bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1830: Phenylacetaldehyde induces apoptosis through endoplasmic reticulum and potentiates the effect of 5-FU treatment in colorectal cancer cells
Authors: Ferrandon Sylvain , Kong Cheng , Fedro Joseph , Aoun Rami-James , Chitchumroonchokchai Chureeporn , Clinton Steven , Kalady Matthew F.
Published: June 2022

Introduction: Phenylacetaldehyde (PAA), a product of phenylalanine metabolism, is generated by microbiota and has demonstrated anticancer properties in breast cancer. As the gut microbiome influences intestinal homeostasis, we investigated the effects of PAA on colorectal cancer (CRC).

Methods: Serum PAA levels and feces microbiota from 30 CRC patients and 30 healthy controls were analyzed by targeted GC-MS/MS and metagenomic sequencing. A validation cohort of 75 CRC patients and 53 healthy controls was analyzed for PAA level in serum. CRC cell lines and CRC-patient derived organoids were cultured and treated with PAA alone and in combination with 5-fluouracil (5-FU, standard chemotherapy used for CRC), then evaluated for viability, proliferation, cell cycle distribution, and cell death in vitro. We interrogated the mechanistic pathways induced by PAA treatment using RNA sequencing and western blot.

Results: PAA levels were significantly decreased in the circulation of CRC patients compared to healthy controls in our study population and the validation cohort. Metagenomic sequencing indicated the bacteria that positively correlated with PAA levels were significantly more abundant in healthy controls; while bacteria that negatively correlated with PAA were more abundant in CRC. In vitro, PAA inhibited the viability and colony formation of CRC cell lines as well as human-derived organoids in a concentration-dependent manner. Mechanistically, PAA treatment induced a P53-dependant G1 cell cycle arrest and cell death in HCT116 and RKO cell lines. Proteomic analysis also demonstrated a downregulation in PI3K/AKT/mTOR and ERK pathways. Investigation by RNA sequencing showed that PAA inhibits CRC by inducing endoplasmic reticulum stress-induced autophagy. When combined with 5-FU, PAA enhanced the potency of 5-FU on viability, cell death, and cell cycle arrest by inducing DNA damage.

Conclusions:The bacteria metabolite PAA increased CRC cell death by endoplasmic reticulum stress-induced autophagy and downregulation of the PI3K/AKT/mTOR and ERK pathways. PAA also potentiated chemotherapeutic efficacy of 5-FU through induction of DNA damages. These findings warrant further exploration of PAA as a potential CRC biomarker and novel therapeutic agent.

Citation Format: Sylvain Ferrandon, Cheng Kong, Joseph Fedro, Rami-James Aoun, Chureeporn Chitchumroonchokchai, Steven Clinton, Matthew F. Kalady. Phenylacetaldehyde induces apoptosis through endoplasmic reticulum and potentiates the effect of 5-FU treatment in colorectal cancer 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 1830.
Abstract 1833: Tumor treating fields induce DNA damage and apoptosis in medulloblastoma
Authors: Nitta Ryan T. , Li Gordon
Published: June 2022

Medulloblastoma (MB) is the most common malignant brain tumor in children and accounts for approximately 20% of all pediatric central nervous system tumors. Current multimodal treatment has led to a 70-90% five-year overall survival rate, however, the prognosis for patients with tumor dissemination and recurrent MB remains poor. Additionally, the majority of survivors exhibit long-term neurocognitive and neuroendocrine complications because of the cytotoxic drugs and high dose radiation. Consequently, more effective and less toxic treatments are necessary in order to raise the quality of life for these young patients. Tumor Treating Fields (TTFields) are an exciting new therapeutic opportunity for MB. TTFields are low intensity, alternating electric fields that disrupt cell division through physical interactions with key molecules during mitosis. The side effects from TTFields treatments are incredibly low, making it an ideal candidate for MB therapy. Currently TTFields were found to increase the survival of adult brain tumor patients by dysregulating the cytoskeletal network leading to mitotic arrest as well as other varied multi-modal mechanisms of action. To determine if TTFields can be an effective treatment for MB, we conducted an in vitro study treating multiple MB cell lines with TTFields. We discovered that TTFields treatment reduced cell growth in three different MB molecular subgroups (SHH, Group 3, and Group 4) with an optimal frequency of 200-300 kHz. We also observed that TTFields treatment dysregulated actin polymerization that corresponded with a reduction in cell motility and invasion. TTFields treatment also increased DNA damage (γH2AX, 53BP1) that correlated with an increase in apoptotic cells. To expand on these initial findings, we conducted a combinatorial study with the small molecule inhibitor, CX-4945. CX-4945 is an inhibitor to casein kinase 2 and is currently being used in a phase I clinical trial to treat recurrent MB (NCT03904862). We discovered that CX-4945 enhanced the growth reduction of TTFields treatment. In addition, combining CX-4945 and TTFields increased the number of cells with dysregulated actin which correlated with a decrease in MB migration and invasion. Our findings demonstrate that TTFields may be a novel and less toxic method to treat MB patients.

