bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2046: Binding of FANCD2 to SRSF1 splicing factor prevents genomic instability through R-loop regulation
Authors: Olazabal-Herrero Anne , Green Allison M. , Chen Xiaoyong , Sung Patrick , Manoj Pillai M. , Kupfer Gary M.
Published: July 2021

BACKGROUND. Fanconi Anemia (FA) is a rare disease characterized by congenital abnormalities, bone marrow failure and cancer susceptibility. The pivotal role of the FA pathway is DNA interstrand crosslink (ICL) repair, where FANCD2-FANCI complex monoubiquitination is the key regulatory step. Recent studies have linked defects in the FA pathway to R-loop metabolism and implicated R-loops as an endogenous source of genomic instability that contributes to the FA phenotype, although the mechanism remains elusive. Splicing factors, such as SRSF1 (ASF/SF2), have also been linked to R-loops, cancer, and myelodysplastic syndrome (MDS), so we hypothesized a link between SRSF1 and FA.

METHODS. Cells: FA-D2 mutant (PD20) and corrected (D2), HeLa. siRNA transfections were used to reduce SRSF1 and FANCD2 protein levels. Survival assays were performed to determine cell sensitivity to DNA damaging agents. In vitro immunoprecipitation was performed using SRSF1 purified from bacteria and FANCD2-FANCI purified from insect cells. R-loops were studied using the Damage At RNA Transcription (DART) assay and S9.6 immunofluorescence. RNA FISH was used to measure mRNA export.

RESULTS. In order to study the involvement of splicing factors in the FA pathway, we knocked down SRSF1 by using siRNA. SRSF1 knockdown leads to impaired FANCD2 monoubiquitination after DNA damage as well as diminished cell survival, thus creating a FA-like phenotype. In addition, immunoprecipitation in cells and in vitro showed direct interaction between FANCD2 and SRSF1. We found that FANCD2 and SRSF1 knockdown leads to increased R-loops visualized by S9.6 immunofluorescence. Besides, DART assay revealed colocalization of SRSF1 and FANCD2 with R-loops in highly transcribed genomic sites, known to be R-loop hotspots. Additionally, we found that SRSF1 interacts with the mRNA export factor NXF1 in a FANCD2-dependent fashion and through RNA. Interestingly, SRSF1-NXF1 interaction is enhanced upon DNA damage. Finally, we found that FANCD2-deficient cells present defects in mRNA export, suggesting that SRSF1 and FANCD2 could cooperate in the prevention of R-loop formation by exporting accumulated RNA in the nucleus.

CONCLUSION. Our findings suggest a novel non-canonical mechanism of FANCD2 via regulation of SRSF1 in R-loop prevention via facilitation of mRNA export, thus preventing genomic instability.

Citation Format: Anne Olazabal-Herrero, Allison M. Green, Xiaoyong Chen, Patrick Sung, Pillai M. Manoj, Gary M. Kupfer. Binding of FANCD2 to SRSF1 splicing factor prevents genomic instability through R-loop regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2046.
Abstract 2048: Property analysis of chromosomal instability-adapted cells using multi-omics approaches
Authors: Morita Tomoko Yamamori , Haeno Hiroshi , Makinoshima Hideki , Suzuki Ayako , Kobayashi Susumu S. , Ohashi Akihiro
Published: July 2021

Background: Chromosomal instability (CIN), which includes polyploidy and aneuploidy, is characterized as the pillar of cancer hallmarks. CIN is observed in >90% solid tumors, being correlated with malignancy grades, poor prognosis, and drug resistance. However, it is still unclear yet how aneuploidy triggers cancer initiation/progression, or how aneuploidy gives negative impacts on cancer therapeutics. In this study, we established our original CIN cell line model, aiming to understand biological significance of cancer aneuploidy on the CIN-mediated stress adaptation.Method: A colorectal cancer cell, HCT116, was treated with an Aurora-B kinase inhibitor for 24 hours to transiently induce CIN in these cells, and then cultured in a drug-free medium for >10 days to establish “CIN-adapted cells”. Cell growth was evaluated by intracellular ATP concentrations. RNAs and DNAs extracted from the cells were subjected to RNA-seq, Exome-seq, and ATAC-seq by the NovaSeq sequencing systems. The cell lysates were subjected to metabolomics analyses by GC-MS system. Bioinformatics analyses were performed using these multiple-omics data.Results: In 10 days after aneuploid formation, 90% aneuploid cells were killed, while 10% aneuploid cells were proliferative with a high growth rate equivalent to that of the parental euploid cells, which appeared to overcome the CIN-mediated antiproliferative pressure. The CIN-adapted cells revealed to be highly sensitive to the low-glucose and hypoxic condition, suggesting that the metabolic pathways would be modulated to adjust the cells to the aneuploid-mediated stress. Transcriptome and metabolome analyses also revealed the gene expressions and the metabolites for the lipid or amino acids metabolisms were significantly changed in the CIN-adapted cells. These findings suggest that the CIN-adapted cells appear to restructure their signaling networks of the lipid and amino acid metabolic pathways, which is expected to play important roles for the aneuploid cells to survive under the CIN-mediated stress. We perform drug screening to identify essential factors in the process leading to CIN-adapted, aiming for a comprehensive understanding of the molecular mechanism and signal networks.Conclusions: We comprehensively characterized the CIN-adapted cancer cell model by the multi-omics approaches. These findings will help us to deeply understand the biological significance of CIN in cancer cells, providing a new insight to novel cancer therapeutics targeting CIN.

