bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 3739: EM-seq permits accurate and precise methylome analysis of cfDNA and FFPE DNA
Authors: Williams Louise 
Published: June 2022

Abstract
The study of DNA methylation from cell-free DNA (cfDNA) and DNA extracted from formalin fixed paraffin embedded (FFPE) tissues are notoriously difficult. The use of these DNA inputs is becoming increasingly important in the field of cancer diagnostics. The cytosine modifications, 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC), are key epigenetic factors that play an important role in cellular processes and their mis-regulation results in diseased states like cancer. Advances in the study of these epigenetic factors in combination with improvements in sample preparation have enabled cancer biomarker identification based on methylation profiling.
The historical gold standard for detecting cytosine methylation has been bisulfite sequencing. This method uses chemical conversion of cytosines into uracils and has been used for both targeted and whole genome methylation analysis. However, bisulfite conversion leads to DNA damage which results in shorter DNA insert sizes as well as biases in the data. It is challenging to make bisulfite libraries from cfDNA and FFPE DNA as typically the DNA is of low quantity and quality.
We developed an enzyme-based methylation detection technology (EM-seq) to address the flaws inherent to bisulfite sequencing. This method, primarily due to the intact nature of DNA after conversion, results in better quality libraries with longer insert sizes, lower duplication rates and minimal GC bias. EM-seq libraries prepared from cfDNA and lung FFPE DNA had longer inserts, lower duplication rates, higher percentages of mapped reads and less GC bias compared to bisulfite libraries. EM-seq libraries also identified a higher number of CpGs with even coverage facilitating accurate methylation calling. EM-seq’s superior sequencing metrics establish it as a standard method for robust methylation profiling, particularly for these types of challenging DNA samples.
Citation Format: Louise Williams. EM-seq permits accurate and precise methylome analysis of cfDNA and FFPE DNA [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 3739.
Abstract 3765: Epigenomics at the speed of sound: High throughput epigenomic mapping of low cell input samples using acoustic enhanced immunoprecipitation
Authors: Alija Besmira , Braunstein Stephanie , Xu Jane , O'Hare Tom , Garding Angela , Viny Aaron 
Published: June 2022

Abstract
Mapping the epigenetic landscape of Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) is essential for understanding gene regulatory mechanisms during cell fate changes, disease onset, progression, as well as for optimizing therapeutic regime. In addition, these information hold immense potential for accurate identification of disease biomarkers, cell of origin, and targets for epigenetic therapy. However, existing chromatin immunoprecipitation sequencing (ChIP-Seq) workflows require significant cellular material and are very time-consuming, requiring cumbersome optimization. Moreover, cellular heterogeneity can influence the accuracy of results from the perspective of sample preparation efficiency and also population-biased effects. Both normal and malignant bone marrow are heterogeneous in cell type and stage of differentiation that leads to imprecise results with regard to epigenetic determinates.In our study, we implemented Covaris’ Adaptive Focused Acoustics® (AFA®) technology to miniaturize sample volumes for both chromatin shearing and immunoprecipitation in a 96-well format. This approach enables a standardized, robust, automatable, half-day workflow for both ChIP and Hi-ChIP applications. Additionally, we evaluated low input in vitro and in vivo samples, where we utilized a range of epitopes including major histone modifications (H3K27ac) and factors (CTCF). AFA was applied to flow-sorted stem and progenitor populations from individual mice. The low cell input capability permitted deconvolution of dynamic epigenetic changes in protein binding and chromatin structure during normal lineage specification of hematopoietic stem cells. Epigenetic remodeling of these sites were consistent with established gene programs essential for myeloid differentiation. Data were found to be in alignment with standard ChIP-seq and Hi-C datasets generated from similar cell populations.The simplified and significantly shortened (Hi)ChIP-seq processes showcased in this study will be highly beneficial for both research and clinical diagnostic applications owing to their requirements for using 96-well plate format and their ability to become integrated into automated workflows. Taken together, this technology and workflow represent a high throughput epigenetic system for small cell numbers of cells with a multitude of applications enabling laboratories focused on cell biology and translational research.
Citation Format: Besmira Alija, Stephanie Braunstein, Jane Xu, Tom O'Hare, Angela Garding, Aaron Viny. Epigenomics at the speed of sound: High throughput epigenomic mapping of low cell input samples using acoustic enhanced immunoprecipitation [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 3765.
Abstract 3767: Identification of regulators of epigenetic silencing of the p16INK4a gene
Authors: Fujita Toshitsugu , Fujii Hodaka 
Published: June 2022

