V01116), HPV-14 (D90261), HPV-20 (D90262), HPV-21 (D90263), HPV-25 (D90264), HPV-5 (M22961), HPV-47 (M32305), HPV-8 (M12737), HPV-2 (X55964), HPV-57 (X55965), HPV-6: X00203), HPV-11 (M14119), HPV-13 (X62843), HPV-43 (HE962420.1 E6 gene), HPV-44 (U31788.1), HPV-42 (HE820226.1 E6 gene for E6 protein), HPV-16 (K02718), HPV 16 (KJ869425.1 E6 and E7 variants), HPV-35 (M74117), HPV-31 (J04353), HPV-52 (KJ869430.1), HPV-33 (M12732), HPV-58 (D90400), HPV-51 (M62877), HPV-18 (X05015), HPV-45 (M27363), HPV-39 (M38185), (ME180, M73258), HPV 16 (KJ869425.1 E6 and E7 variants), Ki-67 (NM_002417.4), p53 (AB082923.1), EGFR (NM_005228.3), MYC (M25803.1), KRAS (EF471957.1), TGF-B (AF254794.1) and RB (AH002963.2) genes. The resulting phylogenetic tree provides a conventional classification of the HPVs and the cancer genes into groups encompassing the known tissue tropism and oncogenic potential of each gene type associated with the disease. The implications of a phylogenetic taxonomy on the diagnostic detection of HPVs as they relate to cervical cancer associate genes are shown to be important in disease management.

Citation Format: John Idoko, Mohammed Faruk, Jigo Yaro, James Enemari, Peter Akpulu. Molecular phylogenetic classification of human papillomaviruses and cancer associated genes: In correlation with cervical cancer manifestation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5339. doi:10.1158/1538-7445.AM2017-5339
Abstract 5341: HPV16 E6 oncoprotein modulates the DPPIV/CD26 in primary keratinocytes
Authors: Beckenkamp Aline , Prati Bruna , Maria-Engler Silvya Stuchi , Lenz Guido , Pilger Diogo André , Pierulivo Enrique Mario , Buffon Andréia
Published: July 2017

Cervical cancer is related to the persistent infection by high-risk human papillomavirus (HPV) subtypes, such as types 16 and 18. The E6 and E7 oncoproteins significantly contribute to the carcinogenic potential of high-risk HPV, through the degradation of p53 and pRb, respectively. The exoprotease dipeptidyl peptidase IV (DPPIV) also known as CD26, is a multifunctional protein involved in several processes related to cancer. DPPIV/CD26 has an intrinsic peptidase activity, able to degrade some bioactive peptides and chemokines, thereby regulating cell migration, proliferation and survival. It has been reported that the reduction in DPPIV/CD26 expression is associated with tumor progression in different types of cancer. Moreover, studies in cervical cancer samples and cell lines point to a reduction in the expression of this protein. However, there are no studies addressing the relationship between HPV infection and DPPIV/CD26 expression. We analyzed the influence of HPV oncoproteins on DPPIV/CD26 expression in human keratinocyte transduced with E6, E7 and E6E7. We observed that HPV16 E6 expression is associated to a reduction in DPPIV/CD26 expression at mRNA and protein levels, whereas HPV16 E7 does not affect DPPIV/CD26 expression. HPV16 E6E7 co-expression leads to a reduction in DPPIV/CD26 expression similar to that found in E6 transduced cells. In addition, we note a more pronounced reduction in DPPIV/CD26 expression in immortalized cells expressing E6E7. Furthermore, keratinocytes expressing HPV16 E6 mutant sequence and HPV11 E6, that do not bind to E6AP and do not degrade p53, have no effect on DPPIV/CD26 expression. Our results suggest that the HPV16 E6 oncoprotein can downregulates DPPIV/CD26. In addition, preliminary results suggest that modulation of DPPIV/CD6 mediated by E6 can be dependent on degradation of p53, since HPV16 E6 mutant and low-risk HPV E6 were not able to affect CD26 expression. Furthermore, the process of cell immortalization also appears to be related to the reduction of DPPIV/CD26 expression. Therefore, these results indicate that HPV oncoproteins promote imbalance DPPIV/CD26 expression which may be relate to cervical carcinogenesis.

