bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 6018: Sequencing of 5-hydroxymethylcytosine to single base resolution
Authors: Evanich Daniel J. , Ponnaluri V. K. Chaithanya , Panchapakesa Vaishnavi , Erijman Ariel , Campbell Matthew A. , Dai Nan , Langhorst Bradley W. , Vaisvila Romualdas , Williams Louise 
Published: April 2023

Abstract
DNA methylation is an epigenetic regulator of gene expression with important functions in development and diseases such as cancer. The modified cytosines, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are identified using Illumina sequencing. Libraries are generated using either NEBNext® EM-seq™, an enzyme-based workflow, or by using sodium bisulfite conversion. These methods, however, cannot differentiate between 5mC and 5hmC. Distinguishing these modifications is important as there is increased interest in 5hmCs role in regulating gene expression. Methods currently exist to enable discrimination of 5mC and 5hmC, for example, oxBS-seq and TAB-seq. These methods still rely upon sodium bisulfite conversion and have reduced data quality due to fragmentation and loss of DNA. Here we describe an enzymatic method that enables specific detection of 5hmC. 5hmC is detected using two enzymatic steps. Firstly, 5hmCs are glucosylated, which protects them from subsequent deamination by APOBEC. In contrast, cytosines and 5mCs are deaminated resulting in their conversion to uracil and thymine, respectively. During Illumina sequencing deaminated cytosine and 5mC are read as thymine and the glucosylated 5hmC are read as cytosine. Subtractive analysis of 5hmC data from EM-seq data, which detects both 5mC and 5hmC, permits the identification of individual 5mC and 5hmC sites. 5hmC data were generated for inputs of 0.1 ng to 200 ng DNA isolated from E14 mouse embryonic stem cells and human brain. The 5hmC libraries had similar characteristics to EM-seq libraries, including expected insert sizes due to intact DNA molecules, low duplication rates and minimal GC bias. T4147 phage DNA was used as an internal control, as all cytosines are 5-hydroxymethylated, with 98-99% of cytosines identified as 5hmC. 5mC and 5hmC levels were also profiled during E14 cell differentiation for 10 days. Interestingly, 5hmC levels decreased whereas 5mC levels increased particularly during the first five days of differentiation. LC-MS/MS quantification of this same DNA mirrored the changes observed by sequencing. The ability to discriminate between 5mC and 5hmC will provide key insights into the role of these cytosine modifications in development and disease.
Citation Format: Daniel J. Evanich, V. K. Chaithanya Ponnaluri, Vaishnavi Panchapakesa, Ariel Erijman, Matthew A. Campbell, Nan Dai, Bradley W. Langhorst, Romualdas Vaisvila, Louise Williams. Sequencing of 5-hydroxymethylcytosine to single base resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6018.
Abstract 6024: Precancerous nature of intestinal metaplasia with accelerated DNA methylation along with altered epigenomic landscape
Authors: Takeuchi Chihiro , Yamashita Satoshi , Liu Yu-Yu , Takeshima Hideyuki , Ushijima Toshikazu 
Published: April 2023

Abstract
Objective. The presence of intestinal metaplasia (IM) is a risk factor for gastric cancer. However, it is still controversial whether IM itself is a precancerous or paracancerous lesion. Here, we aimed to explore the precancerous nature of IM in terms of epigenetic alterations.
Design. Genome-wide DNA methylation analysis was conducted by EPIC BeadArray using IM and non-IM crypts isolated by Alcian blue staining. Organoid establishment, ChIP-seq for H3K27ac, and single-cell ATAC-seq were conducted using IM mucosa. NOS2 was induced using a Tet-on gene expression system in normal cells.