Citation Format: Ryan T. Nitta, Gordon Li. Tumor treating fields induce DNA damage and apoptosis in medulloblastoma [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 1833.
Abstract 1849: Lipid metabolites as potential diagnostic biomarkers for stearoyl-CoA desaturase 1 targeting therapy in hepatocellular carcinoma (HCC)
Authors: Gleba Justyna Joanna
Published: June 2022

New effective therapies should be developed for the treatment of hepatocellular carcinoma (HCC). One of the very promising strategies is blocking the activity of stearoyl-CoA desaturase 1 (SCD1). SCD1 is the enzyme responsible for de novo fatty acid (FAs) synthesis by converting saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs). In our laboratory, we developed a novel, highly specific SCD1inhibitor - SSI-4. We have tested the biological activity of SSI-4 against different HCC cell lines and patient-derived xenografts (PDX) mouse models. Of the twelve tested HCC cell lines, four were highly sensitive to SSI-4 (IC50 1-50 nM). Other cell lines showed moderate or no sensitivity to SSI-4. We conducted analysis of the enzymatic protein levels involved in de novo fatty acid synthesis. Protein expression levels for these enzymes don’t predict response to therapy. To determine whether lipid metabolites predict response to SSI-4 therapy, lipidomic analysis was investigated. Our studies showed thatSSI-4 sensitive cells when exposed to SSI-4 had a higher baseline level of key lipid metabolites such as phospholipids (PL), diglycerides (DAG), diglycerides (TAG), ceramides (CE), and free fatty acids (FFA),compared to insensitive cells. We are currently validating these results in our in vivo models at the tumor tissue and blood levels. Additionally, mechanistic studies are in progress to determine if SREBP1c, an AKT regulated transcription factor known to regulate each of the fatty acid synthetic enzymes plays a role in HCC fatty acid synthesis and sensitivity to SCD1 inhibition.

Citation Format: Justyna Joanna Gleba. Lipid metabolites as potential diagnostic biomarkers for stearoyl-CoA desaturase 1 targeting therapy in hepatocellular carcinoma (HCC) [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 1849.
Abstract 1874: Use of high throughput SPR to characterize antibodies to common cancer biomarkers
Authors: Bibikov Sergei , Rosenfeld John , Harvey Kevin J.
Published: June 2022

As a disease of mis-regulation, cancer is characterized both genetically and proteomically, requiring high quality tools to dissect the change difference between cell states. Among the reagents used to characterize biological systems at the protein level, antibodies are routinely applied, and engineered versions have proven useful for treating multiple therapeutic indications. We have generated a human proteome wide toolbox of polyclonal antibodies, identifying three antigenic 14mer sequences within each of the 20,000+ open reading frames at N-terminal, middle and C-terminal regions. Using a high throughput SPR system, Carterra LSA, we are able to define average on-rates, off-rates, and KD’s for pAb’s generated to important cancer regulators and biomarkers, including KRAS, STAT3, and Gli1. Due to the small size of the peptide targets, the kinetics of the observed interactions with polyclonal antibodies exhibit remarkable 1:1 fit typically observed when measuring monoclonal antibody affinities. The platform is a sensitive system capable of both identifying multiple antibody-antigen interactions with KD in the nanomolar to picomolar range, but also as a mass screening tool for developing de novo antibody pairs. We define the kinetic characteristics of these antibody reagents relative to performance in western blotting specifically. Improving the characterization of such widely used tools is critical for advancing translational opportunities into use in the clinic.

Citation Format: Sergei Bibikov, John Rosenfeld, Kevin J. Harvey. Use of high throughput SPR to characterize antibodies to common cancer biomarkers [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 1874.
Abstract 1882: Passenger gene co-amplification leads to collateral vulnerabilities in cancer
Authors: Bei Yi , Kirchner Marieluise , Brame Luca , Fritsche-Guenther Raphaela , Koeppke Julia , Heeren-Hagemann Anja , Mertins Philipp , Dörr Jan , Henssen Anton George
Published: June 2022

The activation of oncogenes via genomic amplification is a hallmark of the cancer genomes, yet such amplifications frequently also encompass neighboring genes. Such passenger genes are involved in essential cellular activities. We hypothesized that co-amplification of passenger genes create cancer-specific collateral therapeutic vulnerabilities. To test this, we analyzed one of the most commonly amplified gene locus near MYCN, which is amplified in nearly 25% of neuroblastoma and around 5% of all cancers. We observed that DDX1, a DEAD-Box helicase, is the most frequently co-amplified passenger gene in MYCN-amplified cancers. Using CRISPR/Cas9 loss-of-function screen results from the Cancer Dependency Map across 739 human cancer cell lines, we reveal that cells with DDX1 co-amplification are highly dependent on the mTOR complex 1(mTORC1). Mechanistically, western blot and immunoprecipitation analyses shows that DDX1 expression leads to higher mTORC1 pathway activity through the interaction between DDX1 and alpha-ketoglutarate dehydrogenase(alpha-KGDH) in mitochondria. DDX1 hijacks alpha-KGDH complex, thereby impairing the function of the Krebs cycle by interfering with the conversion of alpha-ketoglutarate to succinyl-coA. Live cell metabolomics shows accumulated alpha-ketoglutarate in turn activates mTORC1 pathway to promote the ATP production via oxidative phosphorylation(OXPHOS) to maintain cell survival. Consequently, interference with mTORC1 function reduced ATP production and led to accumulated alpha-KG leading to cell death, specifically in DDX1 expressing cells. This suggests that co-amplification of a passenger gene can result in collateral vulnerabilities in cancers, which has the potential to transform future therapeutic target discovery in oncology.

