bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B21: CRISPR screens provide a comprehensive assessment of cancer vulnerabilities but generate false-positive hits for highly amplified genomic regions
Authors: Munoz Diana , Stegmeier Frank P. , Schlabach Michael
Published: December 2017

CRISPR/Cas9 has emerged as a transformative new tool to systematically probe gene function. In this study, we compare the performance of CRISPR to RNAi-based loss-of-function screens for the identification of cancer dependencies by performing parallel deep-coverage shRNA and CRISPR screens targeting 2722 genes across several cancer cell lines. CRISPR-based dropout screens identified more lethal genes compared to RNAi in all five cancer models, indicating that the identification of many cellular dependencies may require full gene inactivation, as induced by CRISPR but not RNAi. However, in two aneuploid cancer models we found that all genes within highly amplified regions, including non-expressed genes, scored as lethal by CRISPR, revealing an unanticipated class of false-positive hits in CRISPR-based screens. Using a CRISPR tiling array that encompassed all possible sgRNAs against the coding regions of 139 genes, sgRNAs targeting essential domains provide the most robust dropout phenotypes, suggesting that this approach might be used to define the protein domains that are required for cancer dependence. Collectively, these findings demonstrate the utility of CRISPR-based screens in the identification of cancer-dependent genes, but also reveal the need to carefully control for false-positive results especially in chromosomally unstable cancer lines.

Note: This abstract was not presented at the conference.

Citation Format: Diana Munoz, Frank P. Stegmeier, Michael Schlabach. CRISPR screens provide a comprehensive assessment of cancer vulnerabilities but generate false-positive hits for highly amplified genomic regions [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B21.
Abstract B27: Identifying therapeutic combinations with EGFR-targeted therapy through the generation of genome-wide CRISPR-Cas9 knockout libraries in oral cancer cell lines
Authors: Ludwig Megan , Birkeland Andrew , Nimmagadda Sai , Foltin Sue , Jiang Hui , Carey Thomas , Brenner John Chad
Published: December 2017

Oral squamous cell carcinoma (OSCC) remains a disease with poor survival. With recent characterization of the mutational landscape of OSCCs, there is great potential for personalized targeted therapies. However, to date utilization of targeted therapies in OSCC have had limited success with anti-EGFR therapy. Combinations of targeted therapies, which may have greater efficacy by inhibiting compensatory pathways, have not been well studied. To identify synergistic combinations with EGFR-targeted therapy, we introduced Genome-scale CRISPR-Cas9 Knock-Out (GeCKO) libraries into OSCC cell lines. CRISPR-Cas9 generates individual genetic knockouts through targeted gene editing, and by using a pool of CRISPRs targeting over 18,000 genes we can perform genome-scale screening for synergistic targetable pathways. Upon selection of the OSCC GeCKO pool, we identified that knockouts of genes in the FGFR pathway caused sensitivity to the EGFR inhibitor gefitinib. We further investigated the possible synergistic effect of dual EGFR and FGFR inhibition with resazurin viability assays in 14 of our OSCC models. We found 6/14 (43%) of the models were responsive to the combination of EGFR and FGFR inhibition, indicating that the FGFR pathway could be an alternate mechanism of resistance to EGFR-targeted therapy. For further investigation of this combination, we again used CRISPR-Cas9 to generate an EGFR knockout (KO) OSCC cell line. This EGFR KO cell line retains sensitivity to FGFR inhibitors, and we expect that this cell line will be a useful tool in further evaluating the compensatory mechanism of the FGFR pathway. OSCC remains a common and frequently lethal cancer with great potential for the development of personalized targeted therapies. Here, we describe the use of genome-wide CRISPR-Cas9 library to discover the synergistic combination of EGFR and FGFR inhibition. Further investigation suggests the FGFR pathway as a common compensatory mechanism to EGFR inhibition. We hope to use this approach to identify additional compensatory mechanisms of resistance to targeted therapies with the eventual goal of translating these findings to clinic.

Citation Format: Megan Ludwig, Andrew Birkeland, Sai Nimmagadda, Sue Foltin, Hui Jiang, Thomas Carey, John Chad Brenner. Identifying therapeutic combinations with EGFR-targeted therapy through the generation of genome-wide CRISPR-Cas9 knockout libraries in oral cancer cell lines [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B27.
Abstract B30: Novel targets for combination therapy in EGFR mutated NSCLC
Authors: Drobnitzky Neele , Patel Agata , Mumin Amalina , Cahill Fiona , Lourenco Luiza Madia , Olyha Samantha , Corbacioglu Sureyya , Jiang Yanyan , Ryan Anderson J.
Published: December 2017

Non-small cell lung cancer (NSCLC) patients with activating mutations in the epidermal growth factor receptor (EGFR) gene generally show good responses to therapy with EGFR tyrosine kinase inhibitors (EGFR-TKIs). Unfortunately resistance emerges during treatment, driven by different mechanisms. Mutation-driven mechanisms are becoming well understood and include secondary mutations in the EGFR (T790M for 1st generation EGFR-TKIs and C797S for 3rd generation EGFR-TKIs) and activating mutations in NRAS. On the other hand, non-mutation based resistance mechanisms are less well understood but may involve activation of alternative, potentially targetable signalling pathways.