Citation Format: Tomoko Yamamori Morita, Hiroshi Haeno, Hideki Makinoshima, Ayako Suzuki, Susumu S. Kobayashi, Akihiro Ohashi. Property analysis of chromosomal instability-adapted cells using multi-omics approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2048.
Abstract 2050: Comprehensive genomic instability characteristics analysis in Chinese patients with osteosarcoma
Authors: Wang Yonggang , Li Hongtao , Xia Jing , Chen Shiqing , Bai Yuezong
Published: July 2021

Background: Previous study indicated that >80% of osteosarcomas (OSs) have genomic instability characteristics similar to BRCA1/2-deficient cancers. Such BRCA-like traits could manifest a deficiency in the DNA damage repair (DDR) pathway. Patients with alterations in DDR pathway was reported sensitive to platinum-based chemotherapy and PARP inhibitors. In order to seek more precision therapeutic opportunities, we explored the molecular profile of Chinese OS patients to identify whether the analogous phenomena exists as reported.

Material and Methods: 44 archival Chinese OS patients from 3D Medicines and a public dataset including 35 OS patients (download from cBioPortal), were collected and analyzed next-generation sequencing (NGS) results on tumor tissues for gene mutations, copy number alterations (CNAs) and fusions in 33 cancer-associated DDR genes. Simultaneously, 44 Chinese OS patients' tumor analysis generated homologous recombination deficiency (HRD) scores (defined as the unweighted sum of LOH, TAI and LST scores) through the 733 gene panel sequencing.

Results: Among 44 Chinese OS patients, 18 (40.9%) of OS had DDR pathway gene somatic alterations, and TP53 (31.8%) were the most frequently mapped gene. Other DDR somatic alterations were identified in BRCA2 (6.8%), BARD1 (4.5%), BRCA1 (2.3%), PMS2 (2.3%), FANCA (2.3%) and CHEK1 (2.3%). It was worth mention that all gene alterations were copy number reduction except TP53. Collectively, the frequencies of HRR and mismatch repair (MMR) alterations were 15.9% and 2.3%, respectively. HRD score were available in 18 patients, the median was 40.5 (range, 0-81). As to public dataset, 6 (17.1%) patients harbored somatic alterations in DDR genes. The variations were BRCA2 (5.7%), ATR (2.9%), BARD1 (2.9%), FANCC (2.9%), MLH1 (2.9%), MSH6 (2.9%) and PMS2 (2.9%). The HRR mutations frequency was 11% and MMR was 8.6%.

Conclusions: This is the first large-scale comprehensive genomic instability characteristics analysis for Chinese patients with OS. Chinese patients and public dataset patients covers many common features. The DDR gene alteration frequency and HRD score are higher in OS patients, the association of variations in DDR genes and HRD score could be considered substitutable in predicting platinum-based therapy and PARP inhibitor response in clinical practice.

Citation Format: Yonggang Wang, Hongtao Li, Jing Xia, Shiqing Chen, Yuezong Bai. Comprehensive genomic instability characteristics analysis in Chinese patients with osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2050.
Abstract 2060: Therapeutic approaches for CCNE1-amplified HR-proficient ovarian cancer
Authors: Bai Shoumei , Buckanovich Ronald J.
Published: July 2021

Poly (ADP-ribose) polymerase (PARP) inhibitors have shown great success in ovarian cancer patients that are homologous recombination deficient (HRD). However, approximately 50% of high grade serous ovarian cancer (HGSOC) are HR-proficient. HR-proficient Cancer cells evade lethal DNA damage by triggering HR pathways at the cost of genome stability and evolving of more aggressive cancer cells. Cyclin E1 (CCNE1) amplification is a major subset of known HR-proficient HGSOC. This subset of cancer responds poorly to essentially all available ovarian cancer therapies including PARP inhibitors. Our study showed that the mTOR, HR, and checkpoint pathways were enriched in CCNE1-amplifed ovarian cancer patients. Interactome mapping demonstrated that many of the interactive proteins in the mTOR pathway were also components of HR and DNA checkpoint pathways. mTOR inhibition resulted in downregulation of HR and DNA checkpoint proteins. Downregulation of ATR partly mirrored mTOR inhibition. mTOR inhibitors and ATR inhibitors synergized with PARP inhibitors in vitro and in vivo in CCNE1-amplified HGSOC tumors. This was associated with decrease in cancer stem-like cancer cells and loss of polyploid tumor cells, which are vital for evolving of more aggressive, therapy-resistant cancer cells. These studies will have potential impact on further preclinical validation and clinical applications to cure CCNE1-amplifed HRP ovarian cancer.