Citation Format: Yi Bei, Marieluise Kirchner, Luca Brame, Raphaela Fritsche-Guenther, Julia Koeppke, Anja Heeren-Hagemann, Philipp Mertins, Jan Dörr, Anton George Henssen. Passenger gene co-amplification leads to collateral vulnerabilities in cancer [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 1882.
Abstract 1891: Identifying new therapeutic antibodies using the RenMice™ HiTS Platform
Authors: Zhang Xiaoqian , Zhang Shujin , Jin Tianquan , Ji Xin , Zhang Jing
Published: June 2022

Over the past decade, an increasing number of therapeutic antibodies have been approved to target cancer. However, the process of identifying new therapeutic antibodies is routinely hampered by limitations in the traditional discovery process, including immune tolerance of highly homologous genes, and challenges with antibody sequence humanization, clone selection and drug efficacy and safety evaluation. RenMice™ HiTS (Hyperimmune Target Specific) is a collection of chromosome engineered mice with fully human immunoglobulin variable domains replacing mouse counterparts, each with a specific drug target gene knocked out. The goal of the RenMice™ HiTS platform is to overcome the challenges of traditional antibody drug discovery and increase the diversity of antibody paratopes and sequences which recognize functional epitopes. Immunization of target-specific RenMice™ generates a greater diversity of antibodies, including those that recognize the conserved regions between the antigen and the endogenous proteins of the immunized host. The platform is ideal for challenging targets, such as proteins with high homology between human and mouse, or multi-pass transmembrane proteins (eg. GPCRs/ion channels). The platform can be used to generate antibodies that cross-react with human, monkey, dog, and mouse targets, using a hybrid immunization strategy with both human and mouse/dog antigen. Generation of these species cross-reactive antibodies allows for high-throughput in vivo efficacy screening using wild-type mice. It is also possible to predict the preliminary response and toxicity in dogs with these cross-reactive antibodies. Thus, selection of the best antibody candidate based on in vivo efficacy and safety allows for a streamlined and successful preclinical phase. In conclusion, the RenMice™ HiTS platform facilitates the generation of antibodies with better developability properties that recognize challenging targets and novel epitopes.

Citation Format: Xiaoqian Zhang, Shujin Zhang, Tianquan Jin, Xin Ji, Jing Zhang. Identifying new therapeutic antibodies using the RenMice™ HiTS Platform [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 1891.
Abstract 1912: A versatile computational pipeline for the preprocessing of cell-free DNA fragmentation data
Authors: Ivankovic Ivna , Balázs Zsolt , Gitchev Todor , Banos Daniel Trejo , Moldovan Norbert , Balermpas Panagiotis , Willmann Jonas , Andratschke Nicolaus , Moulière Florent , Krauthammer Michael
Published: June 2022

Cell-free DNA (cfDNA) emerges as a promising liquid biopsy biomarker for cancer diagnosis and patient monitoring. Complementing mutation-based assays, cfDNA carries information about epigenetic modifications from decaying cells. This information is encoded in the shape of the cfDNA fragments. Specifically, fragments from cancer tend to be shorter than those originating from other adult cells, enabling a distinction between cancer patients and healthy individuals. Additional cfDNA features such as fragment end motifs and information on nucleosome positioning provide further insight into cancer biology. These cfDNA measures are typically inferred from low-pass whole genome sequencing and subsequent bioinformatics processing. A key bioinformatics step is the alignment of DNA sequencing reads to the reference genome, which critically depends on preprocessing steps such as read trimming and alignment filters. A good understanding of preprocessing settings is thus crucial to derive accurate information from cfDNA. We therefore investigated to what extent preprocessing choices affect cfDNA analysis. To this end, we have built a robust bioinformatics pipeline that evaluates a range of possible preprocessing settings. The pipeline is implemented in a bioinformatics workflow engine, enabling a scalable and reproducible workflow. We are currently evaluating the effect of preprocessing on global and regional fragmentation patterns, detection of nucleosome positioning and identification of fragment end motifs. The different preprocessing settings are also evaluated for their ability to distinguish cancer from control samples using cfDNA fragmentation information. We are benchmarking the pipeline using cfDNA datasets from multiple centres to ensure our findings are generalizable over different platforms and experimental procedures. Our investigations will allow us to build a versatile bioinformatics preprocessing pipeline for the analysis of cell-free DNA fragmentation data.

Citation Format: Ivna Ivankovic, Zsolt Balázs, Todor Gitchev, Daniel Trejo Banos, Norbert Moldovan, Panagiotis Balermpas, Jonas Willmann, Nicolaus Andratschke, Florent Moulière, Michael Krauthammer. A versatile computational pipeline for the preprocessing of cell-free DNA fragmentation data [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 1912.
Abstract 1927: Transcell: In silico characterization of genomic landscape and cellular responses from gene expressions through a two-step transfer learning
Authors: Yeh Shan-Ju , Chen Ruoqiao , Xing Jing , Sun Mengying , Liu Ke , Paithankar Shreya , Zhou Jiayu , Chen Bin
Published: June 2022

Gene expression profiling of new or modified cell lines becomes routine today; however, obtaining comprehensive molecular characterization and cellular responses for a variety of cell lines, including those derived from underrepresented groups, is not trivial when resources are minimal. Using gene expression to predict other measurements has been actively explored; however, systematic investigation of its predictive power in various measurements has not been well studied. We present TransCell, a two-step deep transfer learning framework that utilizes the knowledge derived from pan-cancer tumor samples to predict molecular features and responses. Compared to the five state-of-art methods, TransCell has the best performance in predicting metabolite, gene effect score (or genetic dependency), and drug sensitivity, and has comparable performance in predicting mutation, copy number variation, and protein expression. Notably, TransCell improved the performance by over 50% in drug sensitivity prediction and achieved a correlation of 0.7 in gene effect score prediction. Furthermore, predicted drug sensitivities revealed potential repurposing candidates for new 100 pediatric cancer cell lines, and predicted gene effect scores reflected BRAF resistance in melanoma cell lines. Together, TransCell demonstrates its remarkable predictive power that enables in silico molecular characterization of understudied cell lines.