To identify alternative signalling pathways and potential combination targets, we performed a bioactive small molecule (n=1902) screen to identify targets that could delay the emergence of resistance to sustained inhibition of EGFR in EGFR-mutant NSCLC cells.

9 out of 186 target classes were significantly enriched (p< 0.05) within the top 2.5 % of screen compounds, when ranked for their ability to sensitise cells to the 3rd generation EGFR-TKI AZD9291. We identified expected hits such as AKT, PI3K and mTOR but also novel targets.

To prioritise targets for in vivo validation, compounds were tested in 3D-spheroids, which have a complex microenvironment, providing a more tumour-like model. Most compounds were more cytotoxic in 3D compared to 2D when used individually, preventing or slowing down spheroid growth. EGFR-TKI combination effects in 3D varied when compared to 2D-models. FAK inhibition was less effective in spheroids compared to 2D. AKT or MEK inhibitors showed marked effects in combination, further delaying spheroid growth under EGFR-TKI treatment but also inhibited spheroid growth when used on their own. Inhibitors of novel targets were identified that showed promising combination effects even at sub-toxic single-agent doses.

Our data identify new candidates for combination therapy with EGFR-TKIs and suggest that 3D-spheroids may be a useful approach for prioritising combinations for in vivo studies.

Citation Format: Neele Drobnitzky, Agata Patel, Amalina Mumin, Fiona Cahill, Luiza Madia Lourenco, Samantha Olyha, Sureyya Corbacioglu, Yanyan Jiang, Anderson J. Ryan. Novel targets for combination therapy in EGFR mutated NSCLC [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B30.
Abstract IA14: Understanding the complex biology of KRAS mutant cancers using genetic screens
Authors: L Tina
Published: December 2017

Apart from identifying synthetic lethal genes, genetic screens have the potential to reveal much about the basic biology of cell lines. We utilize two techniques, one analytical and one experimental, to demonstrate how genetic screens can also reveal which pathways are on in cell lines and what are the phenotypic consequences of pathway perturbation. First, we apply a clustering analysis of genome-scale CRISPR screening data to map out active pathways in cancer cell lines and to discover novel members of signaling transduction pathways and protein complexes. Secondly, we performed a combinatorial siRNA screen that knocks out multiple genes at once and measures five functional parameters to comprehensively describe the effects of pathway inhibition. Application of these two techniques to KRAS mutant cancer cell lines reveals a trove of biological information that highlights the vast heterogeneity of this disease and subtype-specific opportunities for therapeutic intervention.

Citation Format: Tina L. Understanding the complex biology of KRAS mutant cancers using genetic screens [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr IA14.
Abstract IA18: Mechanistic participation of cancer testes antigens in tumor initiation and progression
Authors: Whitehurst Angelique
Published: December 2017

Tumors frequently activate genes whose expression is otherwise biased to the testis, collectively known as cancer-testis antigens (CTAs). The extent to which the anomalous expression of CTAs represents epiphenomenon or confers tumorigenic traits is unknown. To gain a systems level view of CTA activity, we implemented a multidimensional functional genomics approach that incorporated seven different phenotypic assays in 11 distinct disease settings. This analysis identified 16 CTAs that are essential for tumor cell viability and 10 CTAs that are pathological drivers of HIF, WNT or TGFb signaling. In particular, we discovered that Fetal and Adult Testis Expressed 1 (FATE1), is a key survival factor in multiple oncogenic backgrounds. FATE1's mechanism of action is to prevent the accumulation of the stress sensing, BH3-only protein, BIK, thereby permitting viability in the presence of toxic stimuli. Importantly, FATE1 expression correlates with significantly decreased survival time in colorectal and lung cancer patients. We also found that the CTA, ZNF165, promotes TGFb signaling by directly inhibiting the expression of negative feedback regulatory pathways. This action is essential for survival of triple negative breast cancer (TNBC) cells in vitro and in vivo. Given these robust phenotypes, we hypothesize that ectopically expressed testes proteins can buttress deviant tumor cell processes that promote tumor initiation and progression.

Citation Format: Angelique Whitehurst. Mechanistic participation of cancer testes antigens in tumor initiation and progression [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr IA18.
Abstract PR04: A CRISPR-based genetic interaction map identifies synergistic drug combinations for cancer
Authors: Han Kyuho , Jeng Edwin , Hess Gaelen , Morgens David , Li Amy , Bassik Michael
Published: December 2017

Identification of effective combination therapies is critical to address the emergence of drug-resistant cancers. Although millions of drug combinations might be created by repurposing existing drugs, direct screening of these combinations is infeasible. Here, we designed a scalable CRISPR-based double knockout (CDKO) system to generate a mammalian genetic interaction (GI) map at unprecedented scale, comprised of 490,000 double-sgRNAs directed against 21,321 pairs of drug targets. We first developed an efficient strategy for cloning and sequencing the libraries, as well as a robust statistical scoring method for calculating GIs from CRISPR-deleted gene pairs. We then extensively validated this system by identifying known and novel genetic interactions in the ricin pathway, and compared the GIs to known protein-protein interactions (PPIs). Using this validated system, we searched for rare synthetic lethal drug target pairs in K562 leukemia cells and identified a number of potent combinations for which corresponding drugs exhibit synergistic killing. Together, this work demonstrates an effective strategy to screen synergistic drug combinations in high throughput, and a powerful CRISPR-based tool to dissect functional genetic interaction networks.