Abstract
Epigenetic silencing of tumor suppressor genes plays critical roles in oncogenesis. The CDKN2A/p16INK4a gene is one of the most frequently silenced tumor suppressor genes. To elucidate molecular mechanisms of maintenance of the silenced status of the p16INK4a gene, we applied the insertional chromatin immunoprecipitation (iChIP) method. The promoter region of the transcriptionally active and silenced alleles of the p16INK4a gene were tagged with a bacterial DNA-binding protein LexA and its binding elements. Subsequently, the promoter regions were affinity purified with antibody against a tag fused with LexA. Proteins more or less abundantly associated with the silenced promoter were identified by stable isotope labeling using amino acids in cell culture (SILAC), one of quantitative mass spectrometry methods. Knock-down experiments of the identified enzymes revealed essential components of maintenance of epigenetic silencing. The essential enzymes can be good targets of development of drugs reactivating expression of the silenced tumor suppressor genes in cancers. We are currently performing drug discovery research to identify small molecules that bind and inhibit those candidate drug target enzymes. Our locus-specific ChIP technology consisting of iChIP and the engineered DNA-binding molecule-mediated ChIP (enChIP) method is a powerful tool to identify novel drug targets for development of drugs regulating gene expression in intractable diseases.
Citation Format: Toshitsugu Fujita, Hodaka Fujii. Identification of regulators of epigenetic silencing of the p16INK4a gene [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 3767.
Abstract 377: Evaluating delivery of TFR1-targeting therapies to the CNS in a novel humanized TFR1 mouse model
Authors: Liu Chang , Shang Chengzhang , Ma Limei , Zhou Xiaofei , Guo Chaoshe 
Published: June 2022

Abstract
Transferrin receptor protein 1 (TFR1), also known as CD71, is a cell surface receptor that plays a major role in cellular iron uptake via receptor-mediated endocytosis. In mammalian species, this receptor is required for erythropoiesis and neural development. The efficient cellular uptake properties of TFR1 have been exploited for site-specific delivery of anti-cancer drugs and biologics, as well as targeted delivery of therapeutic genes to malignant cells overexpressing TFR1. Such delivery is achieved by several methods, including chemical conjugation of transferrin with therapeutic agents, genetic fusion of transferrin with peptides, and anti-TFR1 antibodies. Delivery of therapeutics cross the blood-brain barrier to the central nervous system (CNS) is also greatly facilitated by targeting the TFR1. At Biocytogen, we have successfully generated a humanized B-hTFR1 mouse model for evaluating TFR1-targeted therapeutics in preclinical studies. In this model, the exons 4-19 of mouse Tfr1 gene that encode the extracellular domain of the receptor were replaced by its human counterparts. As the result of this engineering, the ectodomain of the TFR1 is humanized while intracellular domain remains unchanged, maintaining the receptor’s signaling function in mice. Surface staining of erythroid cells from B-hTFR1 mice showed high levels of expression of humanized TFR1. In addition, we were able to detect anti-human TFR1 bispecific antibodies in brain tissues of the B-hTFR1 mouse after intravenous administration of the antibodies, suggesting a robust uptake of the antibodies via the hTFR1 receptor. These results indicate that the B-hTFR1 mouse model is a powerful preclinical tool to evaluate the delivery of therapeutics to the CNS in vivo.
Citation Format: Chang Liu, Chengzhang Shang, Limei Ma, Xiaofei Zhou, Chaoshe Guo. Evaluating delivery of TFR1-targeting therapies to the CNS in a novel humanized TFR1 mouse model [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 377.
Abstract 3774: The effect of CDK5 on mitochondrial function in Glioblastoma Multiforme (GBM)
Authors: Brett-Morris Adina , Mears Jason , Rochon Kristy 
Published: June 2022