Citation Format: Shan-Ju Yeh, Ruoqiao Chen, Jing Xing, Mengying Sun, Ke Liu, Shreya Paithankar, Jiayu Zhou, Bin Chen. Transcell: In silico characterization of genomic landscape and cellular responses from gene expressions through a two-step transfer learning [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 1927.
Abstract 1936: MethylationToRegulation: A deep-learning approach to infer chromatin properties from DNA methylomes
Authors: Putnam Daniel K. , Abraham Brian J. , Chen Xiang
Published: June 2022

Gene regulation is critical for cell identity, and its dysregulation is a defining characteristic of common diseases including cancers. Although promoter activity is a strong predictor of gene expression, activities at other regulatory elements, including enhancers and super-enhancers (SE), are major contributors to gene regulation. For example, distinct transcription factor-regulating SEs define neuroblastoma (NB) subtypes with distinct clinical outcomes. However, technical limitations largely prevent ChIP-seq based enhancer/SE activity profiling in primary patient samples. We previously developed MethylationToActivity (M2A) and demonstrated that high-order DNA methylation (DNAm) features are strong predictors of promoter activity measured by ChIP-seq. This tool is important because genomewide DNAm assays are widely used in clinic to classify tumors and stratify patients on clinical trials. However, it is limited by its reliance on known gene position annotations. Here we present MethylationToRegulation (M2R), a method that utilizes a convolutional neural network (CNN)-based deep learning framework to infer intergenic enhancer/SE activities from DNA methylomes. We obtained paired WGBS and H3K27ac ChIP-seq data for 16 pediatric NB samples profiled in the Pediatric Cancer Genome Project. M2R was trained on 6 samples and tested on the remaining 10 samples. It achieved an average relative prediction accuracy of 84% on test samples when compared to the H3K27ac ChIP-seq replicate consistency from the ENCODE project. We adapted the Rank Ordering of Super-Enhancers algorithm to interpret H3K27ac signals inferred from DNA methylomes to identify SEs. M2R faithfully captured subtype-defining SEs with high specificity, including those associated with master NB transcription factors. Our results demonstrate that M2R accurately quantifies enhancer/SE activities and infers critical epigenetic marks from DNA methylomes. Application of M2R will enable the profiling of epigenetic dysregulation in patient tumor samples, seeking to improve clinical outcomes.

Citation Format: Daniel K. Putnam, Brian J. Abraham, Xiang Chen. MethylationToRegulation: A deep-learning approach to infer chromatin properties from DNA methylomes [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 1936.
Abstract 2: Flavokawain A targets cancer stem cells for inhibiting the growth of prostate cancer
Authors: Song Liankun , Mino Merci , Yamak Jana , Nguyen Vyvyan , Lopez Derron , Pham Victor , Fazelpour Ali , Le Vinh , Fu Dongjun , Tippin Matthew , Zi Xiaolin
Published: June 2022

Prostate cancer stem cells (PCSC) are small tumor subpopulations capable of self-renewal and driving prostate cancer heterogeneity, which is critical for cancer initiation, recurrence, and development of therapeutic resistance. Thus, there’s an urgent need to explore and develop new CSC targeting agents. Flavokawain A (FKA), a novel chalcone from kava plant, can selectively inhibit the growth of pRb deficient cell lines and result in a proteasome-dependent and ubiquitination-mediated Skp2 (S-phase kinase-associated protein 2) degradation, while the potential of FKA to target PCSC is unknown. Therefore, we evaluated the effects of FKA on PCSC self-renewal both in vitro and in vivo. FKA treatment decreased both size and numbers of the tumor spheroids growing from CD44+/CD133+ tumor stem cells, which were sorted out from the prostate tumor cell line DU145 and 22RV1. Expression of the stem cell markers Nanog, Oct4, and Sox2 were all downregulated in the cancer spheroids treated with FKA. Growth of the 22RV1CD44+/CD133+ xenograft tumors was significantly inhibited in FKA diet-fed mice compared to the vehicle control diet-fed mice. Immunohistochemistry analysis revealed less positive Ki67, Nanog, and Oct4 cells in tumor tissues from the FKA dietary group than those from the control dietary group. These results demonstrated that FKA can inhibit prostate cancer stemness and suggested a potential role of FKA for preventing prostate cancer recurrence by targeting PCSC.