Citation Format: Kyuho Han, Edwin Jeng, Gaelen Hess, David Morgens, Amy Li, Michael Bassik. A CRISPR-based genetic interaction map identifies synergistic drug combinations for cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR04.
Abstract PR07: Collateral sensitivity in chemotherapy resistance
Authors: Dalin Simona , Zhao Boyang , Hemann Michael T.
Published: December 2017

The current 5-year survival rate across all cancers is only 68%. The ability of tumors to develop resistance to chemotherapy is a major factor keeping survival rates down. Understanding the evolutionary paths tumors take towards resistance will aid in creating drug regimens designed to avoid resistant tumor states. Research in bacteria suggests that a specific type of drug pair, where resistance to each drug confers sensitivity to the other drug, can slow evolution toward drug resistance. This drug pair relationship, termed collateral sensitivity, has not been systematically investigated in cancer. Here, using a preclinical Eμ-myc; Arf-/- murine model of Burkitt lymphoma we investigate collateral sensitivities in this malignancy. We derived resistant cell lines in vitro by treating the parental cell line with dose escalating concentrations of doxorubicin or cisplatin. We screened a subset of these resistant cell lines against a panel of drugs and found several collateral sensitivities conferred by doxorubicin or cisplatin resistance. We are currently investigating the mechanism of one of these collateral sensitivities. We plan to derive cell lines resistant to a variety of other chemotherapies to learn about collateral sensitivities across many chemotherapeutics. Collateral sensitivity is a promising avenue towards rationally designing combination therapies that reduce the emergence of chemotherapy resistant disease.

Citation Format: Simona Dalin, Boyang Zhao, Michael T. Hemann. Collateral sensitivity in chemotherapy resistance [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR07.
Abstract PR10: ScreenBEAM: a Novel Meta-Analysis Algorithm for Functional Genomics Screens via Bayesian Hierarchical Modeling
Authors: Yu Jiyang , Silva Jose M. , Califano Andrea
Published: December 2017

Motivation: Functional genomics (FG) screens, using RNAi or CRISPR technology, have become a standard tool for systematic, genome-wide loss-of-function studies for therapeutic target discovery. As in many large-scale assays, however, off-target effects, variable reagents' potency, and experimental noise must be accounted for appropriately control for false positives. Indeed, rigorous statistical analysis of high-throughput FG screening data remains challenging, particularly when integrative analyses are used to combine multiple sh/sgRNAs targeting the same gene in the library.

Method: We use large RNAi and CRISPR repositories that are publicly available to evaluate a novel meta-analysis approach for FG screens via Bayesian hierarchical modeling, ScreenBEAM (Screening Bayesian Evaluation and Analysis Method).

Results: Results from our analysis show that the proposed strategy, which seamlessly combines all available data, robustly outperforms classical algorithms developed for microarray data sets as well as recent approaches designed for next generation sequencing technologies (NGS). Remarkably, the ScreenBEAM algorithm works well even when the quality of FG screens is relatively low, which accounts for about 80-95% of the public datasets.

Availability: R package and source code are available at: https://github.com/jyyu/ScreenBEAM.

Citation Format: Jiyang Yu, Jose M. Silva, Andrea Califano. ScreenBEAM: a Novel Meta-Analysis Algorithm for Functional Genomics Screens via Bayesian Hierarchical Modeling [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR10.
Abstract PR13: CRISPRi screening with targeted therapeutics classifies functional long non-coding RNAs in DLBCL
Authors: Webster Dan E. , Phelan James D. , Kasbekar Monica , Shaffer Arthur L. , Staudt Louis M.
Published: December 2017

While tens of thousands of lncRNAs have been detected in the human genome, the functional significance of the majority of these genes remain untested. Here, we use CRISPR interference (CRISPRi) loss-of-function screening combined with targeted drug treatment to identify oncogenic and tumor suppressive lncRNAs. Integrative analysis of 417 primary human tumor and cell line RNA-seq datasets from Diffuse Large B Cell Lymphoma (DLBCL) identified 1,276 intergenic lncRNA candidates, including 59 de novo assembled transcripts. CRISPRi screening in a genetically diverse panel of 4 DLBCL cell lines identified known ABC DLBCL protein-coding oncogenes (BTK, CD79A) and demonstrated oncogenic function of 247 lncRNAs including MIR155HG and MALAT1. To assign functionally-related pathways to unknown lncRNAs, we performed this CRISPRi screen in the context of drugs with known targets: Ibrutinib (BTK), JQ1 (BRD4), and Etoposide (TOP2A). Significant synergy or resistance to drug treatment was observed with 54 lncRNAs. This ongoing work demonstrates a high-throughput pipeline for functional classification of lncRNAs using synthetic lethality screening in cancer.

