bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 379: Application of NanoBRET target engagement cellular assay for measurement of inhibitor binding to wild type and mutant RAS in live cells
Authors: Wu Jianghong , McGinley Shawn , Wang Yuan , Gallagher Peter , Ma Haiching 
Published: June 2022

Abstract
NanoBRET࣪ target engagement (TE) is the first biophysical technique that broadly enables the quantitative determination of protein inhibitor occupancy in live cells, without disruption of cellular membrane integrity. This quantitative capability is achieved in live cells via BRET with an optimized set of cell-permeable tracers, allow the measurement of compound binding to a selected cellular target protein. RAS is a well-known oncogene that is frequently mutated in most lung, pancreatic and colorectal cancers and is associated with poor disease prognosis. Mutated RAS is locked in the activated GTP bound state and facilitates enhanced RAS signaling in cancer cells. While being a desirable target, the absence of good druggable binding pockets has made modulator compound discovery challenging and unsuccessful. The recent discovery of a unique switch II binding pocket and successful inhibition of the KRAS (G12C) mutant by covalent inhibitors have led to the resurgence of interest in the design of inhibitors targeting RAS directly. Here, we performed NanoBRET࣪ TE cellular assay with RAS inhibitors against transfected RAS and its mutants. Our data demonstrate that NanoBRET࣪ TE cellular assay can measure the apparent affinity of RAS inhibitors by competitive displacement of a NanoBRET࣪ RAS switch I/II pocket tracer, reversibly bound to the LgBiT- and SmBiT- KRAS, KRAS (G12C), KRAS (G12D), KRAS (G12V), KRAS (G13D), KRAS (Q61H), KRAS (Q61L), KRAS (Q61R), or HRAS fusion constructs co-transfected in live HEK293 cells. Z factor analysis indicates the assay is High Throughput Screening (HTS) compatible and reproducible. In addition, we are able to confirm the signaling inhibition of downstream phospho-ERK1/2 by KRAS (G12C)-specific inhibitors AMG 510 (Sotorasib), ARS-1620, MRTX849, and MRTX1257 in MIA PaCa-2 pancreas and SW837 colon cancer cells bearing KRAS (G12C) mutation by Western blot assay. Our results suggest NanoBRET࣪ TE cellular assay can serve as a great tool to facilitate RAS pathway drug discovery against human cancers.
Citation Format: Jianghong Wu, Shawn McGinley, Yuan Wang, Peter Gallagher, Haiching Ma. Application of NanoBRET target engagement cellular assay for measurement of inhibitor binding to wild type and mutant RAS in live cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 379.
Abstract 3793: Single-cell transcriptional lineage tracing reveals complexity and plasticity of breast cancer pro-metastatic phenotypes
Authors: Roda Niccolò , Cossa Andrea , Ruscitto Federica , Priami Chiara , Blandano Giada , Dalmasso Alberto , Bertolini Francesco , Pelicci Pier Giuseppe 
Published: June 2022

Abstract
Breast cancer (BC) represents a major clinical hurdle, with metastasis being the most devastating events in patients’ history. Traditional lineage tracing approaches have been widely employed to study the metastatic progression of BC. However, these approaches were limited to the investigation of clonal dynamics and did not provide detailed molecular information underlying the pro-metastatic phenotype. In this work, we exploited a library of degenerated barcodes which were both integrated in the genome and expressed as synthetic transcripts. Coupling this barcoding strategy to scRNA-seq analysis, we tracked the clonal and transcriptional evolution of more than 37,000 MDA-MB-231 (a human triple-negative BC cell line) cells in vivo. Clonal distribution analysis revealed that BC clones display proliferative heterogeneity in primary tumors, with a small fraction of clones outcompeting all the others. Furthermore, primary breast tumors turned out to be composed of two phenotypically distinct groups of clones: highly proliferative, on one side, and migratory/stressed/stem-like, on the other. Then, we showed that the striking majority of metastatic cells derived from a single clone which was under-represented in its primary tumor. Importantly, these pro-metastatic clones upregulated genes and pathways associated with extracellular matrix (ECM) interaction, migration, and type-I interferon (IFN) response. In this scenario, independent pro-metastatic clones heterogeneously upregulated these pathways, thus suggesting a multi-faceted functional phenotype. Despite this variability, we detected a set of genes whose expression was statistically associated with the pro-metastatic phenotype, and we validated the relevance of these molecular features both at the biological and clinical level. First, we observed a reduced metastatic burden upon the in vivo knock-down of genes involved in ECM interaction (e.g., ANGPTL4, KCNQ1OT1, and ITGB4) and type-I IFN response (e.g., LY6E and IFI6). Second, we derived two compact gene signatures showing robust predictive power of BC patient survival, independently of other known clinical factors. In conclusion, our work revealed previously unappreciated cellular mechanisms of BC metastasization, providing new actionable therapeutic targets and new molecular predictors of BC prognosis.
