bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 964: Genome-wide CRISPR screen identifies potential therapeutic combination of EGFR and FGFR inhibitors in oral cancer
Authors: Ludwig Megan , Birkeland Andrew , Nimmagadda Sai , Foltin Sue , Kulkarni Aditi , Jiang Hui , Carey Thomas , Brenner John Chad 
Published: August 2018

Abstract
Oral squamous cell carcinoma (OSCC) has remained a disease with poor survival for decades. Current treatment options such as targeting EGFR has had limited success. We hypothesize that inhibiting compensatory pathways in combination with EGFR signaling may result in greater efficacy. To identify synergistic combinations with EGFR-targeted therapy, we introduced Genome-scale CRISPR-Cas9 KnockOut (GeCKO) libraries into OSCC cell lines. CRISPR-Cas9 generates individual genetic knockouts through targeted gene editing. We used a pool of CRISPRs targeting over 18,000 genes to perform genome-scale screening for drivers of sensitivity to EGFR-targeted therapy. Upon selection of the OSCC GeCKO pool, we identified gene knockouts in the FGFR pathway that increased sensitivity to the EGFR inhibitor gefitinib. Using resazurin viability assays we tested combinations of EGFR and FGFR inhibitors in 14 OSCC cell lines. Six/14 (43%) of the cell lines were responsive to the combination, indicating that the FGFR pathway is an alternate mechanism of resistance to EGFR-targeted therapy in some tumors. We advanced an OSCC cell line to test in vivo by establishing subcutaneous cell line xenografts in mice. After treatment for 21 days, the mice receiving the combination of EGFR and FGFR inhibitors, gefitinib and BGJ398, had significantly smaller tumors than mice receiving vehicle or either monotherapy. These results suggest targeting EGFR in combination with its compensatory pathway such as FGFR signaling could be effective in vivo. While OSCC remains a common and frequently lethal cancer, there is great potential for the development of novel 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 is a common compensatory mechanism to EGFR inhibition, and that targeting EGFR and FGFR in combination has efficacy in an in vivo model. Overall, targeting compensatory mechanisms in combination with EGFR-targeted therapy could generate novel, beneficial treatment plans for patients with OSCC.
Citation Format: Megan Ludwig, Andrew Birkeland, Sai Nimmagadda, Sue Foltin, Aditi Kulkarni, Hui Jiang, Thomas Carey, John Chad Brenner. Genome-wide CRISPR screen identifies potential therapeutic combination of EGFR and FGFR inhibitors in oral cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 964.
Abstract CT104: Evaluation of peripheral T cell subset proliferation as a pharmacodynamic assay to guide the development of anti-CTLA-4 and PD-1 antibody combinations in patients with solid tumors
Authors: Souza Paul De , Malczewski Agnieszka , Proscurshim Igor , Yuan Guojun , Coart Elisabeth , Dupont Christopher , Manrique Mariana , Drouin Elise , Gonzalez Ana , Carini Meg , Shebanova Olga , Cuillerot Jean-Marie 
Published: August 2018

Abstract
The combination of anti-CTLA-4 and anti-PD-1 antibodies has demonstrated significant clinical efficacy in metastatic melanoma, but also significant toxicities. Pharmacodynamic (PD) markers are important tools in optimizing the safety and efficacy of anti-CTLA-4/anti-PD-1 combinations, but have remained elusive to date. To test the hypothesis that blockade of both CTLA-4 and PD-1 together results in increased expression of the proliferation marker Ki67 in CD4+ T cells, we studied two dose regimens of ipilimumab in combination with nivolumab in patients with advanced or metastatic solid tumors, using a validated assay. Subjects received either 1.0 mg or 0.3 mg /kg of ipilimumab every 6 weeks in combination with nivolumab 3mg/kg every 2 weeks for a maximum of 12 weeks (2 cycles). A total of 9 subjects were enrolled at the 1.0 mg/kg of ipilimumab cohort and 12 subjects in the 0.3 mg/kg cohort. Highly statistically significant increases of 4.56 fold (range 3.35-6.20) from baseline at cycle 1 day 8 post-treatment (p &lt; 0.001) in cohort 1, and 3.13 fold (range 2.31-4.33) from baseline at cycle 1 day 8 post-treatment (p &lt; 0.001) in cohort 2, in Ki67 expression were observed in CD4+ T cells. The absolute increase in Ki67 expression was also observed to be dose dependent numerically. Significant increases in Ki67 expression were also observed in CD8+ T cells, regulatory T cells, and natural killer T (NKT) cells following treatment. These results identify and support Ki67 expression as a PD biomarker in the context of CTLA-4 and PD-1 blockade. More work is required to correlate these finding with clinical outcomes in order to establish a benchmark biomarker assay for anti-CTLA-4 and anti-PD-1 combinations. (ACTRN12617001059358)
Citation Format: Paul De Souza, Agnieszka Malczewski, Igor Proscurshim, Guojun Yuan, Elisabeth Coart, Christopher Dupont, Mariana Manrique, Elise Drouin, Ana Gonzalez, Meg Carini, Olga Shebanova, Jean-Marie Cuillerot. Evaluation of peripheral T cell subset proliferation as a pharmacodynamic assay to guide the development of anti-CTLA-4 and PD-1 antibody combinations in patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT104.
