bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5278: NGS-SWIFT: A cloud-based variant analysis framework using control-accessed sequencing data from dbGaP/SRA
Authors: Xiao Chunlin , Yaschenko Eugene , Sherry Stephen
Published: July 2016

Genetic variation analysis plays an important role in elucidating the causes of various human diseases. The drastically reduced costs of genome sequencing driven by next generation sequence technologies now make it possible to analyze genetic variations with hundreds or thousands of samples simultaneously, but with the cost of ever increasing local storage requirements. The tera- and peta-byte scale footprint for sequence data imposes significant technical challenges for data management and analysis, including the tasks of collection, storage, transfer, sharing, and privacy protection. Currently, each analysis group must download all the relevant sequence data into a local file system before variation analysis is initiated. This heavy-weight transaction not only slows down the pace of the analysis, but also creates financial burdens for researchers due to the cost of hardware and time required to transfer the data over typical academic internet connections. To overcome such limitations and explore the feasibility of analyzing control-accessed sequencing data in cloud environment while maintaining data privacy and security, here we introduce a cloud-based analysis framework that facilitates variation analysis using direct access to the NCBI Sequence Read Archive through NCBI SRA Toolkit, which allows the users to programmatically access data housed within SRA with encryption and decryption capabilities and converts it from the SRA format to the desired format for data analysis. A customized machine image (ngs-swift) with preconfigured tools, including NCBI SRA Toolkit and NGS Software Development Kit, and resources essential for variant analysis has been created for instantiating an EC2 instance or instance cluster on Amazon cloud. Performance of this framework has been evaluated using dbGaP study phs000710.v1.p1 (1000Genome Dataset in dbGaP, http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id = phs000710.v1.p1), and compared with that from traditional analysis pipeline, and security handling in cloud environment when dealing with control-accessed sequence data has been addressed. We demonstrate that with this framework, it is cost effective to make variant calls without first transferring the entire set of aligned sequence data into a local storage environment, thereby accelerating variant discovery using control-accessed sequencing data.

Citation Format: Chunlin Xiao, Eugene Yaschenko, Stephen Sherry. NGS-SWIFT: A cloud-based variant analysis framework using control-accessed sequencing data from dbGaP/SRA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5278.
Abstract 528: Identify and prioritize candidate neoantigens from cancer exome sequencing with unmatched accuracy
Authors: White James , Angiuoli Sam , Sausen Mark , Jones Sian , Kann Lisa , Shukla Manish , Sevdali Maria , Velculescu Victor , Diaz Luis , Zhang Theresa
Published: July 2016

Somatic, nonsynonymous genetic alterations present in cancer can lead to the formation of novel protein sequences and thus production of immunogenic “non-self” neoantigens. Some of those neoantigens will be processed, presented on MHC molecules, and induce tumor-specific T cell responses. Because neoantigens play central roles in the cancer-immunity cycle, it is critical to identify the most potent immunogenic neoantigens effectively and accurately.

Combining PGDx's highly accurate cancer exome analyses (CancerXome™) with in silico neoantigen prediction, we have launched ImmunoSelect-R™ that identifies and prioritizes the most relevant mutation-derived neoantigens. To ensure detection of true somatic mutations and prevent false positive mutations from confounding neoantigen identification, ImmunoSelect utilizes CancerXome that delivers unparalleled cancer whole exome sequencing accuracy, achieving 95% sensitivity and 97% positive predictive value at 10% mutant allele frequency with 150x coverage. ImmunoSelect also provides accurate HLA typing from whole exome sequencing with >99.9% sensitivity and specificity. Once exome-based mutations and novel open-reading-frames are identified and HLA genotypes defined, ImmunoSelect utilizes state of art bioinformatics pipelines for prediction and prioritization of the most relevant neoantigens. When applied to a set of experimentally validated neoantigens, ImmunoSelect identified 18 out of 19 of them as being strong neoantigen candidates, suggesting a sensitivity of greater than 90%. Moreover, ImmunoSelect consistently ranked experimentally validated neoantigens within top 20% of all neoantigen candidates derived from whole exome sequencing.

In summary, ImmunoSelect is able to identify and prioritize candidate neoantigens from cancer exome sequencing results effectively and accurately, enabling personalized cancer vaccine development, adoptive T-cell transfer, and prediction of response to checkpoint inhibitors

Citation Format: James White, Sam Angiuoli, Mark Sausen, Sian Jones, Lisa Kann, Manish Shukla, Maria Sevdali, Victor Velculescu, Luis Diaz, Theresa Zhang. Identify and prioritize candidate neoantigens from cancer exome sequencing with unmatched accuracy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 528.
Abstract 5280: A web-based platform for exploring ‘omics and clinical data from healthy breast tissues
Authors: Kuang Xingyan , Marian Catalin , Taslim Cenny , Song Min-Ae , Weng Daniel , Freudenheim Jo L. , Brasky Theodore , Huang Kun , Coombes Kevin , Shields Peter G.
Published: July 2016