Citation Format: Shoumei Bai, Ronald J. Buckanovich. Therapeutic approaches for CCNE1-amplified HR-proficient ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2060.
Abstract 2078: A multiscale map of DNA damage response in human cells
Authors: Kratz Anton , Kim Minkyu , Zheng Fan , Koczor Christopher A. , Fong Samson , Li Jianfeng , Sobol Robert W. , Krogan Nevan , Ideker Trey
Published: July 2021

The DNA damage response (DDR) ensures error-free genome replication and transcription, but is often disrupted in cancer. We developed DDRAM, a multilayered map of DDR derived from our own high-throughput physical interaction screen as well as genome, transcriptome, and proteome datasets from published sources. DDRAM organizes 328 genes into 41 hierarchical pathways. We identify 55 (17%) novel DDR genes with functions distributed across various pathways, and novel and more specific roles for 153 (47%) of established DDR genes. We perform epistasis mapping to support novel pathway assignments of FOXK1 in nucleotide metabolism and XRCC3 in the Fanconi anemia pathway. Using quantitative imaging of a fluorescent probe, in some cases confirmed by epistasis mapping, we experimentally validate members of the Short Patch Base Excision Repair pathway over a time course, revealing a more complete model of poly(ADP-ribose)-dependent recruitment to sites of DNA damage.

Citation Format: Anton Kratz, Minkyu Kim, Fan Zheng, Christopher A. Koczor, Samson Fong, Jianfeng Li, Robert W. Sobol, Nevan Krogan, Trey Ideker. A multiscale map of DNA damage response in human cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2078.
Abstract 2081: Transcriptional silencing of CDK18 in BRG1 mutant lung cancer
Authors: Phillips Loryn Michelle , Johnston Christopher A. , Belinsky Steven A. , Tessema Mathewos
Published: July 2021

In ~10% of lung cancers, the BRG1 gene is mutated often causing a loss of function (LOF). BRG1 is a tumor suppressor that encodes a protein (SMARCA4), which is one of the mutually exclusive ATPase subunits of the Switch/Sucrose Non-fermentable (SWI/SNF) complex. The SWI/SNF complex functions to regulate transcription of many genes including tumor suppressor genes, through mediating chromatin remodeling and nucleosome repositioning by maintaining open chromatin and actively evicting polycomb repressive complexes (PRCs) from target genes and regions. Our previous research showed that BRG1 mutation occurs in approximately 9% of lung adenocarcinomas in smokers and 11% in smokers delineating BRG1 as a clinically relevant mutation. The goal of these studies is to elucidate the downstream effects of BRG1 LOF on gene expression. RNA sequencing of isogenic BRG1 wild type (WT) and knock out (KO) clones from malignant and transformed cell lines identified cell cycle control and chromosomal replication as the most significantly affected pathway. qRT-PCR was used to validate expression changes in 17 genes from this pathway in the WT and KO clones as well as wild type and mutant tumors lines. CDK18 was the most significantly affected target exhibiting a 78% reduction in expression (p<0.001) comparing wild type to mutant cell lines. Ongoing studies are investigating the epigenetic mechanisms leading to transcriptional repression of CDK18 that include cytosine DNA methylation and histone methylation of H3K27. In addition, functional assays (e.g., soft agar, cell migration) are evaluating the role of epigenetically repressed CDK18 in lung carcinogenesis and cell cycle control through siRNA knock down of CDK18 in wild type BRG1 cell lines and carcinogen transformed human bronchial epithelial cells. Together, these studies will provide new insight into the role of LOF of CDK18 in BRG1 mutant cancer. (Supported by P30 CA11800).

Citation Format: Loryn Michelle Phillips, Christopher A. Johnston, Steven A. Belinsky, Mathewos Tessema. Transcriptional silencing of CDK18 in BRG1 mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2081.
Abstract 2084: Single-cell multimodal glioma analyses reveal epigenetic regulators of cellular plasticity and environmental stress response
Authors: Johnson Kevin C. , Anderson Kevin J. , Courtois Elise T. , Gujar Amit D. , Barthel Floris P. , Varn Frederick S. , Luo Diane , Seignon Martine , Yi Eunhee , Kim Hoon , Estecio Marcos R. , Tang Ming , Navin Nicholas E. , Maurya Rahul , Ngan Chew Yee , Verburg Niels , Hamer Philip C. De Witt , Bulsara Ketan , Samuels Michael L. , Das Sunit , Robson Paul , Verhaak Roel G.
Published: July 2021

Glioma intratumoral heterogeneity enables adaptation to challenging microenvironments and contributes to universal therapeutic resistance. Here, we integrated 914 single-cell DNA methylomes, 55,284 single-cell transcriptomes, and bulk multi-omic profiles across 11 adult IDH-mutant or IDH-wild-type gliomas to delineate sources of intratumoral heterogeneity. We found that local DNA methylation instability, or epimutation burden, was elevated in more aggressive tumors, reflected intratumoral variability, linked with transcriptional disruption, and associated with environmental stress response. We show that the activation of cell-state specific transcription factors is impacted by epimutations and that loosened epigenetic control may facilitate cellular plasticity. We validated the impact that the common environmental stressor hypoxia has on the epigenetic stability and shifts in cell states through perturbation-based in vitro experiments coupled with single-cell genomics. Our analyses support that glioma cells under stress hijack epigenetic mechanisms that regulate cell state transitions to overcome challenges posed by nutrient-poor microenvironments and therapeutic insults. We confirmed the link between cellular stress and epigenetic instability by analyzing larger cohorts of bulk longitudinally collected and spatially separated DNA methylation data. Increased DNA methylation instability was associated with accelerated disease progression, and recurrently selected DNA methylation changes were enriched for environmental stress response pathways. Our work provides an integrative framework to better understand glioma evolution and highlights the importance of epigenetic heterogeneity in shaping therapeutic response.