Abstract LB-023: Describing prostate cancer heterogeneity through single cell analysis with mass cytometry
Authors: Roditi Laura De Vargas , Jacobs Andrea , Fankhauser Christian , Poyet Cedric , Wild Peter , Bodenmiller Bernd 
Published: August 2018

Abstract
Background
Single-cell analysis has emerged as a crucial approach to detect variations in genetic and proteomic expression. Prostate cancer is characterized by multiple genomic alterations leading to the complex nature of the disease. The heterogeneity at the proteomic level is not yet well characterized, yet intra-tumor heterogeneity can impair correct diagnosis and treatment, by driving therapy resistance. Meanwhile inter-tumor heterogeneity can strongly impact personalization of treatments highlighting the importance of characterizing both intra- and inter-tumor heterogeneity. Using single-cell mass cytometry data, we identify the proteomic profiles of the different cellular subpopulations found in prostate cancer that are common across a cohort of 50 patients while also describing the variations across patients, thus thoroughly characterizing prostate tumor heterogeneity.
Methods & Results
Mass cytometry experiments allow quantification of dozens of proteins simultaneously at the single-cell level, for thousands of cells. The high dimensionality of this data represents a challenge for data visualization and clustering, which require computational approaches for interpretation of the results. We developed a new computational method to address this task and analyzed tissue sections from surgically removed prostates from 50 patients using mass cytometry with a comprehensive set of 40 antibodies to jointly identify surface markers, enzymes, transcription factors and functional readouts.
Conclusions
Here, we present for the first time an analysis of prostate tumor cell heterogeneity by mass cytometry coupled to a newly developed computational method that improves visualization and clustering of such high-dimensional data. This study was conducted by analyzing prostate cancer samples from fifty patients at the single-cell level using mass cytometry to estimate the abundance of forty different proteins simultaneously for each cell. Our results show that immune, stromal and epithelial cells can be comprehensively distinguished across all patients. A closer look at the epithelial compartment reveals multiple subpopulations which we describe in terms of their protein expression patterns to characterize the intra- and inter-tumor heterogeneity in prostate cancer. This work demonstrates how single cell phenotypic profiles of prostate tumor cells, taken from surgically removed prostates, can be used to quantify the varying heterogeneity during tumor progression.
Citation Format: Laura De Vargas Roditi, Andrea Jacobs, Christian Fankhauser, Cedric Poyet, Peter Wild, Bernd Bodenmiller. Describing prostate cancer heterogeneity through single cell analysis with mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-023.
Abstract LB-040: New conditional Kras-alleles generated by CRISPR-based genome editing to model tumor initiation
Authors: Zafra Maria Paz , Schatoff Emma M. , Han Teng , Dow Lukas E. 