Integrating ‘omics, epidemiology and clinical data from healthy patients and their biologic samples is important for identifying the determinants of early carcinogenesis. In a cross-sectional study of 249 women with no history of cancer and who underwent reduction mammoplasty, whole transcriptome (i.e., gene expression) and epigenome (i.e., methylation and microRNA) data were collected from dissected breast tissues. An extensive epidemiologic questionnaire ascertained data on breast cancer risk factors. Subjects with benign lesions considered at risk for future cancer were excluded. In order to manage, analyze, and share these data efficiently, we organized the data into a relational database to support a series of web-based tools for analyzing and visualizing the multidimensional data in an intuitive graphical user interface (GUI). The current platform improves upon existing web resources that integrate ‘omics and clinical data by including novel features that allow for greater flexibility of bioinformatics analyses. It also focuses on the ‘omics as an outcome, rather, e.g., what epidemiology risk factor or particular gene is associated with downstream effects across the ‘omics. Such features include: 1) a plot function that can visualize up to 4 dimension data chosen by a user from the 3 types of genetic profiles and more than 100 clinical and biomedical variables; 2) a differentiation visualization tool based on the ‘omics data of 2 groups of samples; and 3) miRNA target gene network generated by the correlations between gene expression and miRNA expression of our patient samples and validated miRNA target gene database. The GUI facilitates the discovery of research scientists by reducing complex biological and clinical data into easily understandable views. Even without the expertise of computational biology, researchers can integrate and analyze the high dimensional data that they are interested conveniently on our user-friendly designed platform.

Citation Format: Xingyan Kuang, Catalin Marian, Cenny Taslim, Min-Ae Song, Daniel Weng, Jo L. Freudenheim, Theodore Brasky, Kun Huang, Kevin Coombes, Peter G. Shields. A web-based platform for exploring ‘omics and clinical data from healthy breast tissues. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5280.
Abstract 5284: Characterizing the differences of allelic imbalance between tumor and normal tissues by next-generation sequencing
Authors: Jheng Yao-Wei , Chiu Yu-Chiao , Lu Tzu-Pin , Lai Liang-Chuan , Tsai Mong-Hsun , Hsiao Tzu-Hung , Chuang Eric Y
Published: July 2016

Genetic variation has proved to contribute to tumor formation. One of the phenomenon caused by genetic variation, allelic imbalance, means different expression abundance between two alleles, recently has become a special landmark on cancer biology, with high prevalence in recent cancer studies. However, the present of allelic imbalance has not been fully understood yet. Taking the advantages of next generation sequencing, allelic imbalance can be identified and quantified through bioinformatics algorithms. Here, we developed a mathematical model to identify with allelic imbalance by concurrently analyzing RNA-Seq and DNA-Seq. First we called heterozygous SNP variants from both RNA-Seq and Exome-Seq data. The allele ratio is counted by the ratio of read depth between two alleles after integrating SNPs into gene level. Next, we identify allelic imbalance genes by differences of RNA and DNA allele ratios between tumor and normal samples. As results, a total of 91143 SNPs were identified in Exome-Seq, and about 3% (2586 out of 91143) SNPs were heterozygous in both tumor and normal tissues. We found that about 118 allelic imbalance genes in tumor tissue are significantly different from those in normal tissue. Furthermore, we investigate the enriched function of these allelic imbalance genes. These result showed that these genes are significantly enriched in DNA repair pathway. We speculated that allelic imbalance in tumor tissue may be involved in the dysregulation of DNA repair. Also other enriched functions were identified, such as z-finger protein pathway.

Our goal is to investigate the landscape of allelic imbalance between tumor sample and normal sample. Our results demonstrate the role of allelic imbalance of dysregulation of tumor. Moreover, our method can be applied to uncover more basic mechanisms on allelic imbalance and tumorigenesis.

Citation Format: Yao-Wei Jheng, Yu-Chiao Chiu, Tzu-Pin Lu, Liang-Chuan Lai, Mong-Hsun Tsai, Tzu-Hung Hsiao, Eric Y Chuang. Characterizing the differences of allelic imbalance between tumor and normal tissues by next-generation sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5284.
Abstract 5291: OncoMD: A powerful genomics data analysis and interpretation platform for cancer discovery research
Authors: Gupta Ravi , Paul Kiran V. , Kumaramangalam Kartik , Santhosh Sam , Chaudhuri Amitabha
Published: July 2016

Cancer cells gain fitness advantage over normal cells by accumulating mutations over time. Next Generation DNA sequencing technologies have made the detection of genetic alterations in tumor genomes affordable. Powerful data analysis and interpretation tools enable extraction of disease-relevant information from diverse genomics data to guide diagnosis and treatment decisions. We have built a robust cancer analytics platform OncoMD that combines tumor mutation profiles, expression signatures, copy number variations, epigenetic alterations and drug sensitivity to create a holistic view of human cancer enabling discovery of new targets for therapy and prognosis. We have analyzed the rich mutational data captured in OncoMD to identify potential T-cell neo-epitopes in different cancers and examined their prevalence in the context of the tumor microenvironment. The presence of T-cell neo-epitopes alone is not sufficient to sensitize tumors to immunotherapy drugs and requires cooperation from the tumor microenvironment. Although mutational burden was a strong predictor of T-cell neo-epitopes in our analysis, other genetic alterations such as overexpression, splicing, gene-fusions and insertions/deletions can produce altered peptides exhibiting high affinity binding to HLA class I and II molecules in cancers with low burden of missense mutations. We determined the number of potential T-cell neo-epitopes after classifying tumors based on their epithelial, stromal and immune cell content using gene expression signatures. We observed that the normalized T-cell neo-epitope burden was strongly anti-correlated with the T-cell content of the tumors, compared to their epithelial or stromal content. Together, our findings suggest that tumors enriched in T-cells may be subjected to immunoediting processes and carry relatively fewer T-cell neo-epitopes to avoid elimination. These tumors are likely to respond to immunotherapy treatments.