Citation Format: Kevin C. Johnson, Kevin J. Anderson, Elise T. Courtois, Amit D. Gujar, Floris P. Barthel, Frederick S. Varn, Diane Luo, Martine Seignon, Eunhee Yi, Hoon Kim, Marcos R. Estecio, Ming Tang, Nicholas E. Navin, Rahul Maurya, Chew Yee Ngan, Niels Verburg, Philip C. De Witt Hamer, Ketan Bulsara, Michael L. Samuels, Sunit Das, Paul Robson, Roel G. Verhaak. Single-cell multimodal glioma analyses reveal epigenetic regulators of cellular plasticity and environmental stress response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2084.
Abstract 2089: Epigenetic regulation of sprouty2 in colorectal cancer
Authors: Stuckel Alexei , Khare Sharad
Published: July 2021

Background: The specific role of Sprouty-2 in colorectal cancer (CRC) as either a tumor suppressor or an oncogene is unclear. This is further obscured by the fact that no known driver mutations of Sprouty-2 have been reported. Therefore, for the first time, alterations of DNA 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) were investigated to better understand the regulation of Sprouty-2 in CRC.

Methods: Sprouty-2 transcript expression was evaluated in CRC patient samples and Sprouty-2 protein expression was evaluated in CRC cells. The DNA methylation status spanning Sprouty-2 gene was evaluated using combined bisulfite restriction analysis (COBRA). The 5hmC status in the Sprouty-2 gene body and promoter was analyzed using previously performed nano-hmC-seal data from colon cancers and adjacent normal colonic mucosa. Publicly available RNA-seq and methylation data from The Cancer Genome Atlas (TCGA) were extracted from tumors from CRC patients.

Results: Variable Sprouty-2 mRNA expression was observed among CRC patient tumors compared to adjacent normal appearing colonocytes. For the first time, differential methylation between adjacent normal colon and CRC samples was identified in 2 CTCF binding sites and in the promoter near the Transcriptional Start Site (TSS) of Sprouty-2, where the 5mC status in 1 of the CTCF binding sites correlated with transcript abundance. In CRC, this is the first study to reveal increases of 5hmC deposition in the gene body and promoter region of Sprouty-2 compared to normal colon. In metastatic CRC, an inverse correlation of increased Sprouty-2 promoter 5mC and decreased mRNA expression was found in patients registered in TCGA.

Conclusions: In terms of tumor growth, epigenetic aberrations including Sprouty-2 promoter hypomethylation and increased 5hmC may play a prominent role in positive regulation of Sprouty-2 in CRC. In metastatic stages of CRC, increases in Sprouty-2 promoter 5mC may downregulate Sprouty-2 expression, which may ultimately label Sprouty-2 as a tumor suppressor in CRC.

Citation Format: Alexei Stuckel, Sharad Khare. Epigenetic regulation of sprouty2 in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2089.
Abstract 2090: TP73 expression may be influenced by DNA methylation in human tumorigenesis
Authors: Yao Zhixing , Yao David R. , Yu William , Malki Ahmed , Sherif Zaki A.
Published: July 2021

Epigenetic events that cause alterations in gene expression are common in human cancers. The connection between TP73 expression and its epigenetic functionality in tumorigenesis has scarcely been explored. As a transcription factor, TP73 (aka, p73) like its cousin, p53, is activated in response to DNA damage and regulates the expression of downstream genes involved in cell cycle arrest and apoptosis. However, there are other compounding functions of this gene that reflect its non-tumor-related characteristics, thus making it very difficult to assess its specific role in tumorigenesis. Our previous work showed that DNA methylation activates TP73 expression in hepatocellular carcinoma (HCC) and gastrointestinal cancer. In this study, we determined TP73 expression in normal human mammary epithelial cells (HMECs), human non-cancerous Li-Fraumeni Syndrome (LFS) skin fibroblast cells, human breast cancer cell lines (MCF7, MDA-MB-231 & MDA-MB-468), prostate cancer cell lines (LNCaP & MDA-PCa2b), lung cancer cell lines (A549 & NCI-H596), neuroblastoma cell lines (IMR32 & SK-N-SH) and normal human skin fibroblast cell line (HS27). The results show that TP73 was expressed solely in cancer cells. Subsequent immunohistochemical analyses of TP73 expression in cancerous and adjacent non-cancerous liver tissues of HCC patients clearly showed that TP73 expression was exclusively evident in cancerous tissues but not in the adjacent normal liver tissues. Mechanistically, methylation-specific PCR and bisulfite sequencing studies revealed that TP73 promoter is activated only in cancer cells and this activation occurs via DNA methylation. Moreover, ChIP assays demonstrated that a CTCF (a chromosomal networking protein CCCTC binding factor) and tumor protein p53 (TP53) bind to TP73 promoter and regulate TP73 expression. Our observations demonstrate that a positive correlation in tumorigenesis exists between TP73 expression and DNA methylation in promoter regions of TP73. These findings suggest that TP73's role in carcinogenesis may prove to be pivotal for the development of future diagnostic and therapeutic applications.