Results. IM crypts had a methylation profile unique from that of non-IM crypts, showing extensive DNA hypermethylation in promoter regions with CpG islands, including those of tumor suppressor genes. Also, the IM-specific methylation profile was carried into a fraction of gastric cancers (GC), and the frequency of the IM-type GCs was higher than the fraction of IM cells in the adjacent mucosa, indicating that IM cells have a higher chance of developing into cancer cells than non-IM cells. Moreover, the patients with IM-type GCs showed better overall survival than those without, and IM-type GCs showed good response to chemotherapy. As for the mechanism of accelerated methylation induction, IM organoids had remarkably high endogenous NOS2 expression, and NOS2 induction in normal cells was shown to accelerate aberrant DNA methylation by increasing DNMT activity. IM mucosa underwent dynamic enhancer reprogramming, including the regions involved in abnormal NOS2 expression. Single-cell ATAC-seq also showed NOS2 open chromatin in IM cells but not in gastric cells, and frequent closed chromatin of tumor-suppressor genes in IM cells. NOS2 expression in IM-derived organoids was up-regulated by IL-17A, a cytokine secreted by extracellular bacterial infection.
Conclusions. IM cells were considered to have a strong precancerous nature with an increased chance of converting into cancer cells in terms of the specific methylation profile and the accelerated DNA methylation induction due to abnormal NOS2 expression.
Citation Format: Chihiro Takeuchi, Satoshi Yamashita, Yu-Yu Liu, Hideyuki Takeshima, Toshikazu Ushijima. Precancerous nature of intestinal metaplasia with accelerated DNA methylation along with altered epigenomic landscape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6024.
Abstract 6048: OGT/CDK5/ACSS2 axis regulates breast cancer brain metastatic growth
Authors: Esquea Emily , Ciraku Lorela , Ming Giang Le , Reginato Mauricio 
Published: April 2023

Abstract
Brain metastases (BMs) in breast cancer patients is considered an end-stage event, with no effective drug treatment and a median survival after diagnosis measured in months. Currently, there are no effective drug treatment for BM patients, thus there is an urgent need to develop novel treatment strategies. Breast cancers that metastasize to the brain must adapt to lack of lipid availability in the brain environment and are highly dependent on fatty acid synthesis for growth and survival. However, the signaling pathways that regulate precursors of fatty acid synthesis and lipid metabolism in breast cancer brain metastatic (BCBM) tumors are not known. Here, we show that BCBM cells can generate acetyl-CoA, a major metabolite feeding lipid biosynthesis, via phosphorylation of acetyl-CoA synthetase 2 (ACSS2) by the cyclin-dependent kinse-5 (CDK5) regulated by the nutrient sensor O-GlcNAc transferase (OGT). Breast cancer cells selected to metastasize to the brain contain higher levels of O-GlcNAcylation, OGT. and ACSS2-Ser267 phosphorylation compared to parental cells, and we show that human breast cancer brain metastatic patient samples contain elevated ACSS2-Ser-267 levels. Additionally, overexpression of the OGT, CDK5, or ACSS2-S267D phospho-mimetic mutant confers a growth advantage of breast cancer cells in the brain in vivo. Importantly, we show that ACSS2 is required for breast cancer growth in the brain but not in the mammary fat pad. Pharmacologically targeting the CDK5 or ACSS2 with respective small molecule inhibitors reduces tumor growth in orthotopic ex vivo brain slice model. These results suggest a crucial role for OGT/CDK5/ACSS2 signaling axis to regulate lipid metabolism in BCBM cells and identify CDK5 and ACSS2 as novel therapeutic targets for treatment of breast cancer brain metastatic growth.
Citation Format: Emily Esquea, Lorela Ciraku, Giang Le Ming, Mauricio Reginato. OGT/CDK5/ACSS2 axis regulates breast cancer brain metastatic growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6048.