Citation Format: Ravi Gupta, Kiran V. Paul, Kartik Kumaramangalam, Sam Santhosh, Amitabha Chaudhuri. OncoMD: A powerful genomics data analysis and interpretation platform for cancer discovery research. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5291.
Abstract 536: Excessive HCK kinase activity in the tumor stroma promotes colorectal cancer progression
Authors: Ernst Matthias , Poh Ashleigh , Masson Frederic , Putoczki Tracy , O’Donoghue Robert
Published: July 2016

Excessive activation of the myeloid-specific Src-family kinase Hematopoietic Cell Kinase (HCK) acts as a tumor intrinsic oncogene by promoting proliferation and survival of immune cells, and confers a poor prognosis for leukemia. However, the role of HCK in the tumor stroma of solid malignancies remains unexplored.

We analyzed the expression level of HCK in matched biopsies from sporadic colorectal cancer patients and observed elevated HCK phosphorylation in tumors compared to unaffected colons. Analysis of corresponding RNAseq data revealed a striking correlation between tumors with high HCK gene expression and a gene signature indicative of a tumor-promoting alternatively-activated macrophage (AAM) endotype. To functionally assess this observation, we exploited HckCA mice [Ernst et al., J Exp Med 2002] that express a constitutively active form of Hck as a knockin mutation, and subjected these mice to a chemically-induced model of sporadic colorectal cancer. HckCA mice developed more and larger tumors compared to wild-type (WT) animals, and this was associated with a significant bias towards CD206+ AAMs in tumors of HckCA mice without affecting the total number of tumor-associated leukocyte and lymphocytes. Likewise, adoptive bone-marrow transfer experiments revealed enhanced tumor formation and AAM differentiation in WT mice reconstituted with HckCA bone marrow, with a reciprocal decrease of these parameters in HckCA mice reconstituted with WT marrow. Finally, pharmacologic targeting of the catalytic activity of Hck with a small molecule inhibitor significantly reduced tumor progression and impaired AAM polarisation.

Collectively, our findings suggest that excessive Hck activity in the tumor stroma promotes the progression of solid cancers by modulating the endotype of tumor-associated myeloid cells. Therefore, Hck represents a rational therapeutic target for macrophage re-education in solid cancers by limiting polarization of tumor-promoting AAM.

Citation Format: Matthias Ernst, Ashleigh Poh, Frederic Masson, Tracy Putoczki, Robert O’Donoghue. Excessive HCK kinase activity in the tumor stroma promotes colorectal cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 536.
Abstract 542: Assessing and augmenting the immune response to glioblastoma using repurposed pharmaceuticals
Authors: Brenneman Breanna R. , Floyd Desiree H. , Harris Tajie , Purow Benjamin
Published: July 2016

In order to improve the therapeutic responses to immunotherapies in glioblastoma (GBM) patients, a known pharmaceutical, ritanserin, was repurposed to augment cytotoxic T cell responses and suppress tumor growth. To determine the kinetics of the immune response to GBM, the transplantable syngeneic glioma cell line GL261 was used in C57BL/6 mice to assess the tumor immune response in the brain, spleen, and cervical lymph node via flow cytometry weekly for one month. After identifying the optimal T cell response temporally, mice with established tumors were treated with 100mg/kg of Ritanserin for one week and harvested post treatment. Ritanserin is able to inhibit the activity of diacylglycerol kinase alpha (DGKA), an enzyme that converts diacylglycerol to phosphatidic acid, and can inhibit tumor growth in mice. In addition, DGKA is expressed in T cells, and is more highly expressed in those that have entered an unresponsive (anergic) state, characteristic of dysfunctional T cell responses to tumors. To determine if ritanserin can counteract T cell anergy, T cell responses were determined by assessment of T cell number by subset (CD8+, CD4+, CD4+FoxP3+), by activation (CD62L-CD44+), and by tumor-specific response to ovalbumin-expressing GL261 tumor cells in the brain, spleen, and cervical lymph nodes. In comparison to mock-injected mice, tumor-bearing mice showed a robust T cell response to tumors in the brain, peaking at days 21 and 28 post injection. Treatment with ritanserin resulted in a trend toward increased number and activation of T cells in the brain compared to vehicle-treated mice. In addition, there was a significant increase in OVA-specific T cells in the brain with treatment. In conclusion, T cells respond robustly to brain tumors in mice, and ritanserin treatment appears to increase this response by increased activation and priming of tumor-specific T cells. This indicates that ritanserin may have combinatorial effects on T cells when combined with checkpoint inhibitors such as ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1). Future experiments will focus on combining ritanserin and other repurposed drugs with these checkpoint inhibitors and further characterizing the effect of ritanserin on T cells.