Citation Format: Zhixing Yao, David R. Yao, William Yu, Ahmed Malki, Zaki A. Sherif. TP73 expression may be influenced by DNA methylation in human tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2090.
Abstract 2091: DNA methylation enhances apoptosis resistance of renal cancer cells
Authors: Miyakuni Kosuke , Nishida Jun , Ehata Shogo , Miyazono Kohei
Published: July 2021

Epigenetic modifications are important not only for carcinogenesis but also for metastasis of clear cell renal cell carcinoma (ccRCC). We previously reported that inflammation-related signaling is constitutively activated in advanced ccRCC through the formation of super enhancer. In addition to histone modification, methylation of the primary structure of DNA is implicated in the progression of ccRCC. Recent studies revealed that DNA hypermethylation was frequently observed in ccRCC and correlated with poor prognosis. However, unlike in other cancers, methylated genes responsible for cancer progression is still unclear in ccRCC. In this study, we tried to identify a target(s) for DNA methyltransferases (DNMTs) in ccRCC cells based on the genome-wide analysis. Its function for the regulation of cellular survival was examined. Our preclinical model of ccRCC using the serial orthotopic inoculation model exhibited the up-regulation of DNMT3B in advanced ccRCC. Pre-treatment of advanced ccRCC with 5-aza-dC attenuated a formation of a primary tumor through the induction apoptosis. DNA methylated sites were analyzed using methylation array in reference to RNA-sequencing data. As a result, ubiquinol cytochrome c reductase hinge protein (UQCRH), one of the components of mitochondrial complex III, was extracted as a methylation target in advanced ccRCC. Immunohistochemical analysis revealed that the expression of UQCRH in human ccRCC tissue was lower than normal adjacent tissue. Silencing of UQCRH attenuated the cytochrome c release in response to apoptotic stimuli and resulted in enhancement of primary tumor formation in vivo. Our results imply that DNA methylation induces the decreased expression of potential tumor suppressor gene UQCRH, which is essential for the completion of apoptotic process of ccRCC cells. These findings may contribute to the understanding of molecular mechanisms involved in the drug resistance of ccRCC cells and provide an important insight for the improvement of the efficacy of existing medicines.

Citation Format: Kosuke Miyakuni, Jun Nishida, Shogo Ehata, Kohei Miyazono. DNA methylation enhances apoptosis resistance of renal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2091.
Abstract 2102: Development and validation of Predicine methylation assay towards early cancer detection
Authors: Xiang Binggang , Dai Chao , Wang Ming , Zhao Zhixin , Wang Amy Xiaohong , Zhou Kemin , Jia Shidong , Luo Shujun , Du Pan
Published: July 2021

DNA methylation is one of the earliest signatures during cancer development. DNA methylation patterns from different cancer types can provide distinct epigenetic signatures. Several methods are available for sequencing DNA methylation throughout the genome, including whole genome bisulfite sequencing (WGBS) and antibody-dependent DNA immunoprecipitation (MeDIP). As a non-invasive approach, plasma cfDNA and urinary cfDNA (ucfDNA) have been widely explored for clinical applications. More recently, in virtue of its cancer-specific methylation signatures, cfDNA has been adopted for early cancer detection and further identification of cancer tissue of origin. Predicine has developed a liquid biopsy methylation assay that can robustly detect methylation using genomic and circulating cell-free DNA in body fluids. Predicine's methylation assay has great improvement over WGBS in reducing DNA damage, GC bias and increasing NGS reads mapping rate, and mapping quality score. In this study, we report the clinical utility of Predicine's methylation assay, which efficiently distinguishing healthy donors from patients of various types of cancer. Combined with its genomics NGS counterpart, Predicine's methylation platform is a powerful tool for early cancer detection and real-time monitoring of cancer progression.

Citation Format: Binggang Xiang, Chao Dai, Ming Wang, Zhixin Zhao, Amy Xiaohong Wang, Kemin Zhou, Shidong Jia, Shujun Luo, Pan Du. Development and validation of Predicine methylation assay towards early cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2102.
Abstract 2110: Platinum-inducedBRCA1 promoter DNA hypermethylation and altered metabolic profile promotes chemoresistance in ovarian cancer
Authors: Sood Riddhi , Xiao Sikai , Sriramkumar Shruthi , Hassel Christiane , Nephew Kenneth , O'Hagan Heather
Published: July 2021

High grade serous ovarian cancer (HGSOC) is the most common and aggressive type of ovarian cancer (OC). Platinum resistance is a common occurrence in HGSOC leading to tumor relapse and patient mortality. Multiple mechanisms contribute to platinum resistance in OC, including enrichment of ovarian cancer stem cells (OCSCs), which is observed in relapsed tumors. Platinum treatment activates the DNA damage response (DDR) and the DNA repair protein BRCA1 is an OC susceptibility factor. In our study, we demonstrate that platinum treatment induces a decrease in BRCA1 levels that leads to an increase in activity of ALDH, an OCSC marker. In a parallel pathway that is associated with G2/M arrest, platinum treatment also induces an increase in NAMPT expression and NAD+ levels, the cofactor required for ALDH activity. BRCA1 is repressed through platinum-induced promoter DNA hypermethylation. Importantly, combination of platinum with decitabine, a DNA methyltransferase (DNMT) inhibitor, prevents the platinum-induced increase in OCSCs. This study uniquely provides preclinical support for the use of DNMT inhibitors in the neoadjuvant setting in combination with platinum treatment for OC patients.