Published: August 2018

Abstract
KRAS is the most frequently mutated oncogene in human cancers, and specific cancer types show a clear bias in the types and frequency of Kras alterations. However, we do not have a clear understanding of how distinct Kras mutations dictate tumor biology and response to therapy. The generation of conditional animal models, such as Lox-Stop-Lox (LSL) KrasG12D and LSL-KrasG12Vgeo mice, have provided important tools to dissect the impact of Kras mutation in tumor development, but these models alone do not recapitulate the spectrum of Kras alterations in human cancer. To better reflect the Kras mutational landscape in human cancers, we have developed a series of critical LSL-Kras mouse models that recapitulate common Kras alterations observed in colorectal (G13D), pancreatic (G12R), and lung cancer (G12C), allowing the direct interrogation of the effects of these Kras mutations on disease phenotype and treatment response. To do this, we targeted embryonic stem cells (ESCs) carrying the LSL-KrasG12D allele, by co-transfection of a modified high-fidelity Cas9 (Cas9-HFz), a LSL-Kras-targeted sgRNA, and an HDR template carrying the new mutation. While Cas9-HFz displayed a slight decrease in homology-directed repair (HDR) efficiency in comparison with wildtype Cas9, it enabled high specificity editing of only the LSL allele. Targeted deep sequencing revealed a significant reduction in ‘off-target' events in wildtype Kras locus in Cas9-HFz transfected samples compared to unmodified Cas9. We next generated transgenic mice by blastocyst injection and produced mice carrying each LSL-Kras variant, a pancreas specific Cre (p48-Cre) and a far-red fluorescent Cre-reporter (mKate2). We are now analyzing the impact of specific Kras alterations in tumor initiation in distinct tissues such as the pancreas, colon and lung. Together with available LSL-KrasG12D and LSL-KrasG12V already available, our new alleles will serve as critical tools for better dissect the genetic factors that dictate disease initiation, progression, and response to therapy.
Citation Format: Maria Paz Zafra, Emma M. Schatoff, Teng Han, Lukas E. Dow. New conditional Kras-alleles generated by CRISPR-based genome editing to model tumor initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-040.
Abstract LB-043: Generation of clonal CRISPR/Cas9-edited human iPSC derived cellular models and its applications in physiologically relevant assays
Authors: Revankar Chetana M. , Willems Erik , Lacambacal Rex , gokirmak Tufan , Dizon Jordan , Webb Jacquelyn , Vega Raquel , Piper David 
Published: August 2018

Abstract
Induced pluripotent stem cell (iPSC)-derived cells are widely used in disease models, safety testing and phenotypic and functional assays. The CRISPR/Cas9 system has revolutionized genome editing thus enabling rapid generation of disease models. Cutting edge technologies like genome editing paired with iPSC-derived cell models have reshaped our approach to disease modeling in vitro. Although, CRISPR/Cas9 has had huge impact on generation of complex cellular models, there are challenges in replicating disease-in-a-dish. One of the challenges is the implementation of genome editing tools in iPSC, particularly issues associated with editing tools delivery, cell recovery and clonal isolation of genome-edited cells. Here, we describe a workflow that facilitates the generation and isolation of clonal CRISPR-edited iPSCs through the use of a novel toolset. We observed high genome editing efficiency using an iPSC line that stably expresses the Cas9 protein and obtained improved survival of the iPSCs after delivery of the editing tools through culture in StemFlex™ medium, a novel PSC culture system. Furthermore, we have demonstrated that single edited iPSCs can be isolated through the combination of an optimized cell sorting method and expanded as single cell clones with reagents that support single cell survival. Through this workflow we have generated several iPSC lines carrying disease relevant SNP mutations. These tools and workflows not only contribute to improved success in the derivation of homogenous genome edited human iPSC clones but also provide novel alternatives to study disease-causing mutations in vitro. Additionally, we have also tested these cell models in commonly used cell health assays. These cell models can be further used to generate organoids to mimic conditions found in tissues. iPSC- derived cell models not only permit the study of disease mechanism in the cell types specifically affected in the disease but also deepen our understanding of the molecular mechanisms and in turn help development of novel therapeutics.
Citation Format: Chetana M. Revankar, Erik Willems, Rex Lacambacal, Tufan gokirmak, Jordan Dizon, Jacquelyn Webb, Raquel Vega, David Piper. Generation of clonal CRISPR/Cas9-edited human iPSC derived cellular models and its applications in physiologically relevant assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-043.