Citation Format: Aline Beckenkamp, Bruna Prati, Silvya Stuchi Maria-Engler, Guido Lenz, Diogo André Pilger, Enrique Mario Pierulivo, Andréia Buffon. HPV16 E6 oncoprotein modulates the DPPIV/CD26 in primary keratinocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5341. doi:10.1158/1538-7445.AM2017-5341
Abstract 5343: The HIV shell protein Gp120 stimulates U87 glioma cell proliferation through the activation of glycolysis
Authors: Valentin-Guillama Gabriel , Lopez Sheila , Perez Jose , Cubano Luis , Chorna Natalia , Harrison Jeffrey , Boukli Nawal , Kucheryavykh Lilia
Published: July 2017

Patients infected with human immunodeficiency virus (HIV) are more prone to develop cancers, including glioblastomas (GBM). The median survival of GBM patients with HIV is significantly shorter compared to HIV-negative GBM patients, despite the fact that they receive the same treatments. This indicates that HIV infection is associated with more aggressive tumor behavior and with treatment resistance. Taking into account that HIV itself is not found in GBM specimens, the nature of the GBM-HIV association remains poorly understood and the underlying molecular mechanism is still unknown. Here we study the effect of Gp120, a glycoprotein found in the HIV shell, on GBM cell growth and chemotherapeutic resistance. U87 glioma cells were used in this study. Flow cytometry analyses based on 7-aminoactinomycin D staining and viability assays were used for identification of cell cycle, proliferation rates, and resistance to temozolomide (TMZ). Combined proteomics, metabolomics, and western blot approaches together with colorimetric pyruvate kinase activity assays were used for evaluation of metabolic pathways. U87 cells treated with Gp120 (200ng/ml) for 10 days showed higher proliferation rates and increased cell survival in response to treatment with TMZ (100µM) compared to un-treated cells. Quantitative proteomics studies using isobaric tags and western blot analysis identified expression up-regulation of several glycolytic enzymes, including enolase 2, pyruvate kinase, hexokinase, and glyceraldehyde 3-phosphate dehydrogenase in Gp120 treated U87 cells. Additionally, metabolite analysis revealed an increase of pyruvate, amino acid and lipid synthesis, together with reduction of protein degradation, in response to treatment with Gp120. In summary, we demonstrated that Gp120 promoted proliferation and resistance to TMZ in U87 cells through the activation of glycolysis, resulting in increased protein and lipid synthesis This research was made possible by NIH grant numbers: 1SC2GM102040, R25GM110513, INBRE-PR NIH Grant 8P20GM103475, the UCC RCMI Biomedical Proteomics Facility grant G12MD007583 and US Department of Education Grant number P031S130068.

Citation Format: Gabriel Valentin-Guillama, Sheila Lopez, Jose Perez, Luis Cubano, Natalia Chorna, Jeffrey Harrison, Nawal Boukli, Lilia Kucheryavykh. The HIV shell protein Gp120 stimulates U87 glioma cell proliferation through the activation of glycolysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5343. doi:10.1158/1538-7445.AM2017-5343
Abstract 5351: A simple yet powerful method to purify NGS compatible RNA from formalin fixed paraffin embedded tissues using FormaPure DNA
Authors: Doh Jung H.
Published: July 2017