Citation Format: Dan E. Webster, James D. Phelan, Monica Kasbekar, Arthur L. Shaffer, III, Louis M. Staudt. CRISPRi screening with targeted therapeutics classifies functional long non-coding RNAs in DLBCL [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR13.
Abstract 04: Caspase-8 is a novel candidate driver gene in head and neck squamous cell carcinoma
Authors: Uzunparmak Burak , Lindemann Antje , Frederick Mitchell J. , Myers Jeffrey N. , Pickering Curtis R.
Published: December 2017

Integrated genomic analysis of head and neck squamous cell carcinoma (HNSCC) has identified a heterogeneous spectrum of alterations dominated by tumor suppressor genes. An interesting new candidate driver gene is Caspase-8 (CASP8), which is mutated in 18% of oral cavity HPV-negative tumors. CASP8 is not obviously a part of any well-characterized driver pathway in HNSCC, and its role in this tumor type is unknown. In TCGA genomic data CAPS8 mutant tumors have a unique spectrum of alterations and expression changes, including a dramatic increase in markers of inflammation, which suggest they are a genomic subtype of HNSCC. CAPS8 is a key component of the extrinsic apoptotic pathway, but it also has a role in necrotic death, inflammation, and other pathways. We have characterized human and mouse CASP8 knock-out cell lines in vitro and in vivo in order to begin to understand the function of this gene in HNSCC. Loss of CASP8 function prevents extrinsic apoptosis and alters proliferation and tumorigenicity. It may also regulate the antitumor immune response. CASP8 is an exciting new candidate driver gene in HNSCC that may offer new opportunities for therapeutic intervention.

Citation Format: Burak Uzunparmak, Antje Lindemann, Mitchell J. Frederick, Jeffrey N. Myers, Curtis R. Pickering. Caspase-8 is a novel candidate driver gene in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 04.
Abstract 28: Identifying mechanisms of PD-L1 regulation in HNSCC
Authors: Mann Jacqueline E. , Ludwig Megan , Kulkarni Aditi , Hoesli Rebecca , Kafelghazal Judy , Nesvizhskii Alexey , Brenner J. Chad
Published: December 2017

Evidence of immunosuppression is frequently reported in head and neck squamous cell carcinoma (HNSCC). Thus, there has been growing interest in advancing therapies for HNSCC that relieving immunosuppression to permit anti-tumor immunity. The immune checkpoint inhibitor Pembrolizumab targets the coinhibitory PD-1/PD-L1 pathway, which serves to shield tumor cells from attack by cytotoxic T lymphocytes. While Pembrolizumab was recently approved for the treatment of metastatic and recurrent HNSCC, the initial trial reported overall response rates of only 18%. A more comprehensive understanding of the intrinsic and extrinsic mechanisms inducing PD-L1 expression on tumor cells may reveal therapeutic targets that improve response to immune modulation. We have employed a high-throughput genetic screen to identify targetable signaling pathways that may regulate PD-L1 expression in patients with specific genetic lesions. The Genome-scale CRISPR Knock-Out (GeCKO) library, established in multiple HNSCC cell lines, consists of a pool of stable knockouts targeting >20,000 genes with representation of approximately 300 gRNAs per target gene. Sub-populations with enhanced or diminished expression of PD-L1 were sequenced to identify gRNA targets whose knockout causes a change in PD-L1 expression. We expect that by performing these screens across multiple cell lines with diverse genetic lesions, pathways consistently regulating PD-L1 expression will be revealed, and that targeting such pathways will provide novel avenues for modulating this immunosuppressive mechanism.

Citation Format: Jacqueline E. Mann, Megan Ludwig, Aditi Kulkarni, Rebecca Hoesli, Judy Kafelghazal, Alexey Nesvizhskii, J. Chad Brenner. Identifying mechanisms of PD-L1 regulation in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 28.
Abstract 30: Small molecule profiling uncovers the landscape of combinatorial PI3K inhibitor responses in HNSCC
Authors: Michmerhuizen Nicole L. , Leonard Elizabeth , Harris Micah , Foltin Susan K. , Kulkarni Aditi , Carey Thomas E. , Bradford Carol R. , Hui Jiang , Brenner J. Chad
Published: December 2017

Recent sequencing studies of head and neck squamous cell carcinomas (HNSCCs) have identified the phosphatidylinositol 3-kinase (PI3K) pathway as the most frequently mutated oncogenic pathway in this cancer type. Although activating mutations or amplification in PIK3CA (the gene encoding the catalytic subunit of PI3K) are frequently observed in HNSCC, monotherapies targeting PI3K have shown limited clinical efficacy. We tested a panel of more than 20 patient-derived oral cavity squamous cell carcinoma (OCSCC) cell lines, which were profiled by Nimblegen V3 exome sequencing. Despite PIK3CA copy number amplification and/or mutations, we observed resistance to PI3K inhibitors in these models. Of six inhibitors tested, the only agents with marginal effects were alpha-isoform selective and pan-PI3K agents; these inhibitors, despite on-target and downstream activity, did not cause an appreciable reduction in cell viability when administered at submicromolar concentrations. We hypothesized that other oncogenic pathways, such as the Ras-MEK-ERK pathway, might still be functional in the presence of PI3K inhibitors and serve as compensatory mechanisms of resistance to these drugs. In order to more fully characterize other novel co-dependent pathway in PI3K inhibitor resistant HNSCC models, we have developed a high-throughput screening approach that utilizes a resazurin cell viability assay. This approach is an unbiased means of testing ~1400 inhibitors as monotherapies and in combination with alpha-isoform selective PI3K inhibitor HS-173 and pan-PI3K inhibitor BKM120 and has been utilized in several in vitro models of OCSCC to date. Testing of additional cellular models and validation of “hits” from this screen, which are drugs that are effective in combination but not as monotherapies, may identify additional resistance mechanisms and are currently ongoing. Developing a better understanding of these combination therapies and the patients in which they might be most effective may lead to improved prognosis for this disease.