Citation Format: Riddhi Sood, Sikai Xiao, Shruthi Sriramkumar, Christiane Hassel, Kenneth Nephew, Heather O'Hagan. Platinum-inducedBRCA1 promoter DNA hypermethylation and altered metabolic profile promotes chemoresistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2110.
Abstract 2132: Ezh2-Y641F mutation interacts with Stat3 to regulate the melanoma tumor immune response via disruption of IFN signaling
Authors: Zimmerman Sarah M. , Nixon Samantha , Souroullas George P.
Published: July 2021

Cancer genetic sequencing studies have identified systematic alterations of epigenetic and chromatin modifiers in most cancers, consistent with the fact that chromatin modifiers, such as the Polycomb Repressive Complex 2 (PRC2), regulate the expression of tumor suppressors. In melanoma, 10% of patients exhibit amplifications or point mutations in EZH2, the enzymatic subunit of PRC2; however, the oncogenic mechanisms of EZH2 mutations in melanoma remain elusive. Previously, we observed that expression of Ezh2-Y641F in melanoma cells resulted in upregulation of IFN-related genes. We thus sought to better understand this mutation and the consequences of IFN signaling changes on tumor growth and the tumor immune response. We discovered that Ezh2-Y641F directly interacts with and methylates Stat3, a known regulator of interferon signaling. To understand the effects of this interaction, we performed ChIP-seq for Ezh2 and Stat3 in melanoma cells and found that the binding profiles of Stat3 and Ezh2 are greatly changed in Ezh2-Y641F cells. Furthermore, Ezh2 and Stat3 binding sites co-localized at several genomic loci in Ezh2-Y641F cells, including a region that regulates Ezh2. Knockdown of Stat3 in melanoma cells decreased Ezh2 expression and partially reduced expression of the IFN-related genes that were initially upregulated by Ezh2-Y641F. Analysis of the tumor-infiltrating immune cells by flow cytometry revealed increased CD8+ T cells and decreased myeloid cells in Ezh2-Y641F melanomas, consistent with a more favorable immune response, although tumor growth was unchanged. Genetic inhibition of Stat3 expression via shRNA rescued this phenotype. Our data suggest a novel interaction between the PRC2 complex and Stat3 in melanoma, which has significant physiological consequences on the tumor immune response. Molecular and genomic analysis suggest that Ezh2 and Stat3 together regulate gene expression, including Ezh2 itself. Overall, these data highlight the significant role of chromatin regulators on the tumor immune response.

Citation Format: Sarah M. Zimmerman, Samantha Nixon, George P. Souroullas. Ezh2-Y641F mutation interacts with Stat3 to regulate the melanoma tumor immune response via disruption of IFN signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2132.
Abstract 2135: Functional models of chromosome arm aneuploidies in lung squamous cell carcinoma
Authors: Taylor Alison Marie , Jain Sejal , Shih Juliann , Cherniack Andrew D. , Beroukhim Rameen , Meyerson Matthew
Published: July 2021

Aneuploidy, which we define as whole chromosome or chromosome arm copy number imbalance, is a near-universal characteristic of human cancers. Cancer subtypes are often characterized by tumor specific patterns of chromosome arm copy number alterations; for example, squamous cell carcinomas (SCCs) from different tissues of origin (including lung, head and neck, esophagus, and bladder) have a pattern of chromosome 3p loss and chromosome 3q gain. Although these alterations are frequent, they are not well understood due to difficulty in modeling specific aneuploidy alterations in the matching cell type. However, recent advances in genome engineering allow generation of large chromosomal alterations. We used the CRISPR-Cas9 system to delete one copy of chromosome 3p in human immortalized lung epithelial cells most similar to upper airway basal cells. Deletion of chromosome 3p was validated by whole genome sequencing and karyotyping. Consistent with patient data, expression of 3p genes was decreased upon deletion, as well as increased expression of interferon response genes. Phenotypic characterization revealed that cells with chromosome 3p deletion initially proliferated more slowly than their siblings. These chromosome 3p deleted cells had increased G1 arrest but did not undergo increased apoptosis or cell death. Interestingly, after several passages in culture, the proliferation defect was rescued in chromosome 3p deleted cells. Genome sequencing and karyotype analyses found that evidence of chromosome 3 duplication, transitioning the cell to a state of chromosome 3q gain. We isolated sibling cells with chromosome 3p deletion or chromosome 3q gain and demonstrated that duplication of chromosome 3 could indeed rescue proliferation rates. With our cellular model of chromosome arm-level aneuploidy, we uncovered a selection mechanism that allowed aneuploidy tolerance in vitro, and a possible explanation for joint alteration of both arms of chromosome 3. In conclusion, our genome engineering approach to model chromosome arm-level deletions provides a robust model that will address a gap in our understanding of aneuploidy in cancer.