Extensive archives of formalin-fixed, paraffin-embedded (FFPE) cancer tissues and next generation sequencing (NGS) capabilities provide rich resources for identifying and validating new cancer biomarkers. However, the ability to conduct FFPE-NGS studies has been limited by poor nucleic acid purification methods that are generally difficult and cumbersome to perform, particularly with RNA. FormaPure DNA was recently launched to provide a simplified workflow to extract DNA from FFPE curls. Its nucleic acid binding chemistry employs Beckman Coulter's proprietary SPRI (solid phase reversible immobilization) technology, which purifies nucleic acids without centrifugation or vacuum manifold. The SPRI technology ensures stronger binding and higher quality purification than other silica-coated bead methods that typically lead to lower quality results due to instances of nucleic acid shearing. Here, we present a modified FormaPure DNA workflow to purify RNA from FFPE samples, offering the flexibility to examine both genomic and transcriptomic roles in cancer biomarkers studies. We show that our extractions yield significantly more RNA (up to 2.5 µg per FFPE curl) and take a fraction of the time to perform compared to other benchmarks. Furthermore, we demonstrate that purified RNA can be used to generate high quality NGS data. Lastly, our extraction method can be automated on a variety of Beckman Coulter’s Biomek liquid handling workstations to offer an integrated solution for high-throughput FFPE-RNA-NGS experiments: RNA from 96 FFPE samples can be purified for NGS in less than 5 hours with minimal hands-on time.

Citation Format: Jung H. Doh. A simple yet powerful method to purify NGS compatible RNA from formalin fixed paraffin embedded tissues using FormaPure DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5351. doi:10.1158/1538-7445.AM2017-5351
Abstract 536: Novel identification of STAT1 as a crucial mediator of ETV6-NTRK3-induced tumorigenesis
Authors: Park Jinah , Kim Junil , Sorensen Poul H. , Kim Seong-Jin
Published: July 2017

Chromosomal rearrangements that facilitate tumor formation and progression through activation of oncogenic tyrosine kinase are frequently observed in cancer. The ETV6-NTRK3 (EN) fusion has been implicated in various cancers, including infantile fibrosarcoma, secretory breast carcinoma and acute myeloblastic leukemia, and has exhibited in vivo and in vitro transforming ability. In the present study, we analyzed transcriptome alterations using RNA-Seq in EN-transduced NIH3T3 fibroblasts to identify the mechanisms that are involved in EN-mediated tumorigenesis. Through functional profile assessment of EN-regulated transcriptome alterations, we found that EN up-regulated genes mainly associated with cell motion, membrane invagination, and cell proliferation, while down-regulated genes are involved in cell adhesion, which described the transforming potential and increased proliferation in EN-transduced cells. Notably, KEGG pathway analysis identified the JAK-STAT signaling pathway with the highest statistical significance. Moreover, Ingenuity Pathway Analysis and gene regulatory network analysis identified the Stat1 transcription factor and its target genes as top EN-regulated molecules. We further demonstrated that EN enhanced STAT1 phosphorylation but attenuated STAT1 acetylation, thereby inhibiting the interaction between NF-κB p65 and acetylated STAT1. Consequently, nuclear translocation of NF-κB p65 and subsequent anti-apoptotic NF-κB activity were increased by EN. Taken together, here we report, for the first time, STAT1 as a significantly EN-regulated transcription factor and a crucial mediator of EN-induced tumorigenesis. [This work was supported by a National Research Foundation grant of Korea (NRF-2014M3A9B5073918) funded by the Korea government.]

Citation Format: Jinah Park, Junil Kim, Poul H. Sorensen, Seong-Jin Kim. Novel identification of STAT1 as a crucial mediator of ETV6-NTRK3-induced tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 536. doi:10.1158/1538-7445.AM2017-536
Abstract 5361: Predesigned gene content for rapid deployment of custom oncology panels
Authors: Barry Andrew J. , Emerman Amy , Bowman Sarah , Patel Kruti , Dimalanta Eileen , Adams Scott , Henig Noa , Stewart Fiona , Hendrickson Cynthia , Davis Theodore , Elfe Charles
Published: July 2017

Efficient utilization of targeted gene panels for oncology research is challenged by the wide variation in gene constituents specific to a given study. While focused gene panels efficiently provide the necessary depth of coverage for low frequency variant detection, the high costs and design challenges associated with de novo panel design present challenges.