Citation Format: Niccolò Roda, Andrea Cossa, Federica Ruscitto, Chiara Priami, Giada Blandano, Alberto Dalmasso, Francesco Bertolini, Pier Giuseppe Pelicci. Single-cell transcriptional lineage tracing reveals complexity and plasticity of breast cancer pro-metastatic phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3793.
Abstract 3801: Comprehensive characterization of the biology of inflammatory bowel disease-associated colorectal cancer using the GeoMx® Digital Spatial Profiler (DSP)
Authors: Juncker-Jensen Anna , Chandramohan Lakshmi 
Published: June 2022

Abstract
Inflammatory bowel diseases (IBD) ulcerative colitis (UC) and Crohn's disease (CD) significantly increases the risk for development of colorectal cancer (CRC). However, the exact molecular mechanisms that underlie the transition from inflammation to cancer are not fully understood, and a systematic characterization of the immunopathogenesis of IBD-associated CRC is lacking. To address this issue, we will perform a comprehensive and spatial tissue analysis using the Cancer Transcriptome Atlas (CTA) on the NanoString GeoMx® Digital Spatial Profiler (DSP). The CTA panel is designed to profile the global immune response and all aspects of tumor microenvironment biology, including the 18-gene Tumor Inflammation Signature (TIS).
For DSP analysis, a total of 20 FFPE samples (5 colon cancer patients, 5 UC patients, 5 CD patients, and 5 matched normal samples will be spatially profiled for up to 1,800 genes included on the CTA. Selection of regions of interest (ROI) will be guided by H&E staining and fluorescent markers (CD45, PanCK, Syto13), and profiling of tumor and TME regions will be achieved through segmenting by PanCK+/PanCK- followed by collection of indexed oligonucleotides and sequenced on NextSeq 550 Illumina instrument. NGS reads will be processed into digital counts, normalized and mapped back to each ROI, generating a map of transcript activity within each ROI. For all samples crypt and villus regions will be selected for a detailed spatial analysis. Subsequently, using the proprietary multiplex immunofluorescence (mIF) platform MultiOmyx™ we will characterize the protein immune cell landscape in IBD-associated CRC, using a custom immune panel applied to adjacent slides.
With this study we hope to highlight genes and pathways involved in the transition from inflammation to cancer by combining large-scale spatial gene profiling by DSP CTA with a comprehensive spatial protein profiling by MultiOmyx. It is our hope that this will provide some much-needed understanding of the underlying mechanisms leading from an IBD disorder to the development of colon cancer.
Citation Format: Anna Juncker-Jensen, Lakshmi Chandramohan. Comprehensive characterization of the biology of inflammatory bowel disease-associated colorectal cancer using the GeoMx® Digital Spatial Profiler (DSP) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3801.