Citation Format: Liankun Song, Merci Mino, Jana Yamak, Vyvyan Nguyen, Derron Lopez, Victor Pham, Ali Fazelpour, Vinh Le, Dongjun Fu, Matthew Tippin, Xiaolin Zi. Flavokawain A targets cancer stem cells for inhibiting the growth of prostate cancer [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 2.
Abstract 200: Microtube Array Membrane-Hollow Fiber Assay (MTAM-HFA): A combination of immunocheckpoint blocker (ICBs) and secondary drug anti-cancer drug screening system with significant clinical value propositions for personalized medicine and pre-clinical drug development
Authors: Huang Wan-Ting , Chew Chee Ho , Chen Amanda , Lee Kang-Yuan , Lee Hsing-Lun , Feng Po-Hao , Chiou Jeng-Fong , Lin Kuan-Chou , Wei Po-Li , Chen Chien-Chung
Published: June 2022

Despite modern advances in technology, significant challenges still plague the development of new cancer drugs and cancer care. To address these issue, we are introducing our novel platform technology- Microtube Array Membrane-Hollow Fiber Assay (MTAM-HFA) that is based on combination of the patented MTAM membrane with unique microstructures; replacing the role of traditional hollow fibers enabled the entire HFA assay to be significantly improved in terms of clinical relevance, accuracy, reliability and cost resulting in unprecedented value propositions as an anti-cancer drug screening platform. In the current work, we have clinically demonstrated in several cases where the MTAM-HFA system is capable of accurately predicting clinical response in several Cancers-Lung, Breast and Colon cancers; with unprecedented flexibility of being screened against multiple drug class (including anti-PD1/PDL1) in monotherapy and combination therapy settings. Despite being a phenotypic based assay, the test cycle of MTAM-HFA is only 7-10 days while maintaining a very low cost of up to 60% savings when compared to traditional models such as Patient Derived Xenografts (PDX). When screening ICBs, without the need to use Genetically Engineered Mice (GEM) translates to a cost savings of 98% just for the animal model alone. The combined value proposition of MTAM-HFA makes is a suitable system fulfilling the needs of multiple stakeholders.

Citation Format: Wan-Ting Huang, Chee Ho Chew, Amanda Chen, Kang-Yuan Lee, Hsing-Lun Lee, Po-Hao Feng, Jeng-Fong Chiou, Kuan-Chou Lin, Po-Li Wei, Chien-Chung Chen. Microtube Array Membrane-Hollow Fiber Assay (MTAM-HFA): A combination of immunocheckpoint blocker (ICBs) and secondary drug anti-cancer drug screening system with significant clinical value propositions for personalized medicine and pre-clinical drug development [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 200.
Abstract 2029: Multiomic analysis of whole transcriptome and high plex protein assays on a single FFPE slide
Authors: Bonnett Shilah , Confuorto Nicholas , Fuhrman Kit , Ong Giang , Rosenbloom Alyssa , Lyssand John , Geiss Gary , Beechem Joseph
Published: June 2022

The GeoMx࣪ Digital Spatial Profiler (DSP) enables high-plex, high-throughput spatial profiling and quantification from a single slide for either protein or RNA. To fully understand the interplay between RNA and protein in tissue, we have developed a novel multi analyte assay that allows for the profiling of both analytes from the area of interest (AOI) on a single slide. Here we describe the development and performance of the multi analyte assay on cell pellet array (CPA) and various tissues including tonsil and colorectal cancer (CRC) using a high plex GeoMx Protein Panel and the GeoMx Human Whole Transcriptome Atlas (WTA). We successfully identified conditions to detect RNA and protein simultaneously, with comparable sensitivity and specificity to the single analyte conditions. The data accurately matched cell lines in an unbiased correlation analysis between GeoMx WTA and the entire CCLE RNAseq database. Furthermore, the assay performance was maintained in tissue samples. Tonsil and colorectal cancer biopsies were profiled for RNA (whole transcriptome) and protein from the same AOI, allowing simultaneous measurements of transcriptional and translational regulation of key biological pathways. This multi-analyte profiling enables deeper characterization of precious biological samples that are available in limited quantities.

Citation Format: Shilah Bonnett, Nicholas Confuorto, Kit Fuhrman, Giang Ong, Alyssa Rosenbloom, John Lyssand, Gary Geiss, Joseph Beechem. Multiomic analysis of whole transcriptome and high plex protein assays on a single FFPE slide [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 2029.
Abstract 2068: An ADCC enhanced anti-CCR8 antibody, which preferentially depletes tumor regulatory T cells and inhibits tumor growth
Authors: Haque Jamil A. , Domeier Cristina , Algate Paul A. , Liu Zhi , Yan Wei , Peng YuFeng
Published: June 2022

Tumor associated FOXP3+ regulatory T cells (Tregs) suppress antitumor immunity, thereby hindering protective immunosurveillance of tumors and hampering effective antitumor immune responses in tumor-bearing hosts. Eliminating the suppressive effects of tumor associated Tregs could, therefore, enhance antitumor activity of existing immunotherapies. CCR8 is a seven transmembrane G-coupled protein whose expression is highly upregulated on tumor associated Tregs in several types of tumors, but not on peripheral Tregs. The specific expression on tumor associated Tregs makes CCR8 an ideal target for treating cancers. The chemokine CCL1 interacts with the N terminus and second extracellular domain of CCR8. CCL1 produced by tumor associated macrophages can induce Tregs migration to the tumors. PSB114, a humanized anti-hCCR8 IgG1 antibody, was developed to block CCL1 mediated Tregs chemotaxis and deplete tumor associated CCR8-expressing Tregs. PSB114 was affinity matured by yeast display and glyco-modified to enhance its ADCC activity. PSB114 has high binding affinity for human CCR8 without significant binding to other membrane proteins, resulting in a favorable PK in hFcRn transgenic mice (T1/2 > 13 days). PSB114 is highly specific for the activated Tregs with low picomolar EC50 of depletion activity. When tested in bioassays, PSB114 enhances T cell activation and TNFα production from activated PBMCs. In a humanized mouse model, PSB114 preferentially depletes tumor infiltrating Tregs and increases NK and CD8 T cell infiltration within the tumor. Moreover, PBS114 significantly inhibited MC38 tumor growth in hCCR8 knock-in mice. These results demonstrate the potential of PSB114 for treating cancers in human.