Citation Format: Breanna R. Brenneman, Desiree H. Floyd, Tajie Harris, Benjamin Purow. Assessing and augmenting the immune response to glioblastoma using repurposed pharmaceuticals. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 542.
Abstract 543: Preclinical combination strategies to enhance the efficacy of the anti-PD-1 antibody pembrolizumab
Authors: Pinheiro Elaine M. , Hinton Marlene , Mangadu Ruban , Cai Mingmei , Phan Uyen , Nebozhyn Michael , Hirsch Heather , Loboda Andrey , Javaid Sarah , Wang Yaolin , Sriram Venkataraman , Phillips Joseph H. , McClanahan Terri , Long Brian
Published: July 2016

Pembrolizumab (MK-3475), a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1), has shown activity in multiple types of cancer. Although pembrolizumab is showing a dramatic impact in patients that respond, a large fraction of patients do not respond to single agent therapy. Combination approaches may be the key to improving response rates in these patients. One of the challenges is how best to tailor combination strategies to provide maximal benefit to patients. How we prioritize new combinations will require an understanding of which mechanism is best for which tumor. To elucidate mechanisms that may create an immunogeneic tumor microenvironment and enhance the antitumor activity of PD-1 blockade, we have tested combination strategies with muDX400, a murine anti-PD-1 antibody. The murine preclinical syngeneic tumor models selected for these studies had varied tumor microenvironment compositions and displayed a range of responses to therapy with single agent anti-PD-1. Here we show enhanced anti-tumor activity when PD-1 blockade is combined with conventional chemotherapies, small molecule therapies and additional immunotherapy approaches that target multiple steps in the immune-oncology pathway. Pre and post treatment tumors from these models were extensively characterized by gene expression profiling, whole exome sequencing, flow cytometry and immunohistochemistry analysis. Molecular and cellular correlates of response to anti-PD-1 as a single agent and in combination with these agents were evaluated. These data support potential combination strategies that could either increase efficacy or expand the patient population in which pembrolizumab will demonstrate benefit.

Citation Format: Elaine M. Pinheiro, Marlene Hinton, Ruban Mangadu, Mingmei Cai, Uyen Phan, Michael Nebozhyn, Heather Hirsch, Andrey Loboda, Sarah Javaid, Yaolin Wang, Venkataraman Sriram, Joseph H. Phillips, Terri McClanahan, Brian Long. Preclinical combination strategies to enhance the efficacy of the anti-PD-1 antibody pembrolizumab. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 543.
Abstract 560: Development of a novel class of traps that potently block transforming growth factor-beta (TGF-beta) thereby counteracting TGF-beta mediated immunosuppression and promoting T-cell infiltration into tumors
Authors: O’Connor-McCourt Maureen D. , Lenferink Anne E.G. , Zwaagstra John , Sulea Traian , Baardsnes Jason , Collins Catherine , Cantin Christiane , Durocher Yves , Singh Renu , Koropatnick James
Published: July 2016

Introduction: Elevated TGF-β ligand markedly augments cancer progression primarily by suppressing the immune system in the tumor microenvironment, in particular by suppressing T-cell recruitment and/or activation. We developed a novel class of decoy receptor traps to potently block TGF- β and induce T-cell infiltration into tumors. This promotes the “T-cell-inflamed” tumor state, which is expected to render tumors sensitive to immune checkpoint inhibitors and other immunotherapeutics.

Experimental Procedures: We have computationally designed a class of avidity-enhanced receptor-ectodomain-based traps which bind and neutralize TGF-β. Several trap formats have been produced and tested, with each format exhibiting varying characteristics, including differing circulating half-lives and in vitro blocking potencies (from nM to pM). Representative therapeutic candidates from the different trap formats were evaluated for efficacy in in vivo studies using the syngeneic 4T1 triple negative breast cancer (TNBC) tumor model. Additionally, ex vivo studies were performed on CD4+ and CD8+ T-cells harvested from the draining lymph nodes of treated animals.

Results: In efficacy studies using the syngeneic 4T1 TNBC model, novel TGF-β traps were shown to promote significant T-cell infiltration into tumors. This infiltration resulted in reduced primary tumor growth as well as significant reductions in metastatic lesions. Additionally, ex vivo studies revealed that trap treatment decreased T-cell apoptosis, promoted T-cell proliferation in response to tumor cell lysates in the presence of dendritic cells, as well as increased the capacity of T-cells to specifically lyse 4T1 tumor cells.

Conclusion: Novel computationally-designed TGF-β traps are capable of promoting the “T-cell-inflamed” tumor state. Combination studies in which this novel class of anti-TGF-β immunotherapy is combined with immune checkpoint inhibitors are ongoing.