Citation Format: Nicole L. Michmerhuizen, Elizabeth Leonard, Micah Harris, Susan K. Foltin, Aditi Kulkarni, Thomas E. Carey, Carol R. Bradford, Jiang Hui, J. Chad Brenner. Small molecule profiling uncovers the landscape of combinatorial PI3K inhibitor responses in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 30.
Abstract 48: Targeting IL-1 signaling to improve tumor response to erlotinib in HNSCC
Authors: Simons Andrean , Stanam Aditya , Koch Adam , Espinosa-Cotton Madelyn , Gibson-Corley Katherine
Published: December 2017

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) including erlotinib have demonstrated poor clinical response rates for head and neck squamous cell carcinoma (HNSCC) to date compared to the EGFR monoclonal antibody cetuximab. Therefore we seek to identify molecular mechanisms involved in tumor response to erlotinib and ultimately improve the anti-tumor efficacy of erlotinib for HNSCC patients. We have shown that erlotinib-treated HNSCC cell lines induced the expression and secretion of a wide variety of genes involved in inflammation and immune response including interleukin-1 (IL-1) and interleukin-6 (IL-6) compared to control-treated cells. The secretion of IL-6 from HNSCC cells in response to erlotinib was found to be triggered by MyD88-dependent IL-1 signaling and suppression of IL-1 signaling (using a neutralizing IL-1α antibody) significantly enhanced the anti-tumor efficacy of erlotinib in vivo. In erlotinib-resistant HNSCC cell lines, IL-1 signaling was significantly upregulated compared to their respective parental cell lines. Blockade of IL-1 signaling using a neutralizing IL-1α antibody or an IL-1 receptor (IL-1R) antagonist was able to significantly suppress the growth of erlotinib-resistant HNSCC xenografts as a single agent and in combination with erlotinib which was associated with reduced immune cell recruitment and angiogenesis. Altogether, blockade of the IL-1 pathway may represent a novel strategy to improve tumor response to erlotinib and other EGFR TKIs in HNSCC patients.

Citation Format: Andrean Simons, Aditya Stanam, Adam Koch, Madelyn Espinosa-Cotton, Katherine Gibson-Corley. Targeting IL-1 signaling to improve tumor response to erlotinib in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 48.
Abstract 55: SET protein as a therapeutic target in head and neck squamous cell carcinoma
Authors: Goto Renata Nishida , Stringhetta-Padovani Karina , Vitek Michael , Curti Carlos , Leopoldino Andreia Machado
Published: December 2017

Introduction: The study of cancer molecular profiles allowed the development of several targeting agents, such as OP449 and FTY720/Fingolimod drugs, that can act as PP2A activators. OP449 is a synthetic peptide with antineoplastic action and FTY720 is a potent inhibitor of tumor growth and angiogenesis. SET/I2PP2A, a multifunctional protein, is a potent and specific inhibitor of PP2A and is upregulated in head and neck squamous cell carcinoma (HNSCC). SET has been proposed to be an oxidative stress sensor, promoting cell survival and apoptosis. Both OP449 and FTY720 bind to SET and destabilize SET/PP2A complex, reactivating the PP2A function.

Objectives: The aim was to evaluate the antitumoral effect, in vitro and in vivo, of the association OP449 plus FTY720, in HNSCC.

Material and Methods: HNSCC lineages were analyzed for SET protein status by Western blotting and for cancer stem cell population by using aldehyde dehydrogenase 1 (ALDH1) activity assay. Alterations in SET targets were confirmed by Western blotting. The cytotoxic effects of OP449 and/or FTY720, under different concentrations, were determined after 72 hours through the resazurin assay. The coefficient of drug interaction (CDI) was used to analyze the synergistic inhibitory effect of drug combination. Xenograft tumor model in Balb/c nude mice (n=5 per group), by subcutaneous injection of cells into the flank of the mouse, was used to investigate the antitumoral potential of treatment with OP449 and/or FTY720, every 48 h, for 15 days.

Results and Discussion: HN12 cells were selected due to the higher levels of SET and higher ALDH1 activity compared to other HNSCC cells. The treatment of HN12 cells with OP449 altered the levels of SET targets. A significant synergistic effect was observed when cells were pre-treated with 2 μM OP449 followed by 15 μM FTY720 (CDI=0.27 ± 0.088). Nude mice bearing HN12 human xenograft tumors treated with OP449 (5 mg/kg) and/or FTY720 (2 mg/kg) showed reduction in tumor growth compared to either control (vehicle) or compounds isolated.

Conclusion: Our results indicate that OP449 and FTY720 have a synergistic antitumoral effect, opening perspectives for either alternating or combined therapies in HNSCC, having SET protein as a target.

Financial support: FAPESP, CNPq and CAPES.