The NEBNext DirectTM technology utilizes a novel approach to selectively enrich nucleic acid targets ranging from a single gene to several hundred genes, without sacrificing specificity. The approach rapidly hybridizes both strands of genomic DNA with biotinyated probes prior to strepdavidin bead capture, enzymatic removal of off-target sequence, and conversion of captured molecules into sequencer-ready libraries. This results in a unique read coverage profile that results in uniform coverage across a given target. Unlike alternative hybridization methods, the approach does not necessitate upfront library preparation, and instead converts the captured molecules into dual-indexed illumina sequencer compatible libraries containing an 8 basepair sample ID and a 12bp Unique Molecule Index (UMI). The UMI individually tags each molecule prior to the final PCR amplification of the library, enabling identification of PCR duplicate molecules. The result is a 1-day protocol that enables the preparation of sequence-ready libraries from purified genomic DNA specific to the gene content included in the panel.

We have designed and developed baits specific to the full exonic content of 450 genes associated with cancer. These are designed, balanced, and pooled on a per gene basis, and can be combined into customized panels, allowing rapid turnaround of specific custom gene subsets. Here, we present the ability to rapidly deploy custom gene panels across a variety of panel sizes and content, while maintaining high specificity, uniformity of coverage across target content, and sensitivity to detect nucleic acid variants from tumor samples.

Citation Format: Andrew J. Barry, Amy Emerman, Sarah Bowman, Kruti Patel, Eileen Dimalanta, Scott Adams, Noa Henig, Fiona Stewart, Cynthia Hendrickson, Theodore Davis, Charles Elfe. Predesigned gene content for rapid deployment of custom oncology panels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5361. doi:10.1158/1538-7445.AM2017-5361
Abstract 5362: Targeting BRCA1 and BRCA2 with NEBNext Direct™
Authors: Adams Scott M. , Patel Kruti M. , Emerman Amy B. , Bowman Sarah K. , Elfe Charles D. , Henig Noa , Russello Salvatore , Barry Andrew , Davis Theodore , Hendrickson Cynthia L.
Published: July 2017

The screening and detection of germline BRCA1 and BRCA2 mutations are critical for the effective management of patients with breast or ovarian cancer and for the identification of individuals with a high risk of developing these cancers. In addition, somatic detection of pathogenic variants in BRCA1/2 can influence treatment decisions due to the susceptibility of tumors with BRCA mutations to PARP inhibitors.

Here we introduce the NEBNext DirectTM BRCA enrichment panel for the interrogation of BRCA1 and BRCA2 by Illumina sequencing. NEBNext DirectTM is a novel, hybridization-based method to selectively enrich nucleic acid targets ranging from a single gene to several hundred genes. This approach includes features such as the incorporation of unique molecule identifiers (UMIs) and the ability to capture degraded DNA, enabling accurate detection of low-frequency variants from formalin-compromised DNA and other challenging sample types.

We applied the NEBNext Direct BRCA1/2 panel to frozen tissue and achieved a high specificity for the BRCA targets (96% of the sequenced reads mapped to BRCA1 and BRCA2 and 80% of the sequenced bases were within the targeted regions). In addition, the resulting libraries were highly uniform in coverage, with 91% of the targeted bases covered at a value of at least 50% of the mean depth of coverage and 100% of the bases covered at 20% of the mean or greater. Some variability in the specificity was observed with formalin-fixed, paraffin embedded (FFPE) samples, and this effect was dependent on the quality of the FFPE DNA. The UMIs were used to identify PCR duplicates prior to variant calling, as well as for error correction, enabling the accurate detection of low-frequency variants. The library preparation for all samples was completed in one day, and the entire process of library preparation, sequencing on the Illumina Miseq, and data analysis was completed in a total of two days.