Citation Format: Maureen D. O’Connor-McCourt, Anne E.G. Lenferink, John Zwaagstra, Traian Sulea, Jason Baardsnes, Catherine Collins, Christiane Cantin, Yves Durocher, Renu Singh, James Koropatnick. Development of a novel class of traps that potently block transforming growth factor-beta (TGF-beta) thereby counteracting TGF-beta mediated immunosuppression and promoting T-cell infiltration into tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 560.
Abstract 562: Dinaciclib induces immunogenic cell death and enhances anti-PD-1 mediated tumor suppression
Authors: Hossain Dewan Mohammed Sakib , Ugarte Fernando , Sawant Anandi , Cai Mingmei , Sriram Venkataraman , Pinheiro Elaine , Sadekova Svetlana , Chackerian Alissa
Published: July 2016

Blockade of the checkpoint inhibitor PD-1 has demonstrated remarkable success in the clinic for the treatment of a growing list of different cancers. However, several tumor types are resistant to anti-PD-1 monotherapy. This observation has spurred numerous combination studies to reveal what additional therapeutic interventions may complement anti-PD1 blockade. Recently it has been shown that immunogenic cell death (ICD), induced by radiation and/or chemotherapy, improves T cell responses against different tumor types. ICD is characterized by damage-associated molecular patterns (DAMPs) including surface expression of calreticulin, and release of ATP and HMGB1. Recognition of DAMPs triggers dendritic cell maturation and functions that are critical for tumor antigen-specific T cell activation. Thus therapies that evoke ICD may further augment anti-tumor immunity elicited by anti-PD-1.

In mouse syngeneic tumor models, we observed combinatorial anti-tumor activity of anti-PD1 and the cyclin-dependent kinase inhibitor, dinaciclib. We hypothesized that dinaciclib potentiates the effects of anti-PD-1 by eliciting ICD. Indeed, tumor cells treated with dinaciclib express the hallmarks of ICD including HMGB1 and ATP release and surface expression of calreticulin. Dinaciclib treatment also increases tumor cell phagocytosis and induces dendritic cell maturation. Furthermore, mice immunized with dinaciclib-treated tumor cells are resistant to subsequent tumor challenge. Tumors from mice receiving anti-PD1 and dinaciclib have increased T cell infiltration and dendritic cell activation, indicating the overall quality of the immune response generated may be improved with the combo. Taken together, these findings suggest a potential mechanism for the observed synergy between dinaciclib and anti-PD1. Dinaciclib induces immunogenic cell death, converting the tumor cell into an endogenous vaccine and thereby boosting the effects of anti-PD-1.

Citation Format: Dewan Mohammed Sakib Hossain, Fernando Ugarte, Anandi Sawant, Mingmei Cai, Venkataraman Sriram, Elaine Pinheiro, Svetlana Sadekova, Alissa Chackerian. Dinaciclib induces immunogenic cell death and enhances anti-PD-1 mediated tumor suppression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 562.
Abstract 563: Combined treatment with PD-L1 blockade and a TLR7/8 agonist dramatically enhances antitumor immunity
Authors: Nishii Naoto , Kondo Yuta , Li Lixin , Lau Walter , Harada Hiroyuki , Azuma Miyuki
Published: July 2016

[Aim] Toll like receptor (TLRs) are expressed in many types of immune cells and function as a key sensor of microbial products. Ligand binding to TLRs leads to activation of signaling pathways in innate immune cells, particularly dendritic cells and subsequent induction of adaptive immune responses. TLR7/8 agonists have been shown to enhance antiviral and antitumor immune responses via multiple cytokine production such as IFN-α, IL-1β, IL-6 and TNFα. Resiquimod (R-848) is a synthetic imidazoquinoline-like molecule that binds to TLR7 and TLR8. Presently, resiquimod has been used as a topical drug for skin cancer and viral infection, because of severe side effects of systemic administration. Thus far, the clinical results of anti-PD-1 or anti-PD-L1 therapy have showed great benefits by inducing tumor regression and improving survival. However, the patients obtained the benefits of this treatment are around one third. An additional approach is required to enhance the efficacy of PD-1 blockade therapy. In this study, we examined the effects of combined treatment with anti-PD-L1 antibody and a low dose of resiquimod administration in two murine tumor models (a squamous cell carcinoma cell line, SCCVII and a colon carcinoma cell line, Colon26). [Methods] Anti-PD-L1 mAb (MIH5, 200 μg/mouse) and/or resiquimod (1.73 μg/mouse) were intraperitonealy administrated at three times a week and tumor growth was evaluated. [Results and Discussion] In a Colon26 model, either anti-PD-L1 (MIH5) mAb or resiquimod treatment alone slightly decreased tumor growth, but the combined treatment dramatically reduced the tumor growth and completely eradicated the tumor in some of mice. In a SCCVII model, monotherapy with resiquimod markedly reduced the tumor growth and completely eradicated the tumor in some of mice. The combined treatment further enhanced the efficacy. The delayed combined treatment at day 7 also showed the reduction of tumor size. Functional analyses at 3-4 wks after the tumor inoculation revealed that monotherapy with resiquimod in the SCCVII model and the combined treatment in both models significantly enhanced the percentages of IFN-γ+-expressing CD8+ T cells, but did not clearly affect the percentages of Foxp3+ regulatory T cells. Our results suggest that TLR7/8 agonist administration alone markedly enhanced antitumor responses in a selected tumor model and the combined treatment with PD-L1 blockade and a TLR7/8 agonist further amplify their effects.