Citation Format: Renata Nishida Goto, Karina Stringhetta-Padovani, Michael Vitek, Carlos Curti, Andreia Machado Leopoldino. SET protein as a therapeutic target in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 55.
Abstract 66: c-Fos: A new player in promotion of cancer stem-like cell properties in head and neck squamous cell carcinoma
Authors: Ray Ratna B.
Published: December 2017

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. We have shown that c-Fos was highly up-regulated in the HNSCC sphere forming cells. However, upregulation of c-Fos for the biology of HNSCC-CSCs remains unknown. In this study, we investigated the role of c-Fos in renewal of stemness of HNSCC and tumor growth.

We demonstrated that c-Fos overexpression enhanced the epithelial-mesenchymal transition (EMT) state and expression of CSC markers (Nanog, c-Myc, Sox2 and Notch1). Ectopic expression of c-Fos in HNSCC cells also display increased number of sphere formation. We further observed that overexpression of c-Fos increased the expression of pERK and cyclin D1 in HNSCC cells. Knockdown of c-Fos in Cal27 cells impaired sphere formation. Next we examine the effect of c-Fos expression in vivo. For this, we established stable HNSCC cell line (MDA1386Tu cells) where c-Fos gene is ectopically expressed. Our results demonstrated that exogenous expression of c-Fos in non-tumorigenic MDA1386Tu cells makes these cells tumorigenic in nude mice. Further, subcutaneous transplantation of c-Fos overexpressing Cal27 cells (tumorigenic) into immunocompromised mice enhanced tumor growth as compared to parental cells. Taken together, our results strongly demonstrated a novel role of c-Fos as a regulator of EMT and cancer stem cell reprogramming in HNSCC cells.

Collectively, our results offer that c-Fos may be a new target for developing therapeutic strategies against HNSCC.

Citation Format: Ratna B. Ray. c-Fos: A new player in promotion of cancer stem-like cell properties in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 66.
Abstract IA05: Immunoprevention of oral cancers
Authors: William William N.
Published: December 2017

Oral pre-malignant lesions (OPLs) are usually clinically characterized as leukoplakia or erythhroplakia, and confer an increased risk of oral cancer. Despite decades of clinical investigations with candidate chemopreventive agents such as 13-cis-retinoic acid, beta-carotene, fenretinide, green-tea extract, celecoxib, erlotinib, among others, a standard-of-care chemoprevention strategy for oral cancers has not yet been developed. Importantly, OPLs and oral cavity cancers are not related to HPV, thus HPV vaccines are not expected to be effective in this setting.

One of the greatest challenges in managing patients with OPLs is determining risk for malignant transformation and need for intervention. To that end, retrospective and prospective studies have been completed by our group and others and demonstrate that LOH profiles (primarily at 13p14 and/or 9p21) in OPLs are associated with higher incidence of oral cancers. LOH is now considered the most robust prognostic marker of cancer risk in this setting, and these data now set the stage for development of clinical trials focused on molecularly-defined high risk groups of OPLs.

Immune checkpoint blockade with antibodies directed at programmed cell death protein 1 (PD-1) has recently been successfully developed for the management of advanced head and neck squamous cell carcinomas, validating the importance of immune modulation in the progression of these invasive tumors. Given the favorable toxicity profile, there is interest in studying these agents for prevention of oral cancers in patients with high risk OPLs.

In this presentation, we will review the data that support a role for immune surveillance and immune editing in the development of oral cancers, and describe the rationale and clinical trial design of the Immunoprevention of Oral Cancer (IPOC) study, the first randomized trial to evaluate the PD-1 inhibitor pembrolizumab for oral cancer prevention in patients with molecularly-defined high-risk OPLs.

Citation Format: William N. William, Jr. Immunoprevention of oral cancers [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr IA05.
Abstract PR04: HER3 crosstalk with HPV16-E6E7 is a feedback resistance mechanism to PI3K-targeted therapies in head and neck cancer
Authors: Brand Toni M. , Hartmann Stefan , Bhola Neil E. , Li Hua , Zeng Yan , O'Keefe Rachel , Ranall Max V. , Bandyopadhyay Sourav , Soucheray Margaret , Swaney Danielle L. , Krogan Nevan , Kemp Carolyn , Duvvuri Umamaheswar , Johnson Daniel E. , Ozbun Michelle A. , Bauman Julie E. , Grandis Jennifer R.
Published: December 2017

Human papillomavirus (HPV) 16 plays an etiologic role in a growing subset of head and neck squamous cell carcinoma (HNSCC), where viral expression of the E6 and E7 oncoproteins is necessary for tumor growth and maintenance. Although patients with HPV(+) tumors have a more favorable prognosis, there are currently no HPV-selective therapies. Accumulating evidence indicates that HPV oncoproteins can activate the Phosphoinositol-3-Kinase (PI3K) pathway, which contributes to transformation. Furthermore, PI3K is genomically activated by PIK3CA mutation or amplification in a disproportionately high number of HPV(+) tumors as compared to HPV(-) tumors. Based on this knowledge, we investigated the efficacy of PI3K-targeted therapies in preclinical models of HPV(+) HNSCC. Our results indicate that HPV(+) preclinical models were less sensitive to the PI3K inhibitors BYL719, BKM120, and BEZ235 as compared with HPV(-) models. Sensitivity of HPV(+) cell lines to PI3K inhibitors was increased upon knockdown of the E6 and E7 oncoproteins. Reciprocally, overexpression of E6 and E7 in HPV(-) cells rendered them resistant to PI3K-targeted therapies. Proteomic analyses indicated that treatment of HPV(+) cell lines and patient-derived xenografts (PDXs) with the PI3Kα inhibitor BYL719 induced expression of the receptor tyrosine kinase HER3, as well as E6 and E7. HER3 was found to regulate the abundance of E6 and E7 in the HPV(+) models. Targeting HER3 with siRNAs or the monoclonal antibody, KTN3379, blocked the increase in E6 and E7 protein levels following BYL719 treatment, and enhanced the efficacy of PI3K inhibitors in HPV(+) cell line and PDX models. Taken together, these results suggest that crosstalk between HER3 and HPV16-E6E7 can limit the efficacy of PI3K inhibitors, and that co-targeting HER3 and PI3K may be an effective therapeutic strategy in HPV(+) tumors.