Citation Format: Jamil A. Haque, Cristina Domeier, Paul A. Algate, Zhi Liu, Wei Yan, YuFeng Peng. An ADCC enhanced anti-CCR8 antibody, which preferentially depletes tumor regulatory T cells and inhibits tumor growth [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 2068.
Abstract 2101: RNF2 ablation reprograms the tumor-immune microenvironment and stimulates durable NK and CD4+ T cell-dependent anti-tumor immunity
Authors: Zhang Zhuo , Luo Lin , Xing Chuan , Chen Yu , Xu Peng , Li Mao , Zeng Ling , Li Chao , Ghosh Sadashib , Manna Deborah Della , Townes Tim , Britt William J. , Wajapeyee Narendra , Sleckman Barry P. , Chong Zechen , Leavenworth Jianmei Wu , Yang Eddy
Published: June 2022

Expanding the utility of immune-based cancer treatments is a clinical challenge due to tumor-intrinsic factors that suppress the immune response. Here we report the identification of tumoral Ring Finger Protein 2 (RNF2), the core subunit of the Polycomb Repressor Complex 1 (PRC1), as a negative regulator of antitumor immunity in various human cancers, including breast cancer. In syngeneic murine models of triple negative breast cancer, we found that deleting genes encoding PRC1 subunits Rnf2 or BMI1 proto-oncogene, polycomb ring finger (Bmi1), or the downstream effector of Rnf2, Remodeling and Spacing Factor 1 (Rsf1), was sufficient by itself to induce durable tumor rejection and establish immune memory by enhancing infiltration and activation of NK and CD4+ T-cells, but not CD8+ T-cells, into the tumor and enabled their cooperativity. These findings uncover an epigenetic reprogramming of the tumor-immune microenvironment which fosters durable antitumor immunity and memory.

Citation Format: Zhuo Zhang, Lin Luo, Chuan Xing, Yu Chen, Peng Xu, Mao Li, Ling Zeng, Chao Li, Sadashib Ghosh, Deborah Della Manna, Tim Townes, William J. Britt, Narendra Wajapeyee, Barry P. Sleckman, Zechen Chong, Jianmei Wu Leavenworth, Eddy Yang. RNF2 ablation reprograms the tumor-immune microenvironment and stimulates durable NK and CD4+ T cell-dependent anti-tumor immunity [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 2101.
Abstract 2104: NK cell immunosurveillance of tumors is regulated by NFAT1 and NFAT2
Authors: Märklin Melanie , Holzmayer Samuel , Kaban Kübra , Müller Martin R. , Salih Helmut R.
Published: June 2022

NK cells are lymphoid components of innate immunity and play an important role in tumor immunosurveillance. One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T cells), controlling lymphocyte development and activity. However, while the role of NFAT signaling is well defined in T cells, the cytotoxic lymphocytes of adaptive immunity, surprisingly little is known regarding the relevance of this transcription factor family in NK cells as effector cells of innate immunity. Available data indicate that NFAT activity is dispensable for development of NK cells. However, several lines of evidence including reports on the effects of the immunosuppressive drugs cyclosporin A and tacrolimus, which mediate their effects through inhibition of calcineurin and consecutively NFAT, implicate an involvement of the NFAT family in NK cell function. We here employed different genetic mouse models to unravel the role of NFAT1 (NFATc2) and NFAT2 (NFATc1) in NK cell reactivity.

When NK cells with knockout (KO) of NFAT1 or NFAT2 compared to NK cells of control mice (WT) were employed in in vitro analyses, lack of either NFAT was found to surprisingly result in enhanced NK cell degranulation as well as increased production of granzyme B and perforin upon stimulation of activating receptors like NK1.1 or Nkp46 or upon co-culture with different leukemia and solid tumor tumor cells. In line, cytotoxicity assays revealed increased lysis of YAC-1 and B16F10 tumor cells by both NFAT1- and NFAT2-deficient NK cells as compared to WT controls. The inhibitory effect of NFAT transcription factors on NK cell effector function could also be confirmed in vivo by employing WT and NFAT KO animals in syngeneic B16F10 melanoma and RMA-S flank tumor models, which revealed a significantly reduced tumor burden in NFAT1 and NFAT2 KO mice. Comparative analyses with single NFAT as well as NFAT1+NFAT2 double KO and WT animals further confirmed the inhibitory effect of NFAT1 and NFAT2 and pointed to additive effects of NFAT1 and NFAT2 in NK cell tumor immunosurveillance.

Taken together, our results provide the first evidence for the direct functional involvement of NFAT1 and NFAT2 in NK cell antitumor reactivity and, in contrast to T and B cells, identify NFAT as a negative regulator of NK cell function.