Citation Format: Naoto Nishii, Yuta Kondo, Lixin Li, Walter Lau, Hiroyuki Harada, Miyuki Azuma. Combined treatment with PD-L1 blockade and a TLR7/8 agonist dramatically enhances antitumor immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 563.
Abstract 568: FAK/PYK2 inhibition enhances immune checkpoint inhibitor efficacy
Authors: Wang Yan , Ring Jennifer E. , Sprott Kam , Weaver David T. , Pachter Jonathan A.
Published: July 2016

Although durable responses to single agent immune checkpoint inhibitors have been reported, additional approaches are needed to improve upon this therapeutic benefit. Combinations of immunotherapy agents with tumor microenvironment modulators have the potential to overcome barriers that tumor cells develop to evade the immune system, and provide benefit to a greater proportion of patients. Focal Adhesion Kinase (FAK) and its family member, PYK2, are potentially valuable targets due to their roles in regulating key cellular populations in the tumor microenvironment. In addition to targeting cancer stem cells, the FAK/PYK2 dual inhibitors, VS-6063 and VS-4718, have been shown to inhibit monocyte-derived macrophages, reduce tumor-associated macrophages in xenograft models, and promote a CD8+ T cell-mediated anti-tumor response in squamous cell carcinoma models.

We now report that the combination of VS-4718 with an anti-PD-1 mAb shows improved efficacy over anti-PD-1 mAb alone and extends survival of MC38 syngeneic tumor bearing animals. Analysis of MC38 tumors at day 12 of treatment revealed a significant increase in the CD8+ T cells/Treg ratios in tumors in the VS-4718 + anti-PD-1 combination group, providing a mechanistic understanding for the enhanced efficacy of this combination.

To explore additional combination options, we tested the combination of VS-4718 with anti-4-1BB in the MC38 model. Consistent with what was observed with the anti-PD-1 combination, VS-4718 also enhanced the efficacy of an anti-4-1BB mAb.

To further delineate direct effect of FAK inhibition on human T cells, in vitro T cell proliferation assays were conducted. VS-6063 and VS-4718 dose-dependently stimulated proliferation of CD8+ cytotoxic T cells isolated from healthy donors. This is in distinct contrast to other protein kinase inhibitors, such as the SRC inhibitor dasatinib which impaired the proliferation of CD8+ cytotoxic T cells. In addition, both VS-4718 and VS-6063 decreased CD8+ T cell exhaustion markers, and increased T cell-mediated tumor cell killing in vitro.

These data provide a rationale for clinical trials in cancer patients to test whether a FAK/PYK2 inhibitor in combination with an immune checkpoint inhibitor could increase the breadth of responsive tumor types, increase the number of responders, and confer more durable anti-tumor responses.

Citation Format: Yan Wang, Jennifer E. Ring, Kam Sprott, David T. Weaver, Jonathan A. Pachter. FAK/PYK2 inhibition enhances immune checkpoint inhibitor efficacy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 568.
Abstract 573: Preclinical evaluation of JTX-2011, an anti-ICOS agonist antibody
Authors: Michaelson Jennifer S. , Harvey Christopher J. , Elpek Kutlu G. , Duong Ellen , Wallace Matthew , Shu Chengyi J. , Sathyanarayanan Sriram , Mabry Robert , Shallberg Lindsey , Zi Tong , Deshpande Amit , Sazinsky Stephen L. , Apgar Joshua , Law Deborah
Published: July 2016

ICOS (inducible co-stimulator molecule) is a co-stimulatory molecule and a member of the CD28 superfamily expressed primarily on T lymphocytes. Analysis of cancer patient samples as well as rodent preclinical data have implicated a role for the ICOS pathway in cancer immunotherapy. We have generated a panel of anti-ICOS monoclonal antibodies with in vitro agonistic properties. The anti-ICOS antibodies are efficacious as monotherapies and in combination with anti-PD1 in multiple syngeneic tumor models. Mechanistic studies demonstrate that tumor regression is associated with enhanced ratios of cytotoxic CD8:T regulatory (Treg) cells as well as preferential reduction in ICOS-high Tregs in the tumor microenvironment. JTX-2011, a species cross-reactive high affinity humanized agonist monoclonal antibody, has been selected for development. Evaluation of JTX-2011 in nonhuman primate models will be presented, including data informing safety and PK parameters. Our preclinical data provides rational for clinical development of JTX-2011 as a cancer immunotherapeutic to be tested as both a monotherapy as well as in combination with immunotherapies in solid tumor indications.