This abstract is also being presented as Poster 67.

Citation Format: Toni M. Brand, Stefan Hartmann, Neil E. Bhola, Hua Li, Yan Zeng, Rachel O'Keefe, Max V. Ranall, Sourav Bandyopadhyay, Margaret Soucheray, Danielle L. Swaney, Nevan Krogan, Carolyn Kemp, Umamaheswar Duvvuri, Daniel E. Johnson, Michelle A. Ozbun, Julie E. Bauman, Jennifer R. Grandis. HER3 crosstalk with HPV16-E6E7 is a feedback resistance mechanism to PI3K-targeted therapies in head and neck cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr PR04.
Epilepsy among Systemic Lupus Erythematosus Patients: Insights from a Large Database Analysis
Authors: Watad Abdulla , Tiosano Shmuel , Bragazzi Nicola Luigi , Brigo Francesco , Comaneshter Doron , Cohen Arnon D. , Amital Howard
Published: December 2017

Objective: Epilepsy is characterized by a relevant epidemiological and clinical burden. In the extant literature, an increased risk of seizures has been described in several inflammatory/autoimmune disorders, including systemic lupus erythematosus (SLE). However, so far, relatively few and small size-based studies have been conducted. We aimed to investigate the link between seizure and SLE utilizing a large sample of subjects and extensive data analysis. Methods: Patients with SLE were compared with age- and sex-matched controls regarding the prevalence of epilepsy in a cross-sectional study. Chi-square and t tests were used for univariate analysis and a logistic regression model was used for multivariate analysis. The study was performed utilizing the medical database of Clalit Health Services. Results: The study included 5,018 patients with SLE and 25,090 age- and gender-frequency-matched controls. The proportion of epilepsy was found significantly higher among SLE patients (4.03 vs. 0.87%, p < 0.001). Using logistic regression, adjusting for multiple confounding factors, older age (≥70 years) resulted as negative predictor (OR 0.42 [95% CI 0.27–0.62], p <0.001), whereas the presence of SLE was a positive predictor of epilepsy (OR 4.70 [95% CI 3.94–5.82], p < 0.001). Interaction between SLE and elderly age resulted in high OR of 5.47 for epilepsy (95% CI 2.53–11.9). Conclusion: Our study confirms the higher prevalence of epilepsy in SLE patients. Physicians should be aware of such findings and have a lower threshold for suspecting epileptic seizures in these patients. Further studies are needed to better elucidate the mechanisms by which SLE favors the insurgence of seizures.
MiR-106b-5p Promotes Proliferation and Inhibits Apoptosis by Regulating BTG3 in Non-Small Cell Lung Cancer
Authors: Wei Ke , Pan Chunfeng , Yao Guoliang , Liu Bin , Ma Teng , Xia Yang , Jiang Wei , Chen Liang , Chen Yijiang
Published: December 2017

Background/Aims: MicroRNAs have been validated to play a crucial role in tumorigenesis of non-small cell lung cancer (NSCLC). Although miR-106b-5p has been reported to play a vital role in various malignancies the physiological function of miR-106b-5p in NSCLC still remain unknown. In this study, we investigated the role of miR-106b-5p in NSCLC. Methods: Quantitative real-time polymerase chain reaction was conducted to estimate the expression of miR-106b-5p and BTG3 in both NSCLC tissues and cell lines. The effects of miR-106b-5p on proliferation were determined in vitro using CCK-8 proliferation assays, 5-ethynyl-2’-deoxyuridine (EdU) incorporation, colony formation assays and cell-cycle assays and the in vivo effects were evaluated by a mouse tumorigenicity model. Cell apoptosis and cell cycle was investigated by flow cytometric analysis in vitro. The molecular mechanism underlying the relevance between miR-106b-5p and BTG3 was confirmed by luciferase assay and western blot. Results: In current study, we found a relatively higher miR-106b-5p and lower BTG3 expression in NSCLC specimens and cell lines. BTG3 was verified as a direct target of miR-106b-5p by luciferase assay. In vitro, over-expression of miR-106b-5p promoted proliferation and inhibited apoptosis by down-regulating BTG3 expression. In vivo, miR-106b-5p promoted xenograft tumor formation. Conclusion: Our findings revealed for the first time that miR-106b-5p plays a tumorigenesis role in NSCLC progression by down-regulating BTG3 expression, which may lead to a novel insight to the potential biomarker and novel therapeutic strategies for NSCLC patients.
Functional Characterization of a Novel Truncating Mutation in Lamin A/C Gene in a Family with a Severe Cardiomyopathy with Conduction Defects
Authors: Gerbino Andrea , Bottillo Irene , Milano Serena , Lipari Martina , Zio Roberta De , Morlino Silvia , Mola Maria Grazia , Procino Giuseppe , Re Federica , Zachara Elisabetta , Grammatico Paola , Svelto Maria , Carmosino Monica
Published: December 2017