Citation Format: Alison Marie Taylor, Sejal Jain, Juliann Shih, Andrew D. Cherniack, Rameen Beroukhim, Matthew Meyerson. Functional models of chromosome arm aneuploidies in lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2135.
Abstract 2137: Differential gene expression identifies a transcriptional regulatory network involving ESR1and PITX1 in invasive epithelial ovarian cancer
Authors: Li Yichao , Jaiswal Sushil Kumar , Kaur Rupleen , Alsaadi Dana , Liang Xiaoyu , Drews Frank , DeLoia Julie A. , Krivak Thomas , Petrykowska Hanna M. , Gotea Valer , Welch Lonnie , Laura Elnitski
Published: July 2021

The heterogeneous subtypes and stages of epithelial ovarian cancer (EOC) differ in invasive processes, but the molecular factors that drive aggressive behavior remain nebulous. In this study, we analyzed two types of EOC at opposite ends of the invasiveness spectrum, high-grade serous ovarian cancers (HGSOCs) and serous borderline tumors (SBTs). We identified differentially expressed genes to guide follow on studies of regulatory networks. Initially, in a discovery sample set, we generated transcriptome sequences to identify 11 differentially expressed genes in SBTs and HGSOCs, and a nonoverlapping set of 17 differentially expressed genes stage II and stage III HGSOCs. We validated the classification performance of these gene expression signatures on a validation set of SBT and HGSOC with classification rates of 94 and 98% using the 11-gene and 17-gene sets, respectively. Transcription factor binding sites enriched in the differentially expressed gene promoter regions were examined by Chip-seq analysis in ovarian cancer and other cell lines. We identified a suite of transcription factors implicated in the differential regulation associated with invasiveness, including ERα, RARA, PITX1, FOXA1/FOXF1, and BHLHE41/E40. These data implicate estrogen responsive gene regulatory networks in ovarian cancers and suggest that dynamic regulatory interactions determine malignant EOC outcomes.

Citation Format: Yichao Li, Sushil Kumar Jaiswal, Rupleen Kaur, Dana Alsaadi, Xiaoyu Liang, Frank Drews, Julie A. DeLoia, Thomas Krivak, Hanna M. Petrykowska, Valer Gotea, Lonnie Welch, Elnitski Laura. Differential gene expression identifies a transcriptional regulatory network involving ESR1and PITX1 in invasive epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2137.
Abstract 2140: Delineating a functional link between a phosphoserine aminotransferase (PSAT1)-associated gene signature and EGFR-mutant NSCLC
Authors: Biyik-Sit Rumeysa , Clem Brian F.
Published: July 2021

Phosphoserine aminotransferase 1 (PSAT1) catalyzes the second enzymatic step within the serine synthetic pathway and its expression is elevated in numerous human cancers, such as NSCLC. While PSAT1 suppression decreases SSP activity and cell proliferation, non-canonical activities have recently been suggested that may contribute to its pro-tumorigenic function, as is seen with other metabolic enzymes such as pyruvate kinase. Thus, we conducted a genome wide expression screen to uncover potential unknown cellular processes impacted by PSAT1 and to identify a PSAT1-associated gene signature that might inform patient outcomes in NSCLC. Loss of PSAT1 in EGFR-mutant PC9 lung cancer cells led to 491 differentially expressed genes (279-down; 212-up). KEGG and gene ontology analysis identified biological pathways such as metabolic processes, cell cycle regulation, cell adhesion, and cell motility. Transcript analysis confirmed inhibitory effects on the Rb/E2F1 and β-catenin pathways as well as expression of effectors on the actin cytoskeleton upon loss of PSAT1. Importantly, these could be rescued by PSAT1 restoration, including the loss of F-actin stress fibers as observed with phalloidin staining. Lastly, a bioinformatics approach utilizing transcriptomic data from human lung cancer patients was used to define genes altered between EGFR-mutant lung cancers and normal lung and that were differentially expressed in our PSAT1 depletion RNA-seq study. From this, we identified a PSAT1-associated gene signature that was then used to assign risk survival scores to these NSCLC patients. Subsequent, Kaplan Meier analysis demonstrated the prognostic ability of this gene signature for both relapse free and overall survival in EGFR-mutant NSCLC but was unable to predict risk groups in other NSCLC subtypes. Together, transcriptomic analysis under PSAT1 silencing identified its impact on various biological processes, including actin cytoskeleton rearrangement, and led to identification of a gene signature that has predictive ability towards survival outcomes in EGFR-mutant lung cancer.

Citation Format: Rumeysa Biyik-Sit, Brian F. Clem. Delineating a functional link between a phosphoserine aminotransferase (PSAT1)-associated gene signature and EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2140.
Abstract 2142: Retrospective analysis of 13160 clinically sequenced tumor samples reveals potential cancer drivers
Authors: Yan Cheng , Wu Hongwei , Yang Junhui , Guo Weihua , Yang Yufei
Published: July 2021

Background: High Throughput Sequencing (HTS) has generated tones of sequencing data that is almost unbearable to interpret. Adequate data mining strategy is in need to take huge volume of sequencing data and be able to derive clinically meaningful information. The positional distribution of somatic mutation on corresponding gene has strong implication of oncogenic involvement. We purpose a framework of cancer gene prioritization. Our aim is to derive candidate cancer genes by leveraging large volume of cancer samples.

Methods: We retrospectively analyzed 13160 clinically derived tumor samples consisting of five cancer types. These samples were previously subjected to captured sequencing covering either entire exome or major cancer related genes. To predict genes with oncogenic property, a modified version of 20/20 rule adapted from Bert's study was applied. After pan-cancer analysis, samples of each cancer type were analyzed separately to obtain cancer type specific candidate genes. Candidate genes uniquely predicted by our study were annotated in terms of molecular function and thoroughly assessed through literature curation.