Abstract LB-145: Enhancement of checkpoint inhibitor therapy by beta-alethine
Authors: Taub Floyd , Wright Suzin , Guth Amanda 
Published: August 2018

Abstract
Beta-alethine (BA) is a small molecule drug that has demonstrated impressive anti-tumor immune responses against melanoma, particularly when combine with low dose anti-PD1. Here we report on the dose of BA that optimally enhances anti-PD1, the efficacy of BA combined with various immune checkpoint inhibitors (ICIs) in a murine bladder cancer and we propose a potential mechanism of action of BA to explain its synergistic effect. We tested 3 different doses of BA that were logs apart (30 mg/kg, 30 ug/kg, and 600 ng/kg) either alone or in combination with low dose (10 ug per mouse for a total of 3 doses 3 days apart) anti-PD1 in the DBA mouse Cloudman melanoma mouse model. The lowest dose of BA (600 ng/kg) in combination with anti-PD1 resulted in cures in 33% of the mice when treatment was initiated when tumors were of substantial size (average size of 30 mm2). Previous work starting with smaller tumor sizes (average 20 mm2) resulted in an 80% cure rate when a low dose of BA was used in combination with low dose anti-PD1. Analysis of blood from treated animals demonstrated that the combination led to decreased expression of multiple checkpoint molecules on both T cells and myeloid cells and to a decrease in tumor promoting inflammatory macrophages. We further demonstrated efficacy of combining BA with other ICI therapies in a s.c. MBT2 murine bladder cancer study in C3H mice. In these studies, we saw that BA was synergistic with a-PD1 at all doses tested (30 mg/kg, 30 ug/kg and 600 ng/kg weekly s.c.). The effect was seen even when only 4 weekly doses of BA were given. Interestingly, tumor growth increased when BA was discontinued, a phenomenon observed in previous compassionate use veterinary studies. Synergy with OX40 and CTLA4, were also seen, but further studies of dose and schedule are required for optimization. Our results demonstrate that a low dose of BA is optimal to enhance the efficacy of anti-PD1 and that this observed combinatorial effect is conserved in at least two different tumor types in different mouse strains. Based on analysis of T and myeloid cells in treated mice, we hypothesize that BA works via inducing pleiotropic changes that result in downregulation of multiple immune checkpoint molecules on T cells and on myeloid cells making it less feasible for these cells to use other pathways of immune suppression as compared to single agent anti-PD1 therapy and thus increase potential for cures.
Citation Format: Floyd Taub, Suzin Wright, Amanda Guth. Enhancement of checkpoint inhibitor therapy by beta-alethine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-145.
Abstract LB-177: Quantification of cell signaling proteins by immuno-MALDI
Authors: Popp Robert , Zahedi René P. , LeBlanc André , Mohammed Yassene , Aguilar-Mahecha Adriana , Pötz Oliver , Basik Mark , Batist Gerald , Borchers Christoph H. 
Published: August 2018

Abstract
Introduction. To improve cancer patient stratification and overcome the drawbacks of currently used approaches to assess signaling pathway proteins, we set out to develop immuno-matrix assisted laser ionization/desorption (iMALDI) assays combined with a phosphatase-based phosphopeptide quantitation (PPQ) approach to measure PI3K/AKT/mTOR pathway activity. Specifically, we targeted the C-terminal tryptic peptides of AKT1 and AKT2 due to their involvement in full kinase activation.
Methods. Following trypsin digestion of cell lysate, stable isotope-labeled standard (SIS) peptides are added. After splitting the solution, one aliquot is treated with phosphatase. Bead-coupled antibodies enrich the non-phosphorylated target peptides, which are washed and spotted onto a MALDI plate, and acidic MALDI matrix elutes the peptides. The resulting light/heavy ratios of both aliquots allow the calculation of protein expression levels and peptide phosphorylation stoichiometry.