Abstract 3809: Adaptation of prostate cancer cell lines to tumor microenvironment culture conditions induces expression of androgen receptor splice variant 7 and contributes to drug resistance
Authors: Li Yunmin , Wong Albert , Lu Ann , Lim James 
Published: June 2022

Abstract
The tumor microenvironment (TME) has several characteristics that distinguish it from normal tissue, including elevated interstitial fluid pressures and hypoxic environments. Both pressurized and hypoxic culture conditions can influence gene and protein expression of tumor cells which can result in tumor metastasis and drug resistance. Enzalutamide is effective in treating metastatic castration-resistant prostate cancer (mCRPC), but majority of patients eventually develop resistance to enzalutamide. One of the biomarkers of enzalutamide resistance is the expression of androgen receptor splice variant 7 (AR-V7), but the mechanism of how AR-V7 expression is upregulated in prostate cancer is unclear. To investigate the influence of TME culture conditions on prostate cancer cell lines, 22Rv1 and LNCaP cell lines were incubated under pressurized and hypoxic culture conditions (2 PSI + 1%O2) for a minimum of 7 days. We compared cell growth, morphology changes, gene expression profiles and drug response to enzalutamide treatment. The growth of 22Rv1 and LNCaP cells was markedly inhibited under TME culture conditions, accompanied by distinct changes in cell morphology. 22Rv1 and LNCaP cells cultured under TME conditions exhibited increased resistance to enzalutamide treatment. Higher levels of AR-V7 expression were detected in 22Rv1 and LNCaP cell lines maintained for 7 days in TME culture conditions. RNA-seq data identified 700+ genes that were differentially expressed in 22Rv1 and LNCaP cell lines adapted to TME culture conditions. GSEA analysis identified signaling pathways associated with hypoxia, glycolysis and epithelial-mesenchymal transition were upregulated under TME culture conditions, and the pathways of xenobiotic metabolism and oxidative phosphorylation were downregulated by TME culture conditions. In summary, adaptation to TME culture conditions can promote the expression of AR-V7 in prostate cancer cell lines and can contribute to enzalutamide drug resistance. Adaptation of cell lines to TME culture conditions can provide meaningful insights into mechanisms of drug resistance while providing a novel approach to identify targets and biomarkers of drug resistance.
Citation Format: Yunmin Li, Albert Wong, Ann Lu, James Lim. Adaptation of prostate cancer cell lines to tumor microenvironment culture conditions induces expression of androgen receptor splice variant 7 and contributes to drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3809.
Abstract 3833: The role of chemotherapy-induced fibrosis in the maintenance of tumor initiating cells
Authors: Lujano-Olazaba Omar , Robinson Mikella , Gilbert Samuel F. , Kogan Emily , House Carrie D. 
Published: June 2022

Abstract
Around 80 percent of high-grade serous ovarian cancer patients will relapse with chemotherapy resistant disease. Despite extensive research and developments in immunotherapy, there are limited improvements in overall survival for these patients. Recent research has found that the tumor microenvironment (TME) plays a crucial role in tumor initiation, therapy resistance, and tumor relapse. The TME, which consists of immune cells, blood vessels, and an extracellular matrix (ECM) built primarily by fibroblasts, may enhance the survival and growth of tumor-initiating cells (TICs), chemoresistant stem-like cells thought to be responsible for tumor relapse. Cytotoxic chemotherapies have been shown to modify the TME and this modulation may favor the survival of TICs. For example, carboplatin can activate stromal cells such as fibroblasts to secrete an altered ECM profile, including increases in collagen 1a1 and fibronectin production, which can bind specific integrins upregulated in TICs. Using ex-vivo decellularized peritoneum tissues from mice exposed to chemotherapy or vehicle treatment, we observed a 2-fold increase in growth in peritoneums pre-exposed to chemotherapy relative to those pre-exposed to vehicle. We hypothesize that alterations in the ECM following chemotherapy treatment permit TIC adhesion and growth. To investigate this, mouse embryonic fibroblasts (3T3s) were exposed to chemotherapy during ECM production, decellularized, and reseeded with a panel of ovarian cancer cell lines. Our results suggest a significant increase in cell survival in matrices pre-exposed to chemotherapy compared to control matrices pre-exposed to vehicle. Using this model, we are examining the features of cells that preferentially survive on these matrices. In addition to drug resistance, we are using flow cytometry to assess TIC markers, such as CD117, and CD133, and qRT-PCR to measure stemness genes such as SOX2, OCT4, and NANOG. These studies highlight the need for further investigation into the systemic effects of chemotherapies on the TME and how these changes may be fostering cancer cell adhesion, growth, and survival. Understanding these mechanisms will allow us to develop therapies to achieve better clinical outcomes.