Citation Format: Melanie Märklin, Samuel Holzmayer, Kübra Kaban, Martin R. Müller, Helmut R. Salih. NK cell immunosurveillance of tumors is regulated by NFAT1 and NFAT2 [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 2104.
Abstract 2125: Towards a spatial view of immune cell function in cancer
Authors: Pourmaleki Maryam , Jones Caitlin J. , Greenstein Brian D. , Mellinghoff Sabrina D. , Navarrete Daniel A. , Mehta Smrutiben A. , Socci Nicholas D. , Mellinghoff Ingo K. , Hollmann Travis J.
Published: June 2022

Immunotherapy can result in lasting tumor regressions, but despite its success, only a subset of patients and cancer types benefit from immunotherapy. Thus, uncovering features of the tumor microenvironment (TME) that contribute to this differential response can inform candidates for novel therapies and biomarkers for patient stratification. Recent advances in single-cell technologies allow for profiling of cell states and their spatial interactions within a tumor. Here, we performed multiplexed immunofluorescence (mpIF), a method for in situ single-cell measurement of 30+ proteins, as well as bulk transcriptomics and genomics on 180 tumor samples across 4 immunotherapy-responsive and -resistant solid and liquid cancers: in-transit melanoma (ITM), non-small cell lung cancer (NSCLC), glioblastoma multiforme (GBM), and classical Hodgkin lymphoma (cHL). In aggregate, we measured over 100 million single cells and identified over 1,000 unique tumor and immune cell states. We uncovered immune-suppressive cell states, such as macrophages negative for PD-L1 and B7-H3 in immunotherapy-resistant ITM tumors and all GBM tumors, which generally fail to respond to immunotherapy. In GBM, macrophages exhibited two distinct spatial topologies, infiltrated or excluded. In ITM, we identified pre-treatment cell states and gene expression signatures that associate with immunotherapy response such as MHC class I expression on the tumor cell surface, B cell aggregates, “exhausted” PD-1/LAG-3/TIM-3 triple-positive CD8+ T cells, and expression of interferon-gamma genes. We observed a similar immune checkpoint-rich (PD-1/LAG-3/TIM-3 triple-positive) TME in all cHL tumors, which have remarkably high immunotherapy response rates. We spatially defined the microniche (30-micron radius neighborhood) of “exhausted” CD8+ T cells, and within the microniches, found antigen presentation competent tumor cells, proliferating T cells, and B7-H3 positive macrophages, which together contribute to an activated TME. Together, we present a statistical workflow for the integrated analysis of spatially resolved multidimensional data for cancer target discovery that is tailored towards application in routinely collected formalin-fixed paraffin-embedded cancer biospecimens.

Citation Format: Maryam Pourmaleki, Caitlin J. Jones, Brian D. Greenstein, Sabrina D. Mellinghoff, Daniel A. Navarrete, Smrutiben A. Mehta, Nicholas D. Socci, Ingo K. Mellinghoff, Travis J. Hollmann. Towards a spatial view of immune cell function in cancer [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 2125.
Abstract 2180: Deciphering the Warburg effect: Redox is the key to tumor differentiation
Authors: Haowen Jiang , Greathouse Rachel L. , He Bo , Li Yang , LI Albert M. , Forgo Balint , Banuelos Selene , Gruber Joshua , Shimada Hiroyuki , Chiu Bill , Ye Jiangbin
Published: June 2022

It was proposed by Warburg that the cause and consequence of the Warburg effect were the injury of respiration and cell dedifferentiation, respectively. However, the underlying mechanism was unclear due to limited understanding of metabolic control of epigenetics. Inhibition of mitochondrial electron transport chain (ETC) causes reduction of NAD+/NADH ratio. Recently it was discovered that this redox stress not only drives lactate production from pyruvate to enhance the Warburg effect, but also promotes the conversion of α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), turning the substrate of DNA demethylase TET into an inhibitor of TET, suggesting a potential link between ETC inhibition and dysregulated DNA methylation in cancer. In this study we use a mitochondrial uncoupler niclosamide ethanolamine (NEN) to block the Warburg effect and remodel cancer epigenome. NEN treatment activates ETC and accelerates glutaminolysis to produce αKG, which promotes promoter CpG island demethylation. This epigenomic reprograming not only upregulates tumor suppressor genes such as p53, inhibits N-Myc expression, but also activates neuron differentiation markers to promote differentiation in neuroblastoma cells. Hypoxia is one common microenvironmental stress existing in all solid tumor. NEN treatment reduces 2HG generation from αKG under hypoxia, highlighting its application potential in preventing DNA hypermethylation in vivo. Associated with 2HG reduction, NEN treatment also represses HIF signaling under hypoxia. In addition, NEN treatment inhibits reductive carboxylation flux. Moreover, dietary supplementation with NEN reduces orthotopic neuroblastoma growth and N-Myc expression in vivo. Together, these results validated Warburg’s hypothesis that mitochondrial respiration is the key to maintain normal differentiation. Restoring ETC activity with mitochondrial uncoupler is an effective metabolic/epigenetic therapy against neuroblastoma (Jiang et al., BioRxiv 2021).

Citation Format: Jiang Haowen, Rachel L. Greathouse, Bo He, Yang Li, Albert M. LI, Balint Forgo, Selene Banuelos, Joshua Gruber, Hiroyuki Shimada, Bill Chiu, Jiangbin Ye. Deciphering the Warburg effect: Redox is the key to tumor differentiation [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 2180.
Abstract 2247: Prostate cancer-related mortality risk assessment in a cohort of southern Nigerian men
Authors: Okoye Jude O. , Ogbonnaya Vivian I. , Emeka Chiemeka M. , Obioma Okechi
Published: June 2022

Background: Overexpression of breast cancer susceptibility gene 2 (BRCA2) in patients with prostate cancer is associated with a high Gleason score at diagnosis, poorly differentiated tumors, poor prognosis, and disease recurrence. Such aggressive tumors possess high iron storage which promotes inflammation, angiogenesis, and cell proliferation. Expression of iron progressively increases with cancer grade and stage.