In summary, we demonstrate that application of the NEBNext DirectTM method to the enrichment of BRCA1 and BRCA2 provides a tractable approach for the rapid and highly sensitive analysis of these cancer-associated genes.

Citation Format: Scott M. Adams, Kruti M. Patel, Amy B. Emerman, Sarah K. Bowman, Charles D. Elfe, Noa Henig, Salvatore Russello, Andrew Barry, Theodore Davis, Cynthia L. Hendrickson. Targeting BRCA1 and BRCA2 with NEBNext Direct™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5362. doi:10.1158/1538-7445.AM2017-5362
Abstract 5363: Measuring gene expression at the tumor microenvironment: A comparison between nCounter PanCancer Immune Profiling Panel and Oncomine Immune Response Research Assay
Authors: Mongan Ann , Tom Warren , Au-Young Janice , Pankov Aleksandr , Ganpule Gauri , Hyland Fiona
Published: July 2017

The tumor microenvironment, especially infiltrating T lymphocytes and inflammatory molecules, is believed to be highly relevant to the tumor’s sensitivity to cancer checkpoint blockade therapy. At the same time, the exact markers that are predictive of response for each therapeutic agent are still the subject of active investigations. To address the need for better understanding of the effect of different T cell subsets, antigen presentation, and tumor killing, gene expression profiling presents an attractive means to simultaneously evaluate the tumor microenvironment and cancer cells. In this study we compare the results and performance of the nCounter PanCancer Immune Profiling Panel and the Oncomine Immune Response Research Assay, both of which are designed to measure the expression of genes indicative of an immune response and potential immune-editing activities by tumor cells. The nCounter panel detects gene expression by counting unique probes that hybridize target mRNA, while the Oncomine panel employs NGS to sequence and count reads derived from the targets. While both panels are designed to work with FFPE samples, The nCounter panel expects 100 ng of unamplified RNA, while the Oncomine panel requires only 10 ng total RNA with its AmpliSeq technology. The two panels share 254 common genes, which constitute the basis for this comparison. Across 12 cancer samples (breast, H&N, melanoma, NSCLC, and RCC), results show that the Oncomine panel offers 20% higher dynamic range, thereby providing more robust readouts about the differences among samples. More importantly, with virtually no background noise, the underlying NGS technology provides an absolute zero readout for non-expressing genes which significantly improves the sensitivity for detecting low expressing genes, as their presence can be confirmed by as few as two reads. The two technologies show moderate correlation (R ~ 0.7), with the Oncomine panel more strongly correlating with qPCR (R ~ 0.9). Finally, when clustered using all genes on the panel, only the Oncomine panel provides clear stratification of cancer types, thus allowing the panel to be used for tissue type confirmation in addition to evaluating the immune response.

Citation Format: Ann Mongan, Warren Tom, Janice Au-Young, Aleksandr Pankov, Gauri Ganpule, Fiona Hyland. Measuring gene expression at the tumor microenvironment: A comparison between nCounter PanCancer Immune Profiling Panel and Oncomine Immune Response Research Assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5363. doi:10.1158/1538-7445.AM2017-5363
Abstract 5366: Detection of low-frequency somatic variants using single-molecule, real-time sequencing
Authors: Baybayan Primo , Nolden Laura
Published: July 2017

Detection of somatic mutations, especially in heterogeneous tumor samples where variants may be present at a low level, is challenging. Single Molecule, Real-Time (SMRT®) Sequencing is ideal for minor variant detection because of its ability to sequence single molecules with very high accuracy (>QV40) using the circular consensus sequencing (CCS) approach.