Citation Format: Omar Lujano-Olazaba, Mikella Robinson, Samuel F. Gilbert, Emily Kogan, Carrie D. House. The role of chemotherapy-induced fibrosis in the maintenance of tumor initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3833.
Abstract 3841: SMIM11A, a novel protein involved in the mechanism of vasculogenic mimicry formation in vitro
Authors: Mingo Gabriel , Pradenas Javiera , Babbitt Nicole , González Pamela , Bertocchi Cristina , Owen Gareth 
Published: June 2022

Abstract
Introduction: Vasculogenic mimicry (VM) describes a process by which cancer cells establish an alternative perfusion pathway in an endothelial cell-free manner. Despite the strong correlation with reduced patient survival, the mechanisms by which a tumor can create this self-generated irrigation system are still not fully understood. The process of VM in vitro can occur in laminin-111-containing Matrigel and requires the PI3K pathway. However, the membrane protein component and signaling pathways involved in this process are unknown.
Methods: In an established in vitro model of VM of ovarian and breast cancer cells (HEY and MDA-MB-231, respectively) on Matrigel coating, we utilized gene silencing and blocking antibodies to elucidate the signaling pathways involved in this process. RNASeq was used to identify novel transcripts and siRNA was utilized to verify the requirement of candidate RNA/protein in tubular formation.
Results: siRNA and antibody blocking of integrin β1, but not β3, prevented VM formation in vitro. Individual silencing of cortactin, TKS5, MMP-2, MMP-9 and MMP-14 affected tubular formation. RNAseq analysis suggested that VM has minimal dependence on de novo transcriptional activity yet reported a strong upregulation of small Integral Membrane Protein 11 (SMIM11A). This result was verified by qPCR and siRNA silencing of SMIM11A prevented VM formation.
Discussion: Laminin 111 may interact with integrin β1, and the consequent activation of the PI3K pathway could potentially remodel cytoskeletal proteins and promote the activity of MMPs. We report for the first time a biological role for SMIM11a. This gene is regulated at the RNA level and is required for the formation of tubular structure in vitro. As VM is strongly associated with poor patient survival, understanding the formation of this alternative irrigation system may deliver new druggable targets.
Citation Format: Gabriel Mingo, Javiera Pradenas, Nicole Babbitt, Pamela González, Cristina Bertocchi, Gareth Owen. SMIM11A, a novel protein involved in the mechanism of vasculogenic mimicry formation in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3841.
Abstract 3844: COL11A1: A driver of tumor progression and immune evasion in head and neck squamous cell carcinoma (HNSCC)
Authors: Saunders Harmony Ivanna , Arnold Levi , Markiewicz Mary , Thomas Sufi M. 