Purpose: This study evaluated the expression of BRCA2 protein and Iron expression in Gleason’s graded prostate cancer tissues, in a bid to determine the disease aggressiveness and mortality risk among Southern Nigerian men.

Methods: In this retrospective study, 86 formalin-fixed paraffin-embedded prostate cancer tissues (diagnosed in 2018 and 2019) were reviewed using Gleason’s grading system. Tissues stained for iron and BRCA2 proteins using histochemical and immunohistochemical staining techniques, respectively. Staining intensity was scored using a scale of 0, +1, +2, and +3. For BRCA2 and Iron expressions, score 0 and +1 were considered negative while score +2 and +3 were considered positive. Pearson’s chi-square test was used to determine the association between Gleason’s grade and expression of BRCA2 and iron.

Results: The prevalence of prostate cancer in the age groups of 50-59, 60-69, 70-79, 80-89, and 90-99 years was 10.8%, 32.4%, 40.5%, 13.5% and 2.7%, respectively. Using the Gleason’s grading pattern, the frequency of Grade (mean age in years) I, II, III, IV and V was 9.3% (70.25 ± 6.95), 16.3% (77.29 ± 6.23), 16.3% (75.57 ± 12.59), 25.6% (72.75 ± 7.80) and 32.6% (67.42 ± 9.76), respectively. Higher frequency of BRCA2 and Iron expression were observed in ≥G4 (37.5% and 72.7%) than in ≤G3 (versus 16.7% and 11.8%) at p= 0.191 and p< 0.001, respectively. The prevalence of BRCA2 negative and positive tissues was 65.1% and 34.9%, respectively whereas the prevalence of iron negative and positive tissues was 48.7% and 51.3%, respectively. There was an inverse relationship between tumour grade and age (p= 0.108), and direct relationships between tumour grade and BRCA2 (p= 0.059), tumour grade and iron (p= 0.000), BRCA2 and Iron expressions (p= 0.027), BRCA2 expression and age (p= 0.019), and Iron and age (p= 0.281). Patients with prostate cancer who are within the age group of 50-59 years and those with ≥G4 require close monitoring for improved survival.

Conclusion: This study revealed a high prevalence of aggressive prostate cancer and suggests that there is a high mortality risk among Southern Nigerian men diagnosed with the disease.

Citation Format: Jude O. Okoye, Vivian I. Ogbonnaya, Chiemeka M. Emeka, Okechi Obioma. Prostate cancer-related mortality risk assessment in a cohort of southern Nigerian men [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 2247.
Abstract 2250: Eosinophil-to-lymphocyte ratio and cancer risk in the UK Biobank
Authors: Wang Jeanny , Engels Eric , Song Minkyo
Published: June 2022

Background: Eosinophils exhibit anti-tumor cytotoxic responses in the tumor microenvironment. Some atopic diseases, characterized by elevated peripheral eosinophils, are inversely associated with cancer risk. The balance of atopy and immunity may be estimated by an eosinophil-to-lymphocyte ratio (ELR). This study aims to understand the role of ELR as a biomarker in carcinogenesis.

Methods: We evaluated 445,404 adults aged 37-73 years at baseline in a prospective cohort study, the UK Biobank, who were cancer-free at initial assessment, had more than one year of follow-up, and had baseline white blood cell (WBC) count and differential measurements. ELR was categorized into quartiles. Incident cancers were ascertained from cancer registries from 2006-2015. Multivariable Cox regression was used to estimate per quartile ELR hazard ratios (aHRs) separately for 52 cancer sites, adjusted for age (quartiles), sex, race (white, non-white), smoking/alcohol (never, past, current), body mass index (<25, 25-30, ≥30 kg/m2) and total WBC (quartiles). Multiple comparisons were considered by a Bonferroni-corrected threshold (p<0.00096).

Results: Over a median follow-up time of 6.9 years from baseline, 22,853 incident cancers were diagnosed. The median ELR was 0.07 (interquartile range=0.05-0.12). Higher ELR was inversely associated with overall cancer risk (aHR 0.98, 95%CI: 0.97-0.99; p-trend=0.0001) and chronic lymphocytic leukemia (0.50, 0.44-0.56; <0.0001). We also observed suggestive inverse ELR associations with other lymphocytic leukemia, acute and other myeloid leukemia, myeloma, melanoma and cancers of the nose/middle ear, tongue, prostate (aHR range: 0.63-0.97; p-trend<0.05). Per quartile increase of ELR was suggestively associated with increased risk of diffuse large B-cell lymphoma (aHR 1.13, 95%CI: 1.03-1.25; p-trend=0.01).

Conclusion: We observed inverse associations between ELR and cancer risk in a large general population. Our findings suggest that atopy may be protective against cancer. Future research is warranted to elucidate the relationship between atopy and immunity in tumor surveillance.

Impact/Significance: ELR presents a cost-effective and routinely accessible biomarker that may have implications for carcinogenesis, and may be leveraged to inform early detection of certain cancers.

Citation Format: Jeanny Wang, Eric Engels, Minkyo Song. Eosinophil-to-lymphocyte ratio and cancer risk in the UK Biobank [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 2250.