Abstract 6069: Genomic and transcriptomic characterization of medullary thyroid cancer
Authors: Lee Seong Eun , Park Seongyeol , Yi Shinae , Lim Joonoh , Choi Na Rae , Kwak JungHak , Koh June-Young , Yi Boram , Koo Jaemo , Chang Jae Won , Ju Young Seok , Koo Bon Seok , Kim Jin Man , Park Young Joo , Shong Minho , Lee Eun Kyung , Won Jae Kyung , Kang Yea Eun , Lee Kyu Eun 
Published: April 2023

Abstract
Medullary thyroid carcinoma (MTC) is a rare thyroid malignancy derived from the parafollicular C cells of thyroid gland. It features relatively aggressive biologic behavior among thyroid cancers, but its genomic landscape has not yet been fully explored. Here, we conducted multi-omics data (whole genome sequencing (WGS), and bulk (bulk RNA-seq) or single nucleus RNA sequencing (snRNA-seq)) on ​​MTC, and found genetic characteristics of MTC and a new gene signature predicting aggressiveness of tumors. WGS was performed for 35 pairs of tumor specimens and normal thyroid tissues from 30 patients. And bulk RNA-seq for 59 samples (20 normal thyroid tissues, 26 primary MTCs, 9 recurred lymph nodes, and 4 metastatic lymph nodes) and snRNA-seq for 3 primary MTCs, and 2 recurred/metastatic lymph nodes were also performed. In WGS results, we verified driver mutations, including RET, RAS, and BRAF. Four patients were classified as germline RET mutations based on familial history. Somatic RET M918T mutations were occurred in 8 patients, 9 patients were observed other RET mutations. Also, we observed other somatic mutations such as HRAS and BRAF. Seven patients occurred somatic HRAS mutations (Q61L, Q61R, G13R, A11 and G15), and somatic BRAF mutations (G469A, K601E) were observed in two patients. We found broad-level copy-number alterations in ~50% of patients, and we also found a patient who shows whole-genome duplication. The copy-number alteration was associated with aggressive phenotype in MTC. In addition, we found complex rearrangements such as microhomology-mediated break-induced replication in five patients. Interestingly, we found intra-tumoral heterogeneity of several genes related to C cell differentiation through snRNA-seq analysis by comparing clusters of tumor cells. Using the genes, we calculated the C cell differentiation score of the samples with bulk RNA-seq by GSVA algorithm. This score was related to poor clinical characteristics such as recurrence. In conclusion, multi-omics profiling of MTC reveals that copy number alterations and C cell differentiation status are the important biomarkers for cancer aggressiveness.
Citation Format: Seong Eun Lee, Seongyeol Park, Shinae Yi, Joonoh Lim, Na Rae Choi, JungHak Kwak, June-Young Koh, Boram Yi, Jaemo Koo, Jae Won Chang, Young Seok Ju, Bon Seok Koo, Jin Man Kim, Young Joo Park, Minho Shong, Eun Kyung Lee, Jae Kyung Won, Yea Eun Kang, Kyu Eun Lee. Genomic and transcriptomic characterization of medullary thyroid cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6069.
Abstract 6083: Extensive epigenetic and transcriptomic donor-specific differences observed in iPSC derived allogenic NK (iNK) cells
Authors: Srivas Rohith , Nariya Snehal , Rojsza Melissa , Morse Barry A. , Manor Ohad , Amin Rupesh , Borges Luis 
Published: April 2023

Abstract
Several autologous CAR-T cell therapies have been approved for the treatment of B cell lymphomas and leukemias and a number of trials are in progress to investigate the use of allogeneic, donor derived CAR-T and CAR-NK cell therapies for multiple tumor indications. However, sourcing cell donors, editing cells and generating large batches of clinical grade allogeneic CAR-T and CAR-NK can be challenging. To overcome these difficulties, we are developing NK and T cell-based therapies derived from induced pluripotent stem cells (iPSCs). iPSCs have the capacity to be genetically engineered, enabling precise edits that can enhance the function of cell therapies, and indefinitely expanded in culture, allowing for the production of highly uniform master cell banks. Yet, an open question of this system is the extent to which the donors’ genetic background shapes the phenotype of the iPSC-derived NK and T cells. To answer this question, we measured the genome-wide landscape of chromatin accessibility and gene expression in iNK cells generated from multiple donors. Donor background was linked to significant changes in chromatin accessibility (~30% of regions tested) and expression (~15% genes). We found accessibility changes at binding sites for transcription factors regulating various aspects of NK cell function, including differentiation (GATA2, EOMES, and ETS1), activation (AP-1, T-bet), and cytokine production (STAT3, STAT5), indicating that the donor background could impact the functionality of iPSC-derived NK cells. In particular, we noted that iNK cells derived from one donor, which had decreased effector functions compared to other cell lines, showed increased accessibility at BACH2 binding sites, a key negative regulator of NK cell function. iNK cells derived from this donor also showed an increase in expression of genes linked to NK cell differentiation and cell development, and a decrease in genes involved in cell cycle, cell proliferation, and inflammatory response pathways. Taken together, these data indicate the importance of assessing the impact of donor background on iPSC derived cell therapies through a variety of functional and genomic assays.