Results. The iMALDI assays were validated for linear range and accuracy, as well as interference screening, requiring only ~50 µg of total cell or tissue lysate protein to quantify both AKT1 and AKT2 expression levels and phosphorylation stoichiometry. CVs of technical replicates were found to consistently be below 10%. We were able to quantify AKT1 and AKT2 from various cell lines and fresh frozen tumor samples, including SW480 and HCT116 colon cancer cell lines, MDA-MB-231 breast cancer cells, and colon cancer and breast cancer tumor lysates. Further, a direct comparison of matched fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissues showed that the expression levels and phosphorylation stoichiometry quantified differ depending on the tissue preservation method, and that phosphorylation stoichiometries above 30% occur in only a small subset of samples. A direct comparison of four pairs of normal and adjacent tumor tissues showed elevated AKT1 phosphorylation stoichiometry of ~40% in a colorectal cancer liver metastasis, and significantly elevated AKT1 (3.6-fold) and AKT2 (2.2-fold) expression levels in a surgical breast tumor sample.
Future directions. In a next step, patient-derived mouse xenograft tissue samples, collected after specific drug treatment, will be analyzed, and the AKT results will be correlated to genomic data to answer the hypothesis whether the AKT expression levels and phosphorylation stoichiometries correlate with response to treatment.
Citation Format: Robert Popp, René P. Zahedi, André LeBlanc, Yassene Mohammed, Adriana Aguilar-Mahecha, Oliver Pötz, Mark Basik, Gerald Batist, Christoph H. Borchers. Quantification of cell signaling proteins by immuno-MALDI [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-177.
Abstract LB-292: p95HER2-T cell bispecific antibody for breast cancer treatment
Authors: Vicario Rocío , Ruiz Irene Rius , Morancho Beatriz , Morales Cristina Bernadó , Herter Sylvia , Freimoser-Grundscholber Anne , Somandin Jitka , Sam Johannes , Ast Oliver , Barriocanal Águeda Martinez , Luque Antonio , Escorihuela Marta , Varela Ismael , Nuciforo Paolo , Fasani Roberta , Peg Vicente , Rubio Isabel , Cortés Javier , Serra Violeta , Escrivá Santiago , Sperinde Jeff , Chenna Ahmed , Huang Weidong , Winslow John , Scaltriti Maurizio , Baselga Josep , Tabernero Josep , Umaña Pablo , Bacac Marina , Saura Cristina , Klein Christian , Arribas Joaquín 
Published: August 2018

Abstract
T cell bispecific antibodies (TCBs) are engineered molecules that include, within a single entity, binding sites to the T cell receptor and to a tumor-specific or a tumor-associated antigen. The receptor tyrosine kinase HER2 is a tumor-associated antigen in ~25% of breast cancers. Clinical evidence shows that TCBs targeting HER2 may result in severe toxicities, likely due to the physiologic levels of HER2 in normal epithelia. Approximately 40% of HER2-positive tumors express detectable levels of p95HER2, a carboxy-terminal fragment of HER2. We previously developed different antibodies that specifically recognize p95HER2 but not full-length HER2 and hypothesized that a TCB antibody recognizing p95HER2 would be exquisitely specific for tumor cells. Here, we describe the development of such p95HER2-TCB and show that it has a potent anti-tumor effect on p95HER2-expressing breast cancers. Since p95HER2 is not expressed in normal tissues, in contrast with HER2-TCB, it has no effect on non-transformed cells expressing physiological levels of HER2. These data paves the way for the safe targeting of a subtype of HER2-positive tumors with a novel tumor-specific TCB.
Citation Format: Rocío Vicario, Irene Rius Ruiz, Beatriz Morancho, Cristina Bernadó Morales, Sylvia Herter, Anne Freimoser-Grundscholber, Jitka Somandin, Johannes Sam, Oliver Ast, Águeda Martinez Barriocanal, Antonio Luque, Marta Escorihuela, Ismael Varela, Paolo Nuciforo, Roberta Fasani, Vicente Peg, Isabel Rubio, Javier Cortés, Violeta Serra, Santiago Escrivá, Jeff Sperinde, Ahmed Chenna, Weidong Huang, John Winslow, Maurizio Scaltriti, Josep Baselga, Josep Tabernero, Pablo Umaña, Marina Bacac, Cristina Saura, Christian Klein, Joaquín Arribas. p95HER2-T cell bispecific antibody for breast cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-292.