Abstract
Glioblastoma Multiforme (GBM) is the most common and aggressive form of brain tumors.Despite conventional therapies, average patient survival is less than two years. GBMrecurrence is in part due to self-renewing tumor initiating cells, often called glioma stemcells (GSCs), which are characterized by their proliferation and tumor formation capacity,as well as their metabolic features. Thus, mitochondrial function represents a potentiallink between cellular metabolism and differentiation state. Mitochondrial fission isindispensable for growing and dividing cells. Dynamin-related protein 1 (Drp1) is essentialfor this process. In particular, there is cumulative evidence that links Drp1 with severaltypes of cancers, including GBM. Regulation of Drp1 is mainly controlled by post-translational modifications (PTMs). Importantly, recent studies have shown CDK5-dependent phosphorylation at the activating site serine 616 of Drp1 enhancesmitochondrial fission. CDK5 plays a crucial role in the central nervous system as well as,in tumor progression. Therefore, our lab hypothesized that inhibition of CDK5 would targetmitochondrial function in order to decrease tumor growth and sensitize brain tumors toconventional therapies. Western blot analysis has shown that CDK5-dependentphosphorylation of Drp1 is present in GSCs and GBM cell lines but absent in non-stemtumor cells (NSTC) and astrocytes. Small-molecule CDK5 inhibitors or CDK5 knockdownthrough siRNA in GSCs and GBM cell lines decreased activating phosphorylation of Drp1,leading to elongation of mitochondria morphology and corresponding reductions in cellviability and cell proliferation. Additionally, CDK5 inhibition showed sensitization toionizing radiation (IR) in GSCs and GBM cells. Importantly, CDK5 inhibition or knockdowncombined with IR, showed increased levels of cleaved caspase-7 and cleaved PARPsuggesting a role for apoptosis in decreased cell proliferation and sensitization to IR.Interestingly, CDK5 inhibition also diminished mitochondrial oxidative phosphorylation.Overall, these findings highlight an important role for CDK5 in regulating mitochondrialfunction in GBM, and targeting CDK5 offers a novel strategy to sensitize tumors to chemo-and radiotherapies.
Citation Format: Adina Brett-Morris, Jason Mears, Kristy Rochon. The effect of CDK5 on mitochondrial function in Glioblastoma Multiforme (GBM) [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 3774.
Abstract 3776: Frequent alteration of mitochondria functional pathways in lung cancer
Authors: Hertweck Kate L. , Persing Brian , Prodduturvar Pranitha , Singh Seema , Singh Ajay , Dasgupta Santanu 
Published: June 2022