Citation Format: Rohith Srivas, Snehal Nariya, Melissa Rojsza, Barry A. Morse, Ohad Manor, Rupesh Amin, Luis Borges. Extensive epigenetic and transcriptomic donor-specific differences observed in iPSC derived allogenic NK (iNK) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6083.
Abstract 6104: Prognostic and therapeutic impact of a homologous recombination repair protein TONSL in cancer stem cells
Authors: Lee Hani , Choi SeokGyeong , Ha Sojung , Kim Woo-Young 
Published: April 2023

Abstract
Previously, we found that an important homologous recombination repair protein ‘Tonsoku Like, DNA Repair Protein’ (TONSL) might be required for glioblastoma cancer stem cell (CSC). In this study, we further investigated the role of TONSL in CSC from several cancer cell lines from different tissues. We also analyzed the clinical data available from the public databases to find the clinical impact of TONSL. The higher expression of TONSL is associated with the worse prognosis of lung and gastric cancer. Interestingly, in breast cancer, while its higher expression is also the negative prognostic marker of the luminal A and HER2 positive cancers, that is rather the positive prognostic marker for luminal B and basal cancers. TONSL expression is higher in all 4 subtypes of breast cancer, and ovarian cancer than the corresponding normal tissues. The knockdown of TONSL in an ovarian cancer cell line, OVCAR8, results in the increase of G2/M cells and apoptotic cells in the bulk cultured population while increase also the senescence cells only in the CSC population leading to the severe loss of CSC population. TONSL and the close binding partner in homologous recombination repair (HRR), MMS22L, both were essential for the CSC growth but the bulk cultured cells could survive without those two in a colon cancer cell line, HCT15 suggesting the importance of HRR in colon CSC. Indeed, gemcitabine treatment induced DNA breaks showed a synergistic cytotoxicity with those two HRR factors knockdown in the CSC only. These results suggest that the TONSL and MMS2L mediated HRR is essential for the CSC and while most of bulk cultured cancer cells circumvent the HRR deficit. Collectively, these data showed that the CSCs ultimately depend on the HRR process which requires TONSL and, therefore, CSC population may be more vulnerable to HRR targeting therapeutic approaches.
Citation Format: Hani Lee, SeokGyeong Choi, Sojung Ha, Woo-Young Kim. Prognostic and therapeutic impact of a homologous recombination repair protein TONSL in cancer stem cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6104.
Abstract 6144: Polyploidization enables initial survival of chemotherapy but a progressively increased susceptibility to ferroptosis
Authors: Loftus Luke 
Published: April 2023

Abstract
Once cancer has metastasized it remains incurable owing to evolved resistance to nearly all systemic therapies. Classical views of therapeutic resistance include tumor heterogeneity and genetic instability, but our group and others have identified an unappreciated cancer cell phenotype, the polyaneuploid cancer cell (PACC) state, that provides an alternative model. The PACC state is induced upon exposure to a variety of applied stressors, including multiple different classes of chemotherapy, and is characterized by polyploidization of the aneuploid cancer genome. We have shown that this polyploidization is accomplished through endocycling that leads to an enlarged cell size with increased transcriptomic and protein content, higher oxidative buffer capacity, and increased autophagy. We have demonstrated that PACCs are present in a variety of cell lines, arise in response to multiple stressors in vitro, and are resistant to subsequent treatment with several classes of chemotherapy. Thus, we hypothesize that the PACC state is driving therapeutic resistance to current treatment regimens. Elimination of this overlooked phenotype is critical for sustained anticancer therapy response. Cancer cells, including those in the PACC state, control canonical cell cycle and cell death programs to evade apoptosis from external stressors including chemotherapy, highlighting an opportunity to leverage cell cycle-independent death pathways as an alternative therapeutic strategy1. Ferroptosis is a cell cycle agnostic immunogenic form of cell death that is an ideal pathway for elimination of cells in the PACC state. Characterization of ROS levels, glutathione content, labile iron homeostasis, and lipid content indicate that cells in the PACC state have a mounting susceptibility to ferroptosis. Our data suggests that cells in the PACC state keep ferroptosis in check through elevated levels of reduced glutathione and proficient redox activity, including turn over of GPX4, the sole enzyme counteracting lipid peroxidation. Direct inhibition of GPX4 is highly toxic to cells in the PACC state, particularly at later timepoints post therapy removal when the immediate stress response is largely resolved. These studies will directly lead to development of a therapeutic strategy specifically targeting the PACC state with the intention of eliminating cancer resistance.