Citation Format: Jennifer S. Michaelson, Christopher J. Harvey, Kutlu G. Elpek, Ellen Duong, Matthew Wallace, Chengyi J. Shu, Sriram Sathyanarayanan, Robert Mabry, Lindsey Shallberg, Tong Zi, Amit Deshpande, Stephen L. Sazinsky, Joshua Apgar, Deborah Law. Preclinical evaluation of JTX-2011, an anti-ICOS agonist antibody. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 573.
Abstract 585: Assessing ENPP3 as a renal cancer target for bispecific T-cell engager (BiTE) therapy
Authors: Nolan-Stevaux Olivier , Fajardo Flordeliza , Liu Lily , Coberly Suzanne , McElroy Patricia , Nazarian Aaron , Bott Franziska , Nahrwold Elisabeth , Raum Tobias , Kischel Roman , Lutterbuese Ralf , Hoffmann Patrick , Kufer Peter
Published: July 2016

BiTE® therapeutics are single chain antibody constructs harboring two binding moieties: one directed at a tumor associated antigen and one directed at the CD3e protein, which trigger T cell dependent cellular cytotoxicity (TDCC) against targeted cancer cells. Here, we evaluated the suitability of ENPP3 as a potential BiTE® target. The ENPP3 mRNA is highly differentially expressed in clear cell Renal Cell Carcinoma (ccRCC) and the ENPP3 protein is detected with high uniformity and intensity in ccRCC tumor samples by immunohistochemistry. We demonstrated the surface expression of this protein in Renal Cancer cell lines and confirmed that the ENPP3 protein was highly restricted to the luminal side of normal epithelial layers in which it was detected (proximal renal tubules, bronchial epithelium, salivary glands). We developed highly potent anti-ENPP3 BiTE® molecules and demonstrated the in vitro TDCC activity of these molecules. Two anti-ENPP3 BiTE® molecules further demonstrated tumor formation inhibition activity in a human T cell / human target cell admixture mouse xenograft model.

Citation Format: Olivier Nolan-Stevaux, Flordeliza Fajardo, Lily Liu, Suzanne Coberly, Patricia McElroy, Aaron Nazarian, Franziska Bott, Elisabeth Nahrwold, Tobias Raum, Roman Kischel, Ralf Lutterbuese, Patrick Hoffmann, Peter Kufer. Assessing ENPP3 as a renal cancer target for bispecific T-cell engager (BiTE) therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 585.
Abstract 588: Inhibition of liver inflammation and tumorigenesis by blocking Ccl2-Ccr2 axis in a mouse model
Authors: Teng. Kun-Yu
Published: July 2016

Hepatocellular carcinoma (HCC), the second cancer-related deadly disease, commonly arises in the setting of chronic liver inflammation. CC chemokine ligand 2 (CCL2), a chemokine that recruits CCR2-positive immune cells to promote inflammation, is highly upregulated in HCC patients. Here, we examined the therapeutic efficacy of inhibitors of the Ccl2-Ccr2 axis against hepatitis and HCC in the miR-122 knockout (aka KO) mouse model, in which upregulation of Ccl2 promotes liver inflammation that progresses to HCC with age. To determine whether blocking the Ccl2-Ccr2 axis has any therapeutic potential, we treated KO mice with Ccl2 neutralizing antibody (nab) or a small molecule inhibitor of Ccr2 and monitored liver pathology. Both agents were able to reduce the bridging inflammation in these mice by reducing the population of CD11highGr1+ cells, and expression of IL-6 and TNF-alpha in KO livers. Furthermore, treatment of tumor-bearing KO mice with Ccl2 nab for 8 weeks significantly reduced the recruitment of CD11bhighGr1+ monocytes, IL-6 and TNF-alpha production, and HCC incidence as well as tumor burden. The down-regulation of pStat3 and c-Myc (IL-6 downstream) as well as NF-kappaB (TNF-alpha downstream) upon targeting Ccl2 positively correlated with tumor growth suppression. In addition, inhibition of tumorigenesis by Ccl2 nab also correlated with enhanced cytotoxicity and IFN-gamma expression of NK cells isolated from the tumor microenvironment. Collectively, our results demonstrate that blocking the Ccl2-Ccr2 axis can be an effective therapeutic approach against both hepatitis and HCC.

Citation Format: Kun-Yu Teng. Inhibition of liver inflammation and tumorigenesis by blocking Ccl2-Ccr2 axis in a mouse model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 588.
Abstract 597: Defining the role of macrophage mediated trogocytosis in the clearance of antibody-opsonized tumor cells
Authors: Velmurugan Ramraj , Challa Dilip , Ram Sripad , Ober Raimund J. , Ward E. Sally
Published: July 2016

Understanding the complex behavior of effector cells such as monocytes or macrophages in regulating cancerous growth is of central importance for cancer immunotherapy. Earlier studies using CD20-specific antibodies have demonstrated that the Fcγ receptor (FcγR)-mediated transfer of the targeted receptors from tumor cells to these effector cells through trogocytosis can enable escape from antibody therapy, leading to the viewpoint that this process is pro-tumorigenic. However, there is limited information concerning the role of trogocytosis in antibody-based treatment of solid tumors such as breast cancer.

We have used advanced microscopy methods and quantitative flow cytometric assays to study the effect of antibody-mediated trogocytosis on breast cancer cells and whether they can lead to tumor cell death.

Our results show that long-term coculture with macrophages can reduce cancer cell numbers in an antibody-dependent manner even at low effector:target ratios. Quantitation from simultaneous long-term imaging of macrophage:cancer cell interactions reveals that this cell death is caused by both macrophage-mediated phagocytosis and trogocytosis, with the contribution of each process differing by the phenotype of the effector macrophages.

Together, our results add to our understanding of the numerous interactions macrophages can have with cancer cells, and how therapeutic antibodies modulate their effects. Our recent observations in this area will be presented.