Results: Using the modified 20/20 rule, pan-cancer analysis reported a total number of 186 candidate genes. 29 of them were uniquely reported by our analysis (absence in Bert's list, CGC list and our internal curation list) including 1 oncogene and 28 tumor suppressors. Their oncogenic involvements were further confirmed using random forest classifier 20/20+. 29 novel cancer candidate genes were significantly enriched in well-established cancer pathways described in Sanchez-Vega, et's study (pval<0.01). For instance, Ras GTPase-activating protein 1 (RASA1) is an inhibitory regulator of the Ras-cyclic AMP pathway. Our data revealed that RASA1 is enriched with inactivating mutations across its coding region: a typical mutational pattern of tumor suppressor.

However, literature mining showed limited understanding regarding the its oncogenic rule.

In cancer type specific analysis, 14 additional candidate cancer genes were uniquely reported by our analysis including known driver genes such as HDAC4 in gastric cancer and KLF5 in intestinal cancer. However, some reported candidate genes were less studied, such as oncogene WNT10A in liver cancer, probably due to low mutation frequency in western population (<0.5% according to TCGA). However, it mutated in 5.29% (59/1115) of our liver cancer cohort and highly recurrent mutations were identified at R171C and G213S.

Citation Format: Cheng Yan, Hongwei Wu, Junhui Yang, Weihua Guo, Yufei Yang. Retrospective analysis of 13160 clinically sequenced tumor samples reveals potential cancer drivers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2142.
Abstract 2144: STAT3 regulates the expression of XRCC1 in triple negative breast cancer
Authors: Wright Griffin M. , Gassman Natalie R.
Published: July 2021

Background: Base excision repair (BER) addresses base lesions and abasic sites induced by endogenous and exogenous environmental stresses. XRCC1 is a scaffold protein that facilitates and coordinates the BER repair machinery. Alterations in the protein and gene expression of XRCC1 have been observed in a multitude of cancers, including triple negative breast cancers (TNBC). Increased expression of XRCC1 is associated with aggressiveness and resistance to DNA damaging chemotherapeutics. However, only two transcription factors, E2F1 and Sp1 have been implicated in the transcriptional regulation of XRCC1 to date.

Methods: Utilizing promoter luciferase, transcription factor promoter binding ELISA, and chromatin immunoprecipitation (CHiP), STAT3 binding was identified and mapped in the XRCC1 promoter.

Results: In the current study, we identify a STAT3 binding site within the XRCC1 promoter. Importantly, STAT3 regulation of XRCC1 appears to be constitutive in TNBC cell line models where STAT3 is highly activated. In HEK293T cells, the STAT3 binding site shows low occupancy under basal growth condition, but STAT3 regulation of XRCC1 can be stimulated by cytokine or mitogenic signaling by IL-6 and epidermal growth factor (EGF).

Summary: STAT3 conditionally regulates XRCC1 under specific stress conditions to promote BER. In TNBC, the overexpression of XRCC1 by activated STAT3 likely contributes to increased resistance to DNA damaging chemotherapeutics.

Citation Format: Griffin M. Wright, Natalie R. Gassman. STAT3 regulates the expression of XRCC1 in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2144.
Abstract 2147: Discovering novel targets for adenoid cystic carcinoma of the lacrimal gland
Authors: Moeyersoms Acadia H. , Stathias Vasileios , Zhang Michelle , Gallo Ryan A. , Tse David T. , Pelaez Daniel
Published: July 2021

Lacrimal gland adenoid cystic carcinoma is a rare but highly lethal indolent cancer originating in the cells of secretory glands, primarily of the head and neck. With the 10-year survival rate of 20%, the only life extending technique is to remove the eye and surrounding socket contents entirely. Despite radical treatment, ACC is a very slow growing tumor and most patients will present with metastatic disease to the brain, lung, liver, or bones several years out from initial diagnosis and before symptoms are noticeable. Due to the rarity of LGACC, it is not well understood, but extensive genome analysis of ACC of the head and neck has previously uncovered a high mutation rate of MYB and NOTCH in patient samples, though unfortunately neither can be directly targeted by drugs. We have RNA sequencing data for 7 primary LGACC tumors, mass spectrometry data for 12 primary tumors and RNA sequencing for 15 cell lines derived from tumors to discover possible targetable biomarkers in LGACC and to further understand the biology of these tumors. Our analysis has revealed the signatures and drivers of LGACC, most significantly involving extracellular matrix mechanisms including HAPLN1, FABP7, and VCAN. We compared the genes of proteins differentially expressed in the mass spectrometry and RNA sequencing data and discovered 194 genes that were differentially expressed in both analysis types. With this extensive understanding of the molecular signatures, we used iLINCS integrated web-based platform to determine compounds that would target the differentially expressed genes. We have selected compounds based on a rubric we generated and will test them individually and in combination on LGACC cell lines to determine the best specific treatment for primary tumors and metastatic disease.

Citation Format: Acadia H. Moeyersoms, Vasileios Stathias, Michelle Zhang, Ryan A. Gallo, David T. Tse, Daniel Pelaez. Discovering novel targets for adenoid cystic carcinoma of the lacrimal gland [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2147.