1Loftus, L.V.; Amend, S.R.; Pienta, K.J. Interplay between Cell Death and Cell Proliferation Reveals New Strategies for Cancer Therapy. Int. J. Mol. Sci. 2022, 23, 4723. https://doi.org/10.3390/ijms23094723
Citation Format: Luke Loftus. Polyploidization enables initial survival of chemotherapy but a progressively increased susceptibility to ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6144.
Abstract 6203: CTC1-STN1-TEN1 (CST) complex-independent roles for STN1 in DNA double-strand break repair in pancreatic ductal adenocarcinoma
Authors: Shen Changxian , Yang Linlin , Corrales-Guerrero Sergio , Nair Sindhu , Williams Terence 
Published: April 2023

Abstract
KRAS mutation occurs in 90-95% of pancreatic ductal adenocarcinoma (PDAC) and is a driver of PDAC; however, the underlying molecular mechanisms by which KRAS mutation maintains survival and promotes progression of PDAC need further refinement. We herein report that KRAS mutations increase STN1 (also named OBFC1) expression leading to enhanced repair of DNA double strand breaks (DSBs) in PDAC. Analysis of The Cancer Genome Atlas databases reveal that STN1 is significantly upregulated in PDAC tissue and, is associated with KRAS mutation and poor survival. Silencing KRAS or pharmacologic inhibition of KRAS signaling decreased STN1 expression in PDAC cells. Canonically, STN1 forms a CST (CTC1-STN1-TEN1) complex, which is important for telomere duplication and maintenance. Intriguingly, siRNA-mediated depletion of STN1 (but not CTC1 or TEN1) sensitized PDAC cells to ionizing radiation and chemotherapy as assessed by radiation clonogenic assay and alamarBlue assay, respectively. Consistently, immunoblotting and immunofluorescence analyses of γH2AX (marker of DNA damage) demonstrated that depletion of STN1 (but not CTC1 or TEN1) resulted in DNA damage. To explore the underlying molecular mechanisms, we performed proteomic analysis and found that STN1 physically interacts with many proteins important for DNA repair, replication and cell cycle progression. Of note, we revealed unreported interactions of STN1 with ATM, ATR and DNA-PKcs, key components of DNA damage response signaling. Furthermore, homologous recombination and non-homologous end joining repair reporter assays demonstrated that STN1 silencing reduced both homologous recombination and non-homologous end joining repair of DSBs in U2OS and PDAC cells. Moreover, flow cytometry and immunofluorescence analyses showed that knockdown of STN1 by siRNA impaired cell cycle arrest at S and G2/M phases which was accompanied with mitotic catastrophe in response to ionizing radiation. Our findings have revealed novel CST complex-independent roles for STN1 in DSB repair and suggest STN1 is a novel biomarker for PDAC as well as potentially other cancers.
Citation Format: Changxian Shen, Linlin Yang, Sergio Corrales-Guerrero, Sindhu Nair, Terence Williams. CTC1-STN1-TEN1 (CST) complex-independent roles for STN1 in DNA double-strand break repair in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6203.