Citation Format: Ramraj Velmurugan, Dilip Challa, Sripad Ram, Raimund J. Ober, E. Sally Ward. Defining the role of macrophage mediated trogocytosis in the clearance of antibody-opsonized tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 597.
Abstract 600: Melanoma and ovarian cancer cells tested for drug sensitivity using anchorage-independent growth
Authors: Rotem Asaf , Izar Benjamin , Garraway Levi A.
Published: July 2016

This study aims to test patient-derived melanoma and ovarian cancer cells for their sensitivity to clinically available drugs and drug combinations. The experiments were performed by using the growth in low attachment (GILA) assay, which is equivalent to the classic soft-agar assay and allows screening of 3D ex vivo cultures, which resemble the tumor and its microenvironment.

To assay tumor cells from malignant lesions we isolated ovarian cancer cells from peritoneal fluid (ascites) derived from patients and melanoma cells from metastatic melanoma patients. The fresh samples were enriched for tumor cells and later cultured in an ex vivo manner on either high- or low-attachment surfaces. These cells were subjected to drug treatments and cellular viability was measured at the end of these experiments.

Our experiments demonstrate that the GILA assay is a highly sensitive method for pharmacological tests, as compared to the traditional 2D method (growth on high-attachment surfaces). By using GILA assay we found that melanoma cells are sensitive to drugs that inhibit driver oncogenes, such as BRAF or MEK. In addition, we found interesting targets in metastatic melanoma lesions that carry an unknown mutational background.

Drug sensitivity assays for ovarian cancer cells using the GILA approach showed that cancer cells from various patients with the same cancer type display differential sensitivity to drugs. Additionally, ovarian cancer cells were found to have a different drug-sensitivity threshold when grown in spheres.

Our studies present an approach that utilizes the unique ability of transformed cells to grow in anchorage-independent conditions and enables ex vivo drug sensitivity testing. This phenotypic approach is complementary to a genetic approach that uses DNA sequencing to identify putative oncogenes that confer sensitivity to drugs designed to specifically inhibit the identified oncoprotein.

Citation Format: Asaf Rotem, Benjamin Izar, Levi A. Garraway. Melanoma and ovarian cancer cells tested for drug sensitivity using anchorage-independent growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 600.
Abstract 602: BEZ235 efficacy in 3D-cancer cell line screening
Authors: Irigoyen Macarena , Castillo Gonzalo
Published: July 2016

In vitro efficacy assessment of drugs in panels of tumor-derived cell lines has proven to be a powerful tool for predictive biomarker discovery. However, in the last few years it has become evident that particularly for tumor cell lines derived from solid tumors, the results do not always correlate with what is seen in xenografts as well as clinically. One of the reason is that traditionally these type of studies are done in monolayers (2D), which lack the complexity normally seen in vivo. 3D cultures offer the ability to carry out the same type of studies with the added benefit of incorporating 3-dimensional cellular interactions and physiological gradients, normally seen in a tumor microenvironment. In this study, we evaluated the efficacy of BEZ235, an imidazoquinoline, targeting the phosphatidylinositol 3 kinase (PI3K) and the mammalian target of rapamycin (mTOR). BEZ235 was evaluated in a panel of 60 cell lines grown as spheroids, and this information was used to investigate genes associated with sensitivity. Cell lines grown as spheroids proved to be highly sensitive to the drug, where 45 of 60 cell lines showed an IC50 lower than 1 μM. Biomarker association analysis identified two mutated genes associated to BEZ235 sensitivity. The first biomarker was the phosphatase and tensin homolog (PTEN) a tumor suppressor gene, that when deregulated plays an important role in the development of several cancers. The second biomarker was AXIN1, a negative regulator of the WNT signaling pathway, and its deregulation has been associated with hepatocellular carcinoma. Since both PTEN and AXIN1 are thought to play a role in tissue invasion and metastasis, we studied the ability of BEZ235 to inhibit cellular invasion, and the results are discussed.

Citation Format: Macarena Irigoyen, Gonzalo Castillo. BEZ235 efficacy in 3D-cancer cell line screening. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 602.
Abstract 61: A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A
Authors: Wu Yuanzhong , Kang Tiebang
Published: July 2016

The regulation of stability is particularly crucial for unstable proteins in cells. However, a convenient and unbiased method of identifying regulators of protein stability remains to be generated. Recently, genome-scale CRISPR-Cas9 library has been established as a genetic tool to mediate loss of function screening. Here, we developed a method named Protein Stability Regulators Screening Assay (Pro-SRSA) by combining the whole genome CRISPR-Cas9 library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen regulators of protein stability. Using Cdc25A as an example, Cul4B-DDB1-DCAF8 was determined to be a new E3 ligase for Cdc25A. Moreover, the acetylation of Cdc25A at lysine 150, which was mediated by p300/CBP and deacetylated by HDAC3, was revealed to prevent its ubiquitin-mediated degradation by proteasome. This is the first report that the acetylation as a novel posttranslational modification modulates the Cdc25A stability, and we believe that this efficient and unbiased screening method at the genome-scale will be widely used to globally identify regulators of protein stability.

Citation Format: Yuanzhong Wu, Tiebang Kang. A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 61.