Abstract
Lung cancer killed 1.8 million people globally in 2020. In the United States, there will be over 235,760 cases of lung cancer with an estimated death of 131,880 individuals in 2021. Despite significant improvement in multimodal therapeutic approaches, overall 5-year survival rate is only 21.7%. Lung cancer appears to be mitochondria enriched cancer, however, alteration pattern of hallmark molecular pathways associated with diverse mitochondrial functions remain largely unknown in lung tumorigenesis. Utilizing high-throughput genomic profiling, we identified altered copy number of a panel of 40 mitochondria targeted molecules in primary lung cancer patients and their histologically normal appearing follow up mucosal biopsies. These altered molecules are critical components of the mitochondrial protein carrier, OXPHOS, stress response, amino acid metabolism and apoptosis pathways. This finding suggests for clonal genetic relationship between multifocal lesions and potential role of these alterations in the evolution of second primary tumors, which is an existing problem among lung cancer patients. The Human Cancer Genome Atlas database was utilized to further assess the mRNA expression pattern of these 40 molecules and determine their clinico-pathological correlations. Immunohistochemistry and digital pathology system based evaluations were performed to validate expression pattern of key altered molecules. We identified overexpression of various critical mitochondria pathway molecules including AARS2, AGMAT, SDHA, NDUFB7, LONP1, DGUOK, MRM1 and GCAT in both lung adenocarcinomas (LUAD) and squamous cell carcinomas (LUSC), predicting poor prognosis. Significant loss of expression of ACSF2, ACSS1, MTCH1, SLC25A4, ACAD8 and NAGS was evident in both adenocarcinoma and squamous cell carcinoma subtypes. Molecular characterization of both LUAD and LUSC tumor subtypes identified significant association of various key molecules including ACAD8, AGMAT, DGUOK and NDUFAB1 with proximal-inflammatory, proximal-proliferative and terminal respiratory unit pathways in LUAD; and an association of DGUOK, MDH2, MRM1 and MRPL10 with basal, classical, primitive and secretory pathways in LUSC. Significant loss of SLC25A4 protein expression was evident in lung cancer tissues, which predicted poor survival of the lung cancer patients. Capturing the compendium of hallmark mitochondrial alterations in lung tumorigenesis may open up novel avenues for therapeutic and biomarker interventions to improve the overall survival of the lung cancer patients.
Citation Format: Kate L. Hertweck, Brian Persing, Pranitha Prodduturvar, Seema Singh, Ajay Singh, Santanu Dasgupta. Frequent alteration of mitochondria functional pathways in lung 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 3776.
Abstract 3868: High throughput tissue phenotyping and imaging of the tumor immune microenvironment using novel FlexVUE™ multiplexed immunofluorescence assays
Authors: Cajigas Yvette , Hamer Peter , Ainbinder Alina , Vasaturo Angela , Wood Douglas , Maclean Kirsteen H. , Chatterjee Gourab 
Published: June 2022

Abstract
Immunotherapy has transformed the treatment of metastatic and recurrent solid tumors. Advances in technology in the past few years have created unprecedented opportunities to identify biomarkers of disease processes, especially by using multi-omics technologies and datasets to derive valid and useful signatures of disease. Advances in multiplexed immunofluorescence (mIF) staining and imaging technologies that allow for accurate phenotyping of individual cells while also preserving architectural features of the sample are critical to our understanding of the interplay of tumor and immune cells in the tumor immune microenvironment (TiME).Herein we describe the utility of our new flexible mIF assays (FlexVUE™ panels) to provide the necessary relevant distribution of infiltrating immune cells in tumors coupled to the use of our new UltiStacker™ software. This hardware and software package allows a detailed spatial characterization of specific cell phenotypes defined by co- or lack of expression that may help in predicting clinical responses and mechanisms of resistance to therapy. The FlexVUE™ mIF assay was used to conduct 8-plex immunophenotyping of FFPE tumor sections. Slides were stained with a cocktail of eight primary antibodies using Leica Biosystems BOND RX autostainer and imaged on the Zeiss Axio Scan.Z1. Following the first round of imaging, the slides were decoverslipped and the signal was removed. New targets were probed on the same tissue section in a reagent incubation step on the BOND RX, termed ‘exchange.’ Slides were re-imaged on the Zeiss Axio Scan Z1 using the same dye channels. The image pairs were automatically aligned using UltiStacker™ software and exported for downstream analysis with Indica Labs HALOv3.1 software. Our approach demonstrates a streamlined off the shelf workflow requiring no assay development time that supports whole slide imaging of a flexible high plex panel. In addition, our assays enable traditional same slide H&E fusion on a single tissue slide for comprehensive tissue immunophenotyping analysis of proliferating cells, tumor cells, tumor-infiltrating lymphocytes, macrophages or tertiary lymphoid structures.
Citation Format: Yvette Cajigas, Peter Hamer, Alina Ainbinder, Angela Vasaturo, Douglas Wood, Kirsteen H. Maclean, Gourab Chatterjee. High throughput tissue phenotyping and imaging of the tumor immune microenvironment using novel FlexVUE™ multiplexed immunofluorescence assays [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 3868.
Abstract 3907: A pheno-multiomic integration analysis of primary samples of acute myeloid leukemia reveals biomarkers of cytarabine resistance
Authors: Silberberg Gilad , Vishwakarama Bandana , Walling Brandon , Riveley Chelsea , Audia Alessandra , Zipeto Marianna , Sloma Ido , Wesa Amy , Ritchie Michael 
Published: June 2022