Here, we characterize the Sequel System for the detection of low-frequency somatic variants using constructs containing mutations in coding regions in EGFR, NPM1, AKT1 and JAK2 representing deletion, insertion, substitution and homopolymer variants. Wild type and mutant amplicons, provided by SeraCare, were mixed and serially diluted from 10% down to 0.1% allelic frequency. Independent SMRTbell libraries were constructed for each dilution point, sequenced and analyzed using SMRT Sequencing to identify the variants and determine the observed frequency. The random error profile and high-accuracy CCS reads make it possible to accurately detect low-frequency somatic variants. We will demonstrate sensitivity of the PacBio Systems to detect mutations down to 0.1%.

Citation Format: Primo Baybayan, Laura Nolden. Detection of low-frequency somatic variants using single-molecule, real-time sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5366. doi:10.1158/1538-7445.AM2017-5366
Abstract 5369: Epigenetic, transciptomic and ubiquitomic changes associated with BAP1 loss in uveal melanoma
Authors: Kurtenbach Stefan , Kuznetsov Jeffim N. , Field Matthew G. , Reddy Rohit , Sanchez Margaret I. , Decatur Christina L. , Harbour J William
Published: July 2017

Uveal melanoma (UM) is the most common primary and aggressive ocular cancer. Up to 50% of the patients develop metastasis, which are notoriously resistant to all forms of therapy and despite medical treatment leads to death within a mean time of 5-7 months, with a mortality rate of over 90%. We previously described that gene expression profiling can be used to classy UM tumors into two basic categories, class 1 (low metastatic risk) and class 2 (high metastatic risk). We further described that inactivating mutations in the ubiquitin hydrolase BAP1 are found in over 85% of class 2 tumors. BAP1 is involved in removing ubiquitin from specific proteins to regulate their function, like histone H2A, thereby regulating gene expression.

Building on these findings, we have generated uveal melanoma and melanocyte cell lines that allow for the inducible knockdown of BAP1. First, we performed RNAseq on multiple cell lines before and after BAP1 knockdown and compare the results to gene expression of class 1 and class 2 tumors. We identified significant overlap in key genes linked to metastasis between the primary tumors, uveal melanoma and melanocyte cell lines. Using ChIP-seq to interrogate uveal melanoma cells depleted of BAP1, we identified changes in genome wide histone marks and RNA polymerase localization associated with BAP1. To gain further insight into non-histone deubiquitination targets of BAP1, we performed ubiquitination proteomic profiling using the UbiScan® technology and identified a list of high probability BAP1 substrates.

Taken together, these complementary genome-wide investigations provide a global picture of the cellular functions of BAP1 and they provide novel insights into the metastasis-promoting effect of BAP1 loss in UM.

Citation Format: Stefan Kurtenbach, Jeffim N. Kuznetsov, Matthew G. Field, Rohit Reddy, Margaret I. Sanchez, Christina L. Decatur, J William Harbour. Epigenetic, transciptomic and ubiquitomic changes associated with BAP1 loss in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5369. doi:10.1158/1538-7445.AM2017-5369
Abstract 5373: 3D genome architecture changes during cancer cell migration and metastasis
Authors: McCord Rachel Patton , Golloshi Rosela
Published: July 2017

Metastasis, the most deadly part of cancer, results from cancer cell migration and invasion from a tumor into surrounding tissues. During this migration, the cell often has to squeeze and deform its nucleus through tight spaces. This nuclear squeezing is a rate-limiting step in migration, and the deformations can even rupture the nucleus. The organization of the genome inside the nucleus into loops, domains, and territories is important for proper gene regulation, DNA replication, and genome repair. However, little is known about the characteristics of nuclear organization that might allow for the nuclear deformation of metastatic cancer cells. We are using Chromatin Conformation Capture (Hi-C) and microscopy techniques to study genome organization during cancer cell migration. Using photoconvertible fluorophore Dendra2-H4 we draw and monitor patterns on nuclei of cells migrating through dense collagen matrices, Transwell filters, or microfluidic channels. Our data show changes in these patterns, indicating spatial reorganization of the genome during migration. Previous work has suggested that cancer cell migration requires global condensation of the genome. Some cancer chemotherapeutics act on epigenetic marks to decondense chromatin. We are testing whether such chromatin decondensation affects the physical deformability of the nucleus and thus effective migration. We have used Transwell and wound healing migration assays to monitor migration of cells treated with histone deacetylase inhibitors and histone methyltransferase inhibitors. Our results show that the drugs inhibit migration in a Transwell assay but not in the wound healing assay. This suggests that genome decondensation prevents the passage of the nucleus through narrow 3D spaces. But, a condensed genome organization is not necessary for 2D cell motility, where nuclei are unconfined. Hi-C experiments on migrating cells with deformed nuclei and drug-treated cells will characterize the specific rearrangements in genome structure that occur during cancer cell migration and whether these specific changes are counteracted by epigenetically active drugs.