Published: June 2022

Abstract
We previously reported that collagen type XI alpha 1 (COL11A1) is upregulated in head and neck squamous cell carcinoma (HNSCC) and contributes to tumor progression. Here we tested the hypothesis that COL11A1 regulates the expression of immune regulatory markers in HNSCC. Xena Browser was used to analyze 520 and 42 cancer free normal head and neck tissue samples for COL11A1 expression. The Cancer Genome Atlas (TCGA) RNASeq data from 522 HNSCC samples was downloaded and sorted by clinical parameters and RNA expression for 20,000 genes. COL11A1 expression was correlated with tumor stage (T) and clinical stage (I-IV). Data was sorted by COL11A1 high and low based on the mean COL11A1 expression level and run through the CIBERSORT analysis tool for prediction of immune cell proportions in the tumor. HNSCC lines with COL11A1 knockdown were assessed for immune regulatory markers at the RNA and protein level. COL11A1 was found to be increased in HNSCC relative to normal tissue (tumor vs. normal p&lt;0.0001). COL11A1 expression is highest in HPV negative HNSCC. Its expression correlates with increased tumor (T1 vs T4 p=0.033) and clinical stage (stage III vs. stage IV p=0.023). CIBERSORT analysis demonstrates COL11A1hi patients have significantly decreased active natural killer (NK) cells (active vs. inactive p=0.0166), CD8+ T cells (CD8 low vs. high p&lt;0.0001) and active dendritic cells (active vs. inactive p=0.0035) suggesting immune modulation. COL11A1 targeted lines in vitro show decreased expression of hla-a, -c and cd274 (PD-L1), suggesting COL11A1 expression modulates immune activation by regulating immune suppressive ligands on the tumor cell. COL11A1 is overexpressed in HNSCC and drives tumor proliferation, migration and invasion. Expression levels correlate with tumor and clinical stage. Bioinformatics tool CIBERSORT, predicts decreased immune cell activity in patients with high COL11A1. Targeting COL11A1 in vitro decreased expression of immune suppressive ligands. These preliminary results indicate COL11A1 may drive immune evasion in HPV negative HNSCC. Targeting COL11A1 may improve patient response to immunotherapeutic targets in HNSCC.
Citation Format: Harmony Ivanna Saunders, Levi Arnold, Mary Markiewicz, Sufi M. Thomas. COL11A1: A driver of tumor progression and immune evasion in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3844.
Abstract 3846: Parkin regulates tumor evasion by controlling antigen processing and presentation through the PTEN/AKT network
Authors: Linares Renzo Perales , Mohei Hesham , Leli Nektaria M. , Beghi Silvia , Kostopoulos Nektarios , Maity Amit , Koumenis Costantinos , Facciabene Andrea 
Published: June 2022

Abstract
A growing body of evidence supports the role of Parkin as a tumor suppressor involved in a number of cellular processes, including the downregulation of cell cycle and proliferation, migration, invasion, metastasis, mitophagy, and energy metabolic reprogramming. Indeed, epidemiological, genetic, and bioinformatic studies have shown multiple degrees of defects in the PARK2 gene, localized in human chromosome 6q25-27, a region frequently lost in cancers. In the present study, we show a novel role of Parkin in tumor immune surveillance through dysregulation of the PTEN/PI3k/AKT pathway, involving the modulation of antigen presentation machinery and antitumor-specific CD8+ T cell reactivity. We generated murine and human models that resemble the advanced-stage tumors where Parkin deficiencies are mostly found. In the mouse models, we observed a striking increase of tumor aggressiveness in the absence of Parkin that was associated with loss of antigen presentation and a significant decrease in the infiltration of antitumor CD8+ T cells. Importantly, loss of PARK2 also affected the efficacy of a Tumor-Associated Mitochondria Antigens (TAMAs) based vaccine showing the crucial role of Parkin in tumor development and in the context of antitumor cellular therapeutic strategies. Moreover, we also found that the lack of Parkin in the B16-OVA melanoma model also negatively impacts cytosol-derived antigen presentation and CD8+ T cell immunity. Finally, the analysis of TCGA tumor atlas validates the relevance of PARK2 loss on cancer immunotherapy effectiveness. Thus, during the progression and immunotherapy of cancer, the presence of Parkin mutations that result in loss of its functionality impact antigen presentation and facilitate tumor evasion.
Citation Format: Renzo Perales Linares, Hesham Mohei, Nektaria M. Leli, Silvia Beghi, Nektarios Kostopoulos, Amit Maity, Costantinos Koumenis, Andrea Facciabene. Parkin regulates tumor evasion by controlling antigen processing and presentation through the PTEN/AKT network [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3846.