Abstract
The overall survival of patients diagnosed with Acute Myeloid Leukemia (AML) remains low. While initial responses to therapy are favorable, the duration of response is short and overcoming therapeutic resistance has proven difficult. A better understanding of the tumor cell biology and resistance mechanisms may shed light onto novel therapeutic targets that improve long-term outcome. In this study, we performed an exhaustive analysis to include deep tumor phenotyping, drug sensitivity profiling and comprehensive omic characterization. These datasets were included in integrative pharmaco-phenotypic-multiomic analyses to identify targets and biomarkers associated with cellular phenotype and drug response. Our results reveal that the major cellular discriminant within the cellular phenotype is CD34 expression, which associates with a high PDK-mediated metabolic profile and cytarabine sensitivity. Tumors exhibiting cytarabine resistance associate with a CD34-negative cellular phenotype and molecular characteristics such as MYC copy number gain, and increased expression of SAMDH1, FBP1 and TYMP proteins.
Citation Format: Gilad Silberberg, Bandana Vishwakarama, Brandon Walling, Chelsea Riveley, Alessandra Audia, Marianna Zipeto, Ido Sloma, Amy Wesa, Michael Ritchie. A pheno-multiomic integration analysis of primary samples of acute myeloid leukemia reveals biomarkers of cytarabine 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 3907.
Abstract 393: Targeting MCT inhibition to overcome enzalutamide resistance in prostate cancer
Authors: Bhattacharjee Sayani , Doan Jonathan P. , Wynn Rebecca , Nadiminty Nagalakshmi 
Published: June 2022

Abstract
Prostate cancers (PCa) exhibit a unique metabolic profile with reliance on different forms of glucose metabolism at different stages of disease progression. Early stage PCa cells use the more efficient TCA cycle, while metastatic PCa cells switch to glycolysis (Warburg effect), leading to the accumulation of lactate. Lactate is exported out of the cells by monocarboxylate transporters (MCTs) to maintain redox balance. Such metabolic reprogramming can lead to gain-of-function mutations and affect drug sensitivity. Enzalutamide is a second-generation antiandrogen used for the treatment of metastatic castration-resistant PCa. However, resistance to Enzalutamide develops in most patients within 9-15 months. Since Warburg effect is a hallmark of metastatic PCa, we sought to understand whether lactate transport has any effect on Enzalutamide-resistance in PCa. We measured the expression of MCTs in parental and Enzalutamide-resistant C4-2B and 22RV1 cells and found that they are overexpressed in the Enzalutamide resistant subtypes. Based on this observation, we hypothesized that targeting lactate transport might be a potential strategy to overcome Enzalutamide-resistance in PCa cells. Using cell survival and cell proliferation assays, we found that MCT antagonists resensitized Enzalutamide resistant C4-2B and 22Rv1 cells to treatment with Enzalutamide. Using the Seahorse based glycolytic rate assay we found that these combination treatments significantly reduced the extracellular acidification rate by reducing the level of glycolysis in the Enzalutamide-resistant PCa cells. We also found that treatment with these antagonists either singly, or in combination with Enzalutamide suppressed xenograft growth in SCID mice bearing subcutaneously injected parental or Enzalutamide-resistant C4-2B cells. These findings led to the conclusion that targeting MCTs could be an attractive strategy to overcome Enzalutamide resistance in prostate cancer.
Citation Format: Sayani Bhattacharjee, Jonathan P. Doan, Rebecca Wynn, Nagalakshmi Nadiminty. Targeting MCT inhibition to overcome enzalutamide resistance in 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 393.