Citation Format: Rachel Patton McCord, Rosela Golloshi. 3D genome architecture changes during cancer cell migration and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5373. doi:10.1158/1538-7445.AM2017-5373
Abstract 5376: Requirement of TET2 in MLL-AF9 mediated leukemogenesis
Authors: Nath Dipmoy , Mohi Golam
Published: July 2017

TET methylcytosine dioxygenase 2 (TET2) catalyzes the conversion of 5-methylcytosine to 5-hydroxymethylcytosine resulting in DNA demethylation. Loss-of-function mutations or deletions of TET2 have been found in several myeloid malignancies including myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML). Moreover, deletion of TET2 leads to myeloid malignancies in mice. Therefore, TET2 has been suggested to play a tumor suppressor role in myeloid malignancies. In this study, unexpectedly, we have uncovered a tumor promoter function for TET2 in MLL rearranged leukemia.

Chromosomal rearrangements of the Mixed Lineage Leukemia (MLL) gene have been found in ~10% of acute leukemia and are associated with poor prognosis. We observed that depletion of TET2 significantly inhibited the proliferation of MLL-AF9 positive MOLM14 leukemia cells. Similarly, knockdown of TET2 markedly reduced proliferation in murine Ba/F3 cells stably expressing MLL-AF9. Conversely, overexpression of TET2 significantly increased proliferation in MLL-AF9 expressing MOLM14 and Ba/F3 cells. In addition, deletion of TET2 significantly inhibited MLL-AF9 mediated myeloid colony formation in the bone marrow (BM) cells ex vivo.

Next, we assessed the in vivo effects of TET2 deletion in MLL-AF9-mediated leukemogenesis using retroviral bone marrow transduction and transplantation assays. Whereas recipients of MLL-AF9-transduced wild type BM exhibited marked increase in white blood cells (WBC) and neutrophils in their peripheral blood and significant increase in the spleen size, recipients of MLL-AF9-transduced TET2-deficient BM showed significantly reduced WBC and neutrophil counts and reduced spleen size. Flow cytometric analysis showed significantly increased myeloid precursors (Gr-1+/Mac1+) and granulocyte macrophage progenitors (GMP) in the BM of transplanted animals receiving MLL-AF9 expressing wild type BM compared with recipients of MLL-AF9 expressing TET2-deficient BM. Furthermore, transplanted animals receiving MLL-AF9-transduced wild type BM showed marked increase in hematopoietic stem/progenitor cells (LSK; Lin-Sca-1+c-kit+), which was significantly reduced upon TET2 deletion. Deficiency of TET2 also significantly reduced MLL-AF9-mediated increase in hematopoietic progenitor colony formation in the BM. In addition, deletion of TET2 resulted in increased overall survival in mice expressing MLL-AF9. Together, these data indicate that TET2 is required for leukemic transformation mediated by MLL-AF9. Our results suggest a novel tumor promoter function for TET2 in MLL rearranged leukemia.

Citation Format: Dipmoy Nath, Golam Mohi. Requirement of TET2 in MLL-AF9 mediated leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5376. doi:10.1158/1538-7445.AM2017-5376