Background/Aims: Truncating LMNA gene mutations occur in many inherited cardiomyopathy cases, but the molecular mechanisms involved in the disease they cause have not yet been systematically investigated. Here, we studied a novel frameshift LMNA variant (p.D243Gfs*4) identified in three members of an Italian family co-segregating with a severe form of cardiomyopathy with conduction defects. Methods: HEK293 cells and HL-1 cardiomyocytes were transiently transfected with either Lamin A or D243Gfs*4 tagged with GFP (or mCherry). D243Gfs*4 expression, cellular localization and its effects on diverse cellular mechanisms were evaluated with western blotting, laser-scanning confocal microscopy and video-imaging analysis in single cells. Results: When expressed in HEK293 cells, GFP- (or mCherry)-tagged LMNA D243Gfs*4 colocalized with calnexin within the ER. ER mislocalization of LMNA D243Gfs*4 did not significantly induce ER stress response, abnormal Ca2+ handling and apoptosis when compared with HEK293 cells expressing another truncated mutant of LMNA (R321X) which similarly accumulates within the ER. Of note, HEK293-LMNA D243Gfs*4 cells showed a significant reduction of connexin 43 (CX43) expression level, which was completely rescued by activation of the WNT/β-catenin signaling pathway. When expressed in HL-1 cardiomyocytes, D243Gfs*4 significantly impaired the spontaneous Ca2+ oscillations recorded in these cells as result of propagation of the depolarizing waves through the gap junctions between non-transfected cells surrounding a cell harboring the mutation. Furthermore, mCh-D243Gfs*4 HL-1 cardiomyocytes showed reduced CX43-dependent Lucifer Yellow (LY) loading and propagation. Of note, activation of β-catenin rescued both LY loading and LMNA D243Gfs*4 -HL-1 cells spontaneous activity propagation. Conclusion: Overall, the present results clearly indicate the involvement of the aberrant CX43 expression/activity as a pathogenic mechanism for the conduction defects associated to this LMNA truncating alteration.
Paradigms of Participation in Origen
Authors: Biriukov Dmitry
Published: December 2017

The article provides a classification of different kinds of participation in the conceptual framework by Origen, namely, those of natural participation and those of individual participation. In these concepts, Origen did not always borrow from Platonic philosophy – as it was commonly thought. In fact, Origen used as well Aristotelian and other approaches to the universalia and related questions.
Philoponus’ Dispute Against the Eternity of the World and Its Influence on the Byzantine Philosophy
Authors: Varlamova Maria
Published: December 2017

The article deals with the philosophical and theological context of the dispute against the eternity of the world and studies how Philoponus’ arguments against the eternity of the world were seen in Byzantine philosophy and theology. Special attention is paid to Philoponus’ influence on Byzantine philosophy in such aspects as the concept of matter, the doctrine of the logoi and the relation of the finite to the infinite. In order to demonstrate the influence of Philoponus’ arguments against the eternity of the world the article compares his doctrine with the doctrines of John Damascene and Maximus the Confessor.
Erratum: Erst, A.S., Wang, W., Yu, S.X., Xiang, K.L., Wang, J., Shaulo, D.N., Smirnov, S.V., Kushunina, M., Sukhorukov, A.P. & Nobis, M. (2017) Two new species and four new records of Aquilegia (Ranunculaceae) from China. Phytotaxa 316 (2): 121–137.
Authors: ERST ANDREY S. , WANG WEI , YU SHENG-XIANG , XIANG KUNLI , WANG JIAN , SHAULO DMITRY N. , SMIRNOV SERGEY V. , KUSHUNINA MARIA , SUKHORUKOV ALEXANDER P. , NOBIS MARCIN
Published: December 2017

N/A
Pleroma carajasense (Melastomataceae), a new species endemic to ironstone outcrops in the Brazilian Amazon
Authors: ROCHA KEILA CRISTINA DE JESUS , GOLDENBERG RENATO , VIANA PEDRO LAGE , MEYER FABRÍCIO SCHMITZ
Published: December 2017

A new species of Pleroma from Serra dos Carajás, Pará state, Brazil, is described. Pleroma carajasense occurs in southeastern Amazonia, northern Brazil, in canga vegetation, on ironstone outcrops. It is morphologically related to Tibouchina caatingae, because both are shrubs with elliptic to oval leaves, conspicuous petioles (3–17 mm and 10–15 mm, respectively), a pair of elliptic bracteoles, and purple petals, anthers (both cycles) and style. Pleroma carajasense differs from T. caatingae by its chartaceous leaves (versus coriaceous in T. caatingae), with an obtuse to cuneate base (vs. obtuse to subcordate), hypanthium 4–7 × 1.2–4 mm (vs. 10–15 × 3–5 mm). According to IUCN criteria, this species should be considered endangered (EN). A morphological description, illustrations, photos in the field, scanning electronic micrographs of the leaf indumentum and seeds, information on conservation status